Virus particles retargeted to transferrin receptor 1

Abstract

Provided herein are compositions and methods for retargeting viral particles, e.g., adeno-associated virus (AAV) particles, to TfR1-expressing cells, including blood-brain barrier (BBB) endothelial cells. AAV adapted accordingly can be a viable gene therapy platform for gene therapy of target cells expressing TfR1, e.g., for crossing the blood-brain barrier in patients in need thereof. It is shown herein that AAV capsid proteins can be modified to enable targeted transfer of a nucleotide of interest to mammalian cells expressing transferrin receptor 1 (TfR1; CD71) and / or to enable the modified AAV capsid to cross the blood-brain barrier (BBB) via TfR1-expressing BBB endothelial cells.

Description

[Technical Field]

[0001] The disclosure herein relates to methods of making and using recombinant viral particles that may be useful (a) for infecting (e.g., genetically modifying) cells that express TfR, and / or (b) for transcytosis of AAV particles across cells that express TfR, such as blood-brain barrier endothelial cells in vitro or in vivo, e.g., where the recombinant AAV particles comprise capsid proteins that are retargeted to a cell surface protein that enables the viral particle to bind to transferrin receptor 1 (also referred to in the present application as TfR or CD71, encoded by TFR).

[0002] Sequence Listing The sequence listing in xml format entitled "11081WO01_xml.xml" was created on July 28, 2023, is 461 Kb, and is incorporated herein by reference in its entirety. [Background technology]

[0003] Delivery of genes to specific target cells has become one of the most important technologies in modern medicine for the potential treatment of various chronic and genetic diseases. Ideally, a gene delivery vehicle can stably transfer genetic material into desired cells and avoid transferring genetic material into non-target cells.

[0004] Viral particles, particularly those based on adeno-associated viruses (AAVs), as gene delivery vehicles have been the focus of much research due to AAV's ability to transduce a wide range of primate species and tissues in vivo without overt evidence of pathogenicity (Muzyczka, et al. (1992) Current Topics in Microbiology and Immunology, 158:97-129). Furthermore, AAVs safely transduce postmitotic tissues. The virus can occasionally integrate into host chromosomes, very rarely into the safe-harbor locus on human chromosome 19, but only when replication (Rep) proteins are supplied in trans. The AAV genome rapidly circularizes and concatenates within infected cells, remaining in a stable episomal state within the infected cells and providing long-term, stable expression of the payload.

[0005] More recently, engineering and redirecting AAV infection to specific cells has been achieved. Many of the advances in targeted gene therapy using viral particles can be summarized as non-recombinant (non-genetic) or recombinant (genetic) modifications of viral vectors that result in pseudotyping, expansion, and / or retargeting of the original tropism of the viral particle. (Reviewed in Nicklin and Baker (2002) Curr. Gene Ther. 2:273-93; Verheiji and Rottier (2012) Advances Virol 2012:1-15.)

[0006] In direct recombinant targeting approaches, a targeting ligand is directly inserted into or linked to the viral capsid, i.e., the viral capsid protein gene is modified to express a capsid protein containing a heterologous targeting ligand, which is then redirected to, e.g., binds to, a receptor or marker that is preferentially or exclusively expressed on the target cell. (Stachler et al. (2006) Gene Ther. 13:926-931, White et al. (2004) Circulation 109:513-519, 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).

[0007] In indirect recombinant approaches, viral capsids are modified with heterologous "scaffolds" and then ligated with adapters containing targeting ligands. The adapters bind to the scaffolds and target cells. (See also Arnold et al. (2006) Mol. Ther. 5:125-132; Ponnazhagen et al. (2002) J. Virol. 76:12900-907; WO 97 / 05266). Scaffolds such as (1) Fc-binding molecules (e.g., Fc receptors, protein A, etc.) that bind to the Fc of antibody adapters, (2) (strept)avidin that binds to biotinylated adapters, (3) biotin that binds to adapters fused to (strept)avidin, (4) detectable labels useful for detecting and / or isolating viral particles that are conjugated to bispecific adapters capable of non-covalently binding to the detectable label and target molecules, and recently (5) protein:protein binding pairs that form isopeptide bonds have been reported for various viral particles. (See, e.g., Gigout et al. (2005) Molecular Therapy 11:856-865; Stachler et al. (2008) Molecular Therapy 16:1467-1473; Quetglas et al. (2010) Virus Research 153:179-196; Ohno et al. (1997) Nature Biotechnology 15:763-767; Klimstra et al. (2005) Virology 338:9-21). With advances that provide the ability to direct AAV infection, there remains a need for viral systems that can be adapted for targeted delivery of nucleic acids of interest to target cells or receptors. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Muzyczka, et al. (1992)Current Topics in Microbiology and Immunology,158:97-129 [Non-patent document 2] Nicklin and Baker(2002)Curr.Gene Ther.2:273-93 [Non-patent document 3] Verheiji and Rottier(2012)Advances Virol 2012:1-15 [Non-patent document 4] Stachler et al. (2006) Gene Ther. 13:926-931 [Non-Patent Document 5] White et al. (2004)Circulation 109:513-519 Summary of the Invention [Means for solving the problem]

[0009] It is shown herein that AAV capsid proteins can be modified to enable targeted delivery of a nucleotide of interest to mammalian cells expressing transferrin receptor 1 (TfR1; CD71) and / or to enable the modified AAV capsid to cross the blood-brain barrier (BBB) through BBB endothelial cells expressing TfR1.

[0010] The viral particles described herein are particularly suitable for targeted delivery of a nucleotide of interest specifically to cells expressing transferrin receptor 1 (TfR1; CD71) or across the blood-brain barrier, because the viral capsid or viral capsid protein described herein comprises a first member of a protein:protein binding pair together with its cognate second member of the protein:protein binding pair, where the second member is linked (e.g., fused) to a targeting ligand that binds to transferrin receptor 1 (TfR1) expressed on the cell surface.

[0011] Described herein are recombinant viral capsid proteins and viral particles (e.g., in which the viral capsid proteins encapsidate a nucleic acid of interest), as well as compositions comprising the viral capsid proteins and / or viral particles, e.g., pharmaceutical compositions comprising the viral capsid proteins and / or viral particles, wherein the recombinant viral capsid proteins described herein include (i) a first member of a protein:protein binding pair that is inserted and / or displayed by the viral 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 binding portion thereof that binds to the extracellular domain of transferrin receptor protein 1 (abbreviated as TfR1, TfR, or CD71), wherein the antibody or 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 binding portion thereof, together direct the viral capsid toward cells expressing TfR1, or an extracellular portion thereof, e.g., the amino acid sequence set forth in SEQ ID NO: 436. In some embodiments, the extracellular domain of TfR1 is the extracellular domain of human (h)TfR1.

[0012] In some embodiments, the viral capsid protein / viral particle and / or composition comprising same further comprises a cell expressing TfR1 on its surface, e.g., 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 the cell is in vivo, ex vivo, or in vitro. In some embodiments, the AAV viral capsid protein expressing a TfR1-targeting ligand disclosed herein (including viral particles and / or compositions comprising same) binds to a cell expressing TfR1 on its surface, and the cell is a central nervous system cell such as a cortical neuron, Purkinje cell, glial cell (e.g., astrocyte, oligodendrocyte, etc.). In some embodiments, the cells are blood-brain barrier endothelial cells and / or brain microvascular endothelial cells that express TfR1 on their surface, and the viral capsid binds to the extracellular domain of TfR1 expressed on the surface of the blood-brain barrier endothelial cells and / or brain microvascular endothelial cells, and optionally the blood-brain barrier endothelial cells and / or brain microvascular endothelial cells are in vivo, ex vivo, or in vitro.

[0013] The protein:protein binding pairs described herein are protein:protein binding pairs that spontaneously form an isopeptide bond upon contact. In some embodiments, (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 a targeting ligand; (c) the first member of the protein:protein binding pair and the second member of the protein:protein binding pair are associated by 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 set forth in SEQ ID NO: 326, and (b) the second member of the protein:protein binding pair comprises a bispecific binding protein comprising an anti-c-myc antibody and a targeting ligand.

[0014] In some further embodiments, the viral capsid protein further comprises a linker flanking one or both sides of the first member of the protein:protein binding pair. In some embodiments, the first and / or second linkers operably linking the first member of the protein:protein binding pair to the capsid protein of the viral capsid are each independently at least one amino acid in length (e.g., 10 amino acids in length) and are non-identical or identical. In some embodiments, the first linker is 10 amino acids in length and / or the second linker is 10 amino acids in length, and optionally, the amino acid sequence of the first linker and / or the amino acid sequence of the second linker comprises the amino acid sequence set forth in SEQ ID NO:331 or SEQ ID NO:332.

[0015] 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 mutant cap gene, wherein the mutant cap gene or a portion thereof (e.g., about 15 nucleotides) comprises a nucleotide sequence at least 90% identical to an AAV cap gene or a portion thereof, and wherein the mutant cap gene or portion thereof has been genetically modified to include an insertion of a nucleotide sequence encoding a first member of a protein:protein binding pair, whereby 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 mutant cap gene or portion thereof has been genetically modified to include one or more additional mutations, whereby the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein comprises, in addition to the first member of the protein:protein binding pair, (i) point mutations, e.g., amino acid substitutions, insertions, or deletions; (ii) a chimeric amino acid sequence, or (iii) Both point mutations and chimeric amino acid sequences, including, optionally, amino acid substitutions, insertions, or deletions that reduce the natural tropism of the viral particle and / or create 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 a cap gene or a portion thereof of an AAV, wherein the mutated cap gene or portion thereof has been genetically modified to comprise an insertion of a nucleotide sequence encoding a first member of a protein:protein binding pair, whereby 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 the AAV is selected from the group consisting of AAV1, AAV2, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV rh10, AAV rh32.33, a non-primate AAV listed in Table 5, a combination thereof, and any variant or hybrid thereof.

[0016] In some embodiments, the AAV is AAV2, and optionally the viral capsid comprises a modified AAV2 VP1 capsid protein comprising a first member of a protein:protein binding pair linked, optionally via a linker, to amino acids at positions I453 and / or I587. In some embodiments, the viral capsid comprises a modified AAV2 VP1 capsid protein comprising a first member of a protein:protein binding pair presented at position G453 via a linker, and optionally 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 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.

[0017] In some embodiments, the AAV is AAV9, and optionally the viral capsid comprises a modified AAV9 VP1 capsid protein comprising a first member of a protein:protein binding pair linked, optionally via a linker, to the amino acid at position 1453 or 1589. In some embodiments, the viral capsid comprises a modified AAV9 VP1 capsid protein comprising a first member of a protein:protein binding pair presented at G453, optionally via a linker, and optionally the modified AAV9 VP1 capsid protein further comprises an N272A modification, a W503A modification, or both the N272A modification and the W503A modification. In some embodiments, the recombinant viral capsid of the viral capsid is a mosaic viral capsid comprising a second set of AAV9 VP1 capsid proteins lacking the first member of the protein:protein binding pair, and optionally, the second set of AAV2 VP1 capsid proteins comprises an N272A modification, a W503A modification, or both an N272A modification and a W503A modification.

[0018] In some embodiments, the AAV is AAV1, and optionally the viral capsid comprises a modified AAV1 VP1 capsid protein comprising a first member of a protein:protein binding pair, optionally linked via a linker. In some embodiments, the recombinant viral capsid of the viral capsid is a mosaic viral capsid comprising a second set of AAV1 VP1 capsid proteins lacking the first member of the protein:protein binding pair.

[0019] In some embodiments, the AAV is AAV8, and optionally the viral capsid comprises a modified AAV8 VP1 capsid protein comprising a first member of a protein:protein binding pair, optionally linked via a linker. In some embodiments, the recombinant viral capsid of the viral capsid is a mosaic viral capsid comprising a second set of AAV8 VP1 capsid proteins lacking the first member of the protein:protein binding pair.

[0020] In some embodiments, the AAV is AAVrh32.33, and optionally the viral capsid comprises a modified AAVrh32.33 VP1 capsid protein, optionally comprising a first member of a protein:protein binding pair linked via a linker. In some embodiments, the recombinant viral capsid of the viral capsid is a mosaic viral capsid comprising a second set of AAV rh32.33 VP1 capsid proteins that lack the first member of the protein:protein binding pair.

[0021] In some embodiments, the non-primate AAV is an avian AAV (AAAV), a non-human mammalian AAV, or a Squamata AAV.

[0022] In some embodiments, the non-primate AAV is AAAV, and optionally the viral capsid comprises a modified AAAV VP1 capsid protein comprising a first member of a protein:protein binding pair linked, optionally via a linker, to an amino acid at position 1444 or 1580. In some embodiments, the viral capsid comprises a modified AAAV VP1 capsid protein comprising a first member of a protein:protein binding pair linked, optionally via a linker, to an amino acid at a position selected from the group consisting of 1429, 1430, 1431, 1432, 1433, 1434, 1436, 1437, and 1565.

[0023] In some embodiments, the AAV is a Squamata AAV, e.g., a Bearded Dragon AAV. In some embodiments, the viral capsid comprises a modified Bearded Dragon VP1 capsid protein comprising a first member of a protein:protein binding pair linked, optionally via a linker, to the amino acid at position 1573 or 1436.

[0024] In some embodiments, the AAV is a sea lion AAV.

[0025] 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 expressing TfR (e.g., human TfR). In some embodiments, antibodies or binding portions thereof that bind to the extracellular domain of TfR1 comprise heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) from a heavy chain variable region (HCVR) comprising the amino acid sequence set forth 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 heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) from a heavy chain variable region (HCVR) comprising the amino acid sequence set forth 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). In some embodiments, antibodies or binding portions thereof that bind to TfR1, e.g., human TfR1, and that can be used to retarget AAV viral particles described herein include: (i) an HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 2 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 7 (or a variant thereof); (ii) an HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 12 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 17 (or a variant thereof); (iii) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 22 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 27 (or a variant thereof); (iv) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 32 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 37 (or a variant thereof); (v) an HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 42 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 47 (or a variant thereof); (vi) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 52 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 57 (or a variant thereof); (vii) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 62 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 67 (or a variant thereof); (viii) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 72 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 77 (or a variant thereof); (ix) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 82 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 87 (or a variant thereof); (x) an HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 92 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 97 (or a variant thereof); (xi) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 102 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 107 (or a variant thereof); (xii) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 112 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 117 (or a variant thereof); (xiii) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 122 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 127 (or a variant thereof); (xiv) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 132 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 137 (or a variant thereof); (xv) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 142 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 147 (or a variant thereof); (xvi) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 152 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 157 (or a variant thereof); (xvii) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 162 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 167 (or a variant thereof); (xviii) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 172 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 177 (or a variant thereof); (xix) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 182 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 187 (or a variant thereof); (xx) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 192 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 197 (or a variant thereof); (xxi) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 202 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 207 (or a variant thereof); (xxii) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 212 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 217 (or a variant thereof); (xxiii) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 222 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 227 (or a variant thereof); (xiv) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 232 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 237 (or a variant thereof); (xv) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 242 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 247 (or a variant thereof); (xvi) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 252 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 257 (or a variant thereof); (xvii) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 262 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 267 (or a variant thereof); (xviii) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 272 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 277 (or a variant thereof); (xix) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 282 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 287 (or a variant thereof); (xxx) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 292 (or a variant thereof), and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 297 (or a variant thereof); (xxxi) an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCRV comprising the amino acid sequence set forth in SEQ ID NO: 302 (or a variant thereof), and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCRV comprising the amino acid sequence set forth in SEQ ID NO: 307 (or a variant thereof); and / or (xxxii) A HCVR comprising HCDR1, HCDR2, and HCDR3 of HCRV comprising the amino acid sequence set forth in SEQ ID NO: 312 (or a variant thereof), and a LCVR comprising LCDR1, LCDR2, and LCDR3 of LCRV comprising the amino acid sequence set forth in SEQ ID NO: 317 (or a variant thereof).

[0026] In some embodiments, an antibody or binding portion thereof that binds to a TfR1 protein, e.g., a human TfR1 protein, and can be used to retarget AAV viral particles described herein is (a) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 9 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 10 (or a variant thereof); (b) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20 (or a variant thereof); (c) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30 (or a variant thereof); (d) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 33 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 34 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 35 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 38 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 39 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 40 (or a variant thereof); (e) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 43 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45 (or a variant thereof); and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 48 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 49 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 50 (or a variant thereof); (f) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 53 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 54 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 55 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 58 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 59 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 60 (or a variant thereof); (g) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 63 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 64 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 65 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 68 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 69 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 70 (or a variant thereof); (h) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 73 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 74 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 75 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 78 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 79 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 80 (or a variant thereof); (i) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 83 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 84 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 85 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 88 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 89 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 90 (or a variant thereof); (j) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 93 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 94 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 95 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 98 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 99 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 100 (or a variant thereof); (k) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 103 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 104 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 105 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 108 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 109 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 110 (or a variant thereof); (l) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 113 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 114 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 115 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 118 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 119 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 120 (or a variant thereof); (m) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 123 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 124 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 125 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 128 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 129 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 130 (or a variant thereof); (n) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 133 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 134 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 135 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 138 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 139 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 140 (or a variant thereof); (o) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 143 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 144 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 145 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 148 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 149 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 150 (or a variant thereof); (p) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 153 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 154 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 155 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 158 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 159 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 160 (or a variant thereof); (q) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 163 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 164 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 165 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 168 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 169 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 170 (or a variant thereof); (r) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 173 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 174 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 175 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 178 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 179 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 180 (or a variant thereof); (s) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 183 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 184 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 185 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 188 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 189 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 190 (or a variant thereof); (t) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 193 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 194 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 195 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 198 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 199 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 200 (or a variant thereof); (u) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 203 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 204 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 205 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 208 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 209 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 210 (or a variant thereof); (v) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 213 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 214 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 215 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 218 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 219 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 220 (or a variant thereof); (w) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 223 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 224 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 225 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 228 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 229 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 230 (or a variant thereof); (x) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 233 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 234 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 235 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 238 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 239 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 240 (or a variant thereof); (y) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 243 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 244 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 245 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 248 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 249 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 250 (or a variant thereof); (z) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 253 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 254 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 255 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 258 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 259 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 260 (or a variant thereof); (aa) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 263 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 264 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 265 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 268 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 269 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 270 (or a variant thereof); (ab) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 273 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 274 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 275 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 278 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 279 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 280 (or a variant thereof); (ac) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 283 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 284 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 285 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 288 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 289 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 290 (or a variant thereof); (ad) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 293 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 294 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 295 (or a variant thereof); and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 298 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 299 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 300 (or a variant thereof); (ae) an HCVR comprising an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 303 (or a variant thereof), an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 304 (or a variant thereof), and an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 305 (or a variant thereof), and an LCVR comprising an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 308 (or a variant thereof), an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 309 (or a variant thereof), and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 310 (or a variant thereof); and / or (af) An HCVR comprising an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 313 (or a variant thereof), an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 314 (or a variant thereof), and an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 315 (or a variant thereof), and an LCVR comprising an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 318 (or a variant thereof), an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 319 (or a variant thereof), and an LCDR3 having the amino acid sequence set forth in SEQ ID NO: 320 (or a variant thereof).

[0027] In some embodiments, an antibody or binding portion thereof that binds to a TfR1 protein, e.g., a human TfR1 protein, and can be used to retarget AAV viral particles described herein is (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: 12 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 17 (or a variant thereof); (iii) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 22 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 27 (or a variant thereof); (iv) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 32 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 37 (or a variant thereof); (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); (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); (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); (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); (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); (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); (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); (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); (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); (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); (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); (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); (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); (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); (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); (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); (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); (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); (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); (xxiv) 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); (xxv) 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); (xxvi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 252 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 257 (or a variant thereof); (xxvii) 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); (xxviii) 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); (xxix) 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); (xxx) 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); (xxxi) an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 302 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 307 (or a variant thereof), and / or (xxxii) Comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 312 (or a variant thereof), and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 317 (or a variant thereof).

[0028] In some embodiments, the antibody or binding portion thereof that binds to the extracellular domain of TfR1 is in a bivalent monoclonal antibody or mAb format. In some embodiments, the antibody or binding portion thereof that binds to the extracellular domain of TfR1 is in a Fab format. In some embodiments, the antibody or binding portion thereof that binds to the extracellular domain of TfR1 is in an scFv format. In some embodiments, the antibody or binding portion thereof that binds to the extracellular domain of TfR1 comprises a mutated Ig CH3 domain, which binds to the extracellular domain of TfR1.

[0029] In some embodiments, antibodies or binding portions thereof that bind to a TfR1 protein, e.g., a human TfR1 protein, and can be used to retarget AAV viral particles described herein include (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).

[0030] Also described are viral particles, e.g., viral capsids, comprising the viral capsid proteins described herein. In some embodiments, the viral capsids described herein are mosaic capsids and further comprise a reference viral capsid protein that is at least 95% identical to the recombinant viral capsid protein, wherein the reference viral capsid protein lacks all three of: (i) a first member of a protein:protein binding pair; (ii) a second member of the protein:protein binding pair; and (iii) an antibody or binding portion thereof. In some embodiments, 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, or 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, for example, 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.

[0031] 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 that is organ-specific, tissue-specific, or cell-specific. In some embodiments, the nucleotide of interest encodes a therapeutic moiety that can be secreted from the transduced cell and provide a therapeutic effect to the interstitial space surrounding the transduced cell or to adjacent cells. In some embodiments, the nucleotide of interest encodes a therapeutic moiety that can provide a therapeutic effect in a transduced cell-autonomous manner. In some embodiments, the nucleotide of interest encodes a therapeutic moiety that can provide a therapeutic benefit to the transduced cell, the interstitial space surrounding the transduced cell, and / or to cells adjacent to the transduced cell in an autonomous manner. In some embodiments, the therapeutic moiety is a therapeutic protein, such as a fusion protein, an enzyme, or the like. In some embodiments, the therapeutic moiety is a secreted antibody or a 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, and the like.

[0032] In some embodiments, the promoter is brain-specific. In some embodiments, the promoter is neuron-specific, glial cell-specific, astrocyte-specific, oligodendrocyte-specific, microglia-specific, and / or central nervous system-specific. 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 neuron-, astrocyte-, or oligodendrocyte-specific, or a neuron-, astrocyte-, or oligodendrocyte-preferred promoter. In some embodiments, the promoter is selected from the group consisting of an NSE promoter, a synapsin promoter, an MeCP2 promoter, an oligodendrocyte transcription factor 1 (Olig1) promoter, a chondroitin sulfate proteoglycan (Cspg4) promoter, a CNP (2',3'-cyclic nucleotide 3'-phosphodiesterase) promoter, and a GFAP promoter.

[0033] Also provided herein is a pharmaceutical composition comprising (a) a recombinant viral capsid described herein, and (b) a pharmaceutically acceptable carrier or excipient.

[0034] 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 mammal. In some embodiments, the administering (e.g., contacting) is performed ex vivo. In some embodiments, the administering is performed in a subject, optionally 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, endothelial cells within the mammalian blood-brain barrier express transferrin receptor protein 1 on their cell surface, and (i) a first member of a protein:protein binding pair, (ii) a second member of the protein:protein binding pair, and (iii) an antibody or binding portion thereof, together direct the viral vector to endothelial cells within the mammalian blood-brain barrier. In some embodiments, the viral particles are delivered to the brain by transcytosis after binding to transferrin receptor protein 1 on the surface of endothelial cells, thereby transporting them across the interior of endothelial cells at the blood-brain barrier, thereby preventing the endothelial cells from being infected by the viral particles. In some embodiments, the nucleotide of interest encodes 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 brain-specific promoter, such that the nucleotide of interest is preferentially expressed in the brain over other organs or tissues.

[0035] Also described herein are methods of treating a disease in a patient in need thereof, comprising administering to the patient a viral particle or composition (e.g., a pharmaceutical composition) described herein, wherein the viral particle comprises a nucleotide of interest encapsidated within the viral capsid, and the nucleotide of interest encodes a therapeutic moiety, e.g., 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 therapeutic moiety inhibits, e.g., 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 by a gene encoding an α-synuclein protein, e.g., SNCA, for inhibition via RNA interference. In some embodiments, administration is via intravenous injection. In some embodiments, administration is via intracerebroventricular injection.

[0036] In some embodiments, the targeting ligand is operably linked to a protein (e.g., a second member of a protein:protein binding pair), e.g., optionally fused to the protein via a linker. In some embodiments, the targeting ligand may be a binding moiety, e.g., a natural ligand, an antibody, a multispecific binding molecule, etc. In some embodiments, the targeting ligand is an antibody or 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, e.g., via a linker, to a protein (e.g., a second member of a protein:protein binding pair) that forms an isopeptide covalent bond with the first member. In some embodiments, the capsid proteins described herein comprise a first member comprising a SpyTag operably linked to a viral capsid protein and covalently bound to the SpyTag, and a 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, for example, GSGESG (SEQ ID NO: 433).

