Covalently surface modified adeno-associated virus production by in vitro conjugation, and purification of covalently surface modified adeno-associated virus

Abstract

The present inventions provide covalently surface modified adeno-associated viruses can comprise genes of interest (GOIs) and advantageously can be targeted to certain cell and tissue types for preventative and therapeutic purposes. The covalently surface modified adeno-associated viruses also can be mutated to detarget certain tissues and organs, such as the liver. The present inventions further provide systems and methods for engineering adeno-associated virus (AAV) to create covalently surface modified adeno-associated viruses, and methods of purifying such covalently surface modified adeno-associated viruses. The inventions further include covalently surface modified adeno-associated viruses and preparations and products comprising such covalently surface modified adeno-associated viruses.

Description

REGN 11547 COVALENTLY SURFACE MODIFIED ADENO-ASSOCIATED VIRUS PRODUCTION BY IN VITRO CONJUGATION, AND PURIFICATION OF COVALENTLY SURFACE MODIFIED ADENO-ASSOCIATED VIRUS This application claims priority to U.S. Application Serial No.63 / 713,916, filed October 30, 2024; U.S. Application Serial No.63 / 691,544, filed September 6, 2024; U.S. Application Serial No.63 / 654,478, filed May 31, 2024; U.S. Application Serial No.63 / 620,682, filed January 12, 2024; U.S. Application Serial No.63 / 620,055, filed January 11, 2024; and U.S. Application Serial No.63 / 601,611, filed November 21, 2023. These applications are incorporated by reference in their entirety. FIELD OF THE INVENTIONS

[0001] The present inventions provide systems and methods for engineering adeno-associated virus (AAV) to create covalently surface modified adeno-associated virus species using in vitro conjugation, and methods of purifying such covalently surface modified adeno-associated viruses from unconjugated retargeting molecule species. REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. The .XML copy, created on November 8, 2024, is named “11547.xml” and is 334,915 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety. BACKGROUND OF THE INVENTIONS

[0003] Adeno-associated virus (AAV) is a non-enveloped, single- stranded DNA virus and is used as a gene delivery vector for both research andREGN 11547 therapeutics. Weitzman and Linden, Adeno-Associated Virus Biology (chapter 1), Meth. Molec. Biol.807: 1-23 (2011). There are numerous AAV serotypes and variants thereof. AAV serotypes include, for example, AAV1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, rh74, as well as variants thereof. AAV serotypes share common properties, structure, and genomic sequence and organization. See, for example, Issa et al, Cells 12: 285 (2023); Goedeker et al., Ther. Adv. Neurol. Disord.16: 1–7 (2023).

[0004] Gene transfer vectors based on AAV have demonstrated promise for human gene therapy based on their safety profile and potential to achieve long-term efficacy in animal models. Wang et al., Nature, 18: 358-78 (2019). A major challenge for advancing AAV-based therapies into clinical development is the difficulty and cost of producing sufficient quantities of AAV through transient methodologies. Additionally, recombinant AAVs containing genes of interest (GOIs), but lacking surface modifications, are currently being used in preventative and therapeutic capacities, such as in vaccines and in gene therapy. However, such AAVs have limited tissue specificity and a propensity to accumulate in the liver.

[0005] The wild type AAV genome includes a capsid gene referred to as “Cap” or “cap”. Cap in nature is translated to produce, via alternative start codons and transcript splicing, three size-variant structural proteins referred to as VP1 (about 90 kDa), VP2 (about 72 kDa) and VP3 (about 60 kDa). An AAV capsid contains 60 subunits total of the VP proteins. A ratio of 1:1:10 is considered the most typical ratio for VP1:VP2:VP3, with a stoichiometry of 5 VP1 subunits:5 VP2REGN 11547 subunits:50 VP3 subunits. However, there can be variation. Wörner et al., Nature Communications 12:1642 (2021).

[0006] Recombinant AAV (rAAV) has been produced in HEK 293, HeLa BHK, human amniotic (for example, epithelial cells such as HAEpiC), CHO and Sf9 lines, for example. First generation rAAV was comprised of a GOI replacing the AAV Cap and Rep genes. The GOI would be flanked by AAV inverted terminal repeats (ITRs) so that the GOI could be packaged within an AAV capsid.

[0007] Covalently surface modified AAVs have been produced. See WO 2019 / 006046. However, there exists a need to address by-products, such as excess unconjugated retargeting molecule species, that will exist freely as contaminants. The present invention provides production and purification solutions. SUMMARY OF THE INVENTIONS

[0008] Many potential gene therapy applications would benefit from cell / tissue-specific retargeting and detargeting that is beyond current AAV vector control capabilities. The current inventions advantageously employ covalently surface modified AAV for retargeting. Additionally, according to the inventions, mutations optionally can be introduced into the AAV Cap proteins to detarget tissues and organs, such as the liver.

[0009] The inventions provide methods of producing covalently surface modified adeno-associated virus (AAV) using in vitro conjugation, wherein the method comprises the steps of (I) combining recombinant AAV with retargeting molecules in a ratio selected to achieve a desired level of conjugation; and (II)REGN 11547 incubating the AAV and retargeting molecules for a period of time and under conditions to achieve the desired level of conjugation. The recombinant AAV can obtained by (A) transfecting a cell with: (1) a plasmid comprising a gene of interest flanked by AAV inverted terminal repeats; (2) a plasmid comprising an AAV rep gene and an AAV cap gene; (3) a plasmid comprising AAV rep and cap genes and a polynucleotide sequence encoding a first member of a specific binding pair; and (4) a plasmid comprising adenovirus helper genes E4 and E2, and VA RNA; (B) culturing the transfected cell from step (A) to allow expression of plasmids (1) to (4) and assembly of proteins formed by the expression of plasmids (1) to (4); and (C) harvesting the recombinant AAV. The cell can be a mammalian cell, such as a human cell. Other viruses can be the source of helper polynucleotide sequences encoding helper products, such as herpes simplex virus (UL5, UL8, UL9, UL29, UL30, UL42 and UL52 polynucleotides), human papilloma virus (E1 or E1 carboxyl domain polynucleotides), bocavirus or baculovoirus.

[0010] The inventions also provide methods of producing covalently surface modified adeno-associated virus using in vitro conjugation, wherein the method comprises the steps of (I) combining recombinant AAV (rAAV) with retargeting molecules in a ratio selected to achieve a desired level of conjugation; (II) incubating the AAV and retargeting molecules for a period of time and under conditions to achieve the desired level of conjugation. The rAAV is obtained by (A) transfecting a cell with: (1) a plasmid comprising a gene of interest flanked by adeno- associated virus (AAV) inverted terminal repeats; (2) a plasmid comprising an AAV rep gene and an AAV cap gene; (3) a plasmid comprising AAV rep and cap genes and a polynucleotide sequence encoding a first member of a specific binding pair;REGN 11547 and (4) a plasmid comprising one or more helper polynucleotide sequences; (B) culturing the transfected cell from step (A) to allow expression of plasmids (1) to (4) and assembly of proteins formed by the expression of plasmids (1) to (4); and (C) harvesting the recombinant AAV. The cell can be a mammalian cell, such as a human cell. The recombinant AAV can comprise a gene of interest. The recombinant AAV can comprise a recombinant capsid protein. The recombinant capsid protein can comprise an amino acid sequence of a first member of a specific binding pair, such as SpyTag. The retargeting molecule can be bound to a second cognate member of a specific binding pair, such as SpyCatcher or fragments / derivatives thereof. The retargeting molecule can be an Fc-containing protein, such as a monoclonal antibody, or an Fc-Fusion protein, or a monoclonal antibody fragment, or any molecule containing an antigen-binding region.

[0011] According to the methods of the inventions, the ratio of AAV with FM (First Member) to retargeting molecule with SCM (Second Cognate Member) is 1:20 or a greater ratio difference, for example about 1:30 to about 1:300, about 1:300 to about 1:1000 or about 1:30 to about 1:1000. The populations of FMs and SCMs can be homogenous or heterogeneous as long as the selected FMs can come in contact with appropriate SCMs so that binding can occur.

[0012] Preferred levels of molar ratios of AAV-FM to RM-SCM as follows: 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300, 1:305, 1:310, 1:315, 1:320,REGN 11547 1:325, 1:330, 1:335, 1:340, 1:345, 1:350, 1:355, 1:360, 1:365, 1:370, 1:375, 1:380, 1:385, 1:390, 1:395, 1:400, 1:405, 1:410, 1:415, 1:420, 1:425, 1:430, 1:435, 1:440, 1:445, 1:450, 1:455, 1:460, 1:465, 1:470, 1:475, 1:480, 1:485, 1:490, 1:495, 1:500, 1:505, 1:510, 1:515, 1:520, 1:525, 1:530, 1:535, 1:540, 1:545, 1:550, 1:555, 1:560, 1:565, 1:570, 1:575, 1:580, 1:585, 1:590, 1:595, 1:600, 1:605, 1:610, 1:615, 1:620, 1:625, 1:630, 1:635, 1:640, 1:645, 1:650, 1:655, 1:660, 1:665, 1:670, 1:675, 1:680, 1:685, 1:690, 1:695, 1:700, 1:705, 1:710, 1:715, 1:720, 1:725, 1:730, 1:735, 1:740, 1:745, 1:750, 1:755, 1:760, 1:765, 1:770, 1:775, 1:780, 1:785, 1:790, 1:795, 1:800, 1:805, 1:810, 1:815, 1:820, 1:825, 1:830, 1:835, 1:840, 1:845, 1:850, 1:855, 1:860, 1:865, 1:870, 1:875, 1:880, 1:885, 1:890, 1:895, 1:900, 1:905, 1:910, 1:915, 1:920, 1:925, 1:930, 1:935, 1:940, 1:945, 1:950, 1:955, 1:960, 1:965, 1:970, 1:975, 1:980, 1:985, 1:990, 1:995, 1:1000, 1:>1000 or any ratio thereabout or therebetween.

[0013] More preferred ratios are: 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300 or any ratio thereabout or therebetween.

[0014] The ratio of the plasmids comprising AAV rep and cap genes and a polynucleotide sequence encoding a first member of a specific binding pair to total plasmids comprising AAV rep and cap genes (termed “mosaicisms”) can include the following ratios and those thereabout or therebetween: 1 / 1, 1 / 1.1, 1 / 1.2, 1 / 1.3, 1 / 1.4, 1 / 1.5, 1 / 1.6, 1 / 1.7, 1 / 1.8, 1 / 1.9, 1 / 2, 1 / 2.1, 1 / 2.2, 1 / 2.3, 1 / 2.4, 1 / 2.5, 1 / 2.6, 1 / 2.7, 1 / 2.8, 1 / 2.9, 1 / 3, 1 / 3.1, 1 / 3.2, 1 / 3.3, 1 / 3.4, 1 / 3.5, 1 / 3.6, 1 / 3.7, 1 / 3.8, 1 / 3.9, 1 / 4,REGN 11547 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, / 11.7, 1 / 11.8, 1 / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, / 12.8, 1 / 12.9, 1 / 13, 1 / 13.1, 1 / 13.2, 1 / 13.3, 1 / 13.4, 1 / 13.5, 1 / 13.6, 1 / 13.7, 1 / 13.8, / 13.9, 1 / 14, 1 / 14.1, 1 / 14.2, 1 / 14.3, 1 / 14.4, 1 / 14.5, 1 / 14.6, 1 / 14.7, 1 / 14.8, 1 / 14.9, / 15, 1 / 15.1, 1 / 15.2, 1 / 15.3, 1 / 15.4, 1 / 15.5, 1 / 15.6, 1 / 15.7, 1 / 15.8, 1 / 15.9, 1 / 16, / 16.1, 1 / 16.2, 1 / 16.3, 1 / 16.4, 1 / 16.5, 1 / 16.6, 1 / 16.7, 1 / 16.8, 1 / 16.9, 1 / 17, 1 / 17.1, / 17.2, 1 / 17.3, 1 / 17.4, 1 / 17.5, 1 / 17.6, 1 / 17.7, 1 / 17.8, 1 / 17.9, 1 / 18, 1 / 18.1, 1 / 18.2, / 18.3, 1 / 18.4, 1 / 18.5, 1 / 18.6, 1 / 18.7, 1 / 18.8, 1 / 18.9, 1 / 19, 11 / 9.1, 1 / 19.2, 1 / 19.3, / 19.4, 1 / 91.5, 1 / 19.6, 1 / 19.7, 1 / 19.8, 1 / 19.9, 1 / 20, 1 / 21, 1 / 21.1, 1 / 21.2, 1 / 21.3, / 21.4, 1 / 21.5, 1 / 21.6, 1 / 21.7, 1 / 21.8, 1 / 21.9,1 / 22, 1 / 22.1, 1 / 22.2, 1 / 22.3, 1 / 22.4, / 22.5, 1 / 22.6, 1 / 22.7, 1 / 22.8, 1 / 22.9, 1 / 23, 1 / 23.1, 1 / 23.2, 1 / 23.3, 1 / 23.4, 1 / 23.5, / 23.6, 1 / 23.7, 1 / 23.8, 1 / 23.9, 1 / 24, 1 / 24.1, 1 / 24.2, 1 / 24.3, 1 / 24.4, 1 / 24.5, 1 / 24.6, / 24.7, 1 / 24.8, 1 / 24.9, 1 / 25, 1 / 25.1, 1 / 25.2, 1 / 25.3, 1 / 25.4, 1 / 25.5, 1 / 25.6, 1 / 25.7, / 25.8, 1 / 25.9, 1 / 26, 1 / 26.1, 1 / 26.2, 1 / 26.3, 1 / 26.4, 1 / 26.5, 1 / 26.6, 1 / 26.7, 1 / 26.8,2 / 6.9, 1 / 27, 1 / 27.1, 1 / 27.2, 1 / 27.3, 1 / 27.4, 1 / 27.5, 1 / 27.6, 1 / 27.7, 1 / 27.8, 1 / 27.9, / 28, 1 / 28.1, 1 / 28.2, 1 / 28.3, 1 / 28.4, 1 / 28.5, 1 / 28.6, 1 / 28.7, 1 / 28.8, 1 / 28.9, 1 / 29, / 29.1, 1 / 29.2, 1 / 29.3, 1 / 29.4, 1 / 29.5, 1 / 29.6, 1 / 29.7, 1 / 29.8, 1 / 29.9, 1 / 30, 1 / 30.1, / 30.2, 1 / 30.3, 1 / 30.4, 1 / 30.5, 1 / 30.6, 1 / 30.7, 1 / 30.8, 1 / 30.9, 1 / 31, 1 / 31.1, 1 / 31.2, / 31.3, 1 / 31.4, 1 / 31.5, 1 / 31.6, 1 / 31.7, 1 / 31.8, 1 / 31.9, 1 / 32, 1 / 32.1, 1 / 32.2, 1 / 32.3, / 32.4, 1 / 32.5, 1 / 32.6, 1 / 32.7, 1 / 32.8, 1 / 32.9, 1 / 33, 1 / 33.1, 1 / 33.2, 1 / 33.3, 1 / 33.4,REGN 11547 / 33.5, 1 / 33.6, 1 / 33.7, 1 / 33.8, 1 / 33.9, 1 / 34, 1 / 34.1, 1 / 34.2, 1 / 34.3, 1 / 34.4, 1 / 34.5, / 34.6, 1 / 34.7, 1 / 34.8, 1 / 34.9, 1 / 35, 1 / 35.1, 1 / 35.2, 1 / 35.3, 1 / 35.4, 1 / 35.5, 1 / 35.6, / 35.7, 1 / 35.8, 1 / 35.9, 1 / 36, 1 / 36.1, 1 / 36.2, 1 / 36.3, 1 / 36.4, 1 / 36.5, 1 / 36.6, 1 / 36.7, / 36.8, 1 / 36.9, 1 / 37, 1 / 37.1, 1 / 37.2, 1 / 37.3, 1 / 37.4, 1 / 37.5, 1 / 37.6, 1 / 37.7, 1 / 37.8, / 37.9, 1 / 38, 1 / 38.1, 1 / 38.2, 1 / 38.3, 1 / 38.4, 1 / 38.5, 1 / 38.6, 1 / 38.7, 1 / 38.8, 1 / 38.9, / 39, 1 / 39.1, 1 / 39.2, 1 / 39.3, 1 / 39.4, 1 / 39.5, 1 / 39.6, 1 / 39.7, 1 / 39.8, 1 / 39.9, 1 / 40, / 40.1, 1 / 40.2, 1 / 40.3, 1 / 40.4, 1 / 40.5, 1 / 40.6, 1 / 40.7, 1 / 40.8, 1 / 40.9, 1 / 41, 1 / 41.1, / 41.2, 1 / 41.3, 1 / 41.4, 1 / 41.5, 1 / 41.6, 1 / 41.7, 1 / 41.8, 1 / 41.9, 1 / 42, 1 / 42.1, 1 / 42.2, / 42.3, 1 / 42.4, 1 / 42.5, 1 / 42.6, 1 / 42.7, 1 / 42.8, 1 / 42.9, 1 / 43, 1 / 43.1, 1 / 43.2, 1 / 43.3, / 43.4, 1 / 43.5, 1 / 43.6, 1 / 43.7, 1 / 43.8, 1 / 43.9, 1 / 44, 1 / 44.1, 1 / 44.2, 1 / 44.3, 1 / 44.4, / 44.5, 1 / 44.6, 1 / 44.7, 1 / 44.8, 1 / 44.9, 1 / 45, 1 / 45.1, 1 / 45.2, 1 / 45.3, 1 / 45.4, 1 / 45.5, / 45.6, 1 / 45.7, 1 / 45.8, 1 / 45.9, 1 / 46, 1 / 46.1, 1 / 46.2, 1 / 46.3, 1 / 46.4, 1 / 46.5, 1 / 46.6, / 46.7, 1 / 46.8, 1 / 46.9, 1 / 47, 1 / 47.1, 1 / 47.2, 1 / 47.3, 1 / 47.4, 1 / 47.5, 1 / 47.6, 1 / 47.7, / 47.8, 1 / 47.9, 1 / 48, 1 / 48.1, 1 / 48.2, 1 / 48.3, 1 / 48.4, 1 / 48.5, 1 / 48.6, 1 / 48.7, 1 / 48.8, / 48.9, 1 / 49, 1 / 49.1, 1 / 49.2, 1 / 49.3, 1 / 49.4, 1 / 49.5, 1 / 49.6, 1 / 49.7, 1 / 49.8, 1 / 49.9, / 50, 1 / 50.1, 1 / 50.2, 1 / 50.3, 1 / 50.4, 1 / 50.5, 1 / 50.6, 1 / 50.7, 1 / 50.8, 1 / 50.9, 1 / 51, / 51.1, 1 / 51.2, 1 / 51.3, 1 / 51.4, 1 / 51.5, 1 / 51.6, 1 / 51.7, 1 / 51.8, 1 / 51.9, 1 / 52, 1 / 52.1, / 52.2, 1 / 52.3, 1 / 52.4, 1 / 52.5, 1 / 52.6, 1 / 52.7, 1 / 52.8, 1 / 52.9, 1 / 53, 1 / 53.1, 1 / 53.2, / 53.3, 1 / 53.4, 1 / 53.5, 1 / 53.6, 1 / 53.7, 1 / 53.8, 1 / 53.9, 1 / 54, 1 / 54.1, 1 / 54.2, 1 / 54.3, / 54.4, 1 / 54.5, 1 / 54.6, 1 / 54.7, 1 / 54.8, 1 / 54.9, 1 / 55, 1 / 55.1, 1 / 55.2, 1 / 55.3, 1 / 55.4, / 55.5, 1 / 55.6, 1 / 55.7, 1 / 55.8, 1 / 55.9, 1 / 56, 1 / 56.1, 1 / 56.2, 1 / 56.3, 1 / 56.4, 1 / 56.5, / 56.6, 1 / 56.7, 1 / 56.8, 1 / 56.9, 1 / 57, 1 / 57.1, 1 / 57.2, 1 / 57.3, 1 / 57.4, 1 / 57.5, 1 / 57.6, / 57.7, 1 / 57.8, 1 / 57.9, 1 / 58, 1 / 58.1, 1 / 58.2, 1 / 58.3, 1 / 58.4, 1 / 58.5, 1 / 58.6, 1 / 58.7, / 58.8, 1 / 58.9, 1 / 59, 1 / 59.1, 1 / 59.2, 1 / 59.3, 1 / 59.4, 1 / 59.5, 1 / 59.6, 1 / 59.7, 1 / 59.8, / 59.9 or 1 / 60.REGN 11547

[0015] Preferred mosaicisms are: 1 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, 1 / 12.8, 1 / 12.9 or 1 / 13. More preferred mosaicisms are: 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, or 1 / 12. Still more preferred mosaicisms are 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, or 1 / 11. Ranges of mosaicisms can be 1 / 1 to 1 / 60. Preferred ranges of mosaicisms are 1 / 4 to 1 / 13; 1 / 5 to 1 / 12; 1 / 6 to 1 / 11, 1 / 7 to 1 / 10.5 or 1 / 7.5 to 1 / 10.

[0016] The incubating step can about 4 to 72 hours long. The (I) combining step can further comprise an additive, such as isopropyl alcohol (IPA), preferably at a concentration above 5%. IPA concentrations can be about 6%,REGN 11547 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, 25% or more. Preferably, the concentration can be about 10% to 25%, more preferably 10% to 20%.

[0017] The inventions provide for a desired conjugation of a level of conjugation of at least 10%, at least 20%, at least 30%, and at least 40%. Preferred levels are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100%, any of which can be considered complete depending on the purpose. More preferably, the desired level of conjugation typically will be about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0018] Ranges of desired conjugation levels can be 10% to 100%, 20% to 99%, 30% to 99%, 40% to 99%, 50% to 99%, 60% to 99%, 70% to 99%, 80% to 99%, or 90% to 99%. Preferred ranges of desired conjugation levels can be 40% to 100%, 40% to 99%, 45% to 95%, 50% to 95%, 55% to 90%, 60% to 90%, 65% to 95%, 65% to 90%, 70% to 90%, 75% to 90%, 75% to 85%, 75% to 80%, or 80% to 85%, and any ranges made by combining these ranges, such as 65% to 85%, for example. More preferably, the range is 65% to 95%.REGN 11547

[0019] The helper polynucleotide sequences can encode adenovirus E4, adenovirus E2, and VA RNA, or be from another virus, such as herpes simplex virus (UL5, UL8, UL9, UL29, UL30, UL42 and UL52 polynucleotides), human papilloma virus (E1 or E1 carboxyl domain polynucleotides), bocavirus or baculovoirus.

[0020] The inventions also provide methods of separating a covalently surface modified adeno-associated virus (AAV) species from unconjugated retargeting molecule species, wherein the method comprises the steps of (a) providing a sample comprising the covalently surface modified adeno-associated virus species produced using in vitro conjugation; (b) subjecting the covalently surface modified adeno-associated virus to anion exchange media using a buffering agent and a salt to separate the covalently surface modified AAV species from retargeting molecule species to provide at least one fraction that is enriched in covalently surface modified AAV species; and (c) harvesting the enriched fraction comprising covalently surface modified AAV species with a defined level of covalent surface modifications. The buffering agent can be selected from the group consisting of Bis-Tris-Propane, Bis-Tris, Tris, Glycine, Bicine, Tricine, Acetate, Borate, Citrate, Carbonate, Phosphate, Formate, Sulfate, Succinic acid, Sulfonic acid and variants thereof (for example, MES, PIPES, HEPES, CHES, CAPS, MMS, PBMS), Diethanolamine, and Imidazole, and the salt is selected from the group consisting of NaCl, KCl, MgCl2, CaCl2, NH4Cl, Na2SO4, CaSO4, K2SO4, MgSO4, (NH4)2SO4, sodium citrate, and tetramethylammonium chloride (TMAC). The anion exchange media is selected from the group consisting of CIM QA, CIM DEAE, PRIMA T, Capto Q, Capto Q ImpRes, CAPTO DEAE, POROS HQ, POROS XQ,REGN 11547 POROS PI, POROS D, Fractogel EMD TMAE, Fractogel EMD DEAE, Nuvia Q, Nuvia HP-Q, Sartobind STIC PA, Sartobind Q, Natrix Q. Enrichment can result in greater than 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of unconjugated retargeting molecule species being separated from the covalently surface modified AAV species. Enrichment can result in reducing the presence of unconjugated retargeting molecule species to undetectable levels.

