Virus particles that retarget transferrin receptor 1

Abstract

Provided herein are compositions and methods for retargeting viral particles, such as adeno-associated virus (AAV) particles, to TfR1 expressing cells, including blood brain barrier (BBB) endothelial cells. Thus, the altered AAV may be a viable gene therapy platform for gene therapy of target cells expressing TfR1, e.g., for gene therapy across the blood-brain barrier of a patient in need thereof.

Description

Technical Field

[0001] The present disclosure relates to methods of preparing and using recombinant viral particles (e.g., recombinant AAV particles) that comprise a capsid protein that re-targets a cell surface protein, which permits the viral particle to bind to transferrin receptor 1 (also referred to herein as TfR or CD71; encoded by TFRC), which can be (a) used to infect (e.g., genetically modify) cells that express TfR and / or (b) trans-endocytose AAV particles across cells that express TfR, such as blood-brain barrier endothelial cells, in vitro or in vivo.

[0002] Sequence Listing

[0003] A sequence listing in xml format, named "11081WO01_xml.xml", was created on Jul. 28, 2023, and is 461 Kb in size, and is hereby incorporated by reference in its entirety. Background Art

[0004] Delivery of genes into specific target cells has become one of the most important techniques in modern medicine for the potential treatment of a variety of chronic and genetic diseases. Ideally, a gene delivery vector is able to stably introduce genetic material into the desired cells and avoid introducing genetic material into non-target cells.

[0005] Viral particles, particularly adenovirus-associated virus (AAV)-based viral particles, have been the focus of many studies as gene delivery vectors because AAV is capable of transducing a wide range of primate species and tissues in vivo without evidence of pathogenicity. (Muzyczka et al. (1992) Current Topics in Microbiology and Immunology, 158:97-129). In addition, AAV safely transduces post-mitotic tissues. Although the virus may occasionally integrate into the host chromosome, it rarely integrates and integrates into a safe harbor locus on human chromosome 19, and only when the replication (Rep) proteins are supplied in trans. The AAV genome rapidly circles and concatenates in infected cells and exists in a stable episomal state in infected cells for long-term stable expression of its payload.

[0006] In addition, in recent years, manipulation and redirection of AAV infection to specific cells have been achieved. Many of the advances in targeted gene therapy using viral particles can be summarized as non-recombinant (non-genetic) or recombinant (genetic) modification of viral particles, which results in pseudotyping, amplification, and / or re-targeting of the natural tropism of viral particles. (Reviewed in Nicklin and Baker (2002) Curr. Gene Ther. 2:273-93; Verheiji and Rottier (2012) Advances Virol 2012:1-15).

[0007] In direct recombinant targeting methods, the targeting ligand is directly inserted into or conjugated to the viral capsid, i.e., the protein viral capsid gene is modified to express a capsid protein comprising a heterologous targeting ligand. The targeting ligand binds, for example, to a receptor or marker expressed preferentially or exclusively on the target cell rather than redirecting it. (Stachler et al., (2006) Gene Ther. 13:926–931; White et al., (2004) Circulation 109:513–519; see also Park et al., (2007) Frontiers in Bioscience 13:2653–59; Girod et al., (1999) Nature Medicine 5:1052–56; Grifman et al., (2001) Molecular Therapy 3:964–75; Shi et al., (2001) Human Gene Therapy 12:1697–1711; Shi and Bartlett, (2003) Molecular Therapy 7:515–525).

[0008] In an indirect reconstitution method, a viral capsid is modified with a heterologous "scaffold" and then linked to an adaptor that contains a targeting ligand. The adaptor binds to the scaffold and to the target cell. (Arnold et al., (2006) Mol. Ther. 5:125-132; Ponnazhagen et al., (2002) J. Virol. 76:12900-907; see also WO 97 / 05266) Scaffolds include, for example, (1) an Fc-binding molecule (e.g., an Fc receptor, protein A, etc.) that binds to the Fc of an antibody adaptor, (2) (strept)avidin that binds to a biotinylated adaptor, (3) biotin that binds to an adaptor fused to (strept)avidin, (4) a detectable label that can be used to detect and / or isolate viral particles by binding via a bispecific adaptor that can non-covalently bind the detectable label and a target molecule, and more recently (5) a protein:protein binding pair that forms an isopeptide bond that has been described for various viral particles. (See, e.g., Gigout et al., (2005) Mol. Ther. 11:856–865; Stachler et al., (2008) Mol. Ther. 16:1467–1473; Quetglas et al., (2010) Virus Res. 153:179–196; Ohno et al., (1997) Nat. Biotechnol. 15:763–767; Klimstra et al., (2005) Virology 338:9–21).

[0009] With the advancement of the ability to provide targeted AAV infection, there remains a need for viral systems suitable for targeted transfer of a nucleic acid of interest to a target cell or receptor. SUMMARY OF THE INVENTION

[0010] It is shown herein that an AAV capsid protein can be modified to allow targeted introduction of a nucleotide of interest into mammalian cells expressing transferrin receptor 1 (TfR1; CD71) and / or to allow a modified AAV capsid to cross the blood-brain barrier (BBB) via BBB endothelial cells expressing TfR1.

[0011] The virus particles as described herein are particularly suitable for specifically targeting the introduction of a nucleotide of interest into cells expressing transferrin receptor 1 (TfR1; CD71) or crossing the blood-brain barrier, because the virus capsid or virus capsid protein as described herein comprises a first member of a protein:protein binding pair associated with a second member of the homologous protein:protein binding pair, wherein the second member is linked (e.g., fused) to a targeting ligand that binds to transferrin receptor 1 (TfR1) expressed on the cell surface.

[0012] Recombinant virus capsid proteins and virus particles are described herein (e.g., wherein the virus capsid protein capsidates a nucleic acid of interest), as well as compositions comprising the virus capsid protein and / or virus particle, such as a pharmaceutical composition comprising the virus capsid protein and / or virus particle, wherein the recombinant virus capsid protein as described herein comprises: (i) a first member of a protein:protein binding pair inserted and / or displayed by the virus capsid, (ii) a second member of the protein:protein binding pair, wherein the first member of the protein:protein binding pair and the second member of the protein:protein binding pair associate, and (iii) an antibody or a binding portion thereof that binds to the extracellular domain of the transferrin receptor protein 1 (abbreviated as TfR1, TfR, or CD71), wherein the antibody or the binding portion thereof is fused to the second member of the protein:protein binding pair. In some embodiments, (i) the first member of the protein:protein binding pair, (ii) the second member of the protein:protein binding pair, and (iii) the antibody or the binding portion thereof together direct the tropism of the virus capsid to cells expressing TfR1 or an extracellular portion thereof (e.g., the amino acid sequence shown in SEQ ID NO: 436). In some embodiments, the extracellular domain of TfR1 is the extracellular domain of human (h) TfR1.

[0013] In some embodiments, the viral capsid protein / viral particle and / or composition comprising the same further comprises a cell expressing TfR1 on its surface. For example, the viral capsid binds to the extracellular domain of TfR1 expressed on the surface of the cell. In some embodiments, the cell is a cell selected from the group of cells listed in Table 2, optionally wherein the cell is in vivo, ex vivo or in vitro. In some embodiments, the AAV viral capsid protein (including viral particles and / or compositions comprising the same) displaying a TfR1 targeting ligand as disclosed herein binds to a cell expressing TfR1 on its surface, wherein the cell is a central nervous system cell such as a cortical neuron, a Purkinje cell, a glial cell (e.g., an astrocyte, an oligodendrocyte, etc.). In some embodiments, in some embodiments, the cell is a blood-brain barrier endothelial cell and / or a brain microvascular endothelial cell expressing TfR1 on its surface, wherein the viral capsid binds to the extracellular domain of TfR1 expressed on the surface of the blood-brain barrier endothelial cell and / or the brain microvascular endothelial cell, optionally wherein the blood-brain barrier endothelial cell and / or the brain microvascular endothelial cell is in vivo, ex vivo or in vitro.

[0014] The protein:protein binding pairs described herein are those protein:protein binding pairs that spontaneously form an isopeptide bond upon contact. In some embodiments:

[0015] (a) The first member of the protein:protein binding pair comprises SpyTag, Isopeptag, SnoopTag, SpyTag002, SpyTag003 or a variant thereof,

[0016] (b) The second member of the protein:protein binding pair comprises SpyCatcher, KTag, pilin-C, SnoopCatcher, SpyCatcher002, SpyTag003 or a variant thereof fused to a targeting ligand, and

[0017] (c) The first member of the protein:protein binding pair and the second member of the protein:protein binding pair associate via an isopeptide bond. In some embodiments, (a) the first member of the protein:protein binding pair comprises SpyTag or a variant thereof, and (b) the second member of the protein:protein binding pair comprises SpyCatcher or a variant thereof fused to a targeting ligand. In some embodiments, (a) the first member of the protein:protein binding pair comprises the c-myc amino acid sequence shown in SEQ ID NO:326, and (b) the second member of the protein:protein binding pair comprises a bispecific binding protein containing an anti-c-myc antibody and a targeting ligand.

[0018] In some additional embodiments, the viral capsid protein further comprises a linker located on one or both sides of the first member of a protein:protein binding pair. In some embodiments, the lengths of the first and / or second linkers to which the first member of the protein:protein binding pair is operably linked to the capsid protein of the viral capsid are each independently at least one amino acid (e.g., 10 amino acids in length), and are either different or the same. In some embodiments, the length of the first linker is 10 amino acids and / or the length of the second linker is 10 amino acids, optionally wherein the amino acid sequence of the first linker and / or the amino acid sequence of the second linker comprises the amino acid sequence shown in SEQ ID NO:331 or SEQ ID NO:332.

[0019] In some recombinant viral capsid protein embodiments, the viral capsid protein comprises the amino acid sequence of a modified VP1 capsid protein, a modified VP2 capsid protein, and / or a modified VP3 capsid protein encoded by a mutated cap gene, and the mutated cap gene or a portion thereof (e.g., about 15 nucleotides) comprises a nucleotide sequence that is at least 90% identical to the cap gene of AAV or a portion thereof, wherein the mutated cap gene or a portion thereof is genetically modified to comprise an insertion of a nucleotide sequence encoding the first member of a protein:protein binding pair such that the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein comprises the first member of the protein:protein binding pair. In some embodiments, the mutated cap gene or a portion thereof is genetically modified to comprise one or more additional mutations such that, in addition to the first member of the protein:protein binding pair, the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein comprises:

[0020] (i) point mutations, such as substitutions, insertions, or deletions of amino acids,

[0021] (ii) chimeric amino acid sequences, or

[0022] (iii) Both point mutations and chimeric amino acid sequences, optionally wherein the substitution, insertion, or deletion of an amino acid reduces the natural tropism of the viral particle and / or generates a detectable label. In some recombinant viral capsid protein embodiments, (a) the viral capsid protein comprises the amino acid sequence of a modified VP1 capsid protein, a modified VP2 capsid protein, and / or a modified VP3 capsid protein encoded by a mutated cap gene, (b) the mutated cap gene or a portion thereof (e.g., at least 5 nucleotides, at least 15 nucleotides, at least 30 nucleotides, etc.) comprises a nucleotide sequence that is at least 90% identical to the cap gene of AAV or a portion thereof, wherein the mutated cap gene or a portion thereof is genetically modified to comprise the insertion of a nucleotide sequence encoding the first member of a protein:protein binding pair such that the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein comprises the first member of the protein:protein binding pair, and / or (c) the AAV is selected from the group consisting of AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh10, AAV rh32.33, non-primate AAV, combinations thereof, and any variants or hybrids thereof as listed in Table 5.

[0023] In some embodiments, the AAV is AAV2, and optionally the viral capsid comprises a modified AAV2 VP1 capsid protein that comprises the first member of a protein:protein binding pair optionally linked via a linker to the amino acid at position I453 and / or I587. In some embodiments, the viral capsid comprises a modified AAV2 VP1 capsid protein that comprises the first member of a protein:protein binding pair displayed at position G453 via a linker, optionally wherein the modified AAV2 VP1 capsid protein further comprises an R585A modification, an R588A modification, or both an R585A modification and an R588A modification, and optionally wherein the modified AAV2 VP1 capsid protein further comprises an R484A modification, an R487A modification, an R585A modification, an R588A modification, and a K532A modification, or any combination of an R484A modification, an R487A modification, an R585A modification, an R588A modification, and a K532A modification.

[0024] In some embodiments, the AAV is AAV9, optionally the viral capsid comprises a modified AAV9 VP1 capsid protein, and the modified AAV9 VP1 capsid protein comprises a first member of a protein:protein binding pair optionally linked via a linker to an amino acid at position I453 or I589. In some embodiments, the viral capsid comprises a modified AAV9 VP1 capsid protein, and the modified AAV9 VP1 capsid protein comprises a first member of a protein:protein binding pair displayed at G453 via a linker, optionally wherein the modified AAV9 VP1 capsid protein further comprises an N272A modification, a W503A modification, or both an N272A modification and a W503A modification. In some embodiments, the recombinant viral capsid is a mosaic capsid that comprises a second set of AAV9 VP1 capsid proteins that lack the first member of the protein:protein binding pair, optionally wherein the second set of AAV2 VP1 capsid proteins comprises an N272A modification, a W503A modification, or both an N272A modification and a W503A modification.

[0025] In some embodiments, the AAV is AAV1, optionally the viral capsid comprises a modified AAV1 VP1 capsid protein, and the modified AAV1 VP1 capsid protein comprises a first member of a protein:protein binding pair optionally linked via a linker. In some embodiments, the recombinant viral capsid is a mosaic capsid that comprises a second set of AAV1 VP1 capsid proteins that lack the first member of the protein:protein binding pair.

[0026] In some embodiments, the AAV is AAV8, optionally the viral capsid comprises a modified AAV8 VP1 capsid protein, and the modified AAV8 VP1 capsid protein comprises a first member of a protein:protein binding pair optionally linked via a linker. In some embodiments, the recombinant viral capsid is a mosaic capsid that comprises a second set of AAV8 VP1 capsid proteins that lack the first member of the protein:protein binding pair.

[0027] In some embodiments, the AAV is AAVrh32.33, optionally the viral capsid comprises a modified AAVrh32.33 VP1 capsid protein, and the modified AAVrh32.33 VP1 capsid protein comprises a first member of a protein:protein binding pair optionally linked via a linker. In some embodiments, the recombinant viral capsid is a mosaic capsid that comprises a second set of AAV rh32.33 VP1 capsid proteins that lack the first member of the protein:protein binding pair.

[0028] In some embodiments, non-primate AAV is avian AAV (AAAV), non-primate mammalian AAV, or squamate AAV.

[0029] In some embodiments, non-primate AAV is AAV, optionally the viral capsid comprises a modified AAV VP1 capsid protein, and the modified AAV VP1 capsid protein comprises a first member of a protein:protein binding pair optionally linked via a linker to an amino acid at position I444 or I580. In some embodiments, the viral capsid comprises a modified AAV VP1 capsid protein, and the modified AAV VP1 capsid protein comprises a first member of a protein:protein binding pair optionally linked via a linker to an amino acid at a position selected from the group consisting of I429, I430, I431, I432, I433, I434, I436, I437, and I565.

[0030] In some embodiments, AAV is squamate AAV, such as bearded dragon AAV. In some embodiments, the viral capsid comprises a modified bearded dragon VP1 capsid protein, and the modified bearded dragon VP1 capsid protein comprises a first member of a protein:protein binding pair optionally linked via a linker to an amino acid at position I573 or I436.

[0031] In some embodiments, AAV is sea lion AAV.

[0032] Anti-TfR binding proteins, such as anti-TfR antibodies or binding portions thereof, can be used to retarget recombinant AAV viral capsid proteins / AAV viral particles to cells that express TfR (e.g., human TfR). In some embodiments, an antibody or binding portion thereof that binds to the extracellular domain of TfR1 binds to the same epitope on the extracellular domain of TfR1 as a reference antibody that comprises the HCVR / LCVR amino acid sequence pairs shown in Table 1; comprises heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) of a heavy chain variable region (HCVR) that contains the amino acid sequence shown in SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, 262, 272, 282, 292, 302, or 312 (or variants thereof); and / or comprises light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) of a light chain variable region (LCVR) that contains the amino acid sequence shown in SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, 217, 227, 237, 247, 257, 267, 277, 287, 297, 307, or 317 (or variants thereof). In some embodiments, as described herein, an antibody or binding portion thereof that binds to TfR1 (e.g., human TfR1) and can be used to retarget AAV viral particles comprises:

[0033] (i) an HCVR that comprises HCDR1, HCDR2, and HCDR3 of an HCRV that contains the amino acid sequence shown in SEQ ID NO: 2 (or a variant thereof); and an LCVR that comprises LCDR1, LCDR2, and LCDR3 of an LCRV that contains the amino acid sequence shown in SEQ ID NO: 7 (or a variant thereof);

[0034] (ii) an HCVR that comprises HCDR1, HCDR2, and HCDR3 of an HCRV that contains the amino acid sequence shown in SEQ ID NO: 12 (or a variant thereof); and an LCVR that comprises LCDR1, LCDR2, and LCDR3 of an LCRV that contains the amino acid sequence shown in SEQ ID NO: 17 (or a variant thereof);

[0035] (iii) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:22 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:27 (or a variant thereof);

[0036] (iv) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:32 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:37 (or a variant thereof);

[0037] (v) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:42 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:47 (or a variant thereof);

[0038] (vi) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:52 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:57 (or a variant thereof);

[0039] (vii) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:62 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:67 (or a variant thereof);

[0040] (viii) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:72 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:77 (or a variant thereof);

[0041] (ix) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:82 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:87 (or a variant thereof);

[0042] (x) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:92 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:97 (or a variant thereof);

[0043] (xi) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:102 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:107 (or a variant thereof);

[0044] (xii) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:112 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:117 (or a variant thereof);

[0045] (xiii) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:122 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:127 (or a variant thereof);

[0046] (xiv) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:132 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:137 (or a variant thereof);

[0047] (xv) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 142 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 147 (or a variant thereof);

[0048] (xvi) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 152 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 157 (or a variant thereof);

[0049] (xvii) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 162 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 167 (or a variant thereof);

[0050] (xviii) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 172 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 177 (or a variant thereof);

[0051] (xix) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 182 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 187 (or a variant thereof);

[0052] (xx) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 192 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 197 (or a variant thereof);

[0053] (xxi) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:202 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:207 (or a variant thereof);

[0054] (xxii) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:212 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:217 (or a variant thereof);

[0055] (xxiii) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:222 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:227 (or a variant thereof);

[0056] (xiv) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:232 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:237 (or a variant thereof);

[0057] (xv) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:242 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:247 (or a variant thereof);

[0058] (xvi) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:252 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:257 (or a variant thereof);

[0059] (xvii) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV comprising the amino acid sequence shown in SEQ ID NO: 262 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV comprising the amino acid sequence shown in SEQ ID NO: 267 (or a variant thereof);

[0060] (xviii) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV comprising the amino acid sequence shown in SEQ ID NO: 272 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV comprising the amino acid sequence shown in SEQ ID NO: 277 (or a variant thereof);

[0061] (xix) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV comprising the amino acid sequence shown in SEQ ID NO: 282 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV comprising the amino acid sequence shown in SEQ ID NO: 287 (or a variant thereof);

[0062] (xxx) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV comprising the amino acid sequence shown in SEQ ID NO: 292 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV comprising the amino acid sequence shown in SEQ ID NO: 297 (or a variant thereof);

[0063] (xxxi) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV comprising the amino acid sequence shown in SEQ ID NO: 302 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV comprising the amino acid sequence shown in SEQ ID NO: 307 (or a variant thereof); and / or

[0064] (xxxii) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV comprising the amino acid sequence shown in SEQ ID NO: 312 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV comprising the amino acid sequence shown in SEQ ID NO: 317 (or a variant thereof).

[0065] In some embodiments, as described herein, an antibody or a binding portion thereof that binds to the TfR1 protein (e.g., human TfR1 protein) and can be used to retarget AAV virus particles comprises:

[0066] (a) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 3 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 4 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 5 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 8 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 9 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 10 (or a variant thereof);

[0067] (b) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 13 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 14 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 15 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 18 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 19 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 20 (or a variant thereof);

[0068] (c) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 23 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 24 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 25 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 28 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 29 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 30 (or a variant thereof);

[0069] (d) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 33 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 34 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 35 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 38 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 39 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 40 (or a variant thereof);

[0070] (e) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 43 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 44 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 45 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 48 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 49 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 50 (or a variant thereof);

[0071] (f) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 53 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 54 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 55 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 58 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 59 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 60 (or a variant thereof);

[0072] (g) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 63 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 64 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 65 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 68 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 69 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 70 (or a variant thereof);

[0073] (h) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 73 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 74 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 75 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 78 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 79 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 80 (or a variant thereof);

[0074] (i) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 83 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 84 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 85 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 88 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 89 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 90 (or a variant thereof);

[0075] (j) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 93 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 94 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 95 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 98 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 99 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 100 (or a variant thereof);

[0076] (k) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 103 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 104 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 105 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 108 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 109 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 110 (or a variant thereof);

[0077] (l) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 113 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 114 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 115 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 118 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 119 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 120 (or a variant thereof);

[0078] (m) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 123 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 124 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 125 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 128 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 129 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 130 (or a variant thereof);

[0079] (n) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 133 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 134 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 135 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 138 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 139 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 140 (or a variant thereof);

[0080] (o) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 143 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 144 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 145 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 148 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 149 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 150 (or a variant thereof);

[0081] (p) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 153 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 154 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 155 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 158 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 159 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 160 (or a variant thereof);

[0082] (q) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 163 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 164 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 165 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 168 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 169 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 170 (or a variant thereof);

[0083] (r) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 173 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 174 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 175 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 178 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 179 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 180 (or a variant thereof);

[0084] (s) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 183 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 184 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 185 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 188 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 189 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 190 (or a variant thereof);

[0085] (t) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 193 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 194 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 195 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 198 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 199 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 200 (or a variant thereof);

[0086] (u) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 203 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 204 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 205 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 208 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 209 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 210 (or a variant thereof);

[0087] (v) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 213 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 214 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 215 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 218 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 219 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 220 (or a variant thereof);

[0088] (w) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 223 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 224 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 225 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 228 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 229 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 230 (or a variant thereof);

[0089] (x) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 233 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 234 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 235 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 238 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 239 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 240 (or a variant thereof);

[0090] (y) The HCVR contains the following: HCDR1 having the amino acid sequence shown in SEQ ID NO: 243 (or a variant thereof), HCDR2 having the amino acid sequence shown in SEQ ID NO: 244 (or a variant thereof), and HCDR3 having the amino acid sequence shown in SEQ ID NO: 245 (or a variant thereof); and the LCVR contains the following: LCDR1 having the amino acid sequence shown in SEQ ID NO: 248 (or a variant thereof), LCDR2 having the amino acid sequence shown in SEQ ID NO: 249 (or a variant thereof), and LCDR3 having the amino acid sequence shown in SEQ ID NO: 250 (or a variant thereof);

[0091] (z) The HCVR contains the following: HCDR1 having the amino acid sequence shown in SEQ ID NO: 253 (or a variant thereof), HCDR2 having the amino acid sequence shown in SEQ ID NO: 254 (or a variant thereof), and HCDR3 having the amino acid sequence shown in SEQ ID NO: 255 (or a variant thereof); and the LCVR contains the following: LCDR1 having the amino acid sequence shown in SEQ ID NO: 258 (or a variant thereof), LCDR2 having the amino acid sequence shown in SEQ ID NO: 259 (or a variant thereof), and LCDR3 having the amino acid sequence shown in SEQ ID NO: 260 (or a variant thereof);

[0092] (aa) The HCVR contains the following: HCDR1 having the amino acid sequence shown in SEQ ID NO: 263 (or a variant thereof), HCDR2 having the amino acid sequence shown in SEQ ID NO: 264 (or a variant thereof), and HCDR3 having the amino acid sequence shown in SEQ ID NO: 265 (or a variant thereof); and the LCVR contains the following: LCDR1 having the amino acid sequence shown in SEQ ID NO: 268 (or a variant thereof), LCDR2 having the amino acid sequence shown in SEQ ID NO: 269 (or a variant thereof), and LCDR3 having the amino acid sequence shown in SEQ ID NO: 270 (or a variant thereof);

[0093] (ab) The following HCVR: an HCDR1 containing the amino acid sequence shown in SEQ ID NO: 273 (or a variant thereof), an HCDR2 containing the amino acid sequence shown in SEQ ID NO: 274 (or a variant thereof), and an HCDR3 containing the amino acid sequence shown in SEQ ID NO: 275 (or a variant thereof); and an LCVR containing the following: an LCDR1 containing the amino acid sequence shown in SEQ ID NO: 278 (or a variant thereof), an LCDR2 containing the amino acid sequence shown in SEQ ID NO: 279 (or a variant thereof), and an LCDR3 containing the amino acid sequence shown in SEQ ID NO: 280 (or a variant thereof);

[0094] (ac) The following HCVR: an HCDR1 containing the amino acid sequence shown in SEQ ID NO: 283 (or a variant thereof), an HCDR2 containing the amino acid sequence shown in SEQ ID NO: 284 (or a variant thereof), and an HCDR3 containing the amino acid sequence shown in SEQ ID NO: 285 (or a variant thereof); and an LCVR containing the following: an LCDR1 containing the amino acid sequence shown in SEQ ID NO: 288 (or a variant thereof), an LCDR2 containing the amino acid sequence shown in SEQ ID NO: 289 (or a variant thereof), and an LCDR3 containing the amino acid sequence shown in SEQ ID NO: 290 (or a variant thereof);

[0095] (ad) The following HCVR: an HCDR1 containing the amino acid sequence shown in SEQ ID NO: 293 (or a variant thereof), an HCDR2 containing the amino acid sequence shown in SEQ ID NO: 294 (or a variant thereof), and an HCDR3 containing the amino acid sequence shown in SEQ ID NO: 295 (or a variant thereof); and an LCVR containing the following: an LCDR1 containing the amino acid sequence shown in SEQ ID NO: 298 (or a variant thereof), an LCDR2 containing the amino acid sequence shown in SEQ ID NO: 299 (or a variant thereof), and an LCDR3 containing the amino acid sequence shown in SEQ ID NO: 300 (or a variant thereof);

[0096] (ae) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 303 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 304 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 305 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 308 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 309 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 310 (or a variant thereof); and / or

[0097] (af) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 313 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 314 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 315 (or a variant thereof); and an LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 318 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 319 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 320 (or a variant thereof).

[0098] In some embodiments, as described herein, an antibody or a binding portion thereof that binds to the TfR1 protein (e.g., human TfR1 protein) and can be used to retarget AAV viral particles comprises:

[0099] (i) an HCVR comprising the amino acid sequence shown in SEQ ID NO: 2 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 7 (or a variant thereof);

[0100] (ii) an HCVR comprising the amino acid sequence shown in SEQ ID NO: 12 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 17 (or a variant thereof);

[0101] (iii) an HCVR comprising the amino acid sequence shown in SEQ ID NO: 22 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 27 (or a variant thereof);

[0102] (iv) an HCVR comprising the amino acid sequence shown in SEQ ID NO: 32 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 37 (or a variant thereof);

[0103] (v) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 42 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 47 (or a variant thereof);

[0104] (vi) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 52 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 57 (or a variant thereof);

[0105] (vii) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 62 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 67 (or a variant thereof);

[0106] (viii) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 72 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 77 (or a variant thereof);

[0107] (ix) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 82 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 87 (or a variant thereof);

[0108] (x) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 92 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 97 (or a variant thereof);

[0109] (xi) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 102 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 107 (or a variant thereof);

[0110] (xii) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 112 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 117 (or a variant thereof);

[0111] (xiii) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 122 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 127 (or a variant thereof);

[0112] (xiv) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 137 (or a variant thereof);

[0113] (xv) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 142 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 147 (or a variant thereof);

[0114] (xvi) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 152 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 157 (or a variant thereof);

[0115] (xvii) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 162 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 167 (or a variant thereof);

[0116] (xviii) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 172 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 177 (or a variant thereof);

[0117] (xix) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 182 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 187 (or a variant thereof);

[0118] (xx) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 192 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 197 (or a variant thereof);

[0119] (xxi) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 202 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 207 (or a variant thereof);

[0120] (xxii) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 212 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 217 (or a variant thereof);

[0121] (xxiii) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof);

[0122] (xxiv) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 232 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 237 (or a variant thereof);

[0123] (xxv) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 242 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 247 (or a variant thereof);

[0124] (xxvi) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 252 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 257 (or a variant thereof);

[0125] (xxvii) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 262 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 267 (or a variant thereof);

[0126] (xxviii) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 272 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 277 (or a variant thereof);

[0127] (xxix) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 282 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 287 (or a variant thereof);

[0128] (xxx) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 292 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 297 (or a variant thereof);

[0129] (xxxi) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 302 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 307 (or a variant thereof); and / or

[0130] (xxxii) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 312 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 317 (or a variant thereof).

[0131] In some embodiments, the antibody or binding portion thereof that binds to the extracellular domain of TfR1 is in the form of a bivalent monoclonal antibody or mAb. In some embodiments, the antibody or binding portion thereof that binds to the extracellular domain of TfR1 is in the Fab form. In some embodiments, the antibody or binding portion thereof that binds to the extracellular domain of TfR1 is in the scFv form. In some embodiments, the antibody or binding portion thereof that binds to the extracellular domain of TfR1 comprises a mutated Ig CH3 domain, wherein the mutated Ig CH3 domain binds to the extracellular domain of TfR1.

[0132] In some embodiments, as described herein, an antibody or a binding portion thereof that binds to the TfR1 protein (e.g., human TfR1 protein) and can be used to retarget AAV viral particles comprises: (i) an HCVR comprising the amino acid sequence set forth in SEQ ID NO:2 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:7 (or a variant thereof); (ii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO:42 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:47 (or a variant thereof); (iii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO:122 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:127 (or a variant thereof); (iv) an HCVR comprising the amino acid sequence set forth in SEQ ID NO:132 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:137 (or a variant thereof); (v) an HCVR comprising the amino acid sequence set forth in SEQ ID NO:212 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:217 (or a variant thereof); (vi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO:222 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:227 (or a variant thereof); (vii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO:232 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:237 (or a variant thereof); (viii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO:242 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:247 (or a variant thereof); (ix) an HCVR comprising the amino acid sequence set forth in SEQ ID NO:262 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:267 (or a variant thereof); (x) an HCVR comprising the amino acid sequence set forth in SEQ ID NO:272 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:277 (or a variant thereof); (xi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO:282 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:287 (or a variant thereof); and / or (xii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO:292 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:297 (or a variant thereof).