[0037] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0038] [Figure 1]Histograms obtained from flow cytometry analysis of green fluorescent protein (GFP) expression by cells expressing mTfR ("mTfR+293," "bEnd.3(mTfR+)") or negative control cells that do not express mTfR but do express hASGR1 ("hASGR1+293Ts(ACL18620)") after infection with AAV9 particles are shown. Virus was added at a multiplicity of infection of 1e5 vg / cell. Cells express GFP as a marker of transduction. Cells were transduced with a panel of AAV9-based particles, including wild-type (wt) AAV9, or SpyTag-tagged AAV9-based capsids (AAV9 wt, AAV9 N272A, AAV9 W503A) conjugated with antibody 8D3 targeting mTfR (anti-TfR mAb), or capsids conjugated with an antibody targeting hASGR1 as a negative control. [Figure 2] Immunohistochemical staining of eGFP expression in the cerebellum or liver of wild-type C57BL / 6J mice after injection of 5e10vg / mouse of wild-type (wt) AAV9 or various AAV9-based capsids (AAV9 wt, AAV9 N272A, AAV9 W503A) conjugated with the mTfR-targeting antibody 8D3 (anti-TfR mAb). Representative brain images show coronal sections featuring the caudal plane including the cerebellum at 2.7x magnification. Liver sections are at 8x magnification. [Figure 3] Histograms obtained from flow cytometry assessing green fluorescent protein (GFP) expression by cells expressing mTfR (mTfR+293 and bEnd.3) or cells expressing hASGR1 as a negative control (hASGR1+293(mTfR-)) after infection with AAV9 particles are shown. Virus was added at a multiplicity of infection of 1e5 vg / cell. Cells express GFP as a marker of transduction. Cells were transduced with a panel of AAV9-based particles, including wild-type (wt) AAV9 or SpyTag-tagged AAV9 W503A particles conjugated to various formats of the mTfR-targeting antibody 8D3, either as a bivalent antibody (mAb), Fab, or scFv. [Figure 4]Immunohistochemical staining of eGFP expression in the cerebellum and liver of WT C57BL / 6J mice after injection of wild-type (wt) AAV9 at 7.5e9vg / mouse (150uL injection of 5e10vg / mL solution) and detargeted AAV9 W503A conjugated with an antibody targeting mTfR (anti-TfR mAb) in various antibody formats (bivalent mAb, Fab, and scFv). Representative brain images show coronal sections featuring the caudal plane including the cerebellum at 4.4x magnification. Liver sections are at 14x magnification. [Figure 5A] Figure 5A shows qPCR data measuring viral DNA normalized to β-actin and compared to wild-type (wt) AAV (y-axis) in the brain (Figure 5A) and liver (Figure 5B) of WT C57BL / 6J mice after injection with various antibody formats: wild-type AAV9, detargeted AAV9 W503A conjugated with an antibody targeting mTfR, or hASGR1 as a non-targeting control, at 7.5e9vg / mouse (150uL injection of 5e10vg / mL solution). AAV DNA was measured using a qPCR probe recognizing the eGFP sequence, and levels were normalized to the b-actin housekeeping gene. Mouse TfR-targeted AAVs show enhanced levels of AAV DNA in the brain at 14 days post-injection compared to WT, detargeted, and hASGR1-targeted AAV9. Retargeting to TfR improves CNS transduction regardless of the antibody format used. [Figure 5B] Same as above. [Figure 6]

[0023] Figure 1 shows immunohistochemical staining of eGFP expression in the brains of WT C57BL / 6J mice 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 of planes featuring the hippocampus at 5x magnification, the frontal cortex at 10x magnification, and the cerebellum at 8x magnification. AAV particles targeting human TFR specifically infect hTFR+ cell lines in vitro and exhibit enhanced CNS transduction and reduced liver transduction in vivo after systemic injection, regardless of AAV serotype. [Figure 7] Histograms obtained from flow cytometry assessing green fluorescent protein (GFP) expression by cells expressing hTfR (hTfR+3T3) or, as a negative control, cells not expressing hTfR (3T3) 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 marker of transduction. Cells were transduced with a panel of SpyTag-tagged AAV9 W503A particles conjugated to Fabs targeting hTfR (Fabs H1H12799B, PN69331, H1H12848B, H1H31874B, H1H12843B, H1H12798B, H1H12850B, H1H12847B, H1H12835B, H1H12839B, H1H12841B, H1H12845B) or, as a negative control, Fabs targeting hASGR1. [Figure 8]Immunohistochemical staining of eGFP expression in the brain and liver of TFRC hu / hu mice after injection of 1.5e10 vg / mouse (1e11 vg / mL solution injected in 150 μL) of wild-type (WT) AAV9, an antibody Fab targeting hTfR (H1H12845B), or detargeted AAV9 W503A conjugated with an antibody Fab targeting hASGR1 as a non-targeting control. While AAV9 wild-type particles can transduce the liver of TFRC hu / hu mice, AAV9 W503A particles are detargeted from the liver and do not promote high levels of hepatic eGFP expression. Representative images include higher-magnification coronal sections of planes featuring the hippocampus at 9x magnification, the frontal cortex at 10x magnification, the medial cortex at 10x magnification, and the cerebellum at 10x magnification. [Figure 9A] Immunohistochemical staining of eGFP expression in the brains of TFRC hu / hu mice after injection of 1.5e10 vg / mouse (1e11 vg / mL solution injected in 150 μL) of wild-type (WT) AAV9, antibody Fabs targeting hTfR (Fabs H1H12845B, H1H12850B), or detargeted AAV9 W503A conjugated with antibody Fab targeting hASGR1 as a non-targeting control. Representative images include higher magnification coronal sections of planes featuring the hippocampus, frontal cortex, medial cortex, and cerebellum. [Figure 9B] Similarly, representative IHC staining of eGFP expression in the brains of TFRC hu / hu mice injected with AAV9 W503A conjugated with an additional Fab targeting hTfR is shown (Figure 9B: H1H12841B, H1H12798B, H1H12847B, H1H12839B, H1H12843B; Figure 9C: H1H31874B, H1H12835B, PN69331, H1H12848B, H1H12799B). Representative images include higher-magnification coronal sections of planes featuring the hippocampus at 9x magnification, the frontal cortex at 10x magnification, the medial cortex at 10x magnification, and the cerebellum at 10x magnification. [Figure 9C] Same as above. [Figure 9D]1.5e10vg / mouse of various AAV9s (x-axis): (1) wild-type AAV9, (2) detargeted AAV9 W503A conjugated with antibody Fab targeting hASGR1 as a non-targeting control, or detargeted AAV9 conjugated with antibody Fab targeting hTfR. Quantification of eGFP immunohistochemical staining in the brain (left panel) and liver (right panel) after injection of W503A: (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 areas stained positive for DAB (indicating a positive GFP signal) in the brain and liver was quantified using HALO software. [Figure 10A] (1) Wild-type “WT:AAV9”, (2) Detargeted AAV9 W503A conjugated with an antibody Fab targeting hASGR1 as a non-targeting control, or detargeted AAV9 conjugated with an antibody Fab targeting hTfR. Figure 10A shows qPCR data measuring AAV DNA in the brain (Figure 10A) and liver (Figure 10B) of TFRC hu / hu mice after injection with W503A: (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 levels were normalized to the b-actin housekeeping gene. [Figure 10B] Same as above. [Figure 11A]Figure 1 provides a heatmap showing enhanced in vivo brain and spinal cord transduction in female humanized TFRC mice (TFRChu / hu) after administration of AAV9 particles, each bearing a unique barcode and retargeted with an anti-TfR Fab, compared with wild-type AAV9 viral particles (AAV). Each candidate AAV was packaged with a unique barcoded genome, as described in the Materials and Methods section of the Examples below. After IV administration of the 36 candidate barcoded pools, the indicated tissues were collected, and the relative abundance of each barcode in total RNA purified from each tissue was assessed using next-generation sequencing (NGS). Enrichment values for each virus relative to the input virus pool are shown for tissues of interest: liver (median lobe), left hemisphere of the brain, and spinal cord between the cervical and sacral regions. Data shown are the average of triplicate animals in the study. [Figure 11B] FIG. 11A provides an enrichment plot for the mouse showing enhanced transduction to the brain and spinal cord, and liver detargeting in vivo. [Figure 12A] Immunofluorescence co-staining of eGFP expression and brain cell-specific markers in the brains of TFRC hu / hu mice after intravenous injection (1e11vg / mouse) of detargeted AAV9 W503A conjugated with an antibody Fab targeting hTfR (Fab H1H12845B). Brain transduction was assessed 19 days post-injection. Representative images include 20x magnification planes of sagittal brain sections featuring the cerebellum, cortex, and olfactory bulb. To characterize viral transgene expression by cell type, sections were co-stained for eGFP and cell-specific markers, including microtubule-associated protein 2 (MAP2) and hexaribonucleotide-binding protein 3 (Fox-3 or NeuN) in neurons; glial fibrillary acidic protein (GFAP) in astrocytes; 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) and glutathione S-transferase pi (GST-pi) in oligodendrocytes; cluster of differentiation 31 (CD31) in vascular endothelial cells; and ionized calcium-binding adaptor molecule 1 (Iba1) in microglia. [Figure 12B]Immunofluorescence co-staining of eGFP expression and brain cell-specific markers in the brains of TFRC hu / hu mice after intravenous injection (4e11vg / mouse) of either WT AAV9 (top image) expressing an H2B-eGFP fusion genome for nuclear expression of eGFP, or detargeted AAV9 W503A conjugated with an antibody Fab targeting hTfR (Fab H1H12845B) (bottom image). Brain transduction was assessed 2 weeks after injection. Representative images include 20x magnification planes of sagittal brain sections featuring the cortex, cerebellum, olfactory bulb, and corpus callosum. To characterize viral transgene expression by cell type, sections were co-stained for eGFP and cell-specific markers, including hexaribonucleotide-binding protein 3 (Fox-3 or NeuN) and microtubule-associated protein 2 (MAP2) in neurons; glial fibrillary acidic protein (GFAP) and SRY-box transcription factor 9 (Sox9) in astrocytes; 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) and glutathione S-transferase pi (GST-pi) in oligodendrocytes; cluster of differentiation 31 (CD31) in vascular endothelial cells; and ionized calcium-binding adaptor molecule 1 (Iba1) in microglia. [Figure 13] Immunohistochemical staining of eGFP expression in the brains of TFRC hu / hu mice after intravenous (1e11 vg / mouse) or intracerebroventricular (1e10 vg / mouse) injection of wild-type (WT) AAV9, detargeted AAV9 W503A conjugated with an antibody Fab targeting hTfR (Fab H1H12845B), or detargeted AAV9 W503A conjugated with an antibody Fab targeting hASGR1 as a non-targeting control. Brain transduction was assessed 19 days post-injection. Representative images show sagittal sections of whole brains. [Figure 14A]Figure 14A shows qPCR data measuring viral DNA normalized to β-actin and compared with wild-type (WT) AAV9 (y-axis) in the brain (Figure 14A) and liver (Figure 14B) of TFRC hu / hu mice after intravenous (1e11 vg / mouse) or intracerebroventricular (1e10 vg / mouse) injection of WT AAV9, an antibody Fab targeting hTfR (Fab H1H12845B), or detargeted AAV9 W503A conjugated to hASGR1 as a non-targeting control (x-axis). AAV DNA was measured using a qPCR probe recognizing the eGFP sequence, and levels were normalized to the β-actin housekeeping gene. [Figure 14B] Same as above. [Figure 15] Immunohistochemical staining of eGFP expression in the brains of TFRC hu / hu mice after intravenous injection (1 e11 vg / mouse) of wild-type (WT) AAV9, antibody Fabs targeting hTfR (Fabs H1H12845B, H1H12848B, H1H31874B, H1H12841B, H1H12839B, H1H12835B, H1H12847B, H1H12850B, H1H12798B, H1H12843B, PN69331, H1H12799B), or detargeted AAV9 W503A conjugated to an antibody Fab targeting hASGR1 as a non-targeting control. Representative images include sagittal sections of the whole brain. [Figure 16]Quantification of eGFP expression by immunohistochemistry across brain regions of TFRC hu / hu mice after intravenous injection (1 e11 vg / mouse) of wild-type (WT) AAV9, detargeted AAV9 W503A conjugated with antibody Fab targeting hASGR1 as a non-targeting control, or detargeted AAV9 W503A conjugated with antibody Fab targeting hTfR (H1H12845B, H1H12848B, H1H31874B, H1H12841B, H1H12839B, H1H12835B, H1H12847B, H1H12850B, H1H12798B, H1H12843B, PN69331, and H1H12799B). The percentage of brain areas staining positive for DAB (y-axis, showing positive eGFP signal) 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 demonstrate enhanced brain transduction compared to WT AAV9 in multiple brain regions. [Figure 17] 1e11vg / mouse of the following AAVs (x-axis): (1) wild-type (WT) AAV9, (2) detargeted AAV9 W503A conjugated with an antibody Fab targeting hASGR1 as a non-targeting control, or detargeted AAV9 conjugated with an antibody Fab targeting hTfR. Quantification of eGFP expression by immunohistochemical staining in the cortex, hippocampus, thalamus, olfactory bulb, striatum, cerebellum, brainstem, and hypothalamus of TFRC hu / hu mice after intravenous injection of W503A:(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 area in the brain that stained positively for DAB (y-axis, showing positive eGFP signal) was quantified using HALO software. [Figure 18A]1e11vg / mouse of (1) wild-type (WT) AAV9, (2) detargeted AAV9 W503A conjugated with an antibody Fab targeting hASGR1 as a non-targeting control, or detargeted AAV9 conjugated with an antibody Fab targeting hTfR. Figure 18A shows qPCR data measuring AAV DNA (y-axis) in the brain (Figure 18A) and liver (Figure 18B) of TFRC hu / hu mice after intravenous injection of W503A: (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). AAV DNA was measured using a qPCR probe targeting the eGFP sequence, and levels were normalized to the GAPDH housekeeping gene and compared to wild-type WT AAV9. [Figure 18B] Same as above. [Figure 19A] Figure 19A shows qPCR data measuring AAV DNA (y-axis) in the liver (Figure 19B) 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 to an antibody Fab targeting mTfR ("8D3"), or detargeted AAV9 W503A conjugated to an antibody Fab targeting mTfR ("8D3") (x-axis). AAV DNA was measured using a qPCR probe targeting the eGFP sequence, and levels were normalized to the β-actin housekeeping gene and compared to the highest dose of WT AAV9. [Figure 19B] Same as above. [Figure 20A]Figure 20 shows 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 targeting mTfR ("8D3"), or detargeted AAV9 W503A conjugated with an antibody Fab targeting mTfR ("8D3"). Brain transduction was assessed 16 days post-injection. Representative images show a low-magnification sagittal section of the whole brain (Figure 20A) and a higher-magnification plane from the sagittal section featuring the cortex, hippocampus, and cerebellum (Figure 20B). [Figure 20B] Same as above. [Figure 21A] Figure 21A shows qPCR data measuring viral DNA normalized to b-actin relative to wild-type (wt) AAV9 (y-axis) in the liver (Figure 21A), heart (Figure 21B), quadriceps (Figure 21C), and brain (Figure 21D) of female TFRC hu / hu mice after injection of 1e11vg / mouse of wild-type AAV9, antibodies and Fabs targeting hTfR, or detargeted AAV9 W503A conjugated to hASGR1 as a non-targeting control. AAV DNA was measured using a qPCR probe recognizing the eGFP sequence, and levels were normalized to the b-actin housekeeping gene. [Figure 21B] Same as above. [Figure 21C] Same as above. [Figure 21D] Same as above. [Figure 22A]Immunohistochemical staining of eGFP expression in the brain, heart, quadriceps, and liver of female TFRC hu / hu mice after injection of 1e11vg / mouse wild-type AAV9 and detargeted AAV9 W503A conjugated with H1H12845B Fab or H1H12845B mAb. Representative images include (Figure 22A) the frontal cortex at 12x magnification, a vertical section of the heart at 12x magnification, a section of the quadriceps at 12x magnification, and a horizontal section of the liver at 14x magnification, or (Figure 22B) a sagittal section of the brain at higher magnification featuring the cerebellum at 12x magnification (first row of images from the top), the hippocampus at 3x magnification (second row of images from the top), the cortex at 12x magnification (third row of images from the top), and a horizontal section of the liver at 14x magnification (fourth row of images from the top). [Figure 22B] Same as above. [Figure 23A] Immunohistochemical staining of eGFP expression in the brain, heart, quadriceps, and liver of female TFRC hu / hu mice after injection of 1e11vg / mouse wild-type AAV9 and detargeted AAV9 W503A conjugated with H1H12839B Fab or H1H12839B mAb. Representative images include (FIG. 23A) a sagittal section of the brain at 12x magnification featuring the frontal cortex at 12x magnification, a vertical section of the heart at 12x magnification, a section of the quadriceps at 12x magnification, and a horizontal section of the liver at 14x magnification, or (FIG. 23B) a sagittal section of the brain at higher magnification featuring the cerebellum at 12x magnification, the hippocampus at 12x magnification, the cortex at 12x magnification, and a horizontal section of the liver at 14x magnification. [Figure 23B] Same as above. [Figure 24]We demonstrate that AAV particles targeting the mouse TfR transduce neonatal spinal motor neurons when administered directly into the CNS. Representative immunofluorescence images of GFP expression in the spinal cord of P15 C57BL / 6J mouse pups after a single intracerebroventricular (icv) injection at P0 of 1 e11 vg of the indicated AAV virus expressing GFP driven by the CBh promoter. Images include both a coronal hemispinal view of the lumbar spinal cord (top panel) and a magnified view of the ventral horn region (top panel) where motor neurons reside, visualized using an antibody against the motor neuron marker choline acetyltransferase (ChAT). GFP expression was amplified with an antibody against GFP in these samples. Plotted values represent the mean and standard deviation of independent biological replicates. [Figure 25] We demonstrate that AAV particles targeting the mouse TfR highly transduce neonatal spinal 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 was either virus-free (i.e., PBS) or contained AAV9 WT retargeted to mTfR (8D3 Fab) expressing GFP driven by the CBh promoter at 5e11vg. The images include both a coronal hemispin view of the lumbar spinal cord (top panel) and a magnified view of the ventral horn region (white box in the top panel), where motor neurons reside, visualized using an antibody against the motor neuron marker choline acetyltransferase (ChAT). The GFP signal shown here is native GFP expression without antibody amplification. Each panel represents an independent biological replicate. [Figure 26A]Figure 1 shows qPCR data from the brain, spinal cord, liver, heart, quadriceps, and spleen of WT C57BL / 6J mice measuring the abundance of transcripts encoding antibodies expressed by the indicated AAVs driven by the CAGG promoter after intravenous injection of 5 x 10 vg / mouse of detargeted AAV9 W503A conjugated with 8D3 scFvs targeting mTfR or 5 x 10 vg / mouse of AAV8 (x-axis). AAV RNA was measured using a qPCR probe recognizing the expressed human antibody sequence, and relative mRNA levels were normalized to the β-actin housekeeping gene and compared to the AAV8 control group. [Figure 26B] Figure 1 shows enzyme-linked immunosorbent assay (ELISA) detection of AAV-expressed human antibody titers in brain lysates of WT C57BL / 6J mice after intravenous injection of 5x10vg / mouse of detargeted AAV9 W503A conjugated with 8D3 scFvs targeting mTfR or 5e10vg / mouse of AAV8. Brain antibody concentrations were assessed 12 weeks after injection. [Figure 27] Figure 1 shows qPCR data measuring SNCA mRNA levels (y-axis) in the cortex, midbrain, and striatum of humanized SNCA mice after intravenous injection of 4x10 vg / mouse of detargeted AAV9 W503A expressing SNCA shRNA conjugated with 8D3 Fab targeting the mTfR. SNCA mRNA was measured using a qPCR probe targeting the human SNCA sequence. SNCA mRNA levels were normalized to the GAPDH housekeeping gene and compared to SNCA mRNA levels in naive (untreated) humanized SNCA mice. SNCA mRNA levels in the brain were assessed one month after injection. DETAILED DESCRIPTION OF THE INVENTION

[0039] Transferrin receptor Transferrin (Tf) and its receptor (TfR) are central to the regulation of iron metabolism. There are two transferrin receptors: TfR1, also called cluster of differentiation 71 (CD71), which is widely expressed and binds Tf with high affinity, and the less common TfR2, which is expressed primarily in hepatocytes. As used herein, "TfR" refers to TfR1 (CD71) unless otherwise specified.

[0040] Generally, uptake of Tf-bound iron via TfR1 is the major source of cellular iron import. 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) that contains the Tf-binding site. TfR1 is generally found as a homodimer, with the monomers linked by disulfide bonds on the cell surface and a molecular weight of approximately 180 kDa.

[0041] TfR exists in both human and non-human species, such as non-human primates and rodents. An exemplary amino acid sequence of human (h)TfR1 is set forth as SEQ ID NO: 434, which is identical to the amino acid sequence of the hTfR1 protein represented as Uniprot P02786. The gene encoding TfR, called TFRC, is found on chromosome 3 in humans. An exemplary gene sequence for TFRC, including annotated exons and introns, can be found in the NCBI database (Gene ID: 7037).

[0042] An exemplary amino acid sequence of mouse (m)TfR1 is set forth as SEQ ID NO: 435, which is identical to the amino acid sequence of the mTfR1 protein represented as Uniprot Q62351 and has approximately 77% amino acid sequence identity with hTfR1. The Tfrc gene is found on mouse chromosome 16. The complete gene sequence of mouse Tfrc, including annotated exons and introns, can be found in the NCBI database (Gene ID: 22042).

[0043] 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.

[0044] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps of the kind described herein and / or that will become apparent to those skilled in the art upon reading this disclosure.

[0045] "Percent identity" and the like can be readily determined for amino acid or nucleotide sequences spanning the entire length of a protein or a portion thereof. A portion may be at least about 5 amino acids or 24 nucleotides in length, respectively, and may be up to about 700 amino acids or 2100 nucleotides in length, respectively. Generally, when referring to "identity" between an HCVR and an LCVR, the percent identity refers to identity over the length of HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, and LCDR3. Generally, when referring to "identity," "homology," or "similarity" between two different adeno-associated viruses, the "identity," "homology," or "similarity" is determined with reference to "aligned" sequences. "Aligned" sequences or "alignment" refer to multiple nucleic acid sequences or protein (amino acid) sequences, which often include corrections for missing or added bases or amino acids compared to a reference sequence.

[0046] Alignment may be performed using any of a variety of publicly available or commercially available multiple sequence alignment programs. For amino acid sequences, sequence alignment programs are available, such as "Clustal X," "MAP," "PIMA," "MSA," "BLOCKMAKER," "MEME," and "Match-Box." Generally, one of these programs is used with default settings, but those skilled in the art can change these settings as needed. Alternatively, those skilled in the art can use a different algorithm or computer program that provides at least the same level of identity or alignment as that provided by the referenced algorithm and program. See, for example, J.D. Thomson et al., Nucl. Acids. Res., "A Comprehensive Comparison of Multiple Sequence Alignments," 27(13):2682-2690 (1999).

[0047] For nucleic acid sequences, several sequence alignment programs are also available. Examples of such programs include "Clustal W," "CAP Sequence Assembly," "MAP," and "MEME," which are accessible via web servers on the Internet. Other sources of such programs are known to those of skill in the art. Alternatively, the Vector NTI utility can be used. Numerous algorithms known in the art can also be used to measure nucleotide sequence identity, including the programs mentioned above. As another example, polynucleotide sequences can be compared using FASTA™, a program in GCG version 6.1. FASTA™ provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. For example, percent sequence identity between nucleic acid sequences can be determined using FASTA™ with its default parameters (word size of 6 and NOPAM factor for the scoring matrix) as provided in GCG version 6.1, incorporated herein by reference.

[0048] "Significant identity" includes alignment of amino acid or nucleic acid sequences that are at least 90%, such as at least 93%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98%, for example at least 99%, or such as at least 100% identical.

[0049] The term "chimera" encompasses functional genes or polypeptides comprising nucleic acid or amino acid sequences derived from at least two different organisms, e.g., at least a portion of a gene or polypeptide from a first AAV and a second AAV, wherein at least the first and second portions are operably linked. Unless designated as chimeric, the nucleotide sequences, genes, polypeptides, and amino acids are considered to be non-chimeric, e.g., comprising nucleic acid or amino acid sequences from only a single organism, e.g., only a single AAV.

[0050] The term "antibody" includes immunoglobulin molecules composed of four polypeptide chains: two heavy (H) and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable domain (V H ) and heavy chain constant region (C H The heavy chain constant region comprises at least three domains: C H 1. C H 2. C H Each light chain comprises a light chain variable domain (C H ) and light chain constant region (C L ). The heavy and light chain variable domains can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs), interspersed with relatively conserved regions called framework regions (FRs). Each heavy and light chain variable domain is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2, and HCDR3, and light chain CDRs may be abbreviated as LCDR1, LCDR2, and LCDR3. A typical tetrameric antibody structure contains two identical antigen-binding domains, each of which is represented by a V H Domain and V L It is formed by the association of domains, each of which is a respective C H Domain and C L The domains together form the antibody Fv region. Single domain antibodies contain a single antigen-binding domain, e.g., a V H or VLThe antigen-binding domain of an antibody, e.g., the portion of an antibody that recognizes and binds to the first member of a specific binding pair for an antigen, is also referred to as the "paratope." This is a small region (5-10 amino acids) of the antibody's Fv region, the fragment antigen-binding (Fab region), which may include portions of the antibody's heavy and / or light chains. The term "single-chain variable fragment" or "scFv" includes a single-chain fusion polypeptide comprising an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL). In some embodiments, the VH and VL are connected by a linker sequence of 10-25 amino acids. ScFv polypeptides may also contain other amino acid sequences, such as a CL region or a CH1 region. ScFv molecules may be produced by phage display or by direct subcloning of heavy and light chains from hybridomas or B cells. Ahmad et al., Clinical and Developmental Immunology, volume 2012, article ID 98025, is incorporated herein by reference for methods of generating scFv fragments by phage display and antibody domain cloning. A paratope specifically binds to a first member of a specific binding pair when the paratope binds to the first member of the specific binding pair with high affinity. The term "high affinity" antibody refers to an antibody that binds to a first member of the specific binding pair with a high affinity of about 10 -9 M or less (e.g., about 1 × 10 -9 M, 1 x 10 -10 M, 1 x 10 -11 M, or approximately 1 x 10 -12 K for its target first member of a specific binding pair of M D In one embodiment, K D is measured by surface plasmon resonance, e.g., BIACORE™, and in another embodiment, K D is measured by ELISA.

