AAV capsids comprising Anti-transferrin antibody domains

Abstract

The present disclosure pertains to targeting moieties, (e.g., CNS-targeting moieties) comprising single chain antibody agents which can be inserted in the capsid of a recombinant adeno- associated virus (rAAV) vector. Also disclosed herein are compositions comprising CNS-targeting moieties disclosed herein and methods of making and using the same.

Description

Attorney Docket No.: 2011256-2514 (P1862PCT01) AAV CAPSIDS COMPRISING ANTI-TRANSFERRIN ANTIBODY DOMAINS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No.63 / 570,601, filed on March 27, 2024, the entire contents of which is hereby incorporated by reference in its entirety. BACKGROUND

[0002] Targeted delivery of payloads (e.g., using recombinant adeno-associated viruses) to cells or tissues for treating and / or preventing diseases remains a challenge. SUMMARY

[0003] The present disclosure identifies certain challenges with existing targeted delivery of payloads. For example, the present disclosure identifies that a lack of targeting moieties that can specifically deliver payloads to a target cell or tissue with decreased non-specific delivery to other cells or tissues is a key challenge. Another challenge identified by the present disclosure is the poor potency associated with delivery vectors, e.g., recombinant adeno-associated virus vectors. In some embodiments, improving the potency of delivery vectors could be beneficial in obtaining clinically relevant outcomes.

[0004] Among other things, the present disclosure provides technologies that can address certain limitations identified in existing targeted delivery of payloads. The technologies provided herein are particularly useful for specifically delivering payloads to a target cell or tissue. In some embodiments, by specifically delivering payloads to a target cell or tissue, technologies provided herein can also increase the potency of a payload. In some embodiments, a target cell or tissue expresses one or more receptors (e.g., a human transferrin receptor (hTfR1)) recognized by a variant AAV capsid protein disclosed herein. In some embodiments, a target cell or tissue is a central nervous system (CNS) cell and / or tissue.

[0005] The present disclosure encompasses single chain antibody agents which recognize (e.g., bind to) a hTfR1 or a variant or a fragment thereof. Also disclosed herein are recombinant Page 1 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) adeno-associated virus (rAAV) particles comprising a variant AAV capsid protein comprising a single chain antibody agents which recognizes (e.g., binds to) a hTfR1 or a variant or a fragment thereof. In some embodiments, a targeting moiety in a variant AAV capsid protein is also referred to as a “peptide insertion.” In some embodiments, a single chain antibody agents binds to and / or recognizes a hTfR1 or a variant or a fragment thereof expressed on a cell (e.g., an endothelial cell associated with a blood brain barrier). In some embodiments, a single chain antibody agents which recognizes (e.g., binds to) a hTfR1 or a variant or a fragment thereof provides CNS-cell and / or tissue tropism. In some embodiments, rAAV particles comprising a variant capsid having a single chain antibody agents insertion disclosed herein bind to and / or recognize a target (e.g., hTfR1 or a variant or a fragment thereof) on a cell (e.g., an endothelial cell associated with a blood brain barrier). In some embodiments, binding of rAAV particles comprising a variant capsid having a single chain antibody agents insertion disclosed herein to hTfR1 or a variant or a fragment thereof on a cell, delivers the rAAV particles to said cells. In some embodiments, rAAV particles delivered to cells by binding to hTfR1 or a variant or a fragment thereof allows transcytosis of said rAAV particles across said cell (e.g., to be transported from one surface of said cell to another surface of said cell) into an organ, an intersitial space, and / or an interstitial fluid allowing for transduction of a cell and / or tissue. In some embodiments, delivery of a variant AAV capsid protein to an organ, to an intersitial space, and / or to an interstitial fluid results in transduction of a cell or tissue. Also disclosed herein are compositions comprising rAAV particles disclosed herein, and uses of the same.

[0006] In some embodiments, a single chain antibody agents inserted in an AAV capsid AAV capsid protein provides CNS-cell and / or tissue tropism. In some embodiments, rAAV particles comprising a variant capsid having a single chain antibody agents insertion disclosed herein bind to and / or recognize a target on a cell, e.g., a CNS cell and / or a cell in a CNS tissue, e.g., an endothelial cell associated with a blood brain barrier.

[0007] Without wishing to be bound by any particular theory, in some embodiments, variant AAV capsid proteins disclosed herein are particularly useful for targeting CNS cells and / or tissue expressing human Transferrin receptor (hTfR1) or a variant or a fragment thereof. In some embodiments, a variant AAV capsid protein disclosed herein binds to, e.g., specifically binds, to hTfR1 or a variant or a fragment thereof. Page 2 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0008] Further without wishing to be bound by any particular theory, in some embodiments, variant AAV capsid proteins disclosed herein can traverse a blood brain barrier (e.g., in a human subject) by binding to hTfR1 or a variant or a fragment thereof. In some embodiments, CNS cell and / or tissue tropism of a variant AAV capsid protein disclosed herein is a result of binding to hTfR1 or a variant or a fragment thereof.

[0009] Accordingly, in some embodiments, provided herein is an adeno-associated virus (rAAV) particle comprising: (a) a variant AAV capsid protein comprising a single chain antibody agent that binds to a transferrin receptor (TfR1) or a variant or a fragment thereof, wherein the single chain antibody agent is inserted in a variable region (VR) of a parental AAV capsid protein, and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.

[0010] In some embodiments, a single chain antibody agent comprises one or more complementarity determining regions (CDRs). In some embodiments, a single chain antibody agent comprises a CDR1, a CDR2 and a CDR3.

[0011] In some embodiments, a single chain antibody agent is or comprises a single domain antibody (e.g., a VHH).

[0012] In some embodiments, a single chain antibody agent is or comprises a single chain Fv.

[0013] In some embodiments, a single chain antibody agent is or comprises single chain antibody agent comprises a sequence provided in Table 1. In some embodiments, a single chain antibody agent comprises: (i) a CDR1 sequence provided in Table 1; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a CDR1 sequence provided in Table 1; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to a CDR1 sequence provided in Table 1; (ii) a CDR2 sequence provided in Table 1; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a CDR2 sequence provided in Table 1; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to a CDR2 sequence provided in Table 1; and / or (iii) a CDR3 sequence provided in Table 1; or a sequence with at least 85%, at Page 3 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a CDR3 sequence provided in Table 1; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to a CDR3 sequence provided in Table 1.

[0014] In some embodiments, a single chain antibody agent comprises: (i) a CDR1 sequence of SEQ ID NO: 2; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 2; (ii) a CDR2 sequence of SEQ ID NO: 3; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 3; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 3; and / or (iii) a CDR3 sequence of SEQ ID NO: 4; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 4; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 4.

[0015] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 2; (ii) a CDR2 sequence of SEQ ID NO: 3; and (iii) a CDR3 sequence of SEQ ID NO: 4.

[0016] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 1, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0017] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 158, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0018] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 159, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at Page 4 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0019] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 160, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0020] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 6; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 6; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 6; (ii) a CDR2 sequence of SEQ ID NO: 7; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 7; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 7; and / or (iii) a CDR3 sequence of SEQ ID NO: 8; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 8; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 8.

[0021] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 6; (ii) a CDR2 sequence of SEQ ID NO: 7; and (iii) a CDR3 sequence of SEQ ID NO: 8.

[0022] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 5, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0023] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 122, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. Page 5 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0024] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 135, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0025] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 136, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0026] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 10; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 10; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 10; (ii) a CDR2 sequence of SEQ ID NO: 11; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 11; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 11; and / or (iii) a CDR3 sequence of SEQ ID NO: 12; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 12; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 12.

[0027] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 10; (ii) a CDR2 sequence of SEQ ID NO: 11; and (iii) a CDR3 sequence of SEQ ID NO: 12.

[0028] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 9, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0029] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 14; or a sequence with at least 85%, at least 86%, at least 87%, at least Page 6 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 14; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 14; (ii) a CDR2 sequence of SEQ ID NO: 15; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 15; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 15; and / or (iii) a CDR3 sequence of SEQ ID NO: 16; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 16; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 16.

[0030] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 14; (ii) a CDR2 sequence of SEQ ID NO: 15; and (iii) a CDR3 sequence of SEQ ID NO: 16.

[0031] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 13, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0032] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 18; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 18; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 18; (ii) a CDR2 sequence of SEQ ID NO: 19; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 19; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 19; and / or (iii) a CDR3 sequence of SEQ ID NO: 20; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at Page 7 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 20; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 20.

[0033] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 18; (ii) a CDR2 sequence of SEQ ID NO: 19; and (iii) a CDR3 sequence of SEQ ID NO: 20.

[0034] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 17, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0035] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 22; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 22; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 22; (ii) a CDR2 sequence of SEQ ID NO: 23; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 23; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 23; and / or (iii) a CDR3 sequence of SEQ ID NO: 24; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 24; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 24.

[0036] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 22; (ii) a CDR2 sequence of SEQ ID NO: 23; and (iii) a CDR3 sequence of SEQ ID NO: 24.

[0037] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 21, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. Page 8 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0038] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 26; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 26; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 26; (ii) a CDR2 sequence of SEQ ID NO: 27; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 27; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 27; and / or (iii) a CDR3 sequence of SEQ ID NO: 28; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 28; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 28.

[0039] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 26; (ii) a CDR2 sequence of SEQ ID NO: 27; and (iii) a CDR3 sequence of SEQ ID NO: 28.

[0040] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 25, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0041] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 30; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 30; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 30; (ii) a CDR2 sequence of SEQ ID NO: 31; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 31; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 31; and / or (iii) a CDR3 sequence of SEQ ID NO: 32; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at Page 9 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 32; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 32.

[0042] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 30; (ii) a CDR2 sequence of SEQ ID NO: 31; and (iii) a CDR3 sequence of SEQ ID NO: 32.

[0043] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 29, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0044] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 34; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 34; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 34; (ii) a CDR2 sequence of SEQ ID NO: 35; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 35; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 35; and / or (iii) a CDR3 sequence of SEQ ID NO: 36; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 36; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 36.

[0045] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 34; (ii) a CDR2 sequence of SEQ ID NO: 35; and (iii) a CDR3 sequence of SEQ ID NO: 36.

[0046] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 33, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. Page 10 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0047] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 38; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 38; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 38; (ii) a CDR2 sequence of SEQ ID NO: 39; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 39; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 39; and / or (iii) a CDR3 sequence of SEQ ID NO: 40; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 40; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 40.

[0048] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 38; (ii) a CDR2 sequence of SEQ ID NO: 39; and (iii) a CDR3 sequence of SEQ ID NO: 40.

[0049] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 37, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0050] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 42; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 42; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 42; (ii) a CDR2 sequence of SEQ ID NO: 43; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 43; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 43; and / or (iii) a CDR3 sequence of SEQ ID NO: 44; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at Page 11 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 44; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 44.

[0051] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 42; (ii) a CDR2 sequence of SEQ ID NO: 43; and (iii) a CDR3 sequence of SEQ ID NO: 44.

[0052] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 41, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0053] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 46; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 46; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 46; (ii) a CDR2 sequence of SEQ ID NO: 47; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 47; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 47; and / or (iii) a CDR3 sequence of SEQ ID NO: 48; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 48; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 48.

[0054] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 46; (ii) a CDR2 sequence of SEQ ID NO: 47; and (iii) a CDR3 sequence of SEQ ID NO: 48.

[0055] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 45, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. Page 12 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0056] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 50; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 50; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 50; (ii) a CDR2 sequence of SEQ ID NO: 51; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 51; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 51; and / or (iii) a CDR3 sequence of SEQ ID NO: 52; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 52; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 52.

[0057] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 50; (ii) a CDR2 sequence of SEQ ID NO: 51; and (iii) a CDR3 sequence of SEQ ID NO: 52.

[0058] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 49, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0059] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 2; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 2; (ii) a CDR2 sequence of SEQ ID NO: 117; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 117; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 117; and / or (iii) a CDR3 sequence of SEQ ID NO: 4; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least Page 13 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 4; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 4.

[0060] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 2; (ii) a CDR2 sequence of SEQ ID NO: 117; and (iii) a CDR3 sequence of SEQ ID NO: 4.

[0061] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 116, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0062] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 2; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 2; (ii) a CDR2 sequence of SEQ ID NO: 119; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 119; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 119; and / or (iii) a CDR3 sequence of SEQ ID NO: 4; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 4; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 4.

[0063] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 2; (ii) a CDR2 sequence of SEQ ID NO: 119; and (iii) a CDR3 sequence of SEQ ID NO: 4.

[0064] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 118, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. Page 14 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0065] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 2; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 2; (ii) a CDR2 sequence of SEQ ID NO: 15; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 15; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 15; and / or (iii) a CDR3 sequence of SEQ ID NO: 52; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 52; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 52.

[0066] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 2; (ii) a CDR2 sequence of SEQ ID NO: 15; and (iii) a CDR3 sequence of SEQ ID NO: 52.

[0067] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 120, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0068] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 2; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 2; (ii) a CDR2 sequence of SEQ ID NO: 119; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 119; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 119; and / or (iii) a CDR3 sequence of SEQ ID NO: 52; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at Page 15 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 52; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 52.

[0069] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 2; (ii) a CDR2 sequence of SEQ ID NO: 119; and (iii) a CDR3 sequence of SEQ ID NO: 52.

[0070] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 121, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0071] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 124; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 124; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 124; (ii) a CDR2 sequence of SEQ ID NO: 125; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 125; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 125; and / or (iii) a CDR3 sequence of SEQ ID NO: 8; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 8; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 8.

[0072] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 124; (ii) a CDR2 sequence of SEQ ID NO: 125; and (iii) a CDR3 sequence of SEQ ID NO: 8.

[0073] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 123, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. Page 16 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0074] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 127; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 127; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 127; (ii) a CDR2 sequence of SEQ ID NO: 7; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 7; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 7; and / or (iii) a CDR3 sequence of SEQ ID NO: 128; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 128; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 128.

[0075] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 127; (ii) a CDR2 sequence of SEQ ID NO: 7; and (iii) a CDR3 sequence of SEQ ID NO: 128.

[0076] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 126, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0077] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 130; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 130; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 130; (ii) a CDR2 sequence of SEQ ID NO: 7; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 7; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 7; and / or (iii) a CDR3 sequence of SEQ ID NO: 131; or a sequence with at least 85%, at least 86%, at least 87%, at Page 17 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 131; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 131.

[0078] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 130; (ii) a CDR2 sequence of SEQ ID NO: 7; and (iii) a CDR3 sequence of SEQ ID NO: 131.

[0079] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 129, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0080] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 6; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 6; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 6; (ii) a CDR2 sequence of SEQ ID NO: 133; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 133; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 133; and / or (iii) a CDR3 sequence of SEQ ID NO: 134; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 134; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 134.

[0081] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 6, (ii) a CDR2 sequence of SEQ ID NO: 133; and (iii) a CDR3 sequence of SEQ ID NO: 134.

[0082] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 132, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto. Page 18 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0083] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 6; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 6; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 6; (ii) a CDR2 sequence of SEQ ID NO: 125; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 125; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 125; and / or (iii) a CDR3 sequence of SEQ ID NO: 134; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 134; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 134.

[0084] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 6, (ii) a CDR2 sequence of SEQ ID NO: 125; and (iii) a CDR3 sequence of SEQ ID NO: 134.

[0085] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 137, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0086] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 130; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 130; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 130; (ii) a CDR2 sequence of SEQ ID NO: 7; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 7; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 7; and / or (iii) a CDR3 sequence of SEQ ID NO: 139; or a sequence with at least 85%, at least 86%, at least 87%, at Page 19 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 139; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 139.

[0087] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 130, (ii) a CDR2 sequence of SEQ ID NO: 7; and (iii) a CDR3 sequence of SEQ ID NO: 139.

[0088] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 138, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0089] In some embodiments, a single chain antibody agent binds to a human TfR1 (hTfR1), or a cyno TfR1. In some embodiments, a single chain antibody agent binds to a human TfR1. In some embodiments, a single chain antibody agent binds to cyno TfR1.

[0090] In some embodiments, a single chain antibody agent binds to hTfR1 with an affinity (KD) of about 10 nM to about 2500 nM, e.g., when fused to an Fc domain or when inserted in a variable region (VR) of a parental AAV capsid protein.

[0091] In some embodiments, a single chain antibody agent comprises one or more linkers. In some embodiments, one or more linkers are situated on the N-terminus of the single chain antibody agent and / or the C-terminus of the single chain antibody agent.

[0092] In some embodiments, one or more linkers comprise one or more (GGGGS) repeats. In some embodiments, one or more linkers comprise one or more (GGGGS) repeats on the N-terminus of the single chain antibody agent and / or the C-terminus of the single chain antibody agent.

[0093] In some embodiments, one or more linkers comprise a coiled-coil alpha helix domain or a fragment thereof.

[0094] In some embodiments, a single chain antibody agent comprises one or more coiled-coil alpha helix domains on the N-terminus of the single chain antibody agent and / or the C-terminus of the single chain antibody agent. Page 20 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0095] In some embodiments, a coiled-coil alpha helix domain comprises a leading coil sequence according to SEQ ID NO: 53, or a sequence with at least 90% identity thereto.

[0096] In some embodiments, a coiled-coil alpha helix domain comprises a returning coil sequence according to SEQ ID NO: 54, or a sequence with at least 90% identity thereto.

[0097] In some embodiments, a single chain antibody agent comprises a (GGGGS)5linker according to SEQ ID NO: 59 at the N-terminus and a (GGGGS)1linker according to SEQ ID NO: 55 at the C-terminus.

[0098] In some embodiments, a single chain antibody agent comprises a (GGGGS)4linker according to SEQ ID NO: 58 at the N-terminus.

[0099] In some embodiments, a single chain antibody agent comprises: (i) a leading coiled-coil alpha helix domain and a (GGGGS)5linker according to SEQ ID NO: 60 at the N- terminus; and (ii) a (GGGGS)1linker and a returning coiled-coil alpha helix domain according to SEQ ID NO: 61 at the C-terminus.

[0100] In some embodiments, a single chain antibody agent comprises a linker according to SEQ ID NO: 65, e.g., at the N terminus or the C terminus.

[0101] In some embodiments, a single chain antibody agent insertion site is located between two adjacent amino acids in the variable region of the parental AAV capsid protein.

[0102] In some embodiments, a single chain antibody agent insertion site is located between two non-adjacent amino acids in the variable region of the parental AAV capsid protein.

[0103] In some embodiments, a single chain antibody agent insertion site is in a VP1 of the parental AAV capsid protein. In some embodiments, a single chain antibody agent insertion site is not in a VP2 and / or VP3 of the parental AAV capsid protein.

[0104] In some embodiments, a single chain antibody agent insertion site is in a VP1, VP2 and / or VP3 of the parental AAV capsid protein.

[0105] In some embodiments, insertion of the single chain antibody agent replaces a contiguous stretch of amino acids of the parental AAV capsid protein.

[0106] In some embodiments, insertion of the single chain antibody agent does not replace a contiguous stretch of amino acids of the parental AAV capsid protein. Page 21 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0107] In some embodiments, a single chain antibody agent insertion is in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX of the parental AAV capsid protein.

[0108] In some embodiments, a single chain antibody agent insertion is in VR-IV of the parental AAV capsid protein. In some embodiments, a single chain antibody agent insertion site is in a VP1 of the parental AAV capsid protein. In some embodiments, a single chain antibody agent insertion site is not in a VP2 and / or VP3 of the parental AAV capsid protein.

[0109] In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein and VR-IV comprises amino acids 452-460 of VP1, VP2, or VP3 of the AAV9 capsid protein.

[0110] In some embodiments, a single chain antibody agent insertion site is located between amino acids: (1) 454 and 461of VP1 of the AAV9 capsid protein, (2) 454 and 457 of VP1 of the AAV9 capsid protein; or (3) 451 and 460 of VP1 of the AAV9 capsid protein.

[0111] In some embodiments, a single chain antibody agent insertion is in VR-VIII of the parental AAV capsid protein. In some embodiments, a single chain antibody agent insertion site is in a VP1 of the parental AAV capsid protein. In some embodiments, a single chain antibody agent insertion site is not in a VP2 and / or VP3 of the parental AAV capsid protein.

[0112] In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein and VR-VIII comprises amino acids 580 to 601 of VP1, VP2, or VP3 of the AAV9 capsid protein.

[0113] In some embodiments, a single chain antibody agent insertion site is located between amino acids 588 and 590 of VP1 of an AAV9 capsid protein or the corresponding position in VP1 of another parental AAV capsid protein.

[0114] This disclosure further provides a composition comprising a rAAV particle disclosed herein an a pharmaceutically acceptable excipient.

[0115] In some embodiments, provided herein is an isolated cell transduced with a rAAV particle disclosed herein.

[0116] Further disclosed herein is a an isolated nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a single chain antibody agent that binds to a transferrin receptor (TfR) or a variant or a Page 22 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) fragment thereof, and wherein the single chain antibody agent is inserted in a variable region (VR) of a parental AAV capsid protein.

[0117] In some embodiments, a single chain antibody agent insertion is in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX of the parental AAV capsid protein.

[0118] In some embodiments, a single chain antibody agent insertion is in VR-I of the parental AAV capsid protein.

[0119] In some embodiments, a single chain antibody agent insertion is in VR-II of the parental AAV capsid protein.

[0120] In some embodiments, a single chain antibody agent insertion is in VR-III of the parental AAV capsid protein.

[0121] In some embodiments, a single chain antibody agent insertion is in VR-IV of the parental AAV capsid protein.

[0122] In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein and VR-IV comprises amino acids 452 to 460 of VP1, VP2, or VP3 of the AAV9 capsid protein.

[0123] In some embodiments, a single chain antibody agent insertion is in VR-V of the parental AAV capsid protein.

[0124] In some embodiments, a single chain antibody agent insertion is in VR-VI of the parental AAV capsid protein.

[0125] In some embodiments, a single chain antibody agent insertion is in VR-VII of the parental AAV capsid protein.

[0126] In some embodiments, a single chain antibody agent insertion is in VR-VIII of the parental AAV capsid protein.

[0127] In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein and VR-VIII comprises amino acids 580 to 601 of VP1, VP2, or VP3 of the AAV9 capsid protein.

[0128] In some embodiments, provided herein is an isolated cell comprising a nucleic acid disclosed herein. Page 23 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0129] This disclosure provides a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a single chain antibody agent that binds to a transferrin receptor (TfR) or a variant or a fragment thereof, and wherein the single chain antibody agent is inserted in a variable region (VR) of a parental AAV capsid protein. In some embodiments, a single chain antibody agent insertion is in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX of the parental AAV capsid protein. In some embodiments, a single chain antibody agent insertion is in VR-IV of the parental AAV capsid protein. In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein and VR-IV comprises amino acids 452 to 460 of VP1, VP2, or VP3 of the AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is in VR-VIII of the parental AAV capsid protein. In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein and VR-VIII comprises amino acids 580 to 601 of VP1, VP2, or VP3 of the AAV9 capsid protein.

[0130] Also provided herein is a method of delivering a payload to a CNS cell and / or tissue, comprising administering a pharmaceutical composition disclosed herein to a CNS cell and / or tissue.

[0131] Further provided herein is a composition comprising a rAAV particle disclosed herein for use in delivering a payload to a CNS cell and / or tissue. In some embodiments, delivering comprises administering a composition to a CNS cell and / or tissue.