[0133] Virus particles comprising a viral capsid protein as described herein are also described, such as a viral capsid. In some embodiments, the viral capsid as described herein is a mosaic capsid and further comprises a reference viral capsid protein that is at least 95% identical to the recombinant viral capsid protein, the reference viral capsid protein lacking all three of the following: (i) the first member of a protein:protein binding pair, (ii) the second member of a protein:protein binding pair, and (iii) an antibody or a binding portion thereof. In some embodiments, the mosaic viral capsid comprises the reference capsid protein and the recombinant viral capsid protein in a ratio of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1. In some embodiments, the viral capsid further comprises a nucleotide of interest encapsidated within the viral capsid. In some embodiments, the nucleotide of interest is a reporter gene, such as the nucleotide of interest encodes β-galactosidase, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), MmGFP, blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof.

[0134] In some embodiments, the nucleotide of interest encodes a therapeutic moiety, such as a therapeutic protein, a suicide gene, an antibody or fragment thereof, a CRISPR / Cas system or portion thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule. In some embodiments, the nucleotide of interest is operably linked to a promoter having organ specificity, tissue specificity, or cell specificity. In some embodiments, the nucleotide of interest encodes a therapeutic moiety that is secreted from the transduced cell and provides a therapeutic effect within the interstitial space surrounding the transduced cell or on neighboring cells. In some embodiments, the nucleotide of interest encodes a therapeutic moiety that provides a therapeutic effect to the transduced cell in an autonomous manner. In some embodiments, the nucleotide of interest encodes a therapeutic moiety that provides a therapeutic benefit to the transduced cell in an autonomous manner within the interstitial space surrounding the transduced cell and / or to neighboring cells of the transduced cell. In some embodiments, the therapeutic moiety is a therapeutic protein, such as a fusion protein, an enzyme, etc. In some embodiments, the therapeutic moiety is a secreted antibody or binding portion thereof. In some embodiments, the therapeutic moiety is an RNA molecule, such as an antisense RNA molecule, an RNAi molecule, an shRNA molecule, etc.

[0135] In some embodiments, the promoter has brain specificity. In some embodiments, the promoter has neuron specificity, glial cell specificity, astrocyte specificity, oligodendrocyte specificity, microglia specificity, and / or central nervous system specificity. In some embodiments, the promoter is selected from the group consisting of: human glial fibrillary acidic protein (GFAP) promoter, human synapsin 1 (SYN1) promoter, human synapsin 2 (SYN2) promoter, human metallothionein 3 (MT3) promoter, and human proteolipid protein 1 (PLP1) promoter. In some embodiments, the promoter is a neuron, astrocyte, or oligodendrocyte specific promoter, or a neuron, astrocyte, or oligodendrocyte preferential promoter. In some embodiments, the promoter is selected from the group consisting of: NSE promoter, synapsin promoter, MeCP2 promoter, oligodendrocyte transcription factor 1 (Olig1) promoter, chondroitin sulfate proteoglycan (Cspg4) promoter, CNP (2',3'-cyclic-nucleotide 3'-phosphodiesterase) promoter, and GFAP promoter.

[0136] The present disclosure also provides a pharmaceutical composition comprising (a) a recombinant viral capsid as described herein and (b) a pharmaceutically acceptable carrier or excipient.

[0137] Such pharmaceutical compositions can be used to deliver a nucleotide of interest across the blood-brain barrier in a mammalian subject. Such methods of delivering a nucleotide of interest across the blood-brain barrier in a mammalian subject can include administering (e.g., contacting) the pharmaceutical composition to the mammalian. In some embodiments, the administering (e.g., contacting) is performed ex vivo. In some embodiments, the administering is performed in the subject, optionally wherein the subject is modified to express a targeting ligand, e.g., from a safe harbor locus. In some embodiments, the subject is a primate, preferably a human. In some embodiments, the mammalian blood-brain barrier cells are mammalian brain endothelial cells. In some embodiments, the endothelial cells in the mammalian blood-brain barrier express transferrin receptor protein 1 on the cell surface, and (i) the first member of the protein:protein binding pair, (ii) the second member of the protein:protein binding pair, and (iii) the antibody or its binding portion together direct the tropism of the viral vector to the endothelial cells in the mammalian blood-brain barrier. In some embodiments, after the viral particle binds to transferrin receptor protein 1 on the endothelial cell surface, the viral particle is transported inside the endothelial cells of the blood-brain barrier to be delivered to the brain by transcytosis, such that the endothelial cells are not infected by the viral particle. In some embodiments, the nucleotide of interest encodes a therapeutic protein, a suicide gene, an antibody or a fragment thereof, a CRISPR / Cas system or a portion thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule. In some embodiments, the nucleotide of interest is operably linked to a brain-specific promoter, and the nucleotide of interest is preferentially expressed in the brain relative to other organs or tissues.

[0138] Also described herein is a method of treating a disease in a patient in need thereof, the method comprising administering to the patient the viral particles or compositions (e.g., pharmaceutical compositions) described herein, wherein the viral particles encapsidate a nucleotide of interest within a viral capsid, and wherein the nucleotide of interest encodes a therapeutic moiety, e.g., a therapeutic protein, a suicide gene, an antibody or a fragment thereof, a CRISPR / Cas system or a portion thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule. In some embodiments, the therapeutic moiety targets, e.g., inhibits the action and / or expression of α-synuclein. In some embodiments, the therapeutic moiety comprises an SNCA shRNA molecule, i.e., an shRNA molecule that targets (e.g., is complementary to) an mRNA molecule transcribed from the gene encoding the α-synuclein protein (e.g., SNCA) for inhibition via RNA interference. In some embodiments, the administering is via intravenous injection. In some embodiments, the administering is via intracerebroventricular injection.

[0139] In some embodiments, the targeting ligand is optionally operably linked to a protein (the second member of the protein:protein binding pair), such as fused to the protein. In some embodiments, the targeting ligand can be a binding moiety, such as, a native ligand, an antibody, a multispecific binding molecule, etc. In some embodiments, the targeting ligand is an antibody or a portion thereof. In some embodiments, the targeting ligand is an antibody comprising a variable domain that binds to TfR1 and a heavy chain constant domain. In some embodiments, the targeting ligand is an antibody comprising a variable domain that binds to TfR1 on a target cell and an IgG heavy chain constant domain. In some embodiments, the targeting ligand is an antibody comprising a variable domain that binds to TfR1 on a target cell and an IgG heavy chain constant domain, wherein the IgG heavy chain constant domain is operably linked, such as via a linker, to a protein that forms an isopeptide covalent bond with the first member (e.g., the second member of the protein:protein binding pair). In some embodiments, the capsid proteins described herein comprise a first member and a second member, the first member comprising a SpyTag operably linked to a viral capsid protein and covalently linked to the SpyTag, the second member comprising a SpyCatcher linked to a targeting ligand comprising an antibody variable domain and an IgG heavy chain domain, wherein the SpyCatcher and the IgG heavy chain domain are linked via an amino acid linker, such as GSGESG (SEQ ID NO:433). BRIEF DESCRIPTION OF THE DRAWINGS

[0140] The patent or application file contains at least one color drawing. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent Office upon request and payment of the necessary fee(s).

[0141] Figure 1 Histograms obtained from flow cytometry analysis of green fluorescent protein (GFP) expression after infection of cells expressing mTfR (“mTfR+293”; “bEnd.3(mTf+)”) or negative control cells that do not express mTfR but express hASGR1 (“hASGR1+293T(ACL18620)”) with AAV9 particles are shown. Virus was added at a multiplicity of infection of 1e5 vg / cell. Cells express GFP as a transduction marker. Cells were transduced with a panel of AAV9-based particles, including wild-type (wt) AAV9, or SpyTagged AAV9-based capsids conjugated with an antibody targeting mTfR (anti-TfR mAb) or conjugated with an antibody targeting hASGR1 (as a negative control) (AAV9 wt, AAV9N272A, AAV9 W503A).

[0142] Figure 2Immunohistochemical staining of eGFP expression in the cerebellum or liver of wild-type C57BL / 6J mice is shown after injection of wild-type (wt) AAV9 at 5e10 vg / mouse or various AAV9-based capsids conjugated with an antibody 8D3 targeting mTfR (anti-TfR mAb) (AAV9 wt, AAV9 N272A, AAV9 W503A). Representative brain images depict coronal sections showing the caudal plane including the cerebellum at a magnification of 2.7x. Liver sections are at a magnification of 8x.

[0143] Figure 3 Histograms are shown of green fluorescent protein (GFP) expression obtained from flow cytometry evaluation of cells expressing mTfR (mTfR+293 and bEnd.3) or cells expressing hASGR1 (hASGR1+293 (mTfR-), as a negative control) after infection with AAV9 particles. Virus was added at a multiplicity of infection of 1e5 vg / cell. Cells express GFP as a transduction marker. Cells were transduced with a panel of AAV9-based particles including wild-type (wt) AAV9 or SpyTagged AAV9 W503A particles conjugated with various forms of the mTfR-targeting antibody 8D3 (as a bivalent antibody (mAb), Fab, or scFv).

[0144] Figure 4 Immunohistochemical staining of eGFP expression in the cerebellum and liver of WT C57BL / 6J mice is shown after injection of wild-type (wt) AAV9 at 7.5e9 vg / mouse (injection of 150 uL of a 5e10 vg / mL solution) and off-target AAV9 W503A conjugated with an antibody targeting mTfR (anti-TfR mAb) in various antibody forms (bivalent "mAb", Fab, and scFv). Representative brain images depict coronal sections showing the caudal plane including the cerebellum at a magnification of 4.4x. Liver sections are at a magnification of 14x.

[0145] Figure 5A and 5B shows the measurement of WT C57BL / 6J mice after injection of wild-type AAV9 at 7.5e9 vg / mouse (injection of 150 uL of a 5e10 vg / mL solution), off-target AAV9 W503A conjugated with an antibody targeting mTfR or hASGR1 (as a non-targeting control) in various antibody forms. Figure 5A ) brain and ( Figure 5B) qPCR data of viral DNA in the liver, normalized to β-actin and relative to wild-type (wt) AAV (y-axis). AAV DNA was measured using a qPCR probe that recognizes the eGFP sequence, and the levels were normalized to the β-actin housekeeping gene. At 14 days post-injection, AAV targeting mouse TfR showed enhanced AAV DNA levels in the brain relative to WT, off-target, and targeted hASGR1 AAV9. Retargeting of TfR improved CNS transduction regardless of the antibody format used.

[0146] Figure 6 Immunohistochemical staining of eGFP expression in the brains of WT C57BL / 6J mice is shown after injection with wild-type (“WT”) AAV1, wild-type AAV1 conjugated to an antibody targeting mTfR (“8D3”), wild-type (“WT”) AAV8, or wild-type AAV8 conjugated to an antibody targeting mTfR (“8D3”, 2e12 vg / mouse), or wild-type AAV9 or wild-type AAV9 conjugated to an antibody targeting mTfR (“8D3”, 8e10 vg / mouse”). Representative images include higher magnification coronal sections showing planes of the hippocampus (magnification 5x), frontal cortex (magnification 10x), and cerebellum (magnification 8x). AAV particles targeting human TFR specifically infect hTFR+ cell lines in vitro and exhibit enhanced CNS transduction and reduced liver transduction after systemic injection regardless of the AAV serotype.

[0147] Figure 7 Histograms obtained from flow cytometry evaluation of green fluorescent protein (GFP) expression in cells expressing hTfR (hTfR+ 3T3) or not expressing hTfR (3T3, as a negative control) after infection with AAV9 particles are shown. Virus was added at a multiplicity of infection of 1e5 vg / cell. The virus expresses GFP as a transduction marker. Cells were transduced with a set of SpyTagged AAV9 W503A particles conjugated to Fab targeting hTfR (FabH1H12799B, PN69331, H1H12848B, H1H31874B, H1H12843B, H1H12798B, H1H12850B, H1H12847B, H1H12835B, H1H12839B, H1H12841B, H1H12845B) or Fab targeting hASGR1 (as a negative control).

[0148] Figure 8Immunohistochemical staining of eGFP expression in the brain and liver of mice after injection of wild-type (WT) AAV9 at 1.5e10 vg / mouse (injecting 150 μL of a 1e11 vg / mL solution), off-target AAV9 W503A conjugated with an antibody Fab (H1H12845B) targeting hTfR or an antibody Fab targeting hASGR1 (as an off-target control). AAV9 wild-type particles can transduce TFRC hu / hu in the liver of mice, while AAV9 W503A particles are off-target from the liver and do not promote high levels of liver eGFP expression. Representative images include higher magnification coronal sections showing planes of the hippocampus (magnification 9x), frontal cortex (magnification 10x), medial cortex (magnification 10x), and cerebellum (magnification 10x). hu / hu Immunohistochemical staining of eGFP expression in the brain of mice after injection of wild-type (WT) AAV9 at 1.5e10 vg / mouse (injecting 150 μL of a 1e11 vg / mL solution), off-target AAV9 W503A conjugated with an antibody Fab (Fab H1H12845B, H1H12850B) targeting hTfR or an antibody Fab targeting hASGR1 (as an off-target control). Representative images include higher magnification coronal sections showing planes of the hippocampus, frontal cortex, medial cortex, and cerebellum.

[0149] Figure 9A Immunohistochemical staining of eGFP expression in the brain of mice after injection of wild-type (WT) AAV9 at 1.5e10 vg / mouse (injecting 150 μL of a 1e11 vg / mL solution), off-target AAV9 W503A conjugated with an antibody Fab (Fab H1H12845B, H1H12850B) targeting hTfR or an antibody Fab targeting hASGR1 (as an off-target control). Representative images include higher magnification coronal sections showing planes of the hippocampus, frontal cortex, medial cortex, and cerebellum. hu / hu Representative IHC staining of eGFP expression in the brain of mice injected with AAV9 W503A conjugated with additional Fab targeting hTfR Figure 9B and Figure 9C Similarly shown is the TFRC of mice injected with AAV9 W503A conjugated with additional Fab targeting hTfR hu / hu Representative IHC staining of eGFP expression in the brain of mice ( Figure 9B : H1H12841B, H1H12798B, H1H12847B, H1H12839B, H1H12843B; Figure 9C : H1H31874B, H1H12835B, PN69331, H1H12848B, H1H12799B). Representative images include higher magnification coronal sections showing planes of the hippocampus (magnification 9x), frontal cortex (magnification 10x), medial cortex (magnification 10x), and cerebellum (magnification 10x). Figure 9DQuantification of eGFP immunohistochemical staining in the brain (left panel) and liver (right panel) after injection of various AAV9 (x-axis) at 1.5e10 vg / mouse: (1) wild-type AAV9, (2) off-target AAV9 W503A conjugated with an antibody Fab targeting hASGR1 (as a non-targeting control), or off-target AAV9 W503A conjugated with an antibody Fab targeting hTfR as follows: (3) H1H12799B, (4) PN69331, (5) H1H12848B, (6) H1H31874B, (7) H1H12843B, (8) H1H12798B, (9) H1H12850B, (10) H1H12847B, (11) H1H12835B, (12) H1H12839B, (13) H1H12841B, and (14) H1H12845B. The percentage of the area positive for DAB staining (indicating GFP-positive signal) in the brain and liver was quantified using HALO software.

[0150] Figure 10A and Figure 10B shows the qPCR data of AAV DNA in the ( hu / hu ) brain and ( Figure 10A ) liver of TFRC Figure 10B ) mice, which was after injection of (1) wild-type WT AAV9, (2) off-target AAV9W503A conjugated with an antibody Fab targeting hASGR1 (as a non-targeting control), or off-target AAV9 W503A conjugated with an antibody Fab targeting hTfR as follows: (3) H1H12799B, (4) H1H12848B, (5) H1H31874B, (6) PN69331, (7) H1H12850B, (8) H1H12847B, (9) H1H12839B, (10) H1H12835B, (11) H1H12843B, (12) H1H12798B, (13) H1H12841B, or (14) H1H12845B. AAV DNA was measured using a qPCR probe targeting the eGFP sequence, and the levels were normalized to the housekeeping gene β-actin.

[0151] Figure 11A A heatmap is provided showing, compared to wild-type AAV9 viral particles (AAV), after administration of AAV9 particles (each particle having a unique barcode and re-targeted using anti-TfR Fab) in female humanized TFRC mice (TFRC hu / hu)Enhanced transduction in vivo to the brain and spine. As described in the Materials and Methods of the Examples below, each candidate AAV was packaged with a unique barcoded genome. After intravenous administration of 36 candidate barcode pools, the designated tissues were collected, and the relative abundance of each barcode in the total RNA purified from each tissue was assessed using next-generation sequencing (NGS). Shown are the enrichment values of each virus relative to the input virus pool in the tissues of interest: (liver (mid-lobe), left hemisphere of the brain, and spine between the cervical and sacral regions). The data shown are the averages of three replicate animals in the study. Figure 11B An enrichment plot is provided showing Figure 11A enhanced transduction in vivo to the brain and spine and liver off-targeting in the mice of

[0152] Figure 12A Shown is the immunofluorescence co-staining of eGFP expression and cell-type specific markers in the brain of TFRC hu / hu mice after intravenous injection (1e11 vg / mouse) of off-target AAV9 W503A conjugated with an antibody Fab (FabH1H12845B) targeting hTfR. Brain transduction was evaluated 19 days after injection. Representative images include sagittal brain section planes at 20x magnification showing the cerebellum, cortex, and olfactory bulb. To characterize viral transgene expression by cell type, the sections were co-stained for eGFP and cell-type specific markers, which included: microtubule-associated protein 2 (MAP2) and hexaribonucleotide-binding protein 3 (Fox-3 or NeuN) for neurons; glial fibrillary acidic protein (GFAP) for astrocytes; 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNPase) and glutathione S-transferase pi (GST-pi) for oligodendrocytes; cluster of differentiation 31 (CD31) for vascular endothelial cells; and ionized calcium-binding adaptor molecule 1 (Iba1) for microglia. Figure 12B Shown is TFRC after intravenous injection (4e11 vg / mouse) of WT AAV9 (upper panel) or off-target AAV9 W503A conjugated with an antibody Fab (Fab H1H12845B) targeting hTfR (lower panel) (these viruses express an H2B-eGFP fusion genome for nuclear expression of eGFP) hu / huImmunofluorescence co-staining of eGFP expression and brain cell-specific markers in the brains of mice. Brain transduction was evaluated 2 weeks after injection. Representative images include sagittal brain section planes at 20x magnification showing the cortex, cerebellum, olfactory bulb, and corpus callosum. To characterize viral transgene expression by cell type, the sections were co-stained for eGFP and cell-specific markers, which included: hexaribonucleotide-binding protein 3 (Fox-3 or NeuN) and microtubule-associated protein 2 (MAP2) for neurons; glial fibrillary acidic protein (GFAP) and SRY-box transcription factor 9 (Sox9) for astrocytes; 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNPase) and glutathione S-transferase pi (GST-pi) for oligodendrocytes; cluster of differentiation 31 (CD31) for vascular endothelial cells; and ionized calcium-binding adaptor molecule 1 (Iba1) for microglia.

[0153] Figure 13 TFRC is shown after intravenous (1e11 vg / mouse) or intracerebroventricular (1e10 vg / mouse) injection of wild-type (WT) AAV9, off-target AAV9W503A conjugated with the antibody Fab (Fab H1H12845B) targeting hTfR, or off-target AAV9 W503A conjugated with the antibody Fab targeting hASGR1 (as a non-targeting control). hu / hu Immunohistochemical staining of eGFP expression in the brains of mice. Brain transduction was evaluated 19 days after injection. Representative images show whole-brain sagittal sections.

[0154] Figure 14A and Figure 14B TFRC is shown after intravenous (1e11 vg / mouse) or intracerebroventricular (1e10 vg / mouse) injection of WT AAV9, off-target AAV9 W503A conjugated with the antibody Fab (Fab H1H12845B) targeting hTfR or conjugated with the antibody Fab targeting hASGR1 (as a non-targeting control) (x-axis). hu / hu brains of mice ( Figure 14A ) and livers ( Figure 14B ) of qPCR data of viral DNA, normalized to β-actin and relative to wild-type (WT) AAV9 (y-axis). AAV DNA was measured using a qPCR probe that recognizes the eGFP sequence, and the levels were normalized to the β-actin housekeeping gene.

[0155] Figure 15Immunohistochemical staining of eGFP expression in the brains of mice after intravenous injection (1e11 vg / mouse) of wild-type (WT) AAV9, off-target AAV9 W503A conjugated with antibodies Fab targeting hTfR (Fab H1H12845B, H1H12848B, H1H31874B, H1H12841B, H1H12839B, H1H12835B, H1H12847B, H1H12850B, H1H12798B, H1H12843B, PN69331, H1H12799B), or off-target AAV9 W503A conjugated with antibodies Fab targeting hASGR1 (as a non-targeting control). hu / hu Immunohistochemical staining of eGFP expression in the brains of mice. Representative images include whole-brain sagittal sections.

[0156] Figure 16 Quantification of eGFP expression in discrete brain regions of mice after intravenous injection (1e11 vg / mouse) of wild-type (WT) AAV9, off-target AAV9 W503A conjugated with antibodies Fab targeting hASGR1 (as a non-targeting control), or off-target AAV9 W503A conjugated with antibodies Fab targeting hTfR (H1H12845B, H1H12848B, H1H31874B, H1H12841B, H1H12839B, H1H12835B, H1H12847B, H1H12850B, H1H12798B, H1H12843B, PN69331, and H1H12799B) by immunohistochemical staining. The percentage of the area positive for DAB staining (y-axis, representing eGFP-positive signal) in the brain was quantified using HALO software. Brain regions (x-axis) were defined and analyzed to include: (1) olfactory bulb, (2) cortex, (3) striatum, (4) hippocampus, (5) thalamus, (6) hypothalamus, (7) cerebellum, and (8) brainstem. AAV particles targeting human TfR showed enhanced brain transduction in multiple brain regions compared to WT AAV9. hu / hu Quantification of eGFP expression in the whole discrete brain regions of mice. The percentage of the area positive for DAB staining (y-axis, representing eGFP-positive signal) in the brain was quantified using HALO software. Brain regions (x-axis) were defined and analyzed to include: (1) olfactory bulb, (2) cortex, (3) striatum, (4) hippocampus, (5) thalamus, (6) hypothalamus, (7) cerebellum, and (8) brainstem. AAV particles targeting human TfR showed enhanced brain transduction in multiple brain regions compared to WT AAV9.

[0157] Figure 17 Immunohistochemical staining of TFRC is shown after intravenous injection of the following AAVs (x-axis) at 1e11 vg / mouse. hu / huQuantification of eGFP expression in the cortex, hippocampus, thalamus, olfactory bulb, striatum, cerebellum, brainstem, and hypothalamus of mice: (1) wild-type (WT) AAV9, (2) off-target AAV9 W503A conjugated with an antibody Fab targeting hASGR1 (as a non-targeting control), or off-target AAV9 W503A conjugated with the following antibody Fabs targeting hTfR: (3) H1H12845B, (4) H1H12848B, (5) H1H31874B, (6) H1H12841B, (7) H1H12839B, (8) H1H12835B, (9) H1H12847B, (10) H1H12850B, (11) H1H12798B, (12) H1H12843B, (13) PN69331, and (14) H1H12799B. The percentage of the area in the brain that was DAB staining positive (y-axis, representing eGFP positive signal) was quantified using HALO software.

[0158] Figure 18A and Figure 18B Shows TFRC measured after intravenous injection of (1) wild-type (WT) AAV9, (2) off-target AAV9 W503A conjugated with an antibody Fab targeting hASGR1 (as a non-targeting control), or off-target AAV9 W503A conjugated with the following antibody Fabs targeting hTfR: (3) H1H12845B, (4) H1H12848B, (5) H1H31874B, (6) H1H12841B, (7) H1H12839B, (8) H1H12835B, (9) H1H12847B, (10) H1H12850B, (11) H1H12798B, (12) H1H12843B, (13) PN69331, and (14) H1H12799B (x-axis) at 1e11 vg / mouse. hu / hu Mouse brain ( Figure 18A ) and liver ( Figure 18B ) qPCR data for AAV DNA (y-axis). AAV DNA was measured using a qPCR probe targeting the eGFP sequence and levels were normalized to the GAPDH housekeeping gene and relative to wild-type WT AAV9.

[0159] Figure 19A and Figure 19B Shows WT C57BL / 6J mouse brain (Figure 19A ) and the liver ( Figure 19B ) of qPCR data (y-axis) of AAV DNA. AAV DNA was measured using a qPCR probe targeting the eGFP sequence and the levels were normalized to the β-actin housekeeping gene and relative to the highest dose of WT AAV9.

[0160] Figure 20A and Figure 20B show immunohistochemical staining of eGFP expression in the brains of WT C57BL / 6J mice after intravenous injection of increasing doses (1.6e10, 8e10, 4e11, and 2e12 vg / mouse) of wild-type (WT) AAV9, WT AAV9 conjugated with an antibody Fab against mTfR (“8D3”), or off-target AAV9 W503A conjugated with an antibody Fab against mTfR (“8D3”). Brain transduction was evaluated 16 days after injection. Representative images show low magnification whole brain sagittal sections ( Figure 20A ) and higher magnification planes of sagittal sections showing the cortex, hippocampus, and cerebellum ( Figure 20B ).

[0161] Figure 21A-21D show the measurement of viral DNA by qPCR in the livers ( hu / hu ), hearts ( Figure 21A ), quadriceps muscles ( Figure 21B ), and brains ( Figure 21C ) of female TFRC Figure 21D mice after injection of wild-type AAV9, off-target AAV9 W503A conjugated with an antibody and Fab against hTfR or hASGR1 (as a non-targeting control), normalized to β-actin and relative to wild-type (wt) AAV9 (y-axis). AAV DNA was measured using a qPCR probe that recognizes the eGFP sequence and the levels were normalized to the β-actin housekeeping gene.

[0162] Figure 22A-22B show immunohistochemical staining of eGFP expression in the brains, hearts, quadriceps muscles, and livers of female TFRC hu / hu mice after injection of wild-type AAV9 and off-target AAV9 W503A conjugated with H1H12845B Fab or H1H12845B mAb. Representative images include frontal cortex (magnification 12x), vertical cross-section of the heart (magnification 12x), cross-section of the quadriceps muscle (magnification 12x), and horizontal section of the liver (magnification 14x) ( Figure 22A); or higher magnification brain sagittal sections of the plane of the cerebellum (magnification 12x, image of the first row from the top), hippocampus (magnification 3x, image of the second row from the top), cortex (magnification 12x, image of the third row from the top), and liver horizontal section (magnification 14x, image of the fourth row from the top)( Figure 22B ) of the plane.

[0163] Figure 23A-23B Immunohistochemical staining of eGFP expression in the brain, heart, quadriceps, and liver of female TFRC hu / hu mice after injection of wild-type AAV9 at 1e11 vg / mouse and off-target AAV9 W503A conjugated to H1H12839BFab or H1H12839B mAb. Representative images include frontal cortex (magnification 12x), vertical transverse section of the heart (magnification 12x), transverse section of the quadriceps (magnification 12x), and liver horizontal section (magnification 14x)( Figure 23A ); or higher magnification brain sagittal sections of the plane of the cerebellum (magnification 12x), hippocampus (magnification 3x), cortex (magnification 12x), and liver horizontal section (magnification 14x)( Figure 23B ) of the plane.

[0164] Figure 24 Show that AAV particles targeting mouse TfR transduce neonatal spinal motor neurons when directly administered into the CNS. Representative immunofluorescence images of GFP expression in the spinal cord of P15 C57BL / 6J mouse pups after a single intraventricular (i.c.v.) injection of 1e11 vg of the indicated AAV virus expressing GFP driven by the CBh promoter at P0. Images include a semi-coronal view of the lumbar spinal cord (top panel) and magnified views of the ventral horn region (white box in the top panel), where motor neurons are located, visualized using an antibody against the motor neuron marker choline acetyltransferase (ChAT). GFP expression was amplified with an antibody against GFP in these samples. The values plotted represent the mean and standard deviation of independent biological replicates.

[0165] Figure 25Show that AAV particles targeting mouse TfR are highly transduced into neonatal spinal cord motor neurons when administered intravenously. Representative immunofluorescence images of GFP expression in the spinal cord of P15 C57BL / 6J mouse pups after a single intravenous (i.v.) injection into the facial vein of P0 mouse pups. The injection solution contained virus-free (i.e., PBS) or 5e11 vg of AAV9 WT re-targeted mTfR (8D3 fab) expressing GFP driven by the CBh promoter. Images include semi-coronal views of the lumbar spinal cord (upper panel) and magnified views of the ventral horn region (white box in the upper panel), where motor neurons are located, visualized using an antibody against the motor neuron marker choline acetyltransferase (ChAT). The GFP signal shown here is native GFP expression and is not amplified with an antibody. Each group represents an independent biological replicate.

[0166] Figure 26A Shows qPCR data for the brain, spinal cord, liver, heart, quadriceps, and spleen of WT C57BL / 6J mice measuring the abundance of an antibody-encoding transcript driven by the CAGG promoter expressed by the indicated AAVs after intravenous injection of 5x10 11 vg / mouse of off-target AAV9 W503A conjugated with 8D3 scFv targeting mTfR or 5x10 11 vg / mouse of AAV8 (x-axis). AAV RNA was measured using a qPCR probe that recognizes the expressed human antibody sequence, and the relative mRNA levels were normalized to the β-actin housekeeping gene and relative to the AAV8 control group. Figure 26B Shows enzyme-linked immunosorbent assay (ELISA)-mediated detection (y-axis) of human antibody titers expressed by AAVs in brain lysates of WT C57BL / 6J mice after intravenous injection of 5x10 11 vg / mouse of off-target AAV9 W503A conjugated with 8D3 scFv targeting mTfR or 5e10 vg / mouse of AAV8. Antibody concentrations in the brain were evaluated 12 weeks after injection.

[0167] Figure 27 Shows after intravenous injection of 4x10 11After off-target AAV9 W503A conjugated with 8D3 Fabs targeting mTfR and expressing SNCA shRNA in vg / mice, qPCR data (y-axis) of SNCA mRNA levels in the cortex, midbrain, and striatum of humanized SNCA mice were measured. SNCA mRNA was measured using a qPCR probe targeting the human SNCA sequence, and the SNCA mRNA levels were normalized to the GAPDH housekeeping gene and relative to the SNCA mRNA levels in the initial (untreated) humanized SNCA mouse group. SNCA mRNA levels in the brain were evaluated 1 month after injection. Detailed Description

[0168] Transferrin Receptor

[0169] Transferrin (Tf) and its receptor (TfR) play a central role in regulating iron metabolism. There are two types of transferrin receptors: TfR1, also known as cluster of differentiation 71 (CD71), which is widely expressed and binds Tf with high affinity; TfR2 is less common and is mainly expressed in hepatocytes. Unless otherwise specified, "TfR" as used herein refers to TfR1 (CD71).