[0051] The phrase "complementarity-determining region" or "CDR" includes amino acid sequences encoded by the nucleic acid sequence of an organism's immunoglobulin genes, which amino acid sequences are normally (i.e., in wild-type animals) found between two framework regions in the variable region of the light or heavy chain of an immunoglobulin molecule (e.g., an antibody or T cell receptor). CDRs can be encoded, for example, by germline sequences or rearranged or unrearranged sequences, e.g., by naive or mature B cells, or T cells. CDRs can be somatically mutated (e.g., different from the sequence encoded in the animal germline), humanized, and / or modified with amino acid substitutions, additions, or deletions. Under some circumstances (e.g., with respect to 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, e.g., as a result of splicing or joining of sequences (e.g., VDJ rearrangement to form a heavy chain CDR3).

[0052] The term "light chain" includes immunoglobulin light chain sequences from any organism, and unless otherwise specified, includes human k and l light chains, as well as surrogate light chains, and VpreB. Unless otherwise specified, a 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 carboxyl terminus, a variable domain including FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region. The light chain variable domain is encoded by a light chain variable region gene sequence and generally includes a V segment derived from a repertoire of V and J segments present in the germline. L Segment and J LThe sequences, locations, and nomenclature of V and J light chain segments of various organisms can be found in the IMGT database, www.imgt.org. Light chains include, for example, light chains that selectively bind neither the first member of a specific binding pair nor the second first member of a specific binding pair selectively bound by the first member of the specific binding pair-binding protein in which the light chain appears. Light chains also include light chains that bind to and recognize or assist a heavy chain or another light chain by binding to and recognizing one or more first members of a specific binding pair selectively bound by the first member of the specific binding pair-binding protein in which the light chain appears. Common or universal light chains include light chains derived from the human Vk1-39Jk gene or the human Vk3-20Jk gene, including somatically mutated (e.g., affinity matured) versions thereof. Exemplary human Vk1-39Jk genes include those derived from the human Vk3-20Jk gene and the human Vk3-20Jk gene, including somatically mutated (e.g., affinity matured) versions thereof. L The segments include the human Vk1-39 gene segment, the human Vk3-20 gene segment, the human Vl1-40 gene segment, the human Vl1-44 gene segment, the human Vl2-8 gene segment, the human Vl2-14 gene segment, and the human Vl3-21 gene segment, including somatically mutated (e.g., affinity matured) forms thereof. Light chains can be made that include a variable domain from one organism (e.g., a human or rodent, such as a rat or mouse, or an avian, such as a chicken) and a constant region from the same or a different organism (e.g., a human or rodent, such as a rat or mouse, or an avian, such as a chicken).

[0053] The term "about" or "approximately" includes within a statistically significant range of values. Such a range can be within one order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The allowable variation encompassed by the term "about" or "approximately" will depend on the particular system under study and can be readily appreciated by one of ordinary skill in the art.

[0054] The phrase "heavy chain" or "immunoglobulin heavy chain" includes immunoglobulin heavy chain sequences, including immunoglobulin heavy chain constant region sequences, from any organism. Unless otherwise specified, a heavy chain variable domain contains three heavy chain CDRs and four FR regions. Fragments of heavy chains include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain contains (from N- to C-terminus) the variable domain followed by a C H 1 domain, hinge, C H 2 domain, and C H The functional fragment of the heavy chain has three domains that specifically recognize the first member of the specific binding pair (e.g., a K in the micromolar, nanomolar, or picomolar range). D The heavy chain variable domain is encoded by a variable region nucleotide sequence, which generally corresponds to the V present in germline cells. H , D H , and J H From the segment repertoire, V H , D H , and J H The sequences, locations, and nomenclature of V, D, and J heavy chain segments from 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".

[0055] The terms "heavy chain-only antibody," "heavy chain-only antigen-binding protein," "single domain antigen-binding protein," "single domain binding protein," and the like refer to a monomeric or homodimeric immunoglobulin molecule comprising an immunoglobulin-like chain comprising a variable domain operably linked to a heavy chain constant region, where the heavy chain constant region is typically a functional C H Therefore, the terms "heavy chain-only antibody," "heavy chain-only antigen-binding protein," "single domain antigen-binding protein," "single domain binding protein," and the like refer to antibodies that: (i) lack a functional CH a monomeric single-domain antigen-binding protein comprising one of the immunoglobulin-like chains comprising a variable domain operably linked to a heavy chain constant region lacking one domain; or (ii) a monomeric single-domain antigen-binding protein comprising two immunoglobulin-like chains, each of which is a functional C H In various embodiments, the homodimeric single domain antigen binding protein comprises two identical immunoglobulin-like chains, each of which is functionally linked to a heavy chain constant region lacking one domain, and a homodimeric single domain antigen binding protein comprising a variable domain operably linked to a heavy chain constant region lacking one domain. H In addition, each immunoglobulin-like chain of a single domain antigen-binding protein contains a variable domain, which is derived from a heavy chain variable region gene segment (e.g., V H , D H , J H ), light chain gene segments (e.g., V L , J L ), or a combination thereof, and the sequence of the heavy chain constant region (and optionally the hinge region) gene, e.g., C encoding IgG, IgA, IgE, IgD, or a combination thereof. H heavy chain constant region (C) containing a deletion or inactivating mutation in H A single domain antigen binding protein comprising a variable domain derived from a heavy chain gene segment may be referred to as a "V" H Single domain antibodies or V H Single domain antigen binding proteins comprising a variable domain derived from a light chain gene segment may be referred to as "V single domain antibody binding proteins," see, e.g., U.S. Pat. No. 8,754,287, U.S. Patent Publication Nos. 2014 / 0289876, 2015 / 0197553, 2015 / 0197554, 2015 / 0197555, 2015 / 0196015, 2015 / 0197556, and 2015 / 0197557, each of which is incorporated by reference in its entirety. ... LThese may be referred to as "single domain antigen binding proteins," see, e.g., U.S. Publication No. 2015 / 0289489, incorporated by reference in its entirety.

[0056] The term "light chain" includes immunoglobulin light chain sequences from any organism, and unless otherwise specified, includes human kappa (κ) and lambda (λ) light chains, as well as surrogate light chains, and VpreB. Unless otherwise specified, a 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 carboxyl terminus, a variable domain including FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region amino acid sequence. The light chain variable domain is encoded by a light chain variable region nucleotide sequence and generally includes a light chain V and J gene segment derived from a repertoire of light chain V and J gene segments present in germline cells. L and light chain J L The sequences, locations, and nomenclature of light chain V gene segments and light chain J gene segments of 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, light chains that do not selectively bind either the first member or the second first member of a specific binding pair selectively bound by the first member of the specific binding pair-binding protein in which the light chain appears. Light chains also include light chains that bind to, recognize, or assist heavy chains by binding to and recognizing one or more first members of a specific binding pair selectively bound by the first member of the specific binding pair-binding protein in which the light chain appears. Light chains also include light chains that bind to, recognize, or assist heavy chains by binding to and recognizing one or more first members of a specific binding pair selectively bound by the first member of the specific binding pair-binding protein in which the light chain appears. Common or universal light chains include light chains derived from the human Vk1-39Jk5 gene or the human Vk3-20Jk1 gene, including somatically mutated (eg, affinity matured) versions thereof.

[0057] The phrase "operably linked," as used herein, includes the physical juxtaposition (e.g., in three-dimensional space) of components or elements that interact directly or indirectly with each other or are positioned relative to each other so as to participate in a biological event, which juxtaposition achieves or enables such interaction and / or positioning. For example, a regulatory sequence (e.g., an expression control sequence) in a nucleic acid is said to be "operably linked" to a coding sequence when it 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" includes the covalent linkage of associated components or elements. Those skilled in the art will readily appreciate that in some embodiments, covalent linkage is not required to achieve effective operable linkage. For example, in some embodiments, a nucleic acid regulatory sequence that is operably linked to the coding sequence it controls is contiguous with the nucleotide of interest. Alternatively or additionally, in some embodiments, one or more such regulatory sequences function in trans or at a distance to regulate the coding sequence of interest. In some embodiments, the term "expression control sequences," as used herein, refers to polynucleotide sequences necessary and / or sufficient to affect the expression and processing of coding sequences to which they are linked. In some embodiments, expression control sequences may be or include appropriate transcription initiation, termination, promoter and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation signals, sequences that stabilize cytoplasmic mRNA, sequences that improve translation efficiency (e.g., Kozak consensus sequences), sequences that improve protein stability, and / or, in some embodiments, sequences that improve protein secretion. In some embodiments, one or more control sequences are preferentially or only active in a particular host cell or organism, or type thereof. By way of example, in prokaryotes, control sequences typically include a promoter, a ribosomal binding site, and a transcription termination sequence; in eukaryotes, in many embodiments, control sequences typically include a promoter, an enhancer, and / or a transcription termination sequence.Those skilled in the art will understand that in many embodiments, the term "control sequences" refers to components essential for expression and processing, and in some embodiments includes components whose presence is advantageous for expression (including, for example, leader sequences, targeting sequences, and / or fusion partner sequences).

[0058] "Retargeting" or "redirection" can include scenarios in which wild-type particles target some cells within a tissue and / or some organs within an organism, where general targeting of the tissue or organ is reduced or abolished by the insertion of heterologous amino acids, and retargeting to more specific cells within the tissue or more specific organs within the organism is achieved with (e.g., via) a targeting ligand that binds to a marker expressed by the specific cells. Such retargeting or redirection can also include scenarios in which wild-type particles target a tissue, where tissue targeting is reduced or abolished by the insertion of heterologous amino acids, and retargeting to an entirely different tissue is achieved with a targeting ligand.

[0059] A "specific binding pair," "protein:protein binding pair," and the like, comprises two proteins (e.g., a first member (e.g., a first polypeptide) and a second cognate member (e.g., a second polypeptide)) that interact to form a bond (e.g., a covalent bond between an epitope of a first member and an antigen-binding portion of an antibody that recognizes the epitope) or form a covalent isopeptide bond under conditions that allow or promote bond formation. In some embodiments, the term "cognate" refers to components that function together. Epitopes and their cognate antibodies are known in the art, particularly epitopes that can also serve as detectable labels (e.g., c-myc). Specific protein:protein binding pairs that can interact to form covalent isopeptide bonds 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, a first member of a protein:protein binding pair generally refers to a member of the protein:protein binding pair that is less than 30 amino acids in length and that forms a covalent isopeptide bond with a second cognate protein, which is generally larger but may also be less than 30 amino acids in length, such as the SpyTag:KTag system.

[0060] The term "isopeptide bond" refers to an amide bond between a carboxyl or carboxamide group and an amino group, at least one of which is not derived from the protein backbone or, alternatively, is not found to be part of the protein backbone. An isopeptide bond can form within a single protein, or between two peptides or between a peptide and a protein. Thus, an isopeptide bond can form intramolecularly within a single protein, or intramolecularly, i.e., between two peptide / protein molecules, e.g., between two peptide linkers. Typically, an isopeptide bond 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 between the alpha-amino terminus of a protein or peptide chain and an asparagine, aspartic acid, glutamine, or glutamic acid residue. Each residue of a pair involved in an isopeptide bond is referred to herein as a reactive residue. In a preferred embodiment of the present invention, an isopeptide bond may be formed between a lysine residue and an asparagine residue, or between a lysine residue and an aspartic acid residue. In particular, an isopeptide bond may occur between the side chain amine of a lysine and the carboxamide group of an asparagine or the carboxyl group of an aspartic acid.

[0061] The SpyTag:SpyCatcher system is described in U.S. Patent No. 9,547,003 and Zaveri et al. (2012) 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 the domain, Zakeri et al. obtained the "SpyTag" peptide, which has the sequence AHIVMVDAYKPTK (SEQ ID NO: 321), which forms an amide bond with its cognate protein, "SpyCatcher," a 112-amino acid polypeptide with the amino acid sequence set forth 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) PNAS 111:E1176-1181). SpyLigase (SEQ ID NO: 389) was engineered by deleting the beta strand from SpyCatcher containing the reactive lysine, 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) Angew Chem Int Ed Engl 56:16521-25, the entire contents of which are incorporated herein by reference. SpyTag002 has the amino acid sequence VPTIVMVDAYKRYK, set forth as SEQ ID NO: 324, and binds to SpyCatcher002 (SEQ ID NO: 442).

[0062] The SnoopTag:SnoopCatcher system is described in Veggiani (2016) PNAS 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). Incubation of SnoopTag and SnoopCatcher results in the formation of specific spontaneous isopeptide bonds between the complementary proteins. Veggiani (2016)), supra.

[0063] The isopeptag:pilinC specific binding pair is derived from SpyO128, a major pilin protein from Streptococcus pyogenes (Zakeir and Howarth (2010) J. Am. Chem. Soc. 132:4526-27). The isopeptag has the amino acid sequence TDKDMTITFTNKKDAE, set forth as SEQ ID NO: 325, and binds to pilin-C (residues 18-299 of SpyO128). Incubation of the isopeptag and pilinC results in the formation of a specific spontaneous isopeptide bond between the complementary proteins (Zakeir and Howarth (2010), supra).

[0064] The term "detectable label" includes, for example, a polypeptide sequence that is a member of a specific binding pair that specifically binds with high affinity to another polypeptide sequence, such as an antibody paratope, via a non-covalent bond. Exemplary, non-limiting detectable labels include a hexahistidine tag, a FLAG tag, a Strep II tag, a streptavidin-binding peptide (SBP) tag, a calcitonin-binding peptide (CBP), a glutathione S-transferase (GST), a 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, incorporated herein by reference.) A common detectable label for primate AAV is the B1 epitope (SEQ ID NO: 327). Some AAV capsid proteins described herein do not naturally contain the B1 epitope and can be modified herein to contain the B1 epitope. Generally, the AAV capsid proteins described herein may contain a sequence having substantial homology to the B1 epitope within the last 10 amino acids of the capsid protein. Thus, in some embodiments, non-primate AAV capsid proteins of the present invention may be modified with one or more but fewer than five point mutations within the last 10 amino acids of the capsid protein, thereby causing the AAV capsid protein to contain the B1 epitope.

[0065] The term "target cell" includes any cell in which expression of a nucleotide of interest is desired. Preferably, target cells exhibit receptors on their surface that allow the cells to be targeted by targeting ligands, as described below.

[0066] Terms such as "transduction" or "infection" refer to the introduction of nucleic acid into the nucleus of a target cell by a viral particle. Efficiency related to transduction, e.g., the term "transduction efficiency," refers to the fraction (e.g., percentage) of cells that express a nucleotide of interest after incubation with a set of viral particles containing the nucleotide of interest. Known methods for determining transduction efficiency include flow cytometry of transduced cells using a fluorescent reporter gene, RT-PCR for expression of the nucleotide of interest, etc.

[0067] Generally, the "reference" viral capsid protein / capsid / particle is identical to the test viral capsid protein / capsid / particle, except for the modification to be tested. For example, to determine the effect of inserting a 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 presence or absence of an appropriate targeting ligand) can be compared to the transduction efficiency of a reference viral particle that is identical to the test viral particle in every instance (e.g., additional point mutations, nucleotides of interest, number of viral particles, target cells, etc.) except for the presence of the first member of the specific binding pair (in the absence or presence of an appropriate targeting ligand, as needed). In some embodiments, the reference viral capsid protein can form a capsid with a second viral capsid protein that has been modified to include at least a first member of a protein:protein binding pair, and the reference viral capsid protein does not include the first member of the protein:protein binding pair, and preferably the capsid formed by the reference viral capsid protein and the modified viral capsid protein is a mosaic capsid.

[0068] Adeno-associated virus (AAV) "AAV" is an abbreviation for adeno-associated virus and can 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 from the p5 promoter, while Rep52 and Rep40 are transcribed from the p19 promoter. These proteins primarily function 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). VP3 accounts for over 80% of the total protein in the AAV virion (capsid). In mature virions, VP1, VP2, and VP3 are present in relative amounts of approximately 1:1:10, although ratios as high as 1:1:8 have also been reported. (Padron et al. (2005) J. Virology 79:5047-58)

[0069] The genomic sequences of various AAV serotypes, as well as the sequences of the natural inverted terminal repeats (ITRs), Rep proteins, and capsid subunits, 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 Nos. 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 for their teaching of AAV nucleic acid and amino acid sequences. For example, Srivistava et al. (1983) J. Virology 45:555, Chiorini et al. (1998) J. Virology 71:6823, Chiorini et al. (1999) J. Virology 73:1309, Bantel-Schaal et al. (1999) J. Virology 73:939, Xiao et al. al. (1999) J. Virology 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, Morris et al. al.(2004) Virology 33:375-383, U.S. Patent Publication No. 2017 / 0130245, International Patent Publication Nos. 00 / 28061, 99 / 61601, 98 / 11244, and U.S. Patent No. 6,156,303, each of which is incorporated by reference in its entirety. Table 5 herein provides sequences for various non-primate AAVs.

[0070] "AAV" encompasses all subtypes and both native and modified forms unless otherwise specified. AAVs include 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 type hu11 (AAV hu11), AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAVLK03, AAVrh32.33 (AAVrh.32.33), AAV retro (AAV retro), AAV PHP.B, AAV " Primate AAV " generally refers to the AAV isolated from primates.Such as " non-primate AAV " refers to the AAV isolated from non-primates.Similarly, " non-primate AAV " refers to the AAV isolated from non-primates.

[0071] As used herein, with respect to a gene (e.g., rep, cap, etc.), a capsid protein (e.g., VP1 capsid protein, VP2 capsid protein, VP3 capsid protein, etc.), a region of a capsid protein of a specified AAV (e.g., the PLA2 region, the VP1-u region, the VP1 / VP2 common region, the VP3 region), a nucleotide sequence (e.g., an ITR sequence), e.g., the cap gene or capsid protein of an AAV, "of a [specified] AAV" encompasses not only a gene or polypeptide comprising a nucleic acid sequence or amino acid sequence described herein for the specified AAV, respectively, but also variants of the gene or polypeptide, including variants that include the minimum number of nucleotides or amino acids required to retain one or more biological functions. As used herein, a variant gene or variant polypeptide comprises a nucleic acid sequence or amino acid sequence that differs from the nucleic acid sequence or amino acid sequence described herein for the specified AAV gene or polypeptide, where the difference generally does not alter at least one biological function of the gene or polypeptide and / or does not alter the phylogenetic characteristics of the gene or polypeptide; for example, the difference may be due to degeneracy in the genetic code, isolated variation, sequence length, etc. For example, as used herein, rep and cap genes can encompass rep and cap genes that differ from the wild-type genes in that the genes may encode one or more Rep and Cap proteins, respectively. In some embodiments, the Rep gene encodes at least Rep78 and / or Rep68. In some embodiments, the cap gene may differ from the wild-type in that one or more alternative start codons, or the sequence between one or more alternative start codons, have been removed, such that the cap gene encodes only one Cap protein, e.g., the VP2 and / or VP3 start codons have been removed or replaced, such that the cap gene encodes a functional VP1 capsid protein, but not a VP2 or VP3 capsid protein. Thus, as used herein, a rep gene encompasses any sequence that encodes a functional Rep protein.A cap gene includes any sequence that encodes at least one functional Cap protein.

[0072] It is known that wild-type cap genes express 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," and the like include proteins that are part of the viral capsid. For adeno-associated viruses, the capsid proteins are generally referred to as VP1, VP2, and / or VP3, and they may be encoded by a single cap gene. For AAV, the three AAV capsid proteins are naturally produced in an overlapping manner using alternative translation initiation codons in the cap ORF, but all three proteins use a common stop codon. The wild-type cap gene ORF encodes three alternative initiation codons from 5' to 3': the "VP1 initiation codon," the "VP2 initiation codon," and the "VP3 initiation codon," as well as one "consensus stop codon." VP1, the largest viral protein, is generally encoded from the VP1 start codon to the "consensus stop codon." VP2 is generally encoded from the VP2 start codon to the "consensus stop codon." VP3 is generally encoded from the VP3 start codon to the "consensus stop codon." Therefore, VP1 contains an N-terminal sequence not shared with VP2 or VP3, which is referred to as the VP1-unique region (VP1-u). The VP1-u region is generally encoded by the sequence of the wild-type cap gene, starting from the VP1 start codon to the "VP2 start codon." VP1-u contains a phospholipase A2 domain (PLA2), which may be important for infection and nuclear localization signals that may assist in targeting the virus to 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 is sometimes referred to herein as the VP3 region. The VP3 region is encoded from the VP3 start codon to the consensus stop codon. VP2 shares an additional approximately 60 amino acids with VP1, a region called the VP1 / VP2 common region.

[0073] In some embodiments, one or more of the Cap proteins of the present invention may be encoded by one or more cap genes having one or more ORFs. In some embodiments, the VP proteins of the present invention may be expressed from multiple ORFs containing nucleotide sequences encoding any combination of VP1, VP2, and / or VP3, each producing one or more of the VP1, VP2, and / or VP3 capsid proteins of the present invention, by using separate nucleotide sequences operably linked to at least one expression control sequence for expression in a packaging cell. In some embodiments, the VP capsid proteins of the present invention may be expressed individually from ORFs containing nucleotide sequences encoding any one of VP1, VP2, or VP3, by using separate nucleotide sequences operably linked to a single expression control sequence for expression in a viral replication cell, each producing only one of the VP1, VP2, or VP3 capsid proteins. In another embodiment, the VP proteins may be expressed from a single ORF comprising nucleotide sequences encoding VP1, VP2, and VP3 capsid proteins operably linked to at least one expression control sequence for expression in a viral replicating cell, each producing a VP1, VP2, and VP3 capsid protein. Thus, the amino acid positions provided herein may be provided relative to the VP1 capsid protein of the referenced AAV, and one of skill in the art would readily be able to determine the same amino acid position within the VP2 and / or VP3 capsid proteins of an AAV, and the corresponding amino acid positions among different AAVs, respectively.

[0074] Non-limiting examples of wild-type and / or genetically modified nucleic acid sequences for cap genes and cap proteins useful for retargeting viral particles described herein are set forth in SEQ ID NOs: 392-432.

[0075] The term "inverted terminal repeats" or "ITRs" refers to symmetrical nucleic acid sequences in the genome of adeno-associated viruses that are required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. ITRs function as origins of replication for viral DNA synthesis and are essential cis-elements for AAV particle production, including packaging into AAV particles.

[0076] The AAV ITRs contain recognition sites for the replication proteins Rep78 or Rep68. The "D" region of the ITR contains the DNA nick site where DNA replication is initiated, providing directionality to the nucleic acid replication process. AAV replication in mammalian cells typically involves two ITR sequences.

[0077] A single ITR may be engineered with Rep binding sites on both strands of the "A" region and the two symmetric D regions on either side of the ITR palindrome. On a double-stranded circular DNA template, such engineered constructs allow Rep78- or Rep68-initiated nucleic acid replication to proceed in both directions. A single ITR is sufficient for AAV replication in circular particles. In the methods of generating AAV viral particles of the invention, the rep coding sequence encodes a Rep protein or a Rep protein equivalent, which can bind to the ITRs contained on a transfer plasmid.

[0078] When expressed by a packaging cell together with an appropriate Rep protein, the Cap protein of the present invention can encapsidate a transfer plasmid containing a nucleotide of interest and an even number of two or more ITR sequences. In some embodiments, the transfer plasmid contains one ITR sequence. In some embodiments, the transfer plasmid contains two ITR sequences.

[0079] 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 end of the AAV genome, thereby providing access to the replication machinery of the viral replicating cell. As is known, Rep proteins may be expressed from multiple ORFs containing nucleotide sequences encoding any combination of Rep78, Rep68, Rep52, and / or Rep40, each producing one or more of the Rep78, Rep68, Rep52, and / or Rep40 Rep proteins, by using separate nucleotide sequences operably linked to at least one expression control sequence for expression in the viral replicating cell. Alternatively, the Rep proteins may be individually expressed from ORFs containing nucleotide sequences encoding any one of Rep78, Rep68, Rep52, or Rep40 by using separate nucleotide sequences operably linked to a single expression control sequence for expression in a packaging cell, each producing only one Rep78, Rep68, Rep52, or Rep40 Rep protein. In another embodiment, the Rep proteins may be expressed from a single ORF containing nucleotide sequences encoding the Rep78 and Rep52 Rep proteins operably linked to at least one expression control sequence for expression in a viral replication cell, each producing the Rep78 and Rep52 Rep proteins.

[0080] In the methods of producing AAV virions, e.g., viral particles, of the invention, the rep coding sequence and cap gene of the invention may be provided in a single packaging plasmid. However, one skilled in the art will recognize that this requirement is not required. Such viral particles may or may not contain a genome.

[0081] A "chimeric AAV capsid protein" includes an AAV capsid protein that contains amino acid sequences, e.g., portions, from two or more different AAVs and that has the ability to form and / or forms an AAV viral capsid / virion. A chimeric AAV capsid protein may be encoded by a chimeric AAV capsid gene, e.g., a chimeric nucleotide sequence containing multiple, e.g., at least two, nucleic acid sequences, each of which is identical to a portion of a capsid gene encoding a capsid protein of a separate AAV, and which together encode a functional chimeric AAV capsid protein. Association of a chimeric capsid protein with a particular AAV indicates that the capsid protein contains one or more portions derived from a capsid protein of an AAV and one or more portions derived from a 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 proteins of AAV2 and one or more portions of the VP1, VP2, and / or VP3 capsid proteins of a different AAV.