[0021] The inventions further provide methods of separating a covalently surface modified adeno-associated virus (AAV) species from unconjugated retargeting molecule species, wherein the method comprises the steps of (a) providing a sample comprising the covalently surface modified adeno- associated virus species produced using an in vitro conjugation; (b) subjecting the covalently surface modified adeno-associated virus to cation exchange media using a buffering agent and a salt to separate the covalently surface modified AAV species from unconjugated retargeting molecule species to provide at least one fraction that is enriched in covalently surface modified AAV species; and (c) harvesting the enriched fraction comprising covalently surface modified AAV species. The cation exchange media can be selected from the group consisting of CIM SO3, Capto S, Capto S ImpRes, Capto S ImpAct, Capto SP, POROS HS, POROS XS, CM Sepharose, Nuvia S, Nuvia HR- Sartobind S, Natrix CH. Enrichment can result in greater than 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of unconjugated retargeting molecule species being separated from the covalently surface modified AAV species. Enrichment can result in reducing the presence of unconjugated retargeting molecule species to undetectable levels.REGN 11547

[0022] The inventions further provide methods of separating a covalently surface modified adeno-associated virus (AAV) species from unconjugated retargeting molecule species, wherein the method comprises the steps of (a) providing a sample comprising the covalently surface modified adeno- associated virus species produced using an in vitro conjugation; (b) subjecting the covalently surface modified adeno-associated virus to multimodal ion exchange using a buffering agent and a salt and an inorganic salt for loading and a low salt and low pH buffer for elution in order to separate the covalently surface modified AAV species from unconjugated retargeting molecule species to provide a fraction that is enriched in covalently surface modified AAV species; and (c) harvesting at least one enriched fraction comprising covalently surface modified AAV species. Enrichment can result in greater than 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of unconjugated retargeting molecule species being separated from the covalently surface modified AAV species. Enrichment can result in reducing the presence of unconjugated retargeting molecule species to undetectable levels.

[0023] The inventions further provide methods of separating a covalently surface modified adeno-associated virus (AAV) species from unconjugated retargeting molecule species, wherein the method comprises the steps of (a) providing a sample comprising the covalently surface modified adeno- associated virus species produced using an in vitro conjugation; (b) subjecting the covalently surface modified adeno-associated virus to multimodal binding and size separation bead chromatography using a multimodal buffer comprising in order to separate the covalently surface modified AAV species from unconjugated retargetingREGN 11547 molecule species to provide a fraction that is enriched in covalently surface modified AAV species; and (c) harvesting the enriched fraction comprising covalently surface modified AAV species. The multimodal binding and size separation bead chromatography can be functionalized with an octyl amine that is hydrophobic and positively charged. The beads can comprise a matrix that provides size exclusion, and can be CaptoTMCore 400 or CaptoTMCore 700. Enrichment can result in greater than 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of unconjugated retargeting molecule species being separated from the covalently surface modified AAV species. Enrichment can result in reducing the presence of unconjugated retargeting molecule species to undetectable levels.

[0024] The inventions further provide methods of purifying the covalently surface modified AAV of the inventions described herein using approaches including improved depth filtration, improved tangential flow filtration (such as single-pass tangential flow filtration), improved affinity capture and improved ionic exchange chromatography.

[0025] The inventions further comprise covalently surface modified adeno-associated viruses (AAV) produced and / or purified by the methods.

[0026] The inventions also provide AAV preparations produced by any of the above the methods, and drug products made from the AAV preparations.

[0027] The inventions are amendable to use with all AAV serotypes and variants, including but not limited to AAV1, AAV2, AAV2quad(Y-F), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV rh10, AAV rh39, AAV rh43, and AAV rh74.REGN 11547

[0028] Covalently surface modified adeno-associated viruses (AAV) made according to an example selected from the group consisting of Example 15, Example 16, Example 17, Example 18, Example 19, Example 20, Example 21, Example 22, Example 23, Example 24, Example 25, Example 26, Example 27, Example 28, Example 29, Example 30 and Example 31 also are provided, and can be produced using cells comprising at least one sequence set forth in an example selected from the group consisting of examples selected from the group consisting of Example 32, Example 33, Example 34, Example 35, Example 36, Example 37, Example 38 and Example 39.

[0029] AAV preparations comprising the covalently surface modified adeno-associated viruses and biologic drug products comprising the covalently surface modified adeno-associated virus also are provided. BRIEF DESCRIPTION OF THE FIGURES

[0030] The Figures (Fig. or Figs.) below depict schematic representations of aspects of the inventions and data from the inventions.

[0031] Figure 1A schematically depicts the species heterogeneity involved in in-vitro conjugation reactions between AAV9 comprising a SpyTag peptide (rAAV9-SpyT) and SpyCatcher fused to a monoclonal antibody (SpyC-mAb species). The top row depicts the heterogeneity at the level of the AAV9-SpyT capsid, depicting the “average” AAV species with 6 SpyTags, as well as AAV species with lower (3) and higher number (10) of SpyTags expressed on the capsid surface. SpyTag is an example of a first member (FM). The middle and bottomREGN 11547 rows respectively depict incomplete and optimum conjugation, also referred to as ‘complete conjugation’.

[0032] Optimum (often referred to as ‘complete’ since it is based on the desired purpose) conjugation occurs when a substantial portion, preferably a majority, of accessible first members of a specific binding pair are bound to second cognate members of the specific binding pair, which is the desired outcome. For example, Figure 1A (bottom row) schematically depicts instances of all and nearly all accessible SpyTags bound by SpyCatcher on covalently surface modified AAVs. First member (for example, SpyTag) accessibility in the capsid can have an effect, however. Thus, optimal (complete) conjugation is the point where no unacceptable increase of conjugated species is observed (for example, no unacceptable shift of peaks from unconjugated to conjugated species on analytical size-exclusion chromatography as shown in, for instance, Figure 7G).

[0033] Figure 1B is a graph that depicts retention time (minutes) of high molecular weight (HMV) variants comprising conjugated AAV9-SpyT-SpyC-Ab species, conjugated AAV9-SpyT-SpyC-Ab, unconjugated AAV9-SpyT and unconjugated SpyC-mAb. Ratios of AAV9-SpyT to SpyC-mAb of 1:30 and 1:300 provided the best results.

[0034] Figure 2 schematically depicts and describes quad transfection systems and methods for producing covalently surface modified AAV in a eukaryotic cell, such as HEK 293, for example HEK293F. As described in this figure, the quad transfection system utilizes four plasmids (schematically depicted) to produce AAV comprising SpyTag sequences in the capsids. The plasmids are as follows:REGN 11547 (i) pGOI – a plasmid comprising a GOI (for example, a transgene) flanked by two AAV inverted terminal repeats (ITRs), and optionally a selection marker gene, such as an antibiotic resistance gene (for example, ampicillin resistance); (ii) pRC - a plasmid comprising AAV (here AAV9) rep and cap genes, and optionally a selection marker gene, such as an antibiotic resistance gene (for example, ampicillin resistance); (iii) pRC-SpyT – a plasmid as above in (ii) and further comprising a polynucleotide sequence encoding a 13 amino acid Spy Tag peptide (a first member of a specific binding pair), and optionally a selection marker gene, such as an antibiotic resistance gene (for example, ampicillin resistance). This is an exemplary plasmid pRC-first member (pRC-FM); and (iv) pHELP - a plasmid comprising adenovirus helper genes E4 and E2, and VA RNA (exemplary helper genes, and a optionally selection marker gene, such as an antibiotic resistance gene (for example, ampicillin resistance).

[0035] Figure 3 schematically depicts at the top a pRC-SpyTag plasmid comprising the rep and cap genes and the p40 promoter, and at the bottom schematically shows the insertion of the SpyTag 13 amino acid peptide sequence to form a fusion protein comprising SpyTag and Cap peptide sequences, thereby resulting in mutant VP1, VP2 and VP3 proteins fused to the SpyTag peptide sequence. The AAV still possesses the 5:5:50 stoichiometry of the VP1, 2 and 3 proteins.

[0036] Figure 4 depicts size exclusion chromatograms (SEC) of in vitro conjugation reaction mixtures of rAAV9-FM to antibody-SCM in ratios of 1:1, 1:6, 1:18, 1:30 and 1:300 at pH 8.0 and using rAAV concentration of 5 x 1010to 1 x 1012REGN 11547 capsids (cp) per milliliter on a size exclusion chromatography column with pore size of 1000 Å. The shift in retention time of the AAV peak is highlighted with an arrow showing shifting of the peak maximum to the left indicating conjugation reaction completion. The x-axis is minutes for elution and the y-axis is emission units (EU) for fluorescence.

[0037] Figure 5A depicts analytical size exclusion chromatograms of in-vitro conjugation reaction mixtures of rAAV9-FM to antibody-SCM in ratios of 1:0, 1:1, 1:6, 1:15, 1:30 and 1:300 at pH 5.0, 6.5, 8.0 and 8.5 and using rAAV concentration of 2.3 x 1013cp / mL on a size exclusion chromatography column with pore size of 450 Å. Reaction conditions included brief mixing followed by overnight incubation, resulting in maximum conjugation as shown by the almost complete disappearance of the AAV-SpyT peak and formation of a new conjugated AAV peak at ratios of 1:30 and 1:300. Figure 5B is a preparative scale SEC on a 100 ml SEPAX SRT 500A column to remove unconjugated antibody. UV280 shows the antibody-AAV separation. The multiple overlaid curves are different injections on the same column, as about 30 injections were required to process all of the material.

[0038] Figures 6A and 6B depict size exclusion chromatograms of in- vitro conjugation reaction mixtures of rAAV9 to antibody in ratios of 1:0, 1:1, 1:6, 1:10, 1:20, and 1:30 at pH 5.0 (Figure 6A), and at a 1:30 rAAV9 to antibody ratio and pH values of 5.0, 6.5, 8.0, 8.5, 9.0, 9.5, 10.0 (Figure 6B). Recombinant AAV concentration was 2.3 x 1013cp / mL and separated on a 450 Å size exclusion chromatography column. Reaction conditions included brief mixing followed by 1 hour incubation prior to freezing, resulting in incomplete conjugation shown by the presence of AAV-SpyT peak at all molar ratios.REGN 11547

[0039] Figures 7A to 7K are as follows: Figure 7A depicts exemplaryelution profiles for AAV9 and AAV9-SpyT for empty, partially filled and full capsids, and serves as a model for other AAVs. There will exist empty and partially-filled capsid populations can have isoelectric points (pI) that are above full capsids and other empty and partially-filled capsid populations will have isoelectric points that are below full capsids. Figure 7B is a graph depicting separation empty, partially filled and full AAV9-SpyT capsids using a hybrid gradient combining linear and step gradients. The column was a CIM QA monolith loaded with 5 x 1013to 5 x 1014capsids (cp / ml). The buffer was 20 mM Bis-Tris-Propane, 0.001% P188, pH 9.6 and 1M NaCl and the process resulted in enrichment of full capsids from 14% to 55%. This hybrid gradient enabled improved resolution of the full capsids (central peak) from the empty capsid populations on either side. However, the hybrid gradient is operationally challenging at scale and also does not result in full capsids above the required threshold of 70%.

[0040] Figure 7C depicts data from a two-pass anion exchange chromatogram for a 500L purification of AAV-SpyTag with first pass and second pass microsteps. This process achieved a recovery of 96% full capsids. Partially- filled capsids were only 1% of total and empty capsids were only 3% of total. Figures 7D to 7F depict data from an experiment using anion exchange to separate a covalently surface modified adeno-associated virus species from unconjugated antibody species. A CIM QA 1 mL monolith was used as the anion exchange column, and the column was loaded with an in-vitro conjugation reaction mixture of AAV9-SpyT and SpyC-ASGR1 mAb mixed in a 1:30 molar ratio. In Figure 7D, the AAV9-SpyT capsids were 10% full, whereas in Figure 7E the AAV9-SpyT capsidsREGN 11547 were 70% full. Figure 7F depicts the size exclusion chromatograms combined with multi-angle light scattering (MALS) data of six elution fractions collected from Figure 7E, showing the increasing conjugation level, and increasing mass distribution of the later eluting fractions. Figure 7G is a graph of the retention time of conjugated AAV9 using SpyTag-SpyCatcher with an antibody, unconjugated AAV-SpyTag and unconjugated SpyCatcher-antibody. Figure 7H is a graph depicting the effect of AAV capsid concentration on percent conjugation. Figure 7I is a graph depicting the effect of incubation temperature on percent conjugation, and Figure 7J is a graph depicting the effect of pH on percent conjugation. Figure 7K depicts data for conjugation of AAV-SpyT conjugated to Spy C-Fab (Construct 1) and AAV-SpyT conjugated to Spy C-mAb (Construct 2). Both AAVs have the W503A detargeting mutation and HA-MTM1 as a transgene.

[0041] Figures 8A to 8E depict data from experiments using cation exchange to separate a covalently surface modified adeno-associated virus species from unconjugated antibody species. In Figure 8A, a CIM SO31 mL monolith was used as the cation exchange column, and the column was loaded with an in-vitro conjugation reaction mixture of AAV9-SpyT and SpyC-ASGR1 mAb mixed in a 1:30 molar ratio. Figure 8A depicts a linear gradient elution and highlights the peaks corresponding to the SpyC-Ab and the conjugated AAV9-SpyT-SpyC-Ab species. Figure 8B depicts a step elution in which the in-vitro conjugation reaction mixture was adjusted with sodium chloride solution to a conductivity of 15 mS / cm and the mixture was loaded onto a Poros XS cation exchange column, leading to all SpyC- Ab species not binding onto the column and being removed in the flow-through, while all AAV species bound and were removed later in an isocratic elution at 40REGN 11547 mS / cm conductivity. Figure 8C depicts data showing that Poros XS cation exchange can separate free SpyC-Ab species following the conjugation reaction, shown for a separation of AAV9-SpyT containing CAGG-eGFP transgene conjugated to a SpyC-ASGR1 mAb. Figures 8D and 8E compare CaptoCore400 flow through (size exclusion and anion exchange) (Figure 8D) to Poros XS elution (cation exchange) (Figure 8E).

[0042] Figure 9 depicts an experiment using multimodal anion exchange to separate a covalently surface modified adeno-associated virus species from unconjugated antibody species.

[0043] Figure 10 depicts data from an experiment using multimodal binding and size separation bead chromatography exchange on CaptoCore400 resin to separate a covalently surface modified adeno-associated virus species from unconjugated antibody species. The relative size of species was about 8 nm for the SpyC-Ab, about 15 nm for the AAV9-SpyT, and about 17 nm for the conjugated AAV9-SpyT-SpyC-Ab. The figure includes a size exclusion chromatogram showing that the load material included two peaks (AAV and SpyC-Ab), but the flow through included only one peak (only AAV species), showing removal of free SpyC-Ab species using this technique.

[0044] Figure 11 depicts a multivariate model from experiments using different salt and pH conditions to induce a change in the size and surface characteristics of AAVs, leading to improved yields in the flow through of CaptoCore400 chromatography. The model is a yield profiler and predicts an optimum condition of adjusting the load material with sodium citrate to a finalREGN 11547 concentration of 150 mM at pH 5.5, resulting in a predicted yield of 100% and an experimental yield of 107% as measured by ddPCR quantification.

[0045] Figures 12A-12C depict dynamic light scattering (DLS) data from AAV9-SpyT, SpyC-ASGR1 mAb (Figure 12A), and in-vitro conjugation reactions between these two species under stirred and unstirred conditions in sodium chloride and sodium citrate based buffers. The DLS data shows the formation of large aggregates in the conjugation reaction under stirred conditions (Figure 12B), but no such aggregates in the unstirred condition (Figure 12C) (incubation only).

[0046] Figure 13A shows size exclusion chromatography data tracking the rate of reaction between AAV9-SpyT and SpyC-ASGR1 mAb for three different batches of AAV9-SpyT with different % full capsids. The percentage of conjugated species is measured by relative peak area between the AAV9-SpyT peak at about 8.3 min RT and the conjugated species peaks at 6.5 to 8.0 RT on the size exclusion chromatograms, and tracked in the chart on the right. All batches reached 80% conjugation, but Batch 1 took 48 hours to reach an example of an optimal conjugation level (80%) while Batches 2 and 3 took 24 hours.

[0047] Figure 13B depicts conjugation data with AAV9-SpyT and SpyC-mAb (anti-CACNG1). The mAb has two SpyC. Figure 13C depicts conjugation data with AAV9-SpyT and SpyC-bispecific Ab (anti-CACNG1). This bispecific mAb has one SpyC. Figure 13D depicts conjugation data with AAV9- SpyT and SpyC-Fab (anti-CACNG1). The Fab has one SpyC. Molar ratios ofREGN 11547 AAV:Ab (antibody with 2-SpyC, bispecific antibody with one SpyC, or Fab with one SpyC) ratios of 1:1, 1:6, 1:15, 1:30, 1:300 and 1:1000 were tested.

[0048] Figure 14A is a graph depicting how anion exchange chromatography separates unconjugated AAV from increasingly conjugated species, which are schematically depicted underneath the graph. Figure 14B depicts SEC- MALS data of samples with different levels of conjugation. The samples were obtained from an anion exchange chromatography method similar to the one described in Figures 7D to 7F that can be used to separate populations of differently conjugated species. The chart underneath the graph summarizes the SEC-MALS analysis of the samples which is used to estimate the number of mAbs conjugated on average in each sample, ranging from less than 1 mAb in the first fraction to more than 5 mAbs in the last fraction. Figure 14C shows the results of a transduction assay where these differently conjugated species were compared for transduction ability using cells expressing ASGR1 receptor. It was observed that lower conjugated species resulted in higher transduction efficiency than the combined pool (Load) as well as heavier conjugated species, suggesting a negative impact of higher conjugation levels on transduction ability, despite potential advantages in targeting ability. Figure 14D and Figure 14E collectively concern transduction efficiency at different pHs and percent conjugation. Figure 14D depicts green fluorescent protein (GFP) percentage of bispecific antibody (bsAb or bsmab) with one SpyC, a monospecific monoclonal antibody (mAb) with two SpyCs, or an antigen-binding antibody fragment (Fab) with one SpyC, at reaction pH of 5 or 8. Figure 14E depicts mean fluorescence intensities (MFI) for the bsAb, mAb and Fab at pH 5 or 8.REGN 11547

[0049] Figure 15 schematically depicts the production of an antibody or Fab fused to SpyCatcher.

[0050] Figure 16 schematically depicts the production of AAV comprising SpyTag on the capsid surface using cells transfected with the four plasmids of the quad transfection system and method. See Figure 2.

[0051] Figure 17 schematically depicts the conjugation of AAV-SpyT to an antibody fused to SpyCatcher and then enrichment using chromatography, such as cation exchange chromatography, and tangential flow filtration. See Figure 15 (depicting production of SpyCatcher fused to an antibody or Fab) and Figure 16 (depicting production of AAV-SpyT).

[0052] Figure 18 schematically depicts the effects of different concentrations of additives on conjugation. The selected additives were dimethylsulfoxide (DMSO), L-glutamic acid monosodium salt (L-glutamic acid), arginine, sorbitol, urea, isopropyl alcohol (IPA) and sodium thiocyanate (NaSCN).

[0053] Figure 19 depicts data showing the effects of the additives used in Figure 18.

[0054] Figures 20A to 20H are as follows: Figure 20A schematically depicts a hypothetical readout on the impact that mosaicism has on transduction efficiency (green) and percent (%) conjugation (red) with Fab, bispecific Ab and mAb. In this hypothetical, 80% is the desired limit for conjugated capsids. Mosaicisms selected for testing were 1 / 5, 1 / 7.5, 1 / 10, 1 / 15 and 1 / 30. Figure 20B is a bar graph comparing upstream bioreactor titer and process yield for five differentREGN 11547 mosaicisms (“M”), produced at 2 L scale for 1:5 (M1), 1:7.5 (M2), 1:15 (M4) and 1:30 (M5), and at 50 L scale for 1:10 (M3). Figure 20C is a bar graph comparing percent (%) conjugation vs. time for the five different mosaicisms, as measured using size- exclusion chromatography. The data indicates a maximum conjugation for the 1:7.5 and 1:10 mosaicisms.

[0055] Figures 20D to 20H are graphs schematically depicting data of size-exclusion chromatography overlays showing conjugation process for different mosaicisms M1 (1:5), M2 (1:7.5), M3 (1:10), M4 (1:15), and M5 (1:30). The FM is SpyT and the SCM is SpyC here. Figure 20D depicts an overlay of M1-M5 prior to addition of SpyC-Abs, showing increasing size with increasing level of mosaicism (M1 > M2 > M3 > M4 > M5). The X-axis designates 7.60 to 10.40 minutes and the Y-axis designates 0.00 to 800.00 EU. Figure 20E depicts an overlay of conjugation reactions between M1 and anti-CACNG1 single-SpyC mAb at 2, 8, 24 and 36 h, showing increasing conjugation level over time via reduction of the peak at about 8.5 min RT. The X-axis designates 3.00 to 14.50 minutes and the Y-axis designates 0.00 to 200.00 EU. Figure 20F depicts an overlay of conjugation reaction between M2 and anti-CACNG1 single-SpyC Fab at 2, 8, 24 and 36 h, showing increasing conjugation level over time via reduction of the peak shoulder at about 8.5 min RT. The X-axis designates 1.50 to 14.50 minutes and the Y-axis designates 0.00 to 450.00 EU. Figure 20G schematically depicts an overlay of conjugated species of M1-M5 and anti-CACNG1 single-SpyC Fab after 48 h of reaction, showing increasing conjugation level for higher mosaicisms. The X-axis designates 0.00 to 15.00 minutes and the Y-axis designates 0.00 to 1600.00 EU. Figure 20H depicts in the Final Concentration Pool (FCP) an overlay of conjugated species withREGN 11547 mosaicisms M1-M5 and anti-CACNG1 single-SpyC mAb after 48 h of reaction and removal of majority of excess free SpyC-Ab using preparative size exclusion chromatography, which showed increasing conjugation level for higher mosaicisms. The X-axis designates 0.00 to 15.00 minutes and the Y-axis designates 0.00 to 160.00 EU.

[0056] Figure 21A depicts production purification trains. The top train uses a batch tangential flow filtration unit where repeated passes are required to exchange buffer and concentrate the retentate. The bottom section replaces the batch tangential flow filtration unit with a single pass tangential flow filtration unit. Ionic exchange chromatography of different modalities can be used following TFF, and anion exchange is depicted as an exemplar. Figure 21B depicts a process for purifying retargeted AAV at the 500L production scale.

[0057] Figure 22A schematically shows a batch tangential flow filtration (Batch TFF) (top), where the retentate is repeatedly cycled through a feed tank and pump to repeatedly passed through a membrane, with the concentrated permeate being removed after repeated cycles. A single pass tangential flow unit (Single-Pass TFF or SPTFF) removes material from the feed tank through a pump to a multi-stage membrane module that separate the retentate from the permeate, while concentrating the permeate. Figure 22B is a graph comparing Batch TFF and Single-Pass TFF. Single-Pass TFF achieves higher concentration and is faster as compared to Batch TFF. Single-Pass TFF continuously delivers biological material (such as AAV) to the next operation in the purification train, whereas Batch TFF does not deliver biological material (such as AAV) until the end of the batch cycle.REGN 11547

[0058] Figure 23 schematically compares the batch operation to a continuous operation in terms of Cell lysis, Clarification, TFF (Batch or Single-Pass) and Affinity Capture. The continuous process can be completed in less than a day, whereas the batch process can be multi-day.

[0059] Figure 24 schematically depicts exemplary arrangements for multi-stage membrane module cassettes to be used with Single-Pass TFF. The configurations depict four to seven tiers of membrane module cassettes where the initial tiers (left side) contain more or same number of membrane module cassettes as the succeeding tiers (moving towards the right side). Total area and path length of the membrane module cassettes also are set forth.