[0170] Uptake of Tf-bound iron through TfR1 is usually the main source of cellular iron input. TfR1 is a 90 kDa type II transmembrane protein with 760 amino acids. It contains a cytoplasmic N-terminal domain (amino acids 1 - 67), a transmembrane domain (amino acids 68 - 88), and a large extracellular C-terminal domain (amino acids 89 - 763), which contains the Tf-binding site. TfR1 usually exists as a homodimer, with monomers linked by disulfide bonds on the cell surface and a molecular weight of approximately 180 kDa.

[0171] TfR exists in human and non-human species, such as non-human primates and rodents. An exemplary amino acid sequence of human (h) TfR1 is shown in SEQ ID NO:434, which is identical to the amino acid sequence of the hTfR1 protein shown in Uniprot P02786. The gene encoding TfR is called TFRC and is located on human chromosome 3. An exemplary gene sequence of TFRC (with annotated exons and introns) can be found in the NCBI database (Gene ID: 7037).

[0172] The exemplary amino acid sequence of murine (m) TfR1 is shown as SEQ ID NO: 435, which is identical to the amino acid sequence of the mTfR1 protein shown in Uniprot Q62351 and has approximately 77% amino acid sequence identity with hTfR1. The Tfrc gene is located on chromosome 16 of the mouse. The complete gene sequence of murine Tfrc (with annotated exons and introns) can be found in the NCBI database (Gene ID: 22042).

[0173] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0174] Unless the context clearly dictates otherwise, the singular forms "a" and "the" include plural referents. Thus, for example, reference to "a method" includes one or more methods and / or one or more steps of the type described herein and / or that will become apparent to one of ordinary skill in the art upon reading this disclosure.

[0175] For amino acid or nucleotide sequences, "percent (%) identity" etc. can be readily determined over the full length of a protein or a portion thereof. The length of a portion can be at least about 5 amino acids or 24 nucleotides, respectively, and can be at most about 700 amino acids or 2100 nucleotides, respectively. Generally, when referring to "identity" between an HCVR and an LCVR, the percent identity refers to the identity over the lengths of HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3. Generally, when referring to "identity", "homology", or "similarity" between two different adeno-associated viruses, "identity", "homology", or "similarity" is determined with reference to an "aligned" sequence. An "aligned" sequence or "alignment" refers to a plurality of nucleic acid sequences or protein (amino acid) sequences that, compared to a reference sequence, typically contain corrections for missing or additional bases or amino acids.

[0176] Any of a variety of publicly available or commercially available multiple sequence alignment programs can be used for alignment. Sequence alignment programs can be used for amino acid sequences, such programs as "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME", and "Match-Box" programs. Generally, any of these programs is used with default settings, although those skilled in the art can change these settings as needed. Alternatively, those skilled in the art can utilize another algorithm or computer program that provides at least the same level of identity or alignment as provided by the reference algorithms and programs. See, for example, J.D. Thomson et al., Nucleic Acids Research, "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999).

[0177] Multiple sequence alignment programs can also be used for nucleic acid sequences. Examples of such programs include "Clustal W", "CAP Sequence Assembly", "MAP", and "MEME", which can be accessed through web servers on the Internet. Other sources of such programs are known to those skilled in the art. Alternatively, the Vector NTI utility is also used. There are also a variety of algorithms known in the art that can be used to measure nucleotide sequence identity, including those included in the above programs. As another example, the program FASTA of GCG version 6.1 can be used TM Compare polynucleotide sequences. Fasta TM Provides an alignment of the best overlapping regions between the query sequence and the search sequence and the percent sequence identity. For example, the percent sequence identity between nucleic acid sequences can be that determined using FASTA with its default parameters (font size 6 and NOPAM coefficient of the scoring matrix) as provided in GCG version 6.1 TM which program is incorporated herein by reference.

[0178] "Significant identity" encompasses amino acid or nucleic acid sequence alignments that are at least 90%, such as at least 93%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, or such as at least 100% identical.

[0179] The term "chimeric" encompasses functional genes or polypeptides that respectively include nucleic acid sequences or amino acid sequences from at least two different organisms. For example, a part of the gene or polypeptide of at least a first AAV and at least a second AAV, wherein at least the first part and at least the second part are operably linked. Unless stated to be chimeric, nucleotide sequences, genes, polypeptides, and amino acids are considered non-chimeric, e.g., containing only the nucleic acid sequence or amino acid sequence of a single organism (such as a single AAV).

[0180] The term "antibody" includes immunoglobulin molecules that comprise four polypeptide chains interconnected by disulfide bonds, two heavy (H) chains and two light (L) chains. Each heavy chain includes a heavy chain variable domain (V H ) and a heavy chain constant region (C H ). The heavy chain constant region includes at least three domains C H 1, C H 2, C H 3 and optionally CH 4 . Each light chain includes a light chain variable domain (C H ) and a light chain constant region (C L ). The heavy and light chain variable domains can be further subdivided into hypervariable regions, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each heavy and light chain variable domain contains three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs can be abbreviated as HCDR1, HCDR2, and HCDR3; light chain CDRs can be abbreviated as LCDR1, LCDR2, and LCDR3). A typical tetrameric antibody structure includes two identical antigen-binding domains, each of the antigen-binding domains being formed by the association of V H and V L domains, and each of the antigen-binding domains together with the corresponding C H and C L domains forms the antibody Fv region. A single-domain antibody includes a single antigen-binding domain, e.g., V H or V LThe antigen-binding domain of an antibody, e.g., a portion of an antibody that recognizes and binds to the first member of a specific binding pair that binds to an antigen, is also referred to as a "complementary determining region". It is a small region (5 to 10 amino acids) of the Fv region of an antibody, part of the fragment antigen binding (Fab region), and can contain a portion of the heavy and / or light chains of the antibody. The term "single-chain variable fragment" or "scFv" includes a single-chain fusion polypeptide containing the variable region of the immunoglobulin heavy chain (VH) and the variable region of the immunoglobulin light chain (VL). In some embodiments, VH and VL are linked by a linker sequence of 10 to 25 amino acids. The scFv polypeptide can also contain other amino acid sequences, such as the CL or CH1 regions. ScFv molecules can be prepared by phage display or by direct subcloning of the heavy and light chains of a hybridoma or B cell. Regarding methods for preparing scFv fragments by phage display and antibody domain cloning, Ahmad et al., Clinical and Developmental Immunology, volume 2012, article ID 98025 is incorporated herein by reference in its entirety. When a complementary determining region binds to the first member of a specific binding pair with high affinity, the complementary determining region specifically binds to the first member of the specific binding pair. The term "high-affinity" antibody refers to a K D of about 10 -9 M or lower (e.g., about 1x10 -9 M, 1x10 -10 M, 1x 10 -11 M or about 1x 10 -12 M) antibody. In one embodiment, KD is measured by surface plasmon resonance, e.g., BIACORE TM ; in another embodiment, K D .

[0181] The phrase "complementary determining region" or the term "CDR" encompasses amino acid sequences encoded by the nucleic acid sequences of an organism's immunoglobulin genes, which typically (i.e., in wild-type animals) occur between two framework regions in the variable region of the light or heavy chain of an immunoglobulin molecule (such as an antibody or T cell receptor). CDRs can be encoded by, for example, germline sequences or rearranged or unrearranged sequences, and by, for example, untreated or mature B cells or T cells. CDRs can be somatically mutated (e.g., different from the sequences encoded in the germline of the animal), humanized, and / or modified with amino acid substitutions, additions, or deletions. In some cases (e.g., for CDR3), a CDR can be encoded by two or more sequences (e.g., germline sequences) that are not contiguous (e.g., in an unrearranged nucleic acid sequence), but are contiguous in the B cell nucleic acid sequence, such as due to splicing or joining sequences (e.g., V-D-J recombination to form the heavy chain CDR3).

[0182] The phrase "light chain" encompasses immunoglobulin light chain sequences from any organism and, unless otherwise specified, includes human light chains and VpreB and surrogate light chains. Unless otherwise stated, the light chain variable domain typically includes three light chain CDRs and four framework (FR) regions. Generally, a full-length light chain includes, from the amino terminus to the carboxy terminus, a variable domain and a light chain constant region, the variable domain including FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The light chain variable domain is encoded by a light chain variable region gene sequence that generally includes V L and J L segments derived from the V and J segment repertoires present in the germline. The sequences, locations, and nomenclatures of the V and J light chain segments of various organisms can be found in the IMGT database at www.imgt.org. Light chains include, for example, those that do not selectively bind the first or second first member of a specific binding pair, the members of which are selectively bound by the first member of the specific binding pair binding protein in which they occur. Light chains also include those that bind and recognize one or more first members of a specific binding pair, or assist the heavy chain or another light chain in binding and recognizing the one or more first members, the one or more first members of which are selectively bound by the first member of the specific binding pair binding protein in which they occur. Commonly used or general light chains include those derived from the human V1-39J gene or the human V3-20J gene, and include somatically mutated (e.g., affinity matured) forms thereof. Exemplary human V LThe fragments include the human V1-39 gene segment, the human V3-20 gene segment, the human V1-40 gene segment, the human V1-44 gene segment, the human V2-8 gene segment, the human V2-14 gene segment, and the human V3-21 gene segment, and include their somatically mutated (e.g., affinity matured) forms. Light chains can be prepared that include variable domains from one organism (e.g., human or rodent, e.g., rat or mouse; or avian, e.g., chicken) and constant regions from the same or a different organism (e.g., human or rodent, e.g., rat or mouse; or avian, e.g., chicken).

[0183] The terms “about” or “substantially” encompass a statistically significant range of values. Such ranges can be within an order of magnitude, preferably within 50% of a given value or range, more preferably within 20%, more preferably within 10%, and even more preferably within 5%. The allowable variation covered by the terms “about” or “substantially” depends on the particular system under study and can be readily understood by one of ordinary skill in the art.

[0184] The phrase “heavy chain” or “immunoglobulin heavy chain” encompasses immunoglobulin heavy chain sequences from any organism, including immunoglobulin heavy chain constant region sequences. Unless otherwise stated, the heavy chain variable domain includes three heavy chain CDRs and four FR regions. Fragments of the heavy chain include CDRs, CDRs, and FRs, and combinations thereof. A typical heavy chain has (from the N-terminus to the C-terminus) a C H 1 domain, a hinge, a C H 2 domain, and a C H 3 domain after the variable domain. Functional fragments of the heavy chain include those capable of specifically recognizing the first member of a specific binding pair (e.g., with a K in the micromolar, nanomolar, or picomolar range D for recognizing the first member of a specific binding pair), i.e., fragments that can be expressed and secreted by a cell and include at least one CDR. The heavy chain variable domain is encoded by a variable region nucleotide sequence that generally includes V H derived from a repertoire of V H , D H , and J H , D H , and J H segments present in the germline. The sequences, positions, and nomenclature of the V, D, and J heavy chain segments for various organisms can be found in the IMGT database, which is accessible via the Internet on the World Wide Web (www) at the URL “imgt.org”.

[0185] The terms "heavy-chain only antibody", "heavy-chain only antigen-binding protein", "single-domain antigen-binding protein", "single-domain binding protein", etc. refer to monomeric or homodimeric immunoglobulin molecules comprising an immunoglobulin-like chain that includes a variable domain operably linked to a heavy-chain constant region that cannot bind to a light chain because it typically lacks a functional C H 1 domain. Thus, the terms "heavy-chain only antibody", "heavy-chain only antigen-binding protein", "single-domain antigen-binding protein", "single-domain binding protein", etc. encompass (i) a monomeric single-domain antigen-binding protein comprising one of the immunoglobulin-like chains that includes a variable domain operably linked to a heavy-chain constant region lacking a functional C H 1 domain, or (ii) a homodimeric single-domain antigen-binding protein comprising two immunoglobulin-like chains, each of which includes a variable domain operably linked to a heavy-chain constant region lacking a functional C H 1 domain. In various aspects, the homodimeric single-domain antigen-binding protein comprises two identical immunoglobulin-like chains, each of which includes an identical variable domain operably linked to an identical heavy-chain constant region lacking a functional C H 1 domain. Additionally, each immunoglobulin-like chain of the single-domain antigen-binding protein includes a variable domain that can be derived from a heavy-chain variable region gene segment (e.g., V H , D H , J H ), a light-chain gene segment (e.g., V L , J L ), or a combination thereof, linked to a heavy-chain constant region (C H ) gene sequence, wherein the CH gene sequence includes a deletion or inactivating mutation in the C H 1 coding sequence (and optionally a hinge region) of the heavy-chain constant region gene, e.g., IgG, IgA, IgE, IgD, or a combination thereof. A single-domain antigen-binding protein comprising a variable domain derived from a heavy-chain gene segment can be referred to as a "V H single-domain antibody" or a "V H single-domain antigen-binding protein", see, e.g., U.S. Patent No. 8,754,287; U.S. Patent Publication Nos. 20140289876; 20150197553; 20150197554; 20150197555; 20150196015; 20150197556; and 20150197557, each of which is incorporated herein by reference in its entirety. A single-domain antigen-binding protein comprising a variable domain derived from a light-chain gene segment can be referred to as or a "V L single-domain antigen-binding protein", see, e.g., U.S. Publication No. 20150289489, which is incorporated herein by reference in its entirety.

[0186] The phrase "light chain" includes immunoglobulin light chain sequences from any organism and, unless otherwise specified, includes human kappa (κ) and lambda (λ) light chains, VpreB, and surrogate light chains. Unless otherwise specified, the light chain variable domain generally includes three light chain CDRs and four framework (FR) regions. Generally, a full-length light chain includes, from the amino terminus to the carboxyl terminus, a variable domain and a light chain constant region, the variable domain comprising the FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and light chain constant region amino acid sequences. The light chain variable domain is encoded by a light chain variable region nucleotide sequence that generally includes light chain V L and light chain J L gene segments. The sequences, locations, and nomenclature of light chain V and J gene segments for various organisms can be found in the IMGT database, which is accessible via the Internet on the World Wide Web (www) at the URL "imgt.org". Light chains include, for example, those that do not selectively bind the first or second first member of a specific binding pair, the members being selectively bound by the first member of the specific binding pair binding protein in which they occur. Light chains also include those that bind and recognize one or more first members of a specific binding pair, or assist the heavy chain in binding and recognizing the one or more first members, the one or more first members being selectively bound by the first member of the specific binding pair binding protein in which they occur. Light chains also include those that bind and recognize one or more first members of a specific binding pair, or assist the heavy chain in binding and recognizing the one or more first members, the one or more first members being selectively bound by the first member of the specific binding pair binding protein in which they occur. Commonly used or general light chains include those derived from the human V1-39J5 gene or the human V 3-20J1 gene and include somatically mutated (e.g., affinity matured) forms thereof.

[0187] As used herein, the phrase "operably linked" encompasses the physical juxtaposition (e.g., in three-dimensional space) of components or elements that interact directly or indirectly with one another or otherwise coordinate with one another to participate in a biological event, the juxtaposition enabling or permitting such interaction and / or coordination. By way of example only, a regulatory element is said to be "operably linked" to a coding sequence when a control sequence (e.g., an expression control sequence) in a nucleic acid is positioned relative to the coding sequence such that its presence or absence affects the expression and / or activity of the coding sequence. In many embodiments, "operably linked" involves covalent linkage of the relevant components or elements to one another. Those skilled in the art will readily appreciate that in some embodiments, covalent linkage is not required to effect an effective operable linkage. For example, in some embodiments, a nucleic acid control sequence operably linked to a coding sequence controlled by the nucleic acid control sequence is contiguous with the nucleotide(s) of interest. Alternatively or additionally, in some embodiments, one or more such control sequences act in trans or at a distance to control the coding sequence of interest. In some embodiments, the term "expression control sequence" as used herein refers to a polynucleotide sequence that is necessary and / or sufficient for the expression and processing of a coding sequence to which it is linked. In some embodiments, an expression control sequence may be or include appropriate transcription initiation, termination, promoter, and / or enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequences); sequences that enhance protein stability; and / or, in some embodiments, sequences that enhance protein secretion. In some embodiments, one or more control sequences are active preferentially or solely in a particular host cell or organism or type thereof. By way of example only, in prokaryotes, control sequences typically include a promoter, ribosome binding site, and transcription termination sequence; in eukaryotes, in many embodiments, control sequences typically include a promoter, enhancer, and / or transcription termination sequence. One of ordinary skill in the art will understand from the context that in many embodiments, the term "control sequence" refers to a component whose presence is critical for expression and processing and, in some embodiments, includes components whose presence is beneficial for expression (including, for example, leader sequences, targeting sequences, and / or fusion partner sequences).

[0188] "Retargeting" or "redirecting" can include a situation where a wild-type particle targets several cells within a tissue and / or several organs within an organism, and the normal targeting to the tissue or organ is reduced or eliminated by inserting a heterologous amino acid, and retargeting to more specific cells within the tissue or a specific organ within the organism is achieved by binding to a targeting ligand that binds to a marker expressed by a specific cell (e.g., via a targeting ligand). Such retargeting or redirecting can also include a situation where a wild-type particle targets a tissue, and the targeting to the tissue is reduced or eliminated by inserting a heterologous amino acid, and retargeting to a completely different tissue is achieved with a targeting ligand.

[0189] "Specific binding pair", "protein:protein binding pair", etc. include two proteins that interact to form a bond (e.g., a first member (e.g., a first polypeptide) and a second homologous member (e.g., a second polypeptide)) (e.g., a non-covalent bond between an epitope of the first member and the antigen-binding portion of the second member of an antibody that recognizes the epitope) or a covalent isopeptide bond under conditions that permit or facilitate bond formation. In some embodiments, the term "homologous" refers to components that act together. Epitopes and their homologous antibodies, particularly epitopes that can also serve as detectable labels (e.g., c-myc), are well known in the art. Specific protein:protein binding pairs capable of interacting to form a covalent isopeptide bond are reviewed in Veggiani et al. (2014) Trends Biotechnol. 32:506 and include peptide:peptide binding pairs such as SpyTag:SpyCatcher, SpyTag002:SpyCatcher002, SpyTag:KTag, isopeptag:pilin C, SnoopTag:SnoopCatcher, etc. Generally, the first member of a protein:protein binding pair refers to the member of the protein:protein binding pair that is typically less than 30 amino acids in length and that forms a covalent isopeptide bond with a second homologous protein, where the second homologous protein is typically larger but can also be less than 30 amino acids in length, as in the SpyTag:KTag system.

[0190] The term "isopeptide bond" refers to an amide bond between a carboxyl or formamido group and an amino group, at least one of said groups not being derived from the protein backbone, or, alternatively, not being part of the protein backbone. Isopeptide bonds can form within a single protein or can occur between two peptides or between a peptide and a protein. Thus, isopeptide bonds can form intramolecularly within a single protein or intermolecularly, i.e., between two peptide / protein molecules, such as between two peptide linkers. Generally, isopeptide bonds can occur between a lysine residue and an asparagine, aspartic acid, glutamine or glutamic acid residue or the terminal carboxyl group of a protein or peptide chain, or can occur between the α-amino terminus of a protein or peptide chain and asparagine, aspartic acid, glutamine or glutamic acid. Each residue of the pair involved in an isopeptide bond is referred to herein as a reactive residue. In a preferred embodiment of the present invention, isopeptide bonds can form between a lysine residue and an asparagine residue or between a lysine residue and an aspartic acid residue. Specifically, isopeptide bonds can occur between the side-chain amine of lysine and the formamido group of asparagine or the carboxyl group of aspartic acid.

[0191] The SpyTag:SpyCatcher system is described in U.S. Patent No. 9,547,003 and Zaveri et al. (2012) Proceedings of the National Academy of Sciences of the United States of America (PNAS) 109:E690-E697, each of which is incorporated herein by reference in its entirety, and is derived from the CnaB2 domain of the fibronectin-binding protein FbaB of Streptococcus pyogenes. By splitting this domain, Zakeri et al. obtained the peptide "SpyTag" with the sequence AHIVMVDAYKPTK (SEQ ID NO:321), which forms an amide bond with its cognate protein "SpyCatcher", which is a 112-amino acid polypeptide with the amino acid sequence shown in SEQ ID NO:322. (Zakeri (2012), supra). Another specific binding pair derived from the CnaB2 domain is SpyTag:KTag, which forms an isopeptide bond in the presence of SpyLigase. (Fierer (2014) Proceedings of the National Academy of Sciences of the United States of America (PNAS) 111:E1176-1181). SpyLigase (SEQ ID NO:389) was engineered by excising the β-strand containing the reactive lysine from SpyCatcher, resulting in KTag, the 10-residue first member of a protein:protein binding pair with the amino acid sequence ATHIKFSKRD (SEQ ID NO:323). The SpyTag002:SpyCatcher002 system is described in Keeble et al. (2017) Angewandte Chemie International Edition in English (Angew Chem Int Ed Engl) 56:16521-25, which is incorporated herein by reference in its entirety. SpyTag002 has the amino acid sequence VPTIVMVDAYKRYK shown in SEQ ID NO:324 and binds SpyCatcher002 (SEQ ID NO:442).

[0192] The SnoopTag:SnoopCatcher system is described in Veggiani (2016) Proceedings of the National Academy of Sciences 113:1202-07. The D4 Ig-like domain of RrgA, an adhesin from Streptococcus pneumoniae, was split to form SnoopTag (residues 734-745; SEQ ID NO:390) and SnoopCatcher (residues 749-860; SEQ ID NO:391). Incubating SnoopTag and SnoopCatcher results in a spontaneous isopeptide bond with specificity between the complementary proteins. Veggiani (2016), supra.

[0193] The isopeptag:pilinC specific binding pair is derived from the major pilin protein Spy0128 from Streptococcus pyogenes. (Zakeir and Howarth (2010) J. Am. Chem. Soc. 132:4526-27). The isopeptag has the amino acid sequence TDKDMTITFTNKKDAE shown in SEQ ID NO:325 and binds to pilin-C (residues 18-299 of Spy0128). Incubation of the isopeptag and pilinC generates a spontaneous isopeptide bond that is specific between the complementary proteins. Zakeir and Howarth (2010), supra.

[0194] The term “detectable label” encompasses a polypeptide sequence that is a member of a specific binding pair, e.g., that binds with high affinity via non-covalent bonds to another polypeptide sequence such as an antibody complementarity determining region. Exemplary and non-limiting detectable labels include a hexahistidine tag, a FLAG tag, a Strep II tag, a streptavidin binding peptide (SBP) tag, a calmodulin binding peptide (CBP), glutathione S-transferase (GST), maltose binding protein (MBP), an S-tag, an HA tag, and c-myc (SEQ ID NO:326). (Reviewed in Zhao et al. (2013) J. Analytical Meth. Chem. 1-8; the reference is incorporated herein by reference). A common detectable label for primate AAV is the B1 epitope (SEQ ID NO:327). Some AAV capsid proteins that do not naturally contain the B1 epitope as described herein can be modified herein to contain the B1 epitope. Generally, the AAV capsid proteins described herein can contain a sequence that is substantially homologous to the B1 epitope within the last 10 amino acids of the capsid protein. Thus, in some embodiments, the non-primate AAV capsid proteins of the invention can be modified with one but fewer than five point mutations within the last 10 amino acids of the capsid protein such that the AAV capsid protein includes the B1 epitope.

[0195] The term “target cell” encompasses any cell in which expression of the nucleotide of interest is desired. Preferably, the target cell presents on its surface a receptor that permits the cell to be targeted by a targeting ligand, as described below.

[0196] The terms "transduction" or "infection", etc. refer to the introduction of nucleic acid into the target cell nucleus by viral particles. Terms related to transduction, such as "transduction efficiency", refer to the fraction (e.g., percentage) of cells expressing the nucleotide of interest after incubation with a certain number of viral particles comprising the nucleotide of interest. Well-known methods for determining transduction efficiency include flow cytometry of cells transduced with a fluorescent reporter gene, RT-PCR for expressing the nucleotide of interest, etc.

[0197] Generally, a "reference" viral capsid protein / capsid / particle is the same as the test viral capsid protein / capsid / particle, but the variation in its effect is to be tested. For example, to determine the effect of inserting the first member of a specific binding pair into a test viral particle, e.g., on transduction efficiency, the transduction efficiency of the test viral particle (in the absence or presence of an appropriate targeting ligand) can be compared with the transduction efficiency of a reference viral particle (if desired, in the absence or presence of an appropriate targeting ligand), where the transduction efficiency of the reference viral particle is the same as that of the test viral particle in every case except for the presence of the first member of the specific binding pair (e.g., additional point mutations, the nucleotide of interest, the number of viral particles and target cells, etc.). In some embodiments, the reference viral capsid protein is a reference viral capsid protein capable of forming a capsid with a second viral capsid protein, which is modified to include at least the first member of a protein:protein binding pair, where the reference viral capsid protein does not include the first member of the protein:protein binding pair, preferably where the capsid formed by the reference viral capsid protein and the modified viral capsid protein is a mosaic capsid.

[0198] Adeno-associated virus (AAV)

[0199] "AAV" is an abbreviation for adeno-associated virus and can be used to refer to the virus itself or its derivatives. AAV is a small, non-enveloped, single-stranded DNA virus. Generally, the wild-type AAV genome is 4.7 kb and is characterized by two inverted terminal repeats (ITRs) and two open reading frames (ORFs), rep and cap. The wild-type rep reading frame encodes four proteins with molecular weights of 78 kD ("Rep78"), 68 kD ("Rep68"), 52 kD ("Rep52") and 40 kD ("Rep40"). Rep78 and Rep68 are transcribed by the p5 promoter, and Rep52 and Rep40 are transcribed by the p19 promoter. These proteins are mainly used to regulate the transcription and replication of the AAV genome. The wild-type cap reading frame encodes three structural (capsid) viral proteins (VPs) with molecular weights of 83 - 85 kD (VP1), 72 - 73 kD (VP2) and 61 - 62 kD (VP3). More than 80% of the total protein in the AAV virion (capsid) consists of VP3; the relative abundances of VP1, VP2 and VP3 are found to be approximately 1:1:10 in mature virions, although ratios of 1:1:8 have been reported. Padron et al. (2005) Journal of Virology 79:5047 - 58.

[0200] The genomic sequences of various serotypes of AAV, as well as the native inverted terminal repeat (ITR), Rep protein, and capsid subunit sequences, are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. See, for example, GenBank accession numbers NC_002077 (AAV1), AF063497 (AAV1), NC001401 (AAV-2), AF043303 (AAV2), NC_001729 (AAV3), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF513851 (AAV7), AF513852 (AAV8), and NC_006261 (AAV8); the disclosures of which are incorporated herein by reference to teach AAV nucleic acid and amino acid sequences. See also, for example, Srivistava et al. (1983) J. Virol. 45:555; Chiorini et al. (1998) J. Virol. 71:6823; Chiorini et al. (1999) J. Virol. 73:1309; Bantel-Schaal et al. (1999) J. Virol. 73:939; Xiao et al. (1999) J. Virol. 73:3994; Muramatsu et al. (1996) Virology 221:208; Shade et al., (1986) J. Virol..58:921; Gao et al. (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al. (2004) Virology 33:375-383; U.S. Patent Application 20170130245; International Patent Applications WO 00 / 28061, WO99 / 61601, WO 98 / 11244; and U.S. Patent No. 6,156,303, each of which is incorporated herein by reference in its entirety. Table 5 herein provides the sequences of various non-primate AAVs.

[0201] "AAV" includes all subtypes and naturally occurring and modified forms, unless otherwise specified. AAV includes primate AAVs (e.g., AAV type 1 (AAV1), primate AAV type 2 (AAV2), primate AAV type 3 (AAV3), primate AAV3B, primate AAV type 4 (AAV4), primate AAV type 5 (AAV5), primate AAV type 6 (AAV6), primate AAV6.2, primate AAV type 7 (AAV7), primate AAV type 8 (AAV8), primate AAV type 9 (AAV9), AAV10, AAV hu11 type (AAV hu11), AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAVLK03, AAV type rh32.33 (AAVrh.32.33), AAV retro (AAV retro), AAV PHP.B, AAV PHP.eB, AAV PHP.S, AAVrh.64R1, AAVhu.37, AAVrh.8, AAV2 / 8, etc.); non - primate AAVs (e.g., avian AAV (AAAV)) and other non - primate AAVs such as mammalian AAVs (e.g., bat AAV, sea lion AAV, bovine AAV, canine AAV, equine AAA, caprine AAV, and ovine AAV, etc.), squamate AAVs (e.g., snake AAV and bearded dragon AAV), etc. "Primate AAV" refers to AAVs typically isolated from primates. Similarly, "non - primate AAV" refers to AAVs isolated from non - primates.

[0202] As used herein, with respect to genes (e.g., rep, cap, etc.), capsid proteins (e.g., VP1 capsid protein, VP2 capsid protein, VP3 capsid protein, etc.), regions of the capsid protein of a specific AAV (e.g., PLA 2Regions, VP1-u regions, VP1 / VP2 common regions, VP3 regions), nucleotide sequences (such as ITR sequences), for example, " [specific] AAV" related to the cap gene or capsid protein of AAV, etc. In addition to genes or polypeptides that respectively contain the nucleic acid sequences or amino acid sequences of the specific AAV shown herein, they also include variants of genes or polypeptides, including variants that contain the minimum number of nucleotides or amino acids required to retain one or more biological functions. As used herein, variant genes or variant polypeptides include nucleic acid sequences or amino acid sequences that are different from the nucleic acid sequences or amino acid sequences of the genes or polypeptides of the specific AAV shown herein, where such differences generally do not change at least one biological function of the gene or polypeptide, and / or the phylogenetic characteristics of the gene or polypeptide. For example, the differences may be due to the degeneracy of the genetic code, segregation variation, the length of the sequence, etc. For example, as used herein, the rep gene and the cap gene can cover rep and cap genes that are different from the wild-type genes because the genes can encode one or more Rep proteins and Cap proteins respectively. In some embodiments, the Rep gene encodes at least Rep78 and / or Rep68. In some embodiments, the cap gene contains genes that can be different from the wild-type, where one or more alternative start codons or the sequences between one or more alternative start codons are removed, such that the cap gene encodes only a single cap protein. For example, where the VP2 and / or VP3 start codons are removed or replaced, such that the cap gene encodes a functional VP1 capsid protein instead of a VP2 capsid protein or a VP3 capsid protein. Thus, as used herein, the rep gene covers any sequence that encodes a functional rep protein. The cap gene covers any sequence that encodes at least one functional cap protein.