[0082] 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 to the sequence from which the portion is derived; see Penzes (2015) J. General Virol. 2769. A "portion" encompasses any contiguous segment of amino acids or nucleotides sufficient to determine that the polypeptide or nucleic acid molecule from which the portion is derived is "of a [designated] AAV," or has "significant identity" to a particular AAV, e.g., a non-primate AAV or a distantly related AAV. In some embodiments, a portion comprises at least 5 amino acids or 15 nucleotides that are 100% identical to a sequence associated with the designated AAV. In some embodiments, a portion comprises at least 10 amino acids or 30 nucleotides that are 100% identical to a sequence associated with the designated AAV. In some embodiments, a portion comprises at least 15 amino acids or 45 nucleotides that are 100% identical to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 20 amino acids or 60 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 25 amino acids or 75 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 30 amino acids or 90 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 35 amino acids or 105 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 40 amino acids or 120 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 45 amino acids or 135 nucleotides that have 100% identity to a sequence associated with the designated AAV.In some embodiments, the portion comprises at least 50 amino acids or 150 nucleotides that have 100% identity to a sequence associated with a designated AAV. In some embodiments, the portion comprises at least 60 amino acids or 180 nucleotides that have 100% identity to a sequence associated with a designated AAV. In some embodiments, the portion comprises at least 70 amino acids or 210 nucleotides that have 100% identity to a sequence associated with a designated AAV. In some embodiments, the portion comprises at least 80 amino acids or 240 nucleotides that have 100% identity to a sequence associated with a designated AAV. In some embodiments, the portion comprises at least 90 amino acids or 270 nucleotides that have 100% identity to a sequence associated with a designated AAV. In some embodiments, the portion comprises at least 100 amino acids or 300 nucleotides that have 100% identity to a sequence associated with a designated AAV.

[0083] Modified viral capsid proteins, virus particles, and nucleic acids In some embodiments, a Cap protein, e.g., a VP1 capsid protein described herein, a VP2 capsid protein described herein, and / or a VP3 capsid protein described herein, is modified to include, e.g., a first member of a protein:protein binding pair, a detectable label, a point mutation, etc.

[0084] Chimeras are a type of modification described herein. Generally, modifications of a specified AAV gene or polypeptide or variant thereof result in a nucleic acid or amino acid sequence that differs from the nucleic acid or amino acid sequence described herein for the specified AAV, and the modification alters, imparts, or eliminates one or more biological functions, but does not change the phylogenetic characteristics of the gene or polypeptide. Modifications can include, for example, the insertion of a first member of a protein:protein binding pair and point mutations, e.g., to reduce or abolish the capsid protein's natural tropism and / or to cause the capsid protein to include a detectable label. Preferred modifications include those that do not alter, and preferably reduce low to no recognition, of the modified capsid by pre-existing antibodies present in the general population produced during infection with another AAV, such as, for example, infection with a serotype such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, virions based on such serotypes, virions from currently used AAV gene therapy modalities, or combinations thereof. Other modifications described herein include modifications of the capsid protein, whereby the capsid protein comprises a first member of a protein:protein binding pair, a detectable label, or the like, and generally result from modifications at the genetic level, for example, via modification of the cap gene.

[0085] In some embodiments, a viral capsid comprising a modified viral capsid protein described herein is a mosaic capsid, e.g., comprising at least two pairs of VP1, VP2, and / or VP3 proteins, each pair encoded by a different cap gene. A mosaic capsid herein generally refers to a mosaic of a first viral capsid protein that has been 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. In the context of a mosaic capsid, the second viral capsid protein lacking the first member of the protein:protein binding pair is sometimes referred to as a reference capsid protein and is encoded by a reference cap gene. In some mosaic capsid embodiments, preferably when the VP1, VP2, and / or VP3 capsid protein modified with the first member of the protein:protein pair is not a chimeric capsid protein, the VP1, VP2, and / or VP3 reference capsid protein may comprise an amino acid sequence identical to a viral VP1, VP2, and / or VP3 capsid protein modified with the first member of the protein:protein binding pair, but the reference capsid protein lacks the first member of the protein:protein binding pair. In some mosaic capsid embodiments, the VP1, VP2, and / or VP3 reference capsid protein corresponds to a viral VP1, VP2, and / or VP3 capsid protein modified with the first member of the protein:protein binding pair, but the reference capsid protein lacks the first member of the protein:protein binding pair. In some embodiments, the VP1 reference capsid protein corresponds to a viral VP1 capsid protein modified with a first member of a protein:protein binding pair, where the reference capsid protein lacks the first member of the protein:protein binding pair. In some embodiments, the VP2 reference capsid protein corresponds to a viral VP2 capsid protein modified with a first member of a protein:protein binding pair, where the reference capsid protein lacks the first member of the protein:protein binding pair.In some embodiments, the VP3 reference capsid protein corresponds to a viral VP3 capsid protein modified with a first member of a protein:protein binding pair, but 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 a first member of a protein:protein binding pair, the reference protein may be the corresponding capsid protein, a portion of which forms a part 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 include a first member of a protein:protein binding pair may further comprise as a reference capsid protein an AAV2 VP1 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, or 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 may further comprise, as a reference capsid protein, an AAV2 VP2 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, or 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 may further comprise, as a reference capsid protein, an AAV2 VP2 capsid protein lacking the first member, an AAAV VP1 capsid protein lacking the first member, or a chimeric AAV2 / AAAV VP3 capsid protein lacking the first member.In some mosaic capsid embodiments, the reference capsid protein may be any capsid protein so long as it lacks the first member of the protein:protein binding pair and so long as it is capable of forming a capsid with a first capsid protein modified with the first member of the protein:protein binding pair.

[0086] Generally, mosaic particles may be produced by transfecting a mixture of modified and reference cap genes into producer cells in a specified ratio. The ratio of protein subunits in the particle, e.g., the ratio of modified VP protein:unmodified VP protein, stoichiometrically reflects, but does not necessarily reflect, the ratio of at least two of the cap gene encoding a first capsid protein modified with a first member of a protein:protein binding pair and 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 particle does not stoichiometrically reflect the ratio of modified cap gene:reference cap gene transfected into the packaging cells.

[0087] In some mosaic virus particle embodiments, the ratio of protein subunits ranges from about 1:59 to about 59:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:1 (e.g., a mosaic virus particle includes about 30 modified capsid proteins and about 30 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:2 (e.g., a mosaic virus particle includes about 20 modified capsid proteins and about 40 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 3:5. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:3 (e.g., a mosaic virus particle includes about 15 modified capsid proteins and about 45 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:4 (e.g., the mosaic virus particle comprises about 12 modified capsid proteins and 48 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:5 (e.g., the mosaic virus particle comprises 10 modified capsid proteins and 50 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:6. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:7. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:8. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:9 (e.g., the mosaic virus particle comprises about 6 modified capsid proteins and about 54 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:10.In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:11 (e.g., the mosaic virus particle comprises about 5 modified capsid proteins and about 55 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:12. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:13. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:14 (e.g., the mosaic virus particle comprises about 4 modified capsid proteins and about 56 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:15. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:19 (e.g., the mosaic virus particle comprises about 3 modified capsid proteins and about 57 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:29 (e.g., the mosaic virus particle comprises about 2 modified capsid proteins and about 58 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 1:59. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 2:1 (e.g., the mosaic virus particle comprises about 40 modified capsid proteins and about 20 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 5:3. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 3:1 (e.g., the mosaic virus particle comprises about 45 modified capsid proteins and about 15 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 4:1 (e.g., a mosaic virus particle includes about 48 modified capsid proteins and 12 reference capsid proteins).In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 5:1 (e.g., the mosaic virus particle comprises 50 modified capsid proteins and 10 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 6:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 7:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 8:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 9:1 (e.g., the mosaic virus particle comprises about 54 modified capsid proteins and about 6 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 10:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 11:1 (e.g., the mosaic virus particle comprises about 55 modified capsid proteins and about 5 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 12:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 13:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 14:1 (e.g., a mosaic virus particle comprises about 56 modified capsid proteins and about 4 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 15:1. In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 19:1 (e.g., a mosaic virus particle comprises about 57 modified capsid proteins and about 3 reference capsid proteins). In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 29:1 (e.g., a mosaic virus particle comprises about 58 modified capsid proteins and about 2 reference capsid proteins).In some mosaic virus particle embodiments, the ratio of protein subunits is at least about 59:1.

[0088] In some non-mosaic viral particle embodiments, the ratio of protein subunits may be 1:0, and each capsid protein of the non-mosaic viral particle is modified with a first member of a protein:protein binding pair. In some non-mosaic viral particle embodiments, the ratio of protein subunits may be 0:1, and each capsid protein of the non-mosaic viral particle is not modified with a first member of a protein:protein binding pair.

[0089] In some embodiments, the capsid proteins of the present 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, immobilized divalent cations (e.g., Ni 2+), a biotin moiety that binds to immobilized avidin (e.g., on a biotinylated polypeptide sequence in vivo), a GST (glutathione S-transferase) sequence that binds to immobilized glutathione, an S tag that binds to immobilized S protein, an antigen that binds to an immobilized antibody or domain or fragment thereof (including, for example, T7, myc, FLAG, and B tags that bind to the corresponding antibody), a FLASH tag (specific Examples of detectable labels include a highly detectable label linked to an arsenic-based moiety (e.g., a highly detectable label linked to an arsenic-based moiety), a receptor or receptor domain that binds to an immobilized ligand (or vice versa), protein A or a derivative thereof (e.g., Z) that binds to immobilized IgG, maltose-binding protein (MBP) that binds to immobilized amylose, albumin-binding protein that binds to immobilized albumin, a chitin-binding domain that binds to immobilized chitin, a calmodulin-binding peptide that binds to immobilized calmodulin, and a cellulose-binding domain that binds to immobilized cellulose. Another example of a detectable label is the SNAP-tag, commercially available from Covalys (www.covalys.com). In some embodiments, the detectable labels disclosed herein include detectable labels that are recognized only by an antibody paratope. In some embodiments, the detectable labels disclosed herein include detectable labels that are recognized by an antibody paratope and other specific binding pairs.

[0090] In some embodiments, the detectable label forms a binding pair with an immunoglobulin constant domain. In some embodiments, the detectable label and / or the detectable label binds to a metal ion, e.g., Ni 2+ , Co 2+ , Cu 2+ , Zn 2+ , Fe 3+ In some embodiments, the detectable label is selected from the group consisting of streptavidin, StrepII, HA, L14, 4C-RGD, LH, and protein A.

[0091] 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).

[0092] In some embodiments, the detectable label is a B cell epitope, e.g., about 1 amino acid to about 35 amino acids in length, that forms a binding pair with an antibody paratope, 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.

[0093] In some embodiments, the capsid protein of the invention comprises at least a first member of a peptide:peptide bond pair.

[0094] In some embodiments, the capsid proteins of the present invention comprise a first member of a protein:protein binding pair comprising a detectable label, which may also be used for detection and / or isolation of the Cap protein and / or may be used 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 of a targeting ligand comprising a multispecific binding protein capable of binding both the detectable label and a target expressed by a cell of interest. In some embodiments, the Cap proteins of the present invention comprise a first member of a protein:protein binding pair comprising c-myc (SEQ ID NO: 326). The use of a detectable label as a first member of a protein:protein binding pair is described, for example, in WO2019 / 006043, the entire contents of which are incorporated herein by reference.

[0095] In some embodiments, the capsid protein comprises a first member of a protein:protein binding pair, where the protein:protein binding pair forms a covalent isopeptide bond. In some embodiments, the first member of the protein:protein binding pair is covalently linked to a cognate second member of the protein:protein binding pair via an isopeptide bond, and optionally, the cognate second member of the protein:protein binding pair is fused to a targeting ligand, where the targeting ligand binds to a target expressed by a cell of interest. In some embodiments, the protein:protein binding pair may 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 biologically active portion thereof) and the protein (second cognate member) is SpyCatcher (or a biologically active portion thereof). In some embodiments, the first member is SpyTag (or a biologically active portion thereof) and the protein (second cognate member) is KTag (or a biologically active portion thereof). In some embodiments, the first member is KTag (or a biologically active portion thereof) and the protein (second cognate member) is SpyTag (or a biologically active portion thereof). In some embodiments, the first member is SnoopTag (or a biologically active portion thereof) and the protein (second cognate member) is SnoopCatcher (or a biologically active portion thereof). In some embodiments, the first member is Isopeptag (or a biologically active portion thereof) and the protein (second cognate member) is Pilin-C (or a biologically active portion thereof). In some embodiments, the first member is SpyTag002 (or a biologically active portion thereof) and the protein (second cognate member) is SpyCatcher002 (or a biologically active portion thereof). In some embodiments, the Cap protein of the present invention comprises a SpyTag.The use of a first member of a protein:protein binding pair is described in WO2019 / 006046, which is incorporated herein in its entirety.

[0096] 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 of the invention, chemically attached to the Cap protein of the invention, and / or displayed by the Cap protein of the invention) via a first or second linker, e.g., an amino acid spacer that is at least one amino acid in length. In some embodiments, the first member of the protein:protein binding pair is flanked by first and / or second linkers, e.g., first and / or second amino acid spacers, each of which is at least one amino acid in length.

[0097] In some embodiments, the first and / or second linkers are not identical. In some embodiments, the first and / or second linkers are each independently 1 or 2 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, or 3 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, or 4 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, or 5 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, or 5 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, or 5 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, 5, or 6 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, 5, 6, or 7 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, 5, 6, 7, or 8 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. In some embodiments, the first and / or second linkers are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids or more in length.

[0098] In some embodiments, the first and second linkers are the same sequence and / or length, each being one amino acid long. In some embodiments, the first and second linkers are the same length, each being one amino acid long. In some embodiments, the first and second linkers are the same length, each being two amino acids long. In some embodiments, the first and second linkers are the same length, each being three amino acids long. In some embodiments, the first and second linkers are the same length, each being four amino acids long, e.g., the linkers are GLSG (SEQ ID NO: 328). In some embodiments, the first and second linkers are the same length, each being five amino acids long. In some embodiments, the first and second linkers are the same length, each being six amino acids long, e.g., the first and second linkers each comprise the sequence GLSGSG (SEQ ID NO: 329). In some embodiments, the first and second linkers are the same length, each being seven amino acids long. In some embodiments, the first and second linkers are the same length, each 8 amino acids in length, e.g., the first and second linkers each comprise the sequence GLSGLSGS (SEQ ID NO: 330). In some embodiments, the first and second linkers are the same length, each 9 amino acids in length. In some embodiments, the first and second linkers are the same length, each 10 amino acids in length, e.g., the first and second linkers each comprise GLSGLSGLSG (SEQ ID NO: 331) or GLSGGSGLSG (SEQ ID NO: 332). In some embodiments, the first and second linkers are the same length, each more than 10 amino acids in length.

[0099] Generally, the amino acid sequence of the first member of a protein:protein binding pair described herein, e.g., comprising the first member of a specific binding pair alone or in combination with one or more linkers, is from about 5 amino acids to about 50 amino acids in length. In some embodiments, the amino acid sequence of the first member of a protein:protein binding pair is at least 5 amino acids in length. In some embodiments, the amino acid sequence of the first member of a protein:protein binding pair is 6 amino acids in length. In some embodiments, the amino acid sequence of the first member of a protein:protein binding pair is 7 amino acids in length. In some embodiments, the amino acid sequence of the first member of a protein:protein binding pair is 8 amino acids in length. In some embodiments, the amino acid sequence of the first member of a protein:protein binding pair is 9 amino acids in length. In some embodiments, the amino acid sequence of the first member of a protein:protein binding pair is 10 amino acids in length. In some embodiments, the amino acid sequence of the first member of a protein:protein binding pair is 11 amino acids in length. In some embodiments, the amino acid sequence of the first member of a protein:protein binding pair is 12 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 13 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 14 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 15 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 16 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 17 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 18 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 19 amino acids in length.In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 20 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 21 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 22 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 23 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 24 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 25 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 26 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 27 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 28 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 29 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 30 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 31 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 32 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 33 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 34 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 35 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 36 amino acids in length.In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 37 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 38 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 39 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 40 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 41 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 42 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 43 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 44 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 45 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 46 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 47 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 48 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 49 amino acids in length. In some embodiments, the amino acid sequence of the first member of the protein:protein binding pair is 50 amino acids in length.

[0100] Due to the high degree of conservation, at least among widespread and numerous closely related family members, corresponding insertion sites in AAVs other than those listed can be identified by amino acid alignment or comparison of capsid structure. For example, for exemplary alignments of different AAV capsid proteins, see Rutledge et al. (1998) J. Virol. 72:309-19, Mietzsch et al. (2019) Viruses 11,362, 1-34, and U.S. Patent No. 9,624,274, each of which is incorporated herein by reference in its entirety. For example, Mietzsch et al. (2019) presents an overlay of ribbons from various dependoparvoviruses in Figure 7, depicting variable regions VR I through VR IX. Using structural and sequence analysis as described in the literature, one skilled in the art can determine which amino acids within the variable regions correspond to amino acid sequences of AAV that are amenable to insertion of the first member of a protein:protein binding pair and / or a detectable label.

[0101] Thus, in some embodiments, the first member of the protein:protein binding pair and / or the detectable label is inserted in the VP1 capsid protein of the non-primate AAV after an 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 the protein:protein binding pair and / or the detectable label is inserted in the VP1 capsid protein of the non-primate AAV between amino acids corresponding to N587 and R588 of the AAV2 VP1 capsid. Additional suitable insertion sites for non-primate VP1 capsid proteins include sites corresponding to I-1, I-34, I-138, I-139, I-161, I-261, 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, I-591, I-657, I-664, I-713, and I-716 of the AAV2 VP1 capsid protein (Wu et al. (2000) J. Virol. 74:8635-8647). In some embodiments, the insertion site for the non-primate VP1 capsid protein corresponds to I-453. The modified viral capsid protein described herein can be a non-primate capsid protein comprising a first member of a protein:protein binding pair and / or a detectable label inserted at a position corresponding to a position in an AAV2 capsid protein selected from the group consisting of 1-1, 1-34, 1-138, 1-139, 1-161, 1-261, 1-266, 1-381, 1-447, 1-448, 1-453, 1-459, 1-471, 1-520, 1-534, 1-570, 1-573, 1-584, 1-587, 1-588, 1-591, 1-657, 1-664, 1-713, 1-716, and combinations thereof. In some embodiments, the insertion site in the non-primate VP1 capsid protein corresponds to 1-453.Additional suitable insertion sites in non-primate AAVs include sites corresponding to I-587 in AAV1, I-589 in AAV1, I-585 in AAV3, I-585 in AAV4, and I-585 in AAV5. In some embodiments, a modified viral capsid protein described herein can be a non-primate capsid protein comprising a first member of a protein:protein binding pair and / or a detectable label inserted at a position corresponding to a position selected from the group consisting of I-587(AAV1), I-589(AAV1), I-585(AAV3), I-585(AAV4), I-585(AAV5), and combinations thereof.

[0102] In some embodiments, the first member of the protein:protein binding pair and / or the detectable label is, in the VP1 capsid protein of non-primate AAV, I444 of avian AAV capsid protein VP1, I580 of avian AAV capsid protein VP1, I573 of bearded dragon AAV capsid protein VP1, I436 of bearded dragon AAV capsid protein VP1, I429 of sea lion AAV capsid protein VP1, I4 ... The amino acid sequence is inserted after an amino acid position corresponding to an amino acid position selected from the group consisting of I430 of sea lion AAV capsid protein VP1, I431 of sea lion AAV capsid protein VP1, I432 of sea lion AAV capsid protein VP1, I433 of sea lion AAV capsid protein VP1, I434 of sea lion AAV capsid protein VP1, I436 of sea lion AAV capsid protein VP1, I437 of sea lion AAV capsid protein VP1, and I565 of sea lion AAV capsid protein VP1.

[0103] As used herein, the nomenclature I-###, I#, etc. refers to an insertion site (I) where ### designates the amino acid numbering relative to the VP1 protein of the AAV capsid protein; however, such insertions may be located directly N- or C-terminal, preferably one amino acid C-terminal within a sequence of five amino acids N- or C-terminal to a given amino acid, and preferably three, more preferably two, and particularly one amino acid C-terminal within a sequence N- or C-terminal to a given amino acid. Furthermore, the positions referred to herein are relative to the VP1 protein encoded by the AAV capsid gene; corresponding positions (and point mutations thereof) can be readily identified in the VP2 and VP3 capsid proteins encoded by the capsid genes by performing sequence alignments of the VP1, VP2, and VP3 proteins encoded by the appropriate AAV capsid genes.

[0104] Thus, because capsid proteins are encoded by overlapping reading frames of the same gene with offset start codons, insertion of the coding nucleic acid of one of these sites in the cap gene into the corresponding position also results in insertion of VP1, VP2, and / or VP3. Thus, for example, for AAV2, according to this nomenclature, an insertion of amino acids 1-138 is inserted only into VP1, an insertion of 138-203 is inserted into VP1 and VP2, and an insertion of 203 to the C-terminus is inserted into VP1, VP2, and VP3, and of course, this also applies to insertion site I-587. Thus, the present invention encompasses AAV structural genes with corresponding insertions in the VP1, VP2, and / or VP3 proteins.

[0105] Also provided herein are nucleic acids encoding the VP3 capsid proteins of the present invention. AAV capsid proteins may, but need not, be encoded by overlapping reading frames of the same gene with shifted start codons. In some embodiments, a nucleic acid encoding a VP3 capsid protein of the present invention does not also encode a VP2 capsid protein or a VP1 capsid protein of the present invention. In some embodiments, a nucleic acid encoding a VP3 capsid protein of the present invention may also encode a VP2 capsid protein of the present invention, but not a VP1 capsid of the present invention. In some embodiments, a nucleic acid encoding a VP3 capsid protein of the present invention may also encode a VP2 capsid protein of the present invention and a VP1 capsid of the present invention.

[0106] In some embodiments, a viral capsid comprising a modified viral capsid protein comprising a first and second member of a protein:protein binding pair (e.g., the second member is operably linked to a targeting ligand and comprises a multispecific binding protein) is capable of infecting a particular cell, e.g., has an enhanced ability to target and bind to a particular cell compared to a control viral capsid, which is identical to the modified viral capsid protein except that the control viral capsid lacks either or both of the first and second members of the protein:protein binding pair, e.g., comprises the control capsid protein. In some embodiments, a viral capsid comprising a modified viral capsid protein described herein associated with a first and second member of a protein:protein binding pair linked to a targeting ligand exhibits a detectable transduction efficiency compared to the undetectable transduction efficiency of the control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 10% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 20% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a suitable first and second member of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 30% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein conjugated to appropriate first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 40% greater than the transduction efficiency of a control viral capsid.In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 50% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 60% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 70% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 75% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 80% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 85% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein conjugated to appropriate first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 90% greater than the transduction efficiency of a control capsid.In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to appropriate first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 95% greater than the transduction efficiency of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to appropriate first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 99% greater than the transduction efficiency of a control viral capsid.

[0107] In some embodiments, a viral capsid comprising a modified viral capsid protein comprising a first and second member of a protein:protein binding pair (e.g., the second member is operably linked to a targeting ligand and comprises a multispecific binding protein) is capable of infecting a particular cell, e.g., has an enhanced ability to target and bind to a particular cell compared to a control viral capsid, which is identical to the modified viral capsid protein except that the control viral capsid lacks either or both of the first and second members of the protein:protein binding pair, e.g., comprises the control capsid protein. In some embodiments, a viral capsid comprising a modified viral capsid protein described herein associated with a first and second member of a protein:protein binding pair linked to a targeting ligand exhibits a detectable transduction efficiency compared to the undetectable transduction efficiency of the control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 10% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 20% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a suitable first and second member of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 30% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein conjugated to appropriate first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 40% greater than the transduction efficiency of a control viral capsid.In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 50% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 60% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 70% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 75% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 80% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is 85% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein conjugated to appropriate first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 90% greater than the transduction efficiency of a control capsid.In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to appropriate first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 95% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is 99% greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 1.5-fold greater than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is at least two-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a suitable first and second member of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is at least three-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a suitable first and second member of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is at least four-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein conjugated to appropriate first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 5-fold greater than the transduction efficiency of a control viral capsid.In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is at least six-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is at least seven-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair coupled to a targeting ligand exhibit a transduction efficiency that is at least eight-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 9-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 10-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to suitable first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 20-fold higher than that of a control capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein conjugated to appropriate first and second members of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 30-fold greater than the transduction efficiency of a control viral capsid.In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 40-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 50-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 60-fold greater than the transduction efficiency of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 70-fold higher than the transduction efficiency of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 80-fold higher than the transduction efficiency of a control viral capsid. In some embodiments, viral capsids comprising a modified viral capsid protein described herein coupled to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 90-fold higher than the transduction efficiency of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein described herein bound to a first and second member of a protein:protein binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 100-fold greater than the transduction efficiency of a control viral capsid.In some embodiments, viral particles of the present invention comprising a viral capsid protein comprising an amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof, and optionally a first and second member of a protein:protein binding pair (e.g., the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.), can better evade neutralization by pre-existing antibodies in serum isolated from a human patient compared to a suitable control viral particle (e.g., comprising a viral capsid of an AAV serotype that includes a portion of the viral capsid protein in the viral capsid of the present invention as part of the viral capsid protein comprising the amino acid sequence of a capsid protein of a non-primate AAV, a remote AAV, or a combination thereof), and optionally a first and second member of a protein:protein binding pair (e.g., the second member is operably linked to a targeting ligand, comprises a multispecific binding protein, etc.). In some embodiments, In some embodiments, viral particles of the present invention comprising a viral capsid protein that includes an amino acid sequence of a capsid protein of a non-primate AAV, a distantly related AAV, or a combination thereof, require at least twice as much total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) as appropriate control viral particles (e.g., viral particles of the present invention have an IC50 value that is at least twice that of the control viral particles).

[0108] Targeting Ligands The viral particles described herein may further comprise a targeting ligand. "Retargeting" or "directing" may include a scenario in which a wild-type viral particle targets a tissue and / or some cells in some organs within an organism, the broad targeting of the tissue or organ is reduced or abolished by the insertion of a detectable label or targeting ligand, and retargeting to more specific cells in a tissue or more specific organ within the organism is achieved by a multispecific binding molecule conjugated to a detectable label, and a second domain that binds to a receptor of interest, and / or a targeting ligand that binds to a receptor of interest, respectively. Such retargeting or redirection may also include a scenario in which a wild-type viral particle targets a tissue, the tissue targeting is reduced or abolished by the insertion of a detectable label, and retargeting to an entirely different tissue is achieved with a multispecific binding molecule.

