Viral particles retargeted to skeletal muscle

Abstract

Provided herein are compositions and methods for retargeting viral particles, such as adeno-associated virus (AAV) particles, to muscle cells using muscle-specific surface proteins. AAV adapted accordingly can be a viable gene therapy platform for the treatment of skeletal muscle-related disorders (e.g., X-linked myotubular myopathy (XLMTM), Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM1), facioscapulohumeral muscular dystrophy type 1 (FSHD), congenital muscular dystrophy type 1A (MDC1A), limb-girdle muscular dystrophy, dystroglycopathies, etc.) in patients in need of such treatment.

Description

[Technical field]

[0001] The present disclosure relates to methods of making and using recombinant viral particles, e.g., recombinant AAV particles, that include capsid proteins retargeted to muscle-specific surface proteins, e.g., calcium voltage-gated accessory subunit gamma 1 (CACNG1) or cadherin 15 (CAD15), useful for modifying muscle cells, such as skeletal muscle cells, in vitro or in vivo.

[0002] Sequence Listing The sequence listing in xml format entitled "11074WO01_xml" was created on November 4, 2022, is 252 Kb, and is incorporated by reference in its entirety into this specification. [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 introduce genetic material into desired cells and avoid introducing genetic material into non-target cells.

[0004] Viral particles, particularly those based on adeno-associated virus (AAV), 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 obvious evidence of toxicity or pathogenicity (Muzyczka, et al. (1992) Current Topics in Microbiology and Immunology, 158:97-129). Furthermore, AAV safely transduces postmitotic tissues. The virus can rarely integrate into host chromosomes, with very rare integration into the safe harbor locus of human chromosome 19 occurring only when replication (Rep) proteins are provided in trans. The AAV genome rapidly circularizes and concatenates in infected cells and exists in a stable episomal state in infected cells, providing long-term and stable expression of the payload.

[0005] More recently, it has been achieved to manipulate and redirect AAV infection to specific cells. Many of the advances in targeted gene therapy using viral particles can be summarized as non-recombinant (non-genetic) or recombinant (genetic) modification of viral particles, resulting in pseudotyping, expansion, and / or retargeting of the natural tropism of viral particles (reviewed in Nicklin and Baker (2002) Curr. Gene Ther. 2:273-93, Verheiji and Rottier (2012) Advances Virol 2012:1-15).

[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 is genetically engineered to express a capsid protein that contains a heterologous targeting ligand. The targeting ligand is then redirected, 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 (See also Shi et al. (2001) Human Gene Therapy 12:1697-1711, Shi and Bartlett (2003) Molecular Therapy 7:515-525).

[0007] In the indirect recombinant approach, the viral capsid is engineered with a heterologous "scaffold" and then ligated to an adaptor that contains a targeting ligand, which binds to the scaffold and to the target cell. (Arnold et al. (2006) Mol. Ther. 5:125-132; Ponnazhagen et al. (2002) J. Virol. 76:12900-907; see also WO 97 / 05266.) Scaffolds such as (1) Fc binding molecules (e.g., Fc receptors, protein A, etc.) that bind the Fc of an antibody adaptor, (2) (strept)avidin that binds to a biotinylated adaptor, (3) biotin that binds to an adaptor fused to (strept)avidin, (4) detectable labels useful for detection and / or isolation of virus particles, where the detectable label is attached to a bispecific adaptor that can non-covalently bind to the detectable label and to a target molecule, and more recently (5) protein:protein binding pairs that form isopeptide bonds have been reported for various virus 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 the advances being made in providing the ability to direct AAV infection, there remains a need to discover targets for the specific transfer of a nucleic acid of interest into a cell of interest, such as a mammalian muscle cell. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Muzyczka, et al. (1992) Current Topics in Microbiology and Immunology, 158:97-129

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[0009] It is shown herein that AAV capsid proteins can be modified to allow for targeted introduction of a nucleotide of interest into mammalian skeletal muscle cells.

[0010] The viral particles described herein are particularly suitable for targeted introduction of a nucleotide of interest into muscle cells, in particular because the viral capsids or viral capsid proteins described herein comprise a targeting ligand that binds to a muscle cell-specific surface protein. In some embodiments, the viral capsid or viral capsid protein comprises a first member of a binding pair associated with its cognate second member of the binding pair, the second member being linked (e.g., fused) to a targeting ligand that binds to a muscle cell-specific surface protein. In some embodiments, the targeting ligand is operably linked to the second member, e.g., fused to the second member, optionally 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, and the like. 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 and a heavy chain constant domain that binds to a muscle-specific surface protein on a muscle cell. In some embodiments, the targeting ligand is an antibody comprising a variable domain and an IgG heavy chain constant domain that binds to a muscle-specific surface protein on a target cell. In some embodiments, the targeting ligand is an antibody comprising a variable domain that binds to a muscle-specific surface protein on a target cell and an IgG heavy chain constant domain, where 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 protein described herein comprises a SpyTag operably linked to a viral capsid protein, and comprises a first member that is covalently linked to the SpyTag and a second member that comprises SpyCatcher linked to a targeting ligand comprising an antibody variable domain and an IgG heavy chain domain, where the SpyCatcher and the IgG heavy chain domain are linked via an amino acid linker, such as, e.g., GSGESG (SEQ ID NO: 253). In some embodiments, the muscle-specific surface protein comprises CACNG1.In some embodiments, the targeting ligand binds to CACNG1, for example, human CACNG1. In some embodiments, the targeting ligand comprises a heavy chain variable domain, a light chain variable domain, a heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 comprising the amino acid sequences of a heavy chain variable domain, a light chain variable domain, a heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in any one of SEQ ID NOs: 1-240. [Brief description of the drawings]

[0011] [Figure 1-1] AAV2-based retargeting virus infection delivering GFP to 293 cell lines genetically modified to express ASGR1 or CACNG1 is shown. (A) Scatter plots obtained from flow cytometry evaluating green fluorescent protein (GFP) expression by hASGR1-expressing positive (+) cells after infection with AAV2 WT particles, AAV2 HBM detargeting mutant particles, AAV2 SpyTag anti-ASGR1 particles, or AAV2 SpyTag anti-CACNG1 particles. Also shown are scatter plots obtained from flow cytometry evaluating green fluorescent protein (GFP) expression by hCACNG1-positive (+) cells after infection with AAV2 WT particles, AAV2 HBM detargeting mutant particles, AAV2 SpyTag anti-ASGR1 particles, or AAV2 SpyTag anti-CACNG1 particles. The virus expresses GFP as a marker of transduction. (B) Graph quantifies the percentage of GFP+ cells from the flow cytometry plots in (A). [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 2-1]AAV9-based retargeting virus infection delivering GFP to 293 cell lines genetically modified to express ASGR1 or CACNG1 is shown. (A) Scatter plots obtained from flow cytometry evaluating green fluorescent protein (GFP) expression by hASGR1-expressing positive (+) cells after infection with "AAV9 wt", "AAV9 detargeting mutant" particles, "AAV9 SpyTag anti-ASGR1" particles, or "AAV9 SpyTag anti-CACNG1" particles. Also shown are scatter plots obtained from flow cytometry evaluating green fluorescent protein (GFP) expression by hCACNG1-positive (+) cells after infection with "AAV9 wt", "AAV9 detargeting mutant" particles, "AAV9 SpyTag anti-ASGR1" particles, or "AAV9 SpyTag anti-CACNG1" particles. The virus expresses GFP as a marker of transduction. (B) Graph quantifies the percentage of GFP+ cells from the flow cytometry plots in 2A. [Figure 2-2] Same as above. [Figure 2-3] Same as above. [Figure 2-4] Same as above. [Diagram 3]AAV2-based and AAV9-based retargeting virus infection (MOI 1x106) delivering luciferase to 293 cell lines genetically modified to express ASGR1 or CACNG1 is shown. (A) Luciferase assays were performed to evaluate firefly luciferase expression by hCACNG1 positive (+) cells after infection with AAV2 WT, AAV2 HBM + anti-hASGR1, and AAV2 HBM + anti-hCACNG1 mAb#1 particles. Also shown are the results of luciferase assays evaluating firefly luciferase expression by hASGR1 positive (+) cells after infection with AAV2 WT, AAV2 HBM + anti-hASGR1, and AAV2 HBM + anti-hCACNG1 mAb#1 particles. (B) Luciferase assays were performed to assess firefly luciferase expression by hCACNG1 positive (+) cells following infection with AAV9 WT, AAV9 N272A, AAV9 N272A + anti-hASGR1 full antibody, AAV9 N272A + anti-hASGR1 Fab, AAV9 N272A + anti-CACNG1 mAb#1 full antibody, and AAV9 N272A + anti-hCACNG1 mAb#1 Fab. Also shown are the results of a luciferase assay evaluating firefly luciferase expression by hCACNG1 positive (+) cells following infection with AAV9 WT, AAV9 N272A, AAV9 N272A + anti-hASGR1 full antibody, AAV9 N272A + anti-hASGR1 Fab, AAV9 N272A + anti-CACNG1 mAb#1 full antibody, and AAV9 N272A + anti-hCACNG1 mAb#1 Fab. [Figure 4] Figure 1 shows AAV2-based retargeting viral transduction of human skeletal myotubes. (A) Representative immunofluorescence images are provided, as well as (B) transduction efficiency assessed by quantifying the average GFP expression in myosin heavy chain (MyHC)-positive regions of human skeletal myotubes after 3 days of transduction with 2E+5vg / cell of the indicated AAV expressing eGFP under the control of the CAG promoter. Transduction efficiency was assessed by quantifying the average GFP fluorescence intensity within the myosin heavy chain (MyHC)-positive myotube regions. [Diagram 5]AAV9-based retargeting viral transduction of human skeletal myotubes. (A) Representative immunofluorescence images are provided, as well as (B) transduction efficiency assessed by quantifying the average GFP expression in myosin heavy chain (MyHC)-positive regions of human skeletal myotubes after 3 days of transduction with 2E+5vg / cell of the indicated AAV expressing eGFP under the control of the CAG promoter. [Figure 6] AAV9-based retargeting viral transduction of differentiated mouse C2C12 myotubes. (A) Representative immunofluorescence images as well as (B) transduction efficiency assessed by quantifying the average GFP expression in myosin heavy chain (MyHC)-positive regions of differentiated mouse C2C12 myotubes transduced for 3 days with 2E+5vg / cell of the indicated AAV expressing eGFP under the control of the CAG promoter. [Figure 7-1]Figure 1 shows that systemically delivered AAV2 retargeted to CACNG1 demonstrates antibody-dependent transduction of skeletal muscle in vivo. The graph provides the mean radiance values ​​(photons / sec / cm2 / sr) from luminescence images of (A) liver, (B) tongue, (C) diaphragm, or (D) quadriceps (quad) tissues imaged ex vivo and isolated from mice genetically modified to express human CACNG1 on skeletal muscle cells (CACNG1 humanized mice) and wild-type 50500 mice, which were injected intravenously with phosphate-buffered saline (PBS), or with wild-type (wt) AAV2 particles, AAV2 detargeted particles, or SpyTagged AAV2 particles carrying the firefly luciferase nucleotide of interest and modified with (1) SpyCatcher-anti-human ASGR1 antibody or (2) SpyCatcher-anti-human CACNG1 antibody at 5e11 viral genomes (vg) / animal. These AAV2 viral particles are mosaic viral particles composed of a 1:7 ratio between (a) the "SpyTag" capsid protein (the SpyTag is inserted immediately after residue G453 flanked on either side by a 10 amino acid linker) and (b) a capsid without the SpyTag but containing the R585A and R588A mutations that reduce native receptor binding. The virus expresses firefly luciferase as a marker of transduction. Five weeks after IV injection, mice were anesthetized using isoflurane, injected with luciferin substrate, and euthanized 7–10 min later. Organs were harvested and imaged using an IVIS Spectrum in vivo imaging system (PerkinElmer). Raw data were analyzed using living image software to determine mean radiance (photons / sec / cm2 / sr). [Figure 7-2] Same as above. [Figure 8-1]We show that systemically delivered AAV9 retargeted to CACNG1 demonstrates antibody-dependent transduction of skeletal muscle in vivo. The graphs provide the average radiance values ​​(photons / sec / cm2 / sr) from luminescence images of (A) liver, (B) hind leg, (C) quadriceps (quad), or (D) tongue tissues isolated from mice genetically modified to express human CACNG1 (CACNG1 humanized mice) that were imaged ex vivo and injected intravenously with phosphate buffered saline (PBS) or SpyTagged AAV9 particles carrying 5e10 viral genomes (vg) / animal wild-type (wt) AAV9 particles, AAV9 detargeted particles, or firefly luciferase nucleotides of interest and modified with (1) SpyCatcher-anti-human ASGR1 full antibody, (2) SpyCatcher-anti-human ASGR1 Fab, (3) SpyCatcher-anti-human CACNG1 mAb#1 full antibody, or (4) SpyCatcher-anti-human CACNG1 mAb#1 Fab. These AAV9 viral particles are mosaic viral particles composed of a 1:7 ratio between (a) the "SpyTag" capsid protein (the SpyTag is inserted immediately after residue G453 flanked on either side by a 10 amino acid linker) and (b) a capsid without the SpyTag but containing the N272A mutation that reduces native receptor binding. The virus expresses firefly luciferase as a marker of transduction. Three weeks after IV injection, mice were anesthetized using isoflurane, injected with luciferin substrate, and euthanized 7-10 minutes later. The following organs were harvested for ex vivo imaging: liver, hind limbs, quadriceps, and tongue. Organs were imaged using an IVIS Spectrum in vivo imaging system (PerkinElmer). Raw data was analyzed using living image software to determine mean radiance (photons / sec / cm2 / sr). [Figure 8-2] Same as above. [Figure 9]GFP gene expression analysis in liver and quadriceps of (A) CACNG1hu / hu, (B) WT C57BL / 6, and (C) D2-mdx mice 3 weeks after tail vein injection of 1E+11vg / mouse of wild-type AAV9, detargeted AAV9 N272A, and AAV9 conjugated to antibodies targeting CACNG1 (mAb#1 and mAb#2) or hASGR1 as a non-targeting control. GFP expression was quantified via Taqman-based qPCR assay and normalized to Rplp0 as an endogenous control. GFP mRNA expression is presented relative to WT AAV9 for each tissue / mouse strain. [Figure 10] Representative immunofluorescence images of tibialis anterior and gastrocnemius / plantaris / soleus muscles of D2-mdx mice 3 weeks after tail vein injection of 1E+11vg / mouse of wild-type AAV9, detargeted AAV9 N272A, and AAV9 N272A conjugated to antibodies targeting CACNG1 (mAb#1, mAb#2, and mAb#3) or hASGR1 as a non-targeting control. D2-mdx mice injected with WT AAV9 conjugated to an irrelevant antibody (hASGR1), detargeted AAV9 N272A, AAV9, and AAV9 conjugated to an antibody that binds human and monkey CACNG1 but not mouse CACNG1 (mAb#1) show only limited GFP fluorescence in these muscles, whereas mice injected with AAV9 particles conjugated to antibodies that bind both human and mouse CACNG1 (mAb#2 and mAb#3) show robust GFP fluorescence in patches of myofibers throughout the muscle. [Figure 11]Immunohistochemical staining of eGFP expression in liver and quadriceps of D2-mdx mice after injection of wild-type AAV9, detargeted AAV9 N272A, and AAV9 N272A conjugated to an antibody targeting CACNG1 or hASGR1 as a non-targeting control. AAV9 wild-type particles can transduce the liver of D2-mdx mice, but AAV9 N272A particles are detargeted from the liver and do not express GFP in the liver. D2-mdx mice injected with AAV9 N272A conjugated to an irrelevant antibody (hASGR1) or a CACNG1-targeting antibody (mAb#1) that binds to human and monkey CACNG1 but not mouse CACNG1 show little staining in the liver or quadriceps. AAV9 N272A particles conjugated to antibodies that bind both human and mouse CACNG1 (mAb#2 and mAb#3) show negligible GFP staining in liver but strong GFP staining in quadriceps muscle. [Figure 12] Immunohistochemical staining of eGFP expression in gastrocnemius / plantaris / soleus of D2-mdx mice after injection of wild-type AAV9, detargeted AAV9 N272A, and AAV9 N272A conjugated to antibodies targeting CACNG1 or hASGR1 as a non-targeting control. AAV9 wild-type particles can transduce gastrocnemius / plantaris / soleus of D2-mdx mice at low levels, whereas AAV9 N272A particles transduce gastrocnemius / plantaris / soleus with limited efficiency. D2-mdx mice injected with AAV9 N272A conjugated to an irrelevant antibody (hASGR1) or a CACNG1-targeting antibody (mAb#1) specific for human CACNG1 that does not bind to mouse CACNG1 show little staining in gastrocnemius / plantaris / soleus. AAV9 N272A particles conjugated to antibodies that bind both human and mouse CACNG1 (mAb#2 and mAb#3) show very strong GFP staining in gastrocnemius / plantaris / soleus muscles. [Figure 13]Immunohistochemical staining of eGFP expression in the tibialis anterior muscle of D2-mdx mice after injection of wild-type AAV9, detargeted AAV9 N272A, and AAV9 N272A conjugated to antibodies targeting CACNG1 or hASGR1 as a non-targeting control. AAV9 wild-type particles can transduce the tibialis anterior muscle of D2-mdx mice at low levels. D2-mdx mice injected with AAV9 N272A particles alone or AAV9 N272A conjugated to an irrelevant antibody (hASGR1) or a CACNG1-targeting antibody (mAb#1) that binds to human and monkey CACNG1 but not mouse CACNG1 show little staining in the tibialis anterior muscle. AAV9 N272A particles conjugated to antibodies that bind both human and mouse CACNG1 (mAb#2 and mAb#3) show very strong GFP staining around the tibialis anterior muscle. [Figure 14] Immunohistochemical staining of eGFP expression in the heart and tongue of D2-mdx mice after injection of wild-type AAV9, non-targeted AAV9 N272A, and AAV9 N272A conjugated to antibodies targeting CACNG1 or hASGR1 as a non-targeting control. AAV9 wild-type particles can transduce the tongue of D2-mdx mice at low levels, but can efficiently transduce the heart. D2-mdx mice injected with AAV9 N272A particles alone or AAV9 N272A conjugated to an irrelevant antibody (hASGR1) or a CACNG1-targeting antibody (mAb#1) that binds to human and monkey CACNG1 but not mouse CACNG1 show little staining in the tongue and low levels of staining in the heart. AAV9 N272A particles conjugated to antibodies that bind both human and mouse CACNG1 (mAb#2 and mAb#3) show very strong GFP staining in the tongue and lower, but still detectable, levels of staining in the heart. [Figure 15]1 shows immunohistochemical staining of eGFP expression in the spleen and diaphragm of D2-mdx mice following injection of wild-type AAV9, detargeted AAV9 N272A, and AAV9 N272A conjugated to an antibody targeting CACNG1 or hASGR1 as a non-targeting control. AAV9 wild-type particles are able to transduce the diaphragm of D2-mdx mice at low levels. D2-mdx mice injected with AAV9 N272A particles alone or AAV9 N272A conjugated to an irrelevant antibody (hASGR1 or a CACNG1-targeting antibody (mAb#1) that binds to human and monkey CACNG1 but not mouse CACNG1) show little staining in the diaphragm. AAV9 N272A particles conjugated to antibodies that bind both human and mouse CACNG1 (mAb#2 and mAb#3) show very strong GFP staining in the diaphragm. As expected, little transduction of the spleen is observed with any of the AAVs tested. [Figure 16] FIG. 1 provides an exemplary schematic diagram (not to scale) of a single-stranded (ss) viral genome, including, in the 5' to 3' direction, a 141 base pair inverted terminal repeat (ITR), a CAGG promoter, a sequence encoding enhanced green fluorescent protein (GFP), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), a 32 base pair barcode, a human (h) growth hormone (GH) polyA tail, and the 141 base pair ITR. [Figure 17]FIG. 16 provides a bar graph showing enhanced transduction of various muscles in vivo in non-human primates (cynomolgus monkeys) following administration of AAV9 viral particles containing the viral genomes shown in FIG. 16, each bearing a unique barcode, and retargeted with an anti-CACNG1 antibody, compared to wild-type AAV9 viral particles (AAV) containing the viral genomes shown in FIG. 16. Each candidate AAV was packaged with a unique barcoded genome as described in FIG. 16. Following IV administration of the 12 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). The percentage of NGS reads (Y-axis) that mapped to each barcode and associated capsid in several tissues (x-axis) are shown (normalized to the injected virus pool): liver (lateral left lobe) and a subset of skeletal muscles (diaphragm, biceps brachii, biceps femoris, extensor digitorum longus (EDL), gastrocnemius, intercostal, soleus, tibialis anterior, transverse abdominus, triceps, vastus lateralis, psoas, tongue). Data shown here are the average of two animals in this study. In liver, as expected, AAV9 alone and AAV9 W503A or N272A conjugated to ASGR1 mAb account for the majority of all barcodes present in the tissue. In skeletal muscle tissue, detargeted AAV9 (N272A or W503A) capsids conjugated to a CACNG1-targeting antibody accounted for the majority of all barcodes present in the tissue, outperforming AAV9 alone, which accounted for a small percentage of the total barcodes. [Figure 18]Figure 1 shows AAV9-based retargeted viral transduction of human skeletal myotubes and C2C12 mouse myotubes using vector genome constructs expressing uDys5. The graphs provide transduction efficiency assessed by quantifying relative uDys5 mRNA expression in (A) human myotubes and (B) C2C12 mouse myotubes after 3 days of transduction with 2E+5vg / cell of either AAV9 WT or detargeted AAV9 (N272A) conjugated to an antibody (mAb#3) targeting CACNG1, which expresses uDys5 under the control of the CK8 promoter. uDys5 expression was quantified via Taqman-based qPCR assay and normalized to Hprt as an endogenous control. uDys5 mRNA expression is presented relative to WT AAV9 for each cell type. AAV9 N272A particles conjugated to an antibody that binds to both human and mouse CACNG1 (mAb#3) produce higher levels of uDys5 mRNA in both human and mouse myotubes compared to AAV9 WT. [Figure 19]Figure 1 shows uDys5 gene expression in multiple tissues of D2-mdx mice 5 weeks after tail vein injection of 1E+12vg / mouse of either AAV9 WT or detargeted AAV9 (N272A) conjugated to an antibody targeting CACNG1 (mAb#3), which expresses uDys5 under the control of the CK8 promoter. uDys5 mRNA expression was quantified via Taqman-based qPCR assay and normalized to Rplp0 as an endogenous control. uDys5 mRNA expression is shown relative to WT AAV9 for each tissue. AAV9 N272A particles conjugated to an antibody that binds both human and mouse CACNG1 (mAb#3) show reduced liver transduction compared to AAV9 WT, as expected, producing extremely low levels of uDys5 mRNA in the liver compared to AAV9 WT. AAV9 N272A particles conjugated to an antibody that binds both human and mouse CACNG1 (mAb#3) produce lower, but detectable levels of uDys5 mRNA in the heart compared to AAV9 WT. AAV9 N272A particles conjugated to an antibody that binds both human and mouse CACNG1 (mAb#3) produce higher levels of uDys5 mRNA in all skeletal muscles examined compared to AAV9 WT. [Figure 20] Representative immunofluorescence images of the gastrocnemius muscle and heart of wild-type DBA2 / J and D2-mdx mice 5 weeks after tail vein injection of 1E+12vg / mouse of wild-type AAV9 or detargeted AAV9 N272A particles conjugated to an antibody (mAb#3) targeting CACNG1 expressing uDys5 under the control of the CK8 promoter are presented. D2-mdx mice injected with WT AAV9 show low levels of dystrophin expression in the myofiber membrane of the gastrocnemius muscle and robust expression in the heart. Meanwhile, mice injected with detargeted AAV9 N272A particles conjugated to an antibody (mAb#3) that binds to both human and mouse CACNG1 show robust dystrophin expression in the myofiber membrane of the gastrocnemius muscle and mild expression in the heart. [Figure 21]Figure 1 shows the protein abundance of uDys5 in the quadriceps muscle of D2-mdx mice 5 weeks after tail vein injection of 1E+12vg / mouse of wild-type AAV9 or detargeted AAV9 N272A particles conjugated to an antibody (mAb#3) targeting CACNG1 that expresses uDys5 under the control of the CK8 promoter. β-actin was used as a protein loading control, and protein abundance was quantified and plotted as arbitrary densitometric units (AU). Mice injected with detargeted AAV9 N272A particles conjugated to an antibody (mAb#3) that binds to both human and mouse CACNG1 exhibit substantially more uDys5 protein compared to mice injected with wild-type AAV9. [Figure 22] 1 shows serum creatine kinase (CK) levels in D2-mdx mice 4 weeks after tail vein injection of 1E+12vg / mouse of wild-type or detargeted AAV9 N272A particles conjugated to an antibody (mAb#3) that targets CACNG1 expressing uDys5 under the control of the CK8 promoter. PBS- and wild-type AAV9-treated mice did not show a decrease in serum CK after treatment, whereas all mice treated with detargeted AAV9 N272A particles conjugated to an antibody (mAb#3) that binds both human and mouse CACNG1 showed a decrease in serum CK, indicating a decrease in muscle damage. [Figure 23] Figure 1 shows maximum forelimb grip strength measurements in D2-mdx mice 12 weeks after tail vein injection of 1E+12vg / mouse of wild-type AAV9 or detargeted AAV9 N272A particles conjugated to an antibody (mAb#3) that targets CACNG1 expressing uDys5 under the control of the CK8 promoter. The dotted line represents the grip strength of age-matched control DBA / 2J mice. Mice injected with AAV9 N272A particles conjugated to an antibody (mAb#3) that binds both human and mouse CACNG1 show improved maximum grip strength compared to mice injected with WT AAV9 particles. [Figure 24-1]Figure 1 shows serum levels of liver enzymes and complement pathway biomarkers in non-human primates (cynomolgus monkeys) at the indicated time points after injection of wild-type AAV9 or AAV9 N272A conjugated to an antibody targeting CACNG1 (mAb #3) expressing eGFP under the control of the CAG promoter. Administration of AAV9 wild-type particles resulted in elevations of (A) ALT, (B) AST, (C) Bb, and (D) C3a at 48 hours post-administration, as expected, whereas administration of AAV9 N272A conjugated to an antibody targeting CACNG1 (mAb #3) did not result in elevations of these markers, suggesting that liver-detargeted AAV9 N272A particles conjugated to an antibody targeting CACNG1 offer a safety advantage over liver-tropic wild-type AAV serotypes. [Figure 24-2] Same as above. [Figure 24-3] Same as above. [Figure 24-4] Same as above.