[0132] This disclosure provides use of a composition comprising a rAAV particle disclosed herein in the manufacture of a medicament for delivering a payload to a CNS cell and / or tissue. In some embodiments, delivering comprises administering a composition to a CNS cell and / or tissue.

[0133] In some embodiments, a CNS cell and / or tissue is in vitro.

[0134] In some embodiments, a CNS cell and / or tissue is in vivo.

[0135] In some embodiments, a CNS cell and / or tissue is from a subject that has, or has been determined to have, a CNS disorder.

[0136] In some embodiments, a CNS cell or tissue is chosen from: a CNS epithelial cell, an endothelial cell associated with a blood brain barrier, a nerve cell, a CNS connective tissue cell, a stem cell, a progenitor cell, a CNS immune cell, a spinal cord cell, a cell that lines one or Page 24 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) more brain ventricles, a nerve support cell, a glial cell, a fat cell, a meninges cell, or any combination thereof.

[0137] In some embodiments, a CNS cell comprises an endothelial cell associated with a blood brain barrier. In some embodiments, an endothelial cell associated with a blood brain barrier is part of, or forms a blood brain barrier. In some embodiments, an endothelial cell associated with a blood brain barrier is or comprises a brain capillary endothelial cell (BCEC). In some embodiments, an endothelial cell associated with a blood brain barrier is or comprises a brain microvascular endothelial cell (BMEC). In some embodiments, an endothelial cell associated with a blood brain barrier expresses a hTfR1 or a fragment or variant thereof.

[0138] In some embodiments, a CNS cell comprises a CNS epithelial cell. In some embodiments, a CNS epithelial cell comprises a cell that lines one or more brain ventricles.

[0139] In some embodiments, a CNS cell comprises a nerve cell (neuron). In some embodiments, a neuron comprises a unipolar neuron, a bipolar neuron, a pseudounipolar neuron, a multipolar neuron, or combinations thereof. In some embodiments, a neuron is a motor neuron, a sensory neuron, an interneuron, an excitatory neuron, an inhibitor neuron, a sympathetic neuron, a parasympathetic neuron, or combinations thereof. In some embodiments, a neuron comprises a pyramidal neuron, a dopaminergic neuron, a cholinergic neurons, an adrenergic neuron, a GABAergic neuron, a glutamatergic neuron, a serotonergic neuron, a purinergic neuron, a histaminergic neuron, a lower motor neuron, or combinations thereof.

[0140] In some embodiments, a nerve cell (neuron) comprises nerve support cells. In some embodiments, nerve support cells comprise glial cells. In some embodiments, glial cells comprise astrocytes, microglial cells, ependymal cells, oligodendrocytes, Schwann cells, or combinations thereof.

[0141] In some embodiments, a CNS cell comprises a CNS connective tissue cell. In some embodiments, a CNS connective tissue cell comprises fat cells or meninges cells, or both.

[0142] In some embodiments, a CNS cell comprises a stem cell or progenitor cell. In some embodiments, a stem cell comprises a neural stem cell.

[0143] In some embodiments, a CNS cell comprises a cell that lines one or more brain ventricles. Page 25 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0144] In some embodiments, a CNS cell comprises a meninges cell.

[0145] In some embodiments, a CNS cell comprises a fat cell.

[0146] In some embodiments, a CNS tissue comprises tissue found in: cortex, thalamus, hypothalamus, striatum, putamen, caudate nucleus, hippocampus, entorhinal cortex, basal ganglia, deep cerebellar nuclei, or other parts of the brain and / or spinal cord.

[0147] In some embodiments, CNS tissue comprises tissue found in a frontal cortex, a parietal cortex, an occipital cortex, a temporal cortex or combinations thereof.

[0148] In some embodiments of any of the AAV particles comprising a variant AAV capsid protein, compositions comprising a rAAV particle, or methods of using the same disclosed herein, a variant AAV capsid protein confers increased infectivity and / or transduction of a central nervous system (CNS) cell and / or tissue compared to the infectivity and / or transduction of the CNS cell and / or tissue by a control AAV particle comprising a corresponding parental AAV capsid protein.

[0149] In some embodiments, a rAAV particle comprising a variant AAV capsid protein confers at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 4-fold, at least 5- fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 120-fold, at least 140- fold, at least 160-fold, at least 180-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900- fold, at least 1000-fold, at least 1500-fold, at least 2000-fold, at least 4000-fold increased increased infectivity and / or transduction of a CNS cell compared to: (1) infectivity and / or transduction of a CNS cell by a control AAV particle comprising a corresponding parental AAV capsid protein; or (2) infectivity and / or transduction of a CNS cell which does not express a receptor recognized by a variant AAV capsid protein (e.g., hTfR1) with variant AAV capsid protein.

[0150] In some embodiments, a rAAV particle comprising a variant AAV capsid protein confers about 1.5-fold, about 2-fold, about 2.5-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20- Page 26 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) fold, about 25-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 120-fold, about 140-fold, about 160-fold, about 180-fold, about 200-fold, about 250-fold, about 300-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, about 1000-fold, about 1500- fold, about 2000-fold, about 4000-fold, increased infectivity and / or transduction of a CNS cell or tissue compared to: (1) infectivity and / or transduction of a CNS cell by a control AAV particle comprising a corresponding parental AAV capsid protein; or (2) infectivity and / or transduction of a CNS cell which does not express a receptor recognized by a variant AAV capsid protein (e.g., hTfR1) with variant AAV capsid protein.

[0151] In some embodiments, a rAAV particle comprising a variant AAV capsid protein confers 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15- fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 120- fold, 140-fold, 160-fold, 180-fold, 200-fold, 250-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 1500-fold, 2000-fold, 4000-fold, increased infectivity and / or transduction of a CNS cell or tissue compared to: (1) infectivity and / or transduction of a CNS cell by a control AAV particle comprising a corresponding parental AAV capsid protein; or (2) infectivity and / or transduction of a CNS cell which does not express a receptor recognized by a variant AAV capsid protein (e.g., hTfR1) with variant AAV capsid protein.

[0152] In some embodiments of any of the variant AAV capsid proteins, AAV particles comprising a variant AAV capsid protein, compositions comprising a variant AAV capsid protein, or methods of using the same, a variant AAV capsid protein comprises one or more modifications to an amino acid sequence flanking a single chain antibody agent insertion. In some embodiments, one or more modifications are within about 10 amino acids upstream or downstream of the location of a single chain antibody agent insertion site, e.g., within about 5 amino acids upstream or downstream of the location of the single chain antibody agent insertion site.

[0153] In some embodiments, one or more modifications comprises an insertion, deletion, mutation, or combinations thereof. Page 27 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0154] In some embodiments, a deletion is a deletion of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 amino acids. In some embodiments, a deletion is a deletion of about 1 to about 10 amino acids.

[0155] In some embodiments, when the insertion is in VR-IV of a parental AAV capsid protein the deletion comprises a deletion of amino acids 455 to 460 inclusive of endpoints of VP1 of the AAV9 capsid protein. In some embodiments, when the insertion is in VR-IV of a parental AAV capsid protein the deletion comprises a deletion of amino acids 455 and 456 of VP1 of the AAV9 capsid protein. In some embodiments, when the insertion is in VR-IV of a parental AAV capsid protein the deletion comprises a deletion of amino acids 452 to 459 inclusive of endpoints of VP1 of the AAV9 capsid protein.

[0156] In some embodiments, the single chain antibody agent insertion is in VR-IV of the parental AAV capsid protein and (a) the single chain antibody agent insertion site is located between amino acids 454 and 461 of VP1 of the AAV9 capsid protein; and (b) the deletion is a deletion of amino acids 455 to 460 inclusive of endpoints of VP1 of the AAV9 capsid protein.

[0157] In some embodiments, the single chain antibody agent insertion is in VR-IV of the parental AAV capsid protein and (a) the single chain antibody agent insertion site is located between amino acids 454 and 457 of VP1 of the AAV9 capsid protein; and (b) the deletion is a deletion of amino acids 455 and 456 of VP1 of the AAV9 capsid protein.

[0158] In some embodiments, the single chain antibody agent insertion is in VR-IV of the parental AAV capsid protein and (a) the single chain antibody agent insertion site is located between amino acids 451 and 460 in of VP1 of the AAV9 capsid protein; and (b) the deletion is a deletion of amino acids 452 to 459 inclusive of endpoints of VP1 of the AAV9 capsid protein.

[0159] In some embodiments, when the insertion is in VR-VIII of the parental AAV capsid protein the deletion comprises a deletion of amino acid 589 of VP1 of the AAV9 capsid protein.

[0160] In some embodiments, the single chain antibody agent insertion is in VR-IV of the parental AAV capsid protein and (a) the single chain antibody agent insertion site is located between amino acids 588 and 590 of VP1 of the AAV9 capsid protein; and (b) the deletion is a deletion of amino acid 588 of VP1 of the AAV9 capsid protein. Page 28 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0161] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, one or more modifications are located in a variable region of parental AAV capsid protein. In some embodiments, a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, one or more modifications are located in an AAV9 capsid protein variable region, e.g., VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX, or any combination thereof.

[0162] In some embodiments, one or more modifications are located in: VR-VIII of a VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, one or more modifications are located in: VR-VIII of a VP1, but not in a VP2 and / or VP3 of an AAV9 capsid protein. In some embodiments, one or more modifications are located in: VR-VIII of a VP2 and / or VP3 of an AAV9 capsid protein but not in a VP1.

[0163] In some embodiments, one or more modifications are located in: VR-IV of a VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, one or more modifications are located in: VR- IV of a VP1, but not in a VP2 and / or VP3 of an AAV9 capsid protein. In some embodiments, one or more modifications are located in: VR- IV of a VP2 and / or VP3 of an AAV9 capsid protein but not in a VP1.

[0164] In some embodiments, a variant AAV capsid protein further comprises one or more modifications to an amino acid sequence that is at or near a glycan binding region. In some embodiments, one or more modifications reduces glycan binding. In some embodiments, a glycan is galactose. By way of example, glycan binding residues of an AAV9 capsid protein include, but are not limited to: (a) amino acids 271 and 272 of a VP1, VP2 or VP3; (b) amino acid 446 of a VP1, VP2 or VP3; (c) amino acid 470 of a VP1, VP2 or VP3; (d) amino acids 501 to 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2 or VP3; or (e) amino acids 489 and 545 of a VP1, VP2 or VP3. Based on the exemplary glycan binding domains and / or residues provided herein for an AAV9 capsid protein, those with knowledge in the pertinent field would be able to readily ascertain the corresponding glycan binding domain and / or residues in a different parental AAV capsid protein.

[0165] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, one or more modifications is at a glycan binding domain of a parental AAV capsid protein. In some embodiments, a parental AAV capsid protein Page 29 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) is an AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10 capsid protein. In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein.

[0166] In some embodiments, one or more modifications is at or between amino acids: (a) 271 and 272 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (b) 446 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (c) 470 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (e) 489 and 545 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (f) 591 and 621 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; or (g) any combination or all of (a)-(f).

[0167] In some embodiments of a rAAV particle, a variant AAV capsid protein, a composition, or a method disclosed herein, one or more modifications is at or between amino acids: (a) 271 and 272 of a VP1, VP2 or VP3 of an AAV9 capsid protein; (b) 446 of a VP1, VP2 or VP3 of an AAV9 capsid protein; (c) 470 of a VP1, VP2 or VP3 of an AAV9 capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2 or VP3 of an AAV9 capsid protein; (e) 489 and 545 of VP1of a VP1, VP2 or VP3 of an AAV9 capsid protein; (f) 591 and 621 of a VP1, VP2 or VP3 of an AAV9 capsid protein; or (g) any combination or all of (a)-(f).

[0168] In some embodiments of any of the variant AAV capsid proteins, AAV particles comprising a variant AAV capsid protein, compositions comprising a variant AAV capsid protein, or methods of using the same, a variant AAV capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity relative to a parental AAV capsid protein. Page 30 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0169] In some embodiments, percent identity is determined by comparing the sequence of a variant AAV capsid protein without a single chain antibody agent insertion, with a parental AAV capsid protein.

[0170] In some embodiments, a variant AAV capsid protein and a parental AAV capsid protein have 100% identity when: (a) a single chain antibody agent insertion in a variant AAV capsid protein is not taken into account in the sequence comparison; and (b) a variant AAV capsid protein does not have one or more modifications other than a single chain antibody agent insertion.

[0171] In some embodiments, a variant AAV capsid protein and a parental AAV capsid protein have less than 100% identity when: (a) a single chain antibody agent insertion in a variant AAV capsid protein is not taken into account in the sequence comparison; and (b) a variant AAV capsid protein comprises one or more modifications other than a single chain antibody agent insertion.

[0172] In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein of SEQ ID NO: 2001.

[0173] In some embodiments, a parental AAV capsid protein is an AAV1 capsid protein of SEQ ID NO: 2002.

[0174] In some embodiments, a parental AAV capsid protein is an AAV2 capsid protein of SEQ ID NO: 2003.

[0175] In some embodiments, a parental AAV capsid protein is an AAV3B capsid protein of SEQ ID NO: 2007.

[0176] In some embodiments, a parental AAV capsid protein is an AAV4 capsid protein of SEQ ID NO: 2008.

[0177] In some embodiments, a parental AAV capsid protein is an AAV5 capsid protein of SEQ ID NO: 2004.

[0178] In some embodiments, a parental AAV capsid protein is an AAV6 capsid protein of SEQ ID NO: 2005. Page 31 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0179] In some embodiments, a parental AAV capsid protein is an AAV8 capsid protein of SEQ ID NO: 2006.

[0180] In some embodiments, a parental AAV capsid protein is an AAV7 capsid protein of SEQ ID NO: 2009.

[0181] In some embodiments, a parental AAV capsid protein is an AAV10 capsid protein of SEQ ID NO: 2010.

[0182] In some embodiments, a parental AAV capsid protein is an AAV11 capsid protein of SEQ ID NO: 2011.

[0183] In some embodiments, a parental AAV capsid protein is an AAV12 capsid protein of SEQ ID NO: 2012.

[0184] In some embodiments, a parental AAV capsid protein is an AAV13 capsid protein of SEQ ID NO: 2013.

[0185] In some embodiments, a parental AAV capsid protein is an AAVhu68 capsid protein of SEQ ID NO: 2014.

[0186] In some embodiments, a parental AAV capsid protein is an AAVrh10 capsid protein of SEQ ID NO: 2015.

[0187] In some embodiments of any of the AAV particles comprising a variant AAV capsid protein, compositions comprising a rAAV particle, or methods of using the same disclosed herein, a payload is or comprises a polypeptide that is encoded by a nucleic acid sequence within a rAAV particle.

[0188] In some embodiments, a polypeptide is or comprises a CRISPR-Cas protein, or a variant or fragment thereof. In some embodiments, a CRISPR-Cas protein is chosen from : a Type II, Type V or Type VI CRISPR-Cas protein (e.g., a Cas9 protein), a Cas12a protein, a Cas12b protein, a Cas12c protein, a Cas12d protein, a Cas12e protein, a Cas12f protein, a Cas12g protein, a Cas12h protein, a Cas12i protein, a Cas13a protein, a Cas13b protein or a variant or fragment of any of the foregoing. In some embodiments, the payload also comprises a guide RNA, gRNA, sgRNA, or crRNA / tracrNA that interacts with the CRISPR-Cas protein. In some embodiments a CRISPR-Cas protein is fused with one or more domains, e.g., an activator Page 32 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) domain and / or a repressor domain. In some embodiments a CRISPR-Cas protein is a nuclease. In some embodiment a CRISPR-Cas protein is a nickase and only cleaves one strand of a target nucleic acid molecule. In some embodiment a CRISPR-Cas protein is inactivated and binds to but does not cleave a target nucleic acid molecule.

[0189] In some embodiments, a polypeptide is or comprises a Zinc finger protein, or a variant or fragment thereof. In some embodiments, a Zinc finger protein is chosen from: a Zinc finger nuclease, an artificial restriction enzyme fusion protein, a sequence-targeted zinc-finger DNA-binding unit optionally fused with a nuclease domain (e.g., Fok1 nuclease domain), or a variant or fragment or any combination of any of the foregoing. In some embodiments a Zinc figner protein is fused with one or more domains, e.g., an activator domain and / or a repressor domain.

[0190] In some embodiments, a polypeptide is or comprises a Transcription Activator- Like Effector (TAL) protein, or a variant or fragment thereof. In some embodiments, a TAL comprises: a TAL effector DNA binding domain (e.g., a TAL effector DNA binding domain isolated from Xanthomonas spp.), a Transcription Activator-Like Effector Nuclease (TALEN), e.g., a TAL effector DNA binding domain fused with a nuclease domain (e.g., Fok1 nuclease domain), or a variant or fragment or any combination of any of the foregoing. In some embodiments a TAL protein is fused with one or more domains, e.g., an activator domain and / or a repressor domain.

[0191] In some embodiments, a polypeptide is or comprises a base editor, or a variant or fragment thereof. In some embodiments, a base editor comprises a deaminase, an adenosine deaminase enzyme (ABE), a cytosine deaminase enzyme (CBE), an APOBEC1, an APOBEC3A, an APOBEC3G, an evoAPOBEC, a BE4-YE1, a CDA1, an activation-induced cytidine deaminase (AID), a mutant TadA, an adenosine deaminases (TadA*), an E. coli tRNA-specific adenosine deaminase (TadA), a deaminase associated with a DNA binding domain monomer, a base editing enzyme that is RNA guided, a DNA glyosylase inhibor, one or more DNA glycosylase inhibitor domains, a 5-methylcytosine deaminase, a cytidine deaminase domain, an adenine deaminase domain, an adenosine base editor (ABE), a Target-ACEmax, a synchronous programmable adenine and cytosine editor (SPACE), an A&C-Bemax., a circularly permuted base editor, an adenosine deaminase enzyme (ADAR), a RNA editing for programmable Page 33 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) adenosine to inosine replacement (REPAIR), a leveraging endogenous ADAR for programmable editing of RNA (LEAPER) or a variant or fragment or combination of any of the foregoing. In some embodiments, the payload also comprises a guide RNA, gRNA, sgRNA, or crRNA / tracrNA that interacts with the base editor.

[0192] In some embodiments, a polypeptide is or comprises a prime editor, or a variant or fragment thereof, or a system comprising the same. In some embodiments, a prime editor and / or system comprising the same comprises: a reverse transcriptase, a prime editing enzyme, an editing enzyme that includes a reverse transcriptase domain, an Avian Myeloblastosis Virus (AMV) Reverse Transcriptase, a Murine Leukemia Virus (MLV) Reverse Transcriptase, a HIV- 1 reverse transcriptase, a bacterial reverse transcriptase, a reverse transcriptase associated with a DNA binding domain and / or protein, a reverse transcriptase fused to a DNA binding domain that is a catalytically impaired nuclease domain (e.g., a nickase), a prime editing 1 system (PE1), a prime editing 2 system (PE2), a prime editing 3 system (PE3), a prime editing 3b system (PE3b) or a variant or fragment or any combination of any of the foregoing. In some embodiments, the payload also comprises a prime editing gRNA (pegRNA) or an extended sgRNA that interacts with the prime editor.

[0193] In some embodiments, a polypeptide is or comprises a meganuclease, or a variant or fragment thereof. In some embodiments, a meganuclease is chosen from: a homing endonuclease, a LAGLIDADG family meganuclease, a GIYYIG family meganuclease, a His- Cyst box family meganuclease, or HNH family endonuclease, an I-SeeI, an I-CeuI, a PI-PspI, a PI-SceI, an I-SceIV, an I-CsmI, an I-PanI, an I-SceII, an I-PpoI, an I-SceIII, an I-CreI, an I-TevI, an I-TevII an I-TevIII or a variant or fragment or any combinations of any of the foregoing.

[0194] In some embodiments, a polypeptide is associated with a CNS disorder.

[0195] In some embodiments, a CNS disorder is a result of a genetic abnormality.

[0196] In some embodiments, a CNS disorder is a not a result of a genetic abnormality.

[0197] In some embodiments, a CNS disorder is chosen from: Friedreich’s Ataxia, Dravet Syndrome, Spinocerebellar Ataxia Type 3, Niemann-Pick Type C, Huntington’s Disease, Pompe Disease, Myotonic Dystrophy Type 1, Glut1 Deficiency Syndrome (De Vivo Syndrome), Tay-Sachs, Spinal Muscular Atrophy, Alzheimer's disease, Amyotrophic lateral Page 34 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) sclerosis (ALS), Danon disease, Rett Syndrome, Angelman Syndrome, Parkinson’s disease, tauopathies, genetic epilepsies (e.g., a STXBP1 genetic epilepsy, or a CDKL5 genetic epilepsy), or combinations thereof.

[0198] In some embodiments, a polypeptide is an enzyme.

[0199] In some embodiments, a polypeptide is an antibody.

[0200] In some embodiments, a polypeptide is a secreted protein.

[0201] In some embodiments of any of the rAAV particles comprising a variant AAV capsid protein, compositions comprising a rAAV particle, or methods of using the same disclosed herein, a payload is or comprises an RNA molecule that is encoded by a nucleic acid sequence within a rAAV particle. In some embodiments, an RNA molecule is or comprises an siRNA, a miRNA, a gRNA, an antisense RNA, a circular RNA, an snRNA or an aptamer.

[0202] In some embodiments, an RNA molecule targets a nucleic acid molecule encoding a polypeptide associated with a CNS disorder.

[0203] In some embodiments of any of the rAAV particles comprising a variant AAV capsid protein, compositions comprising a rAAV particle, or methods of using the same disclosed herein, a payload is or comprises a DNA molecule. In some embodiments, a DNA molecule is or comprises a nucleic acid sequence of up to about 5,100 nt in length, e.g., up to about 5,000 nt, up to about 4,900, up to about 4,800, up to about 4,700, up to about 4,600, up to about 4,500, up to about 4,400, etc.

[0204] In some embodiments of any of the rAAV particles comprising a variant AAV capsid protein, compositions comprising a rAAV particle, or methods of using the same disclosed herein, a nucleotide sequence encoding a payload is operably linked to a promoter.

[0205] In some embodiments, a promoter is a CNS promoter or a variant or a fragment thereof. In some embodiments, a CNS promoter is chosen from: a GFAP promoter, a SYN1 promoter, a NSE / RU5’ promoter, a neuroactive peptide cholecystokinin (CCK) promoter, a myelin basic promoter (MBP), a human myelin associated glycoprotein promoter, a phosphate- activated glutaminase (PAG) promoter, a vesicular glutamate transporter (vGLUT) promoter, a glutamic acid decarboxylase (GAD) promoter, Camk2a promoter, TH (tyrosine hydroxylase) Page 35 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) promoter, Hb9 promoter, CNP promoter, NES (nestin) promoter, Tub1a promoter, SST (somatostatin) promoter, MeCP2 promoter, or combinations thereof.

[0206] In some embodiments, a promoter is or comprises a chicken beta actin hybrid (CBh) promoter or a variant or a fragment thereof.

[0207] In some embodiments, a promoter is or comprises a hSyn1 promoter or a variant or a fragment thereof.

[0208] In some embodiments, a promoter is or comprises a GFAP promoter or a variant or a fragment thereof.

[0209] In some embodiments of any of the methods of using a pharmaceutical composition comprising a rAAV particle comprising a variant AAV capsid protein disclosed herein, a pharmaceutical composition is administered via a route of administration chosen from: intravenous, intraarterial, intracoronary, intraparenchymal, subpial, intrathecal, intraocular, intracerebroventricular (ICV), intracisternal magna (ICM), or intramuscular.