[0109] In some embodiments of the invention comprising a detectable label, the targeting ligand comprises a multispecific binding molecule comprising (i) an antibody paratope that specifically binds to the detectable label and (ii) a second binding domain that specifically binds to a receptor that can be bound to the surface of a bead (e.g., for purification) or that can be expressed by a target cell. Thus, multispecific binding molecules include binding molecules that comprise (i) an antibody paratope that specifically binds to the detectable label and (ii) a second binding domain that specifically binds a receptor that targets a viral particle.

[0110] 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 binding portion thereof, e.g., an antibody paratope.

[0111] The antibody paratopes described herein generally comprise, at a minimum, the complementarity determining regions (CDRs) responsible for specific recognition of a target (e.g., a detectable label, a cell surface receptor, etc.), e.g., the CDR3 regions of the heavy and / or light chain variable domains. In some embodiments, the multispecific binding molecule comprises an antibody (or a portion thereof) comprising an antibody paratope that specifically binds to a detectable label.

[0112] One embodiment of the present invention is a multimeric structure comprising the modified viral capsid proteins of the present 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 comprising first members of the specific binding pairs described herein. They can form normal viral capsids (empty viral particles) or viral particles (capsids encapsidating a nucleotide of interest). The formation of viral particles containing a viral genome is a highly desirable characteristic for use with the modified viral capsids described herein.

[0113] A further embodiment of the present invention is the use of at least one modified viral capsid protein and / or nucleic acid encoding same, preferably at least one multimeric structure (e.g., viral particle) for the production and for use in the introduction of a nucleotide of interest into a target cell.

[0114] Generally, the viral capsid proteins described herein can include targeting ligands that target TfR, such as anti-TfR antibodies and binding portions thereof. Antibodies specific for human TfR are well known in the art. Non-limiting exemplary anti-transferrin receptor antibodies are described in, for example, US2017 / 0174778; US2015 / 0196663; US9629801; US2018 / 0002433; WO2016 / 081643; US2018 / 0134797; WO2014 / 189973; US2015 / 0110791; US9708406; US2017 / 0260292; WO2016 / 081640; US2018 / 005760 4;US9611323;WO2012 / 075037;WO2018 / 210898, US2018 / 0344869, US2018 / 0282408, US2017 / 0051071, WO2016 / 207240, WO2015 / 101588, US2016 / 0324984;US2018 / 0222993;WO2017 / 055542;US2018 / 0222992;WO2017 / 055540;Cabezon,I.,et al. Mol Pharm. 2015 Nov 2;12(11):4137-45, Yu YJ, et al. Sci Transl Med (2014) 6:261ra154, Couch, et al. Sci Transl Med. 2013 May 1;5(183):183ra57,1-12.

[0115] Additional nucleic acid and translated amino acid sequences of anti-transferrin antibody and scFv domains that can be used to retarget AAV capsids as described herein are provided as SEQ ID NOs: 1-320 and 333-388.

[0116] Table 1 provides a summary of the SEQ ID NOs for each binding portion (e.g., heavy chain variable domain, light chain variable domain, and CDR1, CDR2, and CDR3) of non-limiting anti-human TfR antibodies that can be used to redirect AAV capsids as described herein. In some embodiments, the AAV capsids described herein comprise a targeting ligand that binds to human TfR, wherein the targeting ligand comprises a heavy chain variable domain, a light chain variable domain, a CDR1, a CDR2, and / or a CDR3 amino acid sequence that is at least 90% identical to the heavy chain variable domain, light chain variable domain, CDR1, CDR2, and / or CDR3 amino acid sequence, respectively, set forth in any one of SEQ ID NOs: 1-320 and 365-388. In some embodiments, the AAV capsids described herein comprise a targeting ligand that binds to human TfR, wherein the targeting ligand comprises a heavy chain variable domain, a light chain variable domain, a CDR1, a CDR2, and / or a CDR3 amino acid sequence that is at least 95% identical to the amino acid sequence of the heavy chain variable domain, the light chain variable domain, CDR1, CDR2, and / or CDR3 set forth in any one of SEQ ID NOs: 1-320 and 365-388, respectively. In some embodiments, the AAV capsids described herein comprise a targeting ligand that binds to human TfR, wherein the targeting ligand comprises a heavy chain variable domain, a light chain variable domain, a CDR1, a CDR2, and / or a CDR3 amino acid sequence that is at least 95% identical, at least 97% identical to the amino acid sequence of the heavy chain variable domain, the light chain variable domain, CDR1, CDR2, and / or CDR3 set forth in any one of SEQ ID NOs: 1-320 and 365-388, respectively. In some embodiments, the AAV capsids described herein comprise a targeting ligand that binds to human TfR, and the targeting ligand comprises a heavy chain variable domain, a light chain variable domain, a CDR1, a CDR2, and / or a CDR3 amino acid sequence that is at least 95% identical, and at least 98% identical, to the amino acid sequences of the heavy chain variable domain, the light chain variable domain, CDR1, CDR2, and / or CDR3 set forth in any one of SEQ ID NOs: 1-320 and 365-388, respectively.In some embodiments, the AAV capsids described herein comprise a targeting ligand that binds to human TfR, and the targeting ligand comprises a heavy chain variable domain, a light chain variable domain, a CDR1, a CDR2, and / or a CDR3 amino acid sequence that is at least 95% identical, or at least 99% identical, to the amino acid sequence of the heavy chain variable domain, the light chain variable domain, CDR1, CDR2, and / or CDR3 set forth in any one of SEQ ID NOs: 1-320 and 365-388, respectively. [Table 1-1] [Table 1-2] [Table 1-3]

[0117] In addition to bivalent monoclonal antibody (mAb) formats, non-limiting examples of targeting ligand formats that bind to TfR 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 consisting of amino acid residues mimicking the hypervariable regions (e.g., isolated complementarity-determining regions (CDRs) such as CDR3 peptides) of an antibody, or constrained FR3-CDR3-FR4 peptides. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "targeting ligand" as used herein. In non-limiting embodiments, anti-TfR targeting ligands that bind to TfR and are useful for retargeting viral capsids described herein include scFvs. As a non-limiting example, Vs that are useful for retargeting viral capsids described herein include scFvs. L -(Gly4Ser)3-V HThe scFv sequence of this format may comprise an amino acid sequence that is 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 333-364.

[0118] In some embodiments, scFvs useful for retargeting viral capsids described herein can comprise an amino acid sequence that is 90%, 95%, 97%, 98%, 99%, or 100% of any one of the amino acid sequences set forth in SEQ ID NOs: 333-364, but is in the form VH-(Gly4Ser)3-VL.

[0119] In some cases, the anti-TfR antigen binding protein is an antibody comprising one or more mutations in a framework region, e.g., the CH1 domain, CH2 domain, CH3 domain, hinge region, or a combination thereof. In some embodiments, the one or more mutations are to stabilize the antibody and / or increase half-life. In some embodiments, the one or more mutations are to modulate Fc receptor interactions, reduce or eliminate Fc effector function, such as FcyR, antibody-dependent cell-mediated cytotoxicity (ADCC), or complement-dependent cytotoxicity (CDC). In additional embodiments, the one or more mutations are to modulate glycosylation.

[0120] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1) and / or the CH3 domain (residues 341-447 of human IgG1) and / or hinge region) of an antibody described herein, numbered according to the Kabat numbering system (e.g., EU index of Kabat), to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. In this embodiment, one, two, or more mutations (e.g., amino acid substitutions) are introduced in the hinge region of the Fc region (CH1 domain) to alter (e.g., increase or decrease) the number of cysteine residues in the hinge region, as described, for example, in U.S. Patent No. 5,677,425. The number of cysteine residues in the hinge region of the CH1 domain can be altered, for example, to facilitate 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.

[0121] In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into an IgG constant domain, or an FcRn-binding fragment thereof (preferably an Fc domain fragment or hinge-Fc domain fragment), to alter (e.g., decrease or increase) the half-life of the antibody in vivo. For examples of mutations that may alter (e.g., decrease or increase) the half-life of an antibody in vivo, 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 comprises a mutation at residue position L234, L235, or a combination thereof. In some embodiments, the mutation comprises L234 and L235. In some embodiments, the mutations include L234A and L235A.

[0122] The anti-TfR antibodies and antigen-binding fragments described herein may be post-translationally modified, for example, glycosylated.

[0123] For example, the antibodies and antigen-binding fragments described herein may be glycosylated (e.g., N-glycosylated and / or O-glycosylated. Typically, the 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 located in one of the following situations: FN 297 S or YN 297 It's in S.

[0124] In one embodiment, the glycosylation is any one or more of the three different N-glycan types: high mannose, complex, and / or hybrid, which are found on IgG with their respective linkages. Complex and hybrid types exist with core fucosylation, the addition of a fucose residue to the innermost N-acetylglucosamine, and without core fucosylation.

[0125] In some cases, the anti-TfR antigen binding protein antibody does not contain a glycosylation sequence that may interfere with transglutamation reactions, e.g., an antibody that does not have a sugar group at N180 and / or N297 on one or more heavy chains. In certain embodiments, the antibody heavy chain has an N180 mutation. In other words, the antibody is mutated to eliminate the asparagine residue at position 180 according to the EU numbering system disclosed by Kabat et al. In certain embodiments, the antibody heavy chain has an N180Q mutation. In certain embodiments, the antibody heavy chain has an N297 mutation. In certain embodiments, the antibody heavy chain has an N297Q or N297D mutation. Antibodies containing such above-mentioned mutations can be prepared by site-directed mutagenesis to remove or disable the glycosylation sequence or by site-directed mutagenesis to insert a glutamine residue at a site away from any interfering glycosylation sites or any other interfering structures. Such antibodies can also be isolated from natural or artificial sources. Glycosylated antibodies also include antibodies containing T299 or S298P or other mutations or combinations of mutations that result in the absence of glycosylation.

[0126] In some cases, the antigen-binding protein is a deglycosylated antibody, i.e., an antibody in which sugar groups have been removed to facilitate transglutaminase-mediated conjugation. Saccharides include, but are not limited to, N-linked oligosaccharides. In some embodiments, deglycosylation is performed at residue N180. In some embodiments, deglycosylation is performed at residue N297. In some embodiments, removal of sugar groups is achieved enzymatically, including, but not limited to, via PNGase.

[0127] In some embodiments, the antibodies or fragments described herein are defucosylated.

[0128] The antibodies and antigen-binding fragments described herein may also be post-translationally modified in other ways, including, for example, Glu or Gln cyclization at the N-terminus, loss of the N-terminal positive charge, Lys variants at the C-terminus, deamidation (Asn to Asp), isomerization (Asp to isoAsp), deamidation (Gln to Glu), oxidation (Cys, His, Met, Tyr, Trp), and / or disulfide bond heterogeneity (shuffling, thioether, and trisulfide formation).

[0129] In some embodiments, the antibodies disclosed herein comprise Q295, which may be naturally occurring in 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.

[0130] Certain embodiments of the present disclosure provide anti-TfR antibodies and antigen-binding fragments comprising an Fc domain containing one or more mutations that enhance or decrease antibody binding to the FcRn receptor, e.g., at acidic pH relative to neutral pH. For example, the present disclosure includes anti-TfR antibodies containing mutations in the CH2 or CH3 region of the Fc domain that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., in endosomes at a pH ranging from about 5.5 to about 6.0). Such mutations may result in an increased serum half-life of the antibody when administered to an animal.

[0131] Non-limiting examples of such Fc modifications include, for example, modifications at the positions: ● 250th place (e.g., E or Q), ● positions 250 and 428 (e.g., L or F); ● 252nd place (e.g., L / Y / F / W or T), position 254 (e.g., S or T), and / or • a modification at position 256 (e.g., S / R / Q / E / D, or T); and / or location: positions 428 and / or 433 (e.g., H / L / R / S / P / Q, or K), and / or • a modification at position 434 (e.g., A, W, H, F, or Y); and / or location: • Modifications at positions 250 and / or 428, and / or location: • Modifications at positions 307 or 308 (e.g., 308F, V308F), and / or 434 are included.

[0132] In one embodiment, the modification is - 428L (e.g., M428L position) and 434S (e.g., N434S) modifications; • 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications; • 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; • 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L), and / or • Contains 307 and / or 308 modifications (e.g., 308F or 308P).

[0133] For example, the present disclosure provides: ● 250Q and 248L (e.g., T250Q and M248L), ● 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E), ● 257I and 311I (e.g., P257I and Q311I), ● 257I and 434H (e.g., P257I and N434H), ● 376V and 434H (e.g., D376V and N434H), ● 307A, 380A, and 434A (e.g., T307A, E380A, and N434A), - 428L and 434S (e.g., M428L and N434S), and - Anti-TfR antibodies comprising an Fc domain containing one or more pairs or groups of mutations selected from the group consisting of 433K and 434F (e.g., H433K and N434F).

[0134] In yet another embodiment, the modifications include a 265A (eg, D265A) and / or a 297A (eg, N297A) modification.

[0135] In one embodiment, the heavy chain constant domain is gamma 4 containing an S228P and / or S108P mutation. See Angal et al., A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody, Mol Immunol. 1993 Jan;30(1):105-108.

[0136] All possible combinations of the aforementioned Fc domain mutations, and other mutations in the antibody variable domains disclosed herein, are contemplated as being within the scope of the present disclosure.

[0137] The anti-TfR antibodies described herein may comprise a modified Fc domain with reduced effector function. As used herein, a "modified Fc domain with reduced effector function" refers to any Fc portion of an immunoglobulin that has been modified, mutated, or truncated relative to a wild-type, native Fc domain such that the molecule comprising the modified Fc exhibits a reduction in the severity or extent of at least one effect selected from the group consisting of cell killing (e.g., ADCC and / or CDC), complement activation, phagocytosis, and opsonization, relative to a comparator molecule comprising a native, wild-type Fc portion. In certain embodiments, a "modified Fc domain with reduced effector function" is an Fc domain that has reduced or attenuated binding to an Fc receptor (e.g., FcγR).

[0138] In certain embodiments, the modified Fc domain is a variant IgG1 Fc or variant IgG4 Fc comprising a substitution in the hinge region. For example, a modified Fc for use in the context of the present disclosure may comprise a variant IgG1 Fc in which at least one amino acid in the IgG1 Fc hinge region is substituted with the corresponding amino acid in an IgG2 Fc hinge region. Alternatively, a modified Fc for use in the context of the present disclosure may comprise a variant IgG4 Fc in which at least one amino acid in the IgG4 Fc hinge region is substituted with the corresponding amino acid in an IgG2 Fc hinge region. Non-limiting exemplary modified Fc regions that may be used in the context of the present disclosure are described in U.S. Patent Application Publication No. 2014 / 0243504, the disclosure of which is incorporated herein by reference in its entirety, as well as any functionally equivalent variants of the modified Fc regions described therein.

[0139] The present disclosure also includes antigen-binding proteins, antibodies, or antigen-binding fragments comprising an HCVR and a chimeric heavy chain constant (CH) region described herein, where 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 comprising part or all of a CH2 domain from a human IgG1, human IgG2, or human IgG4 molecule combined with part or all of a CH3 domain from a human IgG1, human IgG2, or human IgG4 molecule. According to certain embodiments, an antibody of the present disclosure comprises a chimeric CH region with a chimeric hinge region. For example, the chimeric hinge can comprise an "upper hinge" amino acid sequence (amino acid residues 216-227, EU numbering) derived from a human IgG1, IgG2, or IgG4 hinge region combined with a "lower hinge" sequence (amino acid residues 228-236, EU numbering) derived from a human IgG1, IgG2, or IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 upper hinge or a human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. Antibodies comprising the chimeric CH regions described herein, in certain embodiments, exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, e.g., WO2014 / 022540.)

[0140] Other modified Fc domains and Fc modifications that may be used in the context of the present disclosure include any of the modifications described in US2014 / 0171623, US8,697,396, US2014 / 0134162, WO2014 / 043361, the disclosures of which are incorporated herein by reference in their entireties. Methods for constructing antibodies or other antigen-binding fusion proteins comprising the modified Fc domains described herein are known in the art.

[0141] In some embodiments, the anti-TfR antibodies and antigen-binding fragments described herein comprise an Fc domain comprising one or more mutations in the CH2 and / or CH3 regions that create distinct TfR binding sites.

[0142] In one embodiment, the CH2 region has a) position 47 is Glu, Gly, Gln, Ser, Ala, Asn, Tyr, or Trp; position 49 is Ile, Val, Asp, Glu, Thr, Ala, or Tyr; position 56 is Asp, Pro, Met, Leu, Ala, Asn, or Phe; position 58 is Arg, Ser, Ala, or Gly; position 59 is Tyr, Trp, Arg, or Val; position 60 is Glu; position 61 is Trp or Tyr; and position 62 is Gln, Tyr, His, I and position 63 is Leu, Trp, Arg, Asn, Tyr, or Val; b) position 39 is Pro, Phe, Ala, Met, or Asp; position 40 is Gln, Pro, Arg, Lys, Ala, Ile, Leu, Glu, Asp, or Tyr; position 41 is Thr, Ser, Gly, Met, Val, Phe, Trp, or Leu; position 42 is Pro, Val, Ala, Thr, or Asp; and position 43 is Pro, Val, or Phe; position 44 is Trp, Gln, Thr, or Glu; position 68 is Glu, Val, Thr, Leu, or Trp; position 70 is Tyr, His, Val, or Asp; position 71 is Thr, His, Gln, Arg, Asn, or Val; and position 72 is Tyr, Asn, Asp, Ser, or Pro; c) position 41 is Val or Asp; position 42 is Pro, Met, or Asp; position 43 is Pro or Trp; and position 44 is Arg, Trp, Glu, or Thr. position 69 is His, Leu, or Pro; and position 73 is Val or Trp; or d) position 45 is Trp, Val, Ile, or Ala; position 47 is Trp or Gly; position 49 is Tyr, Arg, or Glu; and position 95 is Ser, Arg, or Gln;position 99 is Ile, Ser, or Trp; position 102 is Trp, Thr, Ser, Arg, or Asp; position 103 is Trp; and position 104 is Ser, Lys, Arg, or Val, or a combination thereof, wherein the substitutions and positions are PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH. Determined with reference to amino acids 4 to 113 of EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 446);

[0143] In one embodiment, the CH3 region has a structure in which: a) position 153 is Trp, Leu, or Glu; b) position 157 is Tyr or Phe; c) position 159 is Thr; d) position 160 is Glu; e) position 161 is Trp; f) position 162 is Ser, Ala, Val, or Asn; g) position 163 is Ser or Asn; h) position 186 is Thr or Ser; i) position 188 is Glu or Ser; i) position 189 is Glu; and i) position 194 is Phe; or e; 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, or a combination thereof, wherein the substitutions and positions are determined with reference to amino acids 114 to 220 of SEQ ID NO: 446.

[0144] In some embodiments, the CH3 region comprises one or more mutations selected from: position 384 is Leu, Tyr, Met, or Val; position 386 is Leu, Thr, His, or Pro; position 387 is Val, Pro, or an acidic amino acid; position 388 is Trp; position 389 is Val, Ser, or Ala; position 413 is Glu, Ala, Ser, Leu, Thr, or Pro; position 416 is Thr or an acidic amino acid; and position 421 is Trp, Tyr, His, or Phe, according to EU numbering, or a combination thereof. In one embodiment, the CH3 region comprises one or more amino acid mutations selected from: position 380 is Trp, Leu, or Glu; position 384 is Tyr or Phe; position 386 is Thr; position 387 is Glu; position 388 is Trp; position 389 is Ser, Ala, Val, or Asn; position 390 is Ser or Asn; position 413 is Thr or Ser; position 415 is Glu or Ser; position 416 is Glu; and position 421 is Phe, or a combination thereof.

[0145] In some embodiments, the CH3 region comprises: 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) Position 382 is Phe, position 383 is Tyr, position 384 is Gly, position 385 is N, position 386 is Ala, position 387 is Lys, position 389 is Thr, position 422 is Leu, position 424 is Ala, position 426 is Glu, position 438 is Tyr, and position 440 is Leu;c) Position 382 is Phe, position 383 is Tyr, position 384 is Glu, position 385 is Ala, position 387 is Lys, position 388 is Leu, position 422 is Leu, and position 424 is Ala , 426 is Glu, 438 is Tyr, and 440 is Leu; d) 382 is Phe, 384 is Glu, 386 is Ser, 387 is Lys, 389 is Thr, 422 is Leu, 424 is Ala, 426 is Glu, 438 is Tyr, and 440 is Leu; e) 382 is Phe, 384 is Gly, 385 is Ala, 387 is Lys, 389 is Ser, 422 is Leu, and 424 is A f) one or more mutations selected from 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, and Leu at position 440, or a combination thereof, wherein the positions are determined according to EU numbering.

[0146] Additional mutations in the CH2 and / or CH3 regions that can introduce a non-native TfR binding site into the antigen binding proteins described herein include those described in U.S. Patent Application Publication Nos. 2020 / 0223935, 2020 / 0369746, 2021 / 0130485, 2022 / 0017634, and PCT Patent Application Publication Nos. WO2023 / 279099, WO2023 / 114499, and WO2023 / 114510, which are incorporated by reference in their entireties.

[0147] Use and preparation method A further embodiment of the modified viral capsid proteins described herein is their use to deliver a nucleotide of interest, such as a reporter gene or a therapeutic gene, to target cells. TfR is widely expressed. Table 2 provides a non-limiting list of tissues and relevant cells that may express TfR and thus be targeted by the modified viral capsid proteins described herein and into which a nucleotide of interest, such as a reporter gene or a therapeutic gene, may be inserted. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5]

[0148] Generally, the nucleotide of interest may be a transfer plasmid, which may generally include 5' and 3' inverted terminal repeat (ITR) sequences flanking a reporter gene or therapeutic gene (which, when contained within an AAV vector, may 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', a 5' ITR, a promoter, a gene (e.g., a reporter and / or therapeutic gene), and a 3' ITR.

[0149] Non-limiting examples of useful promoters include the cytomegalovirus (CMV) promoter, the chicken beta-actin (CBA) promoter and its hybrid (CBh), the spleen focus-forming virus (SFFV) promoter, the elongation factor 1 alpha (EF1a) promoter (1.2 kb EF1a promoter or 0.2 kb EF1a promoter), the chimeric EF1a / IF4 promoter, the polyubiquitin C promoter (UbC), and the phosphoglycerate kinase (PGK) promoter. Internal enhancers may also be present in the viral construct to increase expression of the gene of interest. For example, the CMV enhancer (Karasuyama et al. 1989. J. Exp. Med. 169:13, incorporated herein by reference in its entirety) may be used. In some embodiments, the CMV enhancer may be used in combination with the chicken beta-actin promoter, e.g., as a hybrid (CAG). Alternatively, the promoter may be a tissue-specific promoter, i.e., it is active in a specific tissue and / or organ. A tissue-specific promoter comprises one or more tissue-specific promoter and / or enhancer elements, and optionally one or more constitutive promoter and / or enhancer elements as described in US2022 / 0204991, the entire contents of which are incorporated herein by reference. Those skilled in the art will understand that tissue-specific promoter and / or enhancer elements can be isolated from genes that are specifically expressed in tissues by methods well known in the art.

[0150] A variety of reporter genes (or detectable moieties) can be encapsidated into multimeric structures comprising the modified viral capsid proteins described herein. Exemplary reporter genes include, for example, b-galactosidase (encoded by the 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. Although the methods described herein demonstrate the construction of targeted particles that employ the use of a reporter gene encoding green fluorescent protein, one of skill in the art upon reading this disclosure will understand that the viral capsids described herein can be produced in the absence of a reporter gene or with any reporter gene known in the art.

[0151] A variety of therapeutic genes can also be encapsidated into multimeric structures comprising the modified viral capsid proteins described herein, e.g., as part of a transfer particle. Non-limiting examples of therapeutic genes include those encoding toxins (e.g., suicide genes), therapeutic antibodies or fragments thereof, CRISPR / Cas systems or portions thereof, antisense oligonucleotides, antisense RNA, siRNA, shRNA, etc. Tables 3 and 4 provide non-limiting lists of diseases and genes that may be suitable for treatment using the viral particles described herein, may be nucleotides of interest, and / or whose reduction may result in a therapeutic effect. [Table 3-1] [Table 3-2]

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 4-7

Table 4-8

Table 4-9

Table 4-10

Table 4-11

Table 4-12

Table 4-13

Table 4-14

Table 4-15

Table 4-16

Table 4-17

Table 4-18

Table 4-19

[0152] 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. Therefore, such modified capsids can be useful for transporting a nucleotide of interest across the blood-brain barrier for gene therapy of brain disorders, such as central nervous system (CNS) disorders and disorders associated with neurological symptoms. In such cases, expression of a therapeutic gene can be restricted to cells of the central nervous system, such as neurons, by operably linking the nucleotide of interest to a tissue-specific promoter. Non-limiting examples of 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 certain 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 may include, but are not limited to, one or more elements from the human glial fibrillary acidic protein (GFAP) promoter, human synapsin 1 (SYN1) promoter, human synapsin 2 (SYN2) promoter, human metallothionein 3 (MT3) promoter, and / or human proteolipid protein 1 (PLP1) promoter. Other examples of such brain-specific promoters include, but are not limited to, SCG10, tubulin a1 promoter, calcium / calmodulin-dependent protein kinase II (CaMKII) promoter, neuron-specific enolase (NSE) promoter, PDGF (platelet-derived growth factor beta)-b chain promoter, and the like. Additional brain-specific promoter elements are disclosed in WO2016 / 100575A1, which is incorporated herein by reference in its entirety.

[0153] In some embodiments, the brain-specific promoter described herein is 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, and myelin basic protein (MBP) promoter.