[0203] It is well known that the wild-type cap gene expresses all three VP1, VP2, and VP3 capsid proteins from a single open reading frame of the cap gene under the control of the p40 promoter present in the rep ORF. The terms "capsid protein", "Cap protein", etc. include proteins that are part of the viral capsid. For adeno-associated virus, the capsid proteins are commonly referred to as VP1, VP2, and / or VP3, and can be encoded by a single cap gene. For AAV, the three AAV capsid proteins are essentially produced in an overlapping manner using alternative translation initiation codons of the cap ORF, although all three proteins use a common stop codon. The ORF of the wild-type cap gene encodes the following three alternative start codons and one "common stop codon" from 5' to 3': "VP1 start codon", "VP2 start codon", and "VP3 start codon". The largest viral protein, VP1, is typically encoded from the VP1 start codon to the "common stop codon". VP2 is typically encoded from the VP2 start codon to the common stop codon. VP3 is typically encoded from the VP3 start codon to the common stop codon. Thus, VP1 includes a sequence at its N-terminus that is not shared with VP2 or VP3, which is called the VP1-unique region (VP1-u). The VP1-u region is typically encoded by the sequence of the wild-type cap gene that starts from the VP1 start codon until the "VP2 start codon". VP1-u includes a phospholipase A2 domain (PLA 2 ), as well as a nuclear localization signal that may help the virus target the nucleus for uncoating and genome release. The VP1, VP2, and VP3 capsid proteins share the same C-terminal sequence that constitutes the entire VP3, which can also be referred to as the VP3 region in this article. The VP3 region is encoded from the VP3 start codon to the common stop codon. VP2 has an additional approximately 60 amino acids that are shared with VP1. This region is called the VP1 / VP2 common region.

[0204] In some embodiments, one or more of the Cap proteins of the invention can be encoded by one or more cap genes having one or more ORFs. In some embodiments, the VP proteins of the invention can be expressed from more than one ORF comprising nucleotide sequences encoding any combination of VP1, VP2, and / or VP3 using separate nucleotide sequences operably linked to at least one expression control sequence for expression in packaging cells, each cell producing one or more of the VP1, VP2, and / or VP3 capsid proteins of the invention. In some embodiments, the VP capsid proteins of the invention can be expressed individually from an ORF comprising a nucleotide sequence encoding any one of VP1, VP2, or VP3 using a separate nucleotide sequence operably linked to an expression control sequence for expression in virus replication cells, each cell producing only one of the VP1, VP2, or VP3 capsid proteins. In another embodiment, the VP proteins can be expressed from one ORF comprising nucleotide sequences encoding the VP1, VP2, and VP3 capsid proteins, the nucleotide sequences operably linked to at least one expression control sequence for expression in virus replication cells, each cell producing the VP1, VP2, and VP3 capsid proteins. Thus, although the amino acid positions provided herein may be provided relative to the VP1 capsid protein of a reference AAV, one of ordinary skill in the art will be able to readily and separately determine the positions of the same amino acids within the VP2 and / or VP3 capsid proteins of an AAV, as well as the corresponding positions of the amino acids in different AAVs.

[0205] Non-limiting examples of wild-type and / or genetically modified nucleic acid sequences of Cap genes and cap proteins that can be used to retarget virus particles as described herein are shown in SEQ ID NOs: 392 - 432.

[0206] The phrase "inverted terminal repeat" or "ITR" encompasses the symmetric nucleic acid sequences in the genome of adeno-associated virus required for efficient replication. The ITR sequences are located at each end of the AAV DNA genome. The ITR serves as the origin of replication for viral DNA synthesis and is an important cis-component for the production of AAV particles, e.g., packaging into AAV particles.

[0207] The AAV ITR includes recognition sites for the replication proteins Rep78 or Rep68. The "D" region of the ITR includes the DNA nicking site where DNA replication initiates and provides directionality for the nucleic acid replication step. AAVs that replicate in mammalian cells typically include two ITR sequences.

[0208] A single ITR can be engineered with Rep binding sites on both strands in the “A” region and two symmetric D regions on each side of the ITR palindrome. The engineered construct on such a double-stranded circular DNA template allows Rep78- or Rep68-initiated nucleic acid replication to proceed in both directions. A single ITR is sufficient for AAV replication of circular particles. In the method for producing AAV viral particles of the invention, the rep coding sequence encodes a Rep protein or Rep protein equivalent that can bind to the ITR included on the transfer plasmid.

[0209] When expressed with the appropriate Rep protein by a packaging cell, the Cap protein of the invention can encapsidate a transfer plasmid comprising the nucleotide of interest and an even number of two or more ITR sequences. In some embodiments, the transfer plasmid comprises one ITR sequence. In some embodiments, the transfer plasmid comprises two ITR sequences.

[0210] Either Rep78 and / or Rep68 binds to a unique and known site on the ITR hairpin sequence and functions to disrupt and unwind the hairpin structure at the ends of the AAV genome, thereby providing access to the replication machinery of the virus replication cell. It is well known that the Rep protein can be expressed from more than one ORF comprising a nucleotide sequence encoding any combination of Rep78, Rep68, Rep52, and / or Rep40 using separate nucleotide sequences operably linked to at least one expression control sequence for expression in a virus replication cell, each cell producing one or more of the Rep78, Rep68, Rep52, and / or Rep40 Rep proteins. Alternatively, the Rep protein can be expressed individually from an ORF comprising a nucleotide sequence encoding any one of Rep78, Rep68, Rep52, or Rep40 using a separate nucleotide sequence operably linked to an expression control sequence for expression in a packaging cell, each such cell producing only one Rep78, Rep68, Rep52, or Rep40 Rep protein. In another embodiment, the Rep protein can be expressed from one ORF comprising a nucleotide sequence encoding Rep78 and Rep52 Rep proteins, the nucleotide sequence operably linked to at least one expression control sequence for expression in a virus replication cell, each cell producing Rep78 and Rep52 Rep proteins.

[0211] In the method for producing AAV virions (e.g., viral particles) of the invention, the rep coding sequence and the cap gene of the invention can be provided in a single packaging plasmid. However, one of skill in the art will recognize that such conditions are not necessary. Such viral particles can contain or not contain a genome.

[0212] "Chimeric AAV capsid protein" includes an AAV capsid protein that includes an amino acid sequence, e.g., portions from two or more different AAVs and is capable of forming and / or forms an AAV viral capsid / viral particle. The chimeric AAV capsid protein is encoded by a chimeric AAV capsid gene, e.g., a chimeric nucleotide including a plurality of, e.g., at least two, nucleic acid sequences, each of the plurality of nucleic acid sequences being identical to a portion of a capsid gene encoding a capsid protein of a different AAV, and the plurality of nucleic acid sequences together encoding a functional chimeric AAV capsid protein. Association of the chimeric capsid protein with a particular AAV indicates that the capsid protein includes one or more portions of the capsid protein of said AAV and one or more portions of the capsid protein of a different AAV. For example, a chimeric AAV2 capsid protein includes a capsid protein that includes one or more portions of the VP1, VP2, and / or VP3 capsid protein of AAV2 and one or more portions of the VP1, VP2, and / or VP3 capsid protein of a different AAV.

[0213] The term "portion" refers to at least 5 amino acids or at least 15 nucleotides, but less than the full-length polypeptide or nucleic acid molecule, having 100% identity with the sequence from which the portion is derived, see Penzes (2015) Journal of General Virology. 2769. A "portion" encompasses any contiguous segment of amino acids or nucleotides sufficient to identify the form of the polypeptide or nucleic acid molecule from which the portion is derived as "[specific] AAV" or having "substantial identity" with a specific AAV such as non-primate AAV or distant AAV. In some embodiments, a portion comprises at least 5 amino acids or 15 nucleotides having 100% identity with a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 10 amino acids or 30 nucleotides having 100% identity with a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 15 amino acids or 45 nucleotides having 100% identity with a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 20 amino acids or 60 nucleotides having 100% identity with a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 25 amino acids or 75 nucleotides having 100% identity with a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 30 amino acids or 90 nucleotides having 100% identity with a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 35 amino acids or 105 nucleotides having 100% identity with a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 40 amino acids or 120 nucleotides having 100% identity with a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 45 amino acids or 135 nucleotides having 100% identity with a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 50 amino acids or 150 nucleotides having 100% identity with a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 60 amino acids or 180 nucleotides having 100% identity with a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 70 amino acids or 210 nucleotides having 100% identity with a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 80 amino acids or 240 nucleotides having 100% identity with a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 90 amino acids or 270 nucleotides having 100% identity with a sequence associated with a specific AAV. In some embodiments, a portion comprises at least 100 amino acids or 300 nucleotides having 100% identity with a sequence associated with a specific AAV.

[0214] Modified viral capsid proteins, viral particles, nucleic acids

[0215] In some embodiments, the Cap protein (e.g., the VP1 capsid protein as described herein, the VP2 capsid protein as described herein, and / or the VP3 capsid protein as described herein) is modified to include, for example, the first member of a protein:protein binding pair, a detectable label, a point mutation, and the like.

[0216] Chimerism is a modification as described herein. Generally, modification of a gene or polypeptide or a variant thereof of a particular AAV results in a nucleic acid sequence or amino acid sequence that is different from the nucleic acid sequence or amino acid sequence shown herein for the particular AAV, wherein the modification alters, confers, or removes one or more biological functions, but does not alter the phylogenetic identity of the gene or polypeptide. Modifications can include, for example, insertion of the first member of a protein:protein binding pair and point mutations such that the natural tropism of the capsid protein is reduced to eliminated and / or such that the capsid protein includes a detectable label. Preferred modifications include those that do not alter and preferably reduce the low recognition or non-recognition of the modified capsid by pre-existing antibodies present in the general population, which antibodies are generated during infection with another AAV, e.g., an infectious serotype such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, viral particles based on such serotypes, viral particles from currently used AAV gene therapy modalities, or combinations thereof. Other modifications as described herein include modification of the capsid protein such that it includes the first member of a protein:protein binding pair, a detectable label, and the like, which modifications are generally caused by modification at the gene level, e.g., by modification of the cap gene.

[0217] In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein is a mosaic capsid, e.g., comprising at least two sets of VP1, VP2, and / or VP3 proteins, each set of proteins being encoded by a different cap gene. A mosaic capsid herein generally refers to a chimera of a first viral capsid protein modified to include a first member of a protein:protein binding pair and a second corresponding viral capsid protein lacking the first member of the protein:protein binding pair. With respect to a mosaic capsid, the second viral capsid protein lacking the first member of the protein:protein binding pair can be referred to as a reference capsid protein encoded by a reference cap gene. In some mosaic capsid embodiments, preferably when the VP1, VP2, and / or VP3 capsid proteins modified with the first member of the protein:protein pair are not chimeric capsid proteins, the VP1, VP2, and / or VP3 reference capsid proteins can comprise an amino acid sequence identical to the amino acid sequence of the viral VP1, VP2, and / or VP3 capsid proteins modified with the first member of the protein:protein binding pair, except that the reference capsid proteins lack the first member of the protein:protein binding pair. In some mosaic capsid embodiments, the VP1, VP2, and / or VP3 reference capsid proteins correspond to the viral VP1, VP2, and / or VP3 capsid proteins modified with the first member of the protein:protein binding pair, except that the reference capsid proteins lack the first member of the protein:protein binding pair. In some embodiments, the VP1 reference capsid protein corresponds to the viral VP1 capsid protein modified with the first member of the protein:protein binding pair, except that the reference capsid protein lacks the first member of the protein:protein binding pair. In some embodiments, the VP2 reference capsid protein corresponds to the viral VP2 capsid protein modified with the first member of the protein:protein binding pair, except that the reference capsid protein lacks the first member of the protein:protein binding pair. In some embodiments, the VP3 reference capsid protein corresponds to the viral VP3 capsid protein modified with the first member of the protein:protein binding pair, except that the reference capsid protein lacks the first member of the protein:protein binding pair. In some mosaic capsid embodiments comprising chimeric VP1, VP2, and / or VP3 capsid proteins further modified to include the first member of the protein:protein binding pair, the reference protein can be the corresponding capsid protein, and a portion from the corresponding capsid protein forms a portion of the chimeric capsid protein. As a non-limiting example, in some embodiments, a mosaic capsid comprising a chimeric AAV2 / AAAV VP1 capsid protein modified to contain the first member of the protein:protein binding pair can also comprise the following capsid proteins as references: an AAV2 VP1 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, and a chimeric AAV2 / AAAV VP1 capsid protein lacking the first member.Similarly, in some embodiments, a mosaic capsid comprising a chimeric AAV2 / AAAV VP2 capsid protein modified to include a first member of a protein:protein binding pair can further include, as reference capsid proteins: an AAV2 VP2 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, a chimeric AAV2 / AAAV VP2 capsid protein lacking the first member. In some embodiments, a mosaic capsid comprising a chimeric AAV2 / AAAV VP3 capsid protein modified to include a first member of a protein:protein binding pair can further include, as reference capsid proteins: an AAV2 VP2 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, a chimeric AAV2 / AAAV VP3 capsid protein lacking the first member. In some mosaic capsid embodiments, the reference capsid protein can be any capsid protein so long as it lacks the first member of the protein:protein binding pair and is capable of forming a capsid with the first capsid protein modified with the first member of the protein:protein binding pair.

[0218] Generally, mosaic particles can be produced by transfecting a mixture of a modified Cap gene and a reference Cap gene into producer cells at a specified ratio. The ratio of protein subunits in the particles, e.g., the ratio of modified VP proteins:unmodified VP proteins, can but does not necessarily stoichiometrically reflect the ratio of at least two species of the cap gene encoding the first capsid protein modified with the first member of the protein:protein binding pair to one or more reference cap genes, e.g., the ratio of modified cap gene:reference cap gene transfected into the packaging cells. In some embodiments, the ratio of protein subunits in the particles does not stoichiometrically reflect the ratio of modified cap gene:reference cap gene transfected into the packaging cells.

[0219] In some embodiments of the mosaic virus particles, the ratio of protein subunits ranges from about 1:59 to about 59:1. In some embodiments of the mosaic virus particles, the protein subunits are at least about 1:1 (e.g., the mosaic virus particles comprise about 30 modified capsid proteins and about 30 reference capsid proteins). In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 1:2 (e.g., the mosaic virus particles comprise about 20 modified capsid proteins and about 40 reference capsid proteins). In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 3:5. In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 1:3 (e.g., the mosaic virus particles comprise about 15 modified capsid proteins and about 45 reference capsid proteins). In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 1:4 (e.g., the mosaic virus particles comprise about 12 modified capsid proteins and 48 reference capsid proteins). In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 1:5 (e.g., the mosaic virus particles comprise about 10 modified capsid proteins and 50 reference capsid proteins). In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 1:6. In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 1:7. In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 1:8. In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 1:9 (e.g., the mosaic virus particles comprise about 6 modified capsid proteins and about 54 reference capsid proteins). In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 1:10. In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 1:11 (e.g., the mosaic virus particles comprise about 5 modified capsid proteins and about 55 reference capsid proteins). In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 1:12. In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 1:13. In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 1:14 (e.g., the mosaic virus particles comprise about 4 modified capsid proteins and about 56 reference capsid proteins). In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 1:15. In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 1:19 (e.g., the mosaic virus particles comprise about 3 modified capsid proteins and about 57 reference capsid proteins). In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 1:29 (e.g., the mosaic virus particles comprise about 2 modified capsid proteins and about 58 reference capsid proteins). In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 1:59.In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 2:1 (e.g., the mosaic virus particles include about 40 modified capsid proteins and about 20 reference capsid proteins). In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 5:3. In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 3:1 (e.g., the mosaic virus particles include about 45 modified capsid proteins and about 15 reference capsid proteins). In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 4:1 (e.g., the mosaic virus particles include about 48 modified capsid proteins and 12 reference capsid proteins). In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 5:1 (e.g., the mosaic virus particles include about 50 modified capsid proteins and 10 reference capsid proteins). In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 6:1. In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 7:1. In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 8:1. In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 9:1 (e.g., the mosaic virus particles include about 54 modified capsid proteins and about 6 reference capsid proteins). In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 10:1. In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 11:1 (e.g., the mosaic virus particles include about 55 modified capsid proteins and about 5 reference capsid proteins). In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 12:1. In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 13:1. In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 14:1 (e.g., the mosaic virus particles include about 56 modified capsid proteins and about 4 reference capsid proteins). In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 15:1. In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 19:1 (e.g., the mosaic virus particles include about 57 modified capsid proteins and about 3 reference capsid proteins). In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 29:1 (e.g., the mosaic virus particles include about 58 modified capsid proteins and about 2 reference capsid proteins). In some embodiments of the mosaic virus particles, the ratio of protein subunits is at least about 59:1.

[0220] In some non-encapsidated virus particle embodiments, the ratio of protein subunits can be 1:0, where each capsid protein of the non-encapsidated virus particle is modified with the first member of a protein:protein binding pair. In some non-encapsidated virus particle embodiments, the ratio of protein subunits can be 0:1, where each capsid protein of the non-encapsidated virus particle is not modified with the first member of a protein:protein binding pair.

[0221] In some embodiments, the capsid proteins of the invention are modified to include a detectable label. Many detectable labels are known in the art. (See, e.g., Nilsson et al., (1997) "Affinity fusion strategies for detection, purification, and immobilization of modified proteins" Protein Expression and Purification 11:1-16, Terpe et al., (2003) "Overview of tag protein fusions: From molecular and biochemical fundamentals to commercial systems" Applied Microbiology and Biotechnology 60:523-533, and references therein). Detectable labels include, but are not limited to, the following: binding to immobilized divalent cations (e.g., Ni 2+)'s polyhistidine detectable label (e.g., His-6, His-8, or His-10), the biotin moiety that binds to immobilized avidin (e.g., on an in vivo biotinylated polypeptide sequence), the GST (glutathione S-transferase) sequence that binds to immobilized glutathione, the S tag that binds to immobilized S protein, the antigen that binds to immobilized antibody or its domain or fragment (including, for example, T7, myc, FLAG, and B tags that bind to the corresponding antibody), the FLASH tag (a highly detectable tag conjugated to a specific arsenyl moiety), the receptor or receptor domain that binds to immobilized ligand (or vice versa), protein A or its derivative (e.g., Z) that binds to immobilized IgG, the maltose binding protein (MBP) that binds to immobilized amylose, the albumin binding protein that binds to immobilized albumin, the chitin binding domain that binds to immobilized chitin, the calmodulin binding peptide that binds to immobilized calmodulin, and the cellulose binding domain that binds to immobilized cellulose. Another exemplary detectable label is the SNAP-tag, which is commercially available from Covalys Corporation (www.Covalys.com). In some embodiments, the detectable labels disclosed herein include detectable labels recognized only by antibody complementarity-determining regions. In some embodiments, the detectable labels disclosed herein include detectable labels recognized by antibody complementarity-determining regions and other specific binding pairs.

[0222] In some embodiments, the detectable label forms a binding pair with an immunoglobulin constant domain. In some embodiments, the detectable label and / or detectable labels do form a binding pair with a metal ion, e.g., Ni 2+ , Co 2+ , Cu 2+ , Zn 2+ , Fe 3+ , etc. In some embodiments, the detectable label is selected from the group consisting of streptavidin, Strep II, HA, L14, 4C-RGD, LH, and protein A.

[0223] In some embodiments, the detectable label is selected from the group consisting of FLAG, HA, and c-myc (EQKLISEEDL; SEQ ID NO:326). In some embodiments, the detectable label is c-myc (SEQ ID NO:326).

[0224] In some embodiments, the detectable label is a B cell epitope, e.g., having a length between about 1 amino acid and about 35 amino acids and forming a binding pair with an antibody complementarity-determining region, e.g., an immunoglobulin variable domain. In some embodiments, the detectable label comprises the B1 epitope (SEQ ID NO:327). In some embodiments, the capsid protein is modified to include the B1 epitope in the VP3 region.

[0225] In some embodiments, the capsid protein of the present invention comprises at least a first member of a peptide:peptide binding pair.

[0226] In some embodiments, the capsid protein of the present invention comprises a first member of a protein:protein binding pair that includes a detectable label, which can also be used to detect and / or isolate the Cap protein and / or as the first member of a protein:protein binding pair. In some embodiments, the detectable label serves as the first member of a protein:protein binding pair for binding a targeting ligand comprising a multispecific binding protein that can bind both the detectable label and a target expressed by a cell of interest. In some embodiments, the Cap protein of the present invention comprises a first member of a protein:protein binding pair that contains c-myc (SEQ ID NO: 326). The use of the detectable label as the first member of a protein:protein binding pair is described, for example, in WO2019006043, which is incorporated herein by reference in its entirety.

[0227] In some embodiments, the capsid protein comprises a first member of a protein:protein binding pair, wherein the protein:protein binding pair forms a covalent isopeptide bond. In some embodiments, the first member of the protein:protein binding pair is covalently bound via an isopeptide bond to a cognate second member of the protein:protein binding pair, and optionally wherein the cognate second member of the protein:protein binding pair is fused to a targeting ligand that binds to a target expressed by a cell of interest. In some embodiments, the protein:protein binding pair can be selected from the group consisting of SpyTag:SpyCatcher, SpyTag002:SpyCatcher002, SpyTag:KTag, Isopeptag:pilinC, and SnoopTag:SnoopCatcher. In some embodiments, the first member is SpyTag (or a bioactive portion thereof), and the protein (second cognate member) is SpyCatcher (or a bioactive portion thereof). In some embodiments, the first member is SpyTag (or a bioactive portion thereof), and the protein (second cognate member) is KTag (or a bioactive portion thereof). In some embodiments, the first member is KTag (or a bioactive portion thereof), and the protein (second cognate member) is SpyTag (or a bioactive portion thereof). In some embodiments, wherein the first member is SnoopTag (or a bioactive portion thereof), and the protein (second cognate member) is SnoopCatcher (or a bioactive portion thereof). In some embodiments, the first member is Isopeptag (or a bioactive portion thereof), and the protein (second cognate member) is Pilin-C (or a bioactive portion thereof). In some embodiments, the first member is SpyTag002 (or a bioactive portion thereof), and the protein (second cognate member) is SpyCatcher002 (or a bioactive portion thereof). In some embodiments, the Cap protein of the invention comprises SpyTag. The use of the first member of the protein:protein binding pair is described in WO2019006046, which is incorporated herein by reference in its entirety.

[0228] In some embodiments, the first member of the protein:protein binding pair and / or the detectable label is operably linked to the Cap protein of the invention (translated in-frame with the Cap protein, chemically conjugated to the Cap protein, and / or displayed via the Cap protein) via a first linker or a second linker, e.g., an amino acid spacer of at least one amino acid in length. In some embodiments, the first member of the protein:protein binding pair is flanked by a first linker and / or a second linker, e.g., a first and / or second amino acid spacer, wherein each spacer is at least one amino acid in length.

[0229] In some embodiments, the first and / or second linker is / are different. In some embodiments, the length of the first and / or second linker is / are independently one or two amino acids. In some embodiments, the length of the first and / or second linker is / are independently one, two, or three amino acids. In some embodiments, the length of the first and / or second linker is / are independently one, two, three, or four amino acids. In some embodiments, the length of the first and / or second linker is / are independently one, two, three, four, or five amino acids. In some embodiments, the length of the first and / or second linker is / are independently one, two, three, four, five, or six amino acids. In some embodiments, the length of the first and / or second linker is / are independently one, two, three, four, five, six, or seven amino acids. In some embodiments, the length of the first and / or second linker is / are independently one, two, three, four, five, six, seven, or eight amino acids. In some embodiments, the length of the first and / or second linker is / are independently one, two, three, four, five, six, seven, eight, or nine amino acids. In some embodiments, the length of the first and / or second linker is / are independently one, two, three, four, five, six, seven, eight, nine, or ten amino acids. In some embodiments, the length of the first and / or second linker is / are independently one, two, three, four, five, six, seven, eight, nine, ten, or more amino acids.

[0230] In some embodiments, the sequences and / or lengths of the first and second linkers are the same, and the length of each is one amino acid. In some embodiments, the first and second linkers are of the same length, and the length of each is one amino acid. In some embodiments, the first and second linkers are of the same length, and the length of each is two amino acids. In some embodiments, the first and second linkers are of the same length, and the length of each is three amino acids. In some embodiments, the first and second linkers are of the same length, and the length of each is four amino acids. For example, the linker is GLSG (SEQ ID NO:328). In some embodiments, the first and second linkers are of the same length, and the length of each is five amino acids. In some embodiments, the first and second linkers are of the same length, and the length of each is six amino acids. For example, the first and second linkers each comprise the sequence GLSGSG (SEQ ID NO:329). In some embodiments, the first and second linkers are of the same length, and the length of each is seven amino acids. In some embodiments, the first and second linkers are of the same length, and the length of each is eight amino acids. For example, the first and second linkers each comprise the sequence GLSGLSGS (SEQ ID NO:330). In some embodiments, the first and second linkers are of the same length, and the length of each is nine amino acids. In some embodiments, the first and second linkers are of the same length, and the length of each is ten amino acids. For example, the first and second linkers each comprise the sequence GLSGLSGLSG (SEQ ID NO:331) or GLSGGSGLSG (SEQ ID NO:332). In some embodiments, the first and second linkers are of the same length, and the length of each is more than ten amino acids.

[0231] Typically, the first member of the protein:protein binding pair amino acid sequence as described herein, for example, includes the first member of the specific binding pair itself or in combination with one or more linkers, which has a length of from about 5 amino acids to about 50 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is at least 5 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 6 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 7 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 8 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 9 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 10 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 11 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 12 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 13 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 14 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 15 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 16 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 17 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 18 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 19 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 20 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 21 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 22 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 23 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 24 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 25 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 26 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 27 amino acids.In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 28 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 29 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 30 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 31 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 32 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 33 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 34 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 35 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 36 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 37 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 38 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 39 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 40 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 41 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 42 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 43 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 44 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 45 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 46 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 47 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 48 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 49 amino acids. In some embodiments, the length of the first member of the protein:protein binding pair amino acid sequence is 50 amino acids.

[0232] Due to the high conservation of at least large regions and the large number of closely related family members, the corresponding insertion sites of AAVs other than the listed AAVs can be identified by performing amino acid alignments or by comparing capsid structures. See, for example, Rutledge et al. (1998) Journal of Virology 72:309-19; Mietzsch et al. (2019) Virology 11,362,1-34 and U.S. Patent No. 9,624,274 for exemplary alignments of different AAV capsid proteins, each of the references being incorporated herein by reference in its entirety. For example, Mietzcsh et al., (2019) provided band coverage from different parvovirus dependents at Figure 7 depicting variable regions VR I to VR IX. Using such structural and sequence analyses described herein, one of ordinary skill in the art can determine which amino acids within the variable regions correspond to the amino acid sequence of an AAV that can accommodate the insertion of a first member of a protein:protein binding pair and / or a detectable label.

[0233] Thus, in some embodiments, the first member of a protein:protein binding pair and / or a detectable label is inserted into the VP1 capsid protein of a non-primate AAV, after the amino acid position corresponding to an amino acid position selected from the group consisting of: G453 of the AAV2 capsid protein VP1, N587 of the AAV2 capsid protein VP1, G453 of the AAV9 capsid protein VP1, and A589 of the AAV9 capsid protein VP1. In some embodiments, the first member of a protein:protein binding pair and / or a detectable label is inserted into the VP1 capsid protein of a non-primate AAV, between the amino acids corresponding to N587 and R588 of the AAV2 VP1 capsid. Additional suitable insertion sites in the non-primate VP1 capsid protein include those corresponding to the following of the VP1 capsid protein of AAV2: I-1, I-34, I-138, I-139, I-161, I261, I-266, I-381, I-453, I-447, I-448, I-459, I-471, I-520, I-534, I-570, I-573, I-584, I-587, I-588, I591, I-657, I-664, I-713, and I-716 (Wu et al. (2000) Journal of Virology 74:8635-8647). In some embodiments, the insertion site in the non-primate VP1 capsid protein corresponds to I-453. The modified viral capsid protein described herein can be a non-primate capsid protein that comprises the first member of a protein:protein binding pair and / or a detectable label inserted into a position corresponding to a position of the AAV2 capsid protein selected from the group consisting of I-1, I-34, I-138, I-139, I-161, I--266, I-381, I-447, I-448, I-453, I-459, I-471, I-520, I-534, I-570, I-573, I-584, I-587, I-588, I-591, I-657, I-664, I-713, I-716, and combinations thereof. In some embodiments, the insertion site in the non-primate VP1 capsid protein corresponds to I-453. Additional suitable insertion sites in the non-primate AAV include those corresponding to: I-587 of AAV1, I-589 of AAV1, I-585 of AAV3, I-585 of AAV4, and I-585 of AAV5.In some embodiments, a modified viral capsid protein as described herein can be a non-primate capsid protein that includes a protein inserted at a position corresponding to a position selected from the group consisting of: a first member of a protein:protein binding pair and / or a detectable label: I-587 (AAV1), I-589 (AAV1), I-585 (AAV3), I-585 (AAV4), I-585 (AAV5), and combinations thereof.

[0234] In some embodiments, the first member of a protein:protein binding pair and / or a detectable label is inserted into the VP1 capsid protein of a non-primate AAV, after the amino acid position corresponding to the amino acid position selected from the group consisting of: I444 of the VP1 of an avian AAV capsid protein, I580 of the VP1 of an avian AAV capsid protein, I573 of the VP1 of a bearded dragon AAV capsid protein, I436 of the VP1 of a bearded dragon AAV capsid protein, I429 of the VP1 of a sea lion AAV capsid protein, I430 of the VP1 of a sea lion AAV capsid protein, I431 of the VP1 of a sea lion AAV capsid protein, I432 of the VP1 of a sea lion AAV capsid protein, I433 of the VP1 of a sea lion AAV capsid protein, I434 of the VP1 of a sea lion AAV capsid protein, I436 of the VP1 of a sea lion AAV capsid protein, I437 of the VP1 of a sea lion AAV capsid protein, and I565 of the VP1 of a sea lion AAV capsid protein.

[0235] The nomenclature I-, I# etc. herein refers to the insertion site (I) of the amino acid numbering of the VP1 protein relative to the AAV capsid protein named with . However, such an insertion can be directly located at the N-terminus or C-terminus, preferably within 5 amino acids of the N-terminus or C-terminus of a given amino acid, preferably at the C-terminus of one amino acid in the sequence of 3, more preferably 2, and especially 1 amino acid at the N-terminus or C-terminus of the given amino acid. Additionally, the positions mentioned herein are relative to the VP1 protein encoded by the AAV capsid gene, and the corresponding positions (and their point mutations) of the VP2 and VP3 capsid proteins encoded by the capsid gene can be readily identified by performing a sequence alignment of the VP1, VP2, and VP3 proteins encoded by the appropriate AAV capsid gene.