[0210] In some embodiments, a subject is a human.

[0211] Also provided herein is a method of treating a subject having a CNS disorder and / or ameliorating a symptom of a CNS disorder in a subject, the method comprising administering to the subject a pharmaceutical composition disclosed herein.

[0212] In some embodiments, a CNS disorder is chosen from Friedreich’s Ataxia, Dravet Syndrome, Spinocerebellar Ataxia Type 3, Niemann-Pick Type C, Huntington’s Disease, Pompe Disease, Myotonic Dystrophy Type 1, Glut1 Deficiency Syndrome (De Vivo Syndrome), Tay- Sachs, Spinal Muscular Atrophy, Alzheimer’s disease, Amyotrophic lateral sclerosis (ALS), Danon disease, Rett Syndrome, Angelman Syndrome, Parkinson’s disease, tauopathies, genetic epilepsies (e.g., a STXBP1 genetic epilepsy, or a CDKL5 genetic epilepsy), or combinations thereof.

[0213] Other features, objects, and advantages of the present invention are apparent in the detailed description that follows. It should be understood, however, that the detailed description, while indicating embodiments of the present invention, is given by way of illustration only, not limitation. Various changes and modifications within the scope of the invention will become apparent to those skilled in the art from the detailed description. Page 36 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) BRIEF DESCRIPTION OF THE DRAWING

[0214] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings(s) will be provided by the Office upon request and payment of the necessary fee.

[0215] The Figures described below, which together make up the Drawing, are for illustration purposes only, not for limitation.

[0216] FIGS.1A-1C show schematics depicting single variant and barcoded library production schemes for AAV particles displaying VHHs in the capsid (also referred to herein as “AAV-VHH”). FIG.1A shows a schematic detailing the standard triple transfection method for AAV single capsid production. FIG.1B shows a schematic detailing the quadruple transfection method for AAV-VHH production. FIG.1C shows a schematic detailing the transfection method for barcoded AAV-VHH library production, with each capsid packaging a mixture of cis-plasmids consisting of two CAG-driven and two hSyn1-driven H2B-GFP transgenes with unique 16-nt barcodes embedded in the 3’-UTR.

[0217] FIGS.2A-2B show an illustration of designs for inserting an exemplary VHH in an AAV9 VP1 subunit. * indicates ablation of VP2 or VP3 start codons. Bars with a dash represent GGGGS linker. Crossed bars represent VHH sequences. Bars with a circle represent 18-amino acid alpha helix sequences that are predicted to form a coiled-coil structure. Blank bars represent wild-type AAV9 VP1 sequence.

[0218] FIG.3 shows a bar graph depicting transduction of AAV-VHH library variants in WT and TfR1 KO HEK293T cells. Each bar represents a unique capsid, with linker designs designated by numbers on the x-axis, with data grouped by VHH. Y-axis represents Log10 fold enhancement in CAG-driven transgene expression of a AAV capsid displaying an exemplary VHH over AAV9 in WT HEK293T cells (closed bars), and TfR1 KO HEK293T cells (open bars).

[0219] FIG.4 shows an illustration of TfR1 constructs used with in vivo testing of AAV- VHHs. Human, cyno, and murine TfR1 domains are represented as hashed, black, and white regions, respectively.

[0220] FIGS.5A-5B show graphs depicting relative AAV-VHH capsid transduction over AAV9 in WT and hTfR1-KI+ / +mouse models, as determined by barcode expression driven by Page 37 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) CAG or hSyn1 promoter normalized by barcode fractions in the input AAV library. Each symbol represents a unique capsid in the barcoded AAV9-VHH library. Plots show brain transduction enhancement over AAV9 for capsid variants in (FIG.5A) WT mice, and (FIG.5B) hTfR1-KI+ / +model. Linker designs are shown in FIGS.6A-6B.

[0221] FIGS.6A-6B show bar graphs depicting AAV-VHH brain transduction fold enhancement over AAV9 based on CAG-driven barcode expression in (FIG.6A) WT, and (FIG. 6B) hTfR1-KI+ / +models. X axis indicates linker design, and unique anti-TfR VHHs are grouped.

[0222] FIGS.7A-7B depict relative AAV-VHH capsid brain transduction over AAV9 in cynomolgus macaque. FIG.7A: Each symbol represents a unique capsid in the barcoded AAV9- VHH library. X- and y-axes represent CAG- and hSyn1-driven barcode expression enhancement over AAV9 in the brain, respectively. Linker designs are shown in FIG.7B. FIG.7B shows AAV-VHH brain transduction fold enhancement over AAV9 based on CAG-driven barcode expression in the cyno model. X axis indicates linker design, and unique anti-TfR VHHs are grouped.

[0223] FIGS.8A-8B show transgene expression and vector genome biodistribution mediated by Clone A VHH-D2 (Design 2), Clone A VHH-D7 (Design 7) and AAV9 in WT mice. Each symbol represents a biological replicate. FIG.8A represents normalized mCherry mRNA fold enhancement over AAV9 in different tissue types. FIG.8B represents vector genome copies per cell in different tissue types.

[0224] FIGS.9A-9B show mCherry immunohistochemistry images on sagittal brain sections of AAV9, Clone A VHH-D2, or Clone A VHH-D7 treated WT mice. Scale bars represent 5 mm. FIG.9A is in color and FIG.9B is in black and white.

[0225] FIGS.10A-10B show mCherry immunohistochemistry images in brain cortex of AAV9, Clone A VHH-D2, or Clone A VHH-D7 treated WT mice. Scale bars represent 200 microns. FIG.10A is in color and FIG.10B is in black and white.

[0226] FIGS.11A-11B show mCherry immunohistochemistry images in hippocampus of AAV9, Clone A VHH-D2, or Clone A VHH-D7 treated WT mice. Scale bars represent 1 mm. FIG.11A is in color and FIG.11B is in black and white. Page 38 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0227] FIGS.12A-12B show mCherry immunohistochemistry images in cerebellum of AAV9, Clone A VHH-D2, or Clone A VHH-D7 treated WT mice. Scale bars represent 200 microns. FIG.12A is in color and FIG.12B is in black and white.

[0228] FIGS.13A-13B show normalized mCherry mRNA fold enhancement and vector genome copies per cell for different AAV-VHH clones over AAV9 in brain, spinal cord, heart, quad, liver, and spleen (for vector genome only) hTfR1-KI+ / +mice. FIG.13A shows normalized mCherry mRNA fold enhancement and FIG.13B shows vector genome copies per cell. Each dot represents a biological replicate.

[0229] FIGS.14A-14B shows mCherry immunohistochemistry on sagittal brain sections of AAV9, Clone A VHH-D2, Clone A VHH-D7, or Clone B VHH-D1 treated hTfR1-KI+ / +mice. Scale bars represent 5 mm. FIG.14A is in color and FIG.14B is in black and white.

[0230] FIGS.15A-15B shows mCherry immunohistochemistry images in cortical regions of AAV9, Clone A VHH-D2, Clone A VHH-D7, or Clone B VHH-D1 treated hTfR1- KI+ / +mice. Scale bars represent 250 microns. FIG.15A is in color and FIG.15B is in black and white.

[0231] FIGS.16A-16B show mCherry immunohistochemistry images in hippocampus of AAV9, Clone A VHH-D2, Clone A VHH-D7, or Clone B VHH-D1 treated hTfR1-KI+ / +mice. Scale bars represent 1 mm. FIG.16A is in color and FIG.16B is in black and white.

[0232] FIGS.17A-17B show mCherry immunohistochemistry images in cerebellar cortex of AAV9, Clone A VHH-D2, Clone A VHH-D7, or Clone B VHH-D1 treated hTfR1- KI+ / +mice. Scale bars represent 500 microns. FIG.17A is in color and FIG.17B is in black and white.

[0233] FIGS.18A-18B show schematics depicting cryo-EM structures of human TfR1 in complex with Clone A VHH and human holo transferrin. FIG.18A shows a schematic depicting a cryo-EM density map of human TfR1 in complex with Clone A VHH and transferrin at 2.4 Å resolution. FIG.18B shows a schematic depicting an atomic model of a human TfR1 / Clone A VHH / transferrin complex.

[0234] FIG.19 shows a schematic depicting an atomic model of Clone A VHH highlighting paratope residues. O atoms are in white, and N atoms are in black.

[0235] FIG.20 shows a partial alignment of TfR1 apical and helical domain sequences encompassing a predicted Clone A VHH epitope. Amino acid residues within 4 Å of Clone A Page 39 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) VHH, as observed in Clone A VHH-huTfR1 ECD cryo-EM structures, are highlighted in black, with a subset of residues from the other TfR1 protomer (monomer) underlined. The two residues divergent between cyno and human TfR1 within the Clone A VHH epitope are in italics. Strictly conserved residues are denoted by asterisk, strongly conserved residues by colon, and moderately conserved residues by period. Sources of sequences: Human (Homo sapiens, Uniprot P02786.2); Cynomolgus (Macaca fascicularis, NCBI Reference Sequence: XP_045243212.1); Mouse (Mus Musculus, Uniprot Q62351).

[0236] FIG.21 shows a schematic depicting an atomic model of a Clone A VHH-human TfR1 ECD complex, highlighting amino acid residues divergent between cyno and human TfR1 ECD (R325, G724) as well as Clone A VHH residues in close contact (D53, Q99, V101).

[0237] FIGS.22A-22B show schematics depicting a cryo-EM structure of human TfR1 in complex with Clone B VHH and human holo transferrin. FIG.22A shows a schematic depicting a cryo-EM density map of transferrin receptor in complex with Clone B VHH and transferrin at 2.7 Å resolution. FIG.22B shows a schematic depicting an atomic model of TfR1 / Clone B VHH / transferrin complex.

[0238] FIG.23 shows a partial alignment of TfR1 apical and helical domain sequences encompassing a predicted Clone B VHH epitope. The residues within 4 Å of Clone B VHH, as observed in Clone B VHH-huTfR1 ECD cryo-EM structures, are highlighted in black, with a subset of residues from the other TfR1 protomer (monomer) underlined. One residue divergent between cyno and human TfR1 within a Clone B VHH epitope is in italics. Strictly conserved residues are denoted by asterisk, strongly conserved residues by colon, and moderately conserved residues by period. Sources of sequences: Human (Homo sapiens, Uniprot P02786.2); Cynomolgus (Macaca fascicularis, NCBI Reference Sequence: XP_045243212.1); Mouse (Mus Musculus, Uniprot Q62351).

[0239] FIGS.24A-24D depict plots demonstrating monovalent VHH-Fc fusion binding to CHO cells stably expressing the human (circle), cyno (square), murine (triangle), or TfR1 knock-out (diamond) cell lines. EC50 values for (FIG.24A) Clone A VHH, (FIG.24B) Clone B VHH, and (FIG.24C) Clone D VHH binding were determined by a 3-parameter logarithmic fit in the GraphPad Prism software and are summarized in Table 7. FIG.24D shows a schematic depicting the structure of a monovalent VHH-Fc fusion. Page 40 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0240] FIG.25 shows plots depicting kinetic curves for monovalent VHH-Fc binding to surface-immobilized, His-tagged human, cyno, and mouse TfR1 ectodomain. KD values were determined using global fits with a 1:1 kinetics model or with a steady state analysis model (Table 7). Monovalent VHH-Fc concentrations decrease from top to bottom in a 4-fold dilution. Top concentrations are 4000 nM for Clone A VHH and Clone B VHH, 800 nM for Clone D VHH.

[0241] FIG.26 shows a graph depicting correlation between monovalent VHH-TfR1 affinity and AAV-VHH neuronal transduction. Each symbol represents an AAV9 capsid displaying an exemplary VHH. X-axis represents monovalent KDs of the corresponding VHHs against human (circles), cyno (squares), or murine (triangles) TfR1 homologs. Y-axis represents hSyn1-driven transgene expression fold enhancement of exemplary AAV-VHH over AAV9 in the brains of corresponding animal models.

[0242] FIG.27 are graphs showing the results of bio-layer interferometry (BLI) binding analysis to assess binding of exemplary AAV-VHH capsids to recombinant human or cyno TfR1 ectodomain (ECD). Each curve represents an increasing concentration of human or cyno TfR1 ECD (2.7, 8.2, 24.7, 74.1, 222, 667, 2000 nM) in solution. The constructs used were: Clone A- VHH-D2, Clone B-VHH-D1, Clone F-VHH-D2 and Clone M VHH-D2. Data was fit with a 1:2 bivalent analyte model.

[0243] FIG.28 is a correlation plot comparing exemplary VHH-Fc SPR affinity (KD) to human or cynoTfR1 vs. exemplary AAV-VHH BLI affinity (KD) to human or cynoTfR1. Data is presented on a log-log scale with an x=y dashed line. In both the VHH and AAV context, the clones tested were Clones A, B, D and H and in the context of AAV-VHH the construct designs are similar to those in FIG.27. Binding affinity to cynoTfR1 is represented as squares and affinity to human TfR1 is represented as circles.

[0244] FIG.29 is a graph showing transduction by exemplary AAV-VHHs in TfR1 knock-out CHO cells expressing human, cyno or murine TfR1 homologs. Fold transduction in the y-axis is shown as compared to transduction with AAV9.

[0245] FIG.30 is a graph showing transduction by exemplary AAV-VHHs in the brian of B-hTfR1 mice. Transgene expression was driven by either the CAG promoter or hSyn1 promoter. Fold transduction in the y-axis is shown as compared to transduction with AAV9. Page 41 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0246] FIG.31 is a graph showing transduction by exemplary AAV-VHHs in the brian of WT mice. Transgene expression was driven by either the CAG promoter or hSyn1 promoter. Fold transduction in the y-axis is shown as compared to transduction with AAV9.

[0247] FIG.32 is a graph showing vector genome biodistribution in B-hTfR1 mouse brain transdueced with exemplary AAV-VHHs.

[0248] FIG.33 is a graph showing RNA levels of an exemplary transgene in the brain of B-hTfR1 animals administered exemplary AAV-VHHs. Exemplary transgene expression levels in the y-axis are shown in comparison to expression levels of a control RNA, mRPP30, and normalized to expression of the exemplary transgene in the liver of animals administered AAV particles having Clone B-VHH-D1.

[0249] FIG.34 is a graph showing the percentage of cortical neurons positive for expression of an exemplary transgene delivered with exemplary AAV-VHHs in B-hTfR1 mouse brain.

[0250] FIG.35 are panels of representative images showing RNAscope in situ hybridization (ISH) and NeuN antibody immunofluorescent staining in the brain of B-hTfR1 mice administered AAV particles comprising Clone B VHH-D1. The images show AAV-VHH mediated transgene expression in B-hTfR1 mouse brain. The brain slices were stained with an ISH probe recognizing WPRE (as a surrogate for AAV vector; red), a NeuN antibody to label neurons (teal), and DAPI for nuclear labeling; blue. The middle row shows WPRE signal, the top row shows a merge of WPRE ISH and NeuN and DAPI immunofluorescence signals. and the bottom row highlights zoomed-in regions of the cortex outlined in the middle row.Scale bar is 1 mm.

[0251] FIG.36 is a graph showing vector genome biodistribution in the spinal cord of B- hTfR1 mice administered exemplary AAV-VHHs.

[0252] FIG.37 is a graph showing RNA levels of an exemplary transgene in the spinal cord of B-hTfR1 animals administered exemplary AAV-VHHs Exemplary transgene expression levels in the y-axis are shown in comparison to expression levels of a control RNA, murine RPP30 (mRPP30), and normalized to expression of the exemplary transgene in the liver of animals administered AAV particles having Clone B-VHH-D1. Page 42 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0253] FIG.38 is a graph showing vector genome biodistribution in the liver of B-hTfR1 mice administered exemplary AAV-VHHs.

[0254] FIG.39 is a graph showing RNA levels of an exemplary transgene in the liver of B-hTfR1 animals administered exemplary AAV-VHHs. Exemplary transgene expression levels in the y-axis are shown in comparison to expression levels of a control RNA, mRPP30, and normalized to expression of the exemplary transgene in the liver of animals administered AAV particles having Clone B-VHH-D1.

[0255] FIG.40 is a graph showing vector genome biodistribution in various tissues of NHPs administered exemplary AAV-VHHs or AAV9 intravenously. The exemplary AAV- VHHs comprised Clone F VHH-D2 or Clone M VHH-D2. Vector genome distribution from AAV9 (without VHH insertion) is shown as a reference. AAV9 was administered to 10-month old NHPs; and AAV-VHHs were administered to 3+ years old NHPs.

[0256] FIG.41 is a graph showing vector genome biodistribution in various tissues of NHPs administered an exemplary AAV-VHH having Clone B VHH-D1 dosed at two different doses: 3.3 x 10(13) vg / kg (open squares) or 1.1 x 10(14) vg / kg (open triangles). Data from animals administered AAV9 (without VHH insertion) is shown as a reference. AAV9 was administered to 10-month old NHPs; and AAV-VHH was administered to 3+ years old NHPs.

[0257] FIG.42 is a graph showing RNA levels of the WPRE element as a surrogate for transgene expression in NHP administered exemplary AAV-VHHs having Clone F VHH-D2 or Clone M VHH-D2. WPRE RNA expression in various tissue from NHPs administered exemplary AAV-VHHs is compared to WPRE RNA expression in the prefrontal cortex in NHPs administered AAV particles comprising an AAV9 capsid (without VHH) and normalized to expression of a control RNA, hRPP30. AAV9 was administered to 10-month old NHPs; and AAV-VHHs were administered to 3+ years old NHPs.

[0258] FIG.43 is a graph showing RNA levels of the WPRE element as a surrogate for transgene expression in NHP administered exemplary AAV-VHHs having Clone B VHH-D1 in NHPs at two doses: 3.3 x 10(13) vg / kg (open squares) or 1.1 x 10(14) vg / kg (open triangles). WPRE RNA expression in various tissue from NHPs administered exemplary AAV-VHHs is compared to WPRE RNA expression in the prefrontal cortex in NHPs administered AAV particles comprising an AAV9 capsid (without VHH) and normalized to expression of a control Page 43 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) RNA, hRPP30. AAV9 was administered to 10-month old NHPs; and AAV-VHH was administered to 3+ years old NHPs. DEFINITIONS

[0259] In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and / or”; (iii) the terms “comprising” and “including” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps; (iv) the terms “about” and “approximately” may be understood to permit standard variation as would be understood by those of ordinary skill in the art; and (v) where ranges are provided, endpoints are included.

[0260] 5’ and 3’: The terms “5’” and “3’” are relative terms to define a spatial relationship or directionality between two or more segment of a nucleic acid sequence. Thus, 3’ of a nucleic acid indicates a segment of the nucleic acid that is downstream of another segment, while 5’ indicates a segment of the nucleic acid that is upstream of another segment. For example, 3’ may indicate that a segment is in the 3’ half of the nucleic acid sequence or even at the 3’ end of the nucleic acid sequence. Similarly, 5’ may indicate that a segment is in the 5’ half of the nucleic acid sequence or even at the 5’ end of the nucleic acid sequence. Unless indicated otherwise, the directionality of a nucleic acid will be in the 5’ to 3’ direction of translation.

[0261] About or approximately: As used herein, the terms “approximately” or “about” in reference to a number are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0262] Adeno-associated virus (AAV): As used herein, the terms “Adeno-associated virus” and “AAV” refer to viral particles, in whole or in part, of the family Parvoviridae and the genus Dependoparvovirus. AAV is a small replication-defective, nonenveloped virus. AAV includes, but is not limited to, AAV serotype 1, AAV serotype 2, AAV serotype 3 (including serotypes 3A and 3B), AAV serotype 4, AAV serotype 5, AAV serotype 6, AAV serotype 7, AAV serotype 8, AAV serotype 9, AAV serotype 10, AAV serotype 11, AAV serotype 12, AAV Page 44 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) serotype 13, snake AAV, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, goat AAV, shrimp AAV, non-human primate AAV, e.g., from rhesus monkeys, and any variant of any of the foregoing. Wild-type AAV is replication deficient and requires co-infection of cells by a helper virus, e.g., adenovirus, herpes, or vaccinia virus, e.g., an Ad2 or Ad5 virus, or supplementation of helper viral genes, in order to replicate.

[0263] Ad2 helper: As used herein, the term “Ad2 helper” refers to the Adenovirus serotype 2 (Ad2) helper virus (e.g., wildtype or recombinantly engineered Ad2 helper virus) and various Ad2 helper genes and / or Ad2 helper polypeptides or nucleic acids, including, but not limited, to E1a, E1b, E2a, E4Orf6, VA RNA, and any variant or fragment of any of the foregoing. In some embodiments, an Ad2 helper vector (e.g., plasmid) encodes Ad2 helper polypeptides or nucleic acids (e.g., one, two, three, or four of E1 (e.g., E1a and / or E1b), E2a, E4, or VA RNA) necessary to generate functional rAAV particles. In certain embodiments, the Ad2 helper vector is transfected into an E1 complementing cell line (e.g., HEK293). The nucleotide sequence of an Ad2 helper vector and Ad2 helper virus genes can be derived from the Adenovirus 2 genome (GenBank Accession No. J01917.1).

[0264] Ad5 helper: As used herein, the term “Ad5 helper” refers to the Adenovirus serotype 5 (Ad5) helper virus (e.g., wildtype or recombinantly engineered Ad5 helper virus) and various Ad5 helper genes and / or Ad5 helper polypeptides or nucleic acids, including, but not limited, to E1a, E1b, E2a, E4Orf6, and / or VA RNA. In some embodiments, an Ad5 helper vector (e.g., plasmid) encodes Ad5 helper polypeptides or nucleic acids (e.g., one, two, three, or four of E1 (e.g., E1a and / or E1b), E2a, E4, or VA RNA) necessary to generation functional rAAV particles. In certain embodiments, the Ad5 helper vector is transfected into an E1 complementing cell line (e.g., HEK293). The nucleotide sequence of an Ad5 helper vector and Ad5 helper genes can be derived from the Adenovirus 5 genome (GenBank Accession No. AY601635).

[0265] Administration: As used herein, the term “administration” refers to the administration of a composition comprising rAAV particles as described herein to a subject. Administration may be by any appropriate route. For example, in some embodiments, administration may be local or systemic administration (e.g., to a mammal, e.g., to a human, e.g., a patient). A composition of the disclosure may be administered by injection or infusion by any route. For example, a composition may be administered by retinal, subretinal, intravitreal, Page 45 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) suprachoroidal, intraspinal, intracisternal magna, or intrathecal injection or infusion. Additional exemplary routes of administration may include, but are not limited to, bronchial (e.g., bronchial instillation), buccal, enteral, interdermal, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., intratracheal instillation), transdermal, vaginal, and vitreal.

[0266] Bioreactor: The term “bioreactor,” as used herein, refers to any vessel used for the growth of a cell culture (e.g., a mammalian cell culture). The bioreactor can be of any size and / or any shape so long as it is useful for culturing a cell culture (e.g., a mammalian cell culture).