[0154] In other embodiments, the promoter is a neuron-, astrocyte-, or oligodendrocyte-specific, or neuron-, astrocyte-, or oligodendrocyte-preferred promoter, such as a synapsin, MeCP2, oligodendrocyte transcription factor 1 (Olig1), chondroitin sulfate proteoglycan (Cspg4), or CNP (2',3'-cyclic nucleotide 3'-phosphodiesterase) promoter.

[0155] CNS disorders and disorders with neurological symptoms that may be amenable to gene therapy include, but are not limited to, Alzheimer's disease, brain cancer, Behcet's disease, cerebral lupus, Creutzfeldt-Jakob disease, dementia, epilepsy, encephalitis, Friedreich's ataxia, Guillain-Barré syndrome, Gaucher disease, headache, hydrocephalus, Huntington's disease, increased intracranial pressure, cerebral leukodystrophy, migraine, myasthenia gravis, muscular dystrophy, multiple sclerosis, narcolepsy, neuropathy, Prader-Willi syndrome, Parkinson's disease, Rett syndrome, restless legs syndrome, sleep disorders, subarachnoid hemorrhage, stroke, traumatic brain injury, trigeminal neuralgia, transient ischemic attack, and von Hippel-Lindau disease (angiomatosis).

[0156] In some embodiments, the viral capsids described herein are capable of encapsidating a therapeutic gene, the expression of which can prevent, alleviate, or reduce 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, neuraminidase deficiency, sialidosis, aspartylglycosaminuria, mixed saposin deficiency, variant Gaucher disease, Farber lipogranulomatosis, fucosidosis, and beta mannosidosis.

[0157] "Enzyme deficiency diseases" include non-lysosomal storage diseases, such as Krabbe disease (galactosylceramidase), phenylketonuria, galactosemia, maple syrup urine disease, mitochondrial disorders, Friedreich's ataxia, Zellweger syndrome, adrenoleukodystrophy, Wilson's disease, hemochromatosis, ornithine transcarbamylase deficiency, methylmalonic acidemia, propionic acidemia, and lysosomal storage diseases. "Lysosomal storage diseases" include any disorder resulting from defective lysosomal function. Approximately 50 lysosomal storage disorders have been identified, the most well-known of which include Tay-Sachs disease, Gaucher disease, and Niemann-Pick disease. The etiology of these diseases is thought to be the accumulation of defective breakdown products in lysosomes, usually due to loss of protein function. Lysosomal storage diseases are caused by loss-of-function or attenuating variants in proteins that normally degrade or regulate the degradation of lysosomal contents. Proteins closely associated with lysosomal storage diseases include enzymes, receptors, and other transmembrane proteins (e.g., NPC1), post-translational modification proteins (e.g., sulfatases), membrane transport proteins, and non-enzymatic cofactors and other soluble proteins (e.g., GM2 ganglioside activators). Therefore, lysosomal storage diseases not only encompass the aforementioned disorders caused by defective enzymes themselves, but also any disorders caused by any molecular defects. Therefore, as used herein, the term "enzyme" is intended to encompass the aforementioned other proteins associated with lysosomal storage diseases.

[0158] The nature of the molecular damage often influences the severity of the disease; complete loss of function tends to be associated with fetal or neonatal onset and severe symptoms, while partial loss of function is associated with (relatively) milder, later-onset disease. Generally, 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.

[0159] Lysosomal storage diseases are a group of rare disorders that affect the degradation of various substrates in lysosomes. These substrates, including sphingolipids, mucopolysaccharides, glycoproteins, glycogen, and oligosaccharides, can accumulate in the cells of diseased individuals, leading to cell death. Organs affected by lysosomal storage diseases include the central nervous system (CNS), peripheral nervous system (PNS), lung, liver, bone, skeletal and cardiac muscle, and reticuloendothelial system.

[0160] Treatment options for lysosomal storage diseases include enzyme replacement therapy (ERT), substrate reduction therapy, pharmacological chaperone-mediated therapy, hematopoietic stem cell transplantation, and gene therapy. Examples of substrate reduction therapy include the use of miglustat or eliglustat to treat Gaucher disease type 1. These drugs act by blocking synthase activity, thereby reducing substrate production. Hematopoietic stem cell therapy (HSCT) is used, for example, to ameliorate and delay the negative central nervous system phenotype in patients with some forms of MPS. See R.M. Boustany, "Lysosomal storage diseases—the horizon expands," 9(10) Nat. Rev. Neurol. 583-98, October 2013, which are incorporated herein by reference in their entireties.

[0161] Two of the most common LSDs are Pompe disease and Fabry disease. Pompe disease, with an estimated incidence of 1 in 10,000, is caused by a defective lysosomal enzyme, alpha-glucosidase (GAA), resulting in defective glycogen processing in lysosomes. Lysosomal glycogen accumulation occurs primarily in skeletal, cardiac, and hepatic tissues. Onset of Pompe disease in infancy, usually before the age of 2 years, leads to cardiac hypertrophy, hypotonia, hepatomegaly, and death from cardiopulmonary failure. Onset of Pompe disease in adulthood can occur in the teens to fifties and typically involves only skeletal muscle. Currently available treatments include Genzyme's MYOZYME® / LUMIZYME® (alglucosidase alpha), a recombinant human alpha-glucosidase produced in CHO cells and administered by intravenous infusion.

[0162] Fabry disease, which has an estimated overall incidence of 1 in 3,000 people, including milder, later-onset cases, is caused by 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 due to nerve damage and / or small-vessel occlusion, renal insufficiency and failure, cardiac complications such as hypertension and cardiomyopathy, skin symptoms such as the formation of angiokeratoma, anhidrosis or hyperhidrosis, and eye disorders such as cornea verticillata, radial cataracts, and conjunctival and retinal vascular abnormalities. Currently available treatments include Genzyme's FABRAZYME® (agalsidase beta), a recombinant human alpha-galactosidase A produced in CHO cells and administered by intravenous infusion; Shire's REPLAGAL™ (agalsidase alfa), a recombinant human alpha-galactosidase A produced in human fibroblasts and administered by intravenous infusion; and Amicus' GALAFOLD™ (migalastat, 1-deoxygalactonojirimycin), an orally administered small molecule peralone that shifts the folding of abnormal alpha-galactosidase A to a functional conformation.

[0163] A further embodiment of the present invention is a process for the preparation of a modified capsid protein, the method comprising: a) expressing a nucleic acid encoding a modified capsid protein under suitable conditions; b) isolating the expressed capsid protein of step a).

[0164] In some embodiments, the viral particles described herein comprise a mosaic capsid, e.g., the capsid comprises a capsid protein that has been genetically modified as described herein (in the presence or absence of covalent attachment to a targeting ligand) in a specific ratio to a reference capsid protein. Methods for producing such mosaic viral particles include: a) expressing under suitable conditions a nucleic acid encoding a modified capsid protein and a nucleotide encoding a reference capsid protein in a ratio (wt / wt) of at least about 60:1 to 1:60, e.g., 2:1, 1:1, 3:5, 1:2, 1:3, etc.; b) isolating the expressed capsid protein of step a).

[0165] In some embodiments, the compositions described herein comprise, or the methods described herein combine, modified cap genes:reference cap genes (or combinations of reference cap genes) in a ratio ranging from at least about 1:60 to about 60:1, e.g., 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.

[0166] In some embodiments, the ratio of VP protein subunits in a mosaic virus particle stoichiometrically reflects, but is not necessarily, the ratio of modified cap genes to reference cap genes. As a non-limiting illustrative example, a mosaic capsid formed according to the present methods can be considered to have a ratio of modified capsid protein to reference capsid protein similar to, but not necessarily, the ratio (wt:wt) of the nucleic acids encoding the same used to produce the mosaic capsid. In some embodiments, a mosaic capsid comprises a protein subunit ratio of about 1:59 to about 59:1. In some embodiments, a mosaic capsid comprises a modified capsid protein to reference capsid protein ratio of about 7:1.

[0167] A further embodiment of the present invention is a method for modifying the tropism of a virus, the method comprising: (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 packaging cells under conditions sufficient to produce viral particles, wherein the packaging cells comprise the nucleic acid. A further embodiment of the present invention is a method for displaying a targeting ligand on the surface of a capsid protein, the method comprising: (a) expressing under suitable conditions a nucleic acid encoding a modified viral capsid protein described herein (optionally together with nucleotides encoding a reference capsid protein), wherein the nucleic acid encodes a capsid protein comprising a first member of a specific binding pair; (b) isolating the expressed capsid protein comprising the first member of the specific binding pair of step (a), or a capsid comprising the same; and (c) incubating the capsid protein or capsid with a second cognate member of the specific binding pair under suitable conditions to allow formation of an isopeptide bond between the first and second members, wherein the second cognate member of the specific binding pair is fused to a targeting ligand.

[0168] In some embodiments, the packaging cells further comprise a helper plasmid and / or a transfer plasmid containing the nucleotide of interest. In some embodiments, the method further comprises isolating the self-complementing adeno-associated viral particles from the culture supernatant. In some embodiments, the method further comprises lysing the packaging cells and isolating the single-stranded adeno-associated viral particles from the cell lysate. In some embodiments, the method further comprises (a) removing cellular debris, (b) treating the supernatant containing the viral particles with a nuclease, e.g., DNase I and MgCl2, (c) concentrating the viral particles, (d) purifying the viral particles, and (e) any combination of (a)-(d).

[0169] Packaging cells useful for producing the viral particles described herein include, for example, animal cells that are permissive for the virus, or cells modified to be permissive for the virus, or packaging cell constructs using a transforming agent such as, for example, calcium phosphate. Non-limiting examples of packaging cell lines useful for producing the viral particles described herein include, for example, human embryonic kidney 293 (HEK-293) cells (e.g., American Type Culture Collection [ATCC] No. CRL-1573), SV40 Large T-antigen-containing HEK-293 cells (HEK-293T or 293T), HEK293T / 17 cells, human sarcoma cell line HT-1080 (CCL-121), lymphoblastoid cell line Raji (CCL-86), epithelial glioblastoma-astrocytoma 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 Examples of such cells include 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, and CAP-T cells.

[0170] L929 cells, the FLY virus packaging cell line reviewed in Cosset et al (1995) J Virol 69, 7430-7436, NS0 (mouse myeloma) cells, human amniotic cells (e.g., CAP, CAP-T), yeast cells (including but not limited to S. 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 (e.g., High5)), or bacterial cells (including but not limited to E. coli).

[0171] For additional packaging cells and systems, packaging techniques and particles for packaging nucleic acid genomes into pseudotyped viral particles, see, e.g., Polo, et al., Proc Natl Acad Sci USA, (1999) 96:4598-4603. Packaging methods include using packaging cells that permanently express the viral components or transiently transfecting cells with plasmids.

[0172] Further embodiments include methods of redirecting viruses and / or delivering reporter or therapeutic genes to target cells, including methods for transducing cells in vitro (e.g., ex vivo) or in vivo, comprising contacting a target cell with a viral particle comprising a capsid described herein, wherein the capsid comprises a targeting ligand that specifically binds to a receptor expressed by the target cell. In some embodiments, the target cell is in vitro (e.g., ex vivo). In other embodiments, the target cell is in vivo in a subject, e.g., a human.

[0173] target cell A wide variety of cells can be targeted for delivery of a nucleotide of interest using the modified viral particles disclosed herein. Target cells will generally be selected based on the nucleotide of interest and the desired effect.

[0174] In some embodiments, the nucleotide of interest can be delivered to enable the targeted cells to produce a protein that compensates for a defect in the organism, such as an enzyme deficiency or an immune deficiency, such as X-linked severe combined immunodeficiency. Thus, in some embodiments, cells that would normally produce the protein in the animal are targeted. In other embodiments, cells in the region where the protein would be most beneficial are targeted.

[0175] In other embodiments, the nucleotide of interest, such as the gene encoding siRNA, can inhibit the expression of a specific gene in target cells.The nucleotide of interest can, for example, inhibit the expression of a gene involved in the pathogen life cycle.Therefore, cells that are susceptible to infection by pathogens or infected by pathogens can be targeted.In other embodiments, the nucleotide of interest can inhibit the expression of a gene responsible for the production of toxins in target cells.

[0176] In other embodiments, the nucleotide of interest may encode a toxic protein that kills cells that express the toxic protein, in which case tumor cells or other unwanted cells may be targeted.

[0177] In yet 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 to the interstitial space surrounding the transduced cell or to adjacent cells. In some embodiments, the nucleotide of interest encodes a therapeutic protein that can provide a therapeutic effect in a transduced cell-autonomous manner. In some embodiments, the nucleotide of interest encodes a therapeutic protein that can provide a therapeutic benefit to the transduced cell, in the interstitial space surrounding the transduced cell, and / or to cells adjacent to the transduced cell in an autonomous manner.

[0178] Once a specific population of target cells in which expression of a nucleotide of interest is desired is identified, a target receptor that is specifically expressed on that population of target cells is selected. The target receptor may be expressed only in that population of cells, or to a greater extent in that population of cells than in other populations of cells. The more specific the expression, the more specifically delivery can be directed to the target cells. Depending on the context, the desired amount of specificity of the marker (and therefore of gene delivery) may vary. For example, when introducing a toxic gene, high specificity is most preferred to avoid killing non-targeted cells. For expression of a protein for harvesting or expression of a secreted product where a global effect is desired, less marker specificity may be required.

[0179] 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 partner. If the binding partner, e.g., ligand, of the target receptor is known, the ligand can be used as an affinity molecule. However, if the binding molecule is unknown, an antibody against the target receptor can be generated using standard procedures. The antibody can then be used as a targeting ligand.

[0180] Thus, target cells can be selected based on a variety of factors, including, for example, (1) the intended use (e.g., therapy, expression of harvested proteins, and conferring disease resistance), and (2) the expression of a marker with a desired amount of specificity.

[0181] The target cell is not limited in any way and includes both germline cells and cell lines, and somatic cells and cell lines. The target cell can be a stem cell from any source. When the target cell is a germline cell, the target cell is preferably selected from the group consisting of a single-cell embryo and an embryonic stem cell (ES).

[0182] Pharmaceutical Compositions, Dosage Forms, and Administration A further embodiment provides a pharmaceutical product comprising at least one modified viral capsid protein and a suitable targeting ligand according to the invention, and / or a nucleic acid according to the invention. Preferably, such a pharmaceutical product is useful as a gene transfer particle.

[0183] Also disclosed herein are pharmaceutical compositions comprising the viral particles described herein and a pharmaceutically acceptable carrier and / or excipient. Additionally, disclosed herein are pharmaceutical dosage forms comprising the viral particles described herein.

[0184] As discussed herein, the viral particles described herein can be used for a variety of therapeutic applications (in vivo and ex vivo) and as research tools.

[0185] Pharmaceutical compositions based on the viral particles 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, for example, by injection, inhalation, or insulation (either through the mouth or nose), or by oral, buccal, parenteral, or rectal administration, or by administration directly to a tumor.

[0186] Pharmaceutical compositions can be formulated for various 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 intracerebroventricular, intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, pharmaceutical compositions can be formulated in a liquid solution, preferably in a physiologically compatible buffer such as Hank's solution or Ringer's solution. In addition, pharmaceutical compositions can be formulated in solid form and redissolved or suspended immediately before use. Lyophilized forms of pharmaceutical compositions are also suitable.

[0187] For oral administration, pharmaceutical compositions can take 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 hydroxypropyl methylcellulose), 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). Tablets can also be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspensions, or can be presented as a dry product that is constitutional with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifying agents (e.g., lecithin or acacia), non-aqueous vehicles (e.g., oils, oily esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g., methyl- or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring agents, coloring agents, and sweetening agents, as appropriate.

[0188] The pharmaceutical compositions 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, for example, in ampoules or multi-dose containers, optionally with added preservatives. The pharmaceutical compositions can also be formulated as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain other agents, including suspending, stabilizing, and / or dispersing agents.

[0189] In addition, pharmaceutical compositions can be formulated as depot preparations. These long-acting formulations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Thus, for example, the compound can be formulated with a suitable polymeric or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin, or as a sparingly soluble derivative, e.g., as a sparingly soluble salt. Other suitable delivery systems include microspheres, which offer the possibility of localized, non-invasive delivery of drugs over long periods of time. This technology can involve microspheres with precapillary sizes that can be injected into any selected part of an organ via a coronary catheter without causing inflammation or ischemia. The administered therapeutic agent is gradually released from the microspheres and absorbed by surrounding cells present in the selected tissue.

[0190] Systemic administration can also be via transmucosal or transdermal routes. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are 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 may be used to enhance penetration. Transmucosal administration may also be achieved using intranasal sprays or suppositories. For topical administration, the viral particles described herein can be formulated into ointments, salves, gels, or creams, as are commonly known in the art. Cleansing solutions can also be used topically to treat wounds or inflammation to accelerate healing.

[0191] Pharmaceutical forms suitable for injectable use can include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the pharmaceutical form must be sterile and fluid. It must also be stable under the conditions of manufacture and certain storage parameters (e.g., refrigeration and freezing), and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0192] When the formulations disclosed herein are used as therapeutic agents to promote an immune response in a subject, the therapeutic agent can be formulated into a neutral or salt form composition. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) formed with inorganic acids such as hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, or mandelic acids. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, as well as organic bases such as isopropylamine, trimethylamine, histidine, or procaine.

[0193] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of microbial activity can be brought about by various antibacterial and antifungal agents known in the art. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be brought about by the use in the composition of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0194] Sterile injectable solutions can be prepared by incorporating the active compound or construct in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.

[0195] Upon formulation, solutions may be administered in a manner compatible with the dosage formulation, and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, although sustained-release capsules or microparticles and microspheres, etc., can also be used.

[0196] For parenteral administration in aqueous solution, for example, the solution should be suitably buffered if necessary, and the liquid diluent should first be made isotonic with sufficient saline or glucose.These particular aqueous solutions are particularly suitable for intravenous, intratumoral, intramuscular, subcutaneous, and intraperitoneal administration.In this context, the sterile aqueous medium that can be used will be known to those skilled in the art in light of this 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 into the proposed infusion site.

[0197] The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. For example, a subject may be administered the viral particles described herein daily or weekly, or monthly, biennially, or yearly for a period of time, depending on need or exposure to pathogenic microorganisms or to the subject's condition (e.g., cancer).

[0198] In addition to compounds formulated for parenteral administration, such as intravenous, intratumoral, subcutaneous, or intramuscular injection, other pharmaceutically acceptable forms include, for example, tablets or other solids for oral administration, liposomal formulations, sustained release capsules, biodegradable forms, and any other form currently in use.

[0199] Intranasal or inhalable solutions or sprays, aerosols, or inhalants may also be used. Nasal solutions may be aqueous solutions designed to be administered to the nasal passages in drops or sprays. Nasal solutions may be prepared to mimic nasal secretions in many respects. Thus, aqueous nasal solutions are usually isotonic and slightly buffered to maintain a pH of 5.5 to 7.5. In addition, if necessary, antimicrobial preservatives similar to those used in ophthalmic preparations and appropriate drug stabilizers may be included in the formulation. Various commercially available nasal preparations are known and may contain, for example, antibiotics and antihistamines and are used for asthma prophylaxis.

[0200] Oral formulations may contain excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders. In certain defined embodiments, oral pharmaceutical compositions may contain an inert diluent or an assimilable edible carrier, or may be enclosed in a hard or soft shell gelatin capsule, or may be compressed into tablets, or may be incorporated directly with dietary foods. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc.

[0201] Tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, cornstarch, or gelatin; excipients such as dicalcium phosphate; disintegrating agents such as cornstarch, potato starch, or alginic acid; lubricants such as magnesium stearate; and sweeteners such as sucrose, lactose, or saccharin, or flavorings such as peppermint oil, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, a liquid carrier may be contained in addition to materials of the above type. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar, or both. A syrup of elixir may contain the active compound, sucrose as a sweetener, methyl and propylparabens as preservatives, a dye, and a flavoring such as cherry or orange flavor.

[0202] Further embodiments disclosed herein may relate to kits for use in the methods and compositions. The kits may also include suitable containers, such as vials, tubes, minitubes or microtubes, test tubes, flasks, bottles, syringes, or other containers. If additional components or agents are provided, the kits may contain one or more additional containers into which the agents or components can be placed. The kits herein will also typically include a means for containing the viral particles and any other reagent containers in sealed confinement for commercial sale. Such containers may include syringes or blow-molded plastic containers into which the desired vials are retained. Optionally, the compositions described may require one or more additional active agents, such as, for example, anti-inflammatory agents, antiviral agents, antifungal or antibacterial agents, or antitumor agents.

[0203] The compositions disclosed herein may be administered by any means known in the art, including, for example, administering the compositions to a subject intravenously, intratumorally, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intramuscularly, intrathecally, subcutaneously, subconjunctivally, intravesicularly, mucosally, intrapericardially, subumbilically, intraocularly, orally, locally, by inhalation, injection, infusion, continuous infusion, localized perfusion, via catheter, lavage, in a cream, or in a lipid composition.

[0204] Any method known to those skilled in the art can be used for large-scale production of the viral particles, packaging cells, and particle constructs described herein. For example, master seeds and working seed stocks can be prepared under GMP conditions in qualified primary CEFs or by other methods. Packaging cells can be plated into large-surface-area flasks and grown to near confluence, resulting in purified viral particles. Cells can be harvested and the viral particles released into the culture medium, where they are isolated and purified, or intracellular viral particles can be released by mechanical disruption (cell debris can be removed by large-pore depth filtration and endonuclease-digested host cell DNA). The viral particles can then be purified and concentrated by tangential flow filtration, followed by diafiltration. The resulting concentrated bulk can be formulated by diluting with a buffer containing a stabilizer, filling into vials, and lyophilizing. Compositions and formulations can be stored for later use. For use, the lyophilized viral particles can be reconstituted by adding a diluent.

[0205] Certain additional agents used in combination therapy can be formulated and administered by any means known in the art.

[0206] The compositions disclosed herein may also include adjuvants such as aluminum salts and other mineral adjuvants, tensoactive agents, bacterial derivatives, vehicles, and cytokines. Adjuvants may also have antagonistic immunomodulatory properties. For example, adjuvants can stimulate Th1 or Th2 immunity. The compositions and methods disclosed herein may also include adjuvant therapy. [Table 5-1] [Table 5-2] [Table 6-1] [Table 6-2] [Table 6-3] [Table 7-1] [Table 7-2] [Table 8-1] [Table 8-2] [Table 8-3] [Example]

[0207] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0208] result AAV particles can be retargeted to the murine TfR (mTfR) in vitro and in vivo As shown in Figure 1, AAV particles targeting the mouse TfR via a surrogate anti-mTfR (8D3) antibody specifically infect multiple mTfR+ cell lines in vitro. Furthermore, as shown in Figure 2, AAV9 wild-type particles alone or conjugated with the anti-mTfR-targeting antibody 8D3 transduce the liver of WT C57BL / 6J mice in vivo. AAV9 N272A or AAV9 W503A particles conjugated with the anti-mTfR-targeting antibody 8D3 are detargeted from the liver and do not promote high levels of hepatic eGFP expression. Brain sections from mice injected with AAV9 wild-type or detargeted particles conjugated to an antibody that binds mTfR show enhanced eGFP staining in the brain compared to mice injected with AAV9 wild-type.

[0209] Furthermore, as shown in Figure 3, murine surrogate mTfR-binding bivalent mAb, Fab, and scFv conjugated to AAV particles can mediate in vitro transduction in multiple mTfR+ cell lines. Also shown in Figure 4, AAV9 wild-type particles can transduce the liver of WT C57BL / 6J mice, whereas AAV9 W503A particles are retargeted from the liver and do not promote high levels of hepatic eGFP expression. Compared to mice injected with AAV9 wild-type, brain sections from mice injected with AAV9 W503A particles conjugated to an antibody or its variant that binds mTfR show enhanced eGFP staining in the brain. Increased eGFP staining was observed with AAV9 W503A particles conjugated to antibody Fab, scFV, or bivalent antibody (mAb).

[0210] Furthermore, as shown in Figure 5A, AAV DNA from AAV9 W503A particles conjugated with an antibody that binds mTfR was detected at greater levels in the brain compared with AAV9 wild-type or AAV9 W503A particles conjugated with an antibody that binds hASGR1. This was observed in the brains of mice injected with AAV9 W503A particles conjugated with antibody Fab, scFV, or bivalent antibody. In Figure 5B, AAV9 wild-type DNA was detected at high levels in the liver of WT C57BL / 6J mice, while DNA was detected at low levels with various antibody formats from AAV9 W503A particles detargeted from the liver and conjugated with an antibody that targets mTfR or hASGR1 as a non-targeting control. Additionally, brain sections from mice injected with wild-type AAV1, 8, and 9 particles conjugated to Fabs that bind mTfR show enhanced eGFP staining in the brain compared to mice injected with wild-type AAV serotypes 1, 8, and 9 (Figure 6). Thus, retargeting with TfR was observed to improve CNS transduction regardless of the serotype used.

[0211] A dose-escalation study was conducted to determine the efficacy of anti-mTfR Fab conjugated to AAV particles at various doses. As shown in Figure 19A, AAV DNA from WT AAV9 particles conjugated with a Fab that binds mTfR ("8D3") or AAV9 W503A particles conjugated with a Fab that binds mTfR ("8D3") was detected at greater levels in the brain compared to WT AAV9. This increase in AAV DNA in the brain using AAV targeting mTfR was observed at all doses tested. In Figure 19B, WT AAV9 DNA was detected at high levels in the liver, while DNA from AAV9 W503A particles conjugated with a Fab that binds mTfR ("8D3") was detargeted from the liver and detected at lower levels. This decrease in AAV DNA levels in the liver using AAV9 W503A targeting mTfR was observed at all doses tested. Thus, mTfR-retargeted AAV demonstrates enhanced brain and reduced liver delivery of AAV vector DNA compared to WT AAV9 at all doses tested, as measured by qPCR. AAV particles targeting mTfR demonstrate enhanced brain transduction in a dose-dependent manner compared to WT AAV9. Furthermore, compared to WT AAV9 alone, WT AAV9 or AAV9 W503A retargeted to mTfR with Fab ("8D3") results in greater brain transduction at all doses tested (Figures 20A and 20B). Thus, TfR-retargeted WT AAV9 or AAV9 W503A are shown to achieve comparable CNS transduction to wild-type (WT) AAV9 at significantly lower doses.