[0012] In each of the above figures: "WT" refers to wild-type, e.g., AAV capsid protein with no mutations or modifications. "HBM" refers to capsid protein containing R585A and R588A. All "reference / detargeted" AAV2 capsid proteins, e.g., AAV capsid proteins without the SpyTag modification, contain only R585A and R588A. All AAV2 capsid proteins in the above figures that display a SpyTag bound to a SpyCatcher fusion antibody further contain the R484A, R487A, R585A, R588A, and K532A mutations. "N272A" refers to a capsid that contains the N272A mutation. In the diagram above, all "reference" / "detargeted" AAV9 capsid proteins, e.g., AAV9 capsid proteins without the SpyTag modification, contain the N272A mutation. In the diagram above, all AAV9 capsid proteins with the SpyTag modification further contain the W503A mutation, but do not contain the N272A mutation. "Anti-CACNG1", "CACNG1", or "CACNG1 mAb", with reference to AAV, refers to an AAV viral capsid containing an insertion of a SpyTag peptide immediately after residue G453 flanked by 10 amino acid linkers on either side, where the SpyTag peptide is attached by an isopeptide bond to a SpyCatcher fused to an anti-CACNG1 antibody (or a Fab fragment thereof) that specifically binds CACNG1. In the above figure, all AAV particles displaying a SpyTag peptide linked by an isopeptide bond to SpyCatcher fused to an anti-CACNG1 antibody (or its Fab fragment) that specifically binds to CACNG1 contain a mosaic viral capsid containing a 1:7 ratio of SpyTag-modified viral capsid protein to "detargeted" viral protein (shown as "1 / 8"). "CACNG1 mAb#1" refers to an anti-CACNG1 antibody that binds to human and monkey CACNG1 but does not bind to mouse CACNG1. "CACNG1 mAb#2" refers to an anti-CACNG1 antibody that binds to human, monkey, and mouse CACNG1. "CACNG1 mAb#3" refers to an anti-CACNG1 antibody that binds to human, monkey, and mouse CACNG1. "CACNG1 mAb#4" refers to an anti-CACNG1 antibody that binds to human, monkey, and mouse CACNG1. "CACNG1 mAb#5" refers to an anti-CACNG1 antibody that binds to human and monkey but not to mouse CACNG1. "CACNG1" in reference to 293 cells or mice refers to 293 cells or mice, respectively, that have been genetically modified to express human CACNG1. Mice containing homozygous replacement of endogenous Cacng1 with the human Cacng1 sequence were identified as CACNG1 hu / hu "It is called " · Mouse-related "50500" refers to strain-matched controls of CACNG1 mice. "Anti-ASGR1", "ASGR1", or "ASGR1 mAb", with reference to AAV, refers to an AAV viral capsid containing an insertion of a SpyTag peptide immediately after residue G453 flanked on either side by 10 amino acid linkers, where the SpyTag peptide is attached by an isopeptide bond to SpyCatcher fused to an anti-ASGR1 antibody or a Fab fragment thereof that specifically binds ASGR1. In the above figure, all AAV particles displaying a SpyTag peptide linked by an isopeptide bond to SpyCatcher fused to an anti-ASGR1 antibody (or its Fab fragment) that specifically binds to ASGR1 contain a mosaic viral capsid containing a 1:7 ratio of SpyTag-modified viral capsid protein to "detargeted" viral capsid protein (shown as "1 / 8"). "ASGR1" in reference to 293 cells or mice refers to 293 cells or mice, respectively, that have been genetically modified to express human ASGR1. "h" stands for "human" "Vh" refers to the antibody heavy chain "Vk" refers to the antibody light chain "D2-mdx" refers to a mouse model of Duchenne muscular dystrophy DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] Skeletal muscles are the largest organs in the body, accounting for about 40% of total body weight. Skeletal muscles are one of the three major muscle tissues in the human body. Each skeletal muscle is made up of thousands of muscle fibers wrapped together by a connective tissue sheath.

[0014] The primary function of skeletal muscle is through its intrinsic excitation-contraction coupling process. Because muscles are attached to bone tendons, muscle contraction moves bones to perform certain movements. Skeletal muscles also provide structural support and help maintain body posture. Skeletal muscles also act as a storage source of amino acids that can be used by various organs of the body to synthesize organ-specific proteins. Skeletal muscles also serve as a storage source of glucose in the form of glycogen. Skeletal muscles also play a central role in maintaining homeostasis and act as an energy source during starvation. Thus, skeletal muscles play a key role in controlling locomotion, thermoregulation, and whole-body metabolism.

[0015] Many muscle diseases, as well as normal aging, result in a decline in size and function of skeletal muscle tissue, compromising functional mobility and, in severe muscle diseases, leading to long-term disability and premature death.

[0016] Treatment of muscle wasting and genetic muscle diseases typically consists of broad-spectrum acting therapies, such as testosterone therapy for muscle wasting, glucocorticoids for muscular dystrophies, and systemic AAV delivery for the treatment of muscle diseases (e.g., X-linked myotubular myopathy (XLMTM), Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM1), facioscapulohumeral muscular dystrophy type 1 (FSHD), congenital muscular dystrophy type 1A (MDC1A), limb-girdle muscular dystrophy, and dystroglycanopathy, etc.). Non-targeted delivery of these therapies reduces the efficiency of specific muscle uptake while also causing significant deleterious off-target effects in other organs.

[0017] Described herein are viral particles, e.g., AAV viral particles, that target muscle-specific surface proteins, such as calcium-gated auxiliary subunit gamma 1 (CACNG1) or mammalian cadherin 15 (CAD15), to enhance muscle delivery of therapeutic payloads and mitigate off-target effects.

[0018] Voltage-gated calcium channels are generally composed of five subunits. The protein encoded by the CACNG1 gene represents one of these subunits. Furthermore, the protein encoded by the CACNG1 gene, gamma, is one of two known gamma subunit proteins. This particular gamma subunit is part of the skeletal muscle 1,4-dihydropyridine-sensitive calcium channel, an integral membrane protein that plays a role in excitation-contraction coupling. This gene is part of the functionally diverse eight-member protein subfamily of the PMP-22 / EMP / MP20 family, and is located within a cluster with two family members that function as transmembrane AMPA receptor regulatory proteins (TARPs). CACNG1 is highly and specifically expressed in skeletal muscle. The gene encoding human CACNG1 (CACNG1) is located on the long arm of chromosome 17. CACNG1 contains four exons and is approximately 12,244 bases long. An exemplary sequence of the human CACNG1 gene has been assigned NCBI accession number NM_0007582.2 (SEQ ID NO: 241). An exemplary human CACNG1 protein has been assigned UniProt accession number O70578 (SEQ ID NO: 242).

[0019] 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.

[0020] 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 type described herein and / or that will be apparent to those skilled in the art upon reading this disclosure.

[0021] "Percent identity" and the like can be readily determined for amino acid or nucleotide sequences over the entire length of a protein or a portion thereof. The 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", "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" refers to multiple nucleic acid or protein (amino acid) sequences, which often include corrections for missing or added bases or amino acids compared to a reference sequence.

[0022] Alignment may be performed using any of a variety of publicly available or commercially available multiple sequence alignment programs. Sequence alignment programs are available for amino acid sequences, such as the programs "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME" and "Match-Box". Generally, any of these programs are used with default settings, but those skilled in the art may change these settings as needed. Alternatively, those skilled in the art may use another algorithm or computer program that provides at least the same level of identity or alignment as that provided by the referenced algorithms and programs. See, for example, JD Thomson et al, Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999).

[0023] 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 skilled in the art. Alternatively, the Vector NTI utility is used. There are also numerous algorithms known in the art that can be used to measure the identity of nucleotide sequences, 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 of the regions of best overlap between the query and search sequences and the percentage of sequence identity. For example, the 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) provided in GCG version 6.1, which is incorporated herein by reference.

[0024] "Significant identity" includes alignment of amino acid or nucleic acid sequences which are at least 90%, such as at least 93%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, or such as at least 100% identical.

[0025] The term "chimera" encompasses a functional gene or polypeptide that comprises a nucleic acid or amino acid sequence, respectively, from at least two different AAV serotypes, e.g., at least a portion of a gene or polypeptide of a first and a second AAV, where at least the first and second portions are operably linked to form a functional chimeric AAV nucleic acid that encodes a functional amino acid. Unless designated as chimeric, the nucleotide sequences, genes, polypeptides, and amino acids are considered non-chimeric in that the nucleotide sequences, genes, polypeptides, and amino acids comprise a nucleic acid sequence or amino acid sequence that has significant identity, respectively, to a nucleic acid sequence or amino acid sequence of a single AAV serotype.

[0026] The term "antibody" includes immunoglobulin molecules that contain four polypeptide chains, two heavy (H) chains 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, termed complementarity determining regions (CDRs), which are interspersed with more conserved regions, termed framework regions (FRs). Each heavy and light chain variable domain contains 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 also be abbreviated as HCDR1, HCDR2, and HCDR3, and light chain CDRs may also be abbreviated as LCDR1, LCDR2, and LCDR3). A typical tetrameric antibody structure contains two identical antigen-binding domains, each of which is a V H Domain and V L It is formed by the association of domains, each of which is a C HDomain and C L The V domains together form the antibody Fv region. Single domain antibodies contain a single antigen-binding domain, e.g., H Or V L The antigen-binding domain of an antibody, e.g., the portion of an antibody that recognizes and binds to a first member of a specific binding pair for an antigen, is also referred to as a "paratope." A paratope is a small region (5-10 amino acids) of the Fv region of an antibody that is part of the fragment antigen-binding (Fab region) and may contain part of the heavy and / or light chain of the antibody. A paratope specifically binds to a first member of a specific binding pair when it binds to the first member of the specific binding pair with high affinity. The term "high affinity" antibody refers to an antibody that has a high affinity of about 10 to the target first member of the specific binding pair. -9 M or less (for example, about 1×10 -9 M, 1×10 -10 M, 1×10 -11 M, or approximately 1 x 10 -12 M)K D In one embodiment, the antibody has the formula: D is measured by surface plasmon resonance, e.g., BIACORE™, and in another embodiment, K D is measured by ELISA.

[0027] The phrase "complementarity determining region" or "CDR" includes an amino acid sequence encoded by a nucleic acid sequence of an organism's immunoglobulin genes, which amino acid sequence is normally (i.e., in a wild-type animal) found between two framework regions in the variable region of a light or heavy chain of an immunoglobulin molecule (e.g., an antibody or a T cell receptor). A CDR may be encoded, for example, by a germline sequence or a rearranged or unrearranged sequence, for example, by a naive or mature B cell, or a T cell. A CDR may 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. In some circumstances (e.g., for a CDR3), a CDR may 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, for example, as a result of splicing or joining of sequences (e.g., VDJ rearrangement to form a heavy chain CDR3).

[0028] The term "light chain" includes immunoglobulin light chain sequences from any organism, including human kappa and lambda light chains, as well as surrogate light chains, and VpreB, unless otherwise specified. Unless otherwise specified, a light chain variable domain typically includes three light chain CDRs and four framework (FR) regions. In general, 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 is generally a V-segment derived from a repertoire of V and J segments present in the germline. L Segment and J LThe 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 that is selectively bound by the first member of the specific binding pair-binding protein in which the light chain appears. The 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 that is 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 Vκ1-39Jκ gene or the human Vκ3-20Jκ gene, including somatically mutated (e.g., affinity matured) versions thereof. Exemplary human Vκ1-39Jκ gene or human Vκ3-20 ... Vκ3-20Jκ gene or Vκ3-20Jκ gene or Vκ3-20Jκ gene or Vκ3-20Jκ gene or Vκ3-20Jκ gene or Vκ3-20Jκ gene or Vκ3-20Jκ gene or Vκ3-20Jκ gene or Vκ3-20Jκ gene or Vκ3-20Jκ gene or Vκ3-20Jκ gene or Vκ3-20Jκ gene or Vκ L The segments include a human Vκ1-39 gene segment, a human Vκ3-20 gene segment, a human Vλ1-40 gene segment, a human Vλ1-44 gene segment, a human Vλ2-8 gene segment, a human Vλ2-14 gene segment, and a human Vλ3-21 gene segment, including somatically mutated (e.g., affinity matured) versions thereof. Light chains can be made that include a variable domain from one organism (e.g., human or rodent, e.g., rat or mouse, or avian, e.g., chicken) and a constant region from the same or a different organism (e.g., human or rodent, e.g., rat or mouse, or avian, e.g., chicken).

[0029] The term "about" or "approximately" includes within a statistically significant range of values. Such ranges may be within 10-fold, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of a given value or range. The acceptable 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.

[0030] 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 a heavy chain include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain includes (from N-terminus to C-terminus) the variable domain followed by a CDR, a CDR and a FR, and a CDR ... 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 a V domain 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 the V, D, and J heavy chain 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".

[0031] 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, which is typically a functional C HA heavy chain-only antibody is unable to associate with a light chain because it lacks one domain. Thus, 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 an antibody that is capable of binding to a light chain and that lacks (i) a functional C domain. H or (ii) 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 (iii) 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 includes both a homodimeric single domain antigen binding protein, which comprises a variable domain operably linked to a heavy chain constant region lacking one domain, and a homodimeric single domain antigen binding protein, which comprises two identical immunoglobulin-like chains, each of which is a functional C 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 a heavy chain constant region (and optionally a hinge region) gene, such as a C H 1. A heavy chain constant region (C H A single domain antigen binding protein comprising a variable domain derived from a heavy chain gene segment may be linked to a "V H Single domain antibodies or V HSingle domain antigen binding proteins comprising a variable domain derived from a light chain gene segment may be referred to as "single domain antigen binding proteins" and see, e.g., U.S. Patent No. 8,754,287, U.S. Patent Application 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. Single domain antigen binding proteins comprising a variable domain derived from a light chain gene segment may be referred to as "V L These may be referred to as "single domain antigen binding proteins," see, e.g., U.S. Patent Application Publication No. 2015 / 0289489, incorporated by reference in its entirety.

[0032] The term "light chain" includes immunoglobulin light chain sequences from any organism, including 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, unless otherwise specified. 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. A 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 LThe light chain V gene segments and the light chain J gene segments of various organisms include light chain V gene segments and light chain J gene segments. The sequences, locations, and nomenclature of the 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 at 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. A common or universal light chain includes a light chain derived from the human Vκ1-39Jκ5 gene or the human Vκ3-20Jκ1 gene, including somatically mutated (eg, affinity matured) versions thereof.

[0033] The phrase "operably linked" as used herein includes the physical juxtaposition (e.g., in three-dimensional space) of components or elements that are positioned with respect to each other to directly or indirectly interact with each other or participate in a biological event, which juxtaposition achieves or enables such interaction and / or positioning. In one example, a regulatory element (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 covalent binding of components or elements associated with each other. One skilled in the art will readily appreciate that in some embodiments, covalent binding is not required to achieve effective operably linked. For example, proteins that are operably linked together can be associated with each other, for example, via covalent or non-covalent bonds. As a non-limiting example, the capsid proteins described herein may be operably linked to a targeting ligand, where the capsid protein is non-covalently bound to the targeting ligand, optionally with or without a scaffold and / or adaptor between the capsid protein and the targeting ligand, or covalently bound to the targeting ligand. As another example, in some embodiments, a nucleic acid regulatory element that is operably linked to a coding sequence that it controls is contiguous with a nucleotide of interest. Alternatively or in addition, in some embodiments, one or more such regulatory elements act in trans or at a distance to control a coding sequence of interest. In some embodiments, the term "regulatory element" as used herein refers to polynucleotide sequences necessary and / or sufficient to affect the expression and processing of the coding sequence to which they are linked.In some embodiments, the regulatory elements 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 regulatory elements are preferentially or only active in a particular host cell or organism, or type thereof. By way of example, in prokaryotes, regulatory elements may typically include promoters, ribosomal binding sites, and transcription termination sequences, and in eukaryotes, in many embodiments, regulatory elements may typically include promoters, enhancers, and / or transcription termination sequences. Those skilled in the art will appreciate that in many embodiments, the term "regulatory elements" refers to components whose presence is essential for expression and processing, and in some embodiments includes components whose presence is advantageous for expression (e.g., including leader sequences, targeting sequences, and / or fusion partner sequences).

[0034] "Retargeting" or "redirection" may include scenarios where wild-type particles target some cells in a tissue and / or some organs in an organism, where the general targeting of the tissue or organ is reduced or abolished by the insertion of heterologous amino acids, and retargeting to more specific cells in the tissue or more specific organs in an organism is achieved with (e.g., via) a targeting ligand that binds to a marker expressed by a specific cell. Such retargeting or redirection may also include scenarios where wild-type particles target a tissue, where the targeting of the tissue is reduced or abolished by the insertion of heterologous amino acids, and retargeting to an entirely different tissue is achieved with a targeting ligand.