[0236] Thus, the corresponding positions of the coding nucleic acids inserted into one of these sites in the cap gene are such that the insertion is into VP1, VP2, and / or VP3, since the capsid proteins are encoded by overlapping reading frames of the same gene with staggered start codons. Thus, for AAV2, for example, according to this nomenclature, an insertion between amino acids 1 and 138 is an insertion only into VP1, an insertion between 138 and 203 is an insertion into VP1 and VP2, and an insertion between 203 and the C-terminus is an insertion into VP1, VP2, and VP3, and the same is of course true for insertion site I-587. Accordingly, the present invention encompasses the structural genes of AAVs having corresponding insertions in the VP1, VP2, and / or VP3 proteins.

[0237] Also provided herein are nucleic acids encoding the VP3 capsid protein of the invention. The AAV capsid proteins may or may not be encoded by overlapping reading frames of the same gene with staggered start codons. In some embodiments, the nucleic acid encoding the VP3 capsid protein of the invention does not also encode the VP2 capsid protein or the VP1 capsid protein of the invention. In some embodiments, the nucleic acid encoding the VP3 capsid protein of the invention may also encode the VP2 capsid protein of the invention, but does not also encode the VP1 capsid of the invention. In some embodiments, the nucleic acid encoding the VP3 capsid protein of the invention may also encode the VP2 capsid protein of the invention and the VP1 capsid of the invention.

[0238] In some embodiments, the viral capsid comprises a modified viral capsid protein, the modified viral capsid protein comprising a first and a second member of a protein:protein binding pair (e.g., wherein the second member is operably linked to a targeting ligand, including a multispecific binding protein, etc.), the viral capsid being capable of infecting a particular cell, e.g., having an enhanced ability to target and bind to a particular cell as compared to a control viral capsid that is identical to the modified viral capsid protein except that the control viral capsid lacks one or both of the first and second members of the protein:protein binding pair, e.g., comprises a control capsid protein. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a detectable transduction efficiency as compared to an undetectable transduction efficiency of a control viral capsid, the modified viral capsid protein binding to the first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is 10% higher as compared to an undetectable transduction efficiency of a control viral capsid, the modified viral capsid protein binding to the first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is 20% higher as compared to an undetectable transduction efficiency of a control viral capsid, the modified viral capsid protein binding to the first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is 30% higher as compared to the transduction efficiency of a control viral capsid, the modified viral capsid protein binding to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is 40% higher as compared to the transduction efficiency of a control viral capsid, the modified viral capsid protein binding to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is 50% higher as compared to the transduction efficiency of a control viral capsid, the modified viral capsid protein binding to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is 60% higher as compared to the transduction efficiency of a control viral capsid, the modified viral capsid protein binding to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand.In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is 70% higher compared to the transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is 75% higher compared to the transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is 80% higher compared to the transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is 85% higher compared to the transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is 90% higher compared to the transduction efficiency of a control capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is 95% higher compared to the transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is 99% higher compared to the undetectable transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the first and second members of a protein:protein binding pair linked to a targeting ligand.

[0239] In some embodiments, the viral capsid comprises a modified viral capsid protein, the modified viral capsid protein comprising a first and a second member of a protein:protein binding pair (e.g., wherein the second member is operably linked to a targeting ligand, including a multispecific binding protein, etc.), the viral capsid being capable of infecting a particular cell, e.g., having an enhanced ability to target and bind to a particular cell as compared to a control viral capsid that is identical to the modified viral capsid protein except that the control viral capsid lacks one or both of the first and second members of the protein:protein binding pair, e.g., comprises a control capsid protein. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a detectable transduction efficiency as compared to an undetectable transduction efficiency of a control viral capsid, the modified viral capsid protein binding to the first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a 10% higher transduction efficiency as compared to an undetectable transduction efficiency of a control viral capsid, the modified viral capsid protein binding to the first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a 20% higher transduction efficiency as compared to an undetectable transduction efficiency of a control viral capsid, the modified viral capsid protein binding to the first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a 30% higher transduction efficiency as compared to the transduction efficiency of a control viral capsid, the modified viral capsid protein binding to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a 40% higher transduction efficiency as compared to the transduction efficiency of a control viral capsid, the modified viral capsid protein binding to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a 50% higher transduction efficiency as compared to the transduction efficiency of a control viral capsid, the modified viral capsid protein binding to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a 60% higher transduction efficiency as compared to the transduction efficiency of a control viral capsid, the modified viral capsid protein binding to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand.In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is 70% higher compared to the transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is 75% higher compared to the transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is 80% higher compared to the transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is 85% higher compared to the transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is 90% higher compared to the transduction efficiency of a control capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is 95% higher compared to the transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is 99% higher compared to the undetectable transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is at least 1.5-fold higher compared to the undetectable transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, the viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is at least 2-fold higher compared to the undetectable transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the first and second members of a protein:protein binding pair linked to a targeting ligand.In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is at least 3-fold higher compared to the transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is at least 4-fold higher compared to the transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is at least 5-fold higher compared to the transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is at least 6-fold higher compared to the transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is at least 7-fold higher compared to the transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is at least 8-fold higher compared to the transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is at least 9-fold higher compared to the transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is at least 10-fold higher compared to the transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is at least 20-fold higher compared to the transduction efficiency of a control capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand.In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is at least 30-fold higher compared to the transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the appropriate first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is at least 40-fold higher compared to the undetectable transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is at least 50-fold higher compared to the undetectable transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is at least 60-fold higher compared to the undetectable transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is at least 70-fold higher compared to the undetectable transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is at least 80-fold higher compared to the undetectable transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is at least 90-fold higher compared to the undetectable transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the first and second members of a protein:protein binding pair linked to a targeting ligand. In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein exhibits a transduction efficiency that is at least 100-fold higher compared to the undetectable transduction efficiency of a control viral capsid, wherein the modified viral capsid protein binds to the first and second members of a protein:protein binding pair linked to a targeting ligand.In some embodiments, the viral particles of the present invention comprise a viral capsid protein that comprises the amino acid sequence of a non - primate AAV, a remote AAV, or a combination thereof, and optionally comprises the first and second members of a protein:protein binding pair (e.g., wherein the second member is operably linked to a targeting ligand, including a multispecific binding protein, etc.). Compared to a suitable control viral particle (e.g., a viral capsid comprising an AAV serotype, wherein a portion of the viral capsid is included in the viral capsid of the present invention, e.g., as part of the viral capsid protein comprising the amino acid sequence of a non - primate AAV, a remote AAV, or a combination thereof), the viral particle is capable of better evading neutralization by pre - existing antibodies in serum isolated from a human patient. The control viral particle also optionally comprises the first and second members of a protein:protein binding pair (e.g., wherein the second member is operably linked to a targeting ligand, including a multispecific binding protein, etc.). In some embodiments, compared to a suitable control viral particle, the viral particle of the present invention comprising a viral capsid protein requires at least 2 - fold more total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection), e.g., (e.g., the IC50 value of the viral particle of the present invention is at least 2 - fold that of the control viral particle), and the viral capsid protein comprises the amino acid sequence of a non - primate AAV, a remote AAV, or a combination thereof.

[0240] Targeting ligand

[0241] The viral particles described herein can further comprise a targeting ligand. "Retargeting" or "redirecting" can encompass a situation where a wild - type viral particle targets several cells within a tissue and / or several organs within an organism, and by inserting a detectable label or a targeting ligand, the broad targeting of the tissue or organ is reduced or eliminated until it is eliminated, and the retargeting of more specific cells within the tissue or more specific organs within the organism is achieved by respectively using a multispecific binding molecule that binds to the detectable label and a second domain that binds to the receptor of interest and / or by using a targeting ligand that binds to the receptor of interest. Such retargeting or redirecting can also encompass a situation where a wild - type viral particle targets a tissue, and by inserting a detectable label, the targeting of the tissue is reduced or eliminated until it is eliminated, and the retargeting of a completely different tissue is achieved by using a multispecific binding molecule.

[0242] In some embodiments of the invention that include a detectable label, the targeting ligand comprises a multispecific binding molecule that comprises (i) an antibody paratope that specifically binds the detectable label and (ii) a second binding domain that specifically binds a receptor, and the targeting ligand can be conjugated to the surface of a bead (e.g., for purification) or expressed by a target cell. Thus, the multispecific binding molecule comprises those binding molecules that comprise: (i) an antibody paratope that specifically binds the detectable label and (ii) a second binding domain that specifically binds a receptor targeted by a viral particle.

[0243] In some embodiments of the invention, the viral vector comprises a protein:protein binding pair associated by an isopeptide bond as described herein, wherein the second member of the protein:protein binding pair is fused to a targeting ligand. In some embodiments, the targeting ligand fused to the second member of the protein:protein binding pair associated by an isopeptide bond comprises an antibody or a binding portion thereof, such as an antibody paratope.

[0244] An antibody paratope as described herein generally comprises at least complementarity determining regions (CDRs) that participate in specifically recognizing a target (e.g., a detectable label, a cell surface receptor, etc.), e.g., the CDR3 region of the heavy and / or light chain variable domains. In some embodiments, the multispecific binding molecule comprises an antibody (or a portion thereof) that comprises an antibody paratope that specifically binds a detectable label.

[0245] One embodiment of the invention is a multimeric structure comprising a modified viral capsid protein of the invention. The multimeric structure comprises at least 5, preferably at least 10, more preferably at least 30, and most preferably at least 60 modified viral capsid proteins as described herein, the modified viral capsid protein comprising a first member of a specific binding pair. It can form a conventional viral capsid (empty viral particle) or a viral particle (a capsid encapsidating a nucleotide of interest). The formation of a viral particle comprising a viral genome is a highly preferred feature using the modified viral capsids described herein.

[0246] Another embodiment of the invention is the use of at least one modified viral capsid protein and / or a nucleic acid encoding the same, preferably for the manufacture and for use in transferring a nucleotide of interest into a target cell in at least one multimeric structure (e.g., a viral particle).

[0247] Generally, a viral capsid protein as described herein can include a targeting ligand that targets the TfR, such as an anti-TfR antibody and binding portions thereof. Antibodies specific for human TfR are well known in the art. For non-limiting exemplary anti-transferrin receptor antibodies; see, for example, see, for example, US20170174778; US20150196663; US9629801; US20180002433; WO2016081643; US20180134797; WO2014189973; US20150110791; US9708406; US20170260292; WO2016081640; US20180057604; US9611323; WO2012075037; WO2018210898; US20180344869; US20180282408; US20170051071; WO2016207240; WO2015101588; US20160324984, US20180222993, WO2017055542; US20180222992; WO2017055540; Cabezon, I., et al. "Molecular Pharmaceutics (Mol Pharm)". November 2, 2015; 12(11):4137-45; Yu YJ, et al. "Science Translational Medicine (Sci Transl Med)" (2014) 6:261ra154; Couch, et al. "Science Translational Medicine". May 1, 2013; 5(183):183ra57, 1-12.

[0248] Additional nucleic acid sequences and translated amino acid sequences of domains of anti-transferrin antibodies and scFvs that can be used to retarget an AAV capsid as described herein are provided as SEq ID NO: 1-320 and 333-388.

[0249] Table 1 provides an overview of the SEQ ID NOs of each binding portion (e.g., heavy chain variable domain, light chain variable domain, and CDRs 1, 2, and 3) of non-limiting anti-human TfR antibodies that can be used to redirect AAV capsids as described herein. In some embodiments, an AAV capsid as described herein comprises a targeting ligand that binds human TfR, wherein the targeting ligand comprises a heavy chain variable domain, a light chain variable domain, CDR1, CDR2, and / or CDR3 amino acid sequences that are at least 90% identical to the amino acid sequences of the heavy chain variable domain, light chain variable domain, CDR1, CDR2, and / or CDR3 shown in any one of SEQ ID NOs: 1-320 and 365-388. In some embodiments, an AAV capsid as described herein comprises a targeting ligand that binds human TfR, wherein the targeting ligand comprises a heavy chain variable domain, a light chain variable domain, CDR1, CDR2, and / or CDR3 amino acid sequences that are at least 95% identical to the amino acid sequences of the heavy chain variable domain, light chain variable domain, CDR1, CDR2, and / or CDR3 shown in any one of SEQ ID NOs: 1-320 and 365-388. In some embodiments, an AAV capsid as described herein comprises a targeting ligand that binds human TfR, wherein the targeting ligand comprises a heavy chain variable domain, a light chain variable domain, CDR1, CDR2, and / or CDR3 amino acid sequences that are at least 97% identical to the amino acid sequences of the heavy chain variable domain, light chain variable domain, CDR1, CDR2, and / or CDR3 shown in any one of SEQ ID NOs: 1-320 and 365-388. In some embodiments, an AAV capsid as described herein comprises a targeting ligand that binds human TfR, wherein the targeting ligand comprises a heavy chain variable domain, a light chain variable domain, CDR1, CDR2, and / or CDR3 amino acid sequences that are at least 98% identical to the amino acid sequences of the heavy chain variable domain, light chain variable domain, CDR1, CDR2, and / or CDR3 shown in any one of SEQ ID NOs: 1-320 and 365-388. In some embodiments, an AAV capsid as described herein comprises a targeting ligand that binds human TfR, wherein the targeting ligand comprises a heavy chain variable domain, a light chain variable domain, CDR1, CDR2, and / or CDR3 amino acid sequences that are at least 99% identical to the amino acid sequences of the heavy chain variable domain, light chain variable domain, CDR1, CDR2, and / or CDR3 shown in any one of SEQ ID NOs: 1-320 and 365-388.

[0250] Table 1. SEQ ID NOs of domains in antibodies, antigen-binding fragments (e.g., Fab), or scFv molecules that can be used to retarget AAV to human TfR. The SEQ ID NOs in parentheses are the nucleic acids encoding the listed domains.

[0251]

[0252]

[0253]

[0254] Non-limiting examples of forms of targeting ligands that bind to TfR, in addition to the bivalent monoclonal antibody (mAb) form, include: (i) Fab fragments (Fab); (ii) F(ab’)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units composed of amino acid residues that mimic the hypervariable regions of antibodies (e.g., isolated complementarity-determining regions (CDRs), such as CDR3 peptides) or restricted FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetra-bodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also encompassed within the expression “targeting ligand” as used herein. In non-limiting embodiments, the anti-TfR targeting ligand that binds to TfR and can be used to retarget the viral capsid as described herein comprises an scFv. As a non-limiting example, the V L -(Gly 4 Ser) 3 -V H form of the scFv sequence may comprise an amino acid sequence that is 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of the amino acid sequences shown in SEQ ID NOs: 333 - 364.

[0255] In some embodiments, the scFv that can be used to retarget the viral capsid as described herein may comprise an amino acid sequence that is 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of the amino acid sequences shown in SEQ ID NOs: 333 - 364, but is of the VH-(Gly4Ser)3-VL form.

[0256] In some cases, the anti-TfR antigen-binding protein is an antibody that contains one or more mutations in a framework region, such as in the CH1 domain, CH2 domain, CH3 domain, hinge region, or a combination thereof. In some embodiments, one or more mutations are used to stabilize the antibody and / or increase the half-life. In some embodiments, one or more mutations are used to modulate Fc receptor interactions to reduce or eliminate Fc effector functions, such as FcyR, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). In additional embodiments, one or more mutations are used to modulate glycosylation.

[0257] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the antibodies described herein (e.g., numbered according to the Kabat numbering system (e.g., EU index in Kabat), in the CH2 domain (residues 231 - 340 of human IgG1) and / or CH3 domain (residues 341 - 447 of human IgG1) and / or hinge region) to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antigen-dependent cytotoxicity. In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region (CH1 domain) of the Fc region such that the number of cysteine residues in the hinge region is altered (e.g., increased or decreased), as described in U.S. Patent No. 5,677,425. The number of cysteine residues in the hinge region of the CH1 domain can be altered to, for example, facilitate the assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody or to facilitate linker conjugation.

[0258] In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant domain or its FcRn-binding fragment (preferably the Fc or hinge-Fc domain fragment) to alter (e.g., decrease or increase) the in vivo half-life of the antibody. For examples of mutations that alter (e.g., decrease or increase) the in vivo half-life of an antibody, see, e.g., PCT Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745. In some embodiments, the Fc region contains a mutation at residue position L234, L235, or a combination thereof. In some embodiments, the mutation comprises L234 and L235. In some embodiments, the mutation comprises L234A and L235A.

[0259] The anti-TfR antibodies and antigen-binding fragments described herein can be post-translationally modified, such as by glycosylation.

[0260] For example, the antibodies and antigen-binding fragments described herein can be glycosylated (e.g., N-glycosylated and / or O-glycosylated). Typically, antibodies and antigen-binding fragments are glycosylated at the conserved residue N297 of the IgG Fc domain. Some antibodies and fragments contain one or more additional glycosylation sites in the variable region. In one embodiment, the glycosylation site is in the context of FN 297 S or YN 297 S.

[0261] In one embodiment, the glycosylation is any one or more of the following three different N-glycan types: high-mannose type, complex type, and / or hybrid type, which are present on IgG with their respective linkages. The complex type and the hybrid type can have core fucosylation (addition of a fucose residue to the innermost N-acetylglucosamine) and can also have no core fucosylation.

[0262] In some cases, an anti-TfR antigen-binding protein antibody, i.e., an antibody that does not contain a glycosylation sequence that may interfere with the transglutaminase reaction, such as an antibody that does not have sugars at N180 and / or N297 on one or more heavy chains. In a particular embodiment, the heavy chain of the antibody has an N180 mutation. In other words, according to the EU numbering system as disclosed by Kabat et al., the antibody is mutated to no longer have an asparagine residue at position 180. In a particular embodiment, the heavy chain of the antibody has an N180Q mutation. In a particular embodiment, the heavy chain of the antibody has an N297 mutation. In a particular embodiment, the heavy chain of the antibody has an N297Q or N297D mutation. Antibodies containing such above-mentioned mutations can preferably be prepared by site-directed mutagenesis to remove the glycosylation sequence or render the glycosylation sequence ineffective, or by site-directed mutagenesis to insert a glutamine residue at a site remote from any interfering glycosylation site or any other interfering structure. Such antibodies can also be isolated from natural or artificial sources. Non-glycosylated antibodies also include antibodies containing T299 or S298P or other mutations or combinations of mutations that result in a lack of glycosylation.

[0263] In some cases, the antigen-binding protein is a deglycosylated antibody, i.e., an antibody from which sugars are removed to facilitate transglutaminase-mediated conjugation. Sugars include, but are not limited to, N-linked oligosaccharides. In some embodiments, deglycosylation is carried out at residue N180. In some embodiments, deglycosylation is carried out at residue N297. In some embodiments, the removal of sugars is accomplished enzymatically, including but not limited to via PNGase.

[0264] In one embodiment, the antibodies or fragments described herein are non-fucosylated.

[0265] The antibodies and antigen-binding fragments described herein can also be post-translationally modified in other ways, including, for example: Glu or Gln cyclization at the N-terminus; loss of N-terminal positive charge; Lys variants at the C-terminus; deamidation (Asn to Asp); isomerization (Asp to isoAsp); deamidation (Gln to Glu); isomerization (Asp to isoAsp); deamidation (Gln to Glu); oxidation (Cys, His, Met, Tyr, Trp); and / or disulfide bond heterogeneity (rearrangement, thioether, and trisulfide bond formation).

[0266] In some embodiments, the antibodies disclosed herein comprise Q295, which may be native to the antibody heavy chain sequence. In some embodiments, the antibody heavy chains disclosed herein may comprise Q295. In some embodiments, the antibody heavy chains disclosed herein may comprise Q295 and the amino acid substitution N297D.

[0267] According to certain embodiments of the present disclosure, anti-TfR antibodies and antigen-binding fragments are provided, the anti-TfR antibodies and antigen-binding fragments comprising an Fc domain, the Fc domain comprising one or more mutations that, for example, enhance or reduce the binding of the antibody to the FcRn receptor at acidic pH compared to neutral pH. For example, the present disclosure includes anti-TfR antibodies that comprise mutations in the CH2 or CH3 region of the Fc domain, wherein one or more of the mutations increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in endosomes having a pH range from about 5.5 to about 6.0). When administered to an animal, such mutations can result in an increase in the serum half-life of the antibody.

[0268] Non-limiting examples of such Fc modifications include, for example, modifications at the following positions:

[0269] · 250 (e.g., E or Q);

[0270] · 250 and 428 (e.g., L or F);

[0271] · 252 (e.g., L / Y / F / W or T),

[0272] · 254 (e.g., S or T), and / or

[0273] · 256 (e.g., S / R / Q / E / D or T);

[0274] and / or modifications at the following positions:

[0275] · 428 and / or 433 (e.g., H / L / R / S / P / Q or K), and / or

[0276] · 434 (e.g., A, W, H, F or Y);

[0277] and / or modifications at the following positions:

[0278] · 250 and / or 428;

[0279] and / or modifications at the following positions:

[0280] · 307 or 308 (e.g., 308F, V308F), and / or 434.

[0281] In one embodiment, the modifications include:

[0282] · 428L (e.g., M428L) and 434S (e.g., N434S) modifications;

[0283] · 428L, 259I (e.g., V259I) and 308F (e.g., V308F) modifications;

[0284] · 433K (e.g., H433K) and 434 (e.g., 434Y) modifications;

[0285] · 252, 254 and 256 (e.g., 252Y, 254T and 256E) modifications;

[0286] · 250Q and 428L modifications (e.g., T250Q and M428L); and / or

[0287] · 307 and / or 308 modifications (e.g., 308F or 308P).

[0288] For example, the present disclosure includes anti-TfR antibodies, the anti-TfR antibodies comprising an Fc domain, the Fc domain comprising one or more pairs or one or more groups of mutations selected from the group consisting of:

[0289] · 250Q and 248L (e.g., T250Q and M248L);

[0290] · 252Y, 254T and 256E (e.g., M252Y, S254T and T256E);

[0291] · 257I and 311I (e.g., P257I and Q311I);

[0292] · 257I and 434H (e.g., P257I and N434H);

[0293] · 376V and 434H (e.g., D376V and N434H);

[0294] · 307A, 380A and 434A (e.g., T307A, E380A and N434A);

[0295] · 428L and 434S (e.g., M428L and N434S); and

[0296] · 433K and 434F (e.g., H433K and N434F).

[0297] In yet another embodiment, the modification comprises a 265A (e.g., D265A) modification and / or a 297A (e.g., N297A) modification.

[0298] In one embodiment, the heavy chain constant domain is γ4 comprising an S228P and / or S108P mutation. See Angal et al., A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody, Molecular Immunology. January 1993; 30(1):105-108.

[0299] All possible combinations of the foregoing Fc domain mutations and other mutations within the antibody variable domains disclosed herein are encompassed within the scope of the present disclosure.

[0300] The anti-TfR antibodies described herein may comprise a modified Fc domain having reduced effector function. As used herein, "modified Fc domain having reduced effector function" means any Fc portion of an immunoglobulin that has been modified, mutated, truncated, etc. relative to the wild-type naturally occurring Fc domain such that a molecule comprising the modified Fc exhibits a reduction in the severity or degree of action of at least one selected from the group consisting of: cell killing (e.g., ADCC and / or CDC), complement activation, phagocytosis, and opsonization, relative to a comparative molecule of the wild-type naturally occurring form comprising the Fc portion. In certain embodiments, a "modified Fc domain having reduced effector function" is an Fc domain having reduced or diminished binding to an Fc receptor (e.g., FcγR).

[0301] In certain embodiments, the modified Fc domain is a variant IgG1 Fc or variant IgG4 Fc that contains a substitution in the hinge region. For example, the modified Fc for use in the context of the present disclosure may comprise a variant IgG1 Fc, wherein at least one amino acid of the IgG1 Fc hinge region is replaced with the corresponding amino acid from the IgG2 Fc hinge region. Alternatively, the modified Fc for use in the present disclosure may comprise a variant IgG4 Fc, wherein at least one amino acid of the IgG4 Fc hinge region is replaced with the corresponding amino acid from the IgG2 Fc hinge region. Non-limiting exemplary modified Fc regions that can be used in the context of the present disclosure are set forth in U.S. Patent Application Publication No. 2014 / 0243504, the disclosure of which is incorporated herein by reference in its entirety, and any functionally equivalent variants of the modified Fc regions set forth therein.

[0302] The present disclosure also includes antigen-binding proteins, antibodies, or antigen-binding fragments comprising an HCVR and a chimeric heavy chain constant (CH) region as set forth herein, wherein the chimeric CH region comprises segments derived from CH regions of more than one immunoglobulin isotype. For example, an antibody of the present disclosure may comprise a chimeric CH region that comprises a portion or all of the CH2 domain derived from a human IgG1, human IgG2, or human IgG4 molecule, combined with a portion or all of the CH3 domain derived from a human IgG1, human IgG2, or human IgG4 molecule. According to certain embodiments, an antibody of the present disclosure comprises a CH region having a chimeric hinge region. By way of example, the chimeric hinge may comprise the "upper hinge" amino acid sequence (amino acid residues at positions 216 to 227 according to EU numbering) from a human IgG1, human IgG2, or human IgG4 hinge region combined with the "lower hinge" sequence (amino acid residues at positions 228 to 236 according to EU numbering) from a human IgG1, human IgG2, or human IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from the upper hinge of human IgG1 or human IgG4 and amino acid residues derived from the lower hinge of human IgG2. Antibodies comprising a chimeric CH region as described herein may exhibit modified Fc effector functions in certain embodiments without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, e.g., WO2014 / 022540).

[0303] Other modified Fc domains and Fc modifications that can be used in the context of the present disclosure include any of the modifications shown in US2014 / 0171623; US 8,697,396; US2014 / 0134162; WO2014 / 043361, the disclosures of which are incorporated herein by reference in their entirety. Methods of constructing antibodies or other antigen-binding fusion proteins comprising a modified Fc domain as described herein are known in the art.

[0304] In some embodiments, the anti-TfR antibodies and antigen-binding fragments described herein comprise an Fc domain that contains one or more mutations in the CH2 and / or CH3 regions, which mutations create separate TfR-binding sites.

[0305] In one embodiment, the CH2 region comprises one or more amino acid mutations selected from the following, or a combination thereof: a) at position 47 is Glu, Gly, Gln, Ser, Ala, Asn, Tyr or Trp; at position 49 is Ile, Val, Asp, Glu, Thr, Ala or Tyr; at position 56 is Asp, Pro, Met, Leu, Ala, Asn or Phe; at position 58 is Arg, Ser, Ala or Gly; at position 59 is Tyr, Trp, Arg or Val; at position 60 is Glu; at position 61 is Trp or Tyr; at position 62 is Gln, Tyr, His, Ile, Phe, Val or Asp; and at position 63 is Leu, Trp, Arg, Asn, Tyr or Val; b) at position 39 is Pro, Phe, Ala, Met or Asp; at position 40 is Gln, Pro, Arg, Lys, Ala, Ile, Leu, Glu, Asp or Tyr; at position 41 is Thr, Ser, Gly, Met, Val, Phe, Trp or Leu; at position 42 is Pro, Val, Ala, Thr or Asp; at position 43 is Pro, Val or Phe; at position 44 is Trp, Gln, Thr or Glu; at position 68 is Glu, Val, Thr, Leu or Trp; at position 70 is Tyr, His, Val or Asp; at position 71 is Thr, His, Gln, Arg, Asn or Val; and at position 72 is Tyr, Asn, Asp, Ser or Pro; c) at position 41 is Val or Asp; at position 42 is Pro, Met or Asp; at position 43 is Pro or Trp; at position 44 is Arg, Trp, Glu or Thr; at position 45 is Met, Tyr or Trp; at position 65 is Leu or Trp; at position 66 is Thr, Val, Ile or Lys; at position 67 is Ser, Lys, Ala or Leu; at position 69 is His, Leu or Pro; and at position 73 is Val or Trp; or d) at position 45 is Trp, Val, Ile or Ala; at position 47 is Trp or Gly; at position 49 is Tyr, Arg or Glu; at position 95 is Ser, Arg or Gln; at position 97 is Val, Ser or Phe; at position 99 is Ile, Ser or Trp; at position 102 is Trp, Thr, Ser, Arg or Asp; at position 103 is Trp; and at position 104 is Ser, Lys, Arg or Val;wherein the substitutions and positions are determined with reference to amino acids 4-113 of the following sequence: PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:446).;

[0306] In one embodiment, the CH3 region comprises one or more amino acid mutations selected from the following or a combination thereof: position 153 is Trp, Leu or Glu; position 157 is Tyr or Phe; position 159 is Thr; position 160 is Glu; position 161 is Trp; position 162 is Ser, Ala, Val or Asn; position 163 is Ser or Asn; position 186 is Thr or Ser; position 188 is Glu or Ser; position 189 is Glu; and position 194 is Phe; or b) position 118 is Phe or Ile; position 119 is Asp, Glu, Gly, Ala or Lys; position 120 is Tyr, Met, Leu, Ile or Asp; position 122 is Thr or Ala; position 210 is Gly; position 211 is Phe; position 212 is His, Tyr, Ser or Phe; and position 213 is Asp; wherein the substitutions and positions are determined with reference to amino acids 114-220 of SEQ ID NO:446.

[0307] In some embodiments, the CH3 region comprises one or more mutations selected from the following, or combinations thereof: at position 384 is Leu, Tyr, Met or Val; at position 386 is Leu, Thr, His or Pro; at position 387 is Val, Pro or an acidic amino acid; at position 388 is Trp; at position 389 is Val, Ser or Ala; at position 413 is Glu, Ala, Ser, Leu, Thr or Pro; at position 416 is Thr or an acidic amino acid; and at position 421 is Trp, Tyr, His or Phe (according to EU numbering). In some embodiments, the CH3 region comprises one or more amino acid mutations selected from the following, or combinations thereof: at position 380 is Trp, Leu or Glu; at position 384 is Tyr or Phe; at position 386 is Thr; at position 387 is Glu; at position 388 is Trp; at position 389 is Ser, Ala, Val or Asn; at position 390 is Ser or Asn; at position 413 is Thr or Ser; at position 415 is Glu or Ser; at position 416 is Glu; and at position 421 is Phe.