[0212] AAV particles can be retargeted to the human TfR (hTfR) in vitro and in vivo As shown in Figure 7, AAV particles targeting human TFR via conjugation with an anti-hTfR Fab specifically infect hTfR+ cell lines in vitro. Furthermore, as shown in Figure 8, AAV9 wild-type particles specifically infect hTfR+ cell lines. hu / huAAV9 W503A particles can transduce the liver of mice, whereas AAV9 W503A particles are detargeted from the liver and do not promote high levels of eGFP expression in the liver. Compared with wild-type AAV9 and AAV9 W503A particles conjugated with an antibody Fab that binds hASGR1, AAV9 W503A particles conjugated with an antibody Fab that binds hTfR show enhanced GFP staining in the brain. In addition, compared with wild-type AAV9 and AAV9 W503A particles conjugated with an antibody Fab that binds hASGR1, AAV9 W503A particles conjugated with an antibody Fab that binds hTfR show enhanced GFP staining in multiple regions of the brain (Figures 9A-9D). Furthermore, as shown in Figure 10A, AAV DNA from AAV9 W503A particles conjugated with a Fab that binds hTfR is detected at greater levels 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 with AAV9 W503A particles conjugated with multiple antibody Fabs that bind hTFR. In Figure 10B, AAV9 wild-type DNA was detected at greater levels in the brain compared to AAV9 WT or AAV9 W503A particles conjugated with a Fab that binds hASGR1. hu / hu hTfR Fab-retargeted AAV demonstrated enhanced brain and reduced liver delivery of AAV vector DNA, as measured by qPCR.

[0213] To further demonstrate the retargeting of hTfR-conjugated AAV particles, a pool of unique barcoded AAV particles was transfected into female humanized TFRC mice (TFRC hu / hu ) In the liver, as expected, wild-type AAV9 alone accounted for the majority of all barcodes present in the tissue. In the brain and spinal column, detargeted AAV9(W503A) capsids conjugated with TfR-targeting Fab represented the majority of all barcodes present in the tissue, outperforming AAV9 alone, which accounted for a small proportion of all barcodes (Figures 11A and 11B).

[0214] To further understand the tropism of hTfR-retargeted AAV particles, we hu / hu Various brain cell types were examined in mice. As shown in Figures 12A and 12B, AAV particles targeting the human TfR demonstrate transduction across a wide range of brain cell types, including both neuronal and glial cell populations. As shown in Figure 12A, hTfR Fab-retargeted AAVs (WT AAV9 and AAV9 W503A) efficiently transduced multiple neuronal populations, including but not limited to cortical neurons and Purkinje cells. Furthermore, retargeting of both WT AAV9 and AAV9 W503A resulted in transduction of glial cells, including but not limited to astrocytes and oligodendrocytes. Transduction of some brain microvascular endothelial cells was also observed. As shown in Figure 12B, hTfR Fab-retargeted WT AAV9 robustly transduced neurons and astrocytes, and to a lesser extent, oligodendrocytes. Transduction of these various cell types was observed across a wide range of different brain regions, indicating that retargeting with TfR facilitates gene transfer to various cell types in the CNS.

[0215] Next, we compared intravenous and intracerebroventricular administration of hTfR-retargeted AAV particles. After intravenous injection, AAV9 W503A particles conjugated with a Fab that binds hTfR show enhanced eGFP staining in the brain compared with WT AAV9 and AAV9 W503A particles conjugated with a Fab that binds hASGR1. After intracerebroventricular injection, eGFP expression in the brain is comparable between targeted and non-targeted AAVs. Intravenous injection of AAV results in more widespread brain transduction than intracerebroventricular injection, and AAV9 W503A particles conjugated with a Fab that binds hTfR show enhanced transduction across a wide range of brain regions (Figure 13). As shown in Figure 14A, after intravenous delivery, AAV DNA from AAV9 W503A particles conjugated to an antibody that binds hTfR was detected at greater levels in the brain compared to WT AAV9 or AAV9 W503A particles conjugated to an antibody that binds hASGR1. After intracerebroventricular injection, AAV DNA levels in the brain were low and comparable between targeted and non-targeted AAVs. In Figure 14B, after intravenous delivery, WT AAV9 DNA was detected at greater levels in the brain compared to WT AAV9 particles conjugated to an antibody that binds hTfR. hu / hu High levels of AAV DNA were detected in the liver of mice, whereas low levels were detected from AAV9 W503A particles detargeted from the liver and conjugated with Fab targeting hTfR or hASGR1 as a nontargeting control. AAV DNA was detected at higher levels in the liver after intravenous injection compared to intracerebroventricular injection of targeted or nontargeted AAV. AAV particles targeting the human TfR show enhanced brain transduction after systemic intravenous (IV) delivery compared to intracerebroventricular (ICV) delivery.

[0216] To determine the efficacy of various different antibodies, several different anti-hTfR Fabs were conjugated to AAV particles and expressed as TfR. hu / hu Compared with WT AAV9 and AAV9 W503A particles conjugated to a Fab that binds hASGR1, AAV9 W503A particles conjugated to a Fab that binds hTfR showed enhanced eGFP staining in the brain (Figure 15).

[0217] To explore the biodistribution of anti-hTfR Fab conjugated to AAV particles, we performed a TFRC hu / hu We examined eGFP expression in various brain regions in mice. hTfR Fab-retargeted AAV demonstrates enhanced delivery to a wide range of brain regions after systemic injection and enhanced brain transduction compared to WT AAV9 in all brain regions evaluated. For AAV9 W503A particles conjugated with a Fab that binds hTfR, the cortex, hippocampus, and thalamus exhibited the highest levels of eGFP expression, while the hypothalamus consistently exhibited the lowest levels of eGFP expression (Figures 16 and 17). As further confirmation, TFRCC administered with anti-hTfR Fab conjugated to AAV particles exhibited significantly higher levels of eGFP expression in mice. hu / hu AAV DNA was measured in the brain (FIG. 18A) and liver (FIG. 18B) of mice. As shown in FIG. 18A, AAV DNA from AAV9 W503A particles conjugated with a Fab that binds hTfR is detected at greater levels in the brain compared to WT AAV9 or AAV9 W503A particles conjugated with a Fab that binds hASGR1. This increase in AAV DNA was observed with AAV9 W503A particles conjugated with multiple antibody Fabs that bind hTFR. In FIG. 18B, WT AAV9 DNA was detected at greater levels in the brain compared to WT AAV9 W503A particles conjugated with a Fab that binds hASGR1. hu / hu hTfR Fab-retargeted AAV demonstrated enhanced brain and reduced liver delivery of AAV vector DNA, as measured by qPCR.

[0218] Furthermore, TFRC hu / hu hTfR Fab retargeted AAV delivery to various tissues in mice was assessed using qPCR. As shown in Figure 21A, AAV9 wild-type DNA inhibited the delivery of hTfR Fab retargeted AAV to various tissues in female TfR. hu / huHigh levels of AAV9 W503A were detected in the livers of mice, whereas low levels of DNA were detected from AAV9 W503A particles detargeted from the liver and conjugated with hTfR-targeting antibodies and Fabs, or hASGR1 as a non-targeting control. Thus, W503A-detargeted, hTfR Fab-retargeted AAV demonstrates reduced delivery of AAV vector DNA to the liver, as measured by qPCR. In Figure 21B, AAV9 wild-type DNA was detected in the liver of female TFRs compared to AAV9 W503A particles detargeted from the liver and conjugated with hTfR-targeting antibodies and Fabs. hu / hu In the hearts of mice, AAV9 W503A particles conjugated to an antibody that binds hASGR1 were detected at similar levels, but at higher levels than AAV9 W503A particles conjugated to an antibody that binds hASGR1. Thus, W503A-detargeted, hTfR Fab-retargeted AAV demonstrates comparable delivery of AAV vector DNA to the heart, as measured by qPCR. In Figure 21C, AAV9 wild-type DNA was detargeted from the liver and significantly reduced the delivery of AAV9 W503A particles conjugated to an antibody and Fab that target hTfR to female TFR. hu / hu In the quadriceps muscles of mice, W503A particles conjugated to an antibody that binds hASGR1 were detected at similarly higher levels than AAV9 W503A particles conjugated to an antibody that binds hASGR1. Thus, W503A-detargeted, hTfR Fab-retargeted AAV demonstrate comparable delivery of AAV vector DNA to the quadriceps muscles, as measured by qPCR. In Figure 21D, AAV DNA from AAV9 W503A particles conjugated to an antibody or Fab that binds hTfR was detected at greater levels in the brain than AAV9 wild-type or AAV9 W503A particles conjugated to an antibody that binds hASGR1. Thus, W503A-detargeted, hTfR Fab-retargeted AAV demonstrates enhanced delivery of AAV vector DNA to the brain, as measured by qPCR.

[0219] In further experiments, TFRC was assayed using anti-hTfR Fab. hu / huAAV delivery to various tissues in mice was compared with delivery using the corresponding anti-hTfR mAb. As shown in Figure 22A, mice injected with detargeted AAV9 W503A conjugated with H1H12845B Fab or H1H12845B mAb showed comparable eGFP expression in the brain, heart, quadriceps, and liver. Compared with wild-type AAV9, mice injected with detargeted 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 transduce retargeted AAV particles similarly to the brain. As shown in Figure 22B, both groups of mice injected with detargeted AAV9 W503A conjugated with H1H12845B Fab or H1H12845B mAb showed comparable expression of eGFP in the cerebellum, hippocampus, and cortex, as well as the liver. Compared with wild-type AAV9, mice injected with detargeted AAV9 W503A conjugated with H1H12845B Fab or H1H12845B mAb showed higher expression of eGFP in the cerebellum, hippocampus, and cortex, and lower expression of eGFP in the liver. Thus, hTfR Fab and mAb transduce retargeted AAV particles similarly to the brain. As shown in Figure 23A, mice injected with detargeted AAV9 W503A conjugated with H1H12839B Fab or H1H12839B mAb show comparable eGFP expression in the brain, heart, quadriceps, and liver. Compared with wild-type AAV9, mice injected with detargeted 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 transduce retargeted AAV particles similarly to the brain.Also shown in Figure 23B, both groups of mice injected with detargeted AAV9 W503A conjugated with H1H12839B Fab or H1H12839B mAb show comparable expression of eGFP in the cerebellum, hippocampus, and cortex, as well as the liver. Compared with wild-type AAV9, mice injected with detargeted AAV9 W503A conjugated with H1H12839B Fab or H1H12839B mAb showed higher expression of eGFP in the cerebellum, hippocampus, and cortex, and lower expression of eGFP in the liver. Thus, hTfR Fab and mAb transduce retargeted AAV particles similarly to the brain.

[0220] AAV particles can be retargeted to neonatal mouse TfR (mTfR) Finally, we used immunofluorescence imaging to evaluate the delivery of mTfR-retargeted AAV to neonates. As shown in Figure 24, and as expected, both WT AAV9 and the modified version (AAV.PHP.eB) exhibited robust spinal cord and motor neuron transduction. Both WT AAV9 conjugated with TfR antibodies and the W503A capsid variant retained the ability to transduce motor neurons, suggesting that TfR antibody conjugation does not interfere with the tropism of these viruses to neonatal motor neurons. Although non-targeted WT AAV9 and AAV.PHP.eB exhibited stronger GFP signals within motor neurons compared to TfR-targeted AAV9, quantification of the number of transduced motor neurons showed approximately 90% transduction efficiency for all viruses, indicating that the retargeted viruses achieved high transduction efficiency despite lower total GFP signals per cell. Thus, TfR-targeted AAV particles transduce neonatal spinal cord motor neurons when administered directly into the CNS. As shown in Figure 25, neonatal mice injected intravenously with retargeted AAV9 show highly robust and reproducible transduction of lumbar spinal motor neurons when compared to intravenously (iv) injected PBS-negative control mice. Thus, AAV particles targeting TfR highly transduce neonatal spinal motor neurons when administered intravenously.

[0221] AAV particles that target the TfR can deliver functional cargo to the CNS, including nucleic acid sequences encoding therapeutic antibodies and shRNA for targeted gene knockdown. We evaluated the ability of TfR-retargeted AAVs to mediate the expression of therapeutic payloads in the brain via systemic delivery. To evaluate the delivery of vectored antibodies to the brain using mTfR-retargeted AAVs, we compared AAV9 W503A conjugated with an mTfR-targeting scFv with AAV8, which is not expected to cross the blood-brain barrier and transduce cells in the CNS. TfR-targeted AAV particles were packaged with a transgene containing the ubiquitously expressed CAGG promoter and a gene for a human IgG antibody sequence of interest targeting the Pseudomonas aeruginosa type 3 secretion system (PcrV). For each group tested, the heavy and light chain sequences were separated by a distinct 2A motif (P2A, T2A, or F2A), which is essential for the generation of steric hindrance and ribosome skipping, allowing the production of two polypeptides from a single AAV-derived mRNA molecule. As shown in Figure 26A, robust RNA expression of human IgG antibody sequences was observed in the brain and spinal cord of animals treated with TfR-targeted AAV. Thus, AAV targeting the mouse TfR promotes the expression of secretory antibodies in the CNS after IV delivery. AAV RNA from AAV9 W503A particles conjugated with an scFv ("8D3") that binds mTfR was detected at greater levels in the brain and spinal cord compared with AAV8. This increase in AAV RNA in the CNS using AAV targeting mTfR was observed with all 2A sequence variant transgenes tested. AAV RNA levels in non-CNS tissues were minimal in animals injected with AAV9 W503A particles conjugated with an scFv ("8D3") that binds mTfR. Additionally, Figure 26B shows that human antibody protein concentrations in brain lysates from all animals treated with TfR-targeted AAV were higher than those from animals injected with AAV8. Thus, mTfR-retargeted AAV9 W503A facilitates delivery and expression of vectored antibody sequences to the brain via intravenous injection. Thus, AAV targeting the mouse TfR promotes expression of secreted antibodies in the CNS after IV delivery.

[0222] In addition, we evaluated the ability of TfR1-retargeted AAV to mediate shRNA expression and induce downregulation of target mRNA in the brain via systemic delivery. TfR1-targeted AAV particles were packaged with a transgene driving the expression of an shRNA against SNCA. Two SNCA shRNA sequences were tested (SNCA shRNA #1 and SNCA shRNA #2). As shown in Figure 27, SNCA-humanized mice injected with detargeted AAV9 W503A conjugated with mTfR1-Fab and expressing either SNCA shRNA #1 or SNCA shRNA #2 showed a 40-50% reduction in human SNCA mRNA levels in the cortex, midbrain, and striatum compared with untreated SNCA-humanized mice and SNCA-humanized mice injected with detargeted AAV9 W503A conjugated with mTfR1-Fab and expressing a control shRNA. Thus, TfR1-AAV enables the delivery and functional expression of shRNA and efficiently induces downregulation of target mRNA in multiple brain regions. Thus, expression of SNCA shRNA by AAV targeting the mouse TfR reduces SNCA mRNA levels in the CNS after IV delivery.

[0223] Materials and Methods Preparation of AAV viral vectors Virus was produced by transfecting 293T packaging cells using PEI Pro with the following plasmids: pAd helper, an AAV2 ITR-containing genome plasmid encoding a reporter protein, and pAAV-CAP plasmid encoding the AAV Rep and Cap genes, with or without additional plasmids encoding either antibody heavy and light chains, Fab, or scFv. All antibody or Fab heavy chain constructs or scFvs were fused to SpyCatcher at their C-terminus. Transfection complexes were prepared in incomplete DMEM (without additional supplements) and incubated at room temperature for 10 minutes.

[0224] Each virus was generated by transfecting 15 cm plates of 293T packaging cells with the following plasmids and quantities: [Table 9-1] [Table 9-2] [Table 9-3]

[0225] After incubation, the complexes were added to DMEM supplemented with 10% FBS, 1× NEAA, 1% Pen / Strep, and 1% L-glutamine.

[0226] Transfected packaging cells were incubated at 37°C for 3 days, and then virus was recovered from the cell lysate using a standard freeze-thaw protocol. Briefly, packaging cells were lifted by scraping and pelleted. 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). Cell lysis was induced and intracellular virus particles were released by three consecutive freeze-thaw cycles, consisting of transferring the cell suspension between a dry ice / ethanol bath and a 37°C water bath with vigorous vortexing. Viscosity was reduced by treating the lysate with EMD Millipore Benzonase (50 U / ml cell lysate) for 90 minutes at 37°C with occasional mixing. Debris was then pelleted by centrifugation, and the resulting supernatant was filtered through a 0.22 μm PVDF Millex-GV filter. For crude viruses tested in vitro, the crude virus was pipetted into low-protein binding tubes and stored at 4°C. For viruses 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 hours at 10°C using maximum acceleration and deceleration. After ultracentrifugation, iodixanol-purified virions were extracted from the 40-60% interface. AAV in iodixanol solution was diluted with DPBS + / + 0.001% Pluronic® F68 so that the iodixanol concentration was less than 1%. The purified virus was then concentrated to the desired volume using a 100 kDa MWCO Amicon ultrafiltration unit.

[0227] Titers (viral genomes per milliliter; vg / mL) were determined by qPCR using a standard curve of known concentrations of virus.

[0228] Cell line: All 293 cell lines were maintained in DMEM supplemented with 10% FBS, 1x NEAA, 1% Pen / Strep, and 1% L-glutamine. 293 hASGR1 / 2 and 293hTfR cell lines were generated by lentiviral transduction of the parental 293 cell line with vectors expressing the corresponding cDNAs. All cell lines were obtained from the Regeneron TC Core Facility.

[0229] All 3T3 cell lines were maintained in DMEM supplemented with 10% BCS, 1x NEAA, 1% Pen / Strep, and 1% L-glutamine. 3T3 hTfR cell lines were generated by lentiviral transduction of the parental 3T3 cell line with vectors expressing the corresponding cDNAs. All cell lines were obtained from the Regeneron TC Core Facility.

[0230] b. End3 cell line was maintained in DMEM supplemented with 10% FBS, 1x NEAA, 1% Pen / Strep, and 1% L-glutamine. Cell lines were obtained from the Regeneron TC core facility.

[0231] AAV capsid protein constructs GeneBlocks encoding the desired SpyTag insert, flanking linker amino acids, and additional mutations were purchased from IDT and cloned into the corresponding digested pAAV-CAP wt plasmid using Gibson Assembly according to the manufacturer's protocol (NEB).

[0232] Cell infection / transduction and flow cytometry analysis. To infect cells, viral particles were added directly to the medium of cultured cells, and the mixture was incubated at 37°C. Four days after infection, cells were trypsinized and 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.

[0233] Mouse strains Humanized TFRC mice (TFRC hu / hu ) expresses human TfR1 but does not express endogenous TfR1.

[0234] In vivo analysis of AAV9 GFP vector For intravenous (IV) injection, adult (3-4 month old) WT C57BL / 6J mice were injected with 7.5 × 10 9 , 1.6×10 10 , 5×10 10 , 8×10 10 , 4×10 11 , or 2 × 10 12 AAV particles were injected into the tail vein at a dose of 1000 vg / mouse. Adult (3-4 month old) female TFRCs hu / hu For mice, 1.5 x 10 10 , 1×10 11 , or 4 x 10 11 AAV particles were injected into the tail vein of 1000 mg / mouse. For intracerebroventricular (ICV) injection, adult (3-4 month old) humanized TFRC (TFRC hu / hu ) Mice were subjected to stereotaxic surgery and 1 × 10 10 AAV particles were injected into the lateral ventricle at a dose of 1000 mg / mouse. Mice were sacrificed 2–3 weeks after injection and perfused with saline. Brains and livers were harvested for immunohistochemistry, immunofluorescence, and qPCR analysis.

[0235] Barcode analysis Female humanized TFRC mice (TFRC hu / hu ) (7-9 weeks old), 1 × 10 11 vg / mouse or 5 × 10 11AAV9 (WT AAV9, AAV PHP.eB, AAV9 W503A anti-ASGR1 Fab, and 32 AAV9 W503A anti-TfR Fab containing the barcoded pITR-sc-CBh-eGFP-bGHpA plasmid as the viral genome plasmid) was injected retroorbitally (RO) into a mouse. Each of the 36 viruses in the pool was packaged with a version of pITR-sc-CBh-eGFP-bGHpA carrying a unique 32-nucleotide barcode, which was used to quantify transgene expression by capsid variants. Mice were sacrificed 14 days after injection, and the following organs were harvested for RNA extraction: liver, brain, and spinal cord.

[0236] AAV viral vectors were prepared as described above, with one modification: after cell lysis, soluble Spytag peptide (AHIVMVDAYKPTK, SEQ ID NO: 321) was added to the lysed cell suspension at a concentration of 100 μg / mL before centrifugation to pellet debris. The lysed cell suspension and soluble Spytag peptide were incubated at 37°C for 60 minutes. Samples were mixed in equal volumes before centrifugation.

[0237] For barcode analysis, total RNA isolated from MAID7229 mouse tissues and organs was purified using the MagMAX-96 Total RNA Isolation Kit for Microarrays according to the manufacturer's specifications. The RNA was then treated with ezDNase, and cDNA synthesis was performed using SuperScript IV reverse transcriptase and a bGH pA-specific primer (5'-ATCCTCCCCCTTGCTGTCCTGC-3', SEQ ID NO: 443). Barcoded GFP transcripts were amplified from the cDNA samples using Q5 Ultra II 2x Master Mix with primers binding upstream (5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGgcactgacaattccgtggtctagg-3', SEQ ID NO: 444) and downstream (5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGcaaacaacagatggctggcaactag-3', SEQ ID NO: 445) of the barcode. A pooled virus mixture was included in the samples. Each sample was prepared in three technical replicates during library preparation. Amplicons containing Illumina adapters and unique dual indexes (UDI-Illumina) were quantified using Qubit and TapeStation, pooled in equimolar ratios, and sequenced on a Nextseq550 using a medium-output kit for 300 cycles.

[0238] directional research Adult (3-4 month old) humanized TFRC mice (TFRC hu / hu ) mice, 1 × 10 11 vg / mouse AAV particles were injected via the tail vein. Mice were sacrificed 19 days after injection, perfused with phosphate-buffered saline, and brains were harvested for histology.

[0239] For immunofluorescence staining and evaluation of CNS cell type tropism, brains were hemisected in the midsagittal plane, and one half of the brain was fixed in 10% neutral buffered formalin solution and then transferred to 70% ethanol after 24 hours. Brains were stored in 70% ethanol for less than a week before being embedded in paraffin. Brains were embedded in paraffin blocks, cut into 4-μm-thick sections, and mounted on TOMO slides. Slides were dewaxed at 60°C for 1 hour and deparaffinized by washing in xylene for 3 x 10 minutes, followed by 3-minute washes in a descending series of ethanol as follows: 100%, 100%, 95%, 95%, 70%, and 50%. Slides were then incubated in 2 x distilled water, 2 x TBS for 2 minutes. Antigen retrieval in 1x Tris-EDTA, pH 9 (Abcam #ab93684) was performed using a steamer at 98°C for 30 minutes. After antigen retrieval, slides were incubated in 0.3% hydrogen peroxide in TBS to quench endogenous peroxidase activity, washed in TBS for 5 minutes, and blocked in TNB blocking buffer (0.1 M Tris-HCl, 0.15 M NaCl, 0.5% blocking reagent [Akoya, #NEL700A001], 0.3% Triton® X-100) for 30 minutes. The following primary antibodies were used: 0.1 mg / mL GFP (Abcam, #13970), 10 μg / mL GST-pi (MBL International, #MBL-312), 5 μg / mL CD31 (Abcam, #18281), 4 μg / mL MAP2 (Abcam, #ab32454), 2.4 μg / mL Iba1 (Abcam #178847), 2 μg / mL GFAP (Invitrogen #13-0300), 5 μg / mL Sox9 (Abcam #185966), 4 μg / mL CNPase (Sigma #MAB326), and 3.4 μg / mL NeuN (Abcam, #ab177487). Slides were incubated in primary antibodies overnight at 4°C. The primary antibody was rinsed off by 3 x 5 min washes in TBS and incubated with secondary antibody at a concentration of 2 μg / mL and DAPI (Invitrogen, #D3571) in 0.5 x TNB buffer for 1-2 h.The following secondary antibodies were used: donkey anti-chicken A488 (Invitrogen, #A78948), donkey anti-rabbit A647 (Invitrogen, #A31573), donkey anti-rat A594 (Invitrogen, #A21209), donkey anti-mouse A647 (Invitrogen, #A31574), and donkey anti-rabbit A594 (Invitrogen, #A32754). Slides were rinsed with multiple washes of TBS and coverslipped using antifade mounting medium (Invitrogen Anti diamond #P36970). Slides were scanned at 20x magnification on a Zeiss Axio Scan Z1.