[0035] A "specific binding pair," "binding pair," "protein:protein binding pair," and the like, interacts with two members (e.g., a first member (e.g., a first polypeptide) and a second cognate member (e.g., a second polypeptide)) to form a bond (e.g., a non-covalent bond between an epitope of the first member and an antigen-binding portion of the second member of an antibody that recognizes the epitope; a covalent bond between proteins that can form an isopeptide bond, for example; a split intein that recognizes each other and mediates the ligation of adjacent proteins and their own removal through the process of protein trans-splicing). 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, and variants thereof such as SpyTag003:SpyCatcher003. In general, a first member of a protein:protein binding pair generally refers to a member of a protein:protein binding pair that is generally less than 30 amino acids in length and spontaneously forms a covalent isopeptide bond with a second cognate protein, where the second cognate protein is generally larger, but may also be less than 30 amino acids in length, such as the SpyTag:KTag system.

[0036] The term "isopeptide bond" refers to an amide bond between a carboxyl or carboxamide group and an amino group, where at least one of the amino groups is not found to originate from the protein backbone or, alternatively, is not part of the protein backbone. Isopeptide bonds can form within a single protein, or between two peptides or between a peptide and a protein. Thus, isopeptide bonds can form intramolecularly within a single protein, or intermolecularly, i.e., between two peptide / protein molecules, e.g., between two peptide linkers. Typically, isopeptide bonds can occur between a lysine residue and an asparagine, aspartic acid, glutamine, or glutamic acid residue, or the terminal carboxyl group of a protein or peptide chain, or between the alpha-amino terminus of a protein or peptide chain and an asparagine, aspartic acid, glutamine, or glutamic acid residue. Each residue of the pair involved in an isopeptide bond is referred to herein as a reactive residue. In a preferred embodiment of the 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.

[0037] The SpyTag:SpyCatcher system is described in U.S. Pat. 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 a "SpyTag" peptide with the sequence AHIVMVDAYKPTK (SEQ ID NO: 243), 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: 244). (Zakeri (2012), supra). A further specific binding pair derived from the CnaB2 domain is the SpyTag:KTag, which forms an isopeptide bond in the presence of SpyLigase. (Fierer (2014) PNAS 111:E1176-1181). SpyLigase has been engineered by excising a beta strand from SpyCatcher that contains a 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: 245). SpyTag002: The SpyCatcher002 system is described in Keeble et al (2017) Angew Chem Int Ed Engl 56:16521-25, which is incorporated herein by reference in its entirety. SpyTag002 has the amino acid sequence VPTIVMVDAYKRYK, set forth as SEQ ID NO: 255, and binds to SpyCatcher002. SpyTag003 has the amino acid sequence RGVPHIVMVDAYKRYK, set forth as SEQ ID NO: 259, and binds to SpyCatcher003.

[0038] 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) and SnoopCatcher (residues 749-860). Incubation of SnoopTag and SnoopCatcher results in specific spontaneous isopeptide bonds between the complementary proteins. Veggiani (2016) supra.

[0039] The isopeptag:pilin-C specific binding pair was derived from Spy0128, the 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: 254, and binds to pilin-C (residues 18-299 of Spy0128). Incubation of the SnoopTag and SnoopCatcher results in the formation of a specific spontaneous isopeptide bond between the complementary proteins. Zakeir and Howarth (2010), supra.

[0040] 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 non-covalent binding. Exemplary and non-limiting detectable labels include hexahistidine tag, FLAG tag, StrepII tag, streptavidin binding peptide (SBP) tag, calmodulin binding peptide (CBP), glutathione S-transferase (GST), maltose binding protein (MBP), S-tag, HA tag, and myc tag (SEQ ID NO: 246) from c-myc. (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: 247). Some AAV capsid proteins described herein do not naturally contain B1 epitope, and can be modified herein to contain B1 epitope.Generally, the AAV capsid proteins described herein can contain a sequence with substantial homology to the B1 epitope within the last 10 amino acids of capsid protein.Thus, in some embodiments, the non-primate AAV capsid protein of the present invention can be modified with one or more but less than five point mutations within the last 10 amino acids of capsid protein, so that the AAV capsid protein contains B1 epitope.

[0041] 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.

[0042] 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 with a fluorescent reporter gene, RT-PCR for expression of the nucleotide of interest, and the like.

[0043] Generally, the "reference" viral capsid protein / capsid / particle is identical to the test viral capsid protein / capsid / particle, except for the modification to test the effect. For example, to determine the effect of inserting a first member of a specific binding pair into a test viral particle, such as the effect on transduction efficiency, the transduction efficiency of the test viral particle (in the presence or absence of a suitable targeting ligand) can be compared to the transduction efficiency of a reference viral particle (in the absence or presence of a suitable targeting ligand, as required), which is identical to the test viral particle in every instance (e.g., additional point mutations, nucleotides of interest, number of viral particles and target cells, etc.) except for the presence of the first member of the specific binding pair. In some embodiments, the reference viral capsid protein can form a capsid with a second viral capsid protein that is modified to include at least a first member of a protein:protein binding pair, where the reference viral capsid protein does not include the first member of the protein:protein binding pair, and preferably the capsid formed by the reference viral capsid protein and the modified viral capsid protein is a mosaic capsid.

[0044] Adeno-associated virus (AAV) "AAV" is an abbreviation for adeno-associated virus and may be used to refer to the virus itself or its derivatives. AAV is a small, non-enveloped, single-stranded DNA virus. In general, 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). More than 80% of the total protein in the AAV virion (capsid) is composed of VP3, and 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.

[0045] The genomic sequences of various serotypes of AAV, as well as the sequences of 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 numbers NC_002077 (AAV1), AF063497 (AAV1), NC001401 (AAV-2), AF043303 (AAV2), NC_001729 (AAV3), NC_001829 (AAV4), U89790 (AAV4), NC_006152 (AAV5), AF513851 (AAV7), AF513852 (AAV8), and NC_006261 (AAV8). That disclosure is incorporated herein by reference for its 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 Application 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 2 herein provides sequences for various non-primate AAVs.

[0046] "AAV" includes all subtypes known in the art and both naturally occurring and modified forms. AAVs include primate AAVs (e.g., AAV type 1 (AAV1), primate AAV type 2 (AAV2), primate AAV type 3 (AAV3B), primate AAV type 4 (AAV4), primate AAV type 5 (AAV5), primate AAV type 6 (AAV6), primate AAV type 7 (AAV7), primate AAV type 8 (AAV8), primate AAV type 9 (AAV9), AAV10, AAV11, AAV12, AAV13, AAVDJ, Anc80L65, AAV2G9, AAV-LK03, primate AAV type rh10 (AAV rh10), AAV type h10 (AAV h10), AAV type hu11 (AAV hu11), AAV rh32.33 (AAV rh32.33), AAV retro (AAV retro), AAV PHP.B, AAV PHP.eB, AAV PHP.S, AAV2 / 8, non-primate AAV (e.g., avian AAV (AAAV)), and other non-primate AAV, such as mammalian AAV (e.g., bat AAV, sea lion AAV, bovine AAV, canine AAV, equine AAV, caprine AAV, and ovine AAV), squamate AAV (e.g., snake AAV, bearded dragon AAV), and the like. "Primate AAV" generally refers to AAV isolated from primates. Similarly, "non-primate AAV" refers to AAV isolated from non-primate animals.

[0047] 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), such as the cap gene or capsid protein of an AAV, "of a [specified] AAV" encompasses, in addition to a gene or polypeptide that comprises a nucleic acid sequence or amino acid sequence as described herein with respect to the respective specified AAV, 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, e.g., the difference may be due to degeneracy of the genetic code, isolated variation, sequence length, etc. For example, the rep and cap genes as used herein may 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 sequences 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 gene.

[0048] It is known that the wild-type cap gene expresses all three VP1, VP2, and VP3 capsid proteins from one 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 capsid of the virus. For adeno-associated viruses, the capsid proteins are generally referred to as VP1, VP2, and / or VP3, which may be encoded by one cap gene. For AAV, the three AAV capsid proteins are naturally produced in an overlapping manner utilizing alternative translation initiation codons in the cap ORF, but all three proteins use a common stop codon. The ORF of the wild-type cap gene encodes three alternative initiation codons from 5' to 3', namely, the "VP1 initiation codon", the "VP2 initiation codon", and the "VP3 initiation codon", and one "common stop codon". VP1, the largest viral protein, is generally encoded from the VP1 start codon to the "common stop codon." VP2 is generally encoded from the VP2 start codon to the common stop codon. VP3 is generally encoded from the VP3 start codon to the common stop codon. VP1 therefore contains an N-terminal sequence that is 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 as well as a nuclear localization signal that may assist in targeting the virus to the nucleus for uncoating and genome release. 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 common stop codon. VP2 shares an additional approximately 60 amino acids with VP1, a region called the VP1 / VP2 common region.

[0049] In some embodiments, one or more of the Cap proteins of the invention may be encoded by one or more cap genes having one or more ORFs. In some embodiments, the VP proteins of the 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 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 invention may be expressed individually from ORFs containing nucleotide sequences encoding any one of VP1, VP2, or VP3, each producing only one of the VP1, VP2, or VP3 capsid proteins, by using separate nucleotide sequences operably linked to one expression control sequence for expression in a viral replicating cell. In another embodiment, the VP proteins may be expressed from a single ORF that includes 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 will be readily able to determine the position of the same amino acid in the VP2 and / or VP3 capsid proteins of the AAV, and the corresponding amino acid positions among different AAVs, respectively.

[0050] 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 replication origins for viral DNA synthesis and are essential cis elements for AAV particle production, including packaging into AAV particles.

[0051] AAV ITRs contain recognition sites for replication proteins Rep78 or Rep68. The A"D" regions of the ITRs contain DNA nick sites where DNA replication is initiated, providing directionality to the nucleic acid replication process. AAV replication in mammalian cells typically involves two ITR sequences.

[0052] 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 method of making AAV viral particles of the invention, the rep coding sequence encodes a Rep protein or a Rep protein equivalent that can bind to the ITRs contained on the transfer plasmid.

[0053] The Cap protein of the present invention, when expressed together with an appropriate Rep protein by a packaging cell, can encapsidate a transfer plasmid that contains 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.

[0054] Either Rep78 and / or Rep68 binds to a unique and known site on the ITR hairpin sequence and functions to disrupt and unravel the hairpin structure at the end of the AAV genome, thereby providing access to the replication machinery of the virus 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 by using separate nucleotide sequences operably linked to at least one expression control sequence for expression in the virus replicating cell, each producing one or more of Rep78, Rep68, Rep52 and / or Rep40 Rep proteins. Alternatively, the Rep proteins may be expressed individually from ORFs containing nucleotide sequences encoding any one of Rep78, Rep68, Rep52 or Rep40 by using separate nucleotide sequences operably linked to one 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 one ORF containing nucleotide sequences encoding Rep78 and Rep52 Rep proteins, each operably linked to at least one expression control sequence for expression in a viral replication cell producing Rep78 and Rep52 Rep proteins.

[0055] In the methods of making 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 of skill in the art will recognize that such a requirement is not essential. Such viral particles may or may not contain a genome.

[0056] "Chimeric AAV capsid protein" includes AAV capsid proteins that contain amino acid sequences, e.g., portions, from two or more different AAVs and have the ability to form and / or form AAV viral capsids / viral particles. Chimeric AAV capsid proteins may be encoded by chimeric AAV capsid genes, e.g., chimeric nucleotides that contain a plurality, e.g., at least two nucleic acid sequences, each of the plurality being identical to a portion of a capsid gene that encodes a capsid protein of a separate AAV, and the plurality together encoding a functional chimeric AAV capsid protein. Association of a chimeric capsid protein to 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.

[0057] The term "portion" refers to at least 5 amino acids or at least 15 nucleotides, but less than a full length polypeptide or nucleic acid molecule, that has 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 the [designated] AAV" or has "significant identity" to a particular AAV, e.g., a non-primate AAV or a distantly related AAV. In some embodiments, the portion comprises at least 5 amino acids or 15 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 10 amino acids or 30 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 15 amino acids or 45 nucleotides that have 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 20 amino acids or 60 nucleotides with 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 25 amino acids or 75 nucleotides with 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 30 amino acids or 90 nucleotides with 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 35 amino acids or 105 nucleotides with 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 40 amino acids or 120 nucleotides with 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 45 amino acids or 135 nucleotides with 100% identity to a sequence associated with the designated AAV.In some embodiments, the portion comprises at least 50 amino acids or 150 nucleotides with 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 60 amino acids or 180 nucleotides with 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 70 amino acids or 210 nucleotides with 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 80 amino acids or 240 nucleotides with 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 90 amino acids or 270 nucleotides with 100% identity to a sequence associated with the designated AAV. In some embodiments, the portion comprises at least 100 amino acids or 300 nucleotides with 100% identity to a sequence associated with the designated AAV.

[0058] Modified viral capsid proteins, viral particles, and viral nucleic acids In some embodiments, a Cap protein, such as a VP1 capsid protein described herein, a VP2 capsid protein described herein, and / or a VP3 capsid protein described herein, is modified to include any one or combination of, for example, an insertion of a targeting ligand, a chemical modification, a first member of a binding pair, a detectable label, a point mutation, and the like.

[0059] Generally, modification of a specified AAV gene or polypeptide or variant thereof results in a nucleic acid or amino acid sequence that differs from the nucleic acid or amino acid sequence described herein for the specified AAV, in which the modification alters, imparts or eliminates one or more biological functions, but does not change the phylogenetic character of the gene or polypeptide as an AAV gene or AAV polypeptide. Modifications may include any one or combination of the following: replacing a sequence of a first AAV serotype with a sequence of a second AAV serotype to create a chimera, chemical modification, insertion of a first member of a binding pair, and / or point mutations, etc., thereby reducing or abolishing the natural tropism of the capsid protein, making the tropism of the capsid protein more easily redirected, and / or causing the capsid protein to contain a detectable label. The modifications described herein generally do not alter, and preferably include modifications that reduce low recognition to no recognition, of the modified capsid by pre-existing antibodies present in the general population that were produced during infection with another AAV, such as 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.

[0060] Targeting Ligands The modifications described herein may relate to the association (e.g., presentation, operative linkage, binding) of a targeting ligand to a modified capsid protein and / or a capsid comprising the modified capsid protein. Generally, the targeting ligand described herein binds to a surface protein expressed by a mammalian muscle cell, e.g., a protein expressed on the surface of a mammalian muscle cell, e.g., a mammalian muscle cell-specific surface protein. In some embodiments, the modified capsid protein and / or modified capsid comprises a targeting ligand that binds to a mammalian CACNG1, e.g., human CACNG1. [1] Table 1 provides a summary of the sequence numbers of each binding portion (e.g., heavy chain variable domain (HCVR), light chain variable domain (LCVR), and CDR1, CDR2, and CDR3) of non-limiting and exemplary anti-human CACNG1 monoclonal antibodies (mAb IDs) that can be used to redirect the AAV capsids described herein. In some embodiments, the AAV capsid described herein comprises a targeting ligand that binds to human CACNG1, and the targeting ligand comprises a heavy chain variable domain, a light chain variable domain, a heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences that are at least 90% identical to the amino acid sequences of the heavy chain variable domain, the light chain variable domain, the heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, respectively, and / or a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in any one of SEQ ID NOs: 1-240. In some embodiments, the AAV capsid described herein comprises a targeting ligand that binds to human CACNG1, and the targeting ligand comprises a heavy chain variable domain, a light chain variable domain, a heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences that are at least 95% identical to the amino acid sequences of the heavy chain variable domain, the light chain variable domain, the heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, respectively, and / or a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in any one of SEQ ID NOs: 1-240.In some embodiments, the AAV capsid described herein comprises a targeting ligand that binds to human CACNG1, and the targeting ligand comprises a heavy chain variable domain, a light chain variable domain, a heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences that are at least 97% identical to the amino acid sequences of the heavy chain variable domain, the light chain variable domain, the heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in any one of SEQ ID NOs: 1-240. In some embodiments, the AAV capsid described herein comprises a targeting ligand that binds to human CACNG1, and the targeting ligand comprises a heavy chain variable domain, a light chain variable domain, a heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences that are at least 98% identical to the amino acid sequences of the heavy chain variable domain, the light chain variable domain, the heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in any one of SEQ ID NOs: 1-240.In some embodiments, the AAV capsid described herein comprises a targeting ligand that binds to human CACNG1, and the targeting ligand comprises a heavy chain variable domain, a light chain variable domain, a heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, CDR3, LCDR1, LCDR2, LCDR3, and / or a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences that are 99% identical to the amino acid sequences of the heavy chain variable domain, the light chain variable domain, the heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in any one of SEQ ID NOs: 1-240. Provided herein are antibodies or antigen-binding fragments thereof that comprise a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained within a HCVR / LCVR amino acid sequence pair defined by any of the exemplary anti-hCACNG1 antibodies listed in Table 1. In some embodiments, the targeting ligands described herein comprise a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences contained within a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NO:2 / 10, SEQ ID NO:18 / 26, SEQ ID NO:34 / 42, SEQ ID NO:50 / 58, SEQ ID NO:66 / 74, SEQ ID NO:82 / 90, SEQ ID NO:98 / 106, SEQ ID NO:114 / 122, SEQ ID NO:130 / 138, SEQ ID NO:146 / 154, SEQ ID NO:162 / 170, and SEQ ID NO:178 / 186. In certain embodiments, the targeting ligands described herein comprise an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NO:2 / 10, SEQ ID NO:18 / 26, SEQ ID NO:34 / 42, SEQ ID NO:50 / 58, SEQ ID NO:66 / 74, SEQ ID NO:82 / 90, SEQ ID NO:98 / 106, SEQ ID NO:114 / 122, SEQ ID NO:130 / 138, SEQ ID NO:146 / 154, SEQ ID NO:162 / 170, and SEQ ID NO:178 / 186. [Table 1-1] [Table 1-2]