[0060] Figure 25 is a graph depicting volumetric concentration factor (VCF) versus transmembrane pressure (TMP) using the 4-in-series, 5-in-series, 6-in- series and 7-in-series exemplary configurations depicted in Figure 24 with a feed comprising an exemplary AAV, here AAV9 comprising a SpyTag insert.

[0061] Figure 26 depicts data from a 5-in-series configuration according to Figure 24 at flow rates of 90 ml / minute, 120 ml / minute and 150 ml / minute. The log best-fit equation of VCF= A ln (TMP-B) using the values at each flow rate set forth near the plot (and rounded off in the included table) can be used to parameterize the data. At the right side of the figure, there is a graph of parameter value (A, B) and feed flow rate in liters per square meter of membrane per hour (LMH) for 4-in-series and 5-in-series exemplary configurations of Figure 24 and allows optimized conditions to be selected in silico using an exemplary AAV, hereREGN 11547 AAV9 comprising a SpyTag insert. This model can be used to predict the VCF for any flow rate and TMP for an in-series configuration of interest.

[0062] Figure 27A is a design space model based on Figures 25 and 26 using the 5-in-series configuration of Figure 24. Here, the process target was 35 LMH, and the intersecting lines indicate a VCF of 8 and a TMP of 10 psi. An exemplary acceptable zone would be a VCF of 6-10 and a TMP of 7.5 to 12.5 psi. Figure 27B is an exemplary comparison of process parameters between SPTFF and Batch TFF. With Batch TFF, typically there would be one batch before the next operation. However, depending on the scheduling of upstream production bioreactors and bioreactor titers, there could be pooling of multiple batches before the next operation

[0063] Figure 28 depicts data from a bench-scale trial to determine the number of buffer washes need to attain about a 90% recovery of AAV, here AAV9 with integrated SpyTag, in a low-TMP process. On average, the AAV9 here contained an average of 6 SpyTag peptides per capsid. Capsid titer in retentate (cp / ml) versus SPTFF operating time (minutes) was measured using four buffer flushes. As the right side of the figure shows, it was determined that only two buffer flushes were required to achieve about a 90% recovery with a VCF of 8x.

[0064] Figure 29 is a graph depicting Permeate Flux (LMH), Throughput (L / m2), Feed Flow Rate (L / hr) and TMP (psi) in a pilot-scale trial. The data showed flux decline and TMP build up. To mitigate TMP increase beyond 12.5 psi, feed flow rate was slowed. This resulted in a longer process time of 180 minutesREGN 11547 rather than the expected 90 minutes and an overall VCF of 5x was achieved rather than the target of 8x.

[0065] Figure 30 schematically depicts a tween micelle build-up on the TFF membrane, which is believed to be the cause of an unexpected flux decline of about 50%. This figure also set forth the approximate size of AAV, Host Cell protein aggregates (HCP) and Tween-20 micelles. Detergents, such as Tweens, are a common component of cell lysis buffers used in the purification of AAV.

[0066] Figure 31 is a graph depicting fold presence of Tween-20 on the retentate side of membrane and the Permeate side of the membrane for both Batch TFF and SPTFF.

[0067] Figure 32 is a graph depicting the flux decline after two hours with varying percentages of Tween-20 in the lysis buffer. In addition to Tween-20, the buffer contained 20mM Tris, 2mM MgCl2 at a pH of 7.4. The feed flow rate was 35 LMH and the TMP was about 5 to 10 psi.

[0068] Figure 33 compares control with the retentate valve to control with a permeate pump. Option 1 with the retentate valve found that TMP reached 22 psi, and after which the flow had to be reduced from 40 LMH to 30 LMH. VCF dropped from about 10x to about 6x. Option 2 with the permeate pump was superior. TMP was controlled to well under 10 psi and a VCF of 8x was maintained. At the right side of the figure, Option 1 (SPTFF with retentate valve) and Option 2 (SPTFF with permeate pump) were compared to a Batch TFF. Option 1 did not perform as well as Option 2 and Batch TFF. Option 2 was superior to Batch TFF and Option 1 in terms of capsid yield and percent aggregation.REGN 11547

[0069] Figure 34 depicts an overall pilot scale process.

[0070] Figure 35 compares VCFs (1-14), SPTFF retentate flow rates and residence time in affinity capture. VCFs of 7 to 13 and SPTFF retentate flow rates of 75-40 provided an exemplary range of residence time suitable for affinity loading.

[0071] Figure 36 depicts how UV280 profile of affinity capture flow can be used for process monitoring of VCF and process stability using SPTFF for continuous processing. Three different runs were performed for comparison purposes. Run 1 was performed without a permeate pump and achieved a VCF of only 5X. Run 2 was performed with a permeate pump with a feed to retentate flush (with recirculation) and achieved a VCF of 8x. Run 3 was performed with a permeate pump with a feed to retentate flush (with recirculation) and a permeate to retentate flush and achieved a VCF of 10x.

[0072] Figure 37A depicts vector viral titer and viral genome to capsid ratio (Vg:Cp) of different capsid types: a wild type (WT) AAV9, a detargeted AAV9 and a detargeted AAV9 comprising the SpyTag peptide in the viral capsid. Figure 37B depicts virus vector viral titer and viral genome to capsid ratio (Vg:Cp) at different bioreactor scales: 0.2 liters, 2 liters and 50 liters.

[0073] Figure 38 is a graph depicting conjugation reactions between AAV9-SpyT capsids with N272A detargeting mutation, with two different SpyCatcher antibodies, SpyC-ASGR1 mAb and SpyC-FELD1 mAb. Successful conjugation was achieved comparable to results using AAV9-SpyT capsids with W503A detargeting mutations (Figure 5A).REGN 11547 DETAILED DESCRIPTION OF THE INVENTIONS DEFINITIONS

[0074] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the inventions belong.

[0075] The term “about” in the context of numerical values and ranges refers to values or ranges that approximate or are close to the recited values or ranges such that the invention can perform as intended, such as having a desired rate, amount, density, degree, increase, decrease, percentage, ratio, value, purity, pH, concentration, presence of a form or variant, temperature or amount of time, as is apparent from the teachings contained herein. For example, “about” can signify values either above or below the stated value in a range of approx. + / - 10% or more or less depending on the ability to perform. Thus, this term encompasses values beyond those simply resulting from systematic error.

[0076] “Antibodies” (also referred to as "immunoglobulins") are examples of proteins having multiple polypeptide chains and extensive post- translational modifications. The canonical immunoglobulin protein (for example, IgG) comprises four polypeptide chains - two light chains and two heavy chains. Each light chain is linked to one heavy chain via a cysteine disulfide bond, and the two heavy chains are bound to each other via two cysteine disulfide bonds. Immunoglobulins produced in mammalian systems are also glycosylated at various residues (for example, at asparagine residues) with various polysaccharides, andREGN 11547 can differ from species to species, which may affect antigenicity for therapeutic antibodies. Butler and Spearman, "The choice of mammalian cell host and possibilities for glycosylation engineering", Curr. Opin. Biotech.30:107-112 (2014).

[0077] An antibody includes immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains inter- connected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs may be abbreviated as LCDRl, LCDR2 and LCDR3. The term "high affinity" antibody refers to those antibodies having a binding affinity to their target of at least 10-9M, at least 10-10M; at least 10-11M; or at least 10-12M, as measured by surface plasmon resonance, for example, BIACORE™ or solution-affinity ELISA.

[0078] The phrase "bispecific antibody" includes, but is not limited to, an antibody capable of selectively binding two or more epitopes, and can be used as retargeting molecules. In one approach, bispecific antibodies generally comprise two different heavy chains, with each heavy chain specifically binding a different epitopeREGN 11547 -- either on two different molecules (for example, antigens) or on the same molecule (for example, on the same antigen). If a bispecific antibody is capable of selectively binding two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will generally be at least one to two, three or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, and vice versa. The epitopes recognized by the bispecific antibody can be on the same or a different target (for example, on the same or a different protein). Bispecific antibodies can be made, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in a cell that expresses an immunoglobulin light chain. Predominantly, a bispecific antibody has two heavy chains each having three heavy chain CDRs, followed by (N-terminal to C-terminal) a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that either does not confer antigen- binding specificity but that can associate with each heavy chain, or that can associate with each heavy chain and that can bind one or more of the epitopes bound by the heavy chain antigen-binding regions, or that can associate with each heavy chain and enable binding or one or both of the heavy chains to one or both epitopes. In another approach, a bispecific antibody can be an antibody where there are alterations to one or both of the heavy chains on either the variable or constant region.. For example, there can be a bispecific antibody where one of the heavy chains is joined, modified to include, or genetically fused to another molecule, such as a SpyCatcher protein or fragment / derivative thereof, while the other heavy chainREGN 11547 is not .Techniques and components can be referenced in International Patent Publication Nos. WO2010 / 151792, WO2014 / 047231, WO2016 / 018740, WO2016 / 161010, WO2019 / 006046 and U.S. Patent No.8,586,713.

[0079] The term “components” refers to the constituent molecules needed to produce recombinant AAV, such as covalently surface modified adeno- associated viruses and includes, but is not limited to, promoters, polyadenylation signals, transgenes, polynucleotides encoding retargeting molecules, AAV cap genes, AAV rep genes, ITRs, helper polynucleotide sequence(s), polynucleotides encoding a first member of a specific binding pair and a second cognate member of a specific binding pair (for covalently surface modified AAV), as well as peptides encoded by the genes and polynucleotide sequences. Optional components include detargeting mutation sequences, introns, IRESs, RRSs, operators and enhancers.

[0080] The phrase “assembly of components” refers to components that assemble together by way of bonds, forces, interactions and / or attractions. Examples include the assembly of heavy and light chains to form antibodies, capsid proteins, and isopeptide bonds formed during conjugation of specific binding pairs.

[0081] The phrase "heavy chain," or "immunoglobulin heavy chain" includes an immunoglobulin heavy chain constant region sequence from any organism, and unless otherwise specified includes a heavy chain variable domain. Heavy chain variable domains include three heavy chain CDRs and four FR regions, unless otherwise specified. Fragments of heavy chains include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has, following the variable domain (from N-terminal to C-terminal), a CH1 domain, a hinge, a CH2 domain, andREGN 11547 a CH3 domain. A functional fragment of a heavy chain includes a fragment that is capable of specifically recognizing an antigen (for example, recognizing the antigen with a KD in the micromolar, nanomolar, or picomolar range), that is capable of expressing and secreting from a cell, and that comprises at least one CDR.

[0082] The phrase "light chain" includes an immunoglobulin light chain constant region sequence from any organism, and unless otherwise specified includes human kappa and lambda light chains. Light chain variable (VL) domains typically include three light chain CDRs and four framework (FR) regions, unless otherwise specified. Generally, a full-length light chain includes, from amino terminus to carboxyl terminus, a VL domain that includes FR1-CDR1- FR2-CDR2-FR3-CDR3- FR4, and a light chain constant domain. Light chains that can be used with these inventions include those, for example, that do not selectively bind either the first or second antigen selectively bound by the antigen-binding protein. Suitable light chains include those that can be identified by screening for the most commonly employed light chains in existing antibody libraries (wet libraries or in silico), where the light chains do not substantially interfere with the affinity and / or selectivity of the antigen-binding domains of the antigen-binding proteins. Suitable light chains include those that can bind one or both epitopes that are bound by the antigen- binding regions of the antigen-binding protein.

[0083] “Recombinase recognition sites” (RRS), also known as “heterospecific recombination sites,” are used in recombinase mediated cassette exchange (RMCE). Cre / Lox Dre / Rox, Vre / Vlox, SCre / Slox and Flp / Frt are suitable systems, for example. Suitable RRSs for use according to the inventions include Lox P, Lox 66, Lox 71, Lox 511, Lox 2272, Lox 2372, Lox 5171, Lox M2, Lox M3, loxREGN 11547 M7 and Lox M11. These sites can be referred to generically as first (1), second (2), third (3), fourth (4), fifth (5), sixth (6), seventh (7), eighth (8), ninth (9), tenth (10), etc., as is apparent from the context of usage.

[0084] An “intron” is a section of DNA that is not protein encoding, and typically is located between “exons”, which encode protein regions. An intron is removed to form a mature messenger RNA, which is translated to form protein. Some introns are those that can affect the starting point of translation, and exemplars are the hCMV-IE intron (Human cytomegalovirus immediate early protein) and FMDV intron (Foot and Mouth Disease Virus).

[0085] “Intronic selection” refers to the optional use of recombinase recognition sites located in intronic regions to allow for integration of multiple cassettes to form a construct. See Published applications US 2019 / 0263937 A1 and US 2019 / 0233544 A1. For example, selection markers and reporter genes can be engineered to include introns with RRSs contained therein. Intronic selection can be used to create constructs sectionally. For instance, a large construct containing multiple cassettes can be created by using smaller, constituent constructs.

[0086] “Antibody derivatives and fragments” include, but are not limited to: antibody fragments (for example, Fab, ScFv-Fc, dAB-Fc, half antibodies and other combinations of heavy and / or light chains), multispecifics (for example, bispecifics, IgG-ScFv, IgG-dab, ScFV-Fc-ScFV, trispecifics).

[0087] The phrase "Fc-containing protein" includes antibodies, bispecific antibodies, antibody derivatives containing an Fc, antibody fragments containing an Fc, Fc-fusion proteins, immunoadhesins, and other binding proteinsREGN 11547 that comprise at least a functional portion of an immunoglobulin CH2 and CH3 region. A "functional portion" refers to a CH2 and CH3 region that can bind a Fc receptor (for example, an FcyR; or an FcRn, (neonatal Fc receptor), and / or that can participate in the activation of complement. If the CH2 and CH3 region contains deletions, substitutions, and / or insertions or other modifications that render it unable to bind any Fc receptor and also unable to activate complement, the CH2 and CH3 region is not functional. Fc-fusion proteins include, for example, Fc-fusion (N- terminal), Fc-fusion (C-terminal), mono-Fc-fusion and bispecific Fc-fusion proteins.

[0088] “Fc" stands for fragment crystallizable, and is often referred to as a fragment constant. Antibodies contain an Fc region that is made up of two identical protein sequences. IgG has heavy chains known as γ-chains. IgA has heavy chains known as α-chains, IgM has heavy chains known as µ-chains. IgD has heavy chains known as σ-chains. IgE has heavy chains known as ε-chains. In nature, Fc regions are the same in all antibodies of a given class and subclass in the same species. Human IgGs have four subclasses and share about 95% homology amongst the subclasses. In each subclass, the Fc sequences are the same. For example, human IgG1 antibodies will have the same Fc sequences. Likewise, IgG2 antibodies will have the same Fc sequences; IgG3 antibodies will have the same Fc sequences; and IgG4 antibodies will have the same Fc sequences. Alterations in the Fc region can create charge variation.

[0089] "Fc-fusion proteins" comprise part or all of two or more proteins, one of which is an Fc portion of an immunoglobulin molecule, that are not fused in their natural state. Fc-fusion proteins include Fc-Fusion (N-terminal), Fc-Fusion (C- terminal), Mono Fc-Fusion and Bi-specific Fc-Fusion. Preparation of fusion proteinsREGN 11547 comprising certain heterologous polypeptides fused to various portions of antibody- derived polypeptides (including the Fc domain) has been described, for example, by Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88: 10535-39 (1991) ; Byrn et al., Nature 344:677-70, 1990; and Hollenbaugh et al., "Construction of Immunoglobulin Fusion Proteins", in Current Protocols in Immunology, Suppl.4, pages 10.19.1 - 10.19.11 (1992). "Receptor Fc-fusion proteins" comprise one or more of one or more extracellular domain(s) of a receptor coupled to an Fc moiety, which in some aspects comprise a hinge region followed by a CH2 and CH3 domain of an immunoglobulin. In some aspects, the Fc-fusion protein contains two or more distinct receptor chains that bind to a single or more than one ligand(s). Some receptor Fc-fusion proteins may contain ligand binding domains of multiple different receptors. Receptor Fc- fusion proteins are also referred to as "traps," "trap molecules" or “trap proteins.” For example, such trap proteins include an IL-1 trap (for example, Rilonacept, which contains the IL- lRAcP ligand binding region fused to the IL-1R1 extracellular region fused to Fc of hlgGl ; see U.S. Pat. No.6,927,044, or a VEGF Trap (for example, Aflibercept, which contains the Ig domain 2 of the VEGF receptor Fltl fused to the Ig domain 3 of the VEGF receptor Flkl fused to Fc of hlgG1 See U.S. Pat. Nos. 7,087,411 and 7,279,159.

[0090] “Polynucleotide” includes a sequence of nucleotides covalently joined, and includes RNA and DNA. Oligonucleotides are considered shorter polynucleotides. Genes are DNA polynucleotides (polydeoxyribonucleic acid) that ultimately encode polypeptides, which are translated from RNA (polyribonucleic acid) that was typically transcribed from DNA. DNA polynucleotides also can encode RNA polynucleotides that is not translated, but rather function as RNA “products”. TheREGN 11547 type of polynucleotide (that is, DNA or RNA) is apparent from the context of the usage of the term. A polynucleotide referred to or identified by the polypeptide it encodes sets forth and covers all suitable sequences in accordance with codon degeneracy. Polynucleotides, including those disclosed herein, include percent identity sequences and homologous sequences when indicated.

[0091] “Polypeptide” or “peptide” refers to sequence(s) of amino acids covalently joined. Polypeptides include natural, semi-synthetic and synthetic proteins and protein fragments. “Polypeptide” and “protein” can be used interchangeably. Oligopeptides are considered shorter polypeptides.

[0092] A “gene of interest” (GOI) encodes a “protein of interest” or “polypeptide of interest” and optionally can include other associated sequences. The sequences can be natural, semi-synthetic or synthetic. Native sequences, mutant sequences and degenerate sequences can be GOIs. A gene of interest also can be referred to as a “transgene.”

[0093] A “nucleotide of interest” includes GOIs and sequences encoding non-translated RNAs / non-coding RNAs (such as, but not limited to, antisense RNA, micro RNA, small interfering RNA, catalytic RNA and ribozymes). NOIs and GOIs also can be referred to as “payloads.”

[0094] “Protein of interest” or “polypeptide of interest” (POI) can have any amino acid sequence, and includes any protein, polypeptide, or peptide that is desired to be expressed, typically for gene therapy purposes. Protein types can include, but are not limited to, receptors, fusion proteins, agonists, antagonists, activators, inhibitors, enzymes (such as those used in enzyme replacement therapy),REGN 11547 factors and co-factors, repressors, activators, ligands, protein hormones, therapeutic proteins, suicide proteins, structural proteins, storage proteins, transport proteins, signal proteins, neurotransmitters and contractile proteins. Derivatives, components, domains, chains and fragments of the above also are included. The sequences can be natural, semi-synthetic or synthetic.

[0095] “Purification” in its various grammatical forms includes, but is not limited to, the use of one or more procedures such as depth filtration, tangential flow filtration, affinity capture, ionic exchange and the like.

[0096] The term "recombinant capsid protein" includes a capsid protein that has at least one mutation in comparison to the corresponding capsid protein of the wild-type virus, which wild-type may be a reference and / or control virus for comparative study. A recombinant capsid protein includes a capsid protein that comprises a heterologous amino acid sequence, which may be inserted into and / or displayed by the capsid protein. "Heterologous" in a general context means heterologous as compared to the virus, from which the capsid protein is derived. The inserted amino acids can simply be inserted between two given amino acids of the capsid protein. An insertion of amino acids can also go along with a deletion of given amino acids of the capsid protein at the site of insertion, for example, 1 or more capsid protein amino acids are substituted by 5 or more heterologous amino acids). An example of a heterologous amino acid sequence that can be inserted is a member of a specific binding pair, such SpyTag.

[0097] “Detargeting” refers to reducing or abolishing AAV natural preferential transduction, primarily of the liver, by mutating Cap proteins. ForREGN 11547 example, mutations in the galactose binding domain of VP1 assist in detargeting the liver. Other mutations include, but are not limited to, Cap mutations that alter heparin binding or sialic acid binding. These mutations are optional and can be referred to as “detargeting mutations.”

[0098] By way of example, different AAV serotypes are known to preferentially transduce the cells of different tissues. Tissue specificity is limited, and AAV is known to preferentially transduce the liver, which can be a safety and efficacy concern in some contexts. The inventions further provide mutations in the VP1 Protein of AAV9, for example, to lower the AAV preferential transduction of the liver. The AAV9 mutations include N272A and W503A substitutions, where alanine replaces both asparagine at position 272 of VP1 and tryptophan at position 503 of VP1. One or both of the mutations can be undertaken in the VP1 protein. Optionally, other amino acids, such as glutamic acid, serine or others, can be used instead of alanine for substitution. Additional detargeting mutation sites include, but are not limited to, N470, D271, and Y446. The inventions provide exemplary mutations for use with AAVs as follows, which for each serotype can be utilized singly or in combinations : AAV1 – N500E; AAV2 – R585A and R588A; AAV5 – T571S; AAV6 – N500E, K531A and K531E; AAV9 – N272A, W503A, T568P, Q590L, N498Y, L602F, S414N, G453D, K557E, T582I, and combinations include, but are not limited to, T568P + Q590L, N498Y + L602F, S414N + G453D + K557E +T582I.REGN 11547 These and others are set forth in the chart below:REGN 11547Other mutations and detargeting approaches, such as variable loop swaps, are disclosed in publications or otherwise available See, for example, Shen et al., Molecular Therapy 15: 1955–62 (2007). These technologies can be used according to the inventions. Detargeting mutations are optional.REGN 11547

[0099] "Retargeting" or "redirecting" may include a situations in which the wildtype vector targets several cells within a tissue and / or several organs within an organism, which general targeting of the tissue or organs is reduced or abolished by provision of a retargeting molecule, which retargets the covalently surface modified AAV to a different, and optionally more specific cell in the tissue or a specific organ in the organism.

[0100] The term “retargeting molecule” (Rm) is a molecule useful for targeting an antigen, receptor protein, including glycoproteins, and / or ligand (“collectively targets”) found on the surface of a cell, referred to as a “target cell.” The retargeting molecule can be bound to a polypeptide that is part of a specific binding pair. For example, a retargeting molecule could be bound to SpyCatcher (or fragments / derivatives thereof) in order to utilize the SpyTag-SpyCatcher system. The retargeting molecule can target the cell that has the antigen, receptor and / or ligand that the retargeting molecule can bind to, and thereby direct a recombinant AAV to that cell. Fc-containing proteins, such as antibodies, monoclonal antibodies (including derivatives, fragments, half antibodies and other heavy chain and / or light chain combinations), multispecific antibodies (for example, bispecifics and trispecifics), IgG-ScFv, IgG-dab, ScFV-Fc-ScFV, Fc-fusion proteins, receptor-Fc fusion proteins, and trap proteins, are useful as retargeting molecules. Mini-trap proteins also can be used as retargeting molecules. In the literature, the phrase “targeting ligand” has been used for molecules that are useful for targeting. See Yan et al., Pharmaceutics 16: 248 (2024).

[0101] All human and non-human antibody classes can be used as retargeting molecules. IgA, IgD IgE, IgG and IgM can be used as retargetingREGN 11547 molecules. IgG is a preferred class, and includes subclasses IgG1 (including IgG1λ and IgG1κ), IgG2, IgG3, and IgG4. Further antibody types include a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecifϊc antibody, a bispecific antibody, a trispecific antibody, an antigen binding antibody fragment, a single chain antibody, a diabody, triabody, tetrabody, Fab, F(ab’), F(ab')2 and a half antibody.