[0240] For brain biodistribution quantification following immunohistochemical staining, brains were hemisected in the midsagittal plane, and one half of the brain was fixed in 10% neutral buffered formalin solution and then transferred to 70% ethanol after 24 hours. Brains were stored in 70% ethanol for less than a week before being embedded in paraffin. Prior to sectioning, the hemibrains were trimmed to ensure each section included the sagittal plane. Tissues were sectioned at 4 μm thickness using standardized planar sectioning. To obtain representative sagittal brain section images, brain sections were subjected to standardized CC1 antigen retrieval (56 min) and then treated with an antibody against eGFP (Abcam, #ab13970) at a concentration of 2 μg / mL for 5 hours. After secondary antibody incubation, eGFP was detected using the Discovery Dab Map detection kit. Immunohistochemistry was performed on a Ventana Discovery Ultra platform. After staining, slides were imaged at 40x magnification on an Aperio slide scanner. For quantification of GFP expression, 4 μm sagittal sections were taken to coincide with a lateral extension of +1.5 mm from the midline. Brain sections underwent standardized ER2 antigen retrieval (30 min) and then treated with an antibody against eGFP (Invitrogen, #A11122) at a concentration of 2 μg / mL for 1 h. After secondary antibody incubation, eGFP was detected using a Leica Refine detection kit. Immunohistochemistry was performed on the Bond Rx platform. After staining, slides were imaged at 40x magnification with an Aperio slide scanner. Chromogenic IHC images were imported into HALO software for annotation and quantification of the percent area occupied by GFP. Annotations were drawn to allow independent quantification of several regions throughout the brain. These included the olfactory bulb, cortex, striatum, hippocampus, thalamus, hypothalamus, brainstem, and cerebellum. Each set of annotations was manually corrected to match the designated brain region. In the HALO analysis algorithm, GFP signal positivity was assessed by the DAB optical density threshold, which was determined by exceeding the background level of DAB staining. The GFP-positive area was then normalized to the corresponding annotation area and plotted as the % positive area ratio.

[0241] For qPCR, tissues were flash frozen and stored at -80°C before DNA extraction. Viral DNA was measured in each tissue using a probe recognizing eGFP and calculated relative to the β-actin or GAPDH housekeeping genes.

[0242] Dose escalation Adult (3–4 month old) WT C57BL / 6J mice were injected with 1.6 × 10 10 , 8×10 10 , 4×10 11 , 2 × 10 12 AAV particles were injected into the tail vein of mice at a dose of 1000 mg / mouse. Mice were sacrificed 2 weeks after injection and perfused with phosphate-buffered saline. Brains and livers were harvested for immunohistochemistry and qPCR analysis.

[0243] For immunohistochemical staining, brains were hemisected in the midsagittal plane, and one half of the brain was fixed in 10% neutral buffered formalin solution and then transferred to 70% ethanol after 24 hours. Brains were stored in 70% ethanol for less than a week before being embedded in paraffin. Prior to sectioning, the hemibrains were trimmed to ensure each section included the sagittal plane. Tissues were sectioned at 4 μm thickness using standardized planar sectioning. Brain sections underwent standardized ER2 antigen retrieval (30 min) and then treated with an antibody against eGFP (Invitrogen, #A11122) at a concentration of 2 μg / mL for 1 hour. After secondary antibody incubation, eGFP was detected using a Leica Refine detection kit. Immunohistochemistry was performed on a Bond Rx platform. After staining, slides were imaged at 40x magnification using an Aperio slide scanner.

[0244] In vivo analysis of AAV human IgG vectors Adult (3-4 month old) WT C57BL / 6J mice were retroorbitally injected with 5e11 vg / mouse of detargeted AAV9 W503A conjugated with an scFv antibody ("8D3") targeting mTfR, or 5e10 vg / mouse of AAV8. The transgene contained a ubiquitously expressed CAGG promoter and a gene for a human IgG antibody sequence of interest targeting the Pseudomonas aeruginosa type 3 secretion system (PcrV). For each group tested, the heavy and light chain sequences were separated by a distinct 2A motif (P2A, T2A, or F2A), which is essential for steric hindrance and ribosomal skipping, allowing the production of two polypeptides from a single AAV-derived mRNA molecule. Mice were sacrificed 12 weeks after injection and perfused with phosphate-buffered saline (PBS). Brain, spinal cord, liver, heart, quadriceps, and spleen were collected for ELISA and qPCR analysis.

[0245] For ELISA-based detection of human IgG titer in the brain, brains were dissected in the midsagittal plane, and half of the brain was homogenized and lysed in 1x PBS and 2x HALT protease phosphotase inhibitor cocktail (Thermo Scientific, 78440). ELISA plates were coated with 1µg / ml AffiniPure goat anti-human IgG, Fcy fragment (Jackson 109-005-098) overnight at 4°C and then blocked with 3% bovine serum albumin. Diluted mouse brain lysates were applied to the wells at 1mg total protein / mL and incubated for 1 hour at room temperature. Goat anti-human kappa, mouse ads-HRP (Southern Biotech, 2061-05) was used as the secondary antibody. Final detection was performed using SuperSignal ELISA Pico Chemiluminescent Substrate (Thermo, 1856156). Brain lysate sample readouts were interpolated against a purified human IgG standard curve.

[0246] In vivo analysis of AAV vectors expressing SNCA shRNA Adult (2-3 month old) SNCA-humanized mice were treated with 4 × 10 11 Detargeted AAV9 W503A conjugated with a Fab ("8D3") targeting the mTfR of vg / mice was injected via the tail vein. The transgene contained a hU6 promoter driving SNCA shRNA expression and GFP as a fluorescent reporter driven by the Ubc promoter. Two SNCA shRNA sequences were tested (SNCA shRNA #1 and SNCA shRNA #2). Mice were sacrificed 1 month after injection and perfused with phosphate-buffered saline (PBS). Brains were harvested, microdissected, snap-frozen, and stored at -80°C before RNA extraction for qPCR analysis. SNCA mRNA levels were measured in the cortex, midbrain, and striatum using a probe targeting human SNCA mRNA and calculated relative to the GAPDH housekeeping gene.

[0247] Intracerebroventricular (icv) injection in neonatal mice Neonatal pups (P0) were anesthetized on ice for approximately 7–10 minutes until the toe-pinch reflex subsided. The pups were placed under a dissecting microscope with a light source attached, and the injection site was cleaned with an alcohol swab. Injections were performed using a 10 μL Hamilton syringe (Hamilton catalog number 7653-01) with a custom-made detachable needle (32 gauge, 12-degree tip angle, 0.75-inch length). Pups received a single injection into the left ventricle approximately 2 mm lateral to the sup...

Claims

1. Recombinant viral capsid protein, (i) Proteins inserted and / or presented by the recombinant viral capsid protein: the first member of the protein binding pair ("first member"), (ii) The protein: the second member of the protein binding pair ("second member"), the first member and the second member associate with the second member, (iii) A recombinant viral capsid protein comprising an antibody or binding portion thereof that binds to the extracellular domain of transferrin receptor protein 1 (abbreviated as TfR1, TfR, or CD71), wherein the antibody or binding portion thereof is fused with the second member.

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

436.

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

4. It further contains cells that express TfR1 on their surface, The recombinant viral capsid protein according to claim 1, wherein the recombinant viral capsid protein is bound to the extracellular domain of TfR1 expressed on the surface of the cell.

5. The aforementioned cells are the recombinant viral capsid proteins according to claim 4, as listed in Table 2.

6. It further contains blood-brain barrier endothelial cells that express TfR1 on their surface, The recombinant viral capsid protein according to claim 1, wherein the recombinant viral capsid protein is bound to the extracellular domain of TfR1 expressed on the surface of the blood-brain barrier endothelial cells.

7. The recombinant viral capsid protein according to claim 1, wherein the recombinant viral capsid protein is in vitro.

8. The recombinant viral capsid protein according to claim 1, wherein the recombinant viral capsid protein is in vivo.

9. (a) The first member includes SpyTag, IsopTag, SnoopTag, SpyTag002, SpyTag003, or a variant thereof (b) The second member is fused with the antibody or the binding portion thereof to comprise Spycatcher, KTag, pilin-C, SnoopCatcher, SpyCatcher002, SpyTag003, or a variant thereof. (c) The recombinant viral capsid protein according to claim 1, wherein the first member and the second member are associated by an isopeptide bond.

10. (a) The first member includes SpyTag or a variant thereof, (b) The recombinant viral capsid protein according to claim 9, wherein the second member comprises SpyCatcher or a variant thereof fused with the antibody or the binding portion thereof.

11. (a) The first member comprises the c-myc amino acid sequence described in Sequence ID No. 326, (b) The recombinant viral capsid protein according to claim 1, wherein the second member comprises an anti-c-myc antibody and a bispecific binding protein comprising the antibody or the binding portion thereof that binds to the extracellular domain of TfR1.

12. comprising a first linker and / or a second linker that operably links the first member to the recombinant viral capsid protein, The recombinant viral capsid protein according to claim 1, wherein the first linker and the second linker each independently have a length of at least one amino acid.

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

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

15. The recombinant viral capsid protein according to claim 12, wherein the first linker is 10 amino acids long and / or the second linker is 10 amino acids long.

16. The recombinant viral capsid protein according to claim 15, wherein the amino acid sequence of the first linker and / or the amino acid sequence of the second linker comprises the amino acid sequence described in SEQ ID NO: 331 or SEQ ID NO:

332.

17. (a) The recombinant viral capsid protein comprises the amino acid sequences of the modified VP1 capsid protein, modified VP2 capsid protein, and / or modified VP3 capsid protein encoded by the mutant cap gene, (b) The recombinant viral capsid protein according to claim 1, wherein the mutant cap gene or portion thereof contains a nucleotide sequence identical to that of the AAV cap gene or portion thereof by at least 90%, and the mutant cap gene or portion thereof is genetically modified to include the insertion of a nucleotide sequence encoding the first member, thereby the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein containing the first member.

18. The mutated cap gene or a portion thereof is genetically modified to include one or more additional mutations, thereby the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein are added to the first member. (i) Point mutations including amino acid substitutions, insertions, or deletions, (ii) Chimeric amino acid sequence, (iii) The recombinant viral capsid protein according to claim 17, comprising both a point mutation and a chimeric amino acid sequence.

19. The recombinant viral capsid protein according to claim 18, wherein the substitution, insertion, or deletion of amino acids reduces the innate directivity of the viral particle containing the recombinant viral capsid protein and / or produces a detectable label.

20. (a) The recombinant viral capsid protein comprises the amino acid sequences of the modified VP1 capsid protein, modified VP2 capsid protein, and / or modified VP3 capsid protein encoded by the mutant cap gene, (b) The mutant cap gene or portion thereof contains a nucleotide sequence that is at least 90% identical to the cap gene or portion thereof of AAV, and the mutant cap gene or portion thereof is genetically modified to include the insertion of a nucleotide sequence encoding the first member, thereby the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein containing the first member, (c) The recombinant viral capsid protein according to claim 1, wherein 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 animal AAVs listed in Table 5, and combinations thereof.

21. The recombinant viral capsid protein according to claim 20, wherein the AAV is AAV2.

22. The recombinant viral capsid protein according to claim 21, comprising a modified AAV2 VP1 capsid protein, wherein the recombinant viral capsid protein includes the first member linked to the amino acid at position I-453 and / or I-587.

23. The recombinant viral capsid protein according to claim 22, wherein the recombinant viral capsid protein comprises a modified AAV2 VP1 capsid protein having the first member presented at position G453 via a linker.

24. The recombinant viral capsid protein according to claim 23, wherein the modified AAV2 VP1 capsid protein further comprises R585A modification, R588A modification, or both of the R585A modification and the R588A modification.

25. The recombinant viral capsid protein according to claim 23, wherein the modified AAV2 VP1 capsid protein further comprises R484A modification, R487A modification, R585A modification, R588A modification, and K532A modification, or any combination of R484A modification, R487A modification, R585A modification, R588A modification, and K532A modification.

26. The recombinant viral capsid protein according to claim 20, wherein the AAV is AAV9.

27. The recombinant viral capsid protein according to claim 26, comprising a modified AAV9 VP1 capsid protein in which the recombinant viral capsid protein includes the first member linked to an amino acid at position I-453, I-587, or I-589.

28. The recombinant viral capsid protein according to claim 27, wherein the recombinant viral capsid protein comprises a modified AAV9 VP1 capsid protein having the first member presented at G453 via a linker.

29. The recombinant viral capsid protein according to claim 28, wherein the modified AAV9 VP1 capsid protein further comprises N272A modification, W503A modification, or both of the N272A modification and the W503A modification.

30. The recombinant viral capsid protein according to claim 20, wherein the non-primate AAV is an avian AAV (AAAV), a non-human mammal AAV, or a squamate AAV.

31. The recombinant viral capsid protein according to claim 30, wherein the non-primate animal AAV is AAAV.

32. The recombinant viral capsid protein according to claim 31, comprising a modified AAAV VP1 capsid protein in which the recombinant viral capsid protein includes the first member linked to an amino acid at position I-444 or I-580.

33. The recombinant viral capsid protein according to claim 30, wherein the non-primate AAV is a non-human mammalian AAV.

34. The recombinant viral capsid protein according to claim 33, wherein the non-human mammalian AAV is sea lion AAV.

35. The recombinant viral capsid protein according to claim 34, comprising a modified sea lion VP1 capsid protein, wherein the recombinant viral capsid protein includes the first member linked to an amino acid selected from the group consisting of I-429, I-430, I-431, I-432, I-433, I-434, I-436, I-437, and I-565.

36. The recombinant viral capsid protein according to claim 30, wherein the non-primate AAV is a Squamata AAV.

37. The recombinant viral capsid protein according to claim 36, wherein the aforementioned Squamata AAV is the bearded dragon AAV.

38. The recombinant viral capsid protein according to claim 37, comprising a modified bearded dragon VP1 capsid protein, wherein the recombinant viral capsid protein includes the first member linked to an amino acid at position I-573 or I-436.

39. The recombinant viral capsid protein according to claim 1, wherein the antibody or the binding portion thereof that binds to the extracellular domain of TfR1 binds to the same epitope on the extracellular domain of TfR1 as the reference antibody containing the HCVR / LCVR amino acid sequence pair listed in Table 1.

40. The antibody or the binding portion thereof that binds to the extracellular domain of TfR1 is a heavy chain complementarity determining region (HCDR1, HCDR2, and HCDR3) from a heavy chain variable region (HCVR) containing the amino acid sequence (or variant thereof) described in SEQ ID NOs: 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, and / or Recombinant viral capsid protein according to claim 39, comprising light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) from a light chain variable region (LCVR) containing an amino acid sequence (or a variant thereof) as described in SEQ ID NOs: 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.

41. The antibody or the binding portion thereof that binds to the extracellular domain of TfR1 (i) HCVRs comprising HCDR1, HCDR2, and HCDR3 of HCVRs comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 2, and LCVRs comprising LCDR1, LCDR2, and LCDR3 of LCVRs comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 7, (ii) HCVRs comprising HCDR1, HCDR2, and HCDR3 of HCVRs comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 12, and LCVRs comprising LCDR1, LCDR2, and LCDR3 of LCVRs comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 17, (iii) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 22, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 27, (iv) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 32, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 37, (v) HCVRs comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 42, and LCVRs comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 47, (vi) HCVRs comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 52, and LCVRs comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 57, (vii) HCVRs comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 62, and LCVRs comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 67, (viiii) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 72, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 77, (ix) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 82, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 87, (x) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 92, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 97, (xi) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 102, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 107, (xi) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 112, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 117, (xiii) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 122, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 127, (xiv) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 132, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 137, (xv) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 142, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 147, (xvi) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 152, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 157, (xvii) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 162, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 167, (xviiii) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 172, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 177, (xix) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 182, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 187, (xx) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 192, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 197, (xxi) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 202, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 207, (xxii) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 212, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 217, (xxiii) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 222, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 227, (xiv) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 232, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 237, (xv) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 242, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 247, (xvi) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 252, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 257, (xvii) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 262, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 267, (xviiii) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 272, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 277, (xix) HCVR comprising HCDR1, HCDR2, and HCDR3 of HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 282, and LCVR comprising LCDR1, LCDR2, and LCDR3 of LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 287, (xxx) HCVR containing HCDR1, HCDR2, and HCDR3 of HCVR containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 292, and LCVR containing LCDR1, LCDR2, and LCDR3 of LCVR containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 297, (xxxi) HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 302, and LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 307, and / or The recombinant viral capsid protein according to claim 40, comprising an HCVR comprising HCDR1, HCDR2, and HCDR3 of an HCVR comprising the amino acid sequence (or variant thereof) described in (xxxii) SEQ ID NO: 312, and an LCVR comprising LCDR1, LCDR2, and LCDR3 of an LCVR comprising the amino acid sequence (or variant thereof) described in SEQ ID NO:

317.

42. The antibody or the binding portion thereof that binds to the extracellular domain of TfR1 (a) HCVR including HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 3, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 4, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 5, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 8, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 9, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

10. (b) HCVR comprising HCDR1 comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 13, HCDR2 comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 14, and HCDR3 comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 15, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 18, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 19, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

20. (c) HCVR including HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 23, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 24, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 25, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 28, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 29, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

30. (d) HCVR comprising HCDR1 comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 33, HCDR2 comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 34, and HCDR3 comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 35, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 38, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 39, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

40. (e) HCVR comprising HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 43, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 44, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 45, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 48, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 49, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

50. (f) HCVR including HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 53, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 54, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 55, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 58, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 59, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

60. (g) HCVR comprising HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 63, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 64, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 65, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 68, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 69, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

70. (h) HCVR comprising HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 73, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 74, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 75, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 78, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 79, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

80. (i) HCVR including HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 83, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 84, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 85, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 88, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 89, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

90. (j) HCVR comprising HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 93, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 94, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 95, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 98, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 99, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

100. (k) HCVR comprising HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 103, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 104, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 105, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 108, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 109, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

110. (l) HCVR comprising HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 113, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 114, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 115, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 118, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 119, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

120. (m) HCVR including HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 123, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 124, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 125, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 128, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 129, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

130. (n) HCVR comprising HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 133, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 134, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 135, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 138, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 139, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

140. (o) HCVR including HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 143, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 144, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 145, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 148, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 149, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

150. (p) HCVR including HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 153, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 154, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 155, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 158, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 159, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

160. (q) HCVR including HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 163, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 164, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 165, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 168, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 169, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

170. (r) HCVR including HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 173, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 174, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 175, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 178, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 179, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

180. (s) HCVR comprising HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 183, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 184, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 185, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 188, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 189, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

190. (t) HCVR comprising HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 193, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 194, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 195, and LCVR, comprising LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 198, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 199, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 200, (u) HCVR comprising HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 203, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 204, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 205, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 208, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 209, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

210. (v) HCVR comprising HCDR1 comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 213, HCDR2 comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 214, and HCDR3 comprising the amino acid sequence (or variant thereof) described in SEQ ID NO: 215, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 218, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 219, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

220. (w) HCVR including HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 223, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 224, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 225, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 228, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 229, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

230. (x) HCVR comprising HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 233, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 234, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 235, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 238, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 239, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

240. (y) HCVR comprising HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 243, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 244, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 245, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 248, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 249, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

250. (z) HCVR including HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 253, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 254, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 255, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 258, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 259, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

260. (aa) HCVR including HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 263, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 264, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 265, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 268, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 269, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

270. (ab) HCVR comprising HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 273, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 274, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 275, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 278, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 279, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

280. (ac) HCVR including HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 283, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 284, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 285, and LCVR, comprising LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 288, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 289, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 290, (ad) HCVR comprising HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 293, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 294, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 295, and LCVR includes LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 298, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 299, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO:

300. (ae) HCVR comprising HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 303, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 304, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 305, and LCVR, including LCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 308, LCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 309, and LCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 310, and / or (af) HCVR including HCDR1 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 313, HCDR2 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 314, and HCDR3 containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 315, and The recombinant viral capsid protein according to claim 41, comprising LCVR, which includes LCDR1 containing the amino acid sequence (or a variant thereof) described in SEQ ID NO: 318, LCDR2 containing the amino acid sequence (or a variant thereof) described in SEQ ID NO: 319, and LCDR3 containing the amino acid sequence (or a variant thereof) described in SEQ ID NO:

320.

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

317.

44. The antibody or the binding portion thereof that binds to the extracellular domain of TfR1 (i) HCVR containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 2, and LCVR containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 7, (ii) HCVR containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 42, and LCVR containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 47, (iii) HCVR containing the amino acid sequence (or a variant thereof) described in SEQ ID NO: 122, and LCVR containing the amino acid sequence (or a variant thereof) described in SEQ ID NO: 127, (iv) HCVR containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 132, and LCVR containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 137, (v) HCVR containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 212, and LCVR containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 217, (vi) HCVR containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 222, and LCVR containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 227, (vii) HCVR containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 232, and LCVR containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 237, (viiii) HCVR containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 242, and LCVR containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 247, (ix) HCVR containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 262, and LCVR containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 267, (x) HCVR containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 272, and LCVR containing the amino acid sequence (or variant thereof) described in SEQ ID NO: 277, (xi) an HCVR containing the amino acid sequence (or a variant thereof) described in SEQ ID NO: 282, and an LCVR containing the amino acid sequence (or a variant thereof) described in SEQ ID NO: 287, and / or (xi) The recombinant viral capsid protein according to claim 42, comprising an HCVR containing the amino acid sequence (or a variant thereof) described in SEQ ID NO: 292, and an LCVR containing the amino acid sequence (or a variant thereof) described in SEQ ID NO:

297.

45. A recombinant viral capsid comprising the recombinant viral capsid protein described in claim 1.

46. The recombinant viral capsid is a recombinant mosaic viral capsid, and further comprises a reference viral capsid protein that is at least 95% identical to the recombinant viral capsid protein. The recombinant viral capsid according to claim 45, wherein the reference viral capsid protein lacks all three of the following: (i) the first member, (ii) the second member, and (iii) the antibody or its binding site.

47. The recombinant viral capsid according to claim 46, wherein the recombinant mosaic virus capsid comprises the reference viral capsid protein and the recombinant viral capsid protein in at least the ratios of 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, and 20:

1.

48. A viral particle containing a capsidized target nucleotide within the recombinant viral capsid described in Claim 45.

49. The viral particle according to claim 48, wherein the target nucleotide is a reporter gene.

50. The virus particle according to claim 49, wherein the target nucleotide encodes β-galactosidase, green fluorescent protein (GFP), high-sensitivity green fluorescent protein (eGFP), MmGFP, blue fluorescent protein (BFP), high-sensitivity blue fluorescent protein (eBFP), mPlum, mCherry, tdTomato, mStrawberry, J-Red, DsRed, mOrange, mKO, mCitrine, Venus, YPet, yellow fluorescent protein (YFP), high-sensitivity yellow fluorescent protein (eYFP), Emerald, CyPet, cyanide fluorescent protein (CFP), Cerulea, T-Sapphire, luciferase, alkaline phosphatase, or a combination thereof.

51. The viral particle according to claim 48, wherein the target nucleotide 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.

52. The viral particle according to claim 48, wherein the target nucleotide is operably linked to a promoter that is organ-specific, tissue-specific, or cell-specific.

53. The virus particle according to claim 52, wherein the promoter is brain-specific.

54. The virus particle according to claim 52, wherein the promoter is neuron-specific, glial cell-specific, astrocyte-specific, oligodendrocyte-specific, microglia-specific, and / or central nervous system-specific.

55. The virus particle according to claim 54, 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 proteolipide protein 1 (PLP1) promoter.

56. The virus particle according to claim 52, wherein the promoter is specific to neurons, astrocytes, or oligodendrocytes, or preferentially promotes neurons, astrocytes, or oligodendrocytes.

57. The virus particle according to claim 56, wherein the promoter is selected from the group consisting of the 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.

58. A pharmaceutical composition comprising (a) a virus particle according to any one of claims 48 to 57, and (b) a pharmaceutically acceptable carrier or excipient.

59. The pharmaceutical composition according to claim 58 for delivering a target nucleotide across the blood-brain barrier in mammals.

60. The pharmaceutical composition according to claim 59, characterized in that the pharmaceutical composition is administered by intravenous injection.

61. The pharmaceutical composition according to claim 59, wherein the mammalian subject is modified to express the antibody or the binding portion thereof that binds to the extracellular domain of TfR1.

62. The pharmaceutical composition according to claim 61, wherein the mammalian subject is modified to express the antibody or the binding portion thereof from the safe harbor site.

63. The pharmaceutical composition according to claim 59, wherein the target mammal is a primate mammal.

64. The pharmaceutical composition according to claim 63, wherein the primate mammal subject is a human subject.

65. The pharmaceutical composition according to claim 59, wherein the mammalian blood-brain barrier cells of the mammalian blood-brain barrier are mammalian brain endothelial cells.

66. The pharmaceutical composition according to claim 65, wherein the mammalian brain endothelial cells within the mammalian blood-brain barrier express transferrin receptor protein 1 on their cell surface, and (i) the first member, (ii) the second member, and (iii) the antibody or the binding portion thereof together direct the viral particles toward the mammalian brain endothelial cells within the mammalian blood-brain barrier.

67. The pharmaceutical composition according to claim 66, wherein the viral particles are transported across the mammalian blood-brain barrier by transcytosis after binding of the viral particles to the transferrin receptor protein 1 on the surface of the mammalian brain endothelial cells for delivery to the brain.

68. The pharmaceutical composition according to claim 59, wherein the target nucleotide codes for a therapeutic protein, and the therapeutic protein is a soluble protein.

69. The pharmaceutical composition according to claim 68, wherein the therapeutic protein comprises an antibody or a binding portion thereof.

70. The pharmaceutical composition according to claim 69, characterized in that the pharmaceutical composition is administered by intravenous or intraventricular injection.

71. The pharmaceutical composition according to claim 59, wherein the target nucleotide encodes an shRNA molecule.

72. The pharmaceutical composition according to claim 59, wherein the target nucleotide is operably linked to a brain-specific promoter, and the target nucleotide is preferentially expressed in the brain over other organs or tissues.

73. The pharmaceutical composition according to claim 58 for use in a patient who requires treatment for a disease.

74. The pharmaceutical composition according to claim 73, wherein the target nucleotide codes for a therapeutic portion that targets α-synuclein.

75. The pharmaceutical composition according to claim 74, wherein the therapeutic portion comprises an SNCA shRNA molecule.

76. Use of the pharmaceutical composition according to claim 58 for the manufacture of a pharmaceutical for the treatment of a disease.

77. Viral proteins, capsids, genomes, particles, and methods for producing and using them, including the manufacture of pharmaceuticals, as substantially described herein.

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