[0061] 31929 / 10728(wild type hIgG1) / 14647(hIgG1 N180Q) HCVR nucleic acid sequence (SEQ ID NO:1) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTACAGCGTCTGGAATCACCTTCAGAAATTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATGTGGTATGATGGAAGTAATAAGTACT ATGCAGACTCCGTGAAGGCCGTTTCACCATCTCCGGAGACAATTCCAAGGTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTATATTACTGTGCGAGAAGGGGCACTATAAGAACAGCTGCCCCTTTTGACTACTGGGGTCAGGGAACCCTGGTCACCGTCTCCTCA HCVR amino acid sequence (SEQ ID NO:2) QVQLVESGGGVVQPGRSLRLSCTASGITFRNYGMHWVRQAPGKGLEWVAVMWYDGSNKYYADSVKGRFTISGDNSKVYLQMNSLRAEDTAVYYCARRGTIRTAAPFDYWGQGTLVTVSS HCDR1 nucleic acid sequence (SEQ ID NO:3) GGA ATC ACC TTC AGA AAT TAT GGC HCDR1 amino acid sequence (SEQ ID NO:4) GITFRNYG HCDR2 nucleic acid sequence (SEQ ID NO:5) ATG TGG TAT GAT GGA AGT AAT AAG HCDR2 amino acid sequence (SEQ ID NO:6) MWYDGSNK HCDR3 nucleic acid sequence (SEQ ID NO:7) GCG AGA AGG GGC ACT ATA AGA ACA GCT GCC CCT TTT GAC TAC HCDR3 amino acid sequence (SEQ ID NO:8) ARRGTIRTAAPFDY LCVR nucleic acid sequence (SEQ ID NO:9) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA LCVR amino acid sequence (SEQ ID NO: 10) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK LCDR1 nucleic acid sequence (SEQ ID NO:11) CAG AGC ATT AGC AGC TAT LCDR1 amino acid sequence (SEQ ID NO:12) QSISSY LCDR2 nucleic acid sequence (SEQ ID NO:13) GCT GCA TCC LCDR2 amino acid sequence (SEQ ID NO:14) AAS LCDR3 nucleic acid sequence (SEQ ID NO:15) CAA CAG AGT TAC AGT ACC CCT CCG ATC ACC LCDR3 amino acid sequence (SEQ ID NO: 16) QQSYSTPPIT HC nucleic acid alignment (allocation number 193) HC amino acid sequence (SEQ ID NO: 194) [ka] [ka] *The underlined bold asparagine (N) can be mutated to a glutamine (Q) for conjugation by transglutaminase. See, for example, SEQ ID NO: 269. LC nucleic acid sequence (SEQ ID NO: 195) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG LC amino acid sequence (SEQ ID NO: 196) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 10715(wild type hIgG1) / 14570(IgG1 N180Q): HCVR nucleic acid sequence (SEQ ID NO:17) CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGCGACCCTGTCCCGCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAACTGGATCCGCCAGTCCCAGGGAAGGGGCTGGAATGGATTGGGGAAATCCTTCATAGTGGAAGAACCAACTACA ACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGACCTCTGTGACCGCCGCGGACACGGCTGTATATTACTGTGCGGGAAGGATAGCAGCTCGTCACGGCTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA HCVR amino acid sequence (SEQ ID NO: 18) QVQLQQWGAGLLKPSATLSRTCAVYGGSFSGYYWNWIRQSPGKGLEWIGEILHSGRTNYNPSLKSRVTISVDTSKNQFSLKLTSVTAADTAVYYCAGRIAARHGWFDPWGQGTLVTVSS HCDR1 nucleic acid sequence (SEQ ID NO: 19) GGT GGG TCC TTC AGT GGT TAC TAC HCDR1 amino acid sequence (SEQ ID NO:20) GGSFSGYY HCDR2 nucleic acid sequence (SEQ ID NO:21) ATC CTT CAT AGT GGA AGA ACC HCDR2 amino acid sequence (SEQ ID NO:22) ILHSGRT HCDR3 nucleic acid sequence (SEQ ID NO:23) GCG GGA AGG ATA GCA GCT CGT CAC GGC TGG TTC GAC CCC HCDR3 amino acid sequence (SEQ ID NO:24) AGRIAARHGWFDP LCVR nucleic acid sequence (SEQ ID NO:25) GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTACATCTGTAGGAGACAGAGTCACCATCTCTTGTCGGGCGAGTCAGGATATTCGCAAGTGGTTAGCCTGGTATCAACAGAAACCAGGAAAAGCCCCTAAACTCCTGATCTATGCTACATCCAGTT TGCAAAGTGGGGTCCCTTCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAGGATTTTGCAACTTACTTTTGTCAACAGGCTAACAGTTTCCCGTTCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA LCVR amino acid sequence (SEQ ID NO:26) DIQMTQSPSSVSTSVGDRVTISCRASQDIRKWLAWYQQKPGKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPFTFGQGTKLEIK LCDR1 nucleic acid sequence (SEQ ID NO:27) CAG GAT ATT CGC AAG TGG LCDR1 amino acid sequence (SEQ ID NO:28) QDIRKW LCDR2 nucleic acid sequence (SEQ ID NO:29) GCT ACA TCC LCDR2 amino acid sequence (SEQ ID NO:30) ATS LCDR3 nucleic acid sequence (SEQ ID NO:31) CAA CAG GCT AAC AGT TTC CCG TTC ACT LCDR3 amino acid sequence (SEQ ID NO:32) QQANSFPFT HC nucleic acid sequence (SEQ ID NO: 197) HC amino acid sequence (SEQ ID NO: 198) [ka] *The underlined bold asparagine (N) can be mutated to a glutamine (Q) for conjugation by transglutaminase. See, for example, SEQ ID NO: 269. LC nucleic acid sequence (SEQ ID NO: 199) GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTACATCTGTAGGAGACAGAGTCACCATCTCTTGTCGGGCGAGTCAGGATATTCGCAAGTGGTTAGCCTGGTATCAACAGAAACCAGGAAAAGCCCCTAAACTCCTGATCTATGCTACATCCAGTTT GCAAAGTGGGGTCCCTTCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAGGATTTTGCAACTTACTTTTGTCAACAGGCTAACAGTTTCCCGTTCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAAC GAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG LC amino acid sequence (SEQ ID NO: 200) DIQMTQSPSSVSTSVGDRVTISCRASQDIRKWLAWYQQKPGKAPKLLIYATSSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQANSFPFTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 10717(wild type hIgG1) / 14572(hIgG1 N180Q) HCVR nucleic acid sequence (SEQ ID NO:33) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTACATATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATTTGGCATGATGGAAGTGATAAATATT ATGTAGACTCCGTGAAGGGCCGATTCTCCATCGCCAGAGACAATTCCAAGAACACGCTTTATCTGCAAATGAATAGTCTGAGAGTCGAGGACACGGGTATATATTACTGTGCGAGAAGGGGTATACGTGGAACCGTTTTGACCACTGGGGCCTGGGAACCCTGGTCACCGTCTCCTCA HCVR amino acid sequence (SEQ ID NO:34) QVQLVESGGGVVQPGRSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWVAVIWHDGSDKYYVDSVKGRFSIARDNSKNTLYLQMNSLRVEDTGIYYCARRGIRGTVFDHWGLGTLVTVSS HCDR1 nucleic acid sequence (SEQ ID NO:35) GGA TTC ACC TTC AGT ACA TAT GGC HCDR1 amino acid sequence (SEQ ID NO:36) GFTFSTYG HCDR2 nucleic acid sequence (SEQ ID NO:37) ATT TGG CAT GAT GGA AGT GAT AAA HCDR2 amino acid sequence (SEQ ID NO:38) IWHDGSDK HCDR3 nucleic acid sequence (SEQ ID NO:39) GCG AGA AGG GGT ATA CGT GGA ACC GTT TTT GAC CAC HCDR3 amino acid sequence (SEQ ID NO:40) ARRGIRGTVFDH LCVR nucleic acid sequence (SEQ ID NO:41) GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCCTCACTTGTCGGGCCAGTCAGAGTATTAGTAACAAGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCTATAAGGCGTCTAATT TAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA LCVR amino acid sequence (SEQ ID NO:42) DIQMTQSPSTLSASVGDRVTLTCRASQSISNKLAWYQQKPGKAPNLLIYKASNLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSWTFGQGTKVEIK LCDR1 nucleic acid sequence (SEQ ID NO:43) CAG AGT ATT AGT AAC AAG LCDR1 amino acid sequence (SEQ ID NO:44) QSISNK LCDR2 nucleic acid sequence (SEQ ID NO:45) AAG GCG TCT LCDR2 amino acid sequence (SEQ ID NO:46) KAS LCDR3 nucleic acid sequence (SEQ ID NO:47) CAA CAG TAT AAT AGT TAT TCG TGG ACG LCDR3 amino acid sequence (SEQ ID NO:48) QQYNSYSWT HC nucleic acid sequence (SEQ ID NO:201) HC amino acid sequence (SEQ ID NO:202) [ka] *The underlined bold asparagine (N) can be mutated to a glutamine (Q) for conjugation by transglutaminase. See, for example, SEQ ID NO: 269. LC nucleic acid sequence (SEQ ID NO:203) GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCCTCACTTGTCGGGCCAGTCAGAGTATTAGTAACAAGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATCTATAAGGCGTCTAATTT AGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAACAGTATAATAGTTATTCGTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC GAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG LC amino acid sequence (SEQ ID NO:204) DIQMTQSPSTLSASVGDRVTLTCRASQSISNKLAWYQQKPGKAPNLLIYKASNLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYSWTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 10716(wild type hIgG1) / 14571(hIgG1 N180Q) HCVR nucleic acid sequence (SEQ ID NO:49) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGACTCCATCAAATAATTACTACTGGACCTGGCTCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGATTGGTTATATCTATTACAGTGGGAGCGCCAAC TACAACCCCTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTAAATTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGCGAGAGGGGCGGTCAAGTACTTCCGGCATTGGGCCAGGGCACCCTGGTCACCGTCTCCTCA HCVR amino acid sequence (SEQ ID NO:50) QVQLQESGPGLVKPSETLSLTTCTVSGDSINNYYWTWLRQPPGKGLEWIGYIYYSGSANYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCARGAVKYFRHWGQGTLVTVSS HCDR1 nucleic acid sequence (SEQ ID NO:51) GGT GAC TCC ATC AAT AAT TAC TAC HCDR1 amino acid sequence (SEQ ID NO:52) GDSINNYY HCDR2 nucleic acid sequence (SEQ ID NO:53) ATC TAT TAC AGT GGG AGC GCC HCDR2 amino acid sequence (SEQ ID NO:54) IYYSGSA HCDR3 nucleic acid sequence (SEQ ID NO:55) GCG AGA GGG GCG GTC AAG TAC TTC CGG CAT HCDR3 amino acid sequence (SEQ ID NO:56) ARGAVKYFRH LCVR nucleic acid sequence (SEQ ID NO:57) GAAATTGTGTTGACGCAGTCTCCGGGCACCCTCTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGACTATTAACCACAACAACTTAGCCTGGTACCAGCAGAGACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAAC AGGGCCACTGCCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGAAGTGTATTCTTGTCAGCAGTATGGTAGCTTGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA LCVR amino acid sequence (SEQ ID NO:58) EIVLTQSPGTLSLSPGERATLSCRASQTINHNNLAWYQQRPGQAPRLLIYGASNRATAIPDRFSGSGSGTDFTLTISRLEPEDFEVYSCQQYGSLPLTFGGGTKVEIK LCDR1 nucleic acid sequence (SEQ ID NO:59) CAG ACT ATT AAC CAC AAC AAC LCDR1 amino acid sequence (SEQ ID NO:60) QTINHNN LCDR2 nucleic acid sequence (SEQ ID NO:61) GGT GCA TCC LCDR2 amino acid sequence (SEQ ID NO:62) GAS LCDR3 nucleic acid alignment (alignment number 63) CAG CAG TAT GGT AGC TTG CCG CTC ACT LCDR3 アミノ acid arrangement (allocation number 64) QQYGSLPLT HC nucleic acid array (array number 205) HC amino acid sequence (SEQ ID NO:206) [ka] *The underlined bold asparagine (N) can be mutated to a glutamine (Q) for conjugation by transglutaminase. See, for example, SEQ ID NO: 269. LC nucleic acid sequence (SEQ ID NO:207) GAAATTGTGTTGACGCAGTCTCCGGGCACCCTCTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGACTATTAACCACAACAACTTAGCCTGGTACCAGCAGAGACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAAC AGGGCCACTGCCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGAAGTGTATTCTTGTCAGCAGTATGGTAGCTTGCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG LC amino acid sequence (SEQ ID NO:208) EIVLTQSPGTLSLSPGERATLSCRASQTINHNNLAWYQQRPGQAPRLLIYGASNRATAIPDRFSGSGSGTDFTLTISRLEPEDFEVYSCQQYGSLPLTFGGGTKVEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 10783(wild type hIgG1) / 14574(hIgG1 N180Q) HCVR nucleic acid sequence (SEQ ID NO:65) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGACGTCCCTGAGACTCTCCTGTGCAGCGTCAGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGATTGATGGAAGTAATAAATATTATG CAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAAGGGGGGTATAGTAGTAGCTGCCCCCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA HCVR amino acid sequence (SEQ ID NO:66) QVQLVESGGGVVQPGTSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWIDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGGIVVAAPFDYWGQGTLVTVSS HCDR1 nucleic acid sequence (SEQ ID NO:67) GGA TTC ACC TTC AGT AGC TAT GGC HCDR1 amino acid sequence (SEQ ID NO:68) GFTFSSYG HCDR2 nucleic acid sequence (SEQ ID NO:69) ATA TGG ATT GAT GGA AGT AAT AAA HCDR2 amino acid sequence (SEQ ID NO:70) IWIDGSNK HCDR3 nucleic acid sequence (SEQ ID NO:71) GCG AGA AGG GGG GGT ATA GTA GTA GCT GCC CCC TTT GAC TAC HCDR3 amino acid sequence (SEQ ID NO:72) ARRGGIVVAAPFDY LCVR nucleic acid sequence (SEQ ID NO:73) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA LCVR amino acid sequence (SEQ ID NO:74) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK LCDR1 nucleic acid sequence (SEQ ID NO:75) CAG AGC ATT AGC AGC TAT LCDR1 amino acid sequence (SEQ ID NO:76) QSISSY LCDR2 nucleic acid sequence (SEQ ID NO:77) GCT GCA TCC LCDR2 amino acid sequence (SEQ ID NO:78) AAS LCDR3 nucleic acid sequence (SEQ ID NO:79) CAA CAG AGT TAC AGT ACC CCT CCG ATC ACC LCDR3 amino acid sequence (SEQ ID NO:80) QQSYSTPPIT HC nucleic acid sequence (SEQ ID NO:209) HC amino acid sequence (SEQ ID NO:210) [ka] *The underlined bold asparagine (N) can be mutated to a glutamine (Q) for conjugation by transglutaminase. See, for example, SEQ ID NO: 269. LC nucleic acid sequence (SEQ ID NO:211) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG LC amino acid sequence (SEQ ID NO:212) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 31944 HCVR wheelchair (wheelchair 81) CAG GTG CAG TTG GTG GAG TCT GGG GGA GGC GTG GTC CAG CCT GGG AGG TCC CTG AGA CTC TCC TGT GAA GCG TCT GGA ATC ACC TTC AGA AAC TAT GGC ATG CAC TGG GTC CGC CAG GCT CCA GGC AAG GGG CTG GAG TGG GTG GCA GTT ATG TGG TAT GAT GGA AGT AAT AAA TAC TAC GCA GAC TCC GTG AAG GGC CGA TTC ACC ATC TCC AGA GAC AAT TCC AAG AAC ACG GTG TAT CTG CAA ATG AAC AGC CTG AGA GCC GAA GAC ACG GCT GTG TAT TAC TGT GCG AGA CGG GGT CAT ATA GCA ACA GCT GCT CCC TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCA HCVR Railway Railway (Chain Range 82) QVQLVESGGGVVQPGRSLRLSCEASGITFRNYGMHWVRQAPGKGLEWVAVMWYDGSNKYYADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARRGHIATAAPFDYWGQGTLVTVSS HCDR1 Connector(83) GGA ATC ACC TTC AGA AAC TAT GGC HCDR1 amino acid sequence (SEQ ID NO:84) GITFRNYG HCDR2 nucleic acid sequence (SEQ ID NO:85) atg tgg tat gat gga agt aat aaa HCDR2 amino acid sequence (SEQ ID NO:86) MWYDGSN HCDR3 nucleic acid sequence (SEQ ID NO:87) GCG AGA CGG GGT CAT ATA GCA ACA GCT GCT CCC TTT GAC TAC HCDR3 amino acid sequence (SEQ ID NO:88) ARRGHIATAAPFD LCVR nucleic acid sequence (SEQ ID NO:89) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCCGTAGGAGACAGAGTCACCATCAGTTGCCGGGCAAGTCAGAGCATTAGTAGTTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGGTCCTGATGTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAGGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA LCVR amino acid sequence (SEQ ID NO:90) DIQMTQSPSSLSASVGDRVTISCRASQSISSYLNWYQQKPGKAPKVLMYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK LCDR1 nucleic acid sequence (SEQ ID NO:91) CAG AGC ATT AGT AGT TAT LCDR1 amino acid sequence (SEQ ID NO:92) QSISSY LCDR2 nucleic acid sequence (SEQ ID NO:93) GCT GCA TCC LCDR2 amino acid sequence (SEQ ID NO:94) AAS LCDR3 nucleic acid sequence (SEQ ID NO:95) CAA CAG AGT TAC AGT ACC CCT CCG ATC ACC LCDR3 amino acid sequence (SEQ ID NO:96) QQSYSTPPIT HC nucleic acid sequence (SEQ ID NO:213) CAG GTG CAG TTG GTG GAG TCT GGG GGA GGC GTG GTC CAG CCT GGG AGG TCC CTG AGA CTC TCC TGT GAA GCG TCT GGA ATC ACC TTC AGA AAC TAT GGC ATG CAC TGG GTC CGC CAG GCT CCA GGC AAG GGG CTG GAG TGG GTG GCA GTT ATG TGG TAT GAT GGA AGT AAT AAA TAC TAC GCA GAC TCC GTG AAG GGC CGA TTC ACC ATC TCC AGA GAC AAT TCC AAG AAC ACG GTG TAT CTG CAA ATG AAC AGC CTG AGA GCC GAA GAC ACG GCT GTG TAT TAC TGT GCG AGA CGG GGT CAT ATA GCA ACA GCT GCT CCC TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCC TCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAGGTGGACAAGAAAATTGAGCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAATGA HC amino acid sequence (SEQ ID NO:214) QVQLVESGGG VVQPGRSLRL SCEASGITFR NYGMHWVRQA PGKGLEWVAV MWYDGSNKYY ADSVKGRFTI SRDNSKNTVY LQMNSLRAED TAVYYCARRG HIATAAPFDY WGQGTLVTVS S AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEEDDPDVQISWFVNNV EVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK LC nucleic acid sequence (SEQ ID NO:215) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCCGTAGGAGACAGAGTCACCATCAGTTGCCGGGCAAGTCAGAGCATTAGTAGTTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGGTCCTGATGTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAGGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA CGAGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAAC AGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGGGAGAGTGTTGA LC amino acid sequence (SEQ ID NO:216) DIQMTQSPSS LSASVGDRVT ISCRASQSIS SYLNWYQQKP GKAPKVLMYA ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SYSTPPITFG QGTRLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRGEC 31265(wild type hIgG1) / 5972(hIgG1 N180Q) HCVR nucleic acid sequence (SEQ ID NO:97) CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTACAGCGTCTGGATTCACCTTCCGTTCCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGTCAGTTATTTGGATTGATGGAAATAATATATACTATG CAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGGACAGCCTGAGAGCCGAGGACACGGCTGTTTATTACTGTGCGAGAAGACTGGCTATAACATCAGCTGCCCCCTTTGACTACTGGGCCAGGGAACCCTGGTCACCGTCTCCTCA HCVR amino acid sequence (SEQ ID NO:98) QVQLVESGGGVVQPGRSLRLSCTASGFTFRSYGMHWVRQAPGKGLEWVSVIWIDGNNIYYADSVKGRFTISRDNSKNTLYLQMDSLRAEDTAVYYCARRLAITSAAPFDYWGQGTLVTVSS HCDR1 nucleic acid sequence (SEQ ID NO:99) GGA TTC ACC TTC CGT TCC TAT GGC HCDR1 amino acid sequence (SEQ ID NO: 100) GFTFRSYG HCDR2 nucleic acid sequence (SEQ ID NO:101) ATT TGG ATT GAT GGA AAT AAT ATA HCDR2 amino acid sequence (SEQ ID NO: 102) IWIDGNNI HCDR3 nucleic acid sequence (SEQ ID NO: 103) GCG AGA AGA CTG GCT ATA ACA TCA GCT GCC CCC TTT GAC TAC HCDR3 amino acid sequence (SEQ ID NO: 104) ARRLAITSAAPFDY LCVR nucleic acid sequence (SEQ ID NO: 105) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA LCVR amino acid sequence (SEQ ID NO: 106) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEIK LCDR1 nucleic acid sequence (SEQ ID NO: 107) CAG AGC ATT AGC AGC TAT LCDR1 amino acid sequence (SEQ ID NO: 108) QSISSY LCDR2 nucleic acid sequence (SEQ ID NO: 109) GCT GCA TCC LCDR2 amino acid sequence (SEQ ID NO:110) AAS LCDR3 nucleic acid sequence (SEQ ID NO:111) CAA CAG AGT TAC AGT ACC CCT CCG ATC ACC LCDR3 amino acid sequence (SEQ ID NO:112) QQSYSTPPIT HC nucleic acid sequence (SEQ ID NO:217) HC amino acid sequence (SEQ ID NO:218) [ka] *The underlined bold asparagine (N) can be mutated to a glutamine (Q) for conjugation by transglutaminase. See, for example, SEQ ID NO: 269. LC nucleic acid sequence (SEQ ID NO:219) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTG CAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCGGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA CGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAG AGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG LC amino acid sequence (SEQ ID NO:220) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGQGTRLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 31941 HCVR nucleic acid sequence (SEQ ID NO:113) CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACGCCTTCACCACCTATGGTATCACCTGGGTGCGACAGGCCCCTGGACAAGGACTTGAGTGGATGGGATGGATCAGCGCTTACAATGGAAATACAAACTATGCAGAGAAG GTCCAGGGCAGATTCACCATGACCACAGACACATCCACGAATACAGCCTACATGGAGCTGAGGAGCCTGAGATCCGACGACACGGCCGTGTATTTCTGTGCGAGAAAGGGTCACTATGGTTCGGGGACTTATTATAACCCCTTTGGTTTTGATTTTTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA HCVR amino acid sequence (SEQ ID NO:114) QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYGITWVRQAPGQGLEWMGWISAYNGNTNYAEKVQGRFTMTTDTSTNTAYMELRSLRSDDTAVYFCARKGHYGSGTYYNPFGFDFWGQGTMVTVSS HCDR1 nucleic acid sequence (SEQ ID NO:115) ggt tac gcc ttc acc acc tat ggt HCDR1 amino acid sequence (SEQ ID NO:116) GYAFTTYG HCDR2 nucleic acid sequence (SEQ ID NO:117) atc agc gct tac aat gga aat aca HCDR2 amino acid sequence (SEQ ID NO:118) ISAYNGN HCDR3 nucleic acid sequence (SEQ ID NO:119) GCG AGA AAG GGT CAC TAT GGT TCG GGG ACT TAT TAT AAC CCC TTT GGT TTT GAT TTT HCDR3 amino acid sequence (SEQ ID NO: 120) CARKGHYGSGTYYNPFGFD LCVR nucleic acid sequence (SEQ ID NO:121) GAAATTATGTTGATGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGC AGGGCCACTGACATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTTTATTTCTGTCAGCAGTATTATGGCTCACCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAG LCVR amino acid sequence (SEQ ID NO: 122) EIMLMQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATDIPDRFSGSGSGTDFTLTISRLEPEDFAVYFCQQYYGSPWTFGQGTKVEIK LCDR1 nucleic acid sequence (SEQ ID NO: 123) cag agt gtt agc agc agc tac LCDR1 amino acid sequence (SEQ ID NO: 124) QSVSSSY LCDR2 nucleic acid sequence (SEQ ID NO: 125) ggt gca tcc LCDR2 amino acid sequence (SEQ ID NO: 126) GA LCDR3 nucleic acid sequence (SEQ ID NO:127) cag cag tat tat ggc tca cct tgg acg LCDR3 amino acid sequence (SEQ ID NO: 128) CQQYYGSPW HC nucleic acid sequence (SEQ ID NO:221) HC amino acid sequence (SEQ ID NO:222) QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYGITWVRQAPGQGLEWMGWISAYNGNTNYAEKVQGRFTMTTDTSTNTAYMELRSLRSDDTAVYFCARKGHYGSGTYYNPFG FDFWGQGTMVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVP RDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEK TISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK LC nucleic acid sequence (SEQ ID NO:223) GAAATTATGTTGATGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGACATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTTTATTTCTGTCAGCAGTATTATGGCTCACCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAGCGAGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGGGAGAGTGTTGA LC amino acid sequence (SEQ ID NO: 224) EIMLMQSPGT LSLSPGERAT LSCRASQSVS SSYLAWYQQK PGQAPRLLIY GASSRATDIP DRFSGSGSGT DFTLTISRLE PEDFAVYFCQ QYYGSPWTFG QGTKVEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRGEC 7660 HCVR nucleic acid sequence (SEQ ID NO: 129) GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCGGGGGGGTCCCTGAAACTCTCCTGTACAGCCTCTGGGTTGACCCTCAGTGACTCTGCTATGCACTGGGTCCGCCAGGCTTCCGGGAAAGGGCTGGAGTGGGTTGGCCGTATAAGAAATAAGGCTAATAGGTACGCGACA GAATATGCTGCGTCGGTGAAAGGCAGGTTCACCATTTCAAGAGATGATTCAAAGAACACGGCGTATCTACAAATGAACAGCCTGAAAACCGAGGACACGGCCGTGTATTATTGTACTAGAAAACTGGAAGATTTTCCTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA HCVR amino acid sequence (SEQ ID NO: 130) EVQLVESGGGLVQPGGSLKLSCTASGLTLSDSAMHWVRQASGKGLEWVGRIRNKANRYATEYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRNWKIFLFDYWGQGTLVTVSS HCDR1 nucleic acid sequence (SEQ ID NO: 131) GGG TTG ACC CTC AGT GAC TCT GCT HCDR1 amino acid sequence (SEQ ID NO: 132) GLTLSDSA HCDR2 nucleic acid sequence (SEQ ID NO: 133) ATA AGA AAT AAG GCT AAT AGG TAC GCG ACA HCDR2 amino acid sequence (SEQ ID NO: 134) IRNKANRYAT HCDR3 nucleic acid sequence (SEQ ID NO: 135) ACT AGA AAC TGG AAG ATT TTC CTC TTT GAC TAC HCDR3 amino acid sequence (SEQ ID NO: 136) TRNWKIFLFDY LCVR nucleic acid sequence (SEQ ID NO: 137) GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGACTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCAGCAAATACTTAGCCTGGTTCCAGCAGAAACGTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGC AGGACCAGTGGCATCCCCGACAGGATCAGTGGCAGTGGGTCAGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGAAGTTCACCCTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA LCVR amino acid sequence (SEQ ID NO: 138) EIVLTQSPGTLTLSPGERATLSCRASQSVGSKYLAWFQQKRGQAPRLLIYGASSRTSGIPDRISGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK LCDR1 nucleic acid sequence (SEQ ID NO: 139) CAG AGT GTT GGC AGC AAA TAC LCDR1 amino acid sequence (SEQ ID NO: 140) QSVGSKY LCDR2 nucleic acid sequence (SEQ ID NO: 141) GGT GCA TCC LCDR2 amino acid sequence (SEQ ID NO: 142) GAS LCDR3 nucleic acid sequence (SEQ ID NO: 143) CAG CAG TAT GGA AGT TCA CCC TGG ACG LCDR3 amino acid sequence (SEQ ID NO: 144) QQYGSSPWT HC nucleic acid sequence (SEQ ID NO:225) HC amino acid sequence (SEQ ID NO:226) EVQLVESGGGLVQPGGSLKLSCTASGLTLSDSAMHWVRQASGKGLEWVGRIRNKANRYATEYAASVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRNWKIFLFDYWG QGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK LC nucleic acid sequence (SEQ ID NO:227) GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGACTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCAGCAAATACTTAGCCTGGTTCCAGCAGAAACGTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGACCAGTGGCATCCCCGACAGGATCAGTGGCAGTGGGTCAGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATGGAAGTTCACCCTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG LC amino acid sequence (SEQ ID NO: 228) EIVLTQSPGTLTLSPGERATLSCRASQSVGSKYLAWFQQKRGQAPRLLIYGASSRTSGIPDRISGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 9909 HCVR nucleic acid sequence (SEQ ID NO: 145) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCGGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAACAACTATGGCATGAGCTGGGTCCGCCAGGGTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAGTGGTAGTGGTGGTACCACATTCTACGCAGACT CCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGGCAAAGGAGGATATTGTAGTAGTAGCGGCTGCCGTCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA HCVR amino acid sequence (SEQ ID NO: 146) EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYGMSWVRQGPGKGLEWVSSISGSGGTTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGKGGYCSSSGCRHYGMDVWGQGTTVTVSS HCDR1 nucleic acid sequence (SEQ ID NO: 147) GGA TTC ACC TTT AAC AAC TAT GGC HCDR1 amino acid sequence (SEQ ID NO: 148) GFTFNNYG HCDR2 nucleic acid sequence (SEQ ID NO: 149) ATT AGT GGT AGT GGT GGT ACC ACA HCDR2 amino acid sequence (SEQ ID NO: 150) SGSGGT HCDR3 nucleic acid sequence (SEQ ID NO: 151) GGC AAA GGA GGA TAT TGT AGT AGT AGC GGC TGC CGT CAC TAC GGT ATG GAC GTC HCDR3 amino acid sequence (SEQ ID NO: 152) CGKGGYCSSSGCRH LCVR nucleic acid sequence (SEQ ID NO: 153) CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGACCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAATAATTATATATACTGGTACCAGCGGCTCCCAGGAACGACCCCCAAACTCCTCATCTATAGGAATAATCAGCGG CCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACACCCTGAGTGGGTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTA LCVR amino acid sequence (SEQ ID NO: 154) QSVLTQPPSASGTPGQRVTISSCSGSSSNIGNNYIYWYQRLPGTTPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDTLSGYVFGTGTKVTVL LCDR1 nucleic acid sequence (SEQ ID NO: 155) AGC TCC AAC ATC GGA AAT AAT TAT LCDR1 amino acid sequence (SEQ ID NO: 156) SSNIGNNY LCDR2 nucleic acid sequence (SEQ ID NO: 157) agg aat aat LCDR2 amino acid sequence (SEQ ID NO: 158) RN LCDR3 nucleic acid sequence (SEQ ID NO: 159) GCA GCA TGG GAT GAC ACC CTG AGT GGG TAT GTC LCDR3 amino acid sequence (SEQ ID NO: 160) CAAWDDTLSGY HC nucleic acid sequence (SEQ ID NO:229) HC amino acid sequence (SEQ ID NO:230) EVQLLESGGGLVQPGGSLRLSCAASGFTFNNYGMSWVRQGPGKGLEWVSSISGSGGTTYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCGKGGYCSSSGCRHYGMD VWGQGTTVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGP TIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIE RTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK LC nucleic acid sequence (SEQ ID NO:231) CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAATAATTATATATACTGGTACCAGCGGCTCCCAGGAACGACCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACACCCTGAGTGGGTATGTCTTCGGAACTGGGACCAAGGTCACCGTCCTACGAGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGGGAGAGTGTTGA LC amino acid sequence (SEQ ID NO: 232) QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYIYWYQRLPGTTPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDTLSGYVFGTGTKVTVLRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRGEC 10713 (wild-type hIgG1) / 14573 (hIgG1 N180Q) HCVR nucleic acid sequence (SEQ ID NO: 161) GAGGTGCAGCTGGTGGAGTCTGGGGGAAACTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTACCAGCCATGCCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGTTATTACTGGTAGAGGTTTTGACACACACTACG CTGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACATTTCCAAAAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTTTATTACTGTGCGAAAGGTCTCTATGATTCGGGGAATTATTATATCGATTACTGGGCCAGGGAACCCTGGTCACCGTCTCCTCA HCVR amino acid sequence (SEQ ID NO: 162) EVQLVESGGNLVQPGGSLRLSCAASGFTFTSHAMNWVRQAPGKGLEWVSVITGRGFDTHYADSVKGRFTISRDISKNTLYLQMNSLRAEDTAVYYCAKGLYDSGNYYIDYWGQGTLVTVSS HCDR1 nucleic acid sequence (SEQ ID NO: 163) GGA TTC ACC TTT ACC AGC CAT GCC HCDR1 amino acid sequence (SEQ ID NO: 164) GFTFTSHA HCDR2 nucleic acid sequence (SEQ ID NO: 165) ATT ACT GGT AGA GGT TTT GAC ACA HCDR2 amino acid sequence (SEQ ID NO: 166) ITGRGFDT HCDR3 nucleic acid sequence (SEQ ID NO: 167) GCG AAA GGT CTC TAT GAT TCG GGG AAT TAT TAT ATC GAT TAC HCDR3 amino acid sequence (SEQ ID NO: 168) AKGLYDSGNYYIDY LCVR nucleic acid sequence (SEQ ID NO: 169) CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTTTCCTGGTACCAGCAGCTCCCAGGAACAGCCCCCAAACTCCTCATTTATGACAATAATAAGCGAC CCTCAGGGATTCCTGACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCGGACTCCAGACTGGGGACGAGGCCGATTATTACTGCGGAACATGGGATCTCAGCCTGAGTTTCAATTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTA LCVR amino acid sequence (SEQ ID NO: 170) QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDLSLSFNWVFGGGTKLTVL LCDR1 nucleic acid sequence (SEQ ID NO: 171) AGC TCC AAC ATT GGG AAT AAT TAT LCDR1 amino acid sequence (SEQ ID NO: 172) SSNIGNNY LCDR2 nucleic acid sequence (SEQ ID NO: 173) GAC AAT AAT LCDR2 amino acid sequence (SEQ ID NO: 174) DNN LCDR3 nucleic acid sequence (SEQ ID NO: 175) GGA ACA TGG GAT CTC AGC CTG AGT TTC AAT TGG GTG LCDR3 amino acid sequence (SEQ ID NO: 176) GTWDLSLSFNWV HC nucleic acid sequence (SEQ ID NO:233) HC amino acid sequence (SEQ ID NO:234) [ka] *The underlined bold asparagine (N) can be mutated to a glutamine (Q) for conjugation by transglutaminase. See, for example, SEQ ID NO: 269. LC nucleic acid sequence (SEQ ID NO:235) CAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTTTCCTGGTACCAGCAGCTCCCAGGAACAGCCCCCAAACTCCTCATTTATGACAATAATA AGCGACCCTCAGGATTCCTGACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCGGACTCCAGACTGGGGACGAGGCCGATTATTACTGCGGAACATGGGATCTCAGCCTGAGTTTCAATTGGGTGTTCGGCGGAGGGACCAA GCTGACCGTCCTAGGCCAGCCCAAGGCCGCCCCCTCCGTGACCCTGTTCCCCCTCCTCCGAGGAGCTGCAGGCCAACAAGGCCACCCTGGTGTGCCTGATCTCCGACTTCTACCCCGGCGCCGTGACCGTGGCCTGGAAGGCCGACTCCTCCCCGTG AAGGCCGGCGTGGAGACCACCACCCCCTCCAAGCAGTCCAACAACAAGTACGCCGCCTCCTCCTACCTGTCCCTGACCCCCGAGCAGTGGAAGTCCCACCGGTCCTACTCCTGCCAGGTGACCCACGAGGGCTCCACCGTGGAGAAGACCGTGGCCCCCA CCGAGTGCTCCTGA LC amino acid sequence (SEQ ID NO:236) QSVLTQPPSVSAAPGQKVTISCSSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDLSLSFNWVFGGGTKL TVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS 7854 HCVR nucleic acid sequence (SEQ ID NO: 177) CAGGTTCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACGCCTTCACCACCTATGGTATCACCTGGGTGCGACAGGCCCCTGGACAAGGACTTGAGTGGATGGGATGGATCAGCGCTTACAATGGAAATACAAACTATGCAGAGAAG GTCCAGGGCAGATTCACCATGACCACAGACACATCCACGAATACAGCCTACATGGAGCTGAGGAGCCTGAGATCCGACGACACGGCCGTGTATTTCTGTGCGAGAAAGGGTCACTATGGTTCGGGGACTTATTATAACCCCTTTGGTTTTGATTTTTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA HCVR amino acid sequence (SEQ ID NO: 178) QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYGITWVRQAPGQGLEWMGWISAYNGNTNYAEKVQGRFTMTTDTSTNTAYMELRSLRSDDTAVYFCARKGHYGSGTYYNPFGFDFWGQGTMVTVSS HCDR1 nucleic acid sequence (SEQ ID NO: 179) GGT TAC GCC TTC ACC ACC TAT GGT HCDR1 amino acid sequence (SEQ ID NO: 180) GYAFTTYG HCDR2 nucleic acid sequence (SEQ ID NO: 181) ATC AGC GCT TAC AAT GGA AAT ACA HCDR2 amino acid sequence (SEQ ID NO: 182) ISAYNGNT HCDR3 nucleic acid sequence (SEQ ID NO: 183) GCG AGA AAG GGT CAC TAT GGT TCG GGG ACT TAT TAT AAC CCC TTT GGT TTT GAT TTT HCDR3 amino acid sequence (SEQ ID NO: 184) ARKGHYGSGTYYNPFGFDF LCVR nucleic acid sequence (SEQ ID NO: 185) GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGC AGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCTTTGTATTTCTGTCAGCAGTATTATGGCTCACCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA LCVR amino acid sequence (SEQ ID NO: 186) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFALYFCQQYYGSPWTFGQGTKVEIK LCDR1 nucleic acid sequence (SEQ ID NO: 187) CAG AGT GTT AGC AGC AGC TAC LCDR1 amino acid sequence (SEQ ID NO: 188) QSVSSSY LCDR2 nucleic acid sequence (SEQ ID NO: 189) GGT GCA TCC LCDR2 amino acid sequence (SEQ ID NO: 190) GAS LCDR3 nucleic acid sequence (SEQ ID NO: 191) CAG CAG TAT TAT GGC TCA CCT TGG ACG LCDR3 amino acid sequence (SEQ ID NO: 192) QQYYGSPWT HC nucleic acid sequence (SEQ ID NO:237) HC amino acid sequence (SEQ ID NO:238) QVQLVQSGAEVKKPGASVKVSCKASGYAFTTYGITWVRQAPGQGLEWMGWISAYNGNTNYAEKVQGRFTMTTDTSTNTAYMELRSLRSDDTAVYFCARKGHYGSGTYYNPFGF DFWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVES KYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK LC nucleic acid sequence (SEQ ID NO:239) GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCTTTGTATTTCTGTCAGCAGTATTATGGCTCACCTTGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG LC amino acid sequence (SEQ ID NO: 240) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFALYFCQQYYGSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0062] Non-limiting examples of targeting ligands that bind CACNG1 include (i) Fab fragments, (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 that mimic the hypervariable regions of an antibody (e.g., isolated complementarity determining regions (CDRs), such as CDR3 peptides), or constrained FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed by the term "targeting ligand" as used herein. In a non-limiting embodiment, the anti-CACNG1 targeting ligands that bind to CACNG1 useful for retargeting the viral capsids described herein include scFvs. L -(Gly4Ser)3-V H The scFv sequence of this format may comprise a heavy chain variable domain, a light chain variable domain, a heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and / or a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 that is 90%, 95%, 97%, 98%, 99% or 100% identical to any one of the amino acid sequences of the heavy chain variable domain, the light chain variable domain, the heavy chain variable domain / light chain variable domain pair, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, respectively, and / or a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 set forth in any one of SEQ ID NOs: 1 to 240.