[0102] "Specific binding pair”, also referred to a "protein:protein binding pair" and the like include two proteins (that is, a first member, such as a first polypeptide, and a second cognate member, such as a second polypeptide) that interact to form a covalent isopeptide bond under conditions that enable or facilitate isopeptide bond formation, wherein the term "cognate" refers to components that function together by to reacting together to form an isopeptide bond. Thus, two proteins that react together efficiently to form an isopeptide bond under conditions that enable or facilitate isopeptide bond formation can also be referred to as being a "complementary" pair of peptide linkers. Specific binding pairs capable of interacting to form a covalent isopeptide bond are reviewed in Veggiani et al. (2014) Trends Biotechnol.32:506, and include, for example, peptide:peptide binding pairs such as SpyTag:SpyCatcher, SpyTag002:SpyCatcher002, SpyTag:KTag, isopeptag:pilin C, SnoopTag:SnoopCatcher and others. Spy Tag002:SpyCatcher002 and SpyTag003:SpyCatcher003 are different iterations of Spy Tag:Spy Catcher.

[0103] The term "isopeptide bond" refers to an amide bond between a carboxyl or carboxamide group and an amino group at least one of which is not derived from a protein main chain or alternatively viewed is not part of the protein backbone. An isopeptide bond may form within a single protein or may occurREGN 11547 between two peptides or a peptide and a protein. Thus, an isopeptide bond may form intramolecularly within a single protein or intermolecularly, that is between two peptide / protein molecules, such as between two peptide linkers. Typically, an isopeptide bond may occur between a lysine residue and an asparagine, aspartic acid, glutamine, or glutamic acid residue or the terminal carboxyl group of the protein or peptide chain or may occur between the alpha-amino terminus of the protein or peptide chain and an asparagine, aspartic acid, glutamine or glutamic acid. Each residue of the pair involved in the isopeptide bond is referred to herein as a reactive residue. An isopeptide bond may form between a lysine residue and an asparagine residue or between a lysine residue and an aspartic acid residue. Particularly, isopeptide bonds can occur between the side chain amine of lysine and carboxamide group of asparagine or carboxyl group of an aspartate.

[0104] “Mosaicism” refers to the ratio of AAV cap sequence containing plasmids that also contain a sequence encoding a first member as compared to the cap plasmids without the first member sequence. Typically, a cap plasmid will contain both the AAV cap gene and the AAV rep gene. An example is plasmid pRC as taught herein. An example of a plasmid that also encodes a first member is pRC- SpyT as taught herein. Plasmid pRC-SpyT serves as an exemplar of a cap plasmid that comprises a nucleotide sequence of a first member.

[0105] For example, when 1 / 10 of all the pRC plasmids transfected are pRC-SpyT plasmids, the AAV capsid is expected to have an average of 6 SpyT, as 1 / 10 of the 60 viral proteins are expected to have a SpyT (1 pRC-SpyT / 10 total cap- containing plasmids) x 60 = 6). Similarly, a transfection ratio of 1 / 4 is expected to result in 15 SpyT per AAV capsid (1 / 4 x 60 = 15), and a transfection ratio of 1 / 30 isREGN 11547 expected to result in 2 SpyT per AAV capsid (1 / 30 x 60 = 2), as the capsid is stoichiometrically assembled inside the cell using the available viral proteins. The ratio of pRC-SpyT to total pRC plasmids during transfection is referred to as the “mosaicism” of the AAV capsid, with “higher mosaicisms” such as 1 / 4 resulting in higher SpyT on the AAV capsids on average, and “lower mosaicisms” such as 1 / 30 resulting in fewer SpyT on the AAV capsids on average. Thus, where the ratio of pRC-FM to pRC plasmids is a larger fractional value, the mosaicism will be higher. As explained above, 1 / 4 is a larger fractional value than 1 / 30.

[0106] Mosaicisms can be selected by the person skilled in the art in view of the teachings contained herein. Mosaicisms can be ratios as set forth below and those thereabout and therebetween: 1 / 1, 1 / 1.1, 1 / 1.2, 1 / 1.3, 1 / 1.4, 1 / 1.5, 1 / 1.6, 1 / 1.7, 1 / 1.8, 1 / 1.9, 1 / 2, 1 / 2.1, 1 / 2.2, 1 / 2.3, 1 / 2.4, 1 / 2.5, 1 / 2.6, 1 / 2.7, 1 / 2.8, 1 / 2.9, 1 / 3, 1 / 3.1, 1 / 3.2, 1 / 3.3, 1 / 3.4, 1 / 3.5, 1 / 3.6, 1 / 3.7, 1 / 3.8, 1 / 3.9, 1 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, 1 / 12.8, 1 / 12.9, 1 / 13, 1 / 13.1, 1 / 13.2, 1 / 13.3, 1 / 13.4, 1 / 13.5, 1 / 13.6, 1 / 13.7, 1 / 13.8, 1 / 13.9, 1 / 14, 1 / 14.1, 1 / 14.2, 1 / 14.3, 1 / 14.4, 1 / 14.5, 1 / 14.6, 1 / 14.7, 1 / 14.8, 1 / 14.9, 1 / 15, 1 / 15.1, 1 / 15.2, 1 / 15.3, 1 / 15.4, 1 / 15.5, 1 / 15.6, 1 / 15.7, 1 / 15.8, 1 / 15.9, 1 / 16, 1 / 16.1, 1 / 16.2, 1 / 16.3, 1 / 16.4, 1 / 16.5, 1 / 16.6, 1 / 16.7, 1 / 16.8, 1 / 16.9, 1 / 17, 1 / 17.1, 1 / 17.2, 1 / 17.3, 1 / 17.4,REGN 11547 / 17.5, 1 / 17.6, 1 / 17.7, 1 / 17.8, 1 / 17.9, 1 / 18, 1 / 18.1, 1 / 18.2, 1 / 18.3, 1 / 18.4, 1 / 18.5, / 18.6, 1 / 18.7, 1 / 18.8, 1 / 18.9, 1 / 19, 11 / 9.1, 1 / 19.2, 1 / 19.3, 1 / 19.4, 1 / 91.5, 1 / 19.6, / 19.7, 1 / 19.8, 1 / 19.9, 1 / 20, 1 / 21, 1 / 21.1, 1 / 21.2, 1 / 21.3, 1 / 21.4, 1 / 21.5, 1 / 21.6, / 21.7, 1 / 21.8, 1 / 21.9,1 / 22, 1 / 22.1, 1 / 22.2, 1 / 22.3, 1 / 22.4, 1 / 22.5, 1 / 22.6, 1 / 22.7, / 22.8, 1 / 22.9, 1 / 23, 1 / 23.1, 1 / 23.2, 1 / 23.3, 1 / 23.4, 1 / 23.5, 1 / 23.6, 1 / 23.7, 1 / 23.8, / 23.9, 1 / 24, 1 / 24.1, 1 / 24.2, 1 / 24.3, 1 / 24.4, 1 / 24.5, 1 / 24.6, 1 / 24.7, 1 / 24.8, 1 / 24.9, / 25, 1 / 25.1, 1 / 25.2, 1 / 25.3, 1 / 25.4, 1 / 25.5, 1 / 25.6, 1 / 25.7, 1 / 25.8, 1 / 25.9, 1 / 26, / 26.1, 1 / 26.2, 1 / 26.3, 1 / 26.4, 1 / 26.5, 1 / 26.6, 1 / 26.7, 1 / 26.8, 12 / 6.9, 1 / 27, 1 / 27.1, / 27.2, 1 / 27.3, 1 / 27.4, 1 / 27.5, 1 / 27.6, 1 / 27.7, 1 / 27.8, 1 / 27.9, 1 / 28, 1 / 28.1, 1 / 28.2, / 28.3, 1 / 28.4, 1 / 28.5, 1 / 28.6, 1 / 28.7, 1 / 28.8, 1 / 28.9, 1 / 29, 1 / 29.1, 1 / 29.2, 1 / 29.3, / 29.4, 1 / 29.5, 1 / 29.6, 1 / 29.7, 1 / 29.8, 1 / 29.91 / 30, 1 / 30.1, 1 / 30.2, 1 / 30.3, 1 / 30.4, / 30.5, 1 / 30.6, 1 / 30.7, 1 / 30.8, 1 / 30.9, 1 / 31, 1 / 31.1, 1 / 31.2, 1 / 31.3, 1 / 31.4, 1 / 31.5, / 31.6, 1 / 31.7, 1 / 31.8, 1 / 31.9, 1 / 32, 1 / 32.1, 1 / 32.2, 1 / 32.3, 1 / 32.4, 1 / 32.5, 1 / 32.6, / 32.7, 1 / 32.8, 1 / 32.9, 1 / 33, 1 / 33.1, 1 / 33.2, 1 / 33.3, 1 / 33.4, 1 / 33.5, 1 / 33.6, 1 / 33.7, / 33.8, 1 / 33.9, 1 / 34, 1 / 34.1, 1 / 34.2, 1 / 34.3, 1 / 34.4, 1 / 34.5, 1 / 34.6, 1 / 34.7, 1 / 34.8, / 34.9, 1 / 35, 1 / 35.1, 1 / 35.2, 1 / 35.3, 1 / 35.4, 1 / 35.5, 1 / 35.6, 1 / 35.7, 1 / 35.8, 1 / 35.9, / 36, 1 / 36.1, 1 / 36.2, 1 / 36.3, 1 / 36.4, 1 / 36.5, 1 / 36.6, 1 / 36.7, 1 / 36.8, 1 / 36.9, 1 / 37, / 37.1, 1 / 37.2, 1 / 37.3, 1 / 37.4, 1 / 37.5, 1 / 37.6, 1 / 37.7, 1 / 37.8, 1 / 37.9, 1 / 38, 1 / 38.1, / 38.2, 1 / 38.3, 1 / 38.4, 1 / 38.5, 1 / 38.6, 1 / 38.7, 1 / 38.8, 1 / 38.9, 1 / 39, 1 / 39.1, 1 / 39.2, / 39.3, 1 / 39.4, 1 / 39.5, 1 / 39.6, 1 / 39.7, 1 / 39.8, 1 / 39.9, 1 / 40, 1 / 40.1, 1 / 40.2, 1 / 40.3, / 40.4, 1 / 40.5, 1 / 40.6, 1 / 40.7, 1 / 40.8, 1 / 40.9, 1 / 41, 1 / 41.1, 1 / 41.2, 1 / 41.3, 1 / 41.4, / 41.5, 1 / 41.6, 1 / 41.7, 1 / 41.8, 1 / 41.9, 1 / 42, 1 / 42.1, 1 / 42.2, 1 / 42.3, 1 / 42.4, 1 / 42.5, / 42.6, 1 / 42.7, 1 / 42.8, 1 / 42.9, 1 / 43, 1 / 43.1, 1 / 43.2, 1 / 43.3, 1 / 43.4, 1 / 43.5, 1 / 43.6, / 43.7, 1 / 43.8, 1 / 43.9, 1 / 44, 1 / 44.1, 1 / 44.2, 1 / 44.3, 1 / 44.4, 1 / 44.5, 1 / 44.6, 1 / 44.7, / 44.8, 1 / 44.9, 1 / 45, 1 / 45.1, 1 / 45.2, 1 / 45.3, 1 / 45.4, 1 / 45.5, 1 / 45.6, 1 / 45.7, 1 / 45.8,REGN 11547 1 / 45.9, 1 / 46, 1 / 46.1, 1 / 46.2, 1 / 46.3, 1 / 46.4, 1 / 46.5, 1 / 46.6, 1 / 46.7, 1 / 46.8, 1 / 46.9, 1 / 47, 1 / 47.1, 1 / 47.2, 1 / 47.3, 1 / 47.4, 1 / 47.5, 1 / 47.6, 1 / 47.7, 1 / 47.8, 1 / 47.9, 1 / 48, 1 / 48.1, 1 / 48.2, 1 / 48.3, 1 / 48.4, 1 / 48.5, 1 / 48.6, 1 / 48.7, 1 / 48.8, 1 / 48.9, 1 / 49, 1 / 49.1, 1 / 49.2, 1 / 49.3, 1 / 49.4, 1 / 49.5, 1 / 49.6, 1 / 49.7, 1 / 49.8, 1 / 49.9, 1 / 50, 1 / 50.1, 1 / 50.2, 1 / 50.3, 1 / 50.4, 1 / 50.5, 1 / 50.6, 1 / 50.7, 1 / 50.8, 1 / 50.9, 1 / 51, 1 / 51.1, 1 / 51.2, 1 / 51.3, 1 / 51.4, 1 / 51.5, 1 / 51.6, 1 / 51.7, 1 / 51.8, 1 / 51.9, 1 / 52, 1 / 52.1, 1 / 52.2, 1 / 52.3, 1 / 52.4, 1 / 52.5, 1 / 52.6, 1 / 52.7, 1 / 52.8, 1 / 52.9, 1 / 53, 1 / 53.1, 1 / 53.2, 1 / 53.3, 1 / 53.4, 1 / 53.5, 1 / 53.6, 1 / 53.7, 1 / 53.8, 1 / 53.9, 1 / 54, 1 / 54.1, 1 / 54.2, 1 / 54.3, 1 / 54.4, 1 / 54.5, 1 / 54.6, 1 / 54.7, 1 / 54.8, 1 / 54.9, 1 / 55, 1 / 55.1, 1 / 55.2, 1 / 55.3, 1 / 55.4, 1 / 55.5, 1 / 55.6, 1 / 55.7, 1 / 55.8, 1 / 55.9, 1 / 56, 1 / 56.1, 1 / 56.2, 1 / 56.3, 1 / 56.4, 1 / 56.5, 1 / 56.6, 1 / 56.7, 1 / 56.8, 1 / 56.9, 1 / 57, 1 / 57.1, 1 / 57.2, 1 / 57.3, 1 / 57.4, 1 / 57.5, 1 / 57.6, 1 / 57.7, 1 / 57.8, 1 / 57.9, 1 / 58, 1 / 58.1, 1 / 58.2, 1 / 58.3, 1 / 58.4, 1 / 58.5, 1 / 58.6, 1 / 58.7, 1 / 58.8, 1 / 58.9, 1 / 59, 1 / 59.1, 1 / 59.2, 1 / 59.3, 1 / 59.4, 1 / 59.5, 1 / 59.6, 1 / 59.7, 1 / 59.8, 1 / 59.9, or 1 / 60.

[0107] Ranges of ratios of 1 / 1 to 1 / 60 can be employed, and subranges as set forth below.

[0108] Preferred mosaicisms are : 1 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6,REGN 11547 1 / 11.7, 1 / 11.8, 1 / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, 1 / 12.8, 1 / 12.9 or 1 / 13.

[0109] More preferred mosaicisms are: 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, or 1 / 12.

[0110] Still more preferred mosaicisms are: 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, or 1 / 11.

[0111] Preferred ranges of ratios are 1 / 4 to 1 / 13; 1 / 5 to 1 / 12; 1 / 6 to 1 / 11, 1 / 7 to 1 / 10.5 or 1 / 7.5 to 1 / 10.

[0112] “Reporter proteins” as used herein, refers to any protein capable of generating directly or indirectly a detectable signal. Reporter proteins typically fluoresce, or catalyze a colorimetric, bioluminescence, or fluorescent reaction, and often are referred to as “color proteins,” “bioluminescent proteins” or “fluorescent proteins.” However, a reporter protein also can be non-enzymatic and non- fluorescent as long as it can be detected by another protein or moiety, such as a cell surface protein detected with a fluorescent ligand. A reporter protein also can be anREGN 11547 inactive protein that is made functional through interaction with another protein that is fluorescent or catalyzes a reaction. Accordingly, any suitable reporter protein, as understood by one of skill in the art, could be used. The reporter protein can be selected from fluorescent protein, luciferase, alkaline phosphatase, β-galactosidase, β-lactamase, dihydrofolate reductase, ubiquitin, and variants thereof. Fluorescent proteins are useful for the recognition of gene cassettes that have or have not been successfully inserted and / or replaced, as the case may be. Fluid cytometry and fluorescence–activated cell sorting are suitable for detection. Examples of fluorescent proteins are well-known in the art, including, but not limited to Discosoma coral (DsRed), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), cyano fluorescent protein (CFP), enhanced cyano fluorescent protein (eCFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (eYFP) and far-red fluorescent protein (e.g. mKate, mKate2, mPlum, mRaspberry or E2-crimson. See, for example, U.S. Patent Nos.9,816,110. Reporter proteins are encoded by polynucleotides, and are referred to herein as “reporter genes” or “reporter protein genes.” Reporter genes and proteins can be referred to generically as first (1), second (2), third (3), fourth (4), fifth (5), sixth (6), seventh (7), eighth (8), ninth (9), tenth (10), etc., as is apparent from the context of usage. Reporters can be considered a type of marker. “Color” or “fluorescent,” in their various grammatical forms, also can be used the more specifically refer to a reporter protein or gene. Where multiple plasmids are used in a transfection, the plasmids can collectively comprise the same reporter genes or different reporter genes.

[0113] “Selectable” or “selection” marker proteins include proteins conferring certain traits, including but not limited to drug resistance or other selectiveREGN 11547 advantages. Selection markers can give the cell receiving the selectable marker gene resistance towards a certain toxin, drug, antibiotic or other compound and permit the cell to produce protein and propagate in the presence of the toxin, drug, antibiotic or other compound, and are often referred to as “positive selectable markers.” Suitable examples of antibiotic resistance markers include, but are not limited to, proteins that impart resistance to various antibiotics, such as kanamycin, spectinomycin, neomycin, gentamycin (G418), ampicillin, tetracycline, chloramphenicol, puromycin, hygromycin, zeocin, and / or blasticidin. There are other selectable markers, often referred to as ”negative selectable markers,” which cause a cell to stop propagating, stop protein production and / or are lethal to the cell in the presence of the negative selectable marker proteins. Thymidine kinase and certain fusion proteins can serve as negative selectable markers, including but not limited to GyrB-PKR. See White et al., Biotechniques, 50: 303-309 (May 2011). Selectable marker proteins and corresponding genes can be referred to generically as first (1), second (2), third (3), fourth (4), fifth (5), sixth (6), seventh (7), eighth (8), ninth (9), tenth (10), etc., as is apparent from the context of usage. Where multiple plasmids are used in a transfection, the plasmids can collectively comprise the same selection marker genes or different selection marker genes.

[0114] The term "target cells" includes any cells in which expression of a nucleotide of interest is desired or tolerated. Preferably, target cells exhibit a “target,” such as a receptor, ligand, protein, including glycoproteins, and / or antigen, including complexes thereof, on their surface that allows the cell to be targeted. Exemplary targets are calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1), asialoglycoprotein receptor 1 (ASGR1), Fel d 1, ENTPD3, PTPRA,REGN 11547 CD20, CD63 and Her2. Additional targets include GAB A, transferrin receptor, CD3, CD34, integrin, adipophilin, AIM-2, ALDHlAl, alpha-actinin-4, alpha-fetoprotein ("AFP"), ARTC1, B-RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta- catenin, BING-4, CA-125, CALCA, carcinoembryonic antigen ("CEA"), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNKlAl, CTAGl, CTAG2, cyclin Dl, Cyclin-Al, dek-can fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen ("ETA"), ETV6-AML1 fusion protein, EZH2, E6, E7, FGF5, FLT3-ITD, FN1, G250 / MN / CAIX, GAGE-1,2,8, GAGE- 3,4,5,6,7, GAS7, glypican-3, GnTV, gplOO / Pmel 17, GPNMB, HAUS3, Hepsin, HER-2 / neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDOl, IGF2B3, IL13Ralpha2, Intestinal carboxyl esterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, LAGE-1, LDLR-fucosyltransferase AS fusion protein, Lengsin, M-CSF, MAGE-A1, MAGE- A 10, MAGE-A12, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, Mel an- A / MART-1 , Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I, N-raw, NA88- A, neo-PAP, NFYC, NY-BR-1, NY-ESO-l / LAGE-2, OA1, OGT, OS-9, P polypeptide, p53, PAP, PAX5, PBF, pml-RARalpha fusion protein, polymorphic epithelial mucin ("PEM"), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB 38 / N Y-MEL- 1 , RAGE-1, RBAF600, RGS5, RhoC, R F43, RU2AS, SAGE, secernin 1, SIRT2, SNRPD1, SOX10, Spl7, SPA17, SSX-2, SSX-4, STEAPl, survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, Telomerase, TGF- betaRII, TPBG, TRAG-3, Triosephosphate isomerase, TRP-l / gp75, TRP-2, TRP2- INT2, tyrosinase, tyrosinase ("TYR"), VEGF, WT1, XAGE-lb / GAGED2a, Kras, NY-REGN 11547 ESOl, MAGE- A3, HPV E2, HPV E6, HPV E7, WT-1 antigen (in lymphoma and other solid tumors), ErbB receptors, Melan A [MARTI], gp 100, tyrosinase, TRP-l / gp 75, and TRP-2 (in melanoma); MAGE-1 and MAGE-3 (in bladder, head and neck, and non-small cell carcinoma); HPV EG and E7 proteins (in cervical cancer); Mucin [MUC-1] (in breast, pancreas, colon, and prostate cancers); prostate-specific antigen [PSA] (in prostate cancer); carcinoembryonic antigen [CEA] (in colon, breast, and gastrointestinal cancers), and such shared tumor-specific antigens as MAGE-2, MAGE-4, MAGE-6, MAGE- 10, MAGE- 12, BAGE-1, CAGE-1,2,8, CAGE-3 TO 7, LAGE-1, NY-ESO-l / LAGE-2, NA-88, GnTV, TRP2-INT2, E6, E7, human glucagon receptor (hGCGR) and. I human ectonucleoside triphosphate diphosphohydrolase 3 (hENTPD3). Other targets can be selected by the person skilled in the art. See WO 2019 / 006046.

[0115] All numerical limits and ranges set forth herein include all numbers or values thereabout or there between of the numbers of the range or limit. The ranges and limits described herein expressly denominate and set forth all integers, decimals and fractional values defined and encompassed by the range or limit. Thus, a recitation of ranges of values herein are intended to serve as a way of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All recited numbers or values expressly set forth and denominate all ranges created by the recited number or values. DESCRIPTION Challenges in Producing Covalently Surface Modified AAVsREGN 11547

[0116] The Spy:Tag:SpyCatcher system is used to engineer many covalently surface modified AAVs, and is used as an exemplar here.

[0117] In vitro conjugation reactions can occur when (1) AAV having SpyTag sequences in its capsid (“AAV-SpyTag”) and (2) SpyCatcher sequences bound to retargeting molecules (“SpyCatcher-Rm”), such as monoclonal antibodies (“SpyCatcher-Ab”) are mixed to allow formation of a joining isopeptide bond. Typically, the in vitro conjugation reaction is carried out after both the AAV-SpyTag and the SpyCatcher-Rm have been each separately and fully purified to remove contaminants, such as host cell proteins, which typically includes clarification, affinity capture chromatography, non-AAV viral inactivation, polishing chromatography, viral retentive filtration, and / or final formulation. However, following conjugation it is necessary to include an additional processing step to remove the unreacted SpyCatcher-Rm species to prevent them from being present in the final conjugated drug product. These challenges are met and overcome by the present inventions.

[0118] To achieve optimum conjugation (also referred to as “complete conjugation” depending on the purpose) of all available SpyTags on an AAV, such as AAV9, capsid surface with a SpyC-Rm species, reaction conditions should be optimized. Parameters affecting the speed and extent of the reaction are pH, temperature, reaction time, reactant concentration, and molar ratio of reactants. Suboptimal parameters can result in lower conjugation efficiency or degradative effects including aggregation or fragmentation of the AAV9-SpyT, SpyC-Rm, or conjugated AAV9-SpyT-SpyC-Rm species. Thus, there is a need to develop optimal reaction conditions as well as to be able to track the formation of conjugated species.REGN 11547

[0119] Another challenge is the variability in the AAV-SpyTag population in terms of the number of SpyTag peptides on the capsid. This is because AAV capsids are assembled stoichiometrically from 60 total subunits of viral proteins VP1, VP2 and VP3, and a subset of each of VP1, VP2 and VP3 proteins will have a SpyTag insertion. This leads to a distribution in the number of SpyTag-VP proteins present in each assembled capsid. The distribution of species can be either normal or non-normal. For example, if 1 / 10 of the VP proteins have SpyTag insertions, the assembled AAV molecules are expected to have a SpyTag on an estimated 60 / 10 = 6 VP proteins on average, but the actual number of SpyTags expressed can range from 0 to 20 or more. See Figure 1A. This variability is compounded by the variability of the conjugation reaction itself, as all or a subset of the SpyTags can be conjugated to SpyCatcher-Rm species, depending on the conjugation reaction conditions (See, for example, Figure 1A).