[0308] In some embodiments, the CH3 region comprises one or more amino acid mutations selected from the following, or combinations thereof: a) Phe at position 382, Tyr at position 383, Asp at position 384, Asp at position 385, Ser at position 386, Lys at position 387, Leu at position 388, Thr at position 389, Pro at position 419, Arg at position 420, Gly at position 421, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, Leu at position 440, Gly at position 442, and Glu at position 443; b) Phe at position 382, Tyr at position 383, Gly at position 384, N at position 385, Ala at position 386, Lys at position 387, Thr at position 389, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, Leu at position 440; c) Phe at position 382, Tyr at position 383, Glu at position 384, Ala at position 385, Lys at position 387, Leu at position 388, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, Leu at position 440; d) Phe at position 382, Glu at position 384, Ser at position 386, Lys at position 387, Thr at position 389, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, Leu at position 440; e) Phe at position 382, Gly at position 384, Ala at position 385, Lys at position 387, Ser at position 389, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, Leu at position 440; f) Phe at position 382, Gly at position 384, Ala at position 385, Lys at position 387, Leu at position 388, Thr at position 389, Leu at position 422, Ala at position 424, Glu at position 426, Tyr at position 438, Leu at position 440; wherein the positions are determined according to EU numbering.

[0309] Non-natural TfR binding sites can be introduced into the CH2 and / or CH3 regions of the antigen-binding proteins described herein. Additional mutations include those described in US Patent Application Publication Nos. 2020 / 0223935, 2020 / 0369746, 2021 / 0130485, 2022 / 0017634; and PCT Application Publication Nos. WO2023 / 279099, WO2023 / 114499, and WO2023 / 114510, which patent documents are incorporated herein by reference in their entireties.

[0310] Use and manufacturing methods

[0311] Another embodiment of the modified viral capsid proteins described herein is their use for delivering a nucleotide of interest, such as a reporter gene or a therapeutic gene, to a target cell. TfR is widely expressed. Table 2 provides a non-limiting list of tissues and associated cells that can express TfR and can thus be targeted by the modified viral capsid proteins described herein for insertion of a nucleotide of interest (e.g., a reporter gene or a therapeutic gene).

[0312] Table 2

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321] Generally, the nucleotide of interest can be a transfer plasmid, which typically can contain 5' and 3' terminal inverted repeat (ITR) sequences flanking a reporter gene or a therapeutic gene (which, when contained within an AAV particle, can be under the control of a viral or non-viral promoter). In one embodiment, the nucleotide of interest is a transfer plasmid that includes, from 5' to 3', the following: 5' ITR, promoter, gene (e.g., a reporter gene and / or a therapeutic gene), and 3' ITR.

[0322] Non-limiting examples of suitable promoters include, for example, the cytomegalovirus (CMV) promoter, the chicken β-actin (CBA) promoter and its hybrid (CBh), the spleen focus-forming virus (SFFV) promoter, the elongation factor 1α (EF1a) promoter (the 1.2 kb EF1a promoter or the 0.2 kb EF1a promoter), the chimeric EF1a / IF4 promoter, the polyubiquitin C promoter (UbC), and the phosphoglycerate kinase (PGK) promoter. An internal enhancer may also be present in the viral construct to increase the expression of the gene of interest. For example, the CMV enhancer (Karasuyama et al. 1989. J. Exp. Med. 169:13, which is incorporated herein by reference in its entirety) can be used. In some embodiments, the CMV enhancer can be used in combination with the chicken β-actin promoter, for example, as a hybrid (CAG). Alternatively, the promoter can be a tissue-specific promoter, i.e., it is active in a specific tissue and / or organ. Tissue-specific promoters contain one or more tissue-specific promoter and / or enhancer elements, and optionally one or more constitutive promoter and / or enhancer elements, as described in US 2022 / 0204991, which is incorporated herein by reference in its entirety. Those skilled in the art will understand that tissue-specific promoter and / or enhancer elements can be isolated from genes specifically expressed in tissues by methods well known in the art.

[0323] A variety of reporter genes (or detectable moieties) can be encapsulated within a multimeric structure comprising the modified viral capsid proteins described herein. Exemplary reporter genes include, for example, β-galactosidase (the encoded lacZ gene), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), MmGFP, blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or combinations thereof. The methods described herein demonstrate the use of a reporter gene encoding green fluorescent protein to construct targeted particles; however, those skilled in the art will understand, upon reading this disclosure, that the viral particles described herein can be produced in the absence of a reporter gene or in the presence of any reporter gene known in the art.

[0324] It is also possible to encapsulate multiple therapeutic genes in a multimeric structure comprising modified viral capsid proteins as described herein, for example, as part of transfer particles. Non-limiting examples of therapeutic genes include those encoding toxins (e.g., suicide genes), therapeutic antibodies or fragments thereof, CRISPR / Cas systems or parts thereof, antisense oligonucleotides, antisense RNAs, siRNAs, shRNAs, etc. Tables 3 and 4 provide non-limiting lists of diseases and genes that can be the nucleotide of interest and / or whose reduction can be therapeutic and can be applicable for treatment using the viral particles described herein.

[0325] Table 3

[0326]

[0327]

[0328] Table 4

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350] As disclosed herein, the modified capsids disclosed herein can be transported across the blood-brain barrier and used to infect cells of the central nervous system. Thus, such modified capsids can be used to transport a nucleotide of interest across the blood-brain barrier for gene therapy of brain disorders, such as central nervous system (CNS) disorders, disorders with neurological symptoms, and the like. In such cases, expression of the therapeutic gene can be restricted to central nervous system cells (e.g., neurons) by operably linking the nucleotide of interest to a tissue-specific promoter. As non-limiting examples, neuron-specific promoters include, but are not limited to, Syn1, NSE, and MeCP2. Non-limiting examples of oligodendrocyte promoters include, but are not limited to, MBP and MAG. Non-limiting examples of microglia-specific promoters include, but are not limited to, CD68, HEXB, and F4 / 80. Non-limiting examples of astrocyte-specific promoters include, but are not limited to, GFAP and ALDH1L1. In some embodiments, the promoter is brain-specific (e.g., neuron-specific, glial cell-specific, astrocyte-specific, oligodendrocyte-specific, microglia-specific, and / or central nervous system-specific). Exemplary brain-specific promoters can comprise one or more elements from, but not limited to: the human glial fibrillary acidic protein (GFAP) promoter, the human synapsin 1 (SYN1) promoter, the human synapsin 2 (SYN2) promoter, the human metallothionein 3 (MT3) promoter, and / or the human proteolipid protein 1 (PLP1) promoter. Other examples of such brain-specific promoters include, but are not limited to, SCG10, the tubulin a1 promoter, the calcium / calmodulin-dependent protein kinase II (CaMKII) promoter, the neuron-specific enolase (NSE) promoter, the PDGF (platelet-derived growth factor β)-b chain promoter, and the like. More brain-specific promoter elements are disclosed in WO 2016 / 100575A1, which is incorporated herein by reference in its entirety.

[0351] In some embodiments, the brain-specific promoters as described herein are selected from the group consisting of: synapsin 1 promoter; calcium / calmodulin-dependent protein kinase II (CaMKII) promoter; tyrosine hydroxylase (TH) promoter; Forkhead Box A2 (FOXA2) promoter; alpha-internexin (INA) promoter; nestin (NES) promoter; glial fibrillary acidic protein (GFAP) promoter; aldehyde dehydrogenase 1 family member L1 (ALDH1L1) promoter; myelin-associated oligodendrocyte basic protein (MOBP) promoter; myelin basic protein (MBP) promoter.

[0352] In other embodiments, the promoter is a neuron-, astrocyte- or oligodendrocyte-specific promoter, or a neuron-, astrocyte- or oligodendrocyte-preferential promoter, such as synapsin, MeCP2, oligodendrocyte transcription factor 1 (Olig1), chondroitin sulfate proteoglycan (Cspg4) or CNP (2',3'-cyclic-nucleotide 3'-phosphodiesterase) promoter.

[0353] CNS disorders and disorders with neurological symptoms suitable for gene therapy include, but are not limited to: Alzheimer's disease, brain cancer, Behcet's Disease, lupus cerebri, Creutzfeldt-Jakob Disease, dementia, epilepsy, encephalitis, Friedreich's Ataxia, Guillain-Barre Syndrome, Gaucher's disease, headache, hydrocephalus, Huntington's disease, intracranial hypertension, leukodystrophy, migraine, myasthenia gravis, muscular dystrophy, multiple sclerosis, narcolepsy, neuropathy, Prader-Willi Syndrome, Parkinson's disease, Rett Syndrome, restless legs syndrome, sleep disorder, subarachnoid hemorrhage, stroke, traumatic brain injury, trigeminal neuralgia, transient ischemic attack and Von Hippel-Lindau Syndrome (angiomatosis).

[0354] In some embodiments, a viral capsid as described herein can encapsidate a therapeutic gene, the expression of which prevents, alleviates, or reduces one or more symptoms of an enzyme deficiency disease and / or a disease selected from the group consisting of: Fabry disease, Gaucher disease, MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIID, MPS IVB, MPS VI, MPS VII, MPS IX, Pompe disease, lysosomal acid lipase deficiency, metachromatic leukodystrophy, Niemann-Pick disease types A, B, and C2, alpha-mannosidosis, gangliosidosis sialidase deficiency, sialidosis, aspartylglucosaminuria, combined sphingolipid activator protein deficiency, classic Gaucher disease, Farber lipogranulomatosis, fucosidosis, and beta-mannosidosis.

[0355] "Enzyme deficiency disease" includes non-lysosomal storage diseases such as Krabbe disease (galactosylceramidase), phenylketonuria, galactosemia, maple syrup urine disease, mitochondrial disorders, Friedreich ataxia, Zellweger syndrome, adrenoleukodystrophy, Wilson disease, hemochromatosis, ornithine carbamoyltransferase deficiency, methylmalonic acidemia, propionic acidemia, and lysosomal storage diseases. "Lysosomal storage disease" includes any disorder caused by a defect in lysosomal function. Currently, approximately 50 lysosomal storage diseases have been identified, the most well-known of which include Tay-Sachs, Gaucher disease, and Niemann-Pick disease. The pathogenesis of the disease is attributed to the accumulation of incompletely degraded products in lysosomes, usually caused by a loss of protein function. Lysosomal storage diseases are caused by a loss or weakening variant in a protein that normally functions to degrade or coordinate the degradation of lysosomal contents. Proteins associated with lysosomal storage diseases include enzymes, receptors, and other transmembrane proteins (e.g., NPC1), post-translationally modified proteins (e.g., sulfatases), membrane transport proteins, and non-enzyme cofactors and other soluble proteins (e.g., GM2 ganglioside activator). Thus, lysosomal storage diseases encompass more than those disorders caused by the defective enzyme itself and also include any disorder caused by any molecular defect. Accordingly, as used herein, the term "enzyme" is intended to encompass other proteins associated with lysosomal storage diseases.

[0356] In many cases, the nature of the molecular lesion affects the severity of the disease, i.e., complete loss of function is often associated with prenatal or neonatal onset and involves severe symptoms; partial loss of function is associated with milder (relatively mild) and late-onset disease. In general, only a small percentage of activity needs to be restored to correct the metabolic defect in the defective cells. Lysosomal storage diseases are generally described in Desnick and Schuchman, 2012.

[0357] Lysosomal storage diseases are a group of rare diseases that affect the degradation of many substrates in lysosomes. These substrates include sphingolipids, mucopolysaccharides, glycoproteins, glycogen, and oligosaccharides, which can accumulate in the cells of affected individuals, leading to cell death. Organs affected by lysosomal storage diseases include the central nervous system (CNS), peripheral nervous system (PNS), lungs, liver, bone, skeletal and cardiac muscle, and the reticuloendothelial system.

[0358] Options for treating lysosomal storage diseases include enzyme replacement therapy (ERT), substrate reduction therapy, molecular chaperone-mediated therapy, hematopoietic stem cell transplantation therapy, and gene therapy. Examples of substrate reduction therapy include the use of Miglustat or Eliglustat to treat type 1 Gaucher disease. These drugs act by blocking synthase activity, thereby reducing the production of subsequent substrates. For example, the use of hematopoietic stem cell therapy (HSCT) improves and slows the negative central nervous system phenotype in patients with certain forms of MPS. See R.M. Boustany, “Lysosomal storage diseases--the horizon expands”, 9(10) Nat. Rev. Neurol. 583-98, October 2013, which is incorporated herein by reference in its entirety.

[0359] The two most common LSDs are Pompe disease and Fabry disease. The estimated incidence of Pompe disease is 1 / 10,000 and is caused by a defective lysosomal enzyme, α-glucosidase (GAA), resulting in insufficient processing of lysosomal glycogen. Accumulation of lysosomal glycogen occurs mainly in skeletal, cardiac, and liver tissues. Infantile-onset Pompe disease usually leads to cardiomegaly, hypotonia, hepatomegaly, and death due to cardiorespiratory failure before the age of 2. Adult-onset Pompe disease occurs as late as 20 to 60 years of age and usually affects only skeletal muscle. Currently available treatments include Genzyme's (alglucosidase alfa), which is a recombinant human α-glucosidase produced in CHO cells and administered by intravenous infusion.

[0360] Fabry disease includes mild late-onset cases, with an estimated overall incidence of 1 / 3,000, caused by a defective lysosomal enzyme, alpha-galactosidase A (GLA), resulting in the accumulation of globotriaosylceramide in blood vessels and other tissues and organs. Symptoms associated with Fabry disease include pain caused by nerve damage and / or small vessel obstruction; renal insufficiency and eventual failure; cardiac complications such as hypertension and cardiomyopathy; dermatological symptoms such as angiokeratoma formation, anhidrosis or hyperhidrosis; and ocular problems such as corneal vortex dystrophy, spokelike cataracts, and conjunctival and retinal vascular abnormalities. Currently available treatments include Genzyme's (agalsidase beta), a recombinant human alpha-galactosidase A produced in CHO cells and administered by intravenous infusion; Shire's REPLAGAL TM (agalsidase alpha), a recombinant human alpha-galactosidase A produced in human fibroblasts and administered by intravenous infusion; and Amicus's GALAFOLD TM (migalastat or 1-deoxygalactonojirimycin), an orally administered small molecule chaperone that converts the folding of aberrant alpha-galactosidase A into a functional conformation.

[0361] Another embodiment of the present invention is a method for preparing a modified capsid protein, the method comprising the steps of:

[0362] a) expressing a nucleic acid encoding the modified capsid protein under suitable conditions, and

[0363] b) isolating the expressed capsid protein of step a).

[0364] In some embodiments, the viral particles as described herein include chimeric capsids, e.g., capsids comprising a capsid protein that is genetically modified as described herein (in the absence or presence of a covalent bond with a targeting ligand) at a certain ratio with a reference capsid protein. A method for manufacturing such chimeric viral particles includes:

[0365] c) expressing a nucleic acid encoding the modified capsid protein and a nucleotide encoding the reference capsid protein at a ratio of at least about 60:1 to about 1:60, e.g., 2:1, 1:1, 3:5, 1:2, 1:3, etc. (wt / wt) under suitable conditions, and

[0366] d) isolating the expressed capsid protein of step a).

[0367] In some embodiments, the compositions described herein include the following, or the methods described herein combine the following: a modified cap gene: a reference cap gene (or a combination of reference cap genes) in a ratio in the range of at least about 1:60 to about 60:1, such as, for example, within the range of 2:1, 1:1, 3:5, 1:2, 1:3, etc. In some embodiments, the ratio is at least about 1:2. In some embodiments, the ratio is at least about 1:3. In some embodiments, the ratio is at least about 1:4. In some embodiments, the ratio is at least about 1:5. In some embodiments, the ratio is at least about 1:6. In some embodiments, the ratio is at least about 1:7. In some embodiments, the ratio is at least about 1:8. In some embodiments, the ratio is at least about 1:9. In some embodiments, the ratio is at least about 1:10. In some embodiments, the ratio is at least about 1:11. In some embodiments, the ratio is at least about 1:12. In some embodiments, the ratio is at least about 1:13. In some embodiments, the ratio is at least about 1:14. In some embodiments, the ratio is at least about 1:15. In some embodiments, the ratio is at least about 1:16. In some embodiments, the ratio is at least about 1:17. In some embodiments, the ratio is at least about 1:18. In some embodiments, the ratio is at least about 1:19. In some embodiments, the ratio is at least about 1:20. In some embodiments, the ratio is at least about 1:25. In some embodiments, the ratio is at least about 1:30. In some embodiments, the ratio is at least about 1:35. In some embodiments, the ratio is at least about 1:40. In some embodiments, the ratio is at least about 1:45. In some embodiments, the ratio is at least about 1:50. In some embodiments, the ratio is at least about 1:55. In some embodiments, the ratio is at least about 1:60. In some embodiments, the ratio is at least about 2:1. In some embodiments, the ratio is at least about 3:1. In some embodiments, the ratio is at least about 4:1. In some embodiments, the ratio is at least about 5:1. In some embodiments, the ratio is at least about 6:1. In some embodiments, the ratio is at least about 7:1. In some embodiments, the ratio is at least about 8:1. In some embodiments, the ratio is at least about 9:1. In some embodiments, the ratio is at least about 10:1. In some embodiments, the ratio is at least about 11:1. In some embodiments, the ratio is at least about 12:1. In some embodiments, the ratio is at least about 13:1. In some embodiments, the ratio is at least about 14:1.In some embodiments, the ratio is at least about 15:1. In some embodiments, the ratio is at least about 16:1. In some embodiments, the ratio is at least about 17:1. In some embodiments, the ratio is at least about 18:1. In some embodiments, the ratio is at least about 19:1. In some embodiments, the ratio is at least about 20:1. In some embodiments, the ratio is at least about 25:1. In some embodiments, the ratio is at least about 30:1. In some embodiments, the ratio is at least about 35:1. In some embodiments, the ratio is at least about 40:1. In some embodiments, the ratio is at least about 45:1. In some embodiments, the ratio is at least about 50:1. In some embodiments, the ratio is at least about 55:1. In some embodiments, the ratio is at least about 60:1.

[0368] In some embodiments, the ratio of VP protein subunits in the mosaic virus particles can, but need not necessarily, stoichiometrically reflect the ratio of the modified cap gene:reference cap gene. As a non-limiting exemplary embodiment, the ratio of modified capsid protein:reference capsid protein of the mosaic capsid formed according to the method can be considered, but not necessarily be, similar to the ratio (wt:wt) of the nucleic acids encoding them used to produce the mosaic capsid. In some embodiments, the mosaic capsid comprises a protein subunit ratio of from about 1:59 to about 59:1. In some embodiments, the mosaic capsid comprises a ratio of modified capsid protein:reference capsid protein of about 7:1.

[0369] Another embodiment of the invention is a method for altering the tropism of a virus, the method comprising the steps of: (a) inserting a nucleic acid encoding an amino acid sequence into a nucleic acid sequence encoding a viral capsid protein to form a nucleotide sequence encoding a genetically modified capsid protein comprising the amino acid sequence, and / or (b) culturing a packaging cell under conditions sufficient to produce virus particles, wherein the packaging cell comprises the nucleic acid. Another embodiment of the invention is a method for displaying a targeting ligand on the surface of a capsid protein, the method comprising the steps of: (a) expressing, under suitable conditions, a nucleic acid encoding a modified viral capsid protein as described herein (and optionally a nucleotide encoding a reference capsid protein), wherein the nucleic acid pair encodes a capsid protein that is the first member of a specific binding pair, (b) isolating the expressed capsid protein comprising the first member of the specific binding pair or the capsid comprising the expressed capsid protein of step (a), and (c) incubating the capsid protein or capsid with the second homologous member of the specific binding pair under conditions suitable to allow the formation of a isopeptide bond between the first member and the second member, wherein the second homologous member of the specific binding pair is fused to the targeting ligand.

[0370] In some embodiments, the packaging cells further comprise a helper plasmid and / or a transfer plasmid comprising the nucleotide of interest. In some embodiments, the method further comprises isolating self-complementary adeno-associated virus particles from the culture supernatant. In some embodiments, the method further comprises lysing the packaging cells and isolating single-stranded adeno-associated virus particles from the cell lysate. In some embodiments, the method further comprises (a) removing cell debris; (b) treating the supernatant containing the virus particles with a nuclease, such as DNase I and MgCl 2 treating the supernatant containing the virus particles; (c) concentrating the virus particles; (d) purifying the virus particles; and (e) any combination of (a)-(d).

[0371] Packaging cells useful for generating the virus particles described herein include, for example, animal cells that permit the virus or cells that have been modified to permit the virus; or packaging cell constructs, such as using a transfection agent such as calcium phosphate. Non-limiting examples of packaging cell lines useful for generating the virus particles described herein include, for example, human embryonic kidney 293 (HEK-293) cells (e.g., American Type Culture Collection [ATCC] No. CRL-1573), HEK-293 cells containing the SV40 large T-antigen (HEK-293T or 293T), HEK293T / 17 cells, human sarcoma cell line HT-1080 (CCL-121), lymphoblastoid cell line Raji (CCL-86), glioblastoma-astrocytoma epithelial cell line U87-MG (HTB-14), T-lymphoma cell line HuT78 (TIB-161), NIH / 3T3 cells, Chinese hamster ovary cells (CHO) (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), HeLa cells (e.g., ATCC No. CCL-2), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RATI cells, mouse L cells (ATCC No. CCLI.3), HLHepG2 cells, CAP cells, CAP-T cells, etc.

[0372] L929 cells, the FLY viral packaging cell system described in Cosset et al. (1995) Journal of Virology 69, 7430-7436, NS0 (murine myeloma) cells, human amniotic fluid cells (such as CAP, CAP-T), yeast cells (including but not limited to Saccharomyces cerevisiae, Pichia pastoris), plant cells (including but not limited to tobacco NT1, BY-2), insect cells (including but not limited to SF9, S2, SF21, Tni (such as High Five)) or bacterial cells (including but not limited to Escherichia coli).

[0373] For additional packaging cells and systems, packaging techniques and particles for packaging nucleic acid genomes into pseudotyped virus particles, see, for example, Polo et al., Proceedings of the National Academy of Sciences of the United States of America, (1999) 96:4598-4603. Packaging methods include using packaging cells that permanently express viral components, or transiently transfecting cells with plasmids.

[0374] Additional embodiments include methods for redirecting viruses to target cells and / or delivering reporter genes or therapeutic genes to the target cells, the methods including methods for transducing cells in vitro (e.g., ex vivo) or in vivo, the methods including the steps of: contacting the target cells with virus particles comprising a capsid described herein, wherein the capsid comprises a targeting ligand that specifically binds to a receptor expressed by the target cells. In some embodiments, the target cells are in vitro (e.g., ex vivo). In other embodiments, the target cells are in an individual, such as a human, in vivo.

[0375] Target cells

[0376] A variety of cells can be targeted to deliver nucleotides of interest using modified virus particles as disclosed herein. The target cells will generally be selected based on the nucleotide of interest and the desired effect.

[0377] In some embodiments, the nucleotide of interest can be delivered such that the target cells are capable of producing a protein that compensates for a defect in an organism, such as an enzyme defect or an immune defect, such as X-linked severe combined immunodeficiency. Thus, in some embodiments, cells that normally produce a protein in an animal are targeted. In other embodiments, cells in the region where the protein will be most beneficial are targeted.

[0378] In other embodiments, a nucleotide of interest, such as a gene encoding an siRNA, can inhibit the expression of a specific gene in a target cell. The nucleotide of interest can, for example, inhibit the expression of a gene involved in the life cycle of a pathogen. Thus, cells susceptible to or infected by a pathogen can be targeted. In other embodiments, the nucleotide of interest can inhibit the expression of a gene responsible for producing a toxin in a target cell.

[0379] In other embodiments, the nucleotide of interest can encode a cytotoxic protein that kills cells, and the nucleotide of interest is expressed in the cell. In this case, tumor cells or other unwanted cells can be targeted.

[0380] In other embodiments, the nucleotide of interest encodes a therapeutic protein. In some embodiments, the nucleotide of interest encodes a therapeutic protein that can be secreted from the transduced cell and can provide a therapeutic effect within the extracellular space surrounding the transduced cell or on neighboring cells. In some embodiments, the nucleotide of interest encodes a therapeutic protein that can provide a therapeutic effect to the transduced cell in an autonomous manner. In some embodiments, the nucleotide of interest encodes a therapeutic protein that can provide a therapeutic benefit to the transduced cell in an autonomous manner within the extracellular space surrounding the transduced cell and / or to neighboring cells of the transduced cell.

[0381] Once a specific target cell population in which expression of the nucleotide of interest is desired has been identified, a target receptor that is specifically expressed on the target cell population is selected. The target receptor can be expressed only on the cell population or to a greater extent on the cell population than on other cell populations. The more specific the expression, the more specific the delivery can be directed to the target cell. The required amount of specificity of the label (and thus gene delivery) can vary depending on the context. For example, for introducing a toxic gene, high specificity is most preferred to avoid killing non-target cells. For expressing a protein for harvest, or expressing a secreted product in which a global effect is desired, less label specificity may be required.

[0382] As discussed above, the target receptor can be any receptor for which a targeting ligand can be identified or generated. Preferably, the target receptor is a peptide or polypeptide, such as a receptor. However, in other embodiments, the target receptor can be a carbohydrate or other molecule that can be recognized by a binding conjugate. If a binding conjugate for the target receptor is known, such as a ligand, it can be used as an affinity molecule. However, if the binding molecule is unknown, antibodies to the target receptor can be generated using standard procedures. The antibody can then be used as a targeting ligand.

[0383] Thus, target cells can be selected based on a variety of factors, including, for example, (1) the application (e.g., therapy, expression of a protein to be harvested, and conferring disease resistance) and (2) the expression of a marker having the desired amount of specificity.

[0384] The target cells do not limit in any way and include germ cells and cell lines as well as somatic cells and cell lines. The target cells can be stem cells from any source. When the target cells are germ cells, the target cells are preferably selected from the group consisting of single-cell embryos and embryonic stem cells (ES).

[0385] Drug compositions, dosage forms, and administration

[0386] Additional embodiments provide a drug that includes at least one modified viral capsid protein according to the present invention and a suitable targeting ligand and / or nucleic acid according to the present invention. Preferably, such drugs can be used for gene transfer particles.

[0387] Also disclosed herein are drug compositions comprising the viral particles described herein and a pharmaceutically acceptable carrier and / or excipient. Additionally, drug dosage forms comprising the viral particles described herein are disclosed.

[0388] As discussed herein, the viral particles described herein can be used for various therapeutic applications (in vivo and ex vivo) and as research tools.

[0389] The viral particle-based drug compositions disclosed herein can be formulated in any conventional manner using one or more physiologically acceptable carriers and / or excipients. The viral particles can be formulated for administration by, for example, injection, inhalation, or isolation (through the mouth or nose), or by oral, buccal, parenteral, or rectal administration, or by direct administration to a tumor.

[0390] The drug compositions can be formulated for a variety of modes of administration, including systemic, topical, or localized administration. Techniques and formulations can be found, for example, in Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intraventricular, intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection purposes, the drug compositions can be formulated in a liquid solution, preferably in a physiologically compatible buffer, such as Hank's solution or Ringer's solution. Additionally, the drug compositions can be formulated in solid form and redissolved or suspended immediately before use. The lyophilized form of the drug compositions is also suitable.

[0391] For oral administration, the pharmaceutical composition can be in the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients, such as binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets can also be coated by methods well known in the art. Liquid preparations for oral administration can be in the form of, for example, solutions, syrups or suspensions, or they can be presented in dry product form, to be reconstituted with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives, such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or gum arabic); non-aqueous vehicles (e.g., oils, oily esters, ethanol or fractionated vegetable oils); and preservatives (e.g., methyl p-hydroxybenzoate or propyl p-hydroxybenzoate or sorbic acid). The preparations may also contain buffer salts, flavoring agents, coloring agents and sweetening agents, where appropriate.

[0392] The pharmaceutical composition can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form in, for example, ampoules or multi-dose containers, optionally with the addition of a preservative. The pharmaceutical composition can be further formulated as a suspension, solution or emulsion in an oily or aqueous vehicle, and can contain other agents, including suspending agents, stabilizers and / or dispersing agents.

[0393] In addition, the pharmaceutical composition can also be formulated as a depot preparation. These long-acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compound can be formulated with a suitable polymeric material or a hydrophobic material (e.g., in the form of an emulsion in an acceptable oil) or an ion exchange resin, or in the form of a sparingly soluble derivative, such as a sparingly soluble salt. Other suitable delivery systems include microspheres, which make it possible to locally and non-invasively deliver the drug over a long period of time. This technique can involve microspheres of pre-capillary size, which can be injected through a coronary catheter into any selected part of an organ without causing inflammation or ischemia. The therapeutic agent administered is slowly released from the microspheres and is taken up by the surrounding cells present in the selected tissue.

[0394] Systemic administration can also be effected by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the permeation barrier is used in the formulation. Such penetrants are generally known in the art and include, for example, bile salts and fusidic acid derivatives for transmucosal administration. In addition, detergents can be used to facilitate permeation. Transmucosal administration can be effected using a nasal spray or a suppository. For topical administration, the viral particles described herein can be formulated into ointments, salves, gels or creams generally known in the art. A wash solution can also be used topically to treat injuries or inflammation to accelerate healing.

[0395] Pharmaceutical forms suitable for injection use may include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for ready-to-use formulations of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid. It must be stable under the manufacturing conditions and certain storage parameters (such as refrigeration and freezing), and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0396] If the formulations disclosed herein are used as therapeutic agents to enhance the immune response of an individual, the therapeutic agent can be formulated into a composition in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed from the free amino groups of the protein), and are formed from inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed from free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0397] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycols, etc.), suitable mixtures thereof and vegetable oils. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion and by using surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents known in the art. In many cases, an isotonic agent, such as sugar or sodium chloride, will preferably be included. Prolonged absorption of injectable compositions can be achieved by using a delaying absorbent, such as aluminum monostearate and gelatin, in the composition.

[0398] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound or construct into a suitable solvent with the various other ingredients enumerated above, followed by filtration sterilization if desired.

[0399] After formulation, the solution can be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The formulations are readily administered in a variety of dosage forms, such as the types of injectable solutions described above, but can also be in the form of sustained release capsules or microparticles and microspheres, etc.

[0400] For example, for parenteral administration in an aqueous solution, the solution should be buffered appropriately if necessary, and the liquid diluent should first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intratumoral, intramuscular, subcutaneous, and intraperitoneal administration. In this context, the sterile aqueous media that can be employed will be known to those skilled in the art in view of the present disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or injected at the proposed infusion site.

[0401] The person responsible for administration will determine the appropriate dose for an individual in any case. For example, depending on the need or the condition of the individual exposed to a pathogenic organism or a disease (such as cancer), the viral particles described herein can be administered to the individual daily or weekly, or monthly, twice a year, or annually for a certain period of time.

[0402] In addition to compounds formulated for parenteral administration, such as intravenous, intratumoral, intradermal, or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration; liposomal formulations; timed-release capsules; biodegradable and any other currently used forms.