[0063] A targeting ligand that binds to a mammalian muscle cell-specific surface protein can be associated with (e.g., displayed, operably linked, bound by) the modified AAV capsid protein and the resulting AAV capsid according to known methods, e.g., a direct approach in which the targeting ligand is directly inserted (e.g., using recombinant methods) according to known methods. See, e.g., Stachler et al. (2006), supra, White et al. (2004), supra, Girod et al. (1999), supra, Grifman et al. (2001), supra, Shi et al. (2001), supra, Shi and Bartlett (2003). A targeting ligand that binds to a mammalian muscle cell-specific surface protein may be attached to the modified AAV capsid protein and the resulting AAV capsid using known chemical linkers, for example, the AAV capsid protein may be chemically modified to include a dibenzocycotine group or an azide group, and optionally, the targeting ligand described herein may be attached to the dibenzocycotine group or the azide group, see, for example, US Patent Application Publication No. 2022 / 028234, which is incorporated herein by reference in its entirety. Here, the targeting ligand is covalently attached to a primary amino acid group of the AAV capsid protein, for example, via a -CSNH- bond. In some embodiments, the modified capsid described herein includes a targeting ligand, for example, an anti-CACNG1 antibody or a binding portion thereof, that is directly inserted or attached thereto according to well-known direct recombinant methods.

[0064] Bond Pair In some embodiments, a targeting ligand that binds to a mammalian muscle cell-specific surface protein can be associated with (e.g., displayed, operably linked, or bound by) a modified AAV capsid protein and the resulting AAV capsid according to an indirect recombinant approach, where the AAV capsid protein is modified to include a first member of a binding pair (e.g., a heterologous scaffold), optionally the first member of the binding pair is linked (e.g., covalently or non-covalently linked) to a second cognate member of the binding pair (e.g., an adapter), and further optionally the second cognate member of the binding pair is fused to the targeting ligand. Non-limiting exemplary binding pairs are listed in Buning and Srivastava (2019) Mol. Ther. Methods Clin Dev 12:248-265.

[0065] Thus, in some embodiments, the capsid protein modifications described herein include those that typically result from modifications at the genetic level, such as, for example, via modification of the cap gene to insert a first member of a binding pair (e.g., a protein:protein binding pair, a protein:nucleic acid binding pair), a detectable label displayed by the Cap protein, etc.

[0066] In some embodiments, the first member forms a binding pair with an immunoglobulin constant domain. In some embodiments, the first member binds a metal ion, e.g., Ni 2+ , Co 2+ , Cu 2+ , Zn 2+ , Fe 3+ etc. In some embodiments, the first member is selected from the group consisting of streptavidin, StrepII, HA, L14, 4C-RGD, LH, and Protein A.

[0067] In some embodiments, the binding pair comprises an enzyme:nucleic acid binding pair. In some embodiments, the first member comprises a HUH-endonuclease or a HUH tag, and the second member comprises a nucleic acid binding domain. In some embodiments, the first member comprises a HUH tag. See, e.g., US2021 / 0180082, which is incorporated herein by reference in its entirety.

[0068] In some embodiments, a capsid protein of the invention comprises at least a first member of a peptide:peptide binding pair.

[0069] In some embodiments, each of the first and second members of the peptide:peptide binding pair comprises an intein. See, e.g., Wagner et al., (2021) Adv. Sci. 8:2004018(1 / 22); Muik et al. (2017) Biomaterials 144:84, each of which is incorporated herein by reference in its entirety.

[0070] In some embodiments, the first member is a B cell epitope, e.g., from about 1 amino acid to about 35 amino acids in length, which forms a binding pair with an antibody paratope, e.g., an immunoglobulin variable domain. In some embodiments, the capsid proteins of the invention are modified to include a detectable label as the first member of the binding pair. 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 a corresponding antibody), a FLASH® tag (a highly detectable label linked to a specific 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, an 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 a detectable label that is recognized by an antibody paratope, where the detectable label and the antibody paratope form a protein:protein binding pair.

[0071] In some embodiments, the capsid protein of the present invention comprises 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 protein of the present invention comprises a first member of a protein:protein binding pair comprising c-myc (SEQ ID NO: 246). The use of detectable labels as the first member of a protein:protein binding pair is described, for example, in WO2019 / 006043, which is incorporated herein by reference in its entirety.

[0072] In some embodiments, the first member comprises the B1 epitope (SEQ ID NO: 247). In some embodiments, the capsid protein is modified to comprise the B1 epitope in the VP3 region. In some embodiments, the first member is selected from the group consisting of FLAG, HA, and c-myc (EQKLISEEDL, SEQ ID NO: 246).

[0073] 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 peptide:peptide binding pair is covalently linked to a cognate second member of the peptide:peptide binding pair via an isopeptide bond, optionally where the cognate second member of the peptide:peptide 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, SpyTag003:SpyCatcher003, SpyTag:KTag, Isopeptag:pilin-C, and SnoopTag:SnoopCatcher. In some embodiments, the first member is SpyTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is SpyCatcher (or a biologically active portion or variant thereof). In some embodiments, the first member is SpyTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is KTag (or a biologically active portion or variant thereof). In some embodiments, the first member is KTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is SpyTag (or a biologically active portion or variant thereof). In some embodiments, the first member is SnoopTag (or a biologically active portion or variant thereof) and the protein (second cognate member) is SnoopCatcher (or a biologically active portion or variant thereof). In some embodiments, the first member is Isopeptag (or a biologically active portion or variant thereof) and the protein (the second cognate member) is Pilin-C (or a biologically active portion or variant thereof).In some embodiments, the first member is SpyTag002 (or a biologically active portion or variant thereof) and the protein (second cognate member) is SpyCatcher002 (or a biologically active portion or variant thereof). In some embodiments, the first member is SpyTag003 (or a biologically active portion or variant thereof) and the protein (second cognate member) is SpyCatcher003 (or a biologically active portion or variant thereof). In some embodiments, the Cap protein of the invention comprises SpyTag, or a biologically active portion or variant thereof. The use of the first member of a protein:protein binding pair is described in WO2019 / 006046, which is incorporated herein in its entirety.

[0074] 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 added 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.

[0075] 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, 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 linker are each independently 1, 2, 3, 4, 5, 6, 7, or 8 amino acids in length. In some embodiments, the first and / or second linker 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 linker 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 linker are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids or more in length.

[0076] In some embodiments, the first and second linkers are identical in sequence and / or length, each being one amino acid long. In some embodiments, the first and second linkers are identical in length, each being one amino acid long. In some embodiments, the first and second linkers are identical in length, each being two amino acids long. In some embodiments, the first and second linkers are identical in length, each being three amino acids long. In some embodiments, the first and second linkers are identical in length, each being four amino acids long, e.g., the linkers are GLSG (SEQ ID NO: 248). In some embodiments, the first and second linkers are identical in length, each being five amino acids long. In some embodiments, the first and second linkers are identical in length, each being six amino acids long, e.g., the first and second linkers each comprise the sequence of GLSGSG (SEQ ID NO: 249). In some embodiments, the first and second linkers are identical in length, each being seven amino acids long. In some embodiments, the first and second linkers are identical in length, each 8 amino acids in length, e.g., the first and second linkers each comprise the sequence GLSGLSGS (SEQ ID NO: 250). In some embodiments, the first and second linkers are identical in length, each 9 amino acids in length. In some embodiments, the first and second linkers are identical in length, each 10 amino acids in length, e.g., the first and second linkers each comprise the sequence GLSGLSGLSG (SEQ ID NO: 251) or GLSGGSGLSG (SEQ ID NO: 252). In some embodiments, the first and second linkers are identical in length, each greater than 10 amino acids in length.