[0267] Cap polypeptide: As used herein, the term “Cap polypeptide” refers to the structural proteins that form a functional AAV capsid, which can in turn package DNA and infect or transduce a target cell. In some embodiments, a Cap polypeptide comprises a variant AAV capsid protein as disclosed herein. In some embodiments, Cap polypeptides will comprise all of the AAV capsid subunits, but less than all of the capsid subunits may be present as long as a functional capsid is produced. In some embodiments, the nucleic acid sequence encoding Cap polypeptides will be present on a single vector (e.g., plasmid). In some embodiments, the nucleic acid sequence encoding Cap polypeptides will be present on more than one vector (e.g., plasmid), e.g., VP1 encoded by a nucleic acid sequence on a first vector, and VP2 and VP3 encoded by a nucleic acid on a second vector. In some embodiments, the Cap polypeptide comprises an AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10 Cap polypeptide, or a variant of any of the foregoing. AAV capsid genes and proteins have been described in, e.g., Knipe DM. et al., (2001) Fields Virology 6(1), which is hereby incorporated by reference in its entirety.

[0268] Cell Density: As used herein, the term “cell density” refers to that number of cells present in a given volume of medium or the number of cells present in a given surface area. For example, cell density may be represented as viable cells (vc) / cm2of culture medium or vc / mL.

[0269] Corresponding to: As used herein, the term “corresponding to” may be used to designate the position or identity of a structural element in a compound or composition through comparison with an appropriate reference compound or composition. For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or Page 46 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of skill in the art appreciate that residues in a provided polypeptide or polynucleotide sequence are often designated (e.g., numbered or labeled) according to the scheme of a related reference sequence (even if, e.g., such designation does not reflect literal numbering of the provided sequence). By way of illustration, if a reference sequence includes a particular amino acid motif at positions 100-110, and a second related sequence includes the same motif at positions 110-120, the motif positions of the second related sequence can be said to “correspond to” positions 100-110 of the reference sequence. Those of skill in the art appreciate that corresponding positions can be readily identified, e.g., by alignment of sequences, and that such alignment is commonly accomplished by any of a variety of known tools, strategies, and / or algorithms, including without limitation software programs such as, for example, BLAST, CS-BLAST, CUDASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, Hhpred / Hhsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE. Two sequences can be identified as corresponding if they are identical or if they share substantial identity (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity) over a length of (e.g., at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500 or more) units (e.g., nucleotides or amino acids).

[0270] Culture: As used herein, the terms “culture” and “cell culture” refer to a cell population (e.g., a eukaryotic cell population) that is suspended in or covered by a medium under conditions suitable to survival and / or growth of the cell population. As will be clear to those of ordinary skill in the art, these terms can also refer to the combination comprising the cell population and the medium.

[0271] Fragment: As used herein, the terms “fragment” or “portion” refers to a structure that includes a discrete portion of the whole, but lacks one or more moieties found in the whole structure. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole. In some embodiments, a nucleotide fragment comprises or consists of at Page 47 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more monomeric units (e.g., nucleic acids) as found in the whole nucleotide. In some embodiments, a nucleotide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the monomeric units (e.g., residues) found in the whole nucleotide. The whole material or entity may in some embodiments be referred to as the “parent” of the whole.

[0272] Gene: As used herein, the term “gene” refers to a DNA sequence that codes for a product (e.g., an RNA product and / or a polypeptide product). In some embodiments, a gene includes a coding sequence (i.e., a sequence that encodes a particular product). In some embodiments, a gene includes a non-coding sequence. In some particular embodiments, a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequences. In some embodiments, a gene may include one or more regulatory elements that, for example, may control or effect one or more aspects of gene expression (e.g., inducible expression, etc.).

[0273] Gene therapy: As used herein, the term “gene therapy” refers to delivery of a payload with a vector, e.g., a recombinant AAV particle. In some embodiments, gene therapy comprises delivery and / or expression of a payload (e.g., a therapeutic product) to treat or prevent a disorder or condition for which such therapy is sought. In some embodiments, gene therapy comprises: insertion, deletion, or editing (e.g., by mutation, by duplication, by demethylation, by methylation, by upregulation, by downregulation, etc.) of specific genomic DNA sequences to treat or prevent a disorder or condition for which such therapy is sought. In some embodiments, the insertion or deletion of genomic DNA sequences occurs in specific cells (e.g., target cells). Target cells may be from a mammal and / or may be cells in a mammalian subject. Mammals include but are not limited to humans, dogs, cats, cows, sheep, pigs, llamas, etc. In some embodiments, heterologous DNA is transferred to target cells. The heterologous DNA may be introduced into the selected target cells in a manner such that the heterologous DNA is expressed and a therapeutic product encoded thereby is produced. In some embodiments, a therapeutic product is a polypeptide encoded by the heterologous DNA. In some embodiments, a therapeutic product is an RNA encoded by the heterologous DNA. In some embodiments, a therapeutic product is a polypeptide and an RNA encoded by the heterologous DNA. Additionally or Page 48 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) alternatively, the heterologous DNA may in some manner mediate expression of DNA that encodes the therapeutic product, or it may encode a product, such as a polypeptide or RNA that in some manner mediates or modulates, directly or indirectly, expression of a therapeutic product. Genetic therapy may also be used to deliver nucleic acid encoding a gene product that replaces a defective gene or supplements a gene product produced by the mammal or the cell in which it is introduced. The heterologous DNA encoding the therapeutic product may be modified prior to introduction into the cells of the afflicted host in order to enhance or otherwise alter the product or expression thereof. Genetic therapy may also involve delivery of an inhibitor or repressor or other modulator of gene expression. Such an inhibitor or repressor or other modulator can be a polypeptide, peptide, or nucleic acid (e.g., DNA or RNA). Gene therapy may include in vivo or ex vivo techniques. In some embodiments, viral and non-viral based gene transfer methods can be used to introduce a nucleic acid encoding a polypeptide of interest or to introduce a therapeutic nucleic acid into mammalian cells or target tissues. Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as poloxamers or liposomes. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson WF., (1992) Science 256(5058): pp.808- 813; Miller AD., (1992) Nature 357(6378): pp.455-460; Feuerbach FJ. Et al., (1996) Kidney Int. 49(6) : pp.1791-1794 ; Urnov FD. Et al., (2010) Nat. Rev Genet.11(9): pp.636-646; and Collins M. et al., (2015) Proc Biol Sci.282(1821), each of which is hereby incorporated by reference in its entirety.

[0274] Host Cell: As used herein, the term “host cell” refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. Persons of skill upon reading this disclosure will understand that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein. In some embodiments, host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life that are suitable for expressing an exogenous DNA (e.g., a recombinant nucleic acid sequence). Page 49 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0275] Identity: As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “substantially identical” to one another if their sequences are at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0276] Improve, increase, inhibit, or reduce: As used herein the terms “improve”, “increase,” “inhibit,” “reduce,” or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single sample, e.g., of a culture medium) under otherwise comparable conditions absent presence Page 50 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) of (e.g., prior to and / or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.

[0277] Medium: As used herein, the terms “medium,” “culture medium,” and “growth medium” refer to a solution comprising nutrients to nourish cells (e.g., growing cells, e.g., eukaryotic cells). Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cell for survival and / or minimal growth. The solution can also comprise components that enhance survival and / or growth above the minimal rate, including hormones and growth factors. The solution can be formulated to a pI and concentration of one or more salts that are optimal for cellular survival and / or proliferation. For example, the medium can also be a “defined medium” or “chemically defined medium,” e.g., a serum-free medium that contains no proteins, hydrolysates, or components of unknown composition. Defined media are free of animal-derived components and all components have a known chemical structure. One of skill in the art understands a defined medium can comprise recombinant polypeptides, for example, but not limited to, hormones, cytokines, interleukins, and / or other signaling molecules.

[0278] CNS targeting moiety: The phrase “CNS targeting moiety” as used herein refers to a single chain antibody agent which is effective in targeting a central nervous system (CNS) cell and / or tissue, e.g., an endothelial cell associated with a blood brain barrier, a cell or tissue that is present in the brain, spinal cord, or CNS system. In some embodiments, a CNS-targeting moiety can target a CNS cell or tissue by: (i) contacting a CNS cell or tissue (e.g., binding to one or more receptors expressed on a CNS cell or tissue); (ii) contacting a cell in contact with a CNS cell or tissue (e.g., binding to one or more receptors expressed on a cell in contact with a CNS cell or tissue); (iii) delivering a payload to a CNS cell or tissue; or (iv) any combination of (i)- (iii). In some embodiments, a CNS cell or tissue comprises: a CNS epithelial cell, an endothelial cell associated with a blood brain barrier, a nerve cell, a CNS connective tissue cell, a stem cell, a progenitor cell, a CNS immune cell, a spinal cord cell, a cell that lines one or more brain ventricles, a nerve support cell, a glial cell, a fat cell, a meninges cell, or a combination thereof. In some embodiments, a CNS tissue comprises a tissue found in a: cortex, thalamus, hypothalamus, striatum, putamen, caudate nucleus, hippocampus, entorhinal cortex, basal Page 51 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) ganglia, deep cerebellar nuclei, or other parts of a brain and / or spinal cord. In some embodiments, a CNS targeting moiety can be conjugated or fused to a payload. In some embodiments, a CNS targeting moiety can be incorporated into a vector, e.g., a viral vector or a non-viral vector. In some embodiments, a CNS targeting moiety can be inserted in an AAV capsid to form a variant AAV capsid protein as disclosed.

[0279] Nucleic acid: The term “nucleic acid” includes any nucleotides, analogs thereof, and polymers thereof. The term “polynucleotide” as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecules and, thus, include double- and single- stranded DNA, and double- and single-stranded RNA. These terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated, protected and / or capped nucleotides or polynucleotides. The terms encompass poly- or oligo-ribonucleotides (RNA) and poly- or oligo- deoxyribonucleotides (DNA); RNA or DNA derived from N-glycosides or C-glycosides of nucleobases and / or modified nucleobases; nucleic acids derived from sugars and / or modified sugars; and nucleic acids derived from phosphate bridges and / or modified phosphorus-atom bridges (also referred to herein as “internucleotide linkages”). The term encompasses nucleic acids containing any combinations of nucleobases, modified nucleobases, sugars, modified sugars, phosphate bridges or modified phosphorus atom bridges. Examples include, and are not limited to, nucleic acids containing ribose moieties, the nucleic acids containing deoxy-ribose moieties, nucleic acids containing both ribose and deoxyribose moieties, nucleic acids containing ribose and modified ribose moieties. In some embodiments, the prefix poly- refers to a nucleic acid containing 2 to about 10,000, 2 to about 50,000, or 2 to about 100,000 nucleotide monomer units. In some embodiments, the prefix oligo- refers to a nucleic acid containing 2 to about 200 nucleotide monomer units. In accordance with the methods and compositions described herein, in some embodiments, an RNA comprises a short hairpin RNA (shRNA), small interfering RNA (siRNA), mRNA, snRNA, CRISPR / Cas guide RNA, microRNA (miRNA), and / or a precursor thereof.

[0280] Payload: As used herein, the term “payload” refers to a nucleic acid sequence of interest (e.g., comprising a sequence that encodes a target payload, such as a target polypeptide or RNA) that is desired to be introduced into a cell, tissue, organ, organism, and / or system Page 52 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) comprising cells. A target payload can be a heterologous protein with a therapeutic purpose, e.g., an enzyme or antibody. The target payload can be a heterologous nucleic acid with a therapeutic purpose, e.g., an miRNA, siRNA, shRNA, mRNA, snRNA, or CRISPR / Cas guide RNA, or a precursor thereof. One of skill in the art will recognize that the target payload can be selected from any heterologous protein or nucleic acid of interest. As used herein, “encode” or “encodes” means directs the expression of or processed into. For example, as used herein, a nucleic acid encodes a polypeptide sequence if it directs the expression of that polypeptide sequence. As another example, as used herein, a nucleic acid precursor (e.g., a pri-miRNA or pre-miRNA) encodes a further processed version of the nucleic acid (e.g., mature miRNA) if it is processed into the further processed version.

[0281] Pharmaceutical composition: As used herein, the term “pharmaceutical composition” refers to a composition comprising rAAV particles that is suitable for administration to a human or animal subject. In some embodiments, a pharmaceutical composition comprises an active agent formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen. In some embodiments, a therapeutic regimen comprises one or more doses administered according to a schedule that has been determined to achieve a desired therapeutic effect when administered to a subject or population in need thereof (e.g., by a statistically significant probability). A pharmaceutical composition may be specially formulated for administration in solid or liquid form. In some embodiments, a pharmaceutical composition is formulated for administration by parenteral administration, such as by subcutaneous, intramuscular, intravenous or epidural injection. In some embodiments, a pharmaceutical composition is formulated as a sterile solution or suspension, e.g., in a sustained- release formulation. Pharmaceutical compositions of the disclosure may be formulated for administration by injection or infusion (e.g., subcutaneous, intramuscular, intravenous or epidural injection or infusion). For example, compositions may be formulated for administration by retinal, subretinal, intravitreal, suprachoroidal, intraspinal intracisternal magna, or intrathecal injection or infusion. In some embodiments, a pharmaceutical composition is intended and suitable for administration to a human subject. In some embodiments, a pharmaceutical composition is substantially free of contaminants (e.g., sterile and substantially pyrogen-free). Formulations of the pharmaceutical compositions may include, but are not limited to, Page 53 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) formulations for oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; topical application, such as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.

[0282] Polypeptide: The term “polypeptide”, as used herein, generally has its art- recognized meaning of a polymer of at least three amino acids. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional fragments (e.g., fragments retaining at least one activity) of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term “polypeptide” as used herein. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids.

[0283] Recombinant: As used herein, the term “recombinant” is intended to refer to polypeptides that are designed, engineered, prepared, expressed, created, manufactured, and / or or isolated by recombinant means, such as polypeptides expressed using a recombinant expression vector transfected into a host cell; polypeptides isolated from a recombinant, combinatorial human polypeptide library; polypeptides isolated from an animal (e.g., a mouse, rabbit, sheep, fish, etc.) that is transgenic for or otherwise has been manipulated to express a gene or genes, or Page 54 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) gene components that encode and / or direct expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof; and / or polypeptides prepared, expressed, created or isolated by any other means that involves splicing or ligating selected nucleic acid sequence elements to one another, chemically synthesizing selected sequence elements, and / or otherwise generating a nucleic acid that encodes and / or directs expression of the polypeptide or one or more component(s), portion(s), element(s), or domain(s) thereof. In some embodiments, one or more of such selected sequence elements is found in nature. In some embodiments, one or more of such selected sequence elements is designed in silico. In some embodiments, one or more such selected sequence elements results from mutagenesis (e.g., in vivo or in vitro) of a known sequence element, e.g., from a natural or synthetic source such as, for example, in the germline of a source organism of interest (e.g., of a human, a mouse, etc.).

[0284] Recombinant AAV (rAAV) particle: A “recombinant AAV particle”, or “rAAV particle,” as used herein, refers to a transduction-competent, replication-defective viral particle comprising an AAV protein shell (e.g., comprising a capsid as described herein) encapsulating a payload that is flanked on both sides by ITRs. An AAV particle is produced in a suitable host cell (e.g., a HEK293 cell). For example, the host cell is transfected with at least one vector encoding one or more helper polypeptides and nucleic acids (e.g., Ad2 helper polypeptides and nucleic acids), at least one Rep polypeptide, at least one Cap polypeptide, and at least one payload (e.g., for polypeptide expression or a therapeutic nucleic acid), such that the host cell is capable of producing the Rep and Cap polypeptides necessary for packing the rAAV particle. rAAV particles may be used for subsequent gene delivery.

[0285] Rep polypeptide: The term “Rep polypeptide”, as used herein, refers to the AAV non-structural proteins that mediate AAV replication for the production of AAV particles. The AAV replication genes and proteins have been described in, e.g., Knipe 2001, which is hereby incorporated by reference in its entirety.

[0286] Seeding: The term “seeding” as used herein refers to the process of providing a cell culture to a vessel (e.g., a bioreactor or culture flask). For example, the process of providing a cell culture may include propagation of the cells in another bioreactor or vessel before providing to the bioreactor or other vessel. The cells have been frozen and thawed immediately prior to providing them to the bioreactor or vessel. The term “seeding” refers to providing any number of cells, including a single cell. Page 55 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0287] Single chain antibody agent: The term “single chain antibody agent” as used herein refers to a polypeptide that comprises at least one variable domain that includes at least two complementarity determining regions (CDRs) and structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, a single chain antibody agent includes three CDRs. In some embodiments, a single chain antibody agent comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and / or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1-5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, a single chain antibody agent is a single domain antibody. In some embodiments, a single domain antibody is a camelid single domain antibody derived from a camelid heavy-chain antibody (HcAb). In some embodiments, a camelid single domain antibody is also referred to as a VHH. In some embodiments, a single domain antibody is a shark single domain antibody derived from a shark heavy-chain antibody (HcAb). In some embodiments, a shark single domain antibody is also referred to as a VNAR. In some embodiments, a single chain antibody agent is a single chain fragment variable (scFv) which is a single protein chain in which the VHand VLregions pair to form monovalent molecules see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). In some embodiments, a single chain antibody agent may include one or more sequence elements that are humanized, primatized, chimeric, etc, as is known in the art. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc], or other pendant group [e.g., poly-ethylene glycol, etc.]).

[0288] Specifically binds: The term “specifically binds” as used herein refers to an antibody that interacts more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to a particular antigen, epitope, protein, or target molecule than with alternative substances. An antibody that specifically binds an antigen can be Page 56 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) identified, for example, by immunoassays, ELISAs, surface plasmon resonance (SPR), or other techniques known to those of skill in the art. In some embodiments, an antibody that specifically binds an antigen (e.g., human TfR1) can bind related antigens (e.g., cyno TfR1). An antibody that specifically binds an antigen can bind the target antigen at a higher affinity than its affinity for a different antigen. The different antigen can be a related antigen. In some embodiments, an antibody that specifically binds an antigen can bind the target antigen with an affinity that is at least 20 times greater, at least 30 times greater, at least 40 times greater, at least 50 times greater, at least 60 times greater, at least 70 times greater, at least 80 times greater, at least 90 times greater, or at least 100 times greater, than its affinity for a different antigen. In some embodiments, an antibody that specifically binds a particular antigen binds a different antigen at such a low affinity that binding cannot be detected using an assay described herein or otherwise known in the art. In some embodiments, affinity is measured using SPR technology in a Biacore system as described herein or as known to those of skill in the art.

[0289] Subject: As used herein, the term “subject” refers to an organism, for example, a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, a dog). In some embodiments, a human subject is an adult, adolescent, or pediatric subject. In some embodiments, a subject is suffering from a disease, disorder or condition, e.g., a disease, disorder or condition that can be treated as provided herein, e.g., a neurological disease or disorder or a cancer or a tumor listed herein. In some embodiments, a subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is predisposed to and / or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing the disease, disorder or condition. In some embodiments, a subject displays one or more symptoms of a disease, disorder or condition. In some embodiments, a subject does not display a particular symptom (e.g., clinical manifestation of disease) or characteristic of a disease, disorder, or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0290] Titer: As used herein, the term “titer” refers to the quantity of virus in a given volume. Titer, for example, can be expressed as viral genome copies (vg) per given volume or Page 57 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) plaque forming units (pfu) per given volume. In some embodiments, titer can be expressed as number of capsids per given volume.

[0291] Transfection: As used herein, the term “transfection” refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g., mRNA) molecules, into cells, such as eukaryotic cells (e.g., mammalian cells). For example, transfection can include vector-based transfection, viral-based transfection, electroporation, lipofection (e.g., with cationic lipids and / or liposomes), calcium phosphate precipitation, nanoparticle-based transfection, and / or transfection based on cationic polymers (e.g., DEAE-dextran or polyethylenimine). In some embodiments, viral-based transfection is also referred to herein as transduction.

[0292] Treating: As used herein, the term “treating” refers to providing treatment, e.g., providing any type of medical or surgical management of a subject. The treatment can be provided in order to reverse, alleviate, inhibit the progression of, prevent or reduce the likelihood of a disease, disorder, or condition, or in order to reverse, alleviate, inhibit or prevent the progression of, prevent or reduce the likelihood of one or more symptoms or manifestations of a disease, disorder or condition. “Prevent” refers to causing a disease, disorder, condition, or symptom or manifestation of such not to occur for at least a period of time in at least some individuals. Treating can include administering an agent to the subject following the development of one or more symptoms or manifestations indicative of a condition, disease, or disorder, e.g., in order to reverse, alleviate, reduce the severity of, and / or inhibit or prevent the progression of the condition and / or to reverse, alleviate, reduce the severity of, and / or inhibit or one or more symptoms or manifestations of the condition. A composition comprising rAAV particles of the disclosure can be administered to a subject who has developed a disorder or is at increased risk of developing such a disorder relative to a member of the general population. A composition of the disclosure can be administered prophylactically or before development of any symptom or manifestation of the condition. Typically, in this case, the subject will be at risk of developing the condition.

[0293] Variant: As used herein in the context of molecules, e.g., nucleic acids or polypeptides, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference Page 58 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. To give but a few examples, a polypeptide may have a characteristic sequence element that comprises a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and / or contributing to a particular structural motif and / or biological function; a nucleic acid may have a characteristic sequence element that comprises a plurality of nucleotide residues having designated positions relative to on another in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and / or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that are covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. Typically, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant are substituted, inserted, or deleted, as compared to Page 59 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) the reference. In some embodiments, a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference. Often, a variant polypeptide or nucleic acid comprises a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particular biological activity) relative to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference. In some embodiments, a reference polypeptide or nucleic acid is one found in nature.

[0294] Vector: As used herein, the term “vector” refers to a molecule comprising a nucleic acid molecule, where the vector is capable of transporting the nucleic acid molecule into a cell. By way of non-limiting example, one type of vector is a “plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be packaged into a viral capsid and can be transferred into another cell and / or organism. Certain vectors are capable of autonomous replication in a host cell 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) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”

[0295] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacture’s specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods known in the art and as described in various general and more specific references that are cited Page 60 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) and discussed throughout the present specification. See, e.g., Sambrook J. et al., (1989) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press 2ndedition, which is incorporated herein by reference in its entirety.

[0296] VP: As used herein, the term “VP” refers to an AAV VP1 capsid protein, an AAV VP2 capsid protein, an AAV VP3 capsid protein, or variants or fragments or combinations of any of the foregoing. The term “capsid protein” is used interchangeably herein with VP. The numbering used herein in describing exemplary locations of single chain antibody agent insertions in VP1, VP2 or VP3 are used relative to AAV VP1 numbering. For example VP1, VP2 and VP3 of the AAV9 capsid protein correspond to amino acids 1 to 736 of VP1, amino acids 138 to 736 of VP1 and amino acids 203 to 736 of VP1, respectively. Thus, reference to a single chain antibody agent insertion between positions 588 and 589 in an AAV capsid variant refers to positions 588 and 589 in VP1, VP2 or VP3 relative to VP1 numbering. Those with knowledge in the pertinent field would be able to readily ascertain the corresponding position in VP2 and VP3, e.g., by comparing the sequences of VP1, VP2 and VP3 of the parental AAV capsid proteins using methods known in the field such as sequence alignment. In some embodiments, a VP capsid protein is a VP1 capsid protein. In some embodiments, a VP capsid protein is a VP2 capsid protein. In some embodiments, a VP capsid protein is a VP3 capsid protein. In some embodiments, a VP protein comprises a single chain antibody agent insertion disclosed herein.