[0403] Intranasal or inhalable solutions, sprays, aerosols, or inhalants can also be used. An intranasal solution can be an aqueous solution designed to be administered to the nasal passages as drops or a spray. An intranasal solution can be prepared to be similar to nasal secretions in many respects. Thus, an aqueous intranasal solution is usually isotonic and slightly buffered to maintain a pH of 5.5 to 7.5. Additionally, antimicrobial preservatives (similar to those used in ophthalmic preparations) and appropriate pharmaceutical stabilizers can be included in the formulation if necessary. Various commercial intranasal preparations are known and can contain, for example, antibiotics and antihistamines and are used for the prevention of asthma.

[0404] Oral formulations can contain excipients such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions are in the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders. In certain defined embodiments, the oral pharmaceutical composition will contain an inert diluent or an absorbable edible carrier, or it can be enclosed in a hard or soft gelatin capsule, or it can be compressed into a tablet, or it can be directly incorporated into the diet. For oral therapeutic administration, the active compound can be combined with an excipient and used in the form of ingestible tablets, lozenges, dragees, capsules, elixirs, suspensions, syrups, powder tablets, etc.

[0405] Tablets, sugar-coated tablets, pills, capsules, etc. may also contain the following: binders such as tragacanth, gum arabic, corn starch or gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, alginic acid, etc.; lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose or saccharin may be added; or flavoring agents such as peppermint, wintergreen oil or cherry flavoring. When the unit dosage form is a capsule, in addition to the types of materials described above, it may contain a liquid carrier. Various other materials may be present in the form of coatings or used to otherwise condition the physical form of the dosage unit. For example, tablets, pills or capsules may be coated with shellac, sugar or both. The syrup of the elixir may contain the active compound, sucrose as a sweetener, methylparaben and propylparaben as preservatives, dyes, and flavoring agents such as cherry or orange flavoring.

[0406] Other embodiments disclosed herein may relate to kits for use with the methods and compositions. The kits may also comprise suitable containers such as vials, tubes, micro or microcentrifuge tubes, test tubes, flasks, bottles, syringes or other containers. When additional components or reagents are provided, the kits may contain one or more additional containers into which such reagent or component can be placed. The kits herein will generally also comprise means for containing the virus particles and any other sealed reagent containers for commercial sale. Such containers may comprise injection or blow-molded plastic containers in which the required vials may be retained. Optionally, the composition may require one or more additional active agents such as anti-inflammatory agents, antiviral agents, antifungal agents or antibacterial agents or anti-tumor agents.

[0407] The compositions disclosed herein may be administered by any means known in the art. For example, the composition may be administered to an individual intravenously, intratumorally, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, rectally, topically, intratumorally, intramuscularly, intrathecally, subcutaneously, subconjunctivally, intravesicularly, transmucosally, intrapericardially, intraumbilically, intraocularly, orally, topically, by inhalation, by injection, by infusion, by continuous infusion, by topical perfusion, by catheter, by lavage, in a cream or in a lipid composition.

[0408] Any method known to those skilled in the art can be used to generate the virus particles, packaging cells, and particle constructs described herein on a large scale. For example, master and working seed stocks can be prepared in qualified primary CEF under GMP conditions or by other methods. The packaging cells can be plated on flasks with a large surface area, grown to near confluence, and the virus particles purified. The cells can be harvested and the virus particles released into the culture medium, separated and purified, or the intracellular virus particles can be released by mechanical disruption (cell debris can be removed by depth filtration through large pores and the host cell DNA digested with endonucleases). The virus particles can then be purified and concentrated by tangential flow filtration followed by diafiltration. The resulting concentrated bulk can be formulated by dilution with a buffer containing stabilizers, filled into vials, and lyophilized. The compositions and formulations can be stored for subsequent use. For use, the lyophilized virus particles can be reconstituted by addition of a diluent.

[0409] Certain additional agents for combination therapy can be formulated and administered by any method known in the art.

[0410] The compositions disclosed herein can also contain adjuvants (such as aluminum salts and other mineral adjuvants), surfactants, bacterial derivatives, vehicles, and cytokines. The adjuvants can also have immunomodulatory antagonistic properties. For example, the adjuvants can stimulate Th1 or Th2 immunity. The compositions and methods disclosed herein can also contain adjunctive therapies.

[0411] Table 5

[0412]

[0413]

[0414] - Continued - Table 5

[0415]

[0416]

[0417]

[0418]

[0419]

[0420] - Continued - Table 5

[0421]

[0422]

[0423] Brief Description of the Sequences in the Sequence Listing, Table 6

[0424]

[0425]

[0426]

[0427]

[0428]

[0429] Examples

[0430] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[0431] Result

[0432] AAV particles can re-target mouse TfR (mTfR) in vitro and in vivo

[0433] As Figure 1 shown, AAV particles targeted to mouse TfR via an alternative anti-mTfR (8D3) antibody specifically infect multiple mTfR+ cell lines in vitro. In addition, as Figure 2 shown, AAV9 wild-type particles conjugated alone or with the anti-mTfR targeting antibody 8D3 transduce the livers of WT C57BL / 6J mice in vivo. AAV9 N272A or AAV9 W503A particles conjugated with the anti-mTfR targeting antibody 8D3 are off-target from the liver and do not promote high levels of liver eGFP expression. Brain sections of mice injected with AAV9 wild-type conjugated with an antibody that binds mTfR or off-target particles show enhanced eGFP staining in the brain compared to mice injected with AAV9 wild-type.

[0434] In addition, as Figure 3 shown, AAV particles conjugated with murine alternative mTfR-binding bivalent mAb, Fab, and scFv can mediate in vitro transduction in multiple mTfR+ cell lines. And as Figure 4 shown, AAV9 wild-type particles can transduce the livers of WT C57BL / 6J mice, while AAV9 W503A particles are re-targeted from the liver and do not promote high levels of liver eGFP expression. Brain sections of mice injected with AAV9 W503A particles conjugated with an antibody or its variant that binds mTfR show enhanced eGFP staining in the brain compared to mice injected with AAV9 wild-type. Increased eGFP staining was observed for AAV9 W503A particles conjugated with antibody Fab, scFV, or bivalent antibody (mAb).

[0435] In addition, as Figure 5AAs shown, higher levels of AAV DNA of AAV9 W503A particles conjugated with an antibody that binds to mTfR were detected in the brain compared to AAV9 wild-type or AAV9 W503A particles conjugated with an antibody that binds to hASGR1. This was observed in the brains of mice injected with AAV9 W503A particles conjugated with an antibody Fab, scFV, or bivalent antibody. In Figure 5B , high levels of AAV9 wild-type DNA were detected in the livers of WT C57BL / 6J mice, while lower levels of DNA were detected for AAV9 W503A particles that were off-target from the liver and conjugated with antibodies targeting mTfR or hASGR1 (as a non-targeting control) in various antibody formats. Additionally, brain sections from mice injected with wild-type AAV1, 8, and 9 particles conjugated with a Fab that binds to mTfR showed enhanced eGFP staining in the brain compared to mice injected with wild-type AAV serotypes 1, 8, and 9 ( Figure 6 ). Thus, it was observed that re-targeting using TfR improved CNS transduction regardless of the serotype used.

[0436] To determine the efficacy of AAV particles conjugated with anti-mTfR Fab at different doses, a dose escalation study was conducted. As Figure 19A shown, higher levels of AAV DNA of WT AAV9 particles conjugated with a Fab that binds to mTfR (“8D3”) or AAV9 W503A particles conjugated with a Fab that binds to mTfR (“8D3”) were detected in the brain compared to WT AAV9. Increased AAV DNA in the brain of AAV targeting mTfR was observed at all tested doses. In Figure 19B , high levels of WT AAV9 DNA were detected in the liver, while the DNA of AAV9 W503A particles conjugated with a Fab that binds to mTfR (“8D3”) was off-target from the liver and lower levels were detected. Decreased AAV DNA levels in the liver of AAV9 W503A targeting mTfR were observed at all tested doses. Thus, as measured by qPCR, at all tested doses, AAV re-targeting mTfR showed enhanced delivery of the AAV vector DNA to the brain and reduced delivery to the liver compared to WT AAV9. AAV particles targeting mTfR showed enhanced brain transduction in a dose-dependent manner compared to WT AAV9. Additionally, at all tested doses, WT AAV9 or AAV9 W503A re-targeting mTfR with Fab (“8D3”) resulted in higher brain transduction compared to WT AAV9 alone ( Figure 20A and Figure 20B)。Therefore, it was shown that WT AAV9 or AAV9 W503A that re-targets TfR achieved comparable CNS transduction to wild-type (WT) AAV9 at significantly lower doses.

[0437] AAV particles can re-target human TfR (hTfR) in vitro and in vivo

[0438] As Figure 7 shown, AAV particles targeted to human TFR via conjugation with anti-hTfR Fab specifically infect hTfR+ cell lines in vitro. Additionally, as Figure 8 shown, AAV9 wild-type particles can transduce TFRC hu / hu mice livers, while AAV9W503A particles are off-target from the liver and do not promote high levels of liver eGFP expression. Compared to wild-type AAV9 and AAV9W503A particles conjugated with an antibody Fab that binds hASGR1, AAV9 W503A particles conjugated with an antibody Fab that binds hTfR showed enhanced GFP staining in the brain. Additionally, compared to wild-type AAV9 and AAV9 W503A particles conjugated with a Fab that binds hASGR1, AAV9 W503A particles conjugated with a Fab that binds hTfR showed enhanced GFP staining in multiple regions of the brain ( Figure 9A-9D )。Additionally, as Figure 10A shown, higher levels of AAV DNA of AAV9 W503A particles conjugated with a Fab that binds hTfR were detected in the brain compared to AAV9 WT or AAV9 W503A particles conjugated with a Fab that binds hASGR1. This increase in AAV DNA was observed for AAV9 W503A particles conjugated with multiple anti-hTFR antibody Fabs. In Figure 10B it was detected high levels of AAV9 wild-type DNA in the livers of TFRC hu / hu mice, while the DNA of AAV9 W503A particles conjugated with a Fab that binds hTfR or hASGR1 was off-target from the liver and lower levels were detected. Therefore, as measured by qPCR, hTfR Fab re-targeted AAV showed enhanced delivery of AAV vector DNA to the brain and reduced delivery to the liver.

[0439] To further demonstrate the re-targeting of hTfR-conjugated AAV particles, female humanized TFRC mice (TFRC hu / hu) Administration of a unique barcoded pool of AAV particles. As expected, in the liver, single wild-type AAV9 represented the majority of all barcodes present in the tissue. In the brain and spinal cord, the off-target AAV9 (W503A) capsid conjugated to the TfR-targeting Fab represented the majority of all barcodes present in the tissue, outperforming AAV9 alone, which accounted for a small fraction of the total barcodes ( Figure 11A and Figure 11B ).

[0440] To further understand the tropism of AAV particles retargeted to hTfR, various brain cell types of TFRC hu / hu mice were examined. As Figure 12A and Figure 12B shown, AAV particles targeting human TfR exhibited transduction in a wide variety of brain cell types, including neuronal and glial cell populations. As Figure 12A shown, hTfR fab-retargeted AAVs (WT AAV9 and AAV9W503A) efficiently transduced multiple neuronal populations, including but not limited to cortical neurons and Purkinje cells. Additionally, retargeting of WTAAV9 and AAV9 W503A led to transduction of glial cells, including but not limited to astrocytes and oligodendrocytes. Transduction of some brain microvascular endothelial cells was also observed. As Figure 12B shown, hTfR fab-retargeted WT AAV9 strongly transduced neurons and astrocytes and, to a lesser extent, oligodendrocytes. Transduction of these various cell types was observed in many different brain regions, demonstrating that utilization of TfR retargeting facilitated gene transfer to multiple cell types in the CNS.

[0441] Next, a comparison of intravenous injection versus intracerebroventricular administration of AAV particles retargeted to hTfR was performed. After intravenous injection, the AAV9 W503A particle conjugated to the Fab binding hTfR showed enhanced eGFP staining in the brain compared to WT AAV9 and the AAV9 W503A particle conjugated to the Fab binding hASGR1. After intracerebroventricular injection, eGFP expression in the brain was comparable for targeted or non-targeted AAVs. Intravenous injection of AAV led to more widespread brain transduction than intracerebroventricular injection, and the AAV9 W503A particle conjugated to the Fab binding hTfR showed enhanced transduction in many brain regions ( Figure 13 ). As Figure 14AAs shown, after intravenous delivery, higher levels of AAV DNA of AAV9 W503A particles conjugated with an antibody that binds hTfR were detected in the brain compared to WT AAV9 or AAV9 W503A particles conjugated with an antibody that binds hASGR1. After intracerebroventricular injection, the levels of AAV DNA of targeted or non-targeted AAVs in the brain were low and comparable. In Figure 14B In, after intravenous delivery, high levels of WT AAV9 DNA were detected in the livers of TFRC hu / hu mice, while lower levels of DNA were detected for AAV9 W503A particles that were off-target from the liver and conjugated with Fabs that target hTfR or hASGR1 (as a non-targeted control). Higher levels of AAV DNA were detected in the liver after intravenous injection compared to intracerebroventricular injection of targeted or non-targeted AAVs. AAV particles targeting human TfR exhibited enhanced brain transduction after systemic intravenous (IV) delivery compared to intracerebroventricular (ICV) delivery.

[0442] To determine the efficacy of multiple different antibodies, several different anti-hTfR Fabs were conjugated to AAV particles and administered to TFRC hu / hu mice. Compared to WT AAV9 and AAV9 W503A particles conjugated with Fabs that bind hASGR1, AAV9 W503A particles conjugated with Fabs that bind hTfR showed enhanced eGFP staining in the brain ( Figure 15 ).

[0443] To explore the biodistribution of AAV particles conjugated with anti-hTfR Fabs, eGFP expression in multiple brain regions of TFRC hu / hu mice was examined. After systemic injection, hTfR Fab-retargeted AAVs showed enhanced delivery to many brain regions and enhanced brain transduction in all evaluated brain regions relative to WT AAV9. For AAV9 W503A particles conjugated with Fabs that bind hTfR, the cortex, hippocampus, and thalamus showed the highest levels of eGFP expression, while the hypothalamus consistently showed the lowest levels of eGFP expression ( Figure 16 and Figure 17 ). As an additional confirmation, AAV DNA in the brains ( hu / hu ) and livers ( Figure 18A ) of TFRC Figure 18B mice administered AAV particles conjugated with anti-hTfR Fabs was measured. As Figure 18AAs shown, higher levels of AAV DNA of AAV9 W503A particles conjugated with Fab that binds hTfR were detected in the brain compared to WT AAV9 or AAV9 W503A particles conjugated with Fab that binds hASGR1. This increase in AAV DNA was observed for AAV9 W503A particles conjugated with multiple anti-hTFR antibody Fabs. In Figure 18B , high levels of WT AAV9 DNA were detected in the liver of TFRC hu / hu mice, while the DNA of AAV9 W503A particles conjugated with Fab that binds hTfR or hASGR1 was off-target from the liver and lower levels were detected. Thus, as measured by qPCR, hTfR Fab-retargeted AAV showed enhanced delivery of AAV vector DNA to the brain and reduced delivery to the liver.

[0444] In addition, qPCR was used to evaluate the delivery of hTfR Fab-retargeted AAV to multiple tissues in TFRC hu / hu mice. As Figure 21A shown, high levels of AAV9 wild-type DNA were detected in the liver of female TFRC hu / hu mice, while lower levels of DNA were detected for AAV9 W503A particles that were off-target from the liver and conjugated with antibodies and Fabs that target hTfR or hASGR1 (as a non-targeting control). Thus, as measured by qPCR, W503A off-target hTfR Fab-retargeted AAV showed reduced delivery of AAV vector DNA to the liver. In Figure 21B , levels of AAV9 wild-type DNA detected in the heart of female TFRC hu / hu mice were similar to those of AAV9 W503A particles that were off-target from the liver and conjugated with antibodies and Fabs that target hTfR and higher than those of AAV9 W503A particles conjugated with antibodies that bind hASGR1. Thus, as measured by qPCR, W503A off-target hTfR Fab-retargeted AAV showed comparable delivery of AAV vector DNA to the heart. In Figure 21C , levels of AAV9 wild-type DNA detected in the quadriceps of female TFRC hu / hu mice were similar to higher than those of AAV9 W503A particles that were off-target from the liver and conjugated with antibodies and Fabs that target hTfR and higher than those of AAV9 W503A particles conjugated with antibodies that bind hASGR1. Thus, as measured by qPCR, W503A off-target hTfR Fab-retargeted AAV showed comparable delivery of AAV vector DNA to the quadriceps. In Figure 21DIn it, compared with AAV9 wild-type or AAV9 W503A particles conjugated with an antibody that binds to hASGR1, higher levels of AAV DNA of AAV9 W503A particles conjugated with an antibody or Fab that binds to hTfR were detected in the brain. Thus, as measured by qPCR, hTfR Fab re-targeted AAV with W503A off-target showed enhanced delivery of the AAV vector DNA to the brain.

[0445] In further experiments, delivery of AAV with anti-hTfR Fab to various tissues in TFRC hu / hu mice will be compared to delivery with the corresponding anti-hTfR mAb. As Figure 22A shown, groups of mice injected with off-target AAV9 W503A conjugated with H1H12845B Fab or H1H12845B mAb showed comparable eGFP expression in the brain, heart, quadriceps, and liver. Compared to wild-type AAV9, mice injected with off-target AAV9 W503A conjugated with H1H12845B Fab or H1H12845B mAb showed higher eGFP expression in the brain, lower eGFP expression in the heart and liver, and comparable eGFP expression in the quadriceps. Thus, hTfR Fab- and mAb-re-targeted AAV particles transduce the brain similarly. As Figure 22B shown, two groups of mice injected with off-target AAV9 W503A conjugated with H1H12845B Fab or H1H12845B mAb showed comparable eGFP expression in the cerebellum, hippocampus, cortex, and liver. Compared to wild-type AAV9, mice injected with off-target AAV9 W503A conjugated with H1H12845B Fab or H1H12845B mAb resulted in higher eGFP expression in the cerebellum, hippocampus, and cortex, and lower eGFP expression in the liver. Thus, hTfR Fab- and mAb-re-targeted AAV particles transduce the brain similarly. As Figure 23A shown, groups of mice injected with off-target AAV9 W503A conjugated with H1H12839B Fab or H1H12839B mAb showed comparable eGFP expression in the brain, heart, quadriceps, and liver. Compared to wild-type AAV9, mice injected with off-target AAV9 W503A conjugated with H1H12839B Fab or H1H12839B mAb showed higher eGFP expression in the brain, lower eGFP expression in the heart and liver, and comparable eGFP expression in the quadriceps. Thus, hTfR Fab- and mAb-re-targeted AAV particles transduce the brain similarly. And as Figure 23BAs shown, two groups of mice injected with off-target AAV9 W503A conjugated with H1H12839B Fab or H1H12839B mAb showed comparable eGFP expression in the cerebellum, hippocampus, cortex, and liver. Compared with wild-type AAV9, mice injected with off-target AAV9 W503A conjugated with H1H12839B Fab or H1H12839B mAb had higher eGFP expression in the cerebellum, hippocampus, and cortex and lower eGFP expression in the liver. Thus, hTfR Fab- and mAb-retargeted AAV particles transduce the brain similarly.

[0446] AAV particles can retarget the murine TfR (mTfR) in neonates

[0447] Finally, delivery of TfR-retargeted AAV to neonates was evaluated using immunofluorescence imaging. As Figure 24 shown, as expected, both WT AAV9 and the modified form (AAV.PHP.eB) had robust spinal cord and motor neuron transduction. WT AAV9 and the W503A capsid variant conjugated with the TfR antibody both retained the ability to transduce motor neurons, indicating that TfR antibody conjugation does not disrupt the tropism of these viruses for neonatal motor neurons. Although un-targeted WT AAV9 and AAV.PHP.eB showed stronger GFP signals in motor neurons compared with TfR-targeted AAV9, quantification of the number of transduced motor neurons showed that the transduction efficiency of all viruses was approximately 90%, indicating that, despite the lower total GFP signal per cell, the retargeted viruses achieved high transduction efficiency. Thus, TfR-targeted AAV particles transduce neonatal spinal cord motor neurons when administered directly into the CNS. As Figure 25 shown, neonatal mice injected intravenously (i.v.) with retargeted AAV9 demonstrated highly robust and reproducible lumbar spinal cord motor neuron transduction compared with PBS negative control mice injected i.v. Thus, TfR-targeted AAV particles highly transduce neonatal spinal cord motor neurons when administered intravenously.

[0448] TfR-targeted AAV particles can deliver functional cargo to the CNS, including nucleic acid sequences encoding therapeutic antibodies and shRNAs for target gene knockdown.

[0449] The ability of retargeted TfR AAV to mediate therapeutic payload expression in the brain via systemic delivery was evaluated. To evaluate the delivery of vectorized antibodies to the brain using AAV retargeted to mTfR, AAV9W503A conjugated with an scFv targeting mTfR was compared with AAV8, which was not expected to cross the blood-brain barrier and transduce cells in the CNS. AAV particles targeting TfR were packaged with a transgene containing the ubiquitously expressed CAGG promoter and the gene for the target sequence of a human IgG antibody targeting the type III secretion system (PcrV) of Pseudomonas aeruginosa. For each test group, the heavy and light chain sequences were separated by different 2A motifs (P2A, T2A, or F2A), which are crucial for creating steric hindrance and ribosomal skipping to allow the production of two polypeptides from a single AAV-derived mRNA molecule. As Figure 26A shown, robust RNA expression of the human IgG antibody sequence was observed in the brains and spinal cords of animals treated with AAV targeting TfR. Thus, AAV targeting mouse TfR promotes the expression of secreted antibodies in the CNS following intravenous delivery. Higher levels of AAV RNA of AAV9 W503A particles conjugated with an scFv (“8D3”) that binds mTfR were detected in the brain and spinal cord compared to AAV8. This increase in AAV RNA in the CNS of AAV targeting mTfR was observed in all tested 2A sequence variant transgenes. In animals injected with AAV9 W503A particles conjugated with an scFv (“8D3”) that binds mTfR, the levels of AAV RNA in non-CNS tissues were extremely low. Additionally, Figure 26B it was shown that the concentration of human antibody protein in the brain lysates of all animals treated with AAV targeting TfR was higher than that of animals injected with AAV8. Thus, AAV9 W503A retargeted to mTfR promotes the delivery and expression of vectorized antibody sequences to the brain via intravenous injection. Thus, AAV targeting mouse TfR promotes the expression of secreted antibodies in the CNS following intravenous delivery.

[0450] Additionally, the ability of AAV retargeted to TfR1 to mediate shRNA expression and induce target mRNA downregulation in the brain via systemic delivery was evaluated. AAV particles targeting TfR1 were packaged with a transgene driving the expression of shRNA against SNCA. Two SNCA shRNA sequences (SNCA shRNA#1 and SNCA shRNA#2) were tested. As Figure 27As shown, compared with the initial SNCA humanized mice and SNCA humanized mice injected with off-target AAV9 W503A conjugated with mTfR1-Fab and expressing control shRNA, SNCA humanized mice injected with off-target AAV9 W503A conjugated with mTfR1-Fab and expressing SNCA shRNA#1 or SNCA shRNA#2 showed a 40% to 50% reduction in human SNCA mRNA levels in the cortex, midbrain, and striatum. Thus, TfR1-AAV allows for the delivery and functional expression of shRNA and efficiently induces the downregulation of target mRNA in multiple brain regions. Therefore, the expression of SNCA shRNA from AAV targeting mouse TfR reduces SNCA mRNA levels in the CNS after intravenous delivery.

[0451] Materials and Methods

[0452] Preparation of AAV viral vectors

[0453] Viruses were produced by transfecting 293T packaging cells with the following plasmids using PEIPro: pAd helper plasmid, genomic plasmid containing AAV2 ITR encoding a reporter protein, and pAAV-CAP plasmid encoding AAV Rep and CAP genes, with or without an additional plasmid encoding the heavy and light chains of an antibody, Fab, or scFv. The antibody or Fab heavy chain construct or scFv was all fused to SpyCatcher at its C-terminus. Transfection complexes were prepared in incomplete DMEM (without additional supplements) and incubated at room temperature for 10 minutes.

[0454] Each virus was produced by transfecting 15 cm plates of 293T packaging cells with the following plasmids and amounts:

[0455] WT AAV9 / N272A / W503A GFP

[0456] pAd helper plasmid

[0457] 16 ug

[0458] pscAAV-CBh-eGFP 8 ug

[0459] pAAV9-CAP wt or pAAV9 N272A or pAAV9 W503A 8 ug

[0460] WT AAV1 / AAV8 GFP

[0461] pAd helper plasmid

[0462] 16 ug

[0463] pscAAV-CBh-eGFP 8 ug

[0464] pAAV1-CAP wt or pAAV8 CAP wt

[0465] 8 ug

[0466] AAV9 wt anti-human ASGR1 or anti-mTfR mAb GFP

[0467]

[0468] AAV9 W503A anti-human ASGR1 or anti-mTfR mAb GFP

[0469]

[0470] AAV9 N272A anti-human ASGR1 or anti-mTfR mAb GFP

[0471]

[0472] AAV9 W503A anti-human ASGR1 or anti-mTfR 8D3 or anti-hTfR Fab GFP

[0473]

[0474]

[0475] AAV9 W503A anti-mTfR 8D3 scfv GFP

[0476]

[0477] AAV1 anti-mTfR 8D3 Fab GFP

[0478]

[0479] 3 ug of light chain plasmid

[0480]

[0481] AAV9W503A anti-mTfR 8D3scFv anti-PcrVhIgG

[0482]

[0483]

[0484] After incubation, add the complex to DMEM supplemented with 10% FBS, 1X NEAA, 1% Pen / Strep and 1% L-glutamine.

[0485] The transfected packaging cells were incubated at 37 °C for 3 days, and then the virus was collected from the cell lysate using a standard freeze-thaw protocol. Briefly, the packaging cells were detached by scraping and granulated. The supernatant was removed, and the cells were resuspended in a solution of 50 mM Tris-HCl; 150 mM NaCl; and 2 mM MgCl2 [pH 8.0]. Intracellular viral particles were released by inducing cell lysis via three consecutive freeze-thaw cycles, which consisted of shuttling the cell suspension between a dry ice / ethanol bath and a 37 °C water bath under vigorous vortexing. The viscosity was reduced by treating the lysate with EMD Millipore Benzonase (50 U / ml cell lysate) at 37 °C for 90 minutes with occasional mixing. The debris was then pelleted by centrifugation, and the resulting supernatant was filtered through a 0.22 μm PVDF Millex-GV filter. For the crude virus to be tested in vitro, the crude virus was aliquoted into low protein-binding tubes and stored at 4 °C. For the virus to be tested in vivo, the clarified lysate was further purified using a four-step iodixanol density gradient. The gradient was loaded into a Beckman 70Ti rotor and spun at 66,100 rpm for 1.5 h at 10 °C using maximum acceleration and deceleration. After ultracentrifugation, the iodixanol-purified viral particles were extracted from the 40%-60% interface. The iodixanol solution containing AAV was diluted in DPBS+ / +.001% pluronic F68 such that the concentration of iodixanol was less than 1%. The purified virus was then concentrated to the desired volume using a 100 kDa MWCO Amicon ultrafiltration device.

[0486] The titer (viral genomes per milliliter; vg / mL) was determined by qPCR using a standard curve of virus at a known concentration.

[0487] Cell lines:

[0488] All 293 cell lines were maintained in DMEM supplemented with 10% FBS, 1X NEAA, 1% Pen / Strep, and 1% L-glutamine. The 293hASGR1 / 2 and 293hTfR cell lines were generated by lentiviral transduction of the parental 293 cell line with a vector expressing the corresponding cDNA. All cell lines were obtained from the Regeneron TC Core Facility.

[0489] All 3T3 cell lines were maintained in DMEM supplemented with 10% BCS, 1X NEAA, 1% Pen / Strep, and 1% L-glutamine. The 3T3 hTfR cell line was generated by lentiviral transduction of the parental 3T3 cell line with a vector expressing the corresponding cDNA. All cell lines were obtained from the Regeneron TC Core Facility.

[0490] The b.End3 cell line was maintained in DMEM supplemented with 10% FBS, 1X NEAA, 1% Pen / Strep, and 1% L-glutamine. The cell line was obtained from the Regeneron TC Core Facility.

[0491] AAV capsid protein construct

[0492] Gene blocks encoding the desired SpyTag insertions, flanking linker amino acids, and additional mutations were purchased from IDT and cloned into the correspondingly digested pAAV-CAP wt plasmid using Gibson assembly according to the manufacturer's protocol (NEB).

[0493] Cell infection / transduction and flow cytometry analysis.

[0494] To infect cells, viral particles were directly added to the medium of cultured cells, and the mixture was incubated at 37°C. Four days post-infection, the cells were trypsinized, resuspended in PBS containing 2% FBS, and the percentage of GFP+ cells was collected on a BD FACSCanto flow cytometer and analyzed using FlowJo software.

[0495] Mouse strains

[0496] Humanized TFRC mice (TFRC hu / hu ) express human TfR1 and do not express endogenous TfR1.

[0497] In vivo analysis of AAV9 GFP vector

[0498] For intravenous (IV) injection: Adult (3 - 4 months old) WT C57BL / 6J mice were injected via the tail vein with doses of 7.5x10 9 , 1.6x10 10 , 5x10 10 , 8x10 10 , 4x10 11 or 2x10 12 vg / mouse of AAV particles. Adult (3 - 4 months old) female TFRC hu / hu mice were injected via the tail vein with 1.5x10 10 , 1x10 11 or 4x10 11 vg / mouse of AAV particles. For intracerebroventricular (ICV) injection: Adult (3 - 4 months old) humanized TFRC (TFRC hu / hu ) mice underwent stereotaxic surgery and were injected into the lateral ventricle with 1x10 10AAV particles in vg / mice. Mice were sacrificed 2 - 3 weeks after injection and perfused with saline. Brains and livers were harvested for immunohistochemistry, immunofluorescence, and qPCR analysis.

[0499] Barcode analysis

[0500] Female humanized TFRC mice (TFRC hu / hu )(7 - 9 weeks old) were injected retroorbitally (RO) with 1x10 11 vg / mouse or 5x10 11 vg / mouse of AAV9 (WT AAV9, AAV PHP.eB, AAV9 W503A anti - ASGR1 Fab, and 32AAV9 W503A anti - TfR Fab with barcode - tagged pITR - sc - CBh - eGFP - bGHpA plasmid as the viral genome plasmid). Each of the 36 viruses in the pool was packaged with a pITR - sc - CBh - eGFP - bGHpA form carrying a unique 32 - nucleotide - length barcode, which was used to quantify the transgene expression of that capsid variant. Mice were sacrificed 14 days after injection, and the following organs were harvested for RNA extraction: liver, brain, and spinal cord.