[0077] Generally, the first member of the amino acid sequence of a protein:protein binding pair described herein, for example, comprises a first member of a specific binding pair by itself or in combination with one or more linkers, and is from about 5 amino acids to about 50 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is at least 5 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 6 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 7 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 8 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 9 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 10 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 11 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 12 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 13 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 14 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 15 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 16 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 17 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 18 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 19 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 20 amino acids in length.In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 21 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 22 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 23 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 24 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 25 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 26 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 27 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 28 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 29 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 30 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 31 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 32 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 33 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 34 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 35 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 36 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 37 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 38 amino acids in length.In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 39 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 40 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 41 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 42 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 43 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 44 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 45 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 46 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 47 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 48 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 49 amino acids in length. In some embodiments, the first member of the amino acid sequence of the protein:protein binding pair is 50 amino acids in length.

[0078] Modified Capsids Containing Modified Capsid Proteins In some embodiments, a viral capsid comprising a modified viral capsid protein as described herein is a mosaic capsid, e.g., comprising at least two sets of VP1, VP2, and / or VP3 proteins, each set 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 binding pair and a second corresponding viral capsid protein that lacks the first member of the binding pair. In the context of a mosaic capsid, the second viral capsid protein that lacks the first member of the binding pair may also be 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 the amino acid sequence of the viral VP1, VP2, and / or VP3 capsid protein modified with the first member of the binding pair, except that the reference capsid protein lacks the first member of the binding pair. In some mosaic capsid embodiments, the VP1, VP2, and / or VP3 reference capsid protein corresponds to the viral VP1, VP2, and / or VP3 capsid protein modified with the first member of the binding pair, except that the reference capsid protein lacks the first member of the binding pair. In some embodiments, the VP1 reference capsid protein corresponds to the viral VP1 capsid protein modified with the first member of the binding pair, except that the reference capsid protein lacks the first member of the binding pair. In some embodiments, the VP2 reference capsid protein corresponds to a viral VP2 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of the binding pair, hi some embodiments, the VP3 reference capsid protein corresponds to a viral VP3 capsid protein modified with a first member of a binding pair, except that the reference capsid protein lacks the first member of the binding pair.In some mosaic capsid embodiments that include chimeric VP1, VP2, and / or VP3 capsid proteins further modified to include a first member of a binding pair, the reference protein may be a 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 that includes a chimeric AAV2 / AAAV VP1 capsid protein modified to include a first member of a binding pair may further include 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 comprise a first member of a 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 comprise a first member of a 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 binding pair and is capable of forming a capsid with a first capsid protein that is modified with the first member of the binding pair.

[0079] Generally, mosaic particles may be produced by transfecting a mixture of modified and reference Cap genes into a production cell in a specified ratio. The ratio of protein subunits in the particle, e.g., modified VP protein:unmodified VP protein, stoichiometrically reflects, but does not necessarily reflect, the ratio of at least two of the cap genes encoding a first capsid protein modified with a first member of a binding pair and one or more reference cap genes, e.g., modified cap gene:reference cap gene, transfected into a packaging cell. 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 a packaging cell.

[0080] 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 includes about 12 modified capsid proteins and about 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 includes about 10 modified capsid proteins and about 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 includes 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 (eg, a mosaic virus particle includes about 48 modified capsid proteins and about 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 about 50 modified capsid proteins and about 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., the mosaic virus particle includes 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., the mosaic virus particle includes 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., the mosaic virus particle includes 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.

[0081] In some non-mosaic viral particle embodiments, the ratio of protein subunits may be 1:0, in which case each capsid protein of the non-mosaic viral particle is modified with a first member of a binding pair. In some non-mosaic viral particle embodiments, the ratio of protein subunits may be 0:1, in which case each capsid protein of the non-mosaic viral particle is not modified with a first member of a binding pair.

[0082] Insertion site Due to the high degree of conservation of at least a wide range of closely related family members, corresponding insertion sites in AAVs other than those listed can be identified by performing amino acid alignments or comparisons of capsid structures. 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) provide a ribbon overlay of various dependoparvoviruses in Figure 7, depicting the variable regions VR I-VR IX. Using structural analysis, as described in the present specification, and sequence analysis, one of skill in the art can determine which amino acids within the variable regions correspond to amino acid sequences in AAV that can accommodate insertion of, for example, a targeting ligand, a first member of a binding pair and / or a detectable label as described herein.

[0083] Generally, the targeting ligand, first member of the binding pair, and / or the detectable label may be inserted into a variable region or loop of the AAV capsid protein, the GH loop of the AAV capsid protein, or the like.

[0084] In some embodiments, the first member of the 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 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-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 in the VP1 capsid protein of AAV2 (Wu et al. (2000) J. Virol. 74:8635-8647). The modified viral capsid protein described herein may be a non-primate capsid protein comprising a first member of a 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, 1-459, 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. Additional suitable insertion sites in non-primate AAVs include sites corresponding to I-587 or I-590 in AAV1, I-589 in AAV1, I-585 in AAV3, I-584 or I-585 in AAV4, and I-575 or I-585 in AAV5.In some embodiments, the modified viral capsid protein described herein may be a non-primate capsid protein comprising a targeting sequence, a first member of a 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.

[0085] In some embodiments, the first member of the binding pair and / or the detectable label is selected from the group consisting of 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, I435 of sea lion AAV capsid protein VP1, I430 of sea lion AAV capsid protein VP1, I431 of sea lion AAV capsid protein VP1, I432 of sea lion AAV capsid protein VP1, I431 ... and I565 of sea lion AAV capsid protein VP1, or a position corresponding to an amino acid 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.

[0086] As used herein, the designations I-###, I#, etc. refer to an insertion site (I) designating the amino acid numbering with respect to the VP1 protein of the AAV capsid protein as ###, however, such insertions may be located N- or C-terminally, preferably directly C-terminally, of one amino acid in a sequence 5 amino acids N- or C-terminal to a given amino acid, preferably 3 amino acids, more preferably 2 amino acids, especially 1 amino acid 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, and corresponding positions (and point mutations thereof) can be readily identified in the VP2 and VP3 capsid proteins encoded by the capsid genes by performing a sequence alignment of the VP1, VP2, and VP3 proteins encoded by the appropriate AAV capsid genes.

[0087] Thus, since the capsid proteins are encoded by overlapping reading frames of the same gene with shifted start codons, an insertion of the coding nucleic acid of one of these sites in the cap gene at the corresponding position also results in an insertion of VP1, VP2 and / or VP3. Thus, for example, for AAV2, according to this nomenclature, an insertion of amino acids 1-138 is only inserted in VP1, an insertion of 138-203 is inserted in VP1 and VP2, and an insertion of 203 to the C-terminus is inserted in VP1, VP2 and VP3, which of course also applies to the insertion site I-587. Thus, the present invention encompasses structural genes of AAV with corresponding insertions in the VP1, VP2 and / or VP3 proteins.

[0088] Also provided herein is a nucleic acid encoding a VP3 capsid protein of the invention. AAV capsid proteins may, but do not necessarily, 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 invention does not encode a VP2 capsid protein or a VP1 capsid protein of the invention. In some embodiments, a nucleic acid encoding a VP3 capsid protein of the invention may encode a VP2 capsid protein of the invention, but not a VP1 capsid of the invention. In some embodiments, a nucleic acid encoding a VP3 capsid protein of the invention may also encode a VP2 capsid protein of the invention, and a VP1 capsid of the invention.

[0089] In some embodiments, a viral capsid comprising a modified viral capsid protein comprising a first and second member of a binding pair (e.g., where the second member is operably linked to a targeting ligand and comprises a multispecific binding protein) has the ability to infect a particular cell, e.g., has an enhanced ability to target and bind to a particular cell, as compared to the ability of a control viral capsid (which is identical to the modified viral capsid protein except that it lacks either or both of the first and second members of the binding pair, e.g., comprises a control capsid protein). In some embodiments, a viral capsid comprising a modified viral capsid protein described herein bound to a first and second member of a binding pair linked to a targeting ligand exhibits a detectable transduction efficiency, as compared to the undetectable transduction efficiency of the 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 binding pair linked to a targeting ligand exhibits a transduction efficiency that is 10% greater than the transduction efficiency of the control viral capsid. In some embodiments, viral capsids comprising modified viral capsid proteins described herein coupled to a first and second member of a binding pair linked 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 modified viral capsid proteins described herein coupled to a suitable first and second member of a binding pair linked 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 modified viral capsid proteins described herein coupled to a suitable first and second member of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 40% greater than that of a control viral capsid. In some embodiments, viral capsids comprising modified viral capsid proteins described herein coupled to a suitable first and second member of a binding pair linked 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 modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked 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 modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked 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 modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked 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 modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked 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 modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked 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 modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 90% greater than that of a control capsid. In some embodiments, viral capsids comprising modified viral capsid proteins described herein coupled to suitable first and second members of a 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 modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 99% greater than that of a control viral capsid.

[0090] In some embodiments, a viral capsid comprising a modified viral capsid protein comprising a first and second member of a binding pair (e.g., where the second member is operably linked to a targeting ligand and comprises a multispecific binding protein) has the ability to infect a particular cell, e.g., has an enhanced ability to target and bind to a particular cell, as compared to the ability of a control viral capsid (which is identical to the modified viral capsid protein except that it lacks either or both of the first and second members of the binding pair, e.g., comprises a control capsid protein). In some embodiments, a viral capsid comprising a modified viral capsid protein described herein bound to a first and second member of a binding pair linked to a targeting ligand exhibits a detectable transduction efficiency, as compared to the undetectable transduction efficiency of the 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 binding pair linked to a targeting ligand exhibits a transduction efficiency that is 10% greater than the transduction efficiency of the control viral capsid. In some embodiments, viral capsids comprising modified viral capsid proteins described herein coupled to a first and second member of a binding pair linked 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 modified viral capsid proteins described herein coupled to a suitable first and second member of a binding pair linked 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 modified viral capsid proteins described herein coupled to a suitable first and second member of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 40% greater than that of a control viral capsid. In some embodiments, viral capsids comprising modified viral capsid proteins described herein coupled to a suitable first and second member of a binding pair linked 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 modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked 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 modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked 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 modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked 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 modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked 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 modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 85% higher than that of a control viral capsid. In some embodiments, viral capsids comprising modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 90% higher than that of a control capsid. In some embodiments, viral capsids comprising modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 95% higher than that of a control viral capsid. In some embodiments, viral capsids comprising modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is 99% higher than that of a control viral capsid.In some embodiments, viral capsids comprising modified viral capsid proteins described herein coupled to a first and second member of a binding pair linked to a targeting ligand exhibit a transduction efficiency at least 1.5 times higher than that of a control viral capsid. In some embodiments, viral capsids comprising modified viral capsid proteins described herein coupled to a first and second member of a binding pair linked to a targeting ligand exhibit a transduction efficiency at least 2 times higher than that of a control viral capsid. In some embodiments, viral capsids comprising modified viral capsid proteins described herein coupled to a suitable first and second member of a binding pair linked to a targeting ligand exhibit a transduction efficiency at least 3 times higher than that of a control viral capsid. In some embodiments, viral capsids comprising modified viral capsid proteins described herein coupled to a suitable first and second member of a binding pair linked to a targeting ligand exhibit a transduction efficiency at least 4 times higher than that of a control viral capsid. In some embodiments, viral capsids comprising modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 5-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 6-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 7-fold higher than that of a control viral capsid.In some embodiments, viral capsids comprising modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency that is at least 8-fold higher than that of a control viral capsid. In some embodiments, viral capsids comprising modified viral capsid proteins described herein coupled to suitable first and second members of a 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 modified viral capsid proteins described herein coupled to suitable first and second members of a 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 modified viral capsid proteins described herein coupled to suitable first and second members of a 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 modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency at least 30 times higher than that of a control viral capsid. In some embodiments, viral capsids comprising modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency at least 40 times higher than that of a control viral capsid. In some embodiments, viral capsids comprising modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency at least 50 times higher than that of a control viral capsid. In some embodiments, viral capsids comprising modified viral capsid proteins described herein coupled to suitable first and second members of a binding pair linked to a targeting ligand exhibit a transduction efficiency at least 60 times higher than that of a control viral capsid.In some embodiments, a viral capsid comprising a modified viral capsid protein described herein coupled to a first and second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 70 times higher than that of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein described herein coupled to a first and second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 80 times higher than that of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein described herein coupled to a first and second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 90 times higher than that of a control viral capsid. In some embodiments, a viral capsid comprising a modified viral capsid protein described herein coupled to a first and second member of a binding pair linked to a targeting ligand exhibits a transduction efficiency that is at least 100 times higher than that of a control viral capsid. In some embodiments, viral particles of the invention comprising a viral capsid protein comprising an amino acid sequence of a capsid protein of a non-primate AAV, a distantly related AAV, or a combination thereof, and optionally a first and second member of a binding pair (e.g., in which the second member is operably linked to a targeting ligand and comprises a multispecific binding protein, etc.), may be better able to avoid 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 a viral capsid protein of the invention that comprises an amino acid sequence of a capsid protein of a non-primate AAV, a distantly related AAV, or a combination thereof), and optionally comprising a first and second member of a binding pair (e.g., in which the second member is operably linked to a targeting ligand and comprises a multispecific binding protein, etc.).In some embodiments, viral particles of the invention comprising a viral capsid protein comprising an amino acid sequence of a capsid protein of a non-primate AAV, a distantly related AAV, or a combination thereof, require at least two-fold more total IVIG or IgG for neutralization (e.g., 50% or greater inhibition of infection) compared to a suitable control viral particle (e.g., a viral particle of the invention has an IC50 value at least twice that of the control viral particle).

[0091] In some embodiments of the present 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 expressed by a target cell. Thus, the 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 targets the virus particle. Such "targeting" or "directing" may include a scenario in which a wild-type virus particle targets a tissue and / or some cells in some organs within an organism, and the insertion of a detectable label reduces or abolishes the broad targeting of tissues or organs, and retargeting to more specific cells in a tissue or more specific organ within an organism is achieved with the multispecific binding molecule. Such retargeting or redirection may also include scenarios where wild-type viral particles target a tissue, tissue targeting is reduced or abolished by insertion of a detectable label, and retargeting to an entirely different tissue is achieved with the multispecific binding molecule. The antibody paratopes described herein generally comprise, at a minimum, a complementarity determining region (CDR) that specifically recognizes a detectable label, e.g., the CDR3 region of the heavy and / or light chain variable domain. In some embodiments, the multispecific binding molecule comprises an antibody (or a portion thereof) that comprises an antibody paratope that specifically binds to a detectable label. For example, the multispecific binding molecule may comprise a single domain heavy chain variable region or a single domain light chain variable region, in which case the single domain heavy chain variable region or the single domain light chain variable region comprises an antibody paratope that specifically binds to a detectable label. In some embodiments, the multispecific binding molecule may comprise an Fv region, e.g., the multispecific binding molecule may comprise an scFv that comprises an antibody paratope that specifically binds to a detectable label. In some embodiments, the multispecific binding molecules described herein comprise an antibody paratope that specifically binds to c-myc (SEQ ID NO: 246).

[0092] One embodiment of the present invention is a multimeric structure comprising the modified viral capsid protein 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 the first member of the specific binding pair described herein. They can form normal viral capsids (empty viral particles) or viral particles (capsids encapsidating the nucleotide of interest). The formation of viral particles containing the viral genome is a highly favorable property for the use of the modified viral capsids described herein.

[0093] A further embodiment of the present invention is the use of at least one modified viral capsid protein and / or a nucleic acid encoding same, preferably at least one multimeric structure (e.g. a viral particle) for the manufacture and for use in the introduction of a nucleotide of interest into a target cell.

[0094] 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 a target cell. In general, packaging a nucleotide of interest involves replacing the AAV genome with the gene of interest between the AAV ITR sequences to create a transfer plasmid, which is then packaged in an AAV capsid according to known methods. Thus, the modified viral capsids described herein may package a transfer plasmid and / or a nucleotide of interest, which generally may include 5' and 3' inverted repeat (ITR) sequences adjacent to the gene of interest, such as a reporter gene or a therapeutic gene, or a portion of the gene of interest (which may be under the control of a viral or non-viral promoter). According to known methods of packaging AAV viral particles, the modified viral capsid, 5'ITR, and 3'ITR do not need to be of the same AAV serotype. In one embodiment, a transfer plasmid and / or nucleotide of interest comprises, from 5' to 3', a 5' ITR, a promoter, a gene (eg, a reporter and / or therapeutic gene), and a 3' ITR.

[0095] AAV transfer plasmid design consideration is that wild-type AAV genome is about 4.7kb.Therefore, well-known strategies are included herein that provide packaging of target nucleotides that exceed the packaging capacity of individual AAV.Such strategies include, but are not limited to, dual vector strategies that utilize ITR-mediated recombination to express a target gene that is larger than wild-type AAV genome by transcription splicing across the intermolecular recombination ITR from two complementary vector genomes, vector recombination by homology, RNA trans-splicing, and / or protein "trans-splicing" via split intein design. See, e.g., Nakai, H. et al. (2000) Nat. Biotechnol. 18:527-532, Sun, L. (2000) Nat. Med. 6:599-602 (2000), Ghosh, A., et al. (2008) Mol. Ther. 16:124-130 (2008), Lai, Y (2005) Nat. Biotechnol. 23:1435-1439, Chew, W Let al. (2016) Nat. Methods 13:868-874, Li, J. (2008) Hum. Gene Ther. 19:958-964, each of which is incorporated herein by reference in its entirety.

[0096] Dual AAV vector strategies for introducing large genes into target cells have been described that rely on a variety of mechanisms, including but not limited to trans-splicing involving overlapping regions in the dual vector, and hybrids of the two. See also Tornabene and Trapani (2020) Human Gene Ther. 31:47-56; U.S. Patent No. 8,236,557, each of which is incorporated herein by reference in its entirety.

[0097] The trans-splicing approach exploits the ability of AAV ITR sequences to concatenate and reconstitute a full-length genome, with each of two or more viral capsids enclosing one of two or more transfer plasmids, each of which contains a portion of a gene of interest. For example, in a dual vector approach, two transfer plasmids can be designed as follows: a 5'-transfer plasmid contains a promoter, a 5' portion of the coding sequence of the gene of interest, and a splicing donor (SD) signal, and a 3'-transfer plasmid contains a splicing acceptor (SA) signal, a 3' portion of the gene of interest, and a polyA signal. Upon tail-to-head ITR-mediated concatenation of the two AAV genomes, the SD and SA signals allow splicing of the recombinant genome.

[0098] Large genes of interest are also split when taking the overlapping region approach, in which the 5' and 3' portions (hence the 5' and 3' transfer plasmids) share recombinogenic sequences, e.g., homologous regions, e.g., each portion contains overlapping sequences. The gene of interest is made entirely in the target cell through homologous recombination mediated by the recombinogenic sequences, e.g., homologous / overlapping regions.

[0099] In the hybrid approach, the 5'-transfer plasmid and the 3'-transfer plasmid each contain highly recombinogenic sequences, which are located downstream of the SD signal in the 5' portion of the coding sequence of the gene of interest and upstream of the SA signal in the 3' portion of the coding sequence of the gene of interest. In this hybrid system, the gene of interest can be completed either via ITR-mediated concatemerization and splicing and / or by homologous recombination.

[0100] Trans-splicing at the RNA or protein level may also be utilized. In the RNA trans-splicing approach, two transfer plasmids may encode the 5' and 3' fragments, respectively, of the pre-mRNA of a large gene and share an intron hybridization domain that can favor trans-splicing to join the two half-transcripts into a complete full-length mRNA.

[0101] Protein trans-splicing occurs post-translationally and is catalyzed by an intervening protein called a split intein. Split inteins are expressed as two independent polypeptides (N-intein and C-intein) at the termini of two host proteins. The N-intein and C-intein polypeptides remain catalytically inactive until they encounter each other. Upon encountering each other, each intein precisely excises itself from the host protein while mediating the ligation of the N-host polypeptide and the C-host polypeptide via a peptide bond. The use of split inteins has been used for AAV-based delivery of therapeutic genes of interest in muscle, liver, and retinal diseases. For example, simultaneous delivery of two halves of a mini-dystrophin cDNA fused to the N- and C-intein coding sequences showed efficient production of two polypeptides. Li et al.(2008)Hum Gene Ther 19:958-64. Similarly, AAV split inteins have been widely used for expression and ligation of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 nucleases.

[0102] The above dual vector approach is well known in the art. See, for example, Tornabene and Trapani (2020), U.S. Pat. No. 8,236,557, supra. Thus, in some embodiments, the modified viral capsid described herein encapsulates a nucleotide of interest, which comprises a portion of a gene of interest. In some embodiments, the nucleotide of interest comprising a portion of a gene of interest further comprises a splicing donor signal or a splicing acceptor signal and / or a recombination-inducing sequence. In some embodiments, the nucleotide of interest comprising a portion of a gene of interest comprises an intron hybridization domain coding sequence. In some embodiments, the nucleotide of interest comprising a portion of a gene of interest comprises an N-intein or C-intein coding sequence.