[0297] Variant AAV capsid protein: As used herein, the term “variant AAV capsid protein” refers to a VP capsid protein (e.g., a VP1, VP2, or VP3) comprising an insertion of a single chain antibody agent relative to a corresponding parental AAV capsid protein (e.g., a parental VP1, VP2, or VP3). DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0298] The present disclosure provides, inter alia, improved recombinant adeno- associated virus (rAAV) particles that can be used for targeting cells or tissue, e.g., CNS cells and / or tissue. Safe and efficient therapeutic payload delivery to a CNS cell and / or tissue remains a major challenge in gene therapy. Recombinant adeno-associated viruses (rAAVs) have emerged as some of the most promising vectors for in vivo gene therapy, and are currently under clinical evaluation for a number of disorders including CNS disorders. However, naturally Page 61 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) occurring AAV capsids sub-optimally target CNS cells or tissue, and require extremely high doses to achieve minimum effective transgene expression. This poses daunting manufacturing challenges as well as safety concerns.

[0299] The present disclosure is based, in part, on the discovery that AAV tropism to certain cells and / or tissue can be obtained by inserting a single chain antibody agent into an AAV capsid to generate a variant AAV capsid protein and direct said variant AAV capsid protein to certain cells and / or tissue. Without wishing to be bound by any particular theory, in some embodiments, AAV tropism to certain cells and / or tissue can be obtained with a variant AAV capsid protein which binds to one or more cell surface receptors such as hTfR1 or a variant or a fragment thereof.

[0300] Further without wishing to be bound by theory, binding of a variant AAV capsid protein (e.g., in an AAV particle) to hTfR1 or a variant or a fragment thereof, delivers a variant AAV capsid protein to said cell and allows for a variant AAV capsid protein (e.g., in an AAV particle) to transcytose said cell (e.g., to be transported from one surface of said cell to another surface of said cell). In some embodiments, transcytosis of a variant AAV capsid protein (e.g., in an AAV particle) across a cell by binding to a hTfR1 delivers a variant AAV capsid protein (e.g., in an AAV particle) to an organ, to an intersitial space, and / or to an interstitial fluid allowing for transduction of a cell and / or tissue. In some embodiments, delivery of a variant AAV capsid (e.g., in an AAV particle) protein to an organ, to an intersitial space, and / or to an interstitial fluid results in transduction of a cell or tissue.

[0301] For example, when a variant AAV capsid protein (e.g., in an AAV particle) is directed to an endothelial cell which expresses a hTfR1 or a variant or a fragment thereof, said variant AAV capsid protein (e.g., in an AAV particle) can bind to and transcytose the endothelial cell and / or transduce the endothelial cell. In some embodiments, a variant AAV capsid protein (e.g., in an AAV particle) that transcytoses an endothelial cell can be delivered to an organ, an intersitial space, and / or an interstitial fluid.

[0302] As another example example, when a variant AAV capsid protein (e.g., in an AAV particle) is directed to an endothelial cell which is part of, or forms a blood brain barrier (e.g., by binding to a hTfR1 or a fragment or variant thereof), said variant AAV capsid protein (e.g., in an AAV particle) can bind to and transcytose the endothelial cell and be delivered to the brain. In some embodiments, a variant AAV capsid protein (e.g., in an AAV particle) delivered Page 62 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) to a brain transduces one or more cells in a brain. In some embodiments, a variant AAV capsid protein (e.g., in an AAV particle) directed to an endothelial cell transduces an endothelial cell. In some embodiments, transduction of an endothelial cell by a variant AAV capsid protein (e.g., in an AAV particle) results in expression of a payload. In some embodiments, an AAV particle comprises a variant AAV capsid and a heterologous nucleic acid comprising a nucleotide sequence encoding a payload, e.g., as described herein.

[0303] Additionally, this disclosure provides the discovery that AAV CNS cell and / or tissue tropism can be obtained by inserting a single chain antibody agent (e.g., a single domain antibody (e.g., a VHH or a single chain Fv) into an AAV capsid to direct said AAV capsid to a CNS cell and / or tissue. In some embodiments, a variant AAV capsid protein can be directed to a CNS cell and / or tissue (e.g., an endothelial cell associated with a blood brain barrier) by binding to one or more receptors expressed on endothelial cells).

[0304] Accordingly, disclosed herein are technologies for identifying variant AAV capsid proteins with CNS cell and / or tissue tropism by, e.g., binding to hTfR1 or variants or fragments thereof. Also disclosed herein are novel variant AAV capsid proteins that have enhanced CNS cell and / or tissue tropism for example by binding to hTfR1 or variants or fragments thereof.

[0305] In some embodiments, rAAV particles comprising a variant AAV capsid having a single chain antibody agent insertion disclosed herein bind to and / or recognize a target on a CNS cell and / or tissue. Without wishing to be bound by any particular theory, in some embodiments, rAAV particles comprising a variant AAV capsid comprising a single chain antibody agent insertion disclosed herein can enhance vector attachment, internalization, transcytosis, and / or payload expression in a CNS cell and / or tissue.

[0306] This disclosure further provides the surprising insight that binding affinity of a single chain antibody agent described herein (e.g., in an AAV particle comprising a single chain antibody agent inserted in a variable region (VR) of a parental AAV capsid protein) to TfR1 (e.g., human, cyno and / or mosue TfR1) is inversely correlated with the transduction level and / or efficiency of one or more CNS cells and / or tissue by an AAV particle comprising a single chain antibody agent inserted in VR of a parental AAV capsid protein. For example, as shown in Example 1 and FIG.26, exemplary single chain antibody agents that have higher binding affinity (e.g., lower Kd) to human TfR1 (e.g., fused to an Fc domain or in a VR of a parental Page 63 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) AAV capsid protein) showed generally lower transduction of neurons as observed in animals administered an AAV particle comprising a single chain antibody agent inserted in a VR of a parental AAV capsid protein. Without wishing to be bound by any particular theory, it is believed that inserting a single domain antibody agent in a VR of a parental AAV capsid protein can alter the ability of the single domain antibody agent to recognize and / or bind to TfR1, e.g., human, cyno and / or mosue TfR1. Accordingly, the present disclosure provides technologies and methods of identifying single chain antibody agents that bind to TfR1 and are useful for insertion in a variable region of a parental AAV capsid protein, e.g., to transduce CNS cells and / or tissue. Also provided herein are exemplary single chain antibody agents that bind to TfR1 and can be inserted in a variable region of a parental AAV capsid protein, e.g., to transduce CNS cells and / or tissue. Transferrin receptor (hTfR1) binders

[0307] Transferrin receptor (TfR), also known as CD71, is a transmembrane glycoprotein expressed in various sites of the human body at differing levels, whose function is to mediate cellular uptake of iron from a plasma glycoprotein, transferrin. Iron uptake from transferrin involves the binding of transferrin to the transferrin receptor, internalization of transferrin within an endocytic vesicle by receptor-mediated endocytosis and the release of iron from the protein by a decrease in endosomal Ph. Ponka P, Lok CN.. Int J Biochem Cell Biol. 1999 Oct;31(10):1111-37 and Xiaopeng Mo, in Brain Targeted Drug Delivery System, 2019. Apotransferrin (i.e., non-iron conjugate) binds to TfR when bound to two Fe 3+ ions to form holotransferrin (i.e., iron conjugate). The complex of TfR and holotransferrin is translocated into the cell by receptor-mediated endocytosis. Tfr and transferrin dissociate in an endosomal environment, and transferrin moves into the cell while Tfr is recycled to the cell membrane. Transferrin is thought to translocate into cells by binding to TfR and dissociation from TfR.

[0308] In humans and cynomolgus monkeys, two transferrin receptors, TfR1 and TfR2 have been characterized. TfR1 is a high affinity ubiquitously expressed receptor while expression of TfR2 is restricted to certain cell types and is unaffected by intracellular iron concentrations. TfR2 binds to transferrin with a 25-30 fold lower affinity than TfR1. The single chain antibody agents disclosed herein specifically bind to TfR1 (e.g., human and / or cyno TfR1). Page 64 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0309] Human TfR1 is (hTfR1) is a homodimeric type II transmembrane protein composed of a cytoplasmic domain, a single-pass transmembrane region, and an extracellular domain. See e.g., Montemiglio, L.C., Testi, C., Ceci, P. et al. Cryo-EM structure of the human ferritin–transferrin receptor 1 complex. Nat Commun 10, 1121 (2019), the entire contents of which are hereby incorporated by reference. Each monomer of the extracellular domain comprises: (1) a protease-like domain which can be in contact with a cell membrane, (2) a helical domain comprising dimer contact regions, and (3) an apical domain (e.g., as described in FIG. 1A in Montemiglio 2019).

[0310] The sequences for human TfR1, cyno TfR1, and mouse TfR1 are as follows:

[0311] Human TfR1 (UniProt No. P02786.2; SEQ ID NO:2016) MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENADNNTKANVTKPKRC SGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPGEDFPAARRLYWDDLK RKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFKLSKVWRDQHFVKIQV KDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVI VRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFP PSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKEIKILN IFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFAS WSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHP VTGQFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIP ELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYS ARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHT LPALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF

[0312] Cyno TfR1 (UniProt No. G8F602; SEQ ID NO:2017) MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDDEENADNNTKANGTKPKRC GGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEEDFPAAPRLYWDDLK RKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIENQFREFKLSKVWRDQHFVKIQVK DSAQNSVIIVDKNGGLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLDSPVNGSIVIV RAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVKADLSFFGHAHLGTGDPYTPGFPSFNHTQFPP SQSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCKMVTSENKSVKLTVSNVLKETKILN IFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFAS WSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQDVKHP VTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELVERIP ELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLNQYRADVKEMGLSLQWLYS ARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMRVEYYFLSPYVSPKESPFRHVFWGSGSH TLSALLESLKLRRQNNSAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF

[0313] Mouse TfR1 (Genbank No. NP_035768.1; SEQ ID NO: 2018) MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAADEEENADNNMKASVR KPKRFNGRLCFAAIALVIFFLIGFMSGYLGYCKRVEQKEECVKLAETEETDKSETMETED VPTSSRLYWADLKTLLSEKLNSIEFADTIKQLSQNTYTPREAGSQKDESLAYYIENQFHEF KFSKVWRDEHYVKIQVKSSIGQNMVTIVQSNGNLDPVESPEGYVAFSKPTEVSGKLVHA Page 65 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) NFGTKKDFEELSYSVNGSLVIVRAGEITFAEKVANAQSFNAIGVLIYMDKNKFPVVEAD LALFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGKMEGSCPA RWNIDSSCKLELSQNQNVKLIVKNVLKERRILNIFGVIKGYEEPDRYVVVGAQRDALGA GVAAKSSVGTGLLLKLAQVFSDMISKDGFRPSRSIIFASWTAGDFGAVGATEWLEGYLS SLHLKAFTYINLDKVVLGTSNFKVSASPLLYTLMGKIMQDVKHPVDGKSLYRDSNWISK VEKLSFDNAAYPFLAYSGIPAVSFCFCEDADYPYLGTRLDTYEALTQKVPQLNQMVRTA AEVAGQLIIKLTHDVELNLDYEMYNSKLLSFMKDLNQFKTDIRDMGLSLQWLYSARGD YFRATSRLTTDFHNAEKTNRFVMREINDRIMKVEYHFLSPYVSPRESPFRHIFWGSGSHT LSALVENLKLRQKNITAFNETLFRNQLALATWTIQGVANALSGDIWNIDNEF

[0314] In some embodiments, an rAAV particle comprises a variant AAV capsid protein comprising a single chain antibody agent that binds to a transferrin receptor (TfR1) or a variant or a fragment thereof, wherein the single chain antibody agent is inserted in a variable region (VR) of a parental AAV capsid protein. In some embodiments, a single chain antibody agent is or comprises a single domain antibody (e.g., a VHH).

[0315] In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising a single chain antibody agent disclosed herein; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV9 capsid protein. In some embodiments, a single chain antibody agent inserted in a variant AAV capsid protein disclosed herein comprises a a single chain antibody agent sequence provided in Table 1. Table 1: Sequence for exemplary single chain antibody agents Clone Feature SEQ Sequence ID Q T12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) Clone Feature SEQ Sequence ID N Q Q Q Q Q T Q T Q Tage o 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) Clone Feature SEQ Sequence ID N Q T Q T Q T Q T Q T Q T Q Tage o 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) Clone Feature SEQ Sequence ID N Q T Q T Q T R T R T R Tage 69 o 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) Clone Feature SEQ Sequence ID N R T T S R T T S R TPage 70 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0316] In some embodiments, a single chain antibody agent comprises: (i) a CDR1 sequence provided in Table 1; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a CDR1 sequence provided in Table 1; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to a CDR1 sequence provided in Table 1; (ii) a CDR2 sequence provided in Table 1; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a CDR2 sequence provided in Table 1; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to a CDR2 sequence provided in Table 1; and / or (iii) a CDR3 sequence provided in Table 1; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a CDR3 sequence provided in Table 1; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to a CDR3 sequence provided in Table 1.

[0317] In some embodiments, a single chain antibody agent comprises: (i) a CDR1 sequence of SEQ ID NO: 2; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 2; (ii) a CDR2 sequence of SEQ ID NO: 3; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 3; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 3; and / or (iii) a CDR3 sequence of SEQ ID NO: 4; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 4; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 4. Page 71 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0318] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 2; (ii) a CDR2 sequence of SEQ ID NO: 3; and (iii) a CDR3 sequence of SEQ ID NO: 4.

[0319] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 1, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0320] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 158, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0321] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 159, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0322] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 160, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0323] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 6; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 6; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 6; (ii) a CDR2 sequence of SEQ ID NO: 7; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 7; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 7; and / or (iii) a CDR3 sequence of SEQ ID NO: 8; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at Page 72 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 8; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 8.

[0324] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 6; (ii) a CDR2 sequence of SEQ ID NO: 7; and (iii) a CDR3 sequence of SEQ ID NO: 8.

[0325] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 5, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0326] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 122, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0327] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 135, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0328] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 136, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0329] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 10; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 10; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 10; (ii) a CDR2 sequence of SEQ ID NO: 11; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 11; or a sequence Page 73 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 11; and / or (iii) a CDR3 sequence of SEQ ID NO: 12; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 12; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 12.

[0330] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 10; (ii) a CDR2 sequence of SEQ ID NO: 11; and (iii) a CDR3 sequence of SEQ ID NO: 12.

[0331] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 9, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0332] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 14; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 14; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 14; (ii) a CDR2 sequence of SEQ ID NO: 15; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 15; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 15; and / or (iii) a CDR3 sequence of SEQ ID NO: 16; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 16; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 16.

[0333] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 14; (ii) a CDR2 sequence of SEQ ID NO: 15; and (iii) a CDR3 sequence of SEQ ID NO: 16.

[0334] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 13, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at Page 74 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0335] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 18; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 18; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 18; (ii) a CDR2 sequence of SEQ ID NO: 19; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 19; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 19; and / or (iii) a CDR3 sequence of SEQ ID NO: 20; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 20; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 20.

[0336] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 18; (ii) a CDR2 sequence of SEQ ID NO: 19; and (iii) a CDR3 sequence of SEQ ID NO: 20.

[0337] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 17, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0338] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 22; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 22; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 22; (ii) a CDR2 sequence of SEQ ID NO: 23; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 23; or a sequence Page 75 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 23; and / or (iii) a CDR3 sequence of SEQ ID NO: 24; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 24; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 24.

[0339] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 22; (ii) a CDR2 sequence of SEQ ID NO: 23; and (iii) a CDR3 sequence of SEQ ID NO: 24.

[0340] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 21, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0341] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 26; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 26; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 26; (ii) a CDR2 sequence of SEQ ID NO: 27; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 27; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 27; and / or (iii) a CDR3 sequence of SEQ ID NO: 28; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 28; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 28.

[0342] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 26; (ii) a CDR2 sequence of SEQ ID NO: 27; and (iii) a CDR3 sequence of SEQ ID NO: 28.

[0343] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 25, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at Page 76 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0344] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 30; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 30; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 30; (ii) a CDR2 sequence of SEQ ID NO: 31; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 31; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 31; and / or (iii) a CDR3 sequence of SEQ ID NO: 32; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 32; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 32.

[0345] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 30; (ii) a CDR2 sequence of SEQ ID NO: 31; and (iii) a CDR3 sequence of SEQ ID NO: 32.

[0346] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 29, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0347] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 34; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 34; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 34; (ii) a CDR2 sequence of SEQ ID NO: 35; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 35; or a sequence Page 77 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 35; and / or (iii) a CDR3 sequence of SEQ ID NO: 36; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 36; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 36.

[0348] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 34; (ii) a CDR2 sequence of SEQ ID NO: 35; and (iii) a CDR3 sequence of SEQ ID NO: 36.

[0349] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 33, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0350] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 38; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 38; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 38; (ii) a CDR2 sequence of SEQ ID NO: 39; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 39; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 39; and / or (iii) a CDR3 sequence of SEQ ID NO: 40; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 40; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 40.

[0351] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 38; (ii) a CDR2 sequence of SEQ ID NO: 39; and (iii) a CDR3 sequence of SEQ ID NO: 40.

[0352] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 37, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at Page 78 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0353] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 42; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 42; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 42; (ii) a CDR2 sequence of SEQ ID NO: 43; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 43; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 43; and / or (iii) a CDR3 sequence of SEQ ID NO: 44; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 44; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 44.

[0354] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 42; (ii) a CDR2 sequence of SEQ ID NO: 43; and (iii) a CDR3 sequence of SEQ ID NO: 44.

[0355] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 41, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0356] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 46; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 46; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 46; (ii) a CDR2 sequence of SEQ ID NO: 47; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 47; or a sequence Page 79 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 47; and / or (iii) a CDR3 sequence of SEQ ID NO: 48; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 48; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 48.

[0357] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 46; (ii) a CDR2 sequence of SEQ ID NO: 47; and (iii) a CDR3 sequence of SEQ ID NO: 48.

[0358] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 45, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0359] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 50; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 50; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 50; (ii) a CDR2 sequence of SEQ ID NO: 51; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 51; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 51; and / or (iii) a CDR3 sequence of SEQ ID NO: 52; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 52; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 52.

[0360] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 50; (ii) a CDR2 sequence of SEQ ID NO: 51; and (iii) a CDR3 sequence of SEQ ID NO: 52.

[0361] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 49, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at Page 80 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0362] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 2; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 2; (ii) a CDR2 sequence of SEQ ID NO: 117; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 117; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 117; and / or (iii) a CDR3 sequence of SEQ ID NO: 4; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 4; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 4.

[0363] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 2; (ii) a CDR2 sequence of SEQ ID NO: 117; and (iii) a CDR3 sequence of SEQ ID NO: 4.

[0364] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 116, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0365] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 2; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 2; (ii) a CDR2 sequence of SEQ ID NO: 119; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 119; or a sequence Page 81 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 119; and / or (iii) a CDR3 sequence of SEQ ID NO: 4; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 4; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 4.

[0366] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 2; (ii) a CDR2 sequence of SEQ ID NO: 119; and (iii) a CDR3 sequence of SEQ ID NO: 4.

[0367] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 118, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0368] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 2; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 2; (ii) a CDR2 sequence of SEQ ID NO: 15; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 15; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 15; and / or (iii) a CDR3 sequence of SEQ ID NO: 52; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 52; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 52.

[0369] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 2; (ii) a CDR2 sequence of SEQ ID NO: 15; and (iii) a CDR3 sequence of SEQ ID NO: 52.

[0370] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 120, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at Page 82 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0371] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 2; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 2; (ii) a CDR2 sequence of SEQ ID NO: 119; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 119; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 119; and / or (iii) a CDR3 sequence of SEQ ID NO: 52; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 52; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 52.

[0372] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 2; (ii) a CDR2 sequence of SEQ ID NO: 119; and (iii) a CDR3 sequence of SEQ ID NO: 52.

[0373] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 121, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0374] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 124; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 124; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 124; (ii) a CDR2 sequence of SEQ ID NO: 125; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 125; Page 83 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 125; and / or (iii) a CDR3 sequence of SEQ ID NO: 8; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 8; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 8.

[0375] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 124; (ii) a CDR2 sequence of SEQ ID NO: 125; and (iii) a CDR3 sequence of SEQ ID NO: 8.

[0376] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 123, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0377] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 127; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 127; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 127; (ii) a CDR2 sequence of SEQ ID NO: 7; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 7; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 7; and / or (iii) a CDR3 sequence of SEQ ID NO: 128; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 128; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 128.

[0378] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 127; (ii) a CDR2 sequence of SEQ ID NO: 7; and (iii) a CDR3 sequence of SEQ ID NO: 128.

[0379] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 126, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at Page 84 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0380] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 130; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 130; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 130; (ii) a CDR2 sequence of SEQ ID NO: 7; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 7; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 7; and / or (iii) a CDR3 sequence of SEQ ID NO: 131; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 131; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 131.

[0381] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 130; (ii) a CDR2 sequence of SEQ ID NO: 7; and (iii) a CDR3 sequence of SEQ ID NO: 131.

[0382] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 129, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0383] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 6; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 6; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 6; (ii) a CDR2 sequence of SEQ ID NO: 133; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 133; or a sequence Page 85 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 133; and / or (iii) a CDR3 sequence of SEQ ID NO: 134; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 134; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 134.

[0384] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 6, (ii) a CDR2 sequence of SEQ ID NO: 133; and (iii) a CDR3 sequence of SEQ ID NO: 134.

[0385] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 132, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0386] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 6; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 6; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 6; (ii) a CDR2 sequence of SEQ ID NO: 125; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 125; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 125; and / or (iii) a CDR3 sequence of SEQ ID NO: 134; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 134; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 134.

[0387] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 6, (ii) a CDR2 sequence of SEQ ID NO: 125; and (iii) a CDR3 sequence of SEQ ID NO: 134.

[0388] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 137, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at Page 86 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0389] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 130; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 130; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 130; (ii) a CDR2 sequence of SEQ ID NO: 7; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 7; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 7; and / or (iii) a CDR3 sequence of SEQ ID NO: 139; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 139; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 139.

[0390] In some embodiments, a single chain antibody agent comprises (i) a CDR1 sequence of SEQ ID NO: 130, (ii) a CDR2 sequence of SEQ ID NO: 7; and (iii) a CDR3 sequence of SEQ ID NO: 139.

[0391] In some embodiments, a single chain antibody agent comprises the sequence of SEQ ID NO: 138, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

[0392] In some embodiments, a single chain antibody agent binds to hTfR1 with an affinity (KD) of about 10 nM to about 2500 nM, e.g., when fused to an Fc domain or when inserted in a VR region of a parental AAV capsid protein. In some embodiments, binding affinity of a single chain antibody agent to hTfR1 is about 10 nM to about 2500 nM, about 20 nM to about 2500 nM , about 30 nM to about 2500 nM, about 40 nM to about 2500 nM, about 50 nM to about 2500 nM, about 60 nM to about 2500 nM, about 70 nM to about 2500 nM, about 80 nM to about 2500 nM, about 90 nM to about 2500 nM, about 100 nM to about 2500 nM, about 200 nM to about 2500 nM, about 300 nM to about 2500 nM, about 400 nM to about 2500 nM, about 500 Page 87 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) nM to about 2500 nM, about 600 nM to about 2500 nM, about 700 nM to about 2500 nM, about 800 nM to about 2500 nM, about 900 nM to about 2500 nM, about 1000 nM to about 2500 nM, about 1500 nM to about 2500 nM, about 2000 nM to about 2500 nM, about 10 nM to about 2500 nM, about 10 nM to about 2000 nM, about 10 nM to about 1500 nM, about 10 nM to about 1000 nM, about 10 nM to about 900 nM, about 10 nM to about 800 nM, about 10 nM to about 700 nM, about 10 nM to about 600 nM, about 10 nM to about 500 nM, about 10 nM to about 400 nM, about 10 nM to about 300 nM, about 10 nM to about 200 nM, about 10 nM to about 100 nM, about 10 nM to about 90 nM, about 10 nM to about 80 nM, about 10 nM to about 70 nM, about 10 nM to about 60 nM, about 10 nM to about 50 nM, about 10 nM to about 40 nM, about 10 nM to about 30 nM, about 10 nM to about 20 nM, e.g., when fused to an Fc domain or when inserted in a VR region of a parental AAV capsid protein.