[0120] Thus, there are multiple species in the post-processing conjugation reaction mixture with significantly different size and charge characteristics which complicates purification: AAV-SpyTag is about 3700 kDa and each SpyCatcher-Ab, for example, is about 160 kDa (when the Ab is a monoclonal antibody), so a molecule with ten surface conjugations would be of size of about 5300 kDa. Figure 1A schematically depicts the species heterogeneity involved in in- vitro conjugation reactions between rAAV9-SpyT and SpyC-Ab species. This figure depicts the heterogeneity at the level of the AAV9-SpyT capsid, depicting the “average” species with 6 SpyTags, as well as species with lower and higher number of SpyTags expressed on the surface. The figure also depicts the heterogeneity at the level of the conjugated AAV9-SpyT-SpyC-ASGR1 mAb species, where theREGN 11547 number of SpyC-ASGR1 mAb species during the in-vitro conjugation reaction were not in significant molar excess compared to the number of AAV9-SpyT species, which results in incomplete conjugation (showing species with average, low and high levels of conjugation in the middle row). The figure further depicts the heterogeneity at the level of the conjugated AAV9-SpyT-SpyC-ASGR1 mAb species, where the number of SpyC-ASGR1 mAb species during the in-vitro conjugation reaction were in significant molar excess compared to the number of AAV9-SpyT species, resulting in optimal conjugation (showing species with average, low and high levels of conjugation in the bottom row). Differences in size and charge are a result of different amounts of antibody conjugation.

[0121] Figure 1B is a graph that depicts retention time (minutes) of high molecular weight (HMV) variants comprising conjugated AAV9-SpyT-SpyC-Ab species, conjugated AAV9-SpyT-SpyC-Ab, unconjugated AAV9-SpyT and unconjugated SpyC-mAb. Size exclusion chromatography (SEC) was employed. Table 1 below provides percentages of HMW fractions, conjugated fractions and unconjugated fractions for various ratios of AAV to antibody (Fabs also can be used, for example). The data shows that 1:300 ratio of AAV capsid to antibody performs best. Notably, the ratios of 1:6 and 1:15 provided the greatest percentages of HWM variants. Table 1REGN 11547

[0122] Thus, there is a need to establish new approaches suitable for removal of unreacted SpyCatcher-Rm species. Challenges include making the methods applicable to removal of different SpyCatcher-Rm species (including but not limited to mAb, Fab, ScFv, bispecific mAb, trispecific mAb, Fc-fusion proteins and other Ab-related formats, such as half antibodies and other heavy chain and / or light chain combinations), which differ greatly in size, charge, isoelectric point (pI) and structure. A challenge with charge-based methods is that molecules with a high level of surface conjugation typically are more “Rm-like” in terms of their surface charge, and thus tend to co-elute with the SpyCatcher-Rm species in many different types of ion-exchange chromatography. Increasing similarity between the surface charge characteristics of conjugated AAV9-SpyT-SpyC-Rm to the unconjugated SpyC-Rm species is expected with increasing number of conjugations. This presents a significant purification challenge, as applying fractionation criteria to limit the presence of unreacted SpyCatcher-Rm in the elution pool would tend to lose the most heavily conjugated species as well, in addition to reducing the product yield.REGN 11547 Thus, anion exchange chromatography methods, which are traditionally useful for AAV separations cannot be applied to this separation in a straightforward manner. Optimization of the ion-exchange process on different modalities (such as cation exchange or multimodal separations) and with different pH and salt conditions is useful to achieve partitioning between the free SpyC-Rm species and the conjugated AAV9-SpyT-SpyC-Rm species without significant co-elution.

[0123] Size-based methods would seem to be an option, particularly due to the apparently large difference in size between the conjugated AAV species (>3700 kDa) compared to the Ab species (<200 kDa). However, despite this difference, the tightly packed structure of AAV makes it very small compared to other viruses, and it has an effective radius of about 15 to 25 nm, while the SpyCatcher- Rm species are around 10-20 nm in radius. Thus, the difference in actual size is not very significant, leading to ineffectiveness of membrane-based separations on 100, 300 or 500 kDa membranes. Though large-scale size exclusion chromatography is a potential option, the method is difficult to scale due to very long column lengths and long processing times required. Other size-based chromatography resins developed for large-scale purification of viruses include CaptoCore400 and CaptoCore700 resins, but these cannot be directly used in AAV separations due to the small size of AAV (about 15-25 nm) compared to other viruses (such as Adenoviruses or Retroviruses which are greater than 60 nm). The small size of AAV makes it enter the pores of the resins, after which it can only be recovered in denatured form in the strip.

[0124] According to the inventions, buffers comprising at least one buffering agent, for example Bis-Tris-Propane, Bis-Tris, Tris, Glycine, Bicine, Tricine,REGN 11547 Acetate, Borate, Citrate, Carbonate, Phosphate, Formate, Sulfate, Succinic acid, Sulfonic acid and variants thereof (for example, MES, PIPES, HEPES, CHES, CAPS, MMS, PBMS), Diethanolamine, or Imidazole can be used. Buffers also may comprise one or more organic or inorganic salts, such as NaCl, KCl, MgCl2, CaCl2, NH4Cl, Na2SO4, CaSO4, K2SO4, MgSO4, (NH4)2SO4, sodium citrate, and tetramethylammonium chloride (TMAC). The buffer may also comprise additives such as antibiotics and bacteriostatics (for example, sodium azide or AEBSF), protease inhibitors (for example E64), detergents and chaotropes (for example poloxamer, CHAPS, SDS, Triton, Tween, Urea), saturating agents (for example, bovine serum albumin), organic solvents (for example ethanol, isopropanol, acetonitrile), and other additives or excipients including chelatants, stabilizers, reducers (for example, DTT, TCEP, EDTA, Glycerol, Sucrose), and amino acids (for example, Histidine or Arginine).

[0125] The inventions are amenable to the use of anion exchange (AEX) media, which include monoliths, resins and membranes. AEX media includes AEX monoliths (for example, CIM QA, and CIM DEAE), AEX resins (for example, Capto Q, Capto Q ImpRes, CAPTO DEAE, POROS HQ, POROS XQ, POROS PI, POROS D, Fractogel EMD TMAE, Fractogel EMD DEAE, Nuvia Q, Nuvia HP-Q) and AEX membranes (for example, Sartobind STIC PA, Sartobind Q, Natrix Q).

[0126] The inventions also are amenable to the use of cation exchange (CEX) media, which include monoliths, resins and membranes. CEX media includes monoliths (for example CIM SO3), CEX resins (for example Capto S, Capto S ImpRes, Capto S ImpAct, Capto SP, POROS HS, POROS XS, CM Sepharose, Nuvia S, Nuvia HR-S), and CEX membranes (for example, Sartobind S, Natrix CH).REGN 11547

[0127] The inventions are amenable to the use of multimodal media, which includes monoliths and resins. Multimodal monoliths (for example PRIMA T, PRIMA S), multimodal resins (for example Capto Adhere, Capto MMC, CaptoCore400, Capto Adhere ImpRes, Capto MMC ImpRes, HEA HyperCel, PPA HyperCel, CMM HyperCel, MEP HyperCel, HA Ultrogel). Specific Binding Pairs

[0128] The inventions advantageously employ one or more "specific binding pairs," also referred to as "protein:protein binding pairs." An example is the SpyTag:SpyCatcher system. The SpyTag:SpyCatcher system was developed using the Streptococcus pyogenes second immunoglobulin-like collagen adhesion domain (CnaB2) from the fibronectin binding protein FbaB. An isopeptide bond can be formed spontaneously between the SpyTag protein and the SpyCatcher protein. The SpyCatcher peptide weighs about 15 kD. However, the SpyTag protein is only 13 amino acids long. The small size of the SpyTag protein makes it amenable for insertion into the AAV genome, which has a total packing capacity of only about 4.7 kilobases. These systems, such as SpyTag:SpyCatcher, allow a retargeting molecule to be bound to an AAV. See Examples 34, 35 and 36.

[0129] Other systems to facilitate retargeting include the SpyTag:SpyCatcher system is described in U.S. Patent No.9,547,003 and Zakeri et al. (2012) PNAS 109:E690-E697, is derived from the CnaB2 domain of the Streptococcus pyogenes fibronecting-binding protein FbaB. See WO 2019 / 006046.

[0130] SpyTag002:SpyCatcher002 system is described in Keeble et al (2017) Angew. Chem. Int. Ed. Engl 56: 16521-25. See WO 2019 / 006046.REGN 11547

[0131] SpyTag003:Spy Catcher003 also has been created. Spy Tag002:SpyCatcher002 and SpyTag003:SpyCatcher003 are different iterations of Spy Tag:Spy Catcher.

[0132] The SnoopTag:SnoopCatcher system is described in Veggiani (2016) PNAS 113 : 1202-07. The D4 Ig-like domain of RrgA, an adhesion from Streptococcus pneumoniae, was split to form SnoopTag. Incubation of SnoopTag and SnoopCatcher results in a spontaneous isopeptide bond that is specific between the complementary proteins. Veggiani (2016), cited above. See WO 2019 / 006046.

[0133] The Isopeptag:Pilin-C specific binding pair was derived from the major pilin protein Spy0128 from Streptococcus pyogenes. (Zakeir and Howarth (2010) J. Am. Chem. Soc.132:4526-27). See WO 2019 / 006046.

[0134] Other systems to facilitate retargeting can be based upon the splitting and engineering of RegA domain 4. These have led to SnoopTagJr:SnoopCatcher, DogTag:DogCatcher and Snoop Ligase. Other systems include Isopeptag:Pilin-N, SdyTg:SdyCatcher, Jo:In, 3kptTag: 3kptCatcher, 4oq1Taq / 4oq1Catcher, NGTag / Catcher, Rumtrunk / Mooncake, GalacTag, Cpe, Ececo, Corio and all others based upon isopeptide binding pairs or other covalent bonds.

[0135] Systems based upon truncated and / or other variations / derivatives have been developed to lessen the possibility of encountering neutralizing antibodies when the covalently surface modified AAV is administered to a subject. For example, the use of KTag (SEQ ID NO: 85) with SpyTag (SEQ IDREGN 11547 NOS: 54 and 86) can be employed. The bonding between KTag and SpyTag can be undertaken using SpyLigase (SEQ ID NO: 61).

[0136] KTag is made by expressing the β-strand of CnaB2 containing a reactive Lys, which was separately expressed. (10 amino acids). SpyLigase (11 kDa) is based on SpyCatcher and made by removing amino acids from the β-strand that has the reactive Lys and using a circular permutation to replace amino acids from the C-terminus of CnaB2 with a Gly / Ser linker, which is followed by N-terminal CnaB2 amino acids. See Fierer et al., Proc. Nat’l Acad. Sci.131577611. Retargeting molecules

[0137] Retargeting molecules (Rm) include Fc-containing proteins, such as antibodies and Fc-fusion proteins (including receptor Fc fusion proteins such as trap proteins). Fragments and derivatives of antibodies and Fc-fusion proteins also can be used as retargeting molecules.

[0138] All major antibody classes, namely IgG, IgA, IgM, IgD and IgE, can be used as retargeting molecules. IgG is a preferred class, and includes subclasses IgG1 (including IgG1λ and IgG1κ), IgG2, IgG3, and IgG4. Further antibody types include a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecifϊc antibody, a bispecific antibody, a trispecific antibody, an antigen binding antibody fragment, a single chain antibody, a diabody, triabody or tetrabody, a Fab fragment or a F(ab')2 fragment, an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.REGN 11547

[0139] Retargeting molecules bind to targets, which are antigens, receptors and / or ligands found on the surface of a target cell. Exemplary targets are calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1), asialoglycoprotein receptor 1 (ASGR1), Fel d 1, ENTPD3, PTPRA, CD20, CD63 and Her2. Additional targets include GAB A, transferrin. CD3, CD34, integrin, adipophilin, AIM-2, ALDHlAl, alpha-actinin-4, alpha-fetoprotein ("AFP"), ARTC1, B- RAF, BAGE-1, BCLX (L), BCR-ABL fusion protein b3a2, beta-catenin, BING-4, CA- 125, CALCA, carcinoembryonic antigen ("CEA"), CASP-5, CASP-8, CD274, CD45, Cdc27, CDK12, CDK4, CDKN2A, CEA, CLPP, COA-1, CPSF, CSNKlAl, CTAGl, CTAG2, cyclin Dl, Cyclin-Al, dek-can fusion protein, DKK1, EFTUD2, Elongation factor 2, ENAH (hMena), Ep-CAM, EpCAM, EphA3, epithelial tumor antigen ("ETA"), ETV6-AML1 fusion protein, EZH2, E6, E7, FGF5, FLT3-ITD, FN1, G250 / MN / CAIX, GAGE-1,2,8, GAGE- 3,4,5,6,7, GAS7, glypican-3, GnTV, gplOO / Pmel 17, GPNMB, HAUS3, Hepsin, HER-2 / neu, HERV-K-MEL, HLA-A11, HLA-A2, HLA-DOB, hsp70-2, IDOl, IGF2B3, IL13Ralpha2, Intestinal carboxyl esterase, K-ras, Kallikrein 4, KIF20A, KK-LC-1, KKLC1, KM-HN-1, KMHN1 also known as CCDC110, LAGE-1, LDLR- fucosyltransferase AS fusion protein, Lengsin, M-CSF, MAGE-A1, MAGE- A 10, MAGE-A12, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-C1, MAGE-C2, malic enzyme, mammaglobin-A, MART2, MATN, MC1R, MCSP, mdm-2, ME1, Mel an- A / MART-1 , Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, mucin, MUM-1, MUM-2, MUM-3, Myosin, Myosin class I, N-raw, NA88- A, neo-PAP, NFYC, NY-BR-1, NY-ESO-l / LAGE-2, OA1, OGT, OS-9, P polypeptide, p53, PAP, PAX5, PBF, pml-RARalpha fusion protein, polymorphic epithelial mucin ("PEM"), PPP1R3B, PRAME, PRDX5, PSA, PSMA, PTPRK, RAB 38 / N Y-MEL- 1 , RAGE-1, RBAF600, RGS5, RhoC, R F43, RU2AS, SAGE, secernin 1, SIRT2, SNRPD1,REGN 11547 SOX10, Spl7, SPA17, SSX-2, SSX-4, STEAPl, survivin, SYT-SSX1 or -SSX2 fusion protein, TAG-1, TAG-2, Telomerase, TGF-betaRII, TPBG, TRAG-3, Triosephosphate isomerase, TRP-l / gp75, TRP-2, TRP2-INT2, tyrosinase, tyrosinase ("TYR"), VEGF, WT1, XAGE-lb / GAGED2a, Kras, NY- ESOl, MAGE- A3, HPV E2, HPV E6, HPV E7, WT-1 antigen (in lymphoma and other solid tumors), ErbB receptors, Melan A [MARTI], gp 100, tyrosinase, TRP-l / gp 75, and TRP-2 (in melanoma); MAGE-1 and MAGE-3 (in bladder, head and neck, and non-small cell carcinoma); HPV EG and E7 proteins (in cervical cancer); Mucin [MUC-1] (in breast, pancreas, colon, and prostate cancers); prostate-specific antigen [PSA] (in prostate cancer); carcinoembryonic antigen [CEA] (in colon, breast, and gastrointestinal cancers), and such shared tumor-specific antigens as MAGE-2, MAGE-4, MAGE-6, MAGE- 10, MAGE- 12, BAGE-1, CAGE-1,2,8, CAGE-3 TO 7, LAGE-1, NY-ESO-l / LAGE-2, NA- 88, GnTV, TRP2-INT2, E6, E7, human glucagon receptor (hGCGR) and. I human ectonucleoside triphosphate diphosphohydrolase 3 (hENTPD3). Other targets can be selected by the person skilled in the art. See WO 2019 / 006046. Detargeting mutations

[0140] The inventions further provide mutations in the VP1 Protein to lower the AAV preferential transduction of the liver. For AAV9, preferred The mutations include N272A and W503A substitutions, where alanine replaces both asparagine at position 272 of VP1 and tryptophan at position 503 of VP1. One or both of the mutations can be undertaken in the VP1 protein. Optionally, other amino acids, such as glutamic acid, serine or others, can be used instead of alanine for substitution. Other detargeting mutation sites include, but are not limited to, N470, D271, and Y446.REGN 11547

[0141] The inventions provide exemplary mutations for use with AAVs as follows, which for each serotype can be utilized singly or in combinations : AAV1 – N500E; AAV2 – R585A and R588A; AAV5 – T571S; AAV6 – N500E, K531A and K531E; AAV9 – N272A, W503A, T568P, Q590L, N498Y, L602F, S414N, G453D, K557E, T582I, and combinations include, but are not limited to, T568P + Q590L, N498Y + L602F, S414N + G453D + K557E +T582I. These and others are set forth in the chart below:REGN 11547REGN 11547

[0142] Other mutations and detargeting approaches, such as variable loop swaps, are disclosed in publications or otherwise available See, for example, Shen et al., Molecular Therapy 15: 1955–62 (2007). These technologies can be used according to the inventions. Detargeting mutations are optional. Host Cells

[0143] The present inventions are amenable for production in mammalian cell culture. Exemplary cell lines are CHO, Per.C6 cells, Sp2 / 0 cells, HeLa and HEK293 cells. CHO cells include, but are not limited to, CHO-ori, CHO- K1, CHO-s, CHO-DHB11, CHO-DXB11, CHO-K1SV, and mutants and variants thereof. HEK293 cells include, but are not limited, to HEK293, HEK293A, HEK293E, HEK293F, HEK293FT, HEK293FTM, HEK293H, HEK293MSR, HEK293S, HEK293SG, HEK293SGGD, HEK293T and mutants and variants thereof. Adherent HEK 293 cells also can be used for production of covalently surface modified AAV according to the inventions. HEK 293 suspension cultured cells were derived from HEK 293 adherent cells. See Malm et al., Scientific Reports 10: 18996 (2020). Other suitable cells include, but are not limited to BHK (baby hamster kidney) cells, HeLa cells and Human Amniotic cells, such as Human Amniotic Epithelial cells. Other cell types for production include insect cells, such as Sf9. ProductionREGN 11547

[0144] As described above, Figure 2 schematically depicts and describes quad transfection systems and methods for producing covalently surface modified AAV in a eukaryotic cell, such as HEK 293F. As described in Figure 2, the quad transfection system utilizes four plasmids (schematically depicted) to produce AAV comprising SpyTag sequences in the capsids. The plasmids are as follows: (i) pGOI – a plasmid comprising a GOI (for example, a transgene) flanked by two AAV inverted terminal repeats (ITRs), and optionally a selection marker gene, such as an antibiotic resistance gene (for example, ampicillin resistance); (ii) pRC - a plasmid comprising AAV (here AAV9) rep and cap genes, and optionally a selection marker gene, such as an antibiotic resistance gene (for example, ampicillin resistance); (iii) pRC-SpyT – a plasmid as above in (ii) and further comprising a polynucleotide sequence encoding a 13 amino acid Spy Tag peptide (a first member of a specific binding pair), and optionally a selection marker gene, such as an antibiotic resistance gene (for example, ampicillin resistance). This is an exemplary plasmid pRC-first member (pRC-FM); and (iv) pHELP - a plasmid comprising adenovirus helper genes E4 and E2, and VA RNA, (exemplary helper genes) and a optionally selection marker gene, such as an antibiotic resistance gene (for example, ampicillin resistance). The following chart provides a brief summation of this example:REGN 11547

[0145] Promoters that are functional in eukaryotic cells, such as AAV P5, CMV, lac, CAG, CAGG, and SV40 promoters, are provided where needed to initiate transcription, but are not shown. Additionally, internal ribosome entry sites (IRESs), recombinase recognition sites (RRSs), enhancers and operators optionally also can be included, but are not shown. For constructing plasmids having large polynucleotides, such as those encoding antibody chains, intronic selection can be employed, preferably with a second selection marker gene that is different from the first selection marker gene.REGN 11547

[0146] Figure 3 schematically depicts at the top a pRC-SpyTag plasmid comprising the rep and cap genes and the p40 promoter, and at the bottom schematically shows the insertion of the SpyTag 13 amino acid peptide sequence to form a fusion protein comprising SpyTag and Cap peptide sequences, thereby resulting in mutant VP1, VP2 and VP3 proteins fused to the SpyTag peptide sequence. The AAV still possesses the 5:5:50 stoichiometry of the VP1, 2 and 3 proteins.

[0147] Reaction time for the conjugation should be an incubation of at least 0.5, 1, 1.5, 2, 2.5, 3, 3.54, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51, 51.5, 52, 52.5, 53, 54.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 60.5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65, 65.5, 66, 66.5, 67, 67.5, 68, 68.5, 69, 69.5, 70, 70.5, 71, 71.5, 72 or more hours (4 to 72 hours or more). Reaction temperature should be about 4°C to about 40°C, preferably room temperature (19°C to 25°C, preferably about 22°C). Molar ratio of the reactants, namely (AAV-first member) to (retargeting molecule-second cognate member) should be1:20, 1:25 or preferably greater differences. Preferably, the molar ratio should be 1:30 to 1:1000 (includes all ratios there between) to maximize conjugation efficiency. Ratios of 1:30 to 1:300 (includes all ratios there between) are more preferred. See Example 12. Other parameters are set forth below in Table 2: Table 2REGN 11547

[0148] Significant molar excess of SpyCatcher-Rm is typically beneficial to ensure optimum conjugation of SpyTag-AAV species, where the molar excess can range from 20 to 1000-fold, preferably 30 to 300 fold, and most preferably 300 fold. The inventions provide for complete conjugation of first members of a specific binding pair are bound to second cognate members of the specific binding pair. The inventions provide for a desired conjugation of a level of conjugation of at least 10%, at least 20%, at least 30%, and at least 40%. Preferred levels are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100%, any of which can be considered complete depending on the purpose. Optimal conjugation (also known as complete conjugation based on the purpose) is typically desired by the skilled person and preferably will be about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,REGN 11547 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0149] Ranges of desired conjugation levels can be 10% to 100%, 20% to 99%, 30% to 99%, 40% to 99%, 50% to 99%, 60% to 99%, 70% to 99%, 80% to 99%, or 90% to 99%. Preferred ranges of desired conjugation levels can be 40% to 100%, 40% to 99%, 45% to 95%, 50% to 95%, 55% to 90%, 60% to 90%, 65% to 95%, 65% to 90%, 70% to 90%, 75% to 90%, 75% to 85%, 75% to 80%, or 80% to 85%, and any ranges made by combining these ranges, such as 65% to 85%, for example. More preferably, the range is 65% to 95%.

[0150] The percentages of conjugation can be ascertained by a variety of separation techniques, including size exclusion chromatography (SEC) or asymmetrical flow field-flow fractionation (A4F). SEC and A4F would be combined with a detector, such as UV, fluorescence or a multi-angle light scattering (MALS) detector. Due to the molar excess of SpyCatcher-Rm, a significant amount of SpyCatcher-Rm will need to be removed following the conjugation reaction.

[0151] When starting the conjugation reaction, the AAV-SpyT (FM) should be added to the vessel prior to the SpyC (SCM)-RM. AAV-SpyT should be added first but not in ratio excess due to low concentration of AAV to Ab.

[0152] In order to drive the conjugation reaction to completion, it can be beneficial to provide the SpyC-RM in molar excess of the AAV-SpyT, where the excess can be up to 1000-fold. In such cases, once the conjugation reaction is completed, there will remain a significant amount of excess of unconjugated SpyCatcher-Rm following conjugation. This can be due to the SpyCatcher-Rm beingREGN 11547 added in molar excess to the number of available SpyTag binding sites on the AAV and / or due to incomplete reactions.

[0153] Additives can be included in the conjugation reaction to modify conditions and enhance conjugation efficiency. Additives include, but are not limited to, dimethylsulfoxide (DMSO), L-glutamic acid monosodium salt (L-glutamic acid), arginine, sorbitol, urea, isopropyl alcohol, (IPA) and sodium thiocyanate (NaSCN).