[0103] The design of the transfer plasmid / nucleotide of interest includes including one or more regulatory elements, such as promoters and / or enhancer elements, that control the expression of the gene of interest. Non-limiting examples of useful promoters include, for example, the cytomegalovirus (CMV) promoter, 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, and the phosphoglycerate kinase (PGK) promoter. Internal enhancers may also be present in the viral construct to increase the expression of the gene of interest. For example, the CMV enhancer (Karasuyama et al. 1989. J. Exp. Med. 169:13, incorporated herein by reference in its entirety) may be used. In some embodiments, the CMV enhancer may be used in combination with the chicken β-actin promoter. In some embodiments, tissue-specific regulatory elements, such as muscle-specific promoters and / or regulatory elements, may be used to drive the expression of the gene of interest. For example, the use of muscle specific regulatory elements based on the muscle creatine kinase gene has been used in muscle gene therapy treatments such as Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophy (LGMD). See, e.g., Salva, MZ et al. (2007) Mol. Ther. 15:320-329, which is incorporated herein by reference in its entirety. In some embodiments, the transfer plasmid and / or nucleotide of interest herein comprises an enhancer and / or promoter of muscle creatine kinase (MCK), which drives expression of a gene of interest. In some embodiments, the transfer plasmid and / or nucleotide of interest herein comprises an enhancer and / or promoter element that recruits RNA polymerase II, which drives expression of a gene of interest.In some embodiments, the transfer plasmid and / or nucleotide of interest herein comprises an enhancer and / or promoter element that recruits RNA polymerase III, and the enhancer and / or promoter of MCK drives expression of the gene of interest.

[0104] In some embodiments, bidirectional promoter vectors have also been used to deliver dual therapeutic gene cassettes. One example of this is the bidirectional chicken β-actin ubiquitous promoter driving simultaneous expression of the hexosaminidase α and β subunits of the HexA enzyme, two respective genes involved in Tay-Sachs and Sandhoff diseases. Lahey, et al. (2020) Mol. Ther. 28:2150-2160, which is incorporated herein by reference in its entirety. In some embodiments, the transfer plasmids and / or nucleotides of interest herein comprise a bidirectional promoter, which drives the expression of two different genes of interest.

[0105] A variety of reporter genes (or detectable moieties) can be encapsidated into the multimeric structures comprising the modified viral capsid proteins described herein. Exemplary reporter genes include, for example, β-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. The methods described herein demonstrate the construction of targeted particles employing the use of a reporter gene encoding green fluorescent protein. However, upon reading this disclosure, one of skill in the art will understand that the viral capsids described herein can be made in the absence of a reporter gene or can be made using any reporter gene known in the art.

[0106] A variety of therapeutic genes can also be encapsidated into multimeric structures comprising modified viral capsid proteins as 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 RNA, siRNA, shRNA, etc.

[0107] A further embodiment of the 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).

[0108] 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 particular 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.; and isolating the expressed capsid protein of step a).

[0109] In some embodiments, the compositions described herein include, 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.

[0110] In some embodiments, the ratio of VP protein subunits in the mosaic viral particle stoichiometrically reflects, but is not necessarily, the ratio of modified cap genes:reference cap genes. As a non-limiting exemplary embodiment, the mosaic capsid formed according to the present method may be considered to have a ratio of modified capsid protein:reference capsid protein similar to, but not necessarily, the ratio (wt:wt) of the nucleic acids encoding the same used to make the mosaic capsid. In some embodiments, the mosaic capsid comprises a protein subunit ratio of about 1:59 to about 59:1.

[0111] A further embodiment of the invention is a method for altering 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 for the production of viral particles, wherein the packaging cells contain the nucleic acid. A further embodiment of the invention is a method for displaying a targeting ligand on the surface of a capsid protein, the method comprising the steps of: (a) expressing under suitable conditions a nucleic acid encoding a modified viral capsid protein as described herein (optionally together with nucleotides encoding a reference capsid protein), where 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, where the second cognate member of the specific binding pair is fused to the targeting ligand.

[0112] In some embodiments, the packaging cells further comprise a helper plasmid and / or a transfer plasmid comprising the nucleotide of interest. In some embodiments, the method further comprises isolating 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).

[0113] Packaging cells useful for producing the viral particles described herein include, for example, animal cells that are permissive for the virus, or cells that have been 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 vectors 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 numbers CRL9618, CCL61, CRL9096), HeLa cells (e.g., ATCC number 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.

[0114] 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).

[0115] 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.

[0116] Further embodiments include methods comprising contacting a modified Cap protein described herein with a targeting vector via chemical binding and / or association of a first and second member of a specific binding pair under conditions sufficient to operably link the modified Cap protein with the targeting vector, e.g., conditions sufficient to promote association of the targeting vector to the modified Cap protein, where the first member is inserted into the modified Cap protein and the first member and the targeting vector are fused to the second member of the specific binding pair.

[0117] 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. In other embodiments, the target cell is in vivo in a subject, e.g., a human.

[0118] target cell The modified viral particles disclosed herein may be used to target a wide variety of cells for delivery of a nucleotide of interest. The target cell will generally be selected based on the nucleotide of interest and the desired effect.

[0119] In some embodiments, the nucleotide of interest can be delivered to enable the target cells to produce a protein that compensates for an organism's deficiency, 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 area where the protein is most beneficial are targeted.

[0120] In other embodiments, the nucleotide of interest, such as the gene encoding siRNA, can inhibit the expression of a particular gene in target cells.The nucleotide of interest can, for example, inhibit the expression of a gene involved in pathogen life cycle.Thus, 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.

[0121] In other embodiments, the nucleotide of interest may code for a toxic protein that kills cells expressing the toxic protein, in which case tumor cells or other unwanted cells may be targeted.

[0122] In yet another embodiment, the nucleotide of interest encodes a therapeutic protein.

[0123] Once a particular population of target cells in which expression of the nucleotide of interest is desired is identified, a target receptor is selected that is specifically expressed on that population of target cells. The target receptor may be expressed only on that population of cells, or to a greater extent on that population of cells than on 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, to introduce a toxic gene, high specificity is most preferred to avoid killing non-target 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.

[0124] As discussed above, the target receptor can be any receptor for which a targeting ligand can be identified or created. 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.

[0125] Thus, target cells can be selected based on a variety of factors including, for example, (1) the use (e.g., therapy, expression of harvested proteins, and conferring disease resistance), and (2) expression of a marker with a desired amount of specificity.

[0126] The target cells are not limited in any way and include both germline cells and cell lines, and somatic cells and cell lines. When the target cells are germline cells, the target cells are preferably selected from the group consisting of single cell embryos and embryonic stem cells (ES).

[0127] 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.

[0128] Also disclosed herein are pharmaceutical compositions comprising the viral particles described herein and a pharma- ceutically acceptable carrier and / or excipient. Additionally, disclosed herein are pharmaceutical dosage forms comprising the viral particles described herein.

[0129] 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.

[0130] 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 direct administration to a tumor.

[0131] The pharmaceutical composition may be formulated for various modes of administration, including systemic, topical, or localized administration. Techniques and formulations can be found, for example, in Remrnington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the pharmaceutical composition may be formulated in a liquid solution, preferably in a physiologically compatible buffer, such as Hank's solution or Ringer's solution. In addition, the pharmaceutical composition may be formulated in solid form and redissolved or suspended immediately before use. Lyophilized forms of the pharmaceutical composition are also suitable.

[0132] For oral administration, the pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharma- ceutically acceptable excipients, such as binders (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose), fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, or silica), disintegrants (e.g., potato starch or sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulfate). 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 for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means using pharma- ceutically 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 may also contain buffer salts, flavoring, coloring, and sweetening agents, as appropriate.

[0133] The pharmaceutical composition can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, for example, in ampoules or multi-dose containers, optionally with the addition of a preservative. The pharmaceutical composition can further be formulated as a suspension, solution, or emulsion in an oily or aqueous vehicle, and can contain other agents, including suspending, stabilizing, and / or dispersing agents.

[0134] 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 compounds can be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as poorly soluble derivatives, e.g., as poorly soluble salts. 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 include 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.

[0135] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate for the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents can be used to enhance penetration. Transmucosal administration can be performed using intranasal sprays or suppositories. For topical administration, the viral particles described herein can be formulated into ointments, salves, gels, or creams, as generally known in the art. Washing solutions can also be used locally to treat wounds or inflammation to accelerate healing.

[0136] Pharmaceutical forms suitable for injectable use may include sterile aqueous solutions or dispersions; formulations including 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 forms must be sterile and fluid. They must also be stable under the conditions of manufacture and under certain storage parameters (e.g., cooling and freezing), and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0137] When the formulations disclosed herein are used as therapeutic agents to promote immune responses in subjects, the therapeutic agents can be formulated into compositions in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, and the like. Salts formed with 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, procaine, and the like.

[0138] 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 the injectable compositions can be brought about by the use of agents that delay absorption in the composition, for example, aluminum monostearate and gelatin.

[0139] 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.

[0140] 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, and the like, can also be used.

[0141] 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, a 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.

[0142] 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 for a period of time, or monthly, semi-yearly, or annually, depending on need or exposure to pathogenic microorganisms or to the subject's condition (e.g., cancer).

[0143] In addition to compounds formulated for parenteral administration, such as intravenous, intratumoral, subcutaneous, or intramuscular injection, other pharma- ceutically acceptable forms include, for example, tablets or other solids for oral administration, liposomal formulations, sustained release capsules, biodegradable forms, and any other forms currently in use.

[0144] Also, intranasal or inhalable solutions or sprays, aerosols, or inhalants may 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 resemble in many respects nasal secretions. Thus, aqueous nasal solutions are usually isotonic and slightly buffered to maintain a pH of 5.5 to 7.5. In addition, antimicrobial preservatives similar to those used in ophthalmic preparations and appropriate drug stabilizers may be included in the formulation, if necessary. A variety of commercially available nasal preparations are known and may contain, for example, antibiotics and antihistamines and are used for asthma prophylaxis.

[0145] Oral formulations may contain excipients such as pharmaceutical grades of 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 inert diluents or assimilable edible carriers, or may be enclosed in hard or soft shell gelatin capsules, or may be compressed into tablets, or may be directly incorporated with dietary foods. For oral therapeutic administration, active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

[0146] Tablets, troches, pills, capsules and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch, or gelatin, excipients such as dicalcium phosphate, disintegrating agents such as corn starch, potato starch, 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, in addition to the above types of materials, a liquid carrier may be contained. 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.

[0147] 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, mini- or microtubes, test tubes, flasks, bottles, syringes, or other containers. When additional components or agents are provided, the kits may contain one or more additional containers into which the agents or components may be placed. The kits herein will also typically include a means for containing the viral particles and any other reagent containers in sealed containment for commercial sale. Such containers may include syringes or blow-molded plastic containers in which the desired vials are held. Optionally, the compositions described may require one or more additional active agents, such as, for example, anti-inflammatory agents, anti-viral agents, anti-fungal or anti-bacterial agents, or anti-tumor agents.

[0148] The compositions disclosed herein may be administered by any means known in the art. For example, the compositions may include administering to a subject intravenously, intratumorally, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreously, 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.

[0149] 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 in large surface area flasks and grown to near confluence, and viral particles can be purified. Cells can be harvested and the viral particles released into 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 and then diafiltered. The resulting concentrated bulk can be formulated by diluting with a buffer containing a stabilizing agent, filling into vials, and lyophilizing. The compositions and formulations can be stored for later use. For use, the lyophilized viral particles can be reconstituted by adding a diluent.

[0150] Certain additional agents used in combination therapy can be formulated and administered by any means known in the art.

[0151] The compositions disclosed herein may also include adjuvants such as aluminum salts and other mineral adjuvants, tensoactive agents, bacterial derivatives, vehicles, and cytokines. The adjuvants may also have antagonistic immunomodulatory properties. For example, the adjuvants may stimulate Th1 or Th2 immunity. The compositions and methods disclosed herein may also include adjuvant therapy.

[0152] Skeletal muscle-related disorders Also provided herein are methods of treating skeletal muscle-related disorders, e.g., muscle wasting diseases and / or genetic muscle diseases, e.g., X-linked myotubular myopathy (XLMTM), Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM1), facioscapulohumeral muscular dystrophy type 1 (FSHD), congenital muscular dystrophy type 1A (MDC1A), limb-girdle muscular dystrophy, dystroglycanopathies, muscle wasting conditions, metabolic diseases, etc. Generally, such methods comprise administering to a patient suffering from or at risk for such a skeletal-related disorder, a viral particle or pharmaceutical composition as described herein, wherein the viral particle is (i) a viral capsid modified to contain a first member of a protein:protein binding pair; (ii) a second member of the protein:protein binding pair, the second member of the protein:protein binding pair comprising a targeting ligand that binds to a muscle-specific surface protein (e.g., CACNG1) expressed on the surface of a muscle cell; a first member of a protein:protein binding pair and a second member of the protein:protein binding pair that associate to target the viral capsid to muscle cells of the patient; and (iii) a nucleotide of interest encapsidated within the viral capsid.

[0153] In some embodiments, the nucleotide of interest encodes a therapeutic protein, a suicide gene, an antibody or fragment thereof, a CRISPR / Cas system or a portion thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule. For example, in some embodiments, the nucleotide of interest may encode a growth factor, a neurotrophic factor, a disease-modifying muscle protein, a metabolic protein, for example, for a muscle wasting condition or a metabolic disease. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 3-1] [Table 3-2] EXAMPLES

[0154] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0155] method The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.

[0156] Preparation of AAV viral vectors Viruses were generated by transfecting 293T cell packaging cells using PEI Pro with the following plasmids: pAd helper, AAV2 ITR-containing genomic plasmid encoding a reporter protein, and pAAV-CAP plasmid encoding the AAV Rep and Cap genes, with or without additional plasmids encoding either the heavy or light chains of the antibody. All antibody heavy chain constructs are fused at their C-terminus to SpyCatcher as described in WO2019 / 006046, which is incorporated herein by reference in its entirety. Transfection complexes were prepared in incomplete DMEM (without additional supplements) and incubated at room temperature for 10 minutes.

[0157] Each virus was produced by transfecting a 15 cm plate of 293T packaging cells with the following plasmids and quantities: [Table 5-1] [Table 5-2] [Table 5-3]

[0158] CK8-uDys5 is described in US10479821B2, which is incorporated herein by reference in its entirety.

[0159] After incubation, the complexes are added to DMEM supplemented with 10% FBS, 1X NEAA, 1% Pen / Strep, and 1% L-glutamine.

[0160] The transfected packaging cells were incubated at 37°C for 3 days, and then virus was harvested from cell lysates using a standard freeze-thaw protocol. Briefly, packaging cells were lifted and pelleted by scraping. 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 successive 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 60 min 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 to be tested in vitro, the filtered lysate is added directly to an Amicon Ultra-15 centrifugal filter unit equipped with an Ultracel-100 membrane (100 KDa MWCO) filter cartridge. The filter unit is centrifuged at 5-10 min intervals until the desired volume is reached in the upper chamber, and then the concentrated crude virus is pipetted into a low protein binding tube and stored at 4 °C. For viruses to be tested in vivo, the clarified lysate is further purified using a four-step iodixanol density gradient. The gradient is loaded into a Beckman 70Ti rotor and spun at 66,100 rpm, 10 °C, using maximum acceleration and deceleration for 1.5 h. After ultracentrifugation, iodixanol-purified virions are extracted from the 40-60% interface. The AAV in iodixanol solution is diluted with DPBS + / - .001% Pluronic® F68 such that the concentration of iodixanol is less than 1%. The purified virus is then concentrated to the desired volume using a 100 kDa MWCO Amicon ultrafiltration unit.

[0161] Titers (viral genomes per milliliter or vg / mL) were determined by qPCR using a standard curve of known concentrations of virus.

[0162] Cell line: All 293 cell lines were maintained in DMEM supplemented with 10% FBS, 1XNEAA, 1% Pen / Strep, and 1% L-glutamine. 293 hASGR1 / 2 and 293hCACNG1 cell lines were generated by lentiviral transduction of parental 293 cell lines with vectors expressing the corresponding cDNAs. All cell lines were obtained from the Regeneron TC core facility.

[0163] Human skeletal myoblasts were purchased from Cook Myosite (SkMDC; Lot#P01059-14M), maintained in MyoTonic basal medium (MB-2222) supplemented with MyoTonic Growth Supplement (MS-3333), and grown in a 37°C incubator containing 5% CO2.

[0164] C2C12 mouse myoblast cells were purchased from ATCC, maintained in DMEM containing 10% FBS and penicillin-streptomycin supplement, and grown in a 37° C. incubator with 5% CO 2 .

[0165] AAV capsid protein constructs GeneBlocks encoding the desired SpyTag insert, flanking linker amino acids, and additional mutations were purchased from IDT and cloned into BsiWI- and XcmI-digested pAAV9-CAP wt using Gibson Assembly according to the manufacturer's protocol (NEB).

[0166] Cloning SpyCatcher into antibodies GeneBlocks encoding the antibody heavy chain variable region were purchased from IDT and cloned into the CMV hIgG4US Sap1 SpyCatcher backbone using Gibson assembly.

[0167] 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. Three days post-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.

[0168] Cell infection / transduction and luciferase assay readout To infect cells, viral particles were added directly to the medium of cells in culture and the mixture was incubated at 37° C. Three days after infection, the medium was removed and the cells were lysed using Promega Glo lysis buffer. Luciferin substrate (Promega Bright Glo kit) was added to the lysed cells and luminescence was measured using a luminometer.

[0169] Myotube transduction Human skeletal myoblasts were seeded at 8500 cells / well in collagen-coated 96-well plates with clear bottom and black walls. After 24 hours, the growth medium was changed to MyoTonic Differentiation Media (MD-5555) and replaced every 2 days. After 4 days of differentiation, myotubes were formed and differentiation medium supplemented with virus preparations was added to each well. After 3 days, myotubes were fixed with 4% PFA for subsequent GFP detection or lysed with Trizol for detection of uDys5 mRNA.

[0170] C2C12 cells were seeded at 10,000 cells / well in collagen-coated 96-well plates with clear bottom and black walls. After 24 hours, the growth medium was replaced with differentiation medium (DMEM with 2% horse serum) and placed in a 37°C incubator with 7.5% CO2. After 24 hours of differentiation, cells were transduced using virus preparations diluted in differentiation medium. After 3 days, myotubes were fixed with 4% PFA for subsequent GFP detection or lysed with Trizol for detection of uDys5 mRNA.

[0171] Myotube transduction analysis After fixation for 15 min at room temperature, cells were washed with PBS and then blocked with 20% goat serum in PBS containing 0.3% Triton®-X for 1 h at room temperature. Primary antibody against myosin heavy chain (MF20-c, Developmental Studies Hybridoma Bank) was diluted 1:200 in blocking buffer and added to all wells during overnight incubation at 4°C. Wells were then gently washed three times with PBS and Alexa 647-conjugated anti-mouse secondary antibody was diluted 1:500 in blocking buffer and added to all wells for 1 h at room temperature. Cells were then washed with PBS and stained with DAPI before being imaged using an Axio Observer microscope (Ziess). GFP intensity within myosin heavy chain positive regions was analyzed using HALO software (Indica Labs). For analysis of transduction with uDys5, RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. RNA purification was completed according to the manufacturer's protocol and RNA quantity and quality were verified with a NanoDrop 2000. Equal amounts of RNA were reverse transcribed using SuperScript VILO cDNA synthesis MasterMix (ThermoFisher) and diluted 10X in nuclease-free water. Gene expression was assessed using Taqman primers / probes AGGGTAGCTAGCATGGAAAAACA (uDys5 fwd), GGGCTTGTGAGACATGAGTGAT (uDys5 rev), ATTTACATTCTTATGTGCCT (uDys5 probe) and endogenous controls for human and mouse Hprt: Hs02800695_m1 and Mm03024075_m1, respectively (Thermo Fisher).

[0172] Mouse strains Humanized CACNG1 mice (CACNG1 hu / hu) were generated by replacing the coding exon 1, intron 1, coding exons 2-4 (and intervening intron), and 82 bp of the 3' untranslated region (UTR) of mouse Cacng1 with the orthologous partial coding exon 1 sequence, intron 1, coding exons 2-4 (and intervening intron), complete 3'UTR, and an additional 158 bp after the 3'UTR of human CACNG1. Humanized ASGR1 mice were generated according to the method described in WO / 2019 / 006034. Strain-matched (50500) mice were used as controls and bred in-house. Wild-type C57BL / 6, DBA / 2J, and dystrophic D2-mdx mice (MDX) were purchased from Jackson Laboratory (stock numbers 000664, 000671, and 013141, respectively).

[0173] In vivo analysis of AAV2 luciferase vectors Male 50500 and humanized CACNG1 mice ranging in age from 3 to 4 months were injected intravenously via the tail vein with PBS or 5E11 vg of either wild-type AAV2, AAV2 HBM anti-ASGR1, or AAV2 HBM anti-CACNG1 mAb#1 carrying a luciferase reporter. Five weeks after IV injection, mice were anesthetized using isoflurane, injected with luciferin substrate, and euthanized 7 to 10 minutes later. Liver, tongue, diaphragm, and quadriceps were harvested and imaged ex vivo using an IVIS Spectrum in vivo imaging system. Raw data was analyzed using living image software to determine mean radiance (photons / sec / cm2 / sr).

[0174] In vivo analysis of AAV9 luciferase vectors Ten-week-old female humanized CACNG1 and humanized ASGR1 mice were injected intravenously via the tail vein with either PBS or 5E10vg of wild-type AAV9, AAV9 N272A, AAV9 N272A anti-ASGR1 mAb, AAV9 anti-ASGR1 Fab, AAV9 N272A anti-CACNG1 mAb#1, or AAV9 N272A anti-CACNG1 Fab carrying a luciferase reporter. Three weeks after injection, mice were anesthetized using isoflurane, injected with luciferin substrate, and euthanized 7–10 min later. Livers, hind limbs, quadriceps, and tongue were harvested and imaged ex vivo using an IVIS Spectrum in vivo imaging system. Raw data were analyzed using living image software to determine mean radiance (photons / sec / cm2 / sr).