[0393] In some embodiments, binding affinity of a single chain antibody agent to hTfR1 is about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1000 nM, about 1500 nM, about 2000 nM or about 2500 nM, e.g., when fused to an Fc domain or when inserted in a VR region of a parental AAV capsid protein.

[0394] In some embodiments, binding affinity of a single chain antibody agent to hTfR1 is 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM, 1500 nM, 2000 nM or 2500 nM, e.g., when fused to an Fc domain or when inserted in a VR region of a parental AAV capsid protein.

[0395] In some embodiments, a single chain antibody agent binds to cyno TfR1 with an affinity (KD) of about 10 nM to about 2500 nM, e.g., when fused to an Fc domain or when inserted in a VR region of a parental AAV capsid protein. In some embodiments, binding affinity of a single chain antibody agent to cyno TfR1 is about 10 nM to about 2500 nM, about 20 nM to about 2500 nM , about 30 nM to about 2500 nM, about 40 nM to about 2500 nM, about 50 nM to about 2500 nM, about 60 nM to about 2500 nM, about 70 nM to about 2500 nM, about 80 nM to about 2500 nM, about 90 nM to about 2500 nM, about 100 nM to about 2500 nM, about 200 nM to about 2500 nM, about 300 nM to about 2500 nM, about 400 nM to about 2500 nM, about 500 Page 88 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) nM to about 2500 nM, about 600 nM to about 2500 nM, about 700 nM to about 2500 nM, about 800 nM to about 2500 nM, about 900 nM to about 2500 nM, about 1000 nM to about 2500 nM, about 1500 nM to about 2500 nM, about 2000 nM to about 2500 nM, about 10 nM to about 2500 nM, about 10 nM to about 2000 nM, about 10 nM to about 1500 nM, about 10 nM to about 1000 nM, about 10 nM to about 900 nM, about 10 nM to about 800 nM, about 10 nM to about 700 nM, about 10 nM to about 600 nM, about 10 nM to about 500 nM, about 10 nM to about 400 nM, about 10 nM to about 300 nM, about 10 nM to about 200 nM, about 10 nM to about 100 nM, about 10 nM to about 90 nM, about 10 nM to about 80 nM, about 10 nM to about 70 nM, about 10 nM to about 60 nM, about 10 nM to about 50 nM, about 10 nM to about 40 nM, about 10 nM to about 30 nM, about 10 nM to about 20 nM, e.g., when fused to an Fc domain or when inserted in a VR region of a parental AAV capsid protein. In some embodiments, binding affinity of a single chain antibody agent to cyno TfR1 is about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1000 nM, about 1500 nM, about 2000 nM or about 2500 nM, e.g., when fused to an Fc domain or when inserted in a VR region of a parental AAV capsid protein.

[0396] In some embodiments, binding affinity of a single chain antibody agent to cyno TfR1 is 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1000 nM, 1500 nM, 2000 nM or 2500 nM, e.g., when fused to an Fc domain or when inserted in a VR region of a parental AAV capsid protein.

[0397] In some embodiments, a single chain antibody agent is fused to a C terminus of an Fc domain. In some embodiments, a single chain antibody agent is fused to a N terminus of an Fc domain.

[0398] In some embodiments, binding affinity of a single chain antibody agent described herein (e.g., fused to an Fc domain or in an AAV particle comprising a single chain antibody agent inserted in a variable region (VR) of a parental AAV capsid protein) to hTfR1 does not directly correlate with the transduction level and / or efficiency of one or more CNS cells and / or tissue by an AAV particle comprising a single chain antibody agent inserted in VR of a parental AAV capsid protein. Page 89 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) Exemplary TfR1 epitopes

[0399] TfR1 binding single domain antibody agents described herein can bind to one or more epitopes on TfR1 (e.g., human TfR1, cyno TfR1, and / or mouse TfR1). In some embodiments, a TfR1 binding single domain antibody agent described herein binds to one or more epitopes on human TfR1. In some embodiments, a TfR1 binding single domain antibody agent described herein binds to one or more epitopes on cyno TfR1.

[0400] In some embodiments, an epitope on TfR1 (e.g., human TfR1, cyno TfR1, and / or mouse TfR1) bound by a single domain antibody agent described herein is a linear epitope.

[0401] In some embodiments, an epitope on TfR1 (e.g., human TfR1, cyno TfR1, and / or mouse TfR1) bound by a single domain antibody agent described herein is a conformational epitope.

[0402] In some embodiments, an epitope on TfR1 (e.g., human TfR1, cyno TfR1, and / or mouse TfR1) bound by a single domain antibody agent described herein is a discontinuous epitope.

[0403] In some embodiments, an epitope on TfR1 (e.g., human TfR1, cyno TfR1, and / or mouse TfR1) bound by a single domain antibody agent described herein comprises one or more domains of TfR1 (e.g., human TfR1, cyno TfR1, and / or mouse TfR1). In some embodiments, one or more domains of TfR1 (e.g., human TfR1, cyno TfR1, and / or mouse TfR1) is or comprises an extracellular domain of TfR1 (e.g., human TfR1, cyno TfR1, and / or mouse TfR1) or a fragment thereof. In some embodiments, one or more domains of TfR1 (e.g., human TfR1, cyno TfR1, and / or mouse TfR1) is or comprises an apical domain, a helical domain, a protease- like domain of TfR1 (e.g., human TfR1, cyno TfR1, and / or mouse TfR1), and / or fragments of one or more of the foregoing domains.

[0404] In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises an extracellular domain of human TfR1 or a fragment thereof. In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises: (1) an apical domain of human TfR1or a fragment thereof, (2) a helical domain of human TfR1or a fragment thereof, or (3) an apical domain of human TfR1or a fragment thereof, and a helical domain of human TfR1or a fragment thereof. Page 90 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0405] In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises: (1) one or more residues K189, Y309, F321, P322, P323, S324, R325, L329, or L381 from an apical domain of human TfR1, and / or (2) one or more residues E634, M635, G636, R719, N722, N723, G724, A725, F726, N727, E728, T729 or R732 from a helical domain of human TfR1.

[0406] In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises one or more residues K189, Y309, F321, P322, P323, S324, R325, L329, or L381 from an apical domain of human TfR1. In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises all of residues K189, Y309, F321, P322, P323, S324, R325, L329, and L381 from an apical domain of human TfR1.

[0407] In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises one or more residues E634, M635, G636, R719, N722, N723, G724, A725, F726, N727, E728, T729 or R732 from a helical domain of human TfR1. In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises all of residues E634, M635, G636, R719, N722, N723, G724, A725, F726, N727, E728, T729 and R732 from a helical domain of human TfR1.

[0408] In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises all of residues K189, Y309, F321, P322, P323, S324, R325, L329, and L381 from an apical domain of human TfR1, and one or more residues E634, M635, G636, R719, N722, N723, G724, A725, F726, N727, E728, T729 or R732 from a helical domain of human TfR1. In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises one or more residues K189, Y309, F321, P322, P323, S324, R325, L329, or L381 from an apical domain of human TfR1, and all of residues E634, M635, G636, R719, N722, N723, G724, A725, F726, N727, E728, T729 and R732 from a helical domain of human TfR1. In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises all of residues K189, Y309, F321, P322, P323, S324, R325, L329, and L381 from an apical domain of human TfR1, and all of residues E634, M635, G636, R719, N722, N723, G724, A725, F726, N727, E728, T729 and R732 from a helical domain of human TfR1. Page 91 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0409] In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises: (1) one or more residues K189, P323, S324, R325, L381, or E383 from an apical domain of human TfR1, and / or (2) one or more residues E634, M635, R719, N723, N727, E728, T729, or R732 from a helical domain of human TfR1.

[0410] In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises one or more residues K189, P323, S324, R325, L381, or E383 from an apical domain of human TfR1. In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises all of residues K189, P323, S324, R325, L381, and E383 from an apical domain of human TfR1.

[0411] In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises one or more residues E634, M635, R719, N723, N727, E728, T729, or R732 from a helical domain of human TfR1. In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises all of residues E634, M635, R719, N723, N727, E728, T729, and R732 from a helical domain of human TfR1.

[0412] In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises all of residues K189, P323, S324, R325, L381, and E383 from an apical domain of human TfR1 and one or more residues E634, M635, R719, N723, N727, E728, T729, or R732 from a helical domain of human TfR1. In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises one or more residues K189, P323, S324, R325, L381, or E383 from an apical domain of human TfR1 and all of residues E634, M635, R719, N723, N727, E728, T729, and R732 from a helical domain of human TfR1. In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises all of residues K189, P323, S324, R325, L381, and E383 from an apical domain of human TfR1, and all of residues E634, M635, R719, N723, N727, E728, T729, and R732 from a helical domain of human TfR1.

[0413] In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises: (1) one or more residues K189, P323, S324, R325, or L381 from an apical domain of human TfR1, and / or (2) one or more residues E634, M635, R719, N723, N727, E728, T729, or R732 from a helical domain of human TfR1. Page 92 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0414] In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises one or more residues K189, P323, S324, R325, or L381 from an apical domain of human TfR1. In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises all of residues K189, P323, S324, R325, and L381 from an apical domain of human TfR1.

[0415] In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises one or more residues E634, M635, R719, N723, N727, E728, T729, or R732 from a helical domain of human TfR1. In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises all of residues E634, M635, R719, N723, N727, E728, T729, and R732 from a helical domain of human TfR1.

[0416] In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises all of residues K189, P323, S324, R325, and L381 from an apical domain of human TfR1 and one or more residues E634, M635, R719, N723, N727, E728, T729, or R732 from a helical domain of human TfR1. In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises one or more residues K189, P323, S324, R325, or L381 from an apical domain of human TfR1 and all of residues E634, M635, R719, N723, N727, E728, T729, and R732 from a helical domain of human TfR1. In some embodiments, an epitope on human TfR1 bound by a single domain antibody agent described herein comprises all of residues K189, P323, S324, R325, and L381 from an apical domain of human TfR1 and all of residues E634, M635, R719, N723, N727, E728, T729, and R732 from a helical domain of human TfR1.

[0417] In some embodiments, an epitope on cyno TfR1 bound by a single domain antibody agent described herein comprises an extracellular domain of cyno TfR1. In some embodiments, an epitope on cyno TfR1 bound by a single domain antibody agent described herein comprises an apical domain, a helical domain, or an apical domain and a helical domain of cyno TfR1.

[0418] In some embodiments, an epitope on cyno TfR1 bound by a single domain antibody agent described herein comprises: (1) one or more residues K189, Y309, F321, P322, P323, S324, Q325, L329, or L381 from an apical domain of cyno TfR1, and / or (2) one or more Page 93 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) residues E634, M635, G636, R719, N722, N723, S724, A725, F726, N727, E728, T729, or R732 from a helical domain of cyno TfR1.

[0419] In some embodiments, an epitope on cyno TfR1 bound by a single domain antibody agent described herein comprises one or more residues K189, Y309, F321, P322, P323, S324, Q325, L329, or L381 from an apical domain of cyno TfR1. In some embodiments, an epitope on cyno TfR1 bound by a single domain antibody agent described herein comprises all of residues K189, Y309, F321, P322, P323, S324, Q325, L329, and L381 from an apical domain of cyno TfR1.

[0420] In some embodiments, an epitope on cyno TfR1 bound by a single domain antibody agent described herein comprises one or more residues E634, M635, G636, R719, N722, N723, G724, A725, F726, N727, E728, T729, or R732 from a helical domain of cyno TfR1. In some embodiments, an epitope on cyno TfR1 bound by a single domain antibody agent described herein comprises all of residues E634, M635, G636, R719, N722, N723, G724, A725, F726, N727, E728, T729, and R732 from a helical domain of cyno TfR1.

[0421] In some embodiments, an epitope on cyno TfR1 bound by a single domain antibody agent described herein comprises all of residues K189, Y309, F321, P322, P323, S324, Q325, L329, and L381 from an apical domain of cyno TfR1, and one or more residues E634, M635, G636, R719, N722, N723, G724, A725, F726, N727, E728, T729, or R732 from a helical domain of cyno TfR1. In some embodiments, an epitope on cyno TfR1 bound by a single domain antibody agent described herein comprises one or more residues K189, Y309, F321, P322, P323, S324, Q325, L329, or L381 from an apical domain of cyno TfR1, and all of residues E634, M635, G636, R719, N722, N723, G724, A725, F726, N727, E728, T729, and R732 from a helical domain of cyno TfR1. In some embodiments, an epitope on cyno TfR1 bound by a single domain antibody agent described herein comprises all of residues K189, Y309, F321, P322, P323, S324, Q325, L329, and L381 from an apical domain of cyno TfR1, and all of residues E634, M635, G636, R719, N722, N723, G724, A725, F726, N727, E728, T729, and R732 from a helical domain of cyno TfR1.

[0422] In some embodiments, an epitope on cyno TfR1 bound by a single domain antibody agent described herein comprises: (1) one or more residues K189, P323, S324, Q325, Page 94 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) L381, or E383 from an apical domain of cyno TfR1, and / or (2) one or more residues E634, M635, R719, N723, N727, E728, T729, or R732 from a helical domain of cyno TfR1.

[0423] In some embodiments, an epitope on cyno TfR1 bound by a single domain antibody agent described herein comprises one or more residues K189, P323, S324, Q325, L381, or E383 from an apical domain of cyno TfR1. In some embodiments, an epitope on cyno TfR1 bound by a single domain antibody agent described herein comprises all of residues K189, P323, S324, Q325, L381, and E383 from an apical domain of cyno TfR1.

[0424] In some embodiments, an epitope on cyno TfR1 bound by a single domain antibody agent described herein comprises one or more residues E634, M635, R719, N723, N727, E728, T729, or R732 from a helical domain of cyno TfR1. In some embodiments, an epitope on cyno TfR1 bound by a single domain antibody agent described herein comprises all of residues E634, M635, R719, N723, N727, E728, T729, and R732 from a helical domain of cyno TfR1.

[0425] In some embodiments, an epitope on cyno TfR1 bound by a single domain antibody agent described herein comprises all of residues K189, P323, S324, Q325, L381, and E383 from an apical domain of cyno TfR1 and one or more residues E634, M635, R719, N723, N727, E728, T729, or R732 from a helical domain of cyno TfR1. In some embodiments, an epitope on cyno TfR1 bound by a single domain antibody agent described herein comprises one or more residues K189, P323, S324, Q325, L381, or E383 from an apical domain of cyno TfR1 and all of residues E634, M635, R719, N723, N727, E728, T729, and R732 from a helical domain of cyno TfR1. In some embodiments, an epitope on cyno TfR1 bound by a single domain antibody agent described herein comprises all of residues K189, P323, S324, Q325, L381, and E383 from an apical domain of cyno TfR1, and all of residues E634, M635, R719, N723, N727, E728, T729, and R732 from a helical domain of cyno TfR1. Adeno-associated viruses (AAVs)

[0426] Adeno-associated viruses (AAVs) are small, nonenveloped, single-stranded DNA (ssDNA) viruses that belong to the Parvoviridae family. At least twelve distinct AAV serotypes have been identified from human and nonhuman primate sources (see DiMattia M.A. et al., Page 95 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) (2012) J. Virology 86(12): pp.6947-6958, the entire contents of which is hereby incorporated by reference)(hereinafter “DiMattia 2012”). AAV9 is one of the human AAV serotypes that has enhanced transduction efficiency in cardiac and skeletal muscle, liver tissue, pancreatic tissue, and the eye compared to other serotypes (DiMattia 2012).

[0427] The AAV wild-type genome contains at least three genes, rep, cap and X (Büning H. et al., (2019) Molecular Therapy: Methods & Clinical Development vol.12 pages 248-265). The cap gene encodes for viral proteins VP1, VP2, and VP3, and assembly-activating protein (AAP). All three VPs (i.e., viral proteins) are capsid monomers.

[0428] Transcription of the cap gene results in two messenger RNA: a messenger RNA which encodes VP1 and a messenger RNA which encodes VP2 and VP3 (as described in Warrington KH et al., (2004) Journal of Virology volume 78(12) pages 6595-6609). VP1, VP2, and VP3 are present at ratios of 1:1:10, respectively. The VP3 region is observed in all capsid structures of AAV serotypes that have been studied (DiMattia 2012).

[0429] VP proteins comprise beta strands, alpha helical regions, and structurally variable regions (VRs) in the surface loops which connect the beta strands. Without wishing to be bound by any particular theory, it is believed that differences in sequence and / or conformations of VRs contribute to the variability in cellular tropism, differences in tissue transduction efficiency, and / or antigenic reactivity among different AAV serotypes. In some embodiments, differences in VR sequence and / or structure among different AAV serotypes allow for differential recognition of cell surface glycans and / or tissue specific protein or lipid receptor interaction for internalization.

[0430] Wild type AAV9 (WT AAV9) has nine variable regions VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII and VR-IX (DiMattia 2012, see also Table 3 therein). AAV9 VR-I encompasses amino acid positions 262-269. AAV VR-II encompasses amino acid positions 327-332 and has a role, e.g., in genome packaging. AAV9 VR-III encompasses amino acid positions 382-386. AAV9 VR-IV encompasses amino acid positions 452-460 and has a role, e.g., in liver transduction and / or a delayed blood clearance phenotype. AAV9 VR-V encompasses amino acid positions 488-505 and has a role, e.g., in LamR receptor binding, liver and / or muscle-specific transduction, and / or a delayed blood clearance phenotype. AAV9 VR-VI encompasses amino acid positions 527-539 and has a role, e.g., in LamR receptor binding, and / or Page 96 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) a delayed blood clearance phenotype. AAV9 VR-VII encompasses amino acid positions 545-558 and has a role, e.g., in liver transduction and / or delayed blood clearance phenotype. AAV9 VR- VIII encompasses amino acid positions 581-593 and has a role, e.g., in LamR receptor binding and / or transduction. AAV9 VR-IX encompasses amino acid positions 704-714 and has a role, e.g., in heart tropism, melanoma tropism and / or altered tropism.

[0431] In some embodiments, a rAAV particle disclosed herein is a recombinant AAV (rAAV) particle. In some embodiments, a rAAV particle comprises a variant AAV9 capsid protein comprising a single chain antibody agent insertion disclosed herein. In some embodiments, a single chain antibody agent insertion is in any one or all or a combination of VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII and VR-IX of a parental AAV capsid protein.

[0432] In some embodiments, a parental AAV capsid protein comprises the sequence of a wildtype AAV capsid protein, or a sequence having at least 95% identity to the sequence of a wildtype AAV capsid protein, or a sequence having no more than 20 mutations (e.g., substitutions) as compared to the sequence of a wildtype AAV capsid protein. In some embodiments, a parental AAV capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to the sequence of a wildtype AAV capsid protein. In some embodiments, a parental AAV capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to the sequence of a wildtype AAV capsid protein.

[0433] In some embodiments, a parental AAV capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to the sequence of a wildtype AAV capsid protein and one or more mutations, e.g., as disclosed herein.

[0434] In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding Page 97 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3, or any combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation.

[0435] In some embodiments, a parental AAV capsid protein is other than an AAV9 capsid protein and comprises one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) at a position of VP1, VP2 or VP3 corresponding to position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein.

[0436] In some embodiments, one or more mutations comprises a mutation to an amino acid sequence that is at or near a glycan binding region. In some embodiments, one or more mutations reduces glycan binding. In some embodiments, a glycan is galactose.

[0437] In some embodiments, one or more modifications is at or between amino acids: (a) 271 and 272 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (b) 446 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (c) 470 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (e) 489 and 545 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (f) 591 and 621 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; or (g) any combination or all of (a)-(f).

[0438] In some embodiments, one or more mutations comprises a mutation at positions: (a) 271 and 272 of a VP1, VP2 or VP3 of an AAV9 capsid protein; (b) 446 of a VP1, VP2 or VP3 of an AAV9 capsid protein; (c) 470 of a VP1, VP2 or VP3 of an AAV9 capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2 or VP3 of an AAV9 capsid protein; (e) 489 and 545 of a VP1, VP2, or VP3 of an Page 98 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) AAV9 capsid protein; (f) 591 and 621 of a VP1, VP2 or VP3 of an AAV9 capsid protein; or (g) any combination or all of (a)-(f).

[0439] In some embodiments, a parental AAV capsid protein comprises an AAV9 capsid protein, an AAV1 capsid protein, an AAV2 capsid protein, an AAV3B capsid protein, an AAV4 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, an AAV7 capsid protein, an AAV8 capsid protein, an AAV10 capsid protein, an AAV11 capsid protein, an AAV12 capsid protein, an AAV13 capsid protein, an AAVhu68 capsid protein, or an AAVrh10 capsid protein.

[0440] In some embodiments, a parental AAV capsid protein comprises: an AAV9 capsid protein. In some embodiments, an AAV9 capsid protein comprises: the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001.

[0441] In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein.

[0442] In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. Exemplary mutations including liver de-targeting mutations are disclosed in Pulicherla N. et al., (2011) Molecular Therapy volume 19, pages 1070-1078, the entire contents of which are hereby incorporated by reference. Page 99 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0443] In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1of an AAV9 capsid protein or the corresponding position in a VP2 or VP3, or any combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de- targeting mutation) comprises a mutation at position 503, e.g., a W503R mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 595, e.g., a W595C mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 457, e.g., a N457H mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 574, e.g., a T574S mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 592, e.g., a Q592L mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 498, e.g., a N498Y or an N498I mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 602, e.g., a L602F mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 468, e.g., a P468T mutation. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 500, e.g., a E500D mutation.

[0444] In some embodiments, one or more mutations comprises a mutation to an amino acid sequence that is at or near a glycan binding region. In some embodiments, one or more mutations reduces glycan binding. In some embodiments, a glycan is galactose.

[0445] In some embodiments, one or more modifications is at or between amino acids: (a) 271 and 272 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (b) 446 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV Page 100 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) capsid protein; (c) 470 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (e) 489 and 545 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; (f) 591 and 621 of a VP1, VP2, or VP3 of an AAV9 capsid protein or a corresponding position in a capsid protein of another parental AAV capsid protein; or (g) any combination or all of (a)-(f).

[0446] In some embodiments, one or more mutations comprises a mutation at positions: (a) 271 and 272 of a VP1, VP2 or VP3 of an AAV9 capsid protein; (b) 446 of a VP1, VP2 or VP3 of an AAV9 capsid protein; (c) 470 of a VP1, VP2 or VP3 of an AAV9 capsid protein; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of a VP1, VP2 or VP3 of an AAV9 capsid protein; (e) 489 and 545 of a VP1, VP2 or VP3 of an AAV9 capsid protein; (f) 591 and 621 of a VP1, VP2 or VP3 of an AAV9 capsid protein; or (g) any combination or all of (a)-(f).