[0154] The inventions are further described by the following examples, which do not limit the inventions in any manner. The order of performance of the below examples can be altered or combined as determined by the person of skill in the art in view of the teachings and data contained herein.

[0155] The AAV serotype sequences and component sequence types used according to the inventions used in the Description, Summary, Examples and Figures are representative and not limiting. Any AAV serotype sequence and component sequence can be used according to the inventions. These include but are not limited to sequences for promoters, markers, specific binding pairs, helper proteins, AAV sequences (Cap, Rep and ITRs), retargeting molecules, detargeting mutations, IRESs, RRSs, introns, operators, enhancers and polydenylation signals. EXAMPLE 1 Conjugation Using SpyTag:SpyCatcher

[0156] In vitro conjugation can use a four plasmid transfection system (quad transfection) for cells to produce for rAAV comprising SpyTag. For example, the plasmids could be: (1) plasmid containing a GOI flanked by AAV inverted terminal repeats (pGOI); (2) plasmid containing AAV rep and cap genes (pRC); (3)REGN 11547 plasmid containing AAV rep gene and cap gene with a polynucleotide encoding SpyTag peptide inserted in frame into the cap gene (pRC-SpyT); and (4) a plasmid comprising helper polynucleotide sequences, such as adenovirus helper genes, in a first bioreactor (pHELP). See Figure 2. Helper genes also can be obtained from herpesviruses and papilloma viruses.

[0157] For retargeting molecules, such as antibodies, cells (for example, HEK 293) can be transfected with two plasmids, such as: plasmid encoding an antibody heavy chain sequence in frame with a SpyCatcher sequence and (2) a plasmid encoding an antibody light chain sequence in a second bioreactor. The cells will produce an antibody bound to SpyCatcher.

[0158] In addition to the use of monospecific monoclonal antibodies as retargeting molecules, antibody derivatives and fragments can also be employed as retargeting molecules. Antibody fragments and derivatives include, but are not limited to, Fab, ScFv-Fc, dAB-Fc, half antibodies bispecifics, tri-specifics, IgG-ScFv, IgG-dab, ScFV-Fc-ScFV and other combinations of heavy and / or light chains. Fc- fusion proteins, such as trap proteins, and well as mini-trap proteins also can be used a retargeting molecules.

[0159] Eukaryotic cells, preferably mammalian cells, such as human and rodent cells, can be used to produce the rAAV and the retargeting molecules, such as antibodies. The cell types can be the same or different. For example, rAAV can be produced in human cells, such as HEK 293 cells and retargeting molecules in rodent cells, such as CHO cells, and conversely rAAV can be produced in rodent cells (for example, CHO cells) and retargeting molecules in human cells (forREGN 11547 example, HEK 293 cells). In other approaches, both rAAV and retargeting molecules can be both produced in human cells or both produced in rodent cells (for example, CHO cells), depending on the preference of the person skilled in the art. Insect cells such as Sf9 can alternatively be used for producing the rAAV, retargeting molecules, or both.

[0160] The rAAV and retargeting antibody produced as described above can each be purified by one or more of depth filtration, tangential flow filtration (TFF), affinity capture polishing, and viral retentive filtration. TFF is a generic term and includes, but is not limited to, ultrafiltration / diafiltration (UF / DF) and newer approaches such as single-pass tangential flow filtration (SPTFF).

[0161] Conventional TFF can be employed, which uses a batch approach and repeated cycling through the membrane to create a permeate and a retentate. Alternatively, single-pass TFF can be employed to allow for a continuous process. According to the inventions, TFF can advantageously employ permeate pumps to reduce TMP buildup and flux decline. Although not to be bound by any hypothesis or theory, it is believed that detergents, such as Tweens, build up on the retentate side of a TFF membrane and cause TMP buildup and flux decline. Single- Pass TFF units are available from Pall / Cytiva, Repligen and Millipore. Thereafter, the conjugation reaction can be undertaken between the rAAV and the retargeting antibody.

[0162] Figure 1A depicts the species heterogeneity involved in in-vitro conjugation reactions between AAV9 comprising a SpyTag peptide (rAAV9-SpyT) and SpyCatcher fused to a monoclonal antibody (SpyC-mAb species). This figureREGN 11547 depicts the heterogeneity at the level of the AAV9-SpyT capsid, depicting the “average” AAV species with 6 SpyTags, as well as AAV species with lower (3) and higher number (10) of SpyTags expressed on the capsid surface. Heterogeneity at the level of the conjugated AAV9-SpyT-SpyC-ASGR1 monoclonal antibody Ab species (ASGR1 mAb) also is shown where the number of SpyC-ASGR1 mAb species during the in-vitro conjugation reaction were not in a significant molar excess compared to the number of AAV9-SpyT species (1:1 to 1:15 in terms of AAV-SpyT to SpyC-mAb). These 1:1 to 1:15 ratios resulted in incomplete conjugation (species with average, low and high levels of conjugation are depicted). Heterogeneity at the conjugated level also is shown where the number of SpyC-ASGR1 mAb species during the in-vitro conjugation reaction were in significant molar excess compared to the number of AAV9-SpyT species (1:30 to 1:300 in terms of AAV-SpyT to SpyC- mAb). These 1:30 to 1:300 ratios resulted in optimum (complete) conjugation (showing species with average, low and high levels of conjugation).

[0163] Optimal (complete) conjugation occurs when most available first members of a specific binding pair are bound to second cognate members of the specific binding pair. For example, Figure 1A (bottom row) schematically depicts instances of all and nearly all SpyTags bound by SpyCatcher on covalently surface modified AAVs.

[0164] The inventions provide for Optimal conjugation (also referred to as complete conjugation) of first members of a specific binding pair are bound to second cognate members of the specific binding pair. Preferably, optimal conjugation (also known as complete conjugation based on the purpose of the skilled person) is typically desired and preferably will be about 40%, 41%, 42%, 43%, 44%,REGN 11547 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0165] Ranges of desired conjugation levels can be 10% to 100%, 20% to 99%, 30% to 99%, 40% to 99%, 50% to 99%, 60% to 99%, 70% to 99%, 80% to 99%, or 90% to 99%. Preferred ranges of desired conjugation levels can be 40% to 100%, 40% to 99%, 45% to 95%, 50% to 95%, 55% to 90%, 60% to 90%, 65% to 95%, 65% to 90%, 70% to 90%, 75% to 90%, 75% to 85%, 75% to 80%, or 80% to 85%, and any ranges made by combining these ranges, such as 65% to 85%, for example. More preferably, the range is 65% to 95%.

[0166] The percentages of conjugation can be ascertained by a variety of separation techniques, including size exclusion chromatography (SEC) or asymmetrical flow field-flow fractionation (A4F). SEC and A4F would be combined with a detector, such as UV, fluorescence, or a multi-angle light scattering (MALS) detector. EXAMPLE 2 – Separating a covalently surface modified adeno-associated virus species from unconjugated antibody species using size exclusion chromatography

[0167] Figure 4 depicts size exclusion chromatograms (SEC) of in vitro conjugation reaction mixtures of rAAV9 to antibody in ratios of 1:1, 1:6, 1:18, 1:30 and 1:300 at pH 8.0 and using rAAV concentration of 5 x 1010to 1 x 1012capsids (cp)REGN 11547 per milliliter on a size exclusion chromatography column with pore size of 1000 Å. The shift in retention time of the AAV peak is highlighted with an arrow showing shifting of the peak maximum to the left indicating conjugation reaction completion. The x-axis is minutes for elution and the y-axis is emission units (EU) for fluorescence.

[0168] Figure 5A depicts size exclusion chromatograms of in-vitro conjugation reaction mixtures of rAAV9 to antibody in ratios of 1:0, 1:1, 1:6, 1:15, 1:30 and 1:300 at pH 5.0, 6.5, 8.0 and 8.5 and using rAAV concentration of 2.3 x 1013cp / mL on a size exclusion chromatography column with pore size of 450 Å. Reaction conditions included brief mixing followed by overnight incubation, resulting in maximum conjugation as shown by the almost complete disappearance of the AAV-SpyT peak and formation of a new Conjugated AAV peak at ratios of 1:30 and 1:300. Figure 5B is a preparative scale SEC on a 100 ml SEPAX SRT 500A column to remove unconjugated antibody.

[0169] Figures 6A and 6B depict size exclusion chromatograms of in- vitro conjugation reaction mixtures of rAAV9 to antibody in ratios of 1:0, 1:1, 1:6, 1:10, 1:20 (Figure 6A), and 1:30 at pH 5.0, 6.5, 8.0, 8.5, 9.0, 9.5 and 10.0 (Figure 6B), and using rAAV concentration of 2.3 x 1013cp / mL on a 450 Å size exclusion chromatography column. Reaction conditions included brief mixing followed by 1 hour incubation prior to freezing, resulting in incomplete conjugation shown by the presence of AAV-SpyT peak at all molar ratios. EXAMPLE 3 – Separating a covalently surface modified adeno-associated virus species from unconjugated antibody species using anion exchange orREGN 11547 size exclusion chromatography

[0170] Figure 7A depicts exemplary elution profiles for AAV9 and AAV9-SpyT for empty, partially filled and full capsids, and serves as a model for other AAVs. There will exist empty and partially-filled capsid populations can have isoelectric points (pI) that are above full capsids and other empty and partially-filled capsid populations will have isoelectric points that are below full capsids. Figure 7B is a graph depicting separation empty, partially filled and full AAV9-SpyT capsids using a complex hybrid gradient. The column used to obtain the data in Figure 7B was a CIM QA monolith loaded with 5 x 1013to 5 x 1014capsids (cp / )ml. The buffer was 20 mM Bis-Tris-Propane, 0.001% P188, pH 9.6 and 1M NaCl. Figure 7C depicts data from a two-pass anion exchange chromatogram for a 500L purification of AAV-SpyTag. First pass and second pass microsteps. This process achieved a recovery of 96% full capsids. Partially-filled capsids were only 1% of total and empty capsids were only 3% of total. Figures 7D to 7F depict data from an experiment using anion exchange to separate a covalently surface modified adeno-associated virus species from unconjugated antibody species. A CIM QA 1 mL monolith was used as the anion exchange column, and the column was loaded with an in-vitro conjugation reaction mixture of AAV9-SpyT and SpyC-ASGR1 mAb mixed in a 1:30 molar ratio. In Figure 7D, the AAV9-SpyT capsids were 10% full, whereas in Figure 7D the AAV9-SpyT capsids were 70% full. Figure 7F depicts the size exclusion chromatograms combined with multi-angle light scattering (MALS) data of six elution fractions collected from Figure 7E, showing the increasing conjugation level, and increasing mass distribution of the later eluting fractions.REGN 11547

[0171] Figure 7G is a graph depicting the retention time of conjugated AAV9 using SpyTag-SpyCatcher with an antibody, unconjugated AAV-SpyTag and unconjugated SpyCatcher-antibody. Conjugation reaction was studied by tracking percent conjugation over time using size exclusion chromatography according to the following: Peak area of conjugated AAV X 100 Peak area of conjugated plus unconjugated AAV

[0172] Figure 7H is a graph depicting the effect of AAV capsid concentration at 1 x 1012cp / ml, 1 x 1013cp / ml and 3 x 1013cp / ml on percent conjugation. Higher capsid concentration resulted in a greater percentage of conjugation.

[0173] Figure 7I is a graph depicting the effect of incubation temperature at 4°C, 20°C and 37°C on percent conjugation. Higher temperature resulted in a greater percentage of conjugation.

[0174] Figure 7J is a graph depicting the effect of pH on percent conjugation. Strongly basic conditions reduce conjugation percentages.

[0175] Figure 7K depicts data for conjugation of AAV-SpyT conjugated to Spy C-Fab (Construct 1) and AAV-SpyT conjugated to Spy C-mAb (Construct 2). Both AAVs have the W503A detargeting mutation and HA-MTM1 as a transgene.REGN 11547

[0176] The data for the experiment of Figure 7K is set forth below in Table 3. Table 3EXAMPLE 4 – Separating a covalently surface modified adeno-associated virus species from unconjugated antibody species using cation exchange

[0177] Figures 8A and 8B depict data from an experiment using cation exchange to separate a covalently surface modified adeno-associated virus species from unconjugated antibody species. In Figure 8A, a CIM SO31 mL monolith was used as the cation exchange column, and the column was loaded with an in-vitro conjugation reaction mixture of AAV9-SpyT and SpyC-ASGR1 mAb mixed in a 1:30 molar ratio. Figure 8A depicts a linear gradient elution and highlights the peaks corresponding to the SpyC-Ab and the conjugated AAV9-SpyT-SpyC-Ab species. Binding was at pH 5, less than 4mS / cm and elution was at pH 5, 4-80 mS / cm. Figure 8B depicts a step elution in which the in-vitro conjugation reaction mixture was adjusted with sodium chloride solution to a conductivity of 15 mS / cm and the mixture was loaded onto a Poros XS cation exchange column, leading to all SpyC- Ab species not binding into the column and being removed in the flow-through, whileREGN 11547 all AAV species bound and were removed later in an isocratic elution at 40 mS / cm conductivity.

[0178] Figure 8C depicts data showing that Poros XS cation exchange can separate free SpyC-Ab species following the conjugation reaction. Figures 8D and 8E compare CaptoCore400 flow through (size exclusion and anion exchange) (Figure 8D) to Poros XS elution (cation exchange) (Figure 8E). Both provide a large conjugated peak detected with dynamic light scattering (DLS). CaptoCore400 flow though did exhibit aggregates, whereas Poros XS elution did not. EXAMPLE 5 – Separating a covalently surface modified adeno-associated virus species from unconjugated antibody species using multimodal multimodal binding chromatography

[0179] Multimodal binding and size separation bead chromatography exchange to separate a covalently surface modified adeno-associated virus species from unconjugated antibody species. More specifically, the multimodal binding and size separation bead chromatography are functionalized with an octyl amine that is hydrophobic and positively charged, and also include a matrix that provides size exclusion, such as CaptoTMCore 400 or CaptoTMCore 700.

[0180] Covalently surface modified adeno-associated virus species are expected in the flow through and wash (FTW). Unconjugated antibody species are expected to enter resin and be bound tightly, and removed during the strip phase. Loading was 5 x 1013to 1 x 1014cp / ml beads over 3 minutes and room temperature. Figure 9 depicts an experiment using multimodal anion exchange to separate a covalently surface modified adeno-associated virus species from unconjugated antibody species. Table 4 below sets forth the relative percentage of SpyC-Ab in theREGN 11547 flow through and the percentage yield of AAV species in the flow through as measured using size exclusion chromatography when the load material is treated with different amounts of NaCl and adjusted to different levels of pH. The green row in the figure highlights the optimized condition of adjusting the load material to pH 6.0 with 1M NaCl, enabling a complete separation of SpyC-Ab and AAV species in the flow through with 0% SpyC-Ab, along with high yield of AAV species of 85%. Table 4

[0181] Figure 10 depicts data from an experiment using multimodal binding and size separation bead chromatography exchange on CaptoCore400 resin to separate a covalently surface modified adeno-associated virus species from unconjugated antibody species. The relative size of species was ~8 nm for the SpyC-Ab, about 15 nm for the AAV9-SpyT, and about 17 nm for the conjugated AAV9-SpyT-SpyC-Ab. The figure includes a size exclusion chromatogram showing that the load material included two peaks (AAV and SpyC-Ab), but the flow through included only one peak (only AAV, showing removal of free SpyC-Ab species using this approach.REGN 11547 EXAMPLE 6 – Multivariate Modeling

[0182] Figure 11 depicts a multivariate model from experiments using different salt and pH conditions to induce a change in the size and surface characteristics of AAVs, leading to improved yields in the flow through of CaptoCore400 chromatography. The model is a yield profiler and predicts an optimum condition of adjusting the load material with sodium citrate to a final concentration of 150 mM at pH 5.5, resulting in a predicted yield of 100% and an experimental yield of 107% as measured by ddPCR quantification. EXAMPLE 7 – Dynamic Light Scattering of Conjugation Reactions

[0183] Figures 12A-12C depict dynamic light scattering (DLS) data from AAV9-SpyT, SpyC-ASGR1 mAb (Figure 12A), and in-vitro conjugation reactions between these two species under stirred and unstirred conditions in sodium chloride and sodium citrate based buffers. The DLS data shows the formation of large aggregates in the conjugation reaction under stirred conditions (Figure 12B), but no such aggregates in the unstirred condition (Figure 12C) (incubation only). EXAMPLE 8 – Size Exclusion Analysis

[0184] Figure 13A shows size exclusion chromatography data tracking the rate of reaction between AAV9-SpyT and SpyC-ASGR1 mAb for three different batches of AAV9-SpyT with different percentages of full capsids. The molar ratio of AAV9-SpyT to SpyC-ASGR1 mAb was 1:300. The conjugation was performed at aREGN 11547 pH of 5.8 with an overnight hold and light mixing. Here, 80% conjugation was selected as the optimal level.

[0185] The percentage of conjugated species is measured by relative peak area between the AAV9-SpyT peak at about 8.3 min RT and the conjugated species peaks at 6.5 – 8.0 RT on the size exclusion chromatograms, and tracked in the chart on the right. All batches reached 80% conjugation, but Batch 1 took 48 hours to reach 80% conjugation while Batches 2 and 3 took 24 hours.

[0186] Figure 13B depicts conjugation data with AAV9-SpyT and SpyC-mAb (anti-CACNG1). The monospecific mAb has two SpyC.

[0187] Figure 13C depicts conjugation data with AAV9-SpyT and SpyC-bispecific Ab (anti-CACNG1). The bispecific mAb has one SpyC.

[0188] Figure 13D depicts conjugation data with AAV9-SpyT and SpyC-Fab (anti-CACNG1). The Fab has one SpyC.

[0189] Molar AAV:Ab (antibody with two SpyC, bispecific antibody with one SpyC, or Fab with one SpyC, as described above) ratios of 1:1, 1:6, 1:15, 1:30, 1:300 and 1:1000 were tested. The results showed that ratios of 1:30 to 1:1000 of AAV to Ab resulted in greater levels of conjugation. Table 5 below shows that ratios of 1:30 to 1:1000 of AAV to Ab also resulted in lower percentages of high molecular weight species, and that the use of antibodies with only one SpyC (Figures 13C and 13D) resulted in less high molecular weight species than use of an antibody with two SpyC (Figure 13B). See Table 5. Table 5REGN 11547EXAMPLE 9 –SEC MALS and Anion Exchange Analysis

[0190] Figure 14A is a graph depicting how anion exchange chromatography separates unconjugated AAV from more conjugated species, which are schematically depicted underneath the graph. Increasing alt gradients lead to more conjugated AAV. Anion exchange uses positively charged ligands to bind to negatively charged species (at pH greater than pI).

[0191] Figure 14B depicts SEC-MALS data of samples with different levels of conjugation. The samples were obtained from an anion exchange chromatography method similar to the one described in Figures 7A-7D that can be used to separate populations of differently conjugated species. The chart in the lower section of Figure 14B summarizes the SEC-MALS analysis of the samples which is used to estimate the number of mAbs conjugated on average in each sample, ranging from less than 1 mAb in the first fraction to greater than 5 mAbs in the last fraction.

[0192] Figure 14C shows the results of a transduction assay where these differently conjugated species were compared for transduction ability usingREGN 11547 cells expressing ASGR1 receptor. It was observed that lower conjugated species (F1-F2) resulted in higher transduction efficiency than the combined pool (Load) as well as later fractions (F3-F5), suggesting a negative impact of higher conjugation levels on transduction ability, despite potential advantages in targeting ability.

[0193] Figure 14D and Figure 14E collectively concern transduction efficiency at different pHs and percent conjugation. Figure 14D depicts green fluorescent protein (GFP) percentage of bispecific antibody with one SpyC, a monospecific monoclonal antibody (mAb) with two SpyCs, or an antigen-binding antibody fragment (Fab) with one SpyC, conjugated at a reaction pH of 5 or 8. See Figures 13A to 13C and Table 5. Figure 14E depicts mean fluorescence intensities (MFI) for the bsAb, mAb and Fab at pH 5 or 8. Data is depicted for multiplicity of infection (MOI) at 2 x 105(2E5) and 4 x 104(4E4).

[0194] The MOI refers to the ratio of infectious agents (in this case, AAV9-SpyT-SpyC-Ab molecules) to target cells used in the assay. AAV particles are added to the cells in different amounts so that the transduction effect can be compared at different levels of infection, as there may be some levels where no signals are observed. The data in this figure shows results from the 4E4 and 2E5 MOI conditions on this transduction assay, showing that all conjugated viruses performed comparably at both these conditions regardless of antibody format or conjugation reaction pH.

[0195] Additional data is provided in Table 6. Table 6REGN 11547

[0196] The low endotoxin results show that the endotoxin is not confounding the results of cell-based assays (high endotoxin can be detrimental to cells). The data indicate that transduction efficiency post-conjugation was not significantly affected by different pH (5 or 8). However, a higher percentage of conjugation was achieved using pH 5 during the conjugation reaction. EXAMPLE 10 – Large-Scale Production of Covalently Surface Modified AAV

[0197] Figures 15-17 schematically depict exemplary end-to-end processes for production of Covalently Surface Modified AAVs using the in-vitro conjugation reaction and free SpyC-Ab removal steps post-production of a Spy Tagged AAV capsid.

[0198] Figure 15 schematically depicts an exemplary production of retargeting molecules (Rm), here an antibody or Fab fused to SpyCatcher. The antibodies / Fab fused to SpyCatcher are produced in CHO cells in this figure.

[0199] Cell lysis is an early part of AAV production and purification. Detergents can be used to lyse cells to release proteins and viruses contained with the cell. Detergents to lyse cells are usually considered mild detergents and include: sodium dodecyl sulphate (SDS), NP-40, Tweens (for example 20 and 80), TritonsREGN 11547 (for example X-100 and X-114), CHAPS, CHAPSO, Brij (for example, 35 and 58), Octyl thioglucoside, Octyl Glucoside, deoxycholate, and alkyl sulfates, for example.

[0200] Depth filtration typically follows lysis. Downstream processing starts with depth filtration / chromatographic clarification to remove cellular debris after cell lysis, which is followed by affinity capture and non-AAV viral inactivation. Next, polishing 1 and 2 occurs using ion exchange. This is followed by viral retentive filtration and then tangential flow filtration. Here, SpyC-mAb / Fab is formed, which also is depicted in Figure 1A.

[0201] Figure 16 schematically depicts the formation of AAV comprising SpyTag on the capsid surface, specifically AAV9-SpyT. AAV can be produced in human cells (for example, HEK293 or HeLa cells). Cell lysis is undertaken, preferably without Benzonase. Chromatic clarification is performed to remove cell debris following lysis. Next, tangential flow filtration 1 is performed, followed by affinity capture and polishing, here using one, two or three passes on anion exchange chromatographic columns. This is followed by viral retentive filtration and then tangential flow filtration 2. Here, AAV-SpyT is formed. See also Figure 1A.

[0202] Figure 17 schematically depicts the conjugation of SpyCatcher- Rm (for example, mAb / Fab as per Figure 15) to AAV-SpyT (Figure 16). When starting the conjugation reaction, the SpyC-Rm should be added to the vessel prior to the AAV-SpyT. In order to drive the conjugation reaction to completion, it can be beneficial to provide the SpyC-Rm in molar excess of the AAV9-SpyT, where the excess can be up to 1000-fold. In such cases, once the conjugation reaction isREGN 11547 finished, there will remain a significant amount of excess of unconjugated SpyCatcher-Rm following conjugation. This can be due to the SpyCatcher-Rm being added in molar excess to the number of available SpyTag binding sites on the AAV and / or due to incomplete reactions.

[0203] Next, unconjugated retargeting molecules (here mAb or Fab, are removed via chromatography, and then TFF for enrichment of the covalently surface modified AAV . Enrichment can result in greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of unconjugated retargeting molecule species being separated from the covalently surface modified AAV species. Enrichment can result in reducing the presence of unconjugated retargeting molecule species to undetectable levels. The covalently surface modified AAV is schematically depicted in Figure 1A.