[0501] The AAV viral vector was prepared as described above, with one modification: after cell lysis and before centrifuging to pellet debris, soluble Spytag peptide (AHIVMVDAYKPTK; SEQ ID NO:321) was added to the lysed cell suspension to a concentration of 100 μg / mL. The lysed cell suspension and soluble Spytag peptide were incubated at 37 °C for 60 minutes. Before centrifugation, the samples were mixed in equal volumes.

[0502] To analyze the barcode, total RNA isolated from MAID7229 mouse tissues and organs was purified using MagMAX-96 for Microarrays Total RNA isolation kit according to the manufacturer's instructions. The RNA was then treated with ezDNAse and cDNA synthesis was performed using SuperScript IV reverse transcriptase and bGH pA specific primer (5’-ATCCTCCCCCTTGCTGTCCTGC-3'; SEQ ID NO:443). The barcoded GFP transcript was amplified from the cDNA sample using Q5 Ultra II 2x master mix with the upstream (5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGgcactgacaattccgtggtctagg-3’; SEQ ID NO:444) and downstream (5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGcaaacaacagatggctggcaactag-3’; SEQ ID NO:445) primers that bind the barcode. The pooled viral mixture was included in the samples. During library preparation, each sample was prepared in triplicate technical replicates. The amplicons containing Illumina adapters and unique dual indices (UDI-Illumina) were quantified using qubit and Tapestation, pooled at equimolar ratio, and sequenced on Nextseq550 using the 300-cycle mid-output kit.

[0503] Tropism study

[0504] Adult (3 - 4 months old) humanized TFRC mice (TFRC hu / hu ) mice were injected via the tail vein with 1x10 11 vg / mouse of AAV particles. Mice were sacrificed 19 days after injection, perfused with phosphate-buffered saline and the brains were harvested for histological analysis.

[0505] For immunofluorescent staining and evaluation of CNS cell type tropism, the brain was cut in half along the mid-sagittal plane, and one half of the brain was fixed in 10% neutral buffered formalin and then transferred to 70% ethanol after 24 hours. The brain was stored in 70% ethanol for no more than one week and then paraffin-embedded. The brain was embedded in paraffin blocks, sectioned into 4-μm thick slices, and mounted on TOMO slides. The slides were deparaffinized at 60°C for 1 hour by washing in xylene 3 x 10 minutes, followed by washing in the following series of decreasing ethanol concentrations for 3 minutes each: 100%, 100%, 95%,...

Claims

1. A recombinant viral capsid protein, the recombinant viral capsid protein comprising: (i) The first member of a protein:protein binding pair inserted and / or displayed through the viral capsid; (ii) The second member of the protein:protein binding pair, wherein the first member of the protein:protein binding pair and the second member of the protein:protein binding pair associate; and (iii) An antibody or a binding portion thereof that binds to the extracellular domain of the transferrin receptor protein 1 (abbreviated as TfR1, TfR or CD71), wherein the antibody or the binding portion thereof is fused to the second member of the protein:protein binding pair.

2. The recombinant viral capsid protein according to claim 1, wherein the extracellular domain of TfR1 comprises the amino acid sequence shown in SEQ ID NO:

436.

3. The recombinant viral capsid protein according to claim 1 or claim 2, wherein the extracellular domain of TfR1 is the extracellular domain of human (h) TfR1.

4. The recombinant viral capsid protein according to any one of claims 1-3, the recombinant viral capsid protein further comprising a cell expressing TfR1 on its surface, wherein the viral capsid binds to the extracellular domain of TfR1 expressed on the surface of the cell.

5. The recombinant viral capsid protein according to any one of claim 4, wherein the cell is listed in Table 2.

6. The recombinant viral capsid protein according to any one of claims 1-5, the recombinant viral capsid protein further comprising a blood-brain barrier endothelial cell expressing TfR1 on its surface, wherein the viral capsid binds to the extracellular domain of TfR1 expressed on the surface of the blood-brain barrier endothelial cell.

7. The recombinant viral capsid protein according to any one of claims 1-6, wherein the recombinant viral particle or composition is in vitro.

8. The recombinant viral capsid protein according to any one of claims 1-6, wherein the recombinant viral particle or composition is in vivo.

9. The recombinant viral capsid protein according to any one of claims 1-8, wherein: (a) The first member of the protein:protein binding pair comprises SpyTag, Isopeptag, SnoopTag, SpyTag002, SpyTag003 or a variant thereof; (b) The second member of the protein:protein binding pair comprises Spycatcher, KTag, pilin-C, SnoopCatcher, SpyCatcher002, SpyTag003 or a variant thereof fused to the targeting ligand; and (c) The first member of the protein:protein binding pair and the second member of the protein:protein binding pair associate through an isopeptide bond.

10. The recombinant viral capsid protein according to any one of claims 1-9, wherein: (a) The first member of the protein:protein binding pair comprises SpyTag or a variant thereof; and (b) The protein: The second member of the protein:protein binding pair comprises SpyCatcher or a variant thereof fused to the targeting ligand.

11. The recombinant viral capsid protein according to any one of claims 1-8, wherein: (a) The protein: The first member of the protein:protein binding pair comprises the c-myc amino acid sequence shown in SEQ ID NO:326; and (b) The protein: The second member of the protein:protein binding pair comprises a bispecific binding protein comprising an anti-c-myc antibody and the targeting ligand.

12. The recombinant viral capsid protein according to any one of claims 1-11, the recombinant viral capsid protein comprising a first and / or second linker operably linking the first member of the protein:protein binding pair to the recombinant viral capsid protein, wherein each of the first and second linkers independently has a length of at least one amino acid.

13. The recombinant viral capsid protein according to claim 12, wherein the first and second linkers are not the same.

14. The recombinant viral capsid protein according to claim 12, wherein the first and second linkers are the same.

15. The recombinant viral capsid protein according to any one of claims 12-14, wherein the first linker has a length of 10 amino acids and / or the second linker has a length of 10 amino acids, optionally wherein the amino acid sequence of the first linker and / or the amino acid sequence of the second linker comprises the amino acid sequence shown in SEQ ID NO:331 or SEQ ID NO:

332.

16. The recombinant viral capsid protein according to any one of claims 1-15, wherein: (a) The viral capsid protein comprises the amino acid sequence of a modified VP1 capsid protein, a modified VP2 capsid protein, and / or a modified VP3 capsid protein encoded by a mutated cap gene; and (b) The mutated cap gene or a portion thereof comprises a nucleotide sequence that is at least 90% identical to the cap gene of AAV or a portion thereof, wherein the mutated cap gene or a portion thereof is genetically modified to comprise an insertion of a nucleotide sequence encoding the first member of the protein:protein binding pair such that the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein comprises the first member of the protein:protein binding pair.

17. The recombinant viral capsid protein according to any one of claims 1-16, wherein the mutated cap gene or a portion thereof is genetically modified to comprise one or more additional mutations such that, in addition to the first member of the protein:protein binding pair, the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein comprises: (i) Point mutations containing amino acid substitutions, insertions, or deletions; (ii) Chimeric amino acid sequences; or (iii) Both point mutations and chimeric amino acid sequences.

18. The recombinant viral capsid protein according to claim 17, wherein the substitution, insertion or deletion of the amino acid reduces the natural tropism of the viral particle comprising the recombinant viral capsid protein and / or generates a detectable label.

19. The recombinant viral capsid protein according to any one of claims 1-18, wherein: (a) the viral capsid protein comprises the amino acid sequence of a modified VP1 capsid protein, a modified VP2 capsid protein and / or a modified VP3 capsid protein encoded by a mutated cap gene; and (b) the mutated cap gene or a portion thereof comprises a nucleotide sequence that is at least 90% identical to the cap gene of AAV or a portion thereof, wherein the mutated cap gene or a portion thereof is genetically modified to comprise the insertion of a nucleotide sequence encoding the first member of the protein:protein binding pair, such that the modified VP1 capsid protein, the modified VP2 capsid protein and / or the modified VP3 capsid protein comprise the first member of the protein:protein binding pair; and (c) the AAV is selected from the group consisting of AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh10, AAV rh32.33, non-primate AAV, and combinations thereof listed in Table 5.

20. The recombinant viral capsid protein according to any one of claims 16-19, wherein the AAV is AAV2.

21. The recombinant viral capsid protein according to any one of claims 16-20, wherein the viral capsid protein comprises a modified AAV2 VP1 capsid protein, and the modified AAV2 VP1 capsid protein comprises the first member of the protein:protein binding pair optionally linked via a linker to an amino acid at position I453 and / or I587.

22. The recombinant viral capsid protein according to any one of claims 16-21, wherein the viral capsid protein comprises a modified AAV2 VP1 capsid protein, and the modified AAV2 VP1 capsid protein comprises the first member of the protein:protein binding pair displayed at position G453 via a linker, optionally wherein the modified AAV2 VP1 capsid protein further comprises an R585A modification, an R588A modification, or both an R585A modification and an R588A modification, and optionally wherein the modified AAV2 VP1 capsid protein further comprises an R484A modification, an R487A modification, an R585A modification, an R588A modification, and a K532A modification, or any combination of an R484A modification, an R487A modification, an R585A modification, an R588A modification, and a K532A modification.

23. The recombinant viral capsid protein according to any one of claims 16-19, wherein the AAV is AAV9.

24. The recombinant viral capsid protein according to any one of claims 16-19 and 23, wherein the viral capsid protein comprises a modified AAV9 VP1 capsid protein, and the modified AAV9 VP1 capsid protein comprises the first member of the protein:protein binding pair optionally linked via a linker to an amino acid at position I453, I587 or I589.

25. The recombinant viral capsid protein according to any one of claims 16-19 and 23-24, wherein the viral capsid protein comprises a modified AAV9 VP1 capsid protein, and the modified AAV9 VP1 capsid protein comprises the first member of the protein:protein binding pair displayed at G453 via a linker. Optionally, the modified AAV9 VP1 capsid protein further comprises an N272A modification, a W503A modification, or both an N272A modification and a W503A modification.

26. The recombinant viral capsid protein according to any one of claims 16-19, wherein the non-primate AAV is avian AAV (AAAV), non-human mammalian AAV or squamate AAV.

27. The recombinant viral capsid protein according to any one of claims 16-19 and 26, wherein the non-primate AAV is AAV.

28. The recombinant viral capsid protein according to any one of claims 16-19 and 26-27, wherein the viral capsid protein comprises a modified AAV VP1 capsid protein, and the modified AAV VP1 capsid protein comprises the first member of the protein:protein binding pair optionally linked via a linker to an amino acid at position I444 or I580.

29. The recombinant viral capsid protein according to any one of claims 16-19 and 26-28, wherein the viral capsid protein comprises a modified AAV VP1 capsid protein, and the modified AAV VP1 capsid protein comprises the first member of the protein:protein binding pair optionally linked via a linker to an amino acid at a position selected from the group consisting of I429, I430, I431, I432, I433, I434, I436, I437 and I565.

30. The recombinant viral capsid protein according to any one of claims 16-19 and 26, wherein the non-primate AAV is squamate AAV.

31. The recombinant viral capsid protein according to any one of claims 16-19, 26 and 30, wherein the non-primate AAV is bearded dragon AAV.

32. The recombinant viral capsid protein according to any one of claims 16-19, 26 and 30-31, wherein the viral capsid protein comprises a modified bearded dragon VP1 capsid protein, and the modified bearded dragon VP1 capsid protein comprises the first member of the protein:protein binding pair optionally linked via a linker to an amino acid at position I573 or I436.

33. The recombinant viral capsid protein according to any one of claims 16-19 and 26, wherein the non-primate AAV is a non-human mammalian AAV.

34. The recombinant viral capsid protein according to any one of claims 16-19, 26 and 33, wherein the non-primate AAV is a sea lion AAV.

35. The recombinant viral capsid protein according to any one of claims 1-34, wherein the antibody or its binding portion that binds to the extracellular domain of TfR1 binds to the same epitope on the extracellular domain of TfR1 as the reference antibody comprising the HCVR / LCVR amino acid sequence pair shown in Table 1.

36. The recombinant viral capsid protein according to any one of claims 1-35, wherein the antibody or its binding portion that binds to the extracellular domain of TfR1 comprises heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) of a heavy chain variable region (HCVR) containing the amino acid sequence shown in SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, 112, 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, 262, 272, 282, 292, 302, or 312 (or a variant thereof); and / or light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) of a light chain variable region (LCVR) containing the amino acid sequence shown in SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, 217, 227, 237, 247, 257, 267, 277, 287, 297, 307, or 317 (or a variant thereof).

37. The recombinant viral capsid protein according to any one of claims 1-36, wherein the antibody or its binding portion that binds to the extracellular domain of TfR1 comprises: (i) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 2 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 7 (or a variant thereof); (ii) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 12 (or a variant thereof); and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 17 (or a variant thereof); (iii) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:22 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:27 (or a variant thereof); (iv) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:32 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:37 (or a variant thereof); (v) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:42 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:47 (or a variant thereof); (vi) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:52 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:57 (or a variant thereof); (vii) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:62 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:67 (or a variant thereof); (viii) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:72 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:77 (or a variant thereof); (ix) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:82 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:87 (or a variant thereof); (x) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO:92 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO:97 (or a variant thereof); (xi) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV having the amino acid sequence shown in SEQ ID NO: 102 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV having the amino acid sequence shown in SEQ ID NO: 107 (or a variant thereof); (xii) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV having the amino acid sequence shown in SEQ ID NO: 112 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV having the amino acid sequence shown in SEQ ID NO: 117 (or a variant thereof); (xiii) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV having the amino acid sequence shown in SEQ ID NO: 122 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV having the amino acid sequence shown in SEQ ID NO: 127 (or a variant thereof); (xiv) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV having the amino acid sequence shown in SEQ ID NO: 132 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV having the amino acid sequence shown in SEQ ID NO: 137 (or a variant thereof); (xv) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV having the amino acid sequence shown in SEQ ID NO: 142 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV having the amino acid sequence shown in SEQ ID NO: 147 (or a variant thereof); (xvi) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV having the amino acid sequence shown in SEQ ID NO: 152 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV having the amino acid sequence shown in SEQ ID NO: 157 (or a variant thereof); (xvii) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV having the amino acid sequence shown in SEQ ID NO: 162 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV having the amino acid sequence shown in SEQ ID NO: 167 (or a variant thereof); (xviii) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 172 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 177 (or a variant thereof); (xix) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 182 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 187 (or a variant thereof); (xx) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 192 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 197 (or a variant thereof); (xxi) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 202 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 207 (or a variant thereof); (xxii) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 212 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 217 (or a variant thereof); (xxiii) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 222 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 227 (or a variant thereof); (xiv) An HCVR comprising the HCDR1, HCDR2, and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 232 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2, and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 237 (or a variant thereof); (xv) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 242 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 247 (or a variant thereof); (xvi) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 252 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 257 (or a variant thereof); (xvii) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 262 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 267 (or a variant thereof); (xviii) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 272 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 277 (or a variant thereof); (xix) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 282 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 287 (or a variant thereof); (xxx) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 292 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 297 (or a variant thereof); (xxxi) An HCVR comprising the HCDR1, HCDR2 and HCDR3 of an HCRV containing the amino acid sequence shown in SEQ ID NO: 302 (or a variant thereof); and an LCVR comprising the LCDR1, LCDR2 and LCDR3 of an LCRV containing the amino acid sequence shown in SEQ ID NO: 307 (or a variant thereof); and / or (xxxii) An HCVR of HCRV comprising HCDR1, HCDR2 and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 312 (or a variant thereof); and an LCVR of LCRV comprising LCDR1, LCDR2 and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 317 (or a variant thereof).

38. The recombinant viral capsid protein according to any one of claims 1-37, wherein the antibody or its binding portion that binds to the extracellular domain of TfR1 comprises: (a) An HCVR comprising: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 3 (or a variant thereof), HCDR2 containing the amino acid sequence shown in SEQ ID NO: 4 (or a variant thereof), and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 5 (or a variant thereof) ； and An LCVR comprising: LCDR1 containing the amino acid sequence shown in SEQ ID NO: 8 (or a variant thereof), LCDR2 containing the amino acid sequence shown in SEQ ID NO: 9 (or a variant thereof), and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 10 (or a variant thereof); (b) An HCVR comprising: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 13 (or a variant thereof), HCDR2 containing the amino acid sequence shown in SEQ ID NO: 14 (or a variant thereof), and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 15 (or a variant thereof); and An LCVR comprising: LCDR1 containing the amino acid sequence shown in SEQ ID NO: 18 (or a variant thereof), LCDR2 containing the amino acid sequence shown in SEQ ID NO: 19 (or a variant thereof), and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 20 (or a variant thereof); (c) An HCVR comprising: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 23 (or a variant thereof), HCDR2 containing the amino acid sequence shown in SEQ ID NO: 24 (or a variant thereof), and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 25 (or a variant thereof); and An LCVR comprising: LCDR1 containing the amino acid sequence shown in SEQ ID NO: 28 (or a variant thereof), LCDR2 containing the amino acid sequence shown in SEQ ID NO: 29 (or a variant thereof), and LCDR3 containing the amino acid sequence shown in SEQ ID NO: 30 (or a variant thereof); (d) An HCVR comprising: HCDR1 containing the amino acid sequence shown in SEQ ID NO: 33 (or a variant thereof), HCDR2 containing the amino acid sequence shown in SEQ ID NO: 34 (or a variant thereof), and HCDR3 containing the amino acid sequence shown in SEQ ID NO: 35 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 38 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 39 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 40 (or a variant thereof); (e) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 43 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 44 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 45 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 48 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 49 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 50 (or a variant thereof); (f) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 53 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 54 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 55 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 58 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 59 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 60 (or a variant thereof); (g) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 63 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 64 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 65 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 68 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 69 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 70 (or a variant thereof); (h) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 73 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 74 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 75 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO:78 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO:79 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO:80 (or a variant thereof); (i) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO:83 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO:84 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO:85 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO:88 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO:89 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO:90 (or a variant thereof); (j) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO:93 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO:94 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO:95 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO:98 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO:99 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO:100 (or a variant thereof); (k) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO:103 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO:104 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO:105 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO:108 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO:109 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO:110 (or a variant thereof); (l) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO:113 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO:114 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO:115 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 118 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 119 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 120 (or a variant thereof); (m) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 123 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 124 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 125 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 128 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 129 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 130 (or a variant thereof); (n) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 133 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 134 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 135 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 138 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 139 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 140 (or a variant thereof); (o) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 143 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 144 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 145 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 148 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 149 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 150 (or a variant thereof); (p) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 153 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 154 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 155 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 158 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 159 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 160 (or a variant thereof); (q) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 163 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 164 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 165 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 168 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 169 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 170 (or a variant thereof); (r) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 173 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 174 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 175 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 178 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 179 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 180 (or a variant thereof); (s) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 183 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 184 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 185 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 188 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 189 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 190 (or a variant thereof); (t) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 193 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 194 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 195 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 198 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 199 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 200 (or a variant thereof); (u) An HCVR comprising: an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 203 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 204 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 205 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 208 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 209 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 210 (or a variant thereof); (v) An HCVR comprising: an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 213 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 214 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 215 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 218 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 219 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 220 (or a variant thereof); (w) An HCVR comprising: an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 223 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 224 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 225 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 228 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 229 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 230 (or a variant thereof); (x) An HCVR comprising: an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 233 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 234 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 235 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 238 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 239 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 240 (or a variant thereof); (y) An HCVR comprising: an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 243 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 244 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 245 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 248 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 249 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 250 (or a variant thereof); (z) An HCVR comprising: an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 253 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 254 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 255 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 258 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 259 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 260 (or a variant thereof); (aa) An HCVR comprising: an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 263 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 264 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 265 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 268 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 269 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 270 (or a variant thereof); (ab) An HCVR comprising: an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 273 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 274 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 275 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 278 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 279 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 280 (or a variant thereof); (ac) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 283 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 284 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 285 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 288 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 289 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 290 (or a variant thereof); (ad) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 293 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 294 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 295 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 298 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 299 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 300 (or a variant thereof); (ae) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 303 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 304 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 305 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 308 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 309 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 310 (or a variant thereof); and / or (af) An HCVR comprising: an HCDR1 having the amino acid sequence shown in SEQ ID NO: 313 (or a variant thereof), an HCDR2 having the amino acid sequence shown in SEQ ID NO: 314 (or a variant thereof), and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 315 (or a variant thereof); and An LCVR comprising: an LCDR1 having the amino acid sequence shown in SEQ ID NO: 318 (or a variant thereof), an LCDR2 having the amino acid sequence shown in SEQ ID NO: 319 (or a variant thereof), and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 320 (or a variant thereof).

39. The recombinant viral capsid protein according to any one of claims 1-38, wherein the antibody or its binding portion that binds to the extracellular domain of TfR1 comprises: (i) an HCVR having the amino acid sequence shown in SEQ ID NO: 2 (or a variant thereof); and an LCVR having the amino acid sequence shown in SEQ ID NO: 7 (or a variant thereof); (ii) an HCVR having the amino acid sequence shown in SEQ ID NO: 12 (or a variant thereof); and an LCVR having the amino acid sequence shown in SEQ ID NO: 17 (or a variant thereof); (iii) an HCVR having the amino acid sequence shown in SEQ ID NO: 22 (or a variant thereof); and an LCVR having the amino acid sequence shown in SEQ ID NO: 27 (or a variant thereof); (iv) an HCVR having the amino acid sequence shown in SEQ ID NO: 32 (or a variant thereof); and an LCVR having the amino acid sequence shown in SEQ ID NO: 37 (or a variant thereof); (v) an HCVR having the amino acid sequence shown in SEQ ID NO: 42 (or a variant thereof); and an LCVR having the amino acid sequence shown in SEQ ID NO: 47 (or a variant thereof); (vi) an HCVR having the amino acid sequence shown in SEQ ID NO: 52 (or a variant thereof); and an LCVR having the amino acid sequence shown in SEQ ID NO: 57 (or a variant thereof); (vii) an HCVR having the amino acid sequence shown in SEQ ID NO: 62 (or a variant thereof); and an LCVR having the amino acid sequence shown in SEQ ID NO: 67 (or a variant thereof); (viii) an HCVR having the amino acid sequence shown in SEQ ID NO: 72 (or a variant thereof); and an LCVR having the amino acid sequence shown in SEQ ID NO: 77 (or a variant thereof); (ix) an HCVR having the amino acid sequence shown in SEQ ID NO: 82 (or a variant thereof); and an LCVR having the amino acid sequence shown in SEQ ID NO: 87 (or a variant thereof); (x) an HCVR having the amino acid sequence shown in SEQ ID NO: 92 (or a variant thereof); and an LCVR having the amino acid sequence shown in SEQ ID NO: 97 (or a variant thereof); (xi) an HCVR having the amino acid sequence shown in SEQ ID NO: 102 (or a variant thereof); and an LCVR having the amino acid sequence shown in SEQ ID NO: 107 (or a variant thereof); (xii) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 112 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 117 (or a variant thereof); (xiii) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 122 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 127 (or a variant thereof); (xiv) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 132 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 137 (or a variant thereof); (xv) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 142 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 147 (or a variant thereof); (xvi) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 152 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 157 (or a variant thereof); (xvii) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 162 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 167 (or a variant thereof); (xviii) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 172 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 177 (or a variant thereof); (xix) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 182 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 187 (or a variant thereof); (xx) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 192 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 197 (or a variant thereof); (xxi) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 202 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 207 (or a variant thereof); (xxii) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 212 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 217 (or a variant thereof); (xxiii) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 222 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 227 (or a variant thereof); (xxiv) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 232 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 237 (or a variant thereof); (xxv) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 242 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 247 (or a variant thereof); (xxvi) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 252 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 257 (or a variant thereof); (xxvii) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 262 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 267 (or a variant thereof); (xxviii) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 272 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 277 (or a variant thereof); (xxix) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 282 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 287 (or a variant thereof); (xxx) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 292 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 297 (or a variant thereof); (xxxi) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 302 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 307 (or a variant thereof); and / or (xxxii) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 312 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 317 (or a variant thereof).

40. The recombinant viral capsid protein according to any one of claims 1 - 38, wherein the antibody or its binding portion that binds to the extracellular domain of TfR1 comprises: (i) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 2 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 7 (or a variant thereof); (ii) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 42 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 47 (or a variant thereof); (iii) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 122 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 127 (or a variant thereof); (iv) An HCVR comprising the amino acid sequence shown in SEQ ID NO: 132 (or a variant thereof); and an LCVR comprising the amino acid sequence shown in SEQ ID NO: 137 (or a variant thereof); (v) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 212 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 217 (or a variant thereof); (vi) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof); (vii) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 232 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 237 (or a variant thereof); (viii) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof); (ix) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof); (x) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof); (xi) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 282 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 287 (or a variant thereof); and / or (xii) An HCVR comprising the amino acid sequence set forth in SEQ ID NO: 292 (or a variant thereof); and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 297 (or a variant thereof).

41. A recombinant viral capsid, said recombinant viral capsid comprising the recombinant viral capsid protein according to any one of claims 1 - 40.

42. The recombinant viral capsid according to claim 41, wherein the viral capsid is a mosaic viral capsid and further comprises a reference viral capsid protein that is at least 95% identical to the recombinant viral capsid protein, and wherein the reference viral capsid protein lacks all three of the following: (i) the first member of the protein:protein binding pair, (ii) the second member of the protein:protein binding pair, and (iii) the antibody or its binding portion.

43. The recombinant viral capsid according to claim 42, wherein the mosaic viral capsid comprises the reference viral capsid protein and the recombinant viral capsid protein in a ratio of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:

1.

44. The recombinant viral capsid according to any one of claims 41-43, wherein the recombinant viral capsid further comprises a nucleotide of interest encapsidated within the viral capsid.

45. The recombinant viral capsid according to claim 44, wherein the nucleotide of interest is a reporter gene.

46. The recombinant viral capsid according to claim 44 or claim 45, wherein the nucleotide of interest encodes β-galactosidase, green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), MmGFP, blue fluorescent protein (BFP), enhanced blue fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP), Emerald, CyPet, cyan fluorescent protein (CFP), Cerulean, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof.

47. The recombinant viral capsid according to claim 44, wherein the nucleotide of interest encodes a therapeutic protein, a suicide gene, an antibody or a fragment thereof, a CRISPR / Cas system or a portion thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule.

48. The recombinant viral capsid according to any one of claims 44-47, wherein the nucleotide of interest is operably linked to a promoter having organ specificity, tissue specificity, or cell specificity.

49. The recombinant viral capsid according to claim 48, wherein the promoter has brain specificity.

50. The recombinant viral capsid according to claim 48 or claim 49, wherein the promoter has neuron specificity, glial cell specificity, astrocyte specificity, oligodendrocyte specificity, microglia specificity, and / or central nervous system specificity.

51. The recombinant viral capsid according to any one of claims 48-50, wherein the promoter is selected from the group consisting of the human glial fibrillary acidic protein (GFAP) promoter, the human synapsin 1 (SYN1) promoter, the human synapsin 2 (SYN2) promoter, the human metallothionein 3 (MT3) promoter, and the human proteolipid protein 1 (PLP1) promoter.

52. The recombinant viral capsid according to claim 48 or claim 49, wherein the promoter is a neuron, astrocyte, or oligodendrocyte specific promoter, or a neuron, astrocyte, or oligodendrocyte preferential promoter.

53. The recombinant viral capsid according to claim 52, wherein the promoter is selected from the group consisting of: NSE promoter, synaptophysin promoter, MeCP2 promoter, oligodendrocyte transcription factor 1 (Olig1) promoter, chondroitin sulfate proteoglycan (Cspg4) promoter, CNP (2',3'-cyclic-nucleotide 3'-phosphodiesterase) promoter, and GFAP promoter.

54. A pharmaceutical composition, the pharmaceutical composition comprising (a) the recombinant viral capsid according to any one of claims 41-53 and (b) a pharmaceutically acceptable carrier or excipient.

55. A method of delivering a nucleotide of interest across the blood-brain barrier in a mammalian subject, the method comprising administering to the mammalian the pharmaceutical composition according to claim 54.

56. The method according to claim 55, wherein the administration comprises intravenous injection.

57. The method according to claim 56, wherein the subject is modified to express a targeting ligand, such as from a safe harbor locus.

58. The method according to claim 56, wherein the subject is a primate mammal, preferably human.

59. The method according to any one of claims 54-58, wherein the mammalian blood-brain barrier cell is a mammalian brain endothelial cell.

60. The method according to any one of claims 54-59, wherein the endothelial cells in the mammalian blood-brain barrier express transferrin receptor protein 1 on the cell surface, and (i) the first member of the protein:protein binding pair, (ii) the second member of the protein:protein binding pair, and (iii) the antibody or its binding portion together direct the tropism of the viral vector to the endothelial cells in the mammalian blood-brain barrier.

61. The method according to claim 60, wherein after the viral particle binds to transferrin receptor protein 1 on the surface of the endothelial cell, the viral particle is transported inside the endothelial cell of the blood-brain barrier and delivered to the brain by transcytosis, such that the endothelial cell is not infected by the viral particle.

62. The method according to any one of claims 55 to 61, wherein the nucleotide of interest encodes a therapeutic protein, a suicide gene, an antibody or a fragment thereof, a CRISPR / Cas system or a part thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule.

63. The method according to claim 62, wherein the therapeutic protein is a soluble protein.

64. The method according to claim 62 or 63, wherein the therapeutic protein comprises an antibody or a binding portion thereof.

65. The method according to claim 64, wherein the administration comprises intravenous or intraventricular injection.

66. The method according to claim 62, wherein the nucleotide of interest encodes an shRNA molecule.

67. The method according to any one of claims 55 - 66, wherein the nucleotide of interest is operably linked to a brain - specific promoter and is preferentially expressed in the brain relative to other organs or tissues.

68. A method of treating a disease in a patient in need thereof, the method comprising: administering to the patient (a) a viral particle or composition according to any one of claims 1 - 53 or (b) a pharmaceutical composition according to claim 54, wherein the viral particle comprises a nucleotide of interest encapsidated within the viral capsid, and wherein the nucleotide of interest encodes a therapeutic moiety selected from the group consisting of: a therapeutic protein, a suicide gene, an antibody or fragment thereof, a CRISPR / Cas system or portion thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, and a shRNA molecule.

69. The method according to claim 68, wherein the therapeutic moiety targets α - synuclein.

70. The method according to claim 69, wherein the therapeutic moiety comprises an SNCA shRNA molecule.

71. Use of a viral particle or composition according to any one of claims 1 - 53 or a pharmaceutical composition according to claim 54 for the preparation of a medicament for treating a disease.

72. A viral protein, capsid, genome, particle and methods for their preparation and use, including the preparation of a medicament, substantially as herein described.

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