[0447] In some embodiments, a parental AAV capsid protein comprises: an AAV1 capsid protein. In some embodiments, an AAV1 capsid protein sequence is provided in SEQ ID NO: 2002.

[0448] In some embodiments, a parental AAV capsid protein comprises: an AAV2 capsid protein. In some embodiments, an AAV2 capsid protein sequence is provided in SEQ ID NO: 2003.

[0449] In some embodiments, a parental AAV capsid protein comprises: an AAV3B capsid protein. In some embodiments, an AAV3B capsid protein sequence is provided in SEQ ID NO: 2007.

[0450] In some embodiments, a parental AAV capsid protein comprises an AAV4 capsid protein. In some embodiments, an AAV4 capsid protein sequence is provided in SEQ ID NO: 2008. Page 101 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0451] In some embodiments, a parental AAV capsid protein comprises: an AAV5 capsid protein. In some embodiments, an AAV5 capsid protein sequence is provided in SEQ ID NO: 2004.

[0452] In some embodiments, a parental AAV capsid protein comprises: an AAV6 capsid protein. In some embodiments, an AAV6 capsid protein sequence is provided in SEQ ID NO: 2005.

[0453] In some embodiments, a parental AAV capsid protein comprises an AAV7 capsid protein. In some embodiments, an AAV7 capsid protein sequence is provided in SEQ ID NO: 2009.

[0454] In some embodiments, a parental AAV capsid protein comprises: an AAV8 capsid protein. In some embodiments, an AAV8 capsid protein sequence is provided in SEQ ID NO: 2006.

[0455] In some embodiments, a parental AAV capsid protein comprises an AAV10 capsid protein. In some embodiments, an AAV10 capsid protein sequence is provided in SEQ ID NO: 2010.

[0456] In some embodiments, a parental AAV capsid protein comprises an AAV11 capsid protein. In some embodiments, an AAV11 capsid protein sequence is provided in SEQ ID NO: 2011.

[0457] In some embodiments, a parental AAV capsid protein comprises an AAV12 capsid protein. In some embodiments, an AAV12 capsid protein sequence is provided in SEQ ID NO: 2012.

[0458] In some embodiments, a parental AAV capsid protein comprises an AAV13 capsid protein. In some embodiments, an AAV13 capsid protein sequence is provided in SEQ ID NO: 2013.

[0459] In some embodiments, a parental AAV capsid protein comprises an AAVhu68 capsid protein. In some embodiments, an AAVhu68 capsid protein sequence is provided in SEQ ID NO: 2014. Page 102 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0460] In some embodiments, a parental AAV capsid protein comprises an AAVrh10 capsid protein. In some embodiments, an AAVrh10 capsid protein sequence is provided in SEQ ID NO: 2015.

[0461] In some embodiments, a single chain antibody agent insertion is in VR-I of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10.

[0462] In some embodiments, a single chain antibody agent insertion is in VR-II of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10.

[0463] In some embodiments, a single chain antibody agent insertion is in VR-III of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10.

[0464] In some embodiments, a single chain antibody agent insertion is in VR-IV of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10.

[0465] In some embodiments, a single chain antibody agent insertion is in VR-V of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10.

[0466] In some embodiments, a single chain antibody agent insertion is in VR-VI of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10.

[0467] In some embodiments, a single chain antibody agent insertion is in VR-VII of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10.

[0468] In some embodiments, a single chain antibody agent insertion is in VR-VIII of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10. Page 103 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0469] In some embodiments, a single chain antibody agent insertion is in VR-IX of a parental AAV capsid protein, e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10.

[0470] In some embodiments, a parental AAV capsid protein is chosen from an AAV9, AAV1, AAV2, AAV3B, AAV5, AAV6 or AAV8 capsid protein, and VR-VIII comprises amino acids 580 to 601 of a VP1, VP2 or VP3 of an AAV9 capsid protein or the corresponding positions in the capsid proteins of another parental AAV capsid protein, e.g., an AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10capsid protein.

[0471] In some embodiments, a parental AAV capsid protein is an AAV9 capsid protein, and VR-VIII comprises amino acids 580 to 601 of a VP1, VP2 or VP3 of an AAV9 capsid protein.

[0472] In some embodiments, a single chain antibody agent insertion is in a VP (e.g., VP1, VP2, and / or VP3) of a parental AAV capsid protein.

[0473] In some embodiments, a single chain antibody agent insertion site is located between amino acids 588 and 589 of a VP1, VP2 or VP3 of a parental AAV capsid protein (e.g., AAV9, AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV10, AAV11, AAV12, AAV13, AAVhu68, or AAVrh10).

[0474] In some embodiments, a single chain antibody agent insertion is located between amino acids 588 and 590 of a VP1, VP2 or VP3 of an AAV9 capsid protein.

[0475] In some embodiments, a single chain antibody agent insertion is located between amino acids 588 and 590 of a VP1 and VP2 of an AAV9 capsid protein.

[0476] In some embodiments, a single chain antibody agent insertion is located between amino acids 588 and 590 of a VP1 and VP3 of an AAV9 capsid protein.

[0477] In some embodiments, a single chain antibody agent insertion is located between amino acids 588 and 590 of a VP2 and VP3 of an AAV9 capsid protein.

[0478] In some embodiments, a single chain antibody agent insertion is located between amino acids 588 and 590 of a VP1, VP2 and VP3 of an AAV9 capsid protein. Page 104 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0479] In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 461 of a VP1, VP2 or VP3 of an AAV9 capsid protein.

[0480] In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 461 of a VP1 and VP2 of an AAV9 capsid protein.

[0481] In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 461of a VP1 and VP3 of an AAV9 capsid protein.

[0482] In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 461of a VP2 and VP3 of an AAV9 capsid protein.

[0483] In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 461 of a VP1, VP2 and VP3 of an AAV9 capsid protein.

[0484] In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 457 of a VP1, VP2 or VP3 of an AAV9 capsid protein.

[0485] In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 457 of a VP1 and VP2 of an AAV9 capsid protein.

[0486] In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 457 a VP1 and VP3 of an AAV9 capsid protein.

[0487] In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 457 a VP2 and VP3 of an AAV9 capsid protein.

[0488] In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 457 of a VP1, VP2 and VP3 of an AAV9 capsid protein.

[0489] In some embodiments, a single chain antibody agent insertion is located between amino acids 451 and 460 of a VP1, VP2 or VP3 of an AAV9 capsid protein.

[0490] In some embodiments, a single chain antibody agent insertion is located between amino acids 451 and 460of a VP1 and VP2 of an AAV9 capsid protein.

[0491] In some embodiments, a single chain antibody agent insertion is located between amino acids 451 and 460 a VP1 and VP3 of an AAV9 capsid protein. Page 105 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0492] In some embodiments, a single chain antibody agent insertion is located between amino acids 451 and 460 a VP2 and VP3 of an AAV9 capsid protein.

[0493] In some embodiments, a single chain antibody agent insertion is located between amino acids 451 and 460of a VP1, VP2 and VP3 of an AAV9 capsid protein.

[0494] In some embodiments, a single chain antibody agent insertion site is located between two adjacent amino acids in VR-VIII of a parental AAV capsid protein.

[0495] In some embodiments, a single chain antibody agent insertion site is located between two non-adjacent amino acids in VR-VIII of a parental AAV capsid protein.

[0496] In some embodiments, insertion of a heterologous peptide (e.g., a single chain antibody agent, e.g., a VHH) replaces a contiguous stretch of amino acids of a parental AAV capsid protein.

[0497] In some embodiments, insertion of a heterologous peptide (e.g., a single chain antibody agent, e.g., a VHH) does not replace a contiguous stretch of amino acids of a parental AAV capsid protein.

[0498] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV9 capsid protein, e.g., as compared to a WT AAV9 capsid protein. In some embodiments, an AAV9 WT capsid protein sequence is provided in SEQ ID NO: 2001. In some embodiments, a variant AAV9 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV9 capsid protein.

[0499] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV1 capsid protein, e.g., as compared to a WT AAV1 capsid protein. In some embodiments, an AAV1 WT capsid protein is provided in SEQ ID NO: 2002. In some embodiments, a variant AAV1 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV1 capsid.

[0500] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV2 capsid protein, e.g., as compared to a WT AAV2 capsid protein. In some embodiments, an AAV2 WT capsid protein is provided in SEQ ID NO: 2003. In some embodiments, a variant Page 106 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) AAV2 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV2 capsid protein.

[0501] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV3B capsid protein, e.g., as compared to a WT AAV3B capsid protein. In some embodiments, an AAV3B WT capsid protein is provided in SEQ ID NO: 2007. In some embodiments, a variant AAV3B capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV3B capsid protein.

[0502] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV5 capsid protein, e.g., as compared to a WT AAV5 capsid protein. In some embodiments, an AAV5 WT capsid protein is provided in SEQ ID NO: 2004. In some embodiments, a variant AAV5 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV5 capsid protein.

[0503] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV6 capsid protein, e.g., as compared to a WT AAV6 capsid protein. In some embodiments, an AAV6 WT capsid protein is provided in SEQ ID NO: 2005. In some embodiments, a variant AAV6 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV6 capsid protein.

[0504] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV8 capsid protein, e.g., as compared to a WT AAV8 capsid protein. In some embodiments, an AAV8 WT capsid protein is provided in SEQ ID NO: 2006. In some embodiments, a variant AAV8 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV8 capsid protein.

[0505] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV4 capsid protein, e.g., as compared to a WT AAV4 capsid protein. In some embodiments, an AAV4 WT capsid protein is provided in SEQ ID NO: 2008. In some embodiments, a variant Page 107 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) AAV4 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV4 capsid protein.

[0506] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV7 capsid protein, e.g., as compared to a WT AAV7 capsid protein. In some embodiments, an AAV7 WT capsid protein is provided in SEQ ID NO: 2009. In some embodiments, a variant AAV7 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV7 capsid protein.

[0507] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV10 capsid protein, e.g., as compared to a WT AAV10 capsid protein. In some embodiments, an AAV10 WT capsid protein is provided in SEQ ID NO: 2010. In some embodiments, a variant AAV10 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV10 capsid protein.

[0508] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV11 capsid protein, e.g., as compared to a WT AAV11 capsid protein. In some embodiments, an AAV11 WT capsid protein is provided in SEQ ID NO: 2011. In some embodiments, a variant AAV11 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV11 capsid protein.

[0509] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV12 capsid protein, e.g., as compared to a WT AAV12 capsid protein. In some embodiments, an AAV12 WT capsid protein is provided in SEQ ID NO: 2012. In some embodiments, a variant AAV12 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV12 capsid protein.

[0510] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV13 capsid protein, e.g., as compared to a WT AAV13 capsid protein. In some embodiments, an AAV13 WT capsid protein is provided in SEQ ID NO: 2013. In some embodiments, a Page 108 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) variant AAV13 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAV13 capsid protein.

[0511] In some embodiments, a rAAV particle disclosed herein comprises a variant AAVhu68 capsid protein, e.g., as compared to a WT AAVhu68 capsid protein. In some embodiments, an AAVhu68 WT capsid protein is provided in SEQ ID NO: 2014. In some embodiments, a variant AAVhu68 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAVhu68 capsid protein.

[0512] In some embodiments, a rAAV particle disclosed herein comprises a variant AAVrh10 capsid protein, e.g., as compared to a WT AAVrh10 capsid protein. In some embodiments, an AAVrh10 WT capsid protein is provided in SEQ ID NO: 2015. In some embodiments, a variant AAVrh10 capsid protein has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity relative to a WT AAVrh10 capsid protein.

[0513] Additional modifications to an AAV9 capsid protein (not including single chain antibody agent insertions disclosed herein) are possible including, for example, variants disclosed in International Patent Application WO 2003 / 052052 filed on November 12, 2002, the entire contents of which are hereby incorporated by reference. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV9 capsid comprising a single chain antibody agent insertion disclosed herein and one or more AAV9 capsid modifications disclosed in WO 2003 / 052052.

[0514] Several other reports disclose modifications to an AAV9 capsid protein, including: Pulicherla N. et al., (2011) Mol Ther.19(6): pp.1070–1078; Wang D. et al., (2018) Mol Ther Methods Clin Dev. (9): pp.234-246; Adachi K. et al., (2014) Nat. Comm. (5): art. 3075; or Bell CL. Et al., (2012) J Virol.86(13): pp.7326–7333, the entire contents each of which are hereby incorporated by reference. In some embodiments, a rAAV particle disclosed herein comprises a variant AAV9 capsid comprising a single chain antibody agent insertion disclosed herein and one or more AAV9 capsid modifications disclosed in any of the reports referenced herein. Page 109 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) Variant AAV capsid proteins with insertion of single chain antibody agents

[0515] Among other things, disclosed herein, are AAV capsid protein variants, e.g., AAV9 capsid protein variants having one or more single chain antibody agent insertions, e.g., as disclosed herein.

[0516] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein, e.g., a variant AAV9 capsid protein comprising a single chain antibody agent insertion disclosed herein.

[0517] In some embodiments, a single chain antibody agent insertion is in any one or all or a combination of VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII and VR-IX of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0518] In some embodiments, a single chain antibody agent insertion is in VR-I of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0519] In some embodiments, a single chain antibody agent insertion is in VR-II of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0520] In some embodiments, a single chain antibody agent insertion is in VR-III of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0521] In some embodiments, a single chain antibody agent insertion is in VR-IV of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0522] In some embodiments, VR-IV of an AAV9 capsid protein comprises amino acids 452 to 460 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion site is located between amino acids 452 to 460 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion site is located between amino acids 452-455, amino acids 452-450, or amino acids 455-460 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion site is located between amino acids 452-453, 452-454, 452-455, 452-456, 452- 457, 452-458, 452-459, 452-460, 455-460, 455-456, 452-459, of VP1, VP2, or VP3 of an AAV9 capsid protein. Page 110 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0523] In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 461 of a VP1 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 461 of a VP2 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 461 of a VP3 of an AAV9 capsid protein.

[0524] In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 461of a VP1 and VP2 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 461 of a VP2 and VP3 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 461 of a VP1 and VP3 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 461 of a VP1, VP2 and VP3 of an AAV9 capsid protein.

[0525] In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 457 of a VP1 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 457 of a VP2 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 457 of a VP3 of an AAV9 capsid protein.

[0526] In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 457 of a VP1 and VP2 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 457 of a VP2 and VP3 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 457 of a VP1 and VP3 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 454 and 457 of a VP1, VP2 and VP3 of an AAV9 capsid protein.

[0527] In some embodiments, a single chain antibody agent insertion is located between amino acids 451 and 460 of a VP1 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 451 and 460 of a VP2 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 451 and 460 of a VP3 of an AAV9 capsid protein. Page 111 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0528] In some embodiments, a single chain antibody agent insertion is located between amino acids 451 and 460 of a VP1 and VP2 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 451 and 460 of a VP2 and VP3 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 451 and 460 of a VP1 and VP3 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 451 and 460 of a VP1, VP2 and VP3 of an AAV9 capsid protein.

[0529] In some embodiments, a single chain antibody agent insertion site is located between two adjacent amino acids in VR-VIII of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0530] In some embodiments, a single chain antibody agent insertion site is located between two non-adjacent amino acids in VR-VIII of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0531] In some embodiments, a single chain antibody agent insertion is in VR-V of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0532] In some embodiments, a single chain antibody agent insertion is in VR-VI of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0533] In some embodiments, a single chain antibody agent insertion is in VR-VII of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0534] In some embodiments, a single chain antibody agent insertion is in VR-VIII of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0535] In some embodiments, VR-VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion site is located between amino acids 580 to 601 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion site is located between amino acids 580-585, amino acids 585-590, amino acids 590- 595, or amino acids 595-601 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion site is located between amino acids 580- 581, between amino acids 581-582, between amino acids 582-583, between amino acids 583- Page 112 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) 584, between amino acids 584-585, between amino acids 585-586, between amino acids 586- 587, between amino acids 587-588, between amino acids 588-589, between amino acids 589- 590, between amino acids 590-591, between amino acids 591-592, between amino acids 592- 593, between amino acids 593-594, between amino acids 594-595, between amino acids 595- 596, between amino acids 596-597, between amino acids 597-598, between amino acids 598- 599, between amino acids 599-600, or between amino acids 600-601 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion site is located between amino acids 588 and 590 of VP1, VP2, or VP3 of an AAV9 capsid protein.

[0536] In some embodiments, a single chain antibody agent insertion is located between amino acids 588 and 590 of a VP1 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 588 and 590 of a VP2 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 588 and 590 of a VP3 of an AAV9 capsid protein.

[0537] In some embodiments, a single chain antibody agent insertion is located between amino acids 588 and 590 of a VP1 and VP2 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 588 and 590 of a VP2 and VP3 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 588 and 590 of a VP1 and VP3 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 588 and 590 of a VP1, VP2 and VP3 of an AAV9 capsid protein.

[0538] In some embodiments, a single chain antibody agent insertion site is located between two adjacent amino acids in VR-VIII of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0539] In some embodiments, a single chain antibody agent insertion site is located between two non-adjacent amino acids in VR-VIII of an AAV capsid protein, e.g., an AAV9 capsid protein.

[0540] In some embodiments, a single chain antibody agent insertion is in VR-IX of an AAV capsid protein, e.g., an AAV9 capsid protein. Page 113 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0541] In some embodiments, a single chain antibody agent insertion is in VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion site is located between amino acids 588 and 590 of VP1, VP2, or VP3 of an AAV9 capsid protein.

[0542] In some embodiments, insertion of a heterologous peptide (e.g., a single chain antibody agent, e.g., a VHH) replaces a contiguous stretch of amino acids of a parental AAV capsid protein, e.g., an AAV9 parental capsid protein.

[0543] In some embodiments, insertion of a heterologous peptide(e.g., a single chain antibody agent, e.g., a VHH) does not replace a contiguous stretch of amino acids of a parental AAV capsid protein, e.g., an AAV9 parental capsid protein.

[0544] In some embodiments, a recombinant AAV particle (rAAV) disclosed herein comprises a variant AAV capsid protein, e.g., a variant AAV9 capsid protein, comprising a single chain antibody agent insertion disclosed herein, and one or more sequences of VP1, VP2, or VP3 of an AAV9 capsid protein.

[0545] In some embodiments, a recombinant AAV particle (rAAV) disclosed herein comprises a variant AAV capsid protein, e.g., a variant AAV9 capsid protein, comprising a single chain antibody agent insertion disclosed herein. In some embodiments, a single chain antibody agent insertion disclosed herein comprises one or more linkers. In some embodiments, one or more linkers are situated on the N-terminus of a single chain antibody agent and / or the C- terminus of a single chain antibody agent. In some embodiments, one or more linkers are situated on the N-terminus of a single chain antibody agent. In some embodiments, one or more linkers are situated on the C-terminus of a single chain antibody agent. In some embodiments, one or more linkers are situated on the N-terminus and on the C-terminus of a single chain antibody agent.

[0546] In some embodiments, one or more linkers comprise one or more GGGGS repeats. In some embodiments, a single chain antibody agent comprises one GGGGS linker provided in SEQ ID NO: 55 on the N-terminus and / or the C-terminus of a single chain antibody agent. In some embodiments, a single chain antibody agent comprises two GGGGS linkers provided in SEQ ID NO: 56 on the N-terminus and / or the C-terminus of a single chain antibody agent. In some embodiments, a single chain antibody agent comprises three GGGGS linkers Page 114 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) provided in SEQ ID NO: 57 on the N-terminus and / or the C-terminus of a single chain antibody agent. In some embodiments, a single chain antibody agent comprises four GGGGS linkers provided in SEQ ID NO: 58 on the N-terminus and / or the C-terminus of a single chain antibody agent. In some embodiments, a single chain antibody agent comprises five GGGGS linkers provided in SEQ ID NO: 59 on the N-terminus and / or the C-terminus of a single chain antibody agent.

[0547] In some embodiments, one or more linkers comprise a coiled-coil alpha helix domain or a fragment thereof. In some embodments, a single chain antibody agent comprises one or more coiled-coil alpha helix domains on the N-terminus of the single chain antibody agent and / or the C-terminus of the single chain antibody agent. In some embodiments, one or more linkers comprise a coiled-coil alpha helix domain that is or comprises a leading coil according to SEQ ID NO: 53 or a sequence with at least 90% identity thereto. In some embodiments, one or more linkers comprise a coiled-coil alpha helix domain that is or comprises a returning coil according to SEQ ID NO: 54 or a sequence with at least 90% identity thereto.

[0548] In some embodiments, a single chain antibody agent comprises one or more GGGGS linkers as described herein and one or more coiled-coil alpha helix domains as described herein on the N-terminus of the single chain antibody agent. In some embodiments, a single chain antibody agent comprises one or more GGGGS linkers as described herein and one or more coiled-coil alpha helix domains as described herein on the C-terminus of the single chain antibody agent. In some embodiments, a single chain antibody agent comprises one or more GGGGS linkers as described herein and one or more coiled-coil alpha helix domains as described herein on the N-terminus and on the C-terminus of the single chain antibody agent.

[0549] In some embodiments, a single chain antibody agent comprises a (GGGGS)5linker according to SEQ ID NO: 59 at the N-terminus and a (GGGGS)1linker according to SEQ ID NO: 55 at the C-terminus.

[0550] In some embodiments, a single chain antibody agent comprises a (GGGGS)4linker according to SEQ ID NO: 58 at the N-terminus.

[0551] In some embodiments, a single chain antibody agent comprises (i) a leading coiled-coil alpha helix domain and a (GGGGS)5linker according to SEQ ID NO: 60 at the N- Page 115 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) terminus; and (ii) a (GGGGS)1linker and a returning coiled-coil alpha helix domain according to SEQ ID NO: 61 at the C-terminus.

[0552] Exemplary linker sequences described herein are provided in Table 2. Table 2: Exemplary linker sequences SEQ ID Feature Sequence NO

[0553] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV (e.g., AAV9) capsid protein comprising a single chain antibody agent provided in Table 1.

[0554] In some embodiments, a rAAV particle disclosed herein comprises: (1) a variant AAV (e.g., AAV9) capsid protein comprising any one of the single chain antibody agents provided in Table 1; and (2) one or more sequences of a VP (e.g., VP1, VP2, and / or VP3) of an AAV (e.g., AAV9) capsid protein.

[0555] In some embodiments, the single chain antibody agent is inserted in a VP1 of a parental AAV capsid protein.

[0556] In some embodiments, the single chain antibody agent is not inserted in a VP2 and / or VP3 of a parental AAV capsid protein. Page 116 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01)

[0557] In some embodiments, the single chain antibody agent is inserted in a VP1, VP2 and / or VP3 of a parental AAV capsid protein.

[0558] In some embodiments, a rAAV particle disclosed herein comprising a variant AAV (e.g., AAV9) capsid protein comprising a single chain antibody agent provided in Table 1 has an at least 1.5 fold enhanced CNS transduction compared to a rAAV particle comprising a corresponding parental AAV (e.g., AAV9) capsid protein, e.g., without a single chain antibody agent insertion.

[0559] In some embodiments, a rAAV particle disclosed herein comprising a variant AAV (e.g., AAV9) capsid protein comprising a single chain antibody agent provided in Table 1 can be useful for CNS-targeting, e.g., targeting a rAAV particle comprising a variant AAV (e.g., AAV9) capsid protein to a CNS cell and / or tissue.