[0204] An exemplary downstream protocol for a 500L bioreactor would be as follows: 1. Culturing, cell lysis, clarification and affinity capture with a yield of 71%; 2. Two pass AEX with a yield of 80%; 3. Formulation of AAV-SpyT with a yield of 75% -- exemplary formulation processes would include ultrafiltration / diafiltration for concentration and buffer exchange to increase the concentration of AAV capsids to 3 to 8 x 1013cp / mL and to place it into a stable buffer suitable for extended storage, freeze-thaw and reaction incubation; 4. In vitro conjugation AAV-SpyT with SpyC-Rm at 4.0 x 1013vg / ml and using preparative SEC to remove free SpyC-Rm with a yield of 108%; andREGN 11547 5. Final formulation AAV-SpyT-SpyC-Rm at 3 -3.5 x 1013vg / ml at a yield of 81%. The yield percentages are the % of viral genomes (i.e. full capsids) recovered at the end of each unit operation compared to the amounts going into the unit operation. Typically, a yield about 15% is considered acceptable, 30% is considered good and 50% is considered very good.

[0205] Table 7 below identifies titers and yields that would results in worst case for doses per batch. Table 8 below identifies titers and yields per batch under different parameters and conditions that would be expected. Table 71 Assuming 50-fold excess of SpyC-mAb required 2 Assuming systemic 1 x 1013vg / kg dosing in 80 kg adults Table 81 Assuming 300-fold excess of SpyC-mAb required 2 Assuming systemic 5 x 1012vg / kg dosing in 80 kg adults EXAMPLE 11 – The effects of Additives on ConjugationREGN 11547

[0206] The following additives were selected and tested to determine their effects on conjugation. The additives were selected due to potential effects on the charge or hydrophobicity of the reaction molecules or the ionic characteristics of the reaction mixture: 1) 0.5% or 2.5% DMSO. 2) 50 mM or 250 mM L-glutamic acid monosodium salt. 3) 50 mM or 250 mM Arginine. 4) 0.5% or 2.5% w / v Sorbitol. 5) 50 mM or 250 mM Urea. 6) 5% or 25% Isopropyl alcohol (IPA). 7) 5 mM or 25 mM Sodium thiocyanate (NaSCN).

[0207] Figure 18 schematically depicts the effects on conjugation of each of the 7 additives identified above at different concentrations. The addition of 25% IPA during the conjugation reaction yielded 80% conjugation in 4 hours, which was higher than that achieved by a control in 72 hours. The other additives at 4 hours achieved about 70% conjugation in 4 hours, which was the same on controls 1 and 2. Urea at 50 mM achieved about 68% conjugation after 4 hours. The use of additives is optional.

[0208] Isopropyl alcohol should preferably be added at a concentration above 5%. Concentrations include about 5.5%.6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, 25% or more.REGN 11547

[0209] Figure 19 depicts data from a conjugation reaction between AAV-SpyT and SpyC-mAb using the 7 additives. The magnified plot shows the effectiveness of 25% isopropyl alcohol. EXAMPLE 12 - Optimization of mosaicism level and Rm format in Covalently Surface Modified Adeno-Associated Viruses

[0210] The following discussion concerns Covalently Surface Modified Adeno-Associated Viruses and uses AAV with the SpyTag-Spy-Catcher System and having monoclonal antibodies (or fragments or derivative thereof) as retargeting molecules (AAV-SpyT-SpyC-Ab) as an exemplar. Any specific binding pair (that is, a first member and second cognate member) can be used according to the inventions.

[0211] When producing AAV-SpyT-SpyC-Ab, three important aspects are (1) the number of SpyT (an exemplary FM) on the AAV capsid, which directly affects the number of SpyC-Abs that can be covalently attached to the AAV capsid, (2) the format of SpyC-Ab to be covalently attached. Formats include, but are not limited to (i) a mAb with two SpyC in the constant regions, (ii) an antibody with a single SpyC on a constant region (referred to as a “bispecific” here), (iii) a Fab with a single SpyC on the variable heavy chain, or (iv) other options (such as ScFv), and (3) the molar ratio of AAV to retargeting molecule during conjugation.

[0212] To further explain the first aspect, the number of SpyT (an example of a first member (FM)) on the AAV capsid can be modified by adjusting the ratio of the pRC and the pRC-SpyT plasmids that are co-transfected into the bioreactor along with the pGOI and pHelper plasmids. For example, when 1 / 10 of all the pRC plasmids transfected are pRC-SpyT plasmids, the AAV capsid is expected to have an average of 6 SpyT, as 1 / 10 of the 60 viral proteins are expected to have aREGN 11547 SpyT (1 / 10 x 60 = 6). Similarly, a transfection ratio of 1 / 4 is expected to result in 15 SpyT per AAV capsid (1 / 4 x 60 = 15), and a transfection ratio of 1 / 30 is expected to result in 2 SpyT per AAV capsid (1 / 30 x 60 = 2), as the capsid is stoichiometrically assembled inside the cell using the available viral proteins. The ratio of pRC-SpyT to pRC plasmids during transfection is referred to as the “mosaicism” of the AAV capsid, with “higher mosaicisms” such as 1 / 4 resulting in higher SpyT on the AAV capsids on average, and “lower mosaicisms” such as 1 / 30 resulting in fewer SpyT on the AAV capsids on average. Thus, where the ratio of pRC-FM to pRC plasmids is a larger fractional value, the mosaicism will be higher. As explained above, 1 / 4 is a larger fractional value than 1 / 30.

[0213] It is desirable to optimize the mosaicism when producing AAV- SpyT-SpyC-Ab molecules. Firstly, it has shown that when an AAV has too many Abs conjugated on its surface, it can result in a negative impact on transduction efficiency and can lower the potency of the therapeutic (See Example 9 showing how higher levels of Ab conjugation resulted in lower transduction efficiency). However, since the capsid is stoichiometrically assembled, it is desirable for the mosaicism to not be too low in order to prevent the formation of a high proportion of AAV capsids without even a single SpyT, which prevents conjugation. For example, with a mosaicism of 1 / 10, it is difficult to achieve optimal (i.e., ‘complete’ conjugation), which suggests that there is a proportion of AAV capsids without any or without accessible SpyT sites. See, for example, Figure 13A. Lowering the mosaicism yet further to 1 / 20 or 1 / 30 is expected to increase the proportion of capsids that cannot be conjugated at all. Thus, there is a need to optimize mosaicism to achieve both a therapeutic product with maximum potency, as well as a product that does not have a significantREGN 11547 proportion of unconjugated molecules. See Figures 20A to 20H (showing the effect of different mosaicisms on conjugation percentage).

[0214] The second aspect requiring optimization is selecting the format of SpyC-Ab to be conjugated to the AAV capsids. There are advantages and disadvantages to various formats and a combination of in vitro and in vivo data can help to make the decision. For the mAb format, due to the presence of two SpyC, there is the potential for each SpyC to bind to a different AAV capsid, resulting in the formation of “daisy-chain” species such as AAV-SpyT-SpyC-mAb-SpyC-SpyT-AAV-, etc. These species have high molecular weight, are undesirable, and have been observed to form particularly when carrying out the in vitro conjugation reaction between AAV-SpyT and SpyC-mAb in a limiting molar ratio.

[0215] A way to avoid the formation of these species is to use a bispecific format with only one SpyC. However, due to the potential negative effect of high levels of surface conjugation on viral potency, the bispecific format also may not be ideal due to increasing the number of SpyC-Abs conjugated on the surface of each capsid. The Fab format may be beneficial to achieve higher levels of conjugation with maximum potency due to the small size of Fabs (~50 kDa) compared to mAbs (~150 kDa).

[0216] Figures 20A to 20H are as follows: Figure 20A schematically depicts a hypothetical readout on the impact that mosaicism has on transduction efficiency (green) and percent (%) conjugation (red) with Fab, bispecific Ab and mAb. In this hypothetical, 80% is the desired limit for conjugated capsids. Mosaicisms selected were 1 / 5, 1 / 7.5, 1 / 10, 1 / 15 and 1 / 30. Figure 20B is a barREGN 11547 graph comparing upstream bioreactor titer and process yield for five different mosaicisms (“M”), produced at 2 L scale for 1 / 5 (M1), 1 / 7.5 (M2), 1 / 15 (M4) and 1 / 30 (M5), and at 50 L scale for 1 / 10 (M3). Figure 20C is a bar graph comparing percent (%) conjugation vs. time for the five different mosaicisms, as measured using size- exclusion chromatography. The data indicates a maximum conjugation for the 1 / 7.5 and 1 / 10 mosaicisms.

[0217] Figures 20D to 20H are graphs schematically depicting data of size-exclusion chromatography overlays showing conjugation process for different mosaicisms M1 (1 / 5), M2 (1 / 7.5), M3 (1 / 10), M4 (1 / 15), and M5 (1 / 30). The FM is SpyT and the SCM is SpyC here. Figure 20D depicts an overlay of M1-M5 prior to addition of SpyC-Abs, showing increasing size with increasing level of mosaicism (M1 > M2 > M3 > M4 > M5). The X-axis designates 7.60 to 10.40 minutes and the Y-axis designates 0.00 to 800.00 EU. Figure 20E depicts an overlay of conjugation reactions between M1 and anti-CACNG1 bispecific single-SpyC mAb at 2, 8, 24 and 36 h, showing increasing conjugation level over time via reduction of the peak at about 8.5 min RT. The X-axis designates 3.00 to 14.50 minutes and the Y-axis designates 0.00 to 200.00 EU. Figure 20F depicts an overlay of conjugation reaction between M2 and anti-CACNG1 single-SpyC Fab at 2, 8, 24 and 36 h, showing increasing conjugation level over time via reduction of the peak shoulder at about 8.5 min RT. The X-axis designates 1.50 to 14.50 minutes and the Y-axis designates 0.00 to 450.00 EU. Figure 20G schematically depicts an overlay of conjugated species of M1-M5 and anti-CACNG1 single-SpyC Fab after 48 h of reaction, showing increasing conjugation level for higher mosaicisms. The X-axis designates 0.00 to 15.00 minutes and the Y-axis designates 0.00 to 1600.00 EU.REGN 11547 Figure 20H depicts an overlay of conjugated species with mosaicisms M1-M5 and anti-CACNG1 single-SpyC bispecific mAb after 48 h of reaction and removal of majority of excess free SpyC-Ab using preparative size exclusion chromatography, which showed increasing conjugation level for higher mosaicisms. The X-axis designates 0.00 to 15.00 minutes and the Y-axis designates 0.00 to 160.00 EU.

[0218] The third aspect for optimization is the molar ratio of AAV to Retargeting Molecule (for example, an antibody. Figure 5A shows that significant high molecular weight species can be observed in the SEC chromatograms corresponding to molar ratios of 1:6 and 1:15 compared to 1:30 and 1:300. In particular, Figure 5A shows the impact of AAV:antibody molar ratio on a AAV capsid with fixed number of SpyTags (1 / 10 mosaicism level = about 6 SpyTags), while Figures 20A to 20H use a fixed AAV:antibody molar ratio (1:300) but vary the mosaicism, that is the number of SpyTags on the AAV capsid (1 / 30 = about 2 SpyTags to 1 / 5 = about 12 SpyTags).

[0219] Figure 5B is a preparative scale SEC on a 100 ml SEPAX SRT 500A column to remove unconjugated antibody. Figure 5B is an overlay of UV280 profiles over 20 column injections, and clearly depicts the separation of free antibody species (in other words, unconjugated antibody species) from AAV species. The multiple overlaid curves are different injections on the same column, as about 30 injections were required to process all of the material. EXAMPLE 13 – PROCESS ANALYTICAL TECHNOLOGY AND IMPROVED PURIFICATIONREGN 11547

[0220] Figure 21A depicts exemplary production purification trains for AAV, such as recombinant AAV. The top train uses a batch process where repeated passes are required to exchange buffer and concentrate the retentate, which contains the desired biological material, such as AAV. See Adams et al., Biotech. Bioeng.117: 3199-3211 (2020).

[0221] The bottom section of Figure 21A replaces the batch tangential flow filtration unit with a single-pass tangential flow filtration unit (SPTFF unit), which permits a continuous process. It was surprising how well SPTFF performed with AAV, as taught herein,

[0222] The Batch TFF approach can take multiple days (for example, 2 days) due to the repeated cycling through the conventional TFF unit to achieve concentration prior to further purification. The SPTFF approach is a continuous approach, and is significantly faster than the Batch TFF approach, and can be performed in several hours, such as 3 to 5 hours. The SPTFF approach provides faster concentration, while minimizing sheer stress and damage to AAVs. The SPTFF approach also is amenable to the use of Process Analytical Technology (PAT) and automation. Figure 21B depicts a process for purifying retargeted AAV at the 500L production scale.

[0223] For further comparison, Figure 22A schematically shows a Batch TFF (top), where the retentate is repeatedly cycled through a feed tank and pump to repeatedly passed through a membrane, with the concentrated retentate being removed after repeated cycles. A Single-Pass TFF removes biologicalREGN 11547 material from the feed tank through a pump to a multi-stage membrane module that separate the retentate from the permeate, while concentrating the retentate.

[0224] Figure 22B is a graph comparing Batch TFF and Single-Pass TFF. Single-Pass TFF achieves higher concentration and is faster as compared to Batch TFF. Single-Pass TFF continuously sends biological material to the next operation in the purification train, whereas Batch TFF does not send biological material until the end of the batch cycle.

[0225] Figure 23 schematically and qualitatively compares the batch operation to a continuous operation for AAV purification in terms of Cell lysis, Clarification (depth filtration), TFF (Batch or Single-Pass) and Affinity Capture. The continuous process (SPTFF) can be finished in less than a day, whereas the batch process can be multi-day.

[0226] The cell lysis step, typically using a detergent such as Tween- 20, typically takes up to about two hours, and is depicted as the same for both the Batch and the SPTFF (continuous) process. Following lysis, clarification takes about 1 hour. The processes then diverge at the TFF step.

[0227] For the Batch process, TFF takes about 3 hours per batch due to the repeated cycling. Not until a batch is complete can the concentrated biological material in a buffer be passed on the affinity capture, which takes about 2 to 3 hours per batch. Because multiple batches are required, the affinity chromatography is typically not completed until the next day.REGN 11547

[0228] For the Continuous process, clarification, SPTFF and affinity capture can take place substantially simultaneously. Biological material continuously flows to clarification (about 1 hour), SPTFF (about 1 ½ hours) and affinity capture (about 2 hours to 3 hour). Accordingly, when an early portion of biological material is in affinity capture, later portions of biological material are in SPTFF or clarification.

[0229] Figure 24 schematically depicts exemplary arrangements for multi-stage membrane module cassettes to be used with Single-Pass TFF. The configurations depict four to seven tiers of membrane module cassettes where the initial tiers (left side) contain more or same number of membrane module cassettes as the succeeding tiers (moving towards the right side), in the manner suggested by the manufacturer, here Pall / Cytiva. Total area and path length of the membrane module cassettes also are set forth. Other arrangement of membranes, flow rates and transmembrane pressure (TMP) can be selected by the person skilled in the art.

[0230] Figure 25 is a graph depicting volumetric concentration factor (VCF) versus transmembrane pressure (TMP) using the 4-in-series, 5-in-series, 6-in- series and 7-in-series exemplary configurations depicted in Figure 24 with a feed comprising an exemplary AAV, here AAV9 comprising a SpyTag insert. A Batch process target would be 8-10x VCF at a TMP of 5 to 10 psi.

[0231] Figure 26 depicts data from a 5-in-series configuration according to Figure 24 at flow rates of 90 ml / minute, 120 ml / minute and 150 ml / minute. The log best-fit equation of VCF = A ln (TMP-B) using the values at each flow rate set forth near the plot (and rounded off in the included table) can be used toREGN 11547 parameterize the data. At the right side of the figure, there is a graph of parameter value (A, B) and feed flow rate in liters per square meter of membrane per hour (LMH) for 4-in-series and 5-in-series exemplary configurations of Figure 24 and allows optimized conditions to be selected in silico using an exemplary AAV, here AAV9 comprising a SpyTag insert. This model can be used to predict the VCF for any flow rate and TMP for an in-series configuration of interest.

[0232] Figure 27A is a design space model based on Figures 25 and 26 using the 5-in-series configuration of Figure 24. Here, the process target was 35 LMH, and the intersecting lines indicate a VCF of 8 and a TMP of 10 psi. An exemplary acceptable zone would be a VCF of 6-10 and a TMP of 7.5 to 12.5 psi. Figure 27B is an exemplary comparison of process parameters between SPTFF and Batch TFF. With Batch TFF, typically there would be one batch before the next operation. However, depending on the scheduling of upstream production bioreactors and bioreactor titers, there could be pooling of multiple batches before the next operation. Effective residence time of a given portion of biological material in the SPTFF is only about 10 minutes, and the overall time is for all biological material to pass thought the SPTFF.

[0233] Figure 28 depicts data from a bench-scale trial to determine the number of buffer washes need to attain about a 90% recovery of AAV, here AAV9 with integrated SpyTag, in a low-TMP process. On average, the AAV9 here contained 6 SpyTag peptides per viral capsid. The load concentration was 1.7 x 1012capsids / ml (cp / ml). The steady state concentration using SPTFF was 1.6 - 1.9 x 1013cp / ml, yielding a steady state VCF of 10 to 11x. Capsid titer in retentate (cp / ml) versus SPTFF operating time (minutes) was measured using four buffer flushes.REGN 11547 The final pool flush (1 and 2) achieved 1.4 x 1013cp / ml. As the right side of the figure shows, 71% of capsids were recovered in the retentate pool, 11% of capsids were recovered by flush 1 and 6% of capsids were recovered with flush 2. It was determined that only two buffer flushes were required to achieved about a 90% recovery with a VCF of 8x.

[0234] Figure 29 is a graph depicting Permeate Flux (LMH), Throughput (L / m2), Feed Flow Rate (L / hr) and TMP (psi) in a pilot-scale trial. Using continuous SPTFF, the data showed flux decline and TMP build up. To mitigate TMP increase beyond 12.5 psi, feed flow rate was slowed. This resulted in a longer process time of 180 minutes rather than the expected 90 minutes and an overall VCF of 5x was achieved rather than the target VCF of 8x.

[0235] Figure 30 depicts a tween micelle build-up on the TFF membrane. Without being bound by any theory or hypothesis, it is believed that detergent micelle buildup (here, Tween-20) is the cause of an unexpected flux decline of about 50% using SPTFF to concentrate AAV. Typically, a 20% flux decline is expected when concentrating antibodies. This figure also set forth the approximate size of AAV, Host Cell Protein aggregates (HCP) and Tween-20 micelles. The micelle concentration of Tween-20 was determined to be about 0.7%. See Basheva et al, J. Physical Chemistry Chemical Physics, Issue 38 (2007) (discusses properties of films formed by Brij 35 and Tween 20). Detergents, such as Tweens, are a common component of cell lysis buffers used in the production of AAV.REGN 11547

[0236] Figure 31 is a graph depicting fold presence of Tween-20 on the retentate side of membrane and the permeate side of the membrane for both Batch TFF and SPTFF. Most Tween-20 is on the retentate side.

[0237] Figure 32 is a graph depicting the flux decline after two hours with varying percentages of Tween-20 in the lysis buffer. The lower the percentage of Tween-20, the lower the percentage of flux decline encountered. In addition to Tween-20, the buffer contained 20mM Tris, 2mM MgCl2 at a pH of 7.4. The feed flow rate was 35 LMH and the TMP was about 5 to 10 psi.

[0238] With Batch TFF, flux decline can be address by increasing processing time. However, with SPTFF immediate control is desired.

[0239] Figure 33 compares control with the retentate valve to control with a Permeate pump. Option 1 with the retentate valve found that TMP reached 22 psi, and after which the flow had to be reduced from 40 LMH to 30 LMH. VCF dropped from about 10x to about 6x. Option 2 with the permeate pump was superior, which acts as a suction pump. TMP was controlled to well under 10 psi and a VCF of 8x was maintained. At the right side to the figure, Option 1 (SPTFF with retentate valve) and Option 2 (SPTFF with permeate pump) were compared to a Batch TFF. Option 1 did not perform as well as Option 2 and Batch TFF. Option 2 was superior to Batch TFF and Option 1 in terms of capsid yield and percent aggregation. The permeate pump flow should be set within the VCF design space to avoid negative permeate pressure buildup. Thus, the flow rate of operation of the permeate pump should be within the range established in Figure 27A.

[0240] Figure 34 depicts an overall pilot scale process.REGN 11547

[0241] Figure 35 compares VCFs (1-14), SPTFF retentate flow rates and residence time in affinity capture. VCFs of 7 to 13 and SPTFF retentate flow rates of 75-40 provided an exemplary range of residence time suitable for affinity loading. The flow rate should be selected to avoid depleting or overwhelming the affinity column. This calculation was based on a pilot-scale trial with a 525 ml / minute feed flow in a 5-in-one series SPTFF module and then loaded on to a 200 ml POROS CaptureSelect AAV9 column.

[0242] Figure 36 depicts how UV280 profile of affinity capture flow can be used for process monitoring of VCF and process stability using SPTFF for continuous processing. Three different runs were performed for comparison purposes. Run 1 was performed without a permeate pump and achieved a VCF of only 5X. Run 2 was performed with a permeate pump with a feed to retentate flush (with recirculation) and achieved a VCF of 8x. Run 3 was performed with a permeate pump with a feed to retentate flush (with recirculation) and a permeate to retentate flush, which achieved a VCF of 10x. Most chromatography systems have built-in UV280 sensors that can detect load concentration, and provide an indication of VCF and process stability. Any needed correction, such as pump and / or valve control, can be based upon the data received through process analytical technology. See Thakur et al., J. Membrane. Sci.613: 118492 (2020) discuss the use of process analytical technology with SPTFF.

[0243] Advantages and Aspects of SPTFF include: • Greater efficiency in AAV manufacturing from harvest to final capture and purification; • Use of a permeate pump provides real-time control over TMP and maximizesREGN 11547 AAV yield and minimizes AAV aggregation; • Detergents, such as Tween-20, can decrease permeate flux, which can be best managed through use of a permeate pump; and • Volumetric concentration factor (VCF) depends on flow rate, TMP and SPTFF membrane module configuration, which can be addressed by the empirical modeling of VCF vs. TMP curves for optimization based upon the teachings contained herein. EXAMPLE 14 – EFFECT OF RETARGETING CONDITIONS AND BIOREACTOR SCALE ON VECTOR TITER

[0244] Figure 37A depicts vector viral titer and viral genome to capsid ratio (Vg:Cp) of different capsid types: a wild type (WT) AAV9, a detargeted AAV9 and a detargeted AAV9 comprising the SpyTag peptide in the viral capsid. Below each column there is provided a schematic diagram of each capsid type. Vector genome titer was consistent amongst the three capsid types.

[0245] Viral genome to capsid percentage exhibited slightly more variation, and ranged from 11% to 17% full capsids. amongst the three capsid types.

[0246] Figure 37B depicts virus vector viral titer and viral genome to capsid ratio (Vg:Cp) at different bioreactor scales: 0.2 liters, 2 liters and 50 liters. The 0.2 liter exhibited the highest vector genome titer, and was in the middle in terms of viral genome to capsid percentage. The range overall was 13% to 24% viral genome to capsid percentage amongst the three sizes of bioreactors The 2 liter reactor exhibited the largest viral genome to capsid percentage, and the 50 liter reactor exhibited the lowest viral genome to capsid percentage.REGN 11547

[0247] Figure 38 is a graph depicting conjugated AAV9-SpyT-SpyC- Rm species with a detargeting mutation compared to an unconjugated antibody. The detargeting mutation was N272A The retargeting molecules were an ASGR1 monoclonal antibody and a FELD1 monoclonal antibody. The conjugated AAV9- SpyT-SpyC-Rm species were tested. EXAMPLES OF COVALENTLY SURFACE MODIFIED AAV

[0248] The following examples provide teachings on how to produce covalently surface modified AAV of all serotypes using exemplary sequences according to the inventions. Pertinent polynucleotide and amino acid sequences are widely available in the published literature, and therefore the inventions are not limited to the polynucleotide and amino acid sequences set forth in the specification and the sequence listing.