[0175] In vivo analysis of AAV9 GFP vectors Adult (3-7 months old) male CACNG1 hu / hu , WT C57BL / 6, and D2-mdx mice were tail vein injected with 1E +11vg / mouse of AAV9 (WT AAV9, AAV9 N272A, AAV9 N272A anti-ASGR1 mAb, and AAV9 N272A anti-CACNG1 mAb "#1" and "#2", and "#3"). Mice were sacrificed 3 weeks after injection and the following organs were harvested for immunohistochemistry: liver, spleen, heart, tongue, diaphragm, quadriceps, gastrocnemius / plantaris / soleus complex, and tibialis anterior.

[0176] For pooled AAV characterization experiments in cynomolgus monkeys, control AAV and AAV9 variants conjugated to the indicated antibodies were generated individually using the methods described above, but with the barcoded pITR-CAG-GFP-hGHpA plasmid as the viral genome plasmid, and each of the 12 viruses present in the pool was packaged with a version of pITR-CAG-GFP-hGHpA carrying a unique 32 nucleotide long barcode used to quantify transgene expression by the capsid variant. Two male cynomolgus macaques were given an intravenous bolus injection of the pooled virus mixture at 3E+13vg / kg. Two weeks after injection, the animals were euthanized and a series of tissues and organs were harvested for barcode analysis. See, e.g., WO2018 / 144813, Stoeckius et al. (2018) Genome Biol. 19:224, Stoeckius et al. (2017) Nat. Method 9:2579-10, each of which is incorporated by reference in its entirety. Table 4 provides barcode numbers (BC#) associated with various viral particles (e.g., AAV9 cap mutations and corresponding antibody numbers, if applicable). [Table 4-1] [Table 4-2]

[0177] To assess liver health and serum readouts of complement activation (ALT, AST, Bb, and C3a) following administration of wild-type and retargeted AAV, AAV9 and AAV9 N272A anti-CACNG1 mAb#3 were generated as described above and packaged with pAAV CAG eGFP. Male cynomolgus macaques were given an intravenous bolus injection of either 3E+13vg / kg AAV9 wt (2 animals) or AAV9 N272A anti-CACNG1 mAb#3 (2 animals) or saline (1 animal) as a control. Serum readouts of ALT, AST, Bb, and C3a were collected at baseline (10 days prior to administration) and 30 minutes, 6 hours, 24 hours, and 48 hours after administration. Two weeks after injection, animals were euthanized and a series of tissues and organs were harvested for analysis.

[0178] For immunohistochemical staining, tissues were fixed in neutral buffered formalin solution and transferred to 70% ethanol after 24 hours. Organs were stored in 70% ethanol at room temperature prior to paraffin embedding. Tissues were sectioned at 5 μm thickness using standardized planar sectioning. Anti-GFP IHC staining for eGFP expression was performed using a Benchmark ULTRA Ventana IHC / ISH system. Image analysis was performed using HALO analysis software.

[0179] For immunofluorescence staining, muscle tissues were immersed in OCT embedding medium and frozen in liquid nitrogen-cooled isopentane. Tissues were cryosectioned at 12 μm thickness, then fixed in 4% PFA and stained with laminin (Sigma-Aldrich), followed by Alexa 647-conjugated anti-rabbit secondary antibody and DAPI (Thermo Fisher Scientific). Slides were mounted with Fluoromount (Thermo Fisher Scientific) and imaged with an Axioscan slide scanner (Zeiss).

[0180] For barcode analysis, total RNA isolated from cynomolgus monkey tissues and organs was purified using the MagMAX-96 Total RNA Isolation Kit for Microarrays according to the manufacturer's specifications. RNA was then treated with Turbo DNase and cDNA synthesis was performed using SuperScript IV reverse transcriptase and a hGH pA specific primer (5'-GTCATGCATGCCTGGAATC-3', SEQ ID NO: 256). Q5 High Fidelity PCR was used. Barcoded GFP transcripts were amplified from cDNA samples using primers binding upstream (5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCGAGCGCTGCTCGAGAG-3', SEQ ID NO: 257) and downstream (5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGGTCACAGGGATGCCAC-3', SEQ ID NO: 258) of the barcode using a 2x master mix. Pooled virus mixtures were included in the samples. Each sample was prepared in three technical replicates for the duration of the library preparation. Amplicons containing Illumina adapters and unique dual indexes (UDI-Illumina) were quantified using a qubit and tape station, pooled in equimolar ratios, and sequenced on a Nextseq550 using a high-throughput kit for 300 cycles.

[0181] In vivo analysis of AAV9 uDys5 vector Six-week-old male D2-mdx mice were tail vein injected with WT AAV9 expressing uDys5 under the CK8 promoter or AAV9 N272A anti-CACNG1 mAb #3 at 1E+12vg / mouse. Five weeks after injection, mice were sacrificed and the following organs were harvested for qPCR analysis and stored in RNAlater (Thermo Fisher): liver, heart, quadriceps, gastrocnemius, tibialis anterior, soleus, tongue, and diaphragm. Tissues were then homogenized in Trizol, and the aqueous phase was purified using the MagMAX-96 total RNA Isolation kit (Life Technologies), and gDNA was removed using the RNase-free Dnase Set (Qiagen). mRNA was reverse transcribed to cDNA using SuperScript VILO Master Mix (Life Technologies) and qPCR was performed using the following Taqman primers / probes: AGGGTAGCTAGCATGGAAAAACA (uDys5 fwd), GGGCTTGTGAGACATGAGTGAT (uDys5 rev), ATTTACATTCTTATGTGCCT (uDys5 probe), and endogenous control: AAGGCCGTGTGCTGATG (Rplp0 fwd), TCTCCAGAGCTGGGTTGTTCT (Rplp0 rev), AAGAACACCATGATGCGCAAGGC (probe).

[0182] For detection of uDys5 sarcolemmal localization, gastrocnemius and heart cryosections were fixed in 4% PFA and stained with dystrophin (Developmental Studies Hybridoma Bank) followed by Alexa 546-conjugated anti-mouse secondary antibody. Slides were mounted with Fluoromount (Thermo Fisher Scientific) and imaged on an Axioscan slide scanner (Zeiss).

[0183] For detection of uDys5 protein, quadriceps muscles were snap frozen in liquid nitrogen and then homogenized using the mouse leg muscle setting of a Fast Prep-24 5G homogenizer (MP Biomedicals). Muscles were then added to lysis matrix tubes (MP Biomedicals) containing lysis buffer (50 mM Tris HCl, 100 mM NaCl, 1 mM EDTA dihydrate, 1% Triton 100x) containing protease and phosphatase inhibitors (Sigma). After protein quantification using the BCA assay (Thermo Fisher Scientific), lysates were heated at 70°C for 10 min with sample buffer and reducing agent (Thermo Fisher Scientific). Equal amounts of protein were loaded and separated on 4–20% Tris-glycine gels (Bio-Rad) and then transferred to PVDF membranes (BioRad) using the TurboTransfer System (BioRad). Membranes were blocked with 5% non-fat milk (Cell Signaling) and then incubated overnight with the following primary antibodies: dystrophin (Developmental Studies Hybridoma Bank, MANHINGE1A), beta-actin (Abcam, ab8224). After washing, membranes were incubated with appropriate HRP-conjugated secondary antibodies (Cell Signaling). Membranes were incubated in ECL reagent (Cell Signaling) and visualized (Amersham Imager 600). Blot images were quantified using ImageJ.

[0184] To assess the effect of muscle transduction with uDys5 on circulating biomarkers of muscle damage, blood was collected in 1.1 mL Z-Gel microtubes (Sarstedt) before and 4 weeks after dosing and serum was allowed to clot for a minimum of 2 hours. Blood was centrifuged at 12,000 RPM for 10 minutes at 4°C. The supernatant was then collected and frozen at -80°C. Once all samples were collected, they were all thawed and then diluted 1:4 with deionized water. Serum was analyzed using the ADVIA® Chemistry Creatine Kinase (CK_L) Reagent (REF 10729780) on the ADVIA® Chemistry XPT system.

[0185] To evaluate the functional effects of muscle transduction with uDys5, 6-week-old male D2-mdx mice were tail vein injected with WT AAV9 expressing uDys5 under the CK8 promoter or AAV9 N272A anti-CACNG1 mAb #3 at 1E+12vg / mouse and maximal grip strength was assessed 12 weeks after injection. Forelimb grip strength was assessed using a computerized grip strength meter (Bioseb, BIO-GS3) by holding the mouse vertically by the tail and lowering the pull bar until it grasped. The mouse was then pulled slowly upwards until it lost grip strength. The peak force (grams) generated was recorded after five pulls, including a 3-minute rest between trials. After three trials, the maximum force across trials was averaged and recorded as the maximum grip strength.

[0186] Although the present invention has been specifically shown and described with reference to a number of embodiments, those skilled in the art will appreciate that changes in form and detail may be made to the various embodiments disclosed herein without departing from the spirit and scope of the present invention, and that the various embodiments disclosed herein are not intended to serve to limit the scope of the claims. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some suitable methods and materials are now described. All publications cited herein are incorporated by reference in their entirety. 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.

Claims

1. 1. A recombinant adeno-associated virus (AAV) particle comprising: (i) a modified AAV capsid protein; and (ii) a targeting ligand that binds to a mammalian muscle cell-specific surface protein; A recombinant AAV particle, wherein the modified AAV capsid protein is operably linked to the targeting ligand.

2. (a) the modified AAV capsid protein comprises a first member and a second member of a protein:protein binding pair; (b) the second member of the protein:protein binding pair comprises the targeting ligand that binds to the mammalian muscle cell-specific surface protein; The recombinant AAV particle of claim 1, wherein the first member of the protein:protein binding pair and the second member of the protein:protein binding pair are associated to direct the tropism of the AAV particle toward the mammalian muscle cell.

3. The recombinant AAV particle of claim 1, wherein the mammalian muscle cell-specific surface protein is a human muscle cell-specific surface protein.

4. The recombinant AAV particle of claim 1, wherein the mammalian muscle cell is a mammalian skeletal muscle cell.

5. 2. The recombinant AAV particle of claim 1, comprising the modified AAV capsid protein bound to the mammalian muscle cell-specific surface protein expressed on the surface of the mammalian muscle cell.

6. The mammalian muscle cell-specific surface protein, wherein the mammalian muscle cell-specific surface protein is a human muscle cell-specific surface protein; The recombinant AAV particle of claim 5, wherein the mammalian muscle cells are non-human animal muscle cells, rodent muscle cells, rat muscle cells and / or mouse muscle cells, and the mammalian muscle cells have been genetically modified to express the human muscle cell-specific surface protein.

7. The mammalian muscle cell-specific surface protein, wherein the mammalian muscle cell-specific surface protein is a human muscle cell-specific surface protein; The recombinant AAV particle of claim 5, wherein the mammalian muscle cell is a human muscle cell.

8. The recombinant AAV particle of claim 1 , wherein the recombinant AAV particle is in vitro.

9. The recombinant AAV particle of claim 1 , wherein the recombinant AAV particle is in vivo.

10. The recombinant AAV particle of claim 1, wherein the mammalian muscle cell-specific surface protein is mammalian calcium-gated accessory subunit gamma 1 (CACNG1).

11. The recombinant AAV particle of claim 10, wherein the mammalian muscle cell-specific surface protein is human CACNG1.

12. 12. The recombinant AAV particle of any one of claims 1 to 11, wherein the targeting ligand comprises a set of HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences contained within a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, and 178 / 186.

13. (a) the first member of the protein:protein binding pair comprises SpyTag, Isopeptag, SnoopTag, SpyTag002, SpyTag003, or a biologically active portion or variant thereof; (b) the second member of the protein:protein binding pair is (i) SpyCatcher, KTag, Pirin-C, SnoopCatcher, SpyCatcher002, SpyCatcher003, or a biologically active portion or variant thereof; and (ii) said targeting ligand binds to a mammalian muscle cell-specific surface protein; (c) the first member of the protein:protein binding pair and the second member of the protein:protein binding pair are linked by an isopeptide bond.

14. (a) the first member of the protein:protein binding pair comprises a SpyTag, or a biologically active portion or variant thereof; (b) the second member of the protein:protein binding pair comprises SpyCatcher, or a biologically active portion or variant thereof, fused to the targeting ligand that binds to the mammalian muscle cell-specific surface protein.

15. The recombinant AAV particle described in claim 2, wherein the modified AAV capsid protein comprises a first linker and / or a second linker that operably links the first member of the protein:protein binding pair to the modified AAV capsid protein.

16. The recombinant AAV particle of claim 15, wherein the first linker and the second linker are not identical.

17. The recombinant AAV particle of claim 15, wherein the first linker and the second linker are identical.

18. 16. The recombinant AAV particle of claim 15, wherein the first linker is 10 amino acids in length and / or the second linker is 10 amino acids in length.

19. the modified AAV capsid protein comprises a modified VP1 capsid protein, a modified VP2 capsid protein, and / or a modified VP3 capsid protein; the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein comprises an insertion of the first member of the protein:protein binding pair and / or comprises the targeting ligand; The recombinant AAV particle of claim 2, wherein the portion of the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein comprising the insertion of the first member of the protein:protein binding pair and / or comprising the targeting ligand further comprises an amino acid sequence that is at least 90% identical to the corresponding capsid protein of a wild-type AAV.

20. the modified VP1 capsid protein, the modified VP2 capsid protein, and / or the modified VP3 capsid protein, in addition to the insertion of the first member of the protein:protein binding pair and / or the targeting ligand; (i) amino acid substitutions, insertions, or deletions; (ii) a chimeric amino acid sequence, or 20. The recombinant AAV particle of claim 19, further comprising: (iii) a combination of (i) and (ii).

21. The recombinant AAV particle of claim 20, wherein the amino acid substitution, insertion, or deletion reduces the natural tropism of the AAV particle and / or creates a detectable label.

22. The recombinant AAV particle of claim 1, wherein the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, a non-primate AAV listed in Table 2, and any chimera thereof.

23. The recombinant AAV particle of claim 1, wherein the AAV is AAV2.

24. (a) the recombinant AAV particle comprises a modified AAV2 VP1 capsid protein comprising a first member of a protein:protein binding pair, wherein the first member of the protein:protein binding pair is (i) inserted at amino acid position 1-453 and / or 1-587, and / or (ii) linked to the AAV sequences via one or more linkers; and / or (b) the recombinant AAV particles are (i) the first member of the protein:protein binding pair inserted at amino acid position G453; (ii) the first member of the protein:protein binding pair linked to the AAV capsid protein via one or more linkers; and / or (iii) a mutation selected from R585A, R588A, R484A, R487A, and K532A, or any combination thereof a modified AAV2 VP1 capsid protein comprising: The recombinant AAV particle of claim 23.

25. The recombinant AAV particles are (a) (i) a first member of a protein:protein binding pair inserted at amino acid position 1-453 or 1-587; (ii) the first member of the protein:protein binding pair linked to the AAV capsid protein via one or more linkers; and (iii) a mutation selected from the group consisting of R585A, R588A, R484A, R487A, and K532A, or any combination thereof a first set of AAV2 VP1 capsid proteins comprising: (b) a second set of AAV2 VP1 capsid proteins that are identical to said first set of AAV2 VP1 capsid proteins except that they lack said first member of said protein:protein binding pair. A mosaic AAV capsid comprising: The recombinant AAV particle of claim 24.

26. The recombinant AAV particle of claim 1, wherein the AAV is AAV9.

27. (a) the recombinant AAV particle comprises a modified AAV9 VP1 capsid protein comprising a first member of a protein:protein binding pair, wherein the first member of the protein:protein binding pair is (i) inserted at amino acid position 1-453 or 1-589; and / or (ii) linked to an AAV capsid protein via one or more linkers; and / or (b) the recombinant AAV particles are (i) a first member of a protein:protein binding pair inserted at amino acid position G453; (ii) a first member of a protein:protein binding pair linked to the AAV capsid protein via one or more linkers; and / or (iii) a mutation selected from the group consisting of N272A, and W503A, or a combination thereof a modified AAV9 VP1 capsid protein comprising: The recombinant AAV particle of claim 26.

28. The recombinant AAV particles are (a) (i) a first member of a protein:protein binding pair inserted at amino acid position 1-453 or 1-587; (ii) the first member of the protein:protein binding pair linked to the AAV capsid protein via one or more linkers; and (iii) a mutation selected from the group consisting of N272A, and W503A, or a combination thereof a first set of AAV9 VP1 capsid proteins comprising: (b) a second set of AAV9 VP1 capsid proteins that are identical to said first set of AAV9 VP1 capsid proteins except that they lack said first member of said protein:protein binding pair. A mosaic AAV capsid comprising:

28. The recombinant AAV particle of claim 27.

29. The recombinant AAV particle of claim 1, wherein the AAV is a non-primate AAV selected from avian AAV (AAAV), non-primate mammalian AAV, or squamate AAV. (a) The non-primate AAV is an AAAV, and the recombinant AAV particle is (i) a first member of a protein:protein binding pair inserted at amino acid position 1-444 or 1-580, and / or (ii) a first member of a protein:protein binding pair linked to the AAV capsid protein via one or more linkers; a modified AAAV VP1 capsid protein comprising: (b) the non-primate AAV is a Squamate AAV, the Squamate AAV is a bearded dragon AAV, and the recombinant AAV particles are (i) a first member of a protein:protein binding pair inserted at amino acid position 1-573 or 1-436, and / or (ii) a first member of a protein:protein binding pair linked to the AAV capsid protein via one or more linkers; and / or (c) the non-primate AAV is a non-primate mammalian AAV, the non-primate mammalian AAV is a sea lion AAV, and the recombinant AAV particles are (i) a first member of a protein:protein binding pair inserted at an amino acid position 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; and / or (ii) a first member of a protein:protein binding pair linked to the AAV capsid protein via one or more linkers; a modified sea lion AAV VP1 capsid protein comprising:

30. The recombinant AAV particle of claim 29.

31. the recombinant AAV particles are mosaic AAV particles; (a) the mosaic AAV particle comprises (i) a first plurality of reference AAV capsid proteins, each of which is not associated with the targeting ligand, and (ii) a second plurality of AAV capsid proteins, each of which is associated with the targeting ligand; and / or (b) the mosaic AAV particles comprise a first plurality of reference AAV capsid proteins not associated with the targeting ligand and a second plurality of AAV capsid proteins associated with the targeting ligand in a ratio of 1:7; The recombinant AAV particle of claim 1.

32. The recombinant AAV particle of claim 1 , wherein the targeting ligand is an antibody or a portion thereof.

33. A recombinant AAV particle as described in claim 1, further comprising a nucleotide sequence encapsulated within an AAV capsid comprising the modified AAV capsid protein.

34. The recombinant AAV particle of claim 33, wherein the nucleotide sequence is a reporter gene.

35. The recombinant AAV particle of claim 34, wherein the reporter gene encodes a protein selected from the group consisting of 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, and combinations thereof.

36. The recombinant AAV particle of claim 33, wherein the nucleotide sequence encodes a therapeutic protein, a suicide gene, an antibody or fragment thereof, a CRISPR / Cas system or part thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule.

37. A pharmaceutical composition comprising: (a) the recombinant AAV particle of claim 33; and (b) a pharmaceutically acceptable carrier or excipient.

38. The pharmaceutical composition of claim 37 for delivering a nucleotide sequence to a mammalian muscle cell, wherein the mammalian muscle cell is contacted with the pharmaceutical composition; A pharmaceutical composition wherein said mammalian muscle cells express a mammalian muscle cell-specific surface protein.

39. 39. The pharmaceutical composition of claim 38, wherein said contacting is performed ex vivo.

40. 39. The pharmaceutical composition of claim 38, wherein said contacting is performed in a subject.

41. 41. The pharmaceutical composition of claim 40, wherein the subject is a primate and / or the subject is a human.

42. 39. The pharmaceutical composition of claim 38, wherein the mammalian muscle cells are mammalian skeletal muscle cells.

43. 39. The pharmaceutical composition of claim 38, wherein the mammalian muscle cell-specific surface protein is CACNG1.

44. 39. The pharmaceutical composition of claim 38, wherein the nucleotide sequence encodes a therapeutic protein, a suicide gene, an antibody or fragment thereof, a CRISPR / Cas system or part thereof, an antisense oligonucleotide, a ribozyme, an RNAi molecule, or an shRNA molecule.

45. 38. The pharmaceutical composition of claim 37 for treating muscle wasting or a genetic muscle disorder in a subject in need thereof, comprising: The recombinant AAV particles or the pharmaceutical composition are administered to the subject, The pharmaceutical composition, wherein the nucleotide sequence 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.

46. 38. Use of the pharmaceutical composition of claim 37 for the manufacture of a medicament for the treatment of muscle wasting or a genetic muscle disorder.

47. 47. The pharmaceutical composition of claim 45 or the use of claim 46, wherein the muscle wasting or genetic muscle disease is selected from the group consisting of X-linked myotubular myopathy (XLMTM), Duchenne muscular dystrophy (DMD), myotonic dystrophy (DM1), facioscapulohumeral muscular dystrophy type 1 (FSHD), congenital muscular dystrophy type 1A (MDC1A), limb-girdle muscular dystrophy, and dystroglycanopathy.

48. The pharmaceutical composition of claim 45 or the use of claim 46, wherein the administration of the recombinant AAV particles or the pharmaceutical composition to the subject does not result in an increase in the level of the liver enzyme or complement component that is more than three-fold higher than the corresponding level of the liver enzyme or complement component before the administration.

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