[0560] In some embodiments, a single chain antibody agent insertion in a variant AAV capsid protein disclosed herein does not comprise an additional sequence N-terminal of a single chain antibody agent sequence provided in Table 1.

[0561] In some embodiments, a single chain antibody agent insertion in a variant AAV capsid protein disclosed herein does not comprise an additional sequence C-terminal of a single chain antibody agent sequence provided in Table 1.

[0562] In some embodiments, a single chain antibody agent insertion in a variant AAV capsid protein disclosed herein does not comprise an additional sequence N-terminal and C- terminal of a single chain antibody agent sequence provided in Table 1. Exemplary capsid variants with single chain antibody agent insertions in VR-VIII

[0563] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a single chain antibody agent insertion relative to a parental AAV capsid protein. In some embodiments, (1) a single chain antibody agent insertion comprises any one of the single chain antibody agent insertions disclosed herein; (2) a single chain antibody agent comprises one or more linkers disclosed herein; (3) a single chain antibody agent insertion site is in a VR-VIII or VR-IV of a parental AAV capsid protein; (4) at least one amino acid is deleted in a parental AAV capsid protein (e.g., a sequence between the insertion site of a single Page 117 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) chain antibody agent is deleted in a parental AAV9 capsid protein); and (5) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the single chain antibody agent is inserted in a VP1 of a parental AAV capsid protein. In some embodiments, the single chain antibody agent is not inserted in a VP2 and / or VP3 of a parental AAV capsid protein.

[0564] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a single chain antibody agent insertion relative to a parental AAV capsid protein. In some embodiments, (1) a single chain antibody agent insertion comprises a single chain antibody agent insertion provided in Table 1; (2) a single chain antibody agent comprises one or more linkers disclosed herein; (3) a single chain antibody agent insertion site is in a VR-VIII of a parental AAV capsid protein; (4) at least one amino acid is deleted in a parental AAV capsid protein; and (5) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the single chain antibody agent is inserted in a VP1 of a parental AAV capsid protein. In some embodiments, the single chain antibody agent is not inserted in a VP2 and / or VP3 of a parental AAV capsid protein. In some embodiments, a single chain antibody agent insertion is in VR-VIII of an AAV9 capsid protein. In some embodiments, a VR- VIII of an AAV9 capsid protein comprises amino acids 580 to 601 of VP1, VP2 or VP3. In some embodiments, a single chain antibody agent insertion is located between amino acids 588 and 590 of VP1, VP2, or VP3 of an AAV9 capsid protein. In some embodiments, a sequence between the insertion site of a single chain antibody agent is deleted in a parental AAV9 capsid protein, e.g., amino acids 589 is deleted. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at Page 118 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation.

[0565] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a single chain antibody agent insertion relative to a parental AAV capsid protein. In some embodiments, (1) a single chain antibody agent insertion comprises a single chain antibody agent insertion provided in Table 1; (2) a single chain antibody agent comprises one or more linkers disclosed herein; (3) a single chain antibody agent insertion site is in a VR-IV of a parental AAV capsid protein; (4) at least one amino acid is deleted in a parental AAV capsid protein; and (5) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the single chain antibody agent is inserted in a VP1 of a parental AAV capsid protein. In some embodiments, the single chain antibody agent is not inserted in a VP2 and / or VP3 of a parental AAV capsid protein. In some embodiments, a single chain antibody agent insertion is in VR- IV of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 454 to 461 of VP1, VP2, or VP3 of a parental AAV9 capsid protein. In some embodiments, a sequence between the insertion site of a single chain antibody agent is deleted in a parental AAV9 capsid protein, e.g., amino acids 455 to 460 are deleted. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at Page 119 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation.

[0566] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a single chain antibody agent insertion relative to a parental AAV capsid protein. In some embodiments, (1) a single chain antibody agent insertion comprises a single chain antibody agent insertion provided in Table 1; (2) a single chain antibody agent comprises one or more linkers disclosed herein; (3) a single chain antibody agent insertion site is in a VR-IV of a parental AAV capsid protein; (4) at least one amino acid is deleted in a parental AAV capsid protein; and (5) a parental AAV capsid protein comprises an AAV9 capsid protein. In some embodiments, the single chain antibody agent is inserted in a VP1 of a parental AAV capsid protein. In some embodiments, the single chain antibody agent is not inserted in a VP2 and / or VP3 of a parental AAV capsid protein. In some embodiments, a single chain antibody agent insertion is in VR- IV of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 454 to 457 of VP1, VP2, or VP3 of a parental AAV9 capsid protein. In some embodiments, a sequence between the insertion site of a Page 120 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) single chain antibody agent is deleted in a parental AAV9 capsid protein, e.g., amino acids 455 and 456 are deleted. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a W503R mutation.

[0567] In some embodiments, a rAAV particle disclosed herein comprises a variant AAV capsid protein comprising a single chain antibody agent insertion relative to a parental AAV capsid protein. In some embodiments, (1) a single chain antibody agent insertion comprises a single chain antibody agent insertion provided in Table 1; (2) a single chain antibody agent comprises one or more linkers disclosed herein; (3) a single chain antibody agent insertion site is in a VR-IV of a parental AAV capsid protein; (4) at least one amino acid is deleted in a parental AAV capsid protein; and (5) a parental AAV capsid protein comprises an AAV9 capsid protein. Page 121 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) In some embodiments, the single chain antibody agent is inserted in a VP1 of a parental AAV capsid protein. In some embodiments, the single chain antibody agent is not inserted in a VP2 and / or VP3 of a parental AAV capsid protein. In some embodiments, a single chain antibody agent insertion is in VR- IV of an AAV9 capsid protein. In some embodiments, a single chain antibody agent insertion is located between amino acids 451 to 460 of VP1, VP2, or VP3 of a parental AAV9 capsid protein. In some embodiments, a sequence between the insertion site of a single chain antibody agent is deleted in a parental AAV9 capsid protein, e.g., amino acids 452 to 459 are deleted. In some embodiments, an AAV9 capsid protein comprises the sequence of a wild-type AAV9 capsid protein provided in SEQ ID NO: 2001; or a sequence having at least 95% identity to SEQ ID NO: 2001; or a sequence having no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having no more than 1, no more than 2, no more than 5, no more than 10, or no more than 20 mutations (e.g., substitutions) as compared to SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises the sequence provided in SEQ ID NO: 2001. In some embodiments, an AAV9 capsid protein comprises a sequence having at least 95% identity (e.g., at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or at least 99.5% identity) to SEQ ID NO: 2001, and one or more mutations, e.g., as disclosed herein. In some embodiments, one or more mutations comprises a mutation that alters a binding profile of a parental AAV capsid protein (e.g., binding to one or more tissues). In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein comprises a mutation that reduces binding to one or more tissues such as liver tissue, e.g., a liver-detargeting mutation. In some embodiments, one or more mutations that alters a binding profile of a parental AAV capsid protein (e.g., a liver de-targeting mutation) comprises a mutation at position 503, 595, 457, 574, 592, 498, 602, 468, or 500 of a VP1 of an AAV9 capsid protein or the corresponding position in a VP2 or VP3 protein or a combination thereof. In some embodiments, a mutation that alters a binding profile of a pa...

Claims

Attorney Docket No.: 2011256-2514 (P1862PCT01) CLAIMS We claim:

1. A recombinant adeno-associated virus (rAAV) particle comprising: (a) a variant AAV capsid protein comprising a single chain antibody agent that binds to a transferrin receptor (TfR1) or a variant or a fragment thereof, wherein the single chain antibody agent is inserted in a variable region (VR) of a parental AAV capsid protein, and (b) a heterologous nucleic acid comprising a nucleotide sequence encoding a payload.

2. The rAAV particle of claim 1, wherein the single chain antibody agent comprises one or more complementarity determining regions (CDRs), optionally wherein the single chain antibody agent comprises a CDR1, a CDR2 and a CDR3.

3. The rAAV particle of claim 1 or 2, wherein the single chain antibody agent is or comprises: (i) a single domain antibody (e.g., a VHH); or (ii) a single chain Fv.

4. The rAAV particle of any one of the preceding claims, wherein the single chain antibody agent comprises a sequence provided in Table 1.

5. The rAAV particle of any one of the preceding claims, wherein the single chain antibody agent comprises: (i) a CDR1 sequence provided in Table 1; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a CDR1 sequence provided in Table 1; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to a CDR1 sequence provided in Table 1; (ii) a CDR2 sequence provided in Table 1; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a Page 376 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) CDR2 sequence provided in Table 1; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to a CDR2 sequence provided in Table 1; and / or (iii) a CDR3 sequence provided in Table 1; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a CDR3 sequence provided in Table 1; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to a CDR3 sequence provided in Table 1.

6. The rAAV particle of any one of claims 1-5, wherein the single chain antibody agent comprises: (i) a CDR1 sequence of SEQ ID NO: 2; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 2; (ii) a CDR2 sequence of SEQ ID NO: 3; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 3; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 3; and / or (iii) a CDR3 sequence of SEQ ID NO: 4; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 4; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO:

4.

7. The rAAV particle of claim 6, wherein the single chain antibody agent comprises the sequence of SEQ ID NO: 1, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

8. The rAAV particle of any one of claims 1-5, wherein the single chain antibody agent comprises: Page 377 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) (i) a CDR1 sequence of SEQ ID NO: 6; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 6; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 6; (ii) a CDR2 sequence of SEQ ID NO: 7; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 7; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 7; and / or (iii) a CDR3 sequence of SEQ ID NO: 8; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 8; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO:

8.

9. The rAAV particle of claim 8, wherein the single chain antibody agent comprises the sequence of SEQ ID NO: 5, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

10. The rAAV particle of any one of claims 1-5, wherein the single chain antibody agent comprises: (i) a CDR1 sequence of SEQ ID NO: 22; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 22; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 22; (ii) a CDR2 sequence of SEQ ID NO: 23; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 23; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 23; and / or (iii) a CDR3 sequence of SEQ ID NO: 24; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at Page 378 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 24; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO:

24.

11. The rAAV particle of claim 10, wherein the single chain antibody agent comprises the sequence of SEQ ID NO: 21, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

12. The rAAV particle of any one of claims 1-5, wherein the single chain antibody agent comprises: (i) a CDR1 sequence of SEQ ID NO: 50; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 50; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 50; (ii) a CDR2 sequence of SEQ ID NO: 51; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 51; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO: 51; and / or (iii) a CDR3 sequence of SEQ ID NO: 52; or a sequence with at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 52; or a sequence having 1, 2, 3, 4 or 5 substitutions relative to SEQ ID NO:

52.

13. The rAAV particle of claim 12, wherein the single chain antibody agent comprises the sequence of SEQ ID NO: 49, or a sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity thereto.

14. The rAAV particle of any one of the preceding claims, wherein the single chain antibody agent binds to a human TfR (hTfR), or a cyno TfR. Page 379 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) 15. The rAAV particle of any one of the preceding claims, wherein the single chain antibody agent binds to hTfR1 with an affinity (KD) of about 10 nM to about 25000 nM.

16. The rAAV particle of any one of the preceding claims, wherein the single chain antibody agent comprises one or more linkers.

17. The rAAV particle of claim 16, wherein the one or more linkers are situated on the N- terminus of the single chain antibody agent and / or the C-terminus of the single chain antibody agent.

18. The rAAV particle of claim 16 or 17, wherein the one or more linkers comprise: (i) one or more (GGGGS) repeats, and / or (ii) a coiled-coil alpha helix domain or a fragment thereof, optionally wherein the coiled- coil alpha helix domain comprises a leading coil sequence according to SEQ ID NO: 53, or a sequence with at least 90% identity thereto, and / or the coiled-coil alpha helix domain comprises a returning coil sequence according to SEQ ID NO: 54, or a sequence with at least 90% identity thereto.

19. The rAAV particle of any one of the preceding claims, wherein the single chain antibody agent insertion site is located between two adjacent amino acids in the variable region of the parental AAV capsid protein.

20. The rAAV particle of any one of claims 1-18, wherein the single chain antibody agent insertion site is located between two non-adjacent amino acids in the variable region of the parental AAV capsid protein.

21. The rAAV particle of any one of the proceeding claims, wherein the single chain antibody agent insertion site is in a VP1 of the parental AAV capsid protein.

22. The rAAV particle of any one of the proceeding claims, wherein the single chain antibody agent insertion site is not in a VP2 and / or VP3 of the parental AAV capsid protein. Page 380 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) 23. The rAAV particle of any one of the preceding claims, wherein the insertion of the single chain antibody agent replaces a contiguous stretch of amino acids of the parental AAV capsid protein.

24. The rAAV particle of any one of claims 1-22, wherein the insertion of the single chain antibody agent does not replace a contiguous stretch of amino acids of the parental AAV capsid protein.

25. The rAAV particle of any one of the preceding claims, wherein the single chain antibody agent insertion is in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VIII or VR-IX of the parental AAV capsid protein.

26. The rAAV particle of any one of the preceding claims, wherein the single chain antibody agent insertion is in VR-IV of the parental AAV capsid protein.

27. The rAAV particle of claim 26, wherein the parental AAV capsid protein is an AAV9 capsid protein and VR-IV comprises amino acids 452-460 of VP1, VP2, or VP3 of the AAV9 capsid protein.

28. The rAAV particle of claim 27, wherein the single chain antibody agent insertion site is located between amino acids: (1) 454 and 461 of VP1 of the AAV9 capsid protein, (2) 454 and 457 of VP1 of the AAV9 capsid protein; or (3) 451 and 460 of VP1 of the AAV9 capsid protein.

29. The rAAV particle of any one of claims 1-25, wherein the single chain antibody agent insertion is in VR-VIII of the parental AAV capsid protein. Page 381 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) 30. The rAAV particle of claim 29, wherein the parental AAV capsid protein is an AAV9 capsid protein and VR-VIII comprises amino acids 580 to 601 of VP1, VP2, or VP3 of the AAV9 capsid protein.

31. The rAAV particle of claim 30, wherein the single chain antibody agent insertion site is located between amino acids 588 and 590 of VP1 of an AAV9 capsid protein or the corresponding position in VP1 of another parental AAV capsid protein.

32. The rAAV particle of any one of the preceding claims, wherein the variant AAV capsid protein confers at least 1.5-fold increased infectivity and / or transduction of a central nervous system (CNS) cell compared to infectivity and / or transduction of: (i) the CNS cell by a control AAV particle comprising the corresponding parental AAV capsid protein without an insertion of a single chain antibody agent in the AAV capsid protein; or (ii) a CNS cell which does not express a receptor recognized by the variant AAV capsid protein (e.g., hTfR) by the variant AAV capsid protein.

33. The rAAV particle of claim 32, wherein the CNS cell comprises a CNS epithelial cell, an endothelial cell associated with a blood brain barrier, a nerve cell, a CNS connective tissue cell, a stem cell or progenitor cell, a CNS immune cell, a spinal cord cell, or combinations thereof.

34. The rAAV particle of claim 33, wherein: (i) the CNS epithelial cell comprises a cell that lines one or more brain ventricles, (ii) the nerve cell comprises nerve support cells, and / or (iii) the CNS connective tissue cell comprises fat cells, meninges cells, or both.

35. The rAAV particle of any one of the preceding claims, wherein the variant AAV capsid protein further comprises one or more modifications to an amino acid sequence flanking the single chain antibody agent insertion site.

36. The rAAV particle claim 35, wherein the one or more modifications are: Page 382 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) (a) between the two amino acids flanking the insertion site; (b) within about 10 amino acids upstream or downstream of the location of the single chain antibody agent insertion site, e.g., within about 5 amino acids upstream or downstream of the location of the single chain antibody agent insertion site.

37. The rAAV particle of 35 or 36, wherein the one or more modifications comprises an insertion, deletion, mutation, or combinations thereof.

38. The rAAV particle of claim 37, wherein the deletion is between two amino acids flanking the insertion site, optionally wherein the deletion is a deletion of: (i) at least 1 amino acid, or (ii) about 1 to 10 amino acids.

39. The rAAV particle of claim 37 or 38, wherein when the insertion is in VR-IV of the parental AAV capsid protein the deletion comprises: (1) a deletion of amino acids 455 to 460 inclusive of endpoints of VP1 of the AAV9 capsid protein; (2) a deletion of amino acids 455 and 456 of VP1 of the AAV9 capsid protein; or (3) a deletion of amino acids 452 to 459 inclusive of endpoints of VP1 of the AAV9 capsid protein.

40. The rAAV particle of claim 37 or 38, wherein when the insertion is in VR-VIII of the parental AAV capsid protein the deletion comprises a deletion of amino acid 589 of VP1 of the AAV9 capsid protein.

41. The rAAV particle of any one of the preceding claims wherein the variant AAV capsid protein further comprises one or more modifications to an amino acid sequence that is at or near a glycan binding region, optionally wherein the one or more modifications reduces glycan binding. Page 383 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) 42. The rAAV particle of any one of claims 35-41, wherein the one or more modifications is at or between amino acids: (a) 271 and 272 of VP1 of the AAV9 capsid protein or the corresponding position in the capsid protein of another AAV serotype; (b) 446 of VP1 of the AAV9 capsid protein or the corresponding position in the capsid protein of another AAV serotype; (c) 470 of VP1 of the AAV9 capsid protein or the corresponding position in the capsid protein of another AAV serotype; (d) 501 and 505 (e.g., at any one or all or a combination of residues 501, 502, 503, 504 or 505) of VP1 of the AAV9 capsid protein or the corresponding position in the capsid protein of another AAV serotype; (e) 489 and 545 of VP1 of the AAV9 capsid protein or the corresponding position in the capsid protein of another AAV serotype; (f) 591 and 621 of VP1 of the AAV9 capsid protein or the corresponding position in the capsid protein of another AAV serotype; or (g) any combination, or all, of (a)-(f).

43. The rAAV particle of any one of the preceding claims, wherein the variant AAV capsid protein has at least 90% identity relative to a parental AAV capsid protein, wherein percent identity is determined by comparing the sequence of the variant AAV capsid protein without the single chain antibody agent insertion, with the parental AAV capsid protein.

44. The rAAV particle of any one of the preceding claims, wherein the parental AAV capsid protein is an AAV9 capsid protein of SEQ ID NO: 2001.

45. The rAAV particle of any one of the preceding claims, wherein the payload is or comprises a polypeptide.

46. The rAAV particle of claim 45, wherein the polypeptide is or comprises: (i) a CRISPR-Cas protein, optionally, wherein the CRISPR-Cas protein is a Type II, Type V or Type VI CRISPR-Cas protein, e.g., a Cas9 protein, a Cas12a protein, a Cas12b protein, a Page 384 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) Cas12c protein, a Cas12d protein, a Cas12e protein, a Cas12f protein, a Cas12g protein, a Cas12h protein, a Cas12i protein, a Cas13a protein, a Cas13b protein or a variant or fragment of any of the foregoing; (ii) a Zinc finger protein, or a variant or a fragment thereof; (iii) a TALEN protein, or a variant or a fragment thereof; (iv) a base editor, or a variant or a fragment thereof; (v) a prime editor, or a variant or a fragment thereof; (vi) a meganuclease or a variant or a fragment thereof; (vii) an enzyme; (viii) an antibody; and / or (ix) a secreted protein.

47. The rAAV particle of claim 45 or 46, wherein the polypeptide is associated with a CNS disorder.

48. The rAAV particle of any one of claims 1-44, wherein the payload is or comprises an RNA molecule, optionally wherein the RNA molecule is a mRNA, an siRNA, a miRNA, a gRNA, an antisense RNA, a circular RNA, an snRNA, or an aptamer.

49. The rAAV particle of claim 48, wherein the RNA molecule targets a nucleic acid molecule encoding a polypeptide associated with a CNS disorder.

50. The rAAV particle of claim 47 or 49, wherein the CNS disorder is chosen from Friedreich’s Ataxia, Dravet Syndrome, Spinocerebellar Ataxia Type 3, Niemann-Pick Type C, Huntington’s Disease, Pompe Disease, Myotonic Dystrophy Type 1, Glut1 Deficiency Syndrome (De Vivo Syndrome), Tay-Sachs, Spinal Muscular Atrophy, Alzheimer's disease, Amyotrophic lateral sclerosis (ALS), Danon disease, Rett Syndrome, Angelman Syndrome, Parkinson’s disease, tauopathies, genetic epilepsies, or combinations thereof.

51. The rAAV particle of any one of claims 1-44, wherein the payload is or comprises a DNA molecule. Page 385 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) 52. The rAAV particle of any one of the preceding claims, wherein the nucleotide sequence encoding a payload is operably linked to a promoter, optionally wherein the promoter is a CNS promoter.

53. The rAAV particle of claim 52, wherein the CNS promoter is chosen from: a GFAP promoter, a SYN1 promoter, a NSE / RU5’ promoter, a neuroactive peptide cholecystokinin (CCK) promoter, a myelin basic promoter (MBP), a human myelin associated glycoprotein promoter, a phosphate-activated glutaminase (PAG) promoter, a vesicular glutamate transporter (vGLUT) promoter, a glutamic acid decarboxylase (GAD) promoter, Camk2a promoter, TH (tyrosine hydroxylase) promoter, Hb9 promoter, CNP promoter, NES (nestin) promoter, Tub1a promoter, SST (somatostatin) promoter, MeCP2 promoter, or any combination thereof.

54. A pharmaceutical composition comprising: (a) a rAAV particle of any one of the preceding claims; and (b) a pharmaceutically acceptable excipient.

55. A method of delivering a payload to a CNS cell, comprising administering the pharmaceutical composition of claim 54 to the CNS cell.

56. A method of treating a subject having a CNS disorder and / or ameliorating a symptom of a CNS disorder in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 54.

57. The method of claim 56, wherein the CNS disorder is chosen from Friedreich’s Ataxia, Dravet Syndrome, Spinocerebellar Ataxia Type 3, Niemann-Pick Type C, Huntington’s Disease, Pompe Disease, Myotonic Dystrophy Type 1, Glut1 Deficiency Syndrome (De Vivo Syndrome), Tay-Sachs, Spinal Muscular Atrophy, Alzheimer's disease, Amyotrophic lateral sclerosis (ALS), Danon disease, Rett Syndrome, Angelman Syndrome, Parkinson’s disease, tauopathies, genetic epilepsies, or any combination thereof. Page 386 of 388 12609165v1Attorney Docket No.: 2011256-2514 (P1862PCT01) 58. The method of any one of claims 55-57, wherein the pharmaceutical composition is administered via a route of administration chosen from: intravenous, intraarterial, intracoronoary, intraparenchymal, subpial, intrathecal, intraocular, intracerebroventricular (ICV), intracisternal magna (ICM), or intramuscular.

59. The method of any one of claims 55-58, wherein the subject is a human.

60. An isolated cell transduced with the rAAV particle of any one of claims 1-53.

61. An isolated nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein, wherein the variant AAV capsid protein comprises a single chain antibody agent that binds to a transferrin receptor (TfR) or a variant or a fragment thereof, and wherein the single chain antibody agent is inserted in a variable region (VR) of a parental AAV capsid protein.

62. An isolated cell comprising the nucleic acid of claim 61.

63. A variant AAV capsid protein, wherein the variant AAV capsid protein comprises a single chain antibody agent that binds to a transferrin receptor (TfR) or a variant or a fragment thereof, and wherein the single chain antibody agent is inserted in a variable region (VR) of a parental AAV capsid protein. Page 387 of 388 12609165v1

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