[0249] The below examples set forth rAAV comprising VP1, VP2 and / or VP3 protein fused to a first member of a specific binding pair. The rAAV can be later conjugated to a retargeting molecule fused to a second cognate member of the specific binding pair. Use of SpyTag and SpyCatcher as specific binding pairs as disclosed herein provides teachings and data that is applicable to the use of other specific binding pairs, including fragments and derivative of specific binding pairs, disclosed herein or otherwise known in the field. Conjugation is disclosed throughout, and an exemplary overview can be found at Figures 12A-C, 13A-B, 14A-E, 15, 16, 17, 18, 19, 20 A-H and 21A-B and associated text. Rep and cap genes from the same serotype are exemplified below, but the inventions also provide for use or rep and cap genes from different serotypes.REGN 11547 EXAMPLE 15 – AAV1

[0250] Covalently surface modified AAV1 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and harvesting the resulting AAV, and then conjugating the resulting AAV with a second cognate member (SCM) that is fused to a retargeting molecule The first chart below uses SpyTag as the first member of a specific binding pair, and Adenovirus Helper Genes. Modifications can be undertaken as disclosed below:(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV1.REGN 11547 (2) Plasmids pRC-FM can be modified for other specific binding pairs. The first member (FM) should be complementary to a second cognate member (SCM) that is fused to a retargeting molecule (for example, an antibody, as well derivatives and fragments thereof, and still other molecules). See Definitions and Description sections of the Detailed Description, the Figures and associated text for “retargeting” and “retargeting molecules”. The populations of FMs and SCMs can be homogenous or heterogeneous as long as the selected FMs can come in contact with appropriate SCMs so that binding can occur. (3) Detargeting mutations are optional and can be undertaken as disclosed herein and in publications. See Definitions and Description sections of the Detailed Description, the Figures and associated text for “detargeting” and “detargeting mutations”. (4) To lessen the possibility of encountering neutralizing antibodies when the covalently surface modified AAV is administered to a subject, KTag can be used as an SCM to bind with SpyTag (FM) (SEQ ID NOS: 54 and 86). KTag and SpyLigase are based upon SpyCatcher. KTag (SEQ ID NO: 85) has only 10 amino acids, which renders it less immunogenic. KTag can be bound to SpyTag in the presence of SpyLigase (SEQ ID NO: 61). The SpyLigase can be removed by subsequent purification, such as by chromatography and / or filtration so that the SpyLigase will not have an appreciable presence in the final drug substance. (5) Preferred mosaicisms are as follows: 1 / 1, 1 / 1.1, 1 / 1.2, 1 / 1.3, 1 / 1.4, 1 / 1.5, 1 / 1.6, 1 / 1.7, 1 / 1.8, 1 / 1.9, 1 / 2, 1 / 2.1, 1 / 2.2, 1 / 2.3, 1 / 2.4, 1 / 2.5, 1 / 2.6, 1 / 2.7, 1 / 2.8, 1 / 2.9, 1 / 3, 1 / 3.1, 1 / 3.2, 1 / 3.3, 1 / 3.4, 1 / 3.5, 1 / 3.6, 1 / 3.7, 1 / 3.8, 1 / 3.9, 1 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5,REGN 11547 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, 1 / 12.8, 1 / 12.9, / 13, 1 / 13.1, 1 / 13.2, 1 / 13.3, 1 / 13.4, 1 / 13.5, 1 / 13.6, 1 / 13.7, 1 / 13.8, 1 / 13.9, 1 / 14, / 14.1, 1 / 14.2, 1 / 14.3, 1 / 14.4, 1 / 14.5, 1 / 14.6, 1 / 14.7, 1 / 14.8, 1 / 14.9, 1 / 15, 1 / 15.1, / 15.2, 1 / 15.3, 1 / 15.4, 1 / 15.5, 1 / 15.6, 1 / 15.7, 1 / 15.8, 1 / 15.9, 1 / 16, 1 / 16.1, 1 / 16.2, / 16.3, 1 / 16.4, 1 / 16.5, 1 / 16.6, 1 / 16.7, 1 / 16.8, 1 / 16.9, 1 / 17, 1 / 17.1, 1 / 17.2, 1 / 17.3, / 17.4, 1 / 17.5, 1 / 17.6, 1 / 17.7, 1 / 17.8, 1 / 17.9, 1 / 18, 1 / 18.1, 1 / 18.2, 1 / 18.3, 1 / 18.4, / 18.5, 1 / 18.6, 1 / 18.7, 1 / 18.8, 1 / 18.9, 1 / 19, 11 / 9.1, 1 / 19.2, 1 / 19.3, 1 / 19.4, 1 / 91.5, / 19.6, 1 / 19.7, 1 / 19.8, 1 / 19.9, 1 / 20, 1 / 21, 1 / 21.1, 1 / 21.2, 1 / 21.3, 1 / 21.4, 1 / 21.5, / 21.6, 1 / 21.7, 1 / 21.8, 1 / 21.9,1 / 22, 1 / 22.1, 1 / 22.2, 1 / 22.3, 1 / 22.4, 1 / 22.5, 1 / 22.6, / 22.7, 1 / 22.8, 1 / 22.9, 1 / 23, 1 / 23.1, 1 / 23.2, 1 / 23.3, 1 / 23.4, 1 / 23.5, 1 / 23.6, 1 / 23.7, / 23.8, 1 / 23.9, 1 / 24, 1 / 24.1, 1 / 24.2, 1 / 24.3, 1 / 24.4, 1 / 24.5, 1 / 24.6, 1 / 24.7, 1 / 24.8, / 24.9, 1 / 25, 1 / 25.1, 1 / 25.2, 1 / 25.3, 1 / 25.4, 1 / 25.5, 1 / 25.6, 1 / 25.7, 1 / 25.8, 1 / 25.9, / 26, 1 / 26.1, 1 / 26.2, 1 / 26.3, 1 / 26.4, 1 / 26.5, 1 / 26.6, 1 / 26.7, 1 / 26.8, 12 / 6.9, 1 / 27, / 27.1, 1 / 27.2, 1 / 27.3, 1 / 27.4, 1 / 27.5, 1 / 27.6, 1 / 27.7, 1 / 27.8, 1 / 27.9, 1 / 28, 1 / 28.1, / 28.2, 1 / 28.3, 1 / 28.4, 1 / 28.5, 1 / 28.6, 1 / 28.7, 1 / 28.8, 1 / 28.9, 1 / 29, 1 / 29.1, 1 / 29.2, / 29.3, 1 / 29.4, 1 / 29.5, 1 / 29.6, 1 / 29.7, 1 / 29.8, 1 / 29.9, 1 / 30, 1 / 30.1, 1 / 30.2, 1 / 30.3, / 30.4, 1 / 30.5, 1 / 30.6, 1 / 30.7, 1 / 30.8, 1 / 30.9, 1 / 31, 1 / 31.1, 1 / 31.2, 1 / 31.3, 1 / 31.4, / 31.5, 1 / 31.6, 1 / 31.7, 1 / 31.8, 1 / 31.9, 1 / 32, 1 / 32.1, 1 / 32.2, 1 / 32.3, 1 / 32.4, 1 / 32.5, / 32.6, 1 / 32.7, 1 / 32.8, 1 / 32.9, 1 / 33, 1 / 33.1, 1 / 33.2, 1 / 33.3, 1 / 33.4, 1 / 33.5, 1 / 33.6, / 33.7, 1 / 33.8, 1 / 33.9, 1 / 34, 1 / 34.1, 1 / 34.2, 1 / 34.3, 1 / 34.4, 1 / 34.5, 1 / 34.6, 1 / 34.7,REGN 11547 1 / 34.8, 1 / 34.9, 1 / 35, 1 / 35.1, 1 / 35.2, 1 / 35.3, 1 / 35.4, 1 / 35.5, 1 / 35.6, 1 / 35.7, 1 / 35.8, 1 / 35.9, 1 / 36, 1 / 36.1, 1 / 36.2, 1 / 36.3, 1 / 36.4, 1 / 36.5, 1 / 36.6, 1 / 36.7, 1 / 36.8, 1 / 36.9, 1 / 37, 1 / 37.1, 1 / 37.2, 1 / 37.3, 1 / 37.4, 1 / 37.5, 1 / 37.6, 1 / 37.7, 1 / 37.8, 1 / 37.9, 1 / 38, 1 / 38.1, 1 / 38.2, 1 / 38.3, 1 / 38.4, 1 / 38.5, 1 / 38.6, 1 / 38.7, 1 / 38.8, 1 / 38.9, 1 / 39, 1 / 39.1, 1 / 39.2, 1 / 39.3, 1 / 39.4, 1 / 39.5, 1 / 39.6, 1 / 39.7, 1 / 39.8, 1 / 39.9, 1 / 40, 1 / 40.1, 1 / 40.2, 1 / 40.3, 1 / 40.4, 1 / 40.5, 1 / 40.6, 1 / 40.7, 1 / 40.8, 1 / 40.9, 1 / 41, 1 / 41.1, 1 / 41.2, 1 / 41.3, 1 / 41.4, 1 / 41.5, 1 / 41.6, 1 / 41.7, 1 / 41.8, 1 / 41.9, 1 / 42, 1 / 42.1, 1 / 42.2, 1 / 42.3, 1 / 42.4, 1 / 42.5, 1 / 42.6, 1 / 42.7, 1 / 42.8, 1 / 42.9, 1 / 43, 1 / 43.1, 1 / 43.2, 1 / 43.3, 1 / 43.4, 1 / 43.5, 1 / 43.6, 1 / 43.7, 1 / 43.8, 1 / 43.9, 1 / 44, 1 / 44.1, 1 / 44.2, 1 / 44.3, 1 / 44.4, 1 / 44.5, 1 / 44.6, 1 / 44.7, 1 / 44.8, 1 / 44.9, 1 / 45, 1 / 45.1, 1 / 45.2, 1 / 45.3, 1 / 45.4, 1 / 45.5, 1 / 45.6, 1 / 45.7, 1 / 45.8, 1 / 45.9, 1 / 46, 1 / 46.1, 1 / 46.2, 1 / 46.3, 1 / 46.4, 1 / 46.5, 1 / 46.6, 1 / 46.7, 1 / 46.8, 1 / 46.9, 1 / 47, 1 / 47.1, 1 / 47.2, 1 / 47.3, 1 / 47.4, 1 / 47.5, 1 / 47.6, 1 / 47.7, 1 / 47.8, 1 / 47.9, 1 / 48, 1 / 48.1, 1 / 48.2, 1 / 48.3, 1 / 48.4, 1 / 48.5, 1 / 48.6, 1 / 48.7, 1 / 48.8, 1 / 48.9, 1 / 49, 1 / 49.1, 1 / 49.2, 1 / 49.3, 1 / 49.4, 1 / 49.5, 1 / 49.6, 1 / 49.7, 1 / 49.8, 1 / 49.9, 1 / 50, 1 / 50.1, 1 / 50.2, 1 / 50.3, 1 / 50.4, 1 / 50.5, 1 / 50.6, 1 / 50.7, 1 / 50.8, 1 / 50.9, 1 / 51, 1 / 51.1, 1 / 51.2, 1 / 51.3, 1 / 51.4, 1 / 51.5, 1 / 51.6, 1 / 51.7, 1 / 51.8, 1 / 51.9, 1 / 52, 1 / 52.1, 1 / 52.2, 1 / 52.3, 1 / 52.4, 1 / 52.5, 1 / 52.6, 1 / 52.7, 1 / 52.8, 1 / 52.9, 1 / 53, 1 / 53.1, 1 / 53.2, 1 / 53.3, 1 / 53.4, 1 / 53.5, 1 / 53.6, 1 / 53.7, 1 / 53.8, 1 / 53.9, 1 / 54, 1 / 54.1, 1 / 54.2, 1 / 54.3, 1 / 54.4, 1 / 54.5, 1 / 54.6, 1 / 54.7, 1 / 54.8, 1 / 54.9, 1 / 55, 1 / 55.1, 1 / 55.2, 1 / 55.3, 1 / 55.4, 1 / 55.5, 1 / 55.6, 1 / 55.7, 1 / 55.8, 1 / 55.9, 1 / 56, 1 / 56.1, 1 / 56.2, 1 / 56.3, 1 / 56.4, 1 / 56.5, 1 / 56.6, 1 / 56.7, 1 / 56.8, 1 / 56.9, 1 / 57, 1 / 57.1, 1 / 57.2, 1 / 57.3, 1 / 57.4, 1 / 57.5, 1 / 57.6, 1 / 57.7, 1 / 57.8, 1 / 57.9, 1 / 58, 1 / 58.1, 1 / 58.2, 1 / 58.3, 1 / 58.4, 1 / 58.5, 1 / 58.6, 1 / 58.7, 1 / 58.8, 1 / 58.9, 1 / 59, 1 / 59.1, 1 / 59.2, 1 / 59.3, 1 / 59.4, 1 / 59.5, 1 / 59.6, 1 / 59.7, 1 / 59.8, 1 / 59.9, 1 / 60 or ratios thereabout or therebetween.REGN 11547 Ranges of ratios of 1 / 1 to 1 / 60 can be employed, and subranges as set forth below. More preferred mosaicisms are: 1 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, 1 / 12.8, 1 / 12.9 or 1 / 13. Further preferred mosaicisms are: 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, or 1 / 12. Still more preferred mosaicisms are: 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, or 1 / 11.REGN 11547 Preferred ranges of mosaicisms are 1 / 4 to 1 / 13; 1 / 5 to 1 / 12; 1 / 6 to 1 / 11, 1 / 7 to 1 / 10.5. or 1 / 7.5 to 1 / 10. (6) Preferred molar ratios of AAV-FM to SCM-RM are as follows: 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300, 1:305, 1:310, 1:315, 1:320, 1:325, 1:330, 1:335, 1:340, 1:345, 1:350, 1:355, 1:360, 1:365, 1:370, 1:375, 1:380, 1:385, 1:390, 1:395, 1:400, 1:405, 1:410, 1:415, 1:420, 1:425, 1:430, 1:435, 1:440, 1:445, 1:450, 1:455, 1:460, 1:465, 1:470, 1:475, 1:480, 1:485, 1:490, 1:495, 1:500, 1:505, 1:510, 1:515, 1:520, 1:525, 1:530, 1:535, 1:540, 1:545, 1:550, 1:555, 1:560, 1:565, 1:570, 1:575, 1:580, 1:585, 1:590, 1:595, 1:600, 1:605, 1:610, 1:615, 1:620, 1:625, 1:630, 1:635, 1:640, 1:645, 1:650, 1:655, 1:660, 1:665, 1:670, 1:675, 1:680, 1:685, 1:690, 1:695, 1:700, 1:705, 1:710, 1:715, 1:720, 1:725, 1:730, 1:735, 1:740, 1:745, 1:750, 1:755, 1:760, 1:765, 1:770, 1:775, 1:780, 1:785, 1:790, 1:795, 1:800, 1:805, 1:810, 1:815, 1:820, 1:825, 1:830, 1:835, 1:840, 1:845, 1:850, 1:855, 1:860, 1:865, 1:870, 1:875, 1:880, 1:885, 1:890, 1:895, 1:900, 1:905, 1:910, 1:915, 1:920, 1:925, 1:930, 1:935, 1:940, 1:945, 1:950, 1:955, 1:960, 1:965, 1:970, 1:975, 1:980, 1:985, 1:990, 1:995, 1:1000, 1:>1000 or any ratio thereabout or therebetween.

[0251] More preferred ratios AAV-FM to SCM-RM are: 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230,REGN 11547 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300 or any ratio thereabout or therebetween.

[0252] Preferred ranges of AAV-FM to SCM-RM are 1:20 to 1:1000, and more preferred ranges are: 1:30 to 1:100, 1:40 to 1:100, 1:50 to 1:1001:60 to 1:100, 1:70 to 1:100, 1:80 to 1:100, 1:90:1:100, 1:30 to 1:150, 1:40 to 1:150, 1:50 to 1:1501:60 to 1:150, 1:70 to 1:150, 1:80 to 1:150, 1:90 to :150, 1:100 to 1:150, 1:30 to 1:200, 1:40 to 1:200, 1:50 to 1:2001:60 to 1:200, 1:70 to 1:200, 1:80 to 1:200, 1:90:1:200, 1:100 to 1:200, 1:30 to 1:250, 1:40 to 1:250, 1:50 to 1:2501:60 to 1:250, 1:70 to 1:250, 1:80 to 1:250, 1:90 to :250, 1:100 to 1:250, 1:30 to 1:300, 1:40 to 1:300, 1:50 to 1:3001:60 to 1:300, 1:70 to 1:300, 1:80 to 1:300, 1:90:1:300, or 1:100 to 1:300. (7) The inventions provide for a desired conjugation of a level of conjugation of at least 10%, at least 20%, at least 30%, and at least 40%. Preferred levels are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100%, any of which can be considered complete depending on the purpose. More preferably, the desired level of conjugation typically will be about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Ranges of desired conjugation levels can be 10% to 100%, 20% to 99%, 30% 99%, 40% to 99%, 50% to 99%, 60% to 99%, 70% to 99%, 80% to 99%, or 90% toREGN 11547 99%. Preferred ranges of desired conjugation levels can be 40% to 100%, 40% to 99%, 45% to 95%, 50% to 95%, 55% to 90%, 60% to 90%, 65% to 95%, 65% to 85%, 65% to 90%, 70% to 90%, 75% to 90%, 75% to 85%, 75% to 80%, or 80% to 85%. More preferably, the range is 65% to 95%.

[0253] The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein. Points 1-7 above also apply to the second chart below:EXAMPLE 16 – AAV2REGN 11547

[0254] Covalently surface modified AAV2 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and harvesting the resulting AAV, and then conjugating the resulting AAV with a second cognate member (SCM) that is fused to a retargeting molecule The first chart below uses SpyTag as the first member of a specific binding pair, and Adenovirus Helper Genes. Modifications can be undertaken as disclosed below:(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV2. (2) Plasmids pRC-FM can be modified for other specific binding pairs. The first member (FM) should be complementary to a second cognate member (SCM) that is fused to a retargeting molecule (for example, an antibody, as well derivatives andREGN 11547 fragments thereof, and still other molecules). See Definitions and Description sections of the Detailed Description, the Figures and associated text for “retargeting” and “retargeting molecules”. The populations of FMs and SCMs can be homogenous or heterogeneous as long as the selected FMs can come in contact with appropriate SCMs so that binding can occur. (3) Detargeting mutations are optional and can be undertaken as disclosed herein and in publications. See Definitions and Description sections of the Detailed Description, the Figures and associated text for “detargeting” and “detargeting mutations”. (4) To lessen the possibility of encountering neutralizing antibodies when the covalently surface modified AAV is administered to a subject, KTag can be used as an SCM to bind with SpyTag (FM) (SEQ ID NOS: 54 and 86). KTag and SpyLigase are based upon SpyCatcher. KTag (SEQ ID NO: 85) has only 10 amino acids, which renders it less immunogenic. KTag can be bound to SpyTag in the presence of SpyLigase (SEQ ID NO: 61). The SpyLigase can be removed by subsequent purification, such as by chromatography and / or filtration so that the SpyLigase will not have an appreciable presence in the final drug substance. (5) Preferred mosaicisms are as follows: 1 / 1, 1 / 1.1, 1 / 1.2, 1 / 1.3, 1 / 1.4, 1 / 1.5, 1 / 1.6, 1 / 1.7, 1 / 1.8, 1 / 1.9, 1 / 2, 1 / 2.1, 1 / 2.2, 1 / 2.3, 1 / 2.4, 1 / 2.5, 1 / 2.6, 1 / 2.7, 1 / 2.8, 1 / 2.9, 1 / 3, 1 / 3.1, 1 / 3.2, 1 / 3.3, 1 / 3.4, 1 / 3.5, 1 / 3.6, 1 / 3.7, 1 / 3.8, 1 / 3.9, 1 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5,REGN 11547 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, 1 / 12.8, 1 / 12.9, / 13, 1 / 13.1, 1 / 13.2, 1 / 13.3, 1 / 13.4, 1 / 13.5, 1 / 13.6, 1 / 13.7, 1 / 13.8, 1 / 13.9, 1 / 14, / 14.1, 1 / 14.2, 1 / 14.3, 1 / 14.4, 1 / 14.5, 1 / 14.6, 1 / 14.7, 1 / 14.8, 1 / 14.9, 1 / 15, 1 / 15.1, / 15.2, 1 / 15.3, 1 / 15.4, 1 / 15.5, 1 / 15.6, 1 / 15.7, 1 / 15.8, 1 / 15.9, 1 / 16, 1 / 16.1, 1 / 16.2, / 16.3, 1 / 16.4, 1 / 16.5, 1 / 16.6, 1 / 16.7, 1 / 16.8, 1 / 16.9, 1 / 17, 1 / 17.1, 1 / 17.2, 1 / 17.3, / 17.4, 1 / 17.5, 1 / 17.6, 1 / 17.7, 1 / 17.8, 1 / 17.9, 1 / 18, 1 / 18.1, 1 / 18.2, 1 / 18.3, 1 / 18.4, / 18.5, 1 / 18.6, 1 / 18.7, 1 / 18.8, 1 / 18.9, 1 / 19, 11 / 9.1, 1 / 19.2, 1 / 19.3, 1 / 19.4, 1 / 91.5, / 19.6, 1 / 19.7, 1 / 19.8, 1 / 19.9, 1 / 20, 1 / 21, 1 / 21.1, 1 / 21.2, 1 / 21.3, 1 / 21.4, 1 / 21.5, / 21.6, 1 / 21.7, 1 / 21.8, 1 / 21.9,1 / 22, 1 / 22.1, 1 / 22.2, 1 / 22.3, 1 / 22.4, 1 / 22.5, 1 / 22.6, / 22.7, 1 / 22.8, 1 / 22.9, 1 / 23, 1 / 23.1, 1 / 23.2, 1 / 23.3, 1 / 23.4, 1 / 23.5, 1 / 23.6, 1 / 23.7, / 23.8, 1 / 23.9, 1 / 24, 1 / 24.1, 1 / 24.2, 1 / 24.3, 1 / 24.4, 1 / 24.5, 1 / 24.6, 1 / 24.7, 1 / 24.8, / 24.9, 1 / 25, 1 / 25.1, 1 / 25.2, 1 / 25.3, 1 / 25.4, 1 / 25.5, 1 / 25.6, 1 / 25.7, 1 / 25.8, 1 / 25.9, / 26, 1 / 26.1, 1 / 26.2, 1 / 26.3, 1 / 26.4, 1 / 26.5, 1 / 26.6, 1 / 26.7, 1 / 26.8, 12 / 6.9, 1 / 27, / 27.1, 1 / 27.2, 1 / 27.3, 1 / 27.4, 1 / 27.5, 1 / 27.6, 1 / 27.7, 1 / 27.8, 1 / 27.9, 1 / 28, 1 / 28.1, / 28.2, 1 / 28.3, 1 / 28.4, 1 / 28.5, 1 / 28.6, 1 / 28.7, 1 / 28.8, 1 / 28.9, 1 / 29, 1 / 29.1, 1 / 29.2, / 29.3, 1 / 29.4, 1 / 29.5, 1 / 29.6, 1 / 29.7, 1 / 29.8, 1 / 29.9, 1 / 30, 1 / 30.1, 1 / 30.2, 1 / 30.3, / 30.4, 1 / 30.5, 1 / 30.6, 1 / 30.7, 1 / 30.8, 1 / 30.9, 1 / 31, 1 / 31.1, 1 / 31.2, 1 / 31.3, 1 / 31.4, / 31.5, 1 / 31.6, 1 / 31.7, 1 / 31.8, 1 / 31.9, 1 / 32, 1 / 32.1, 1 / 32.2, 1 / 32.3, 1 / 32.4, 1 / 32.5, / 32.6, 1 / 32.7, 1 / 32.8, 1 / 32.9, 1 / 33, 1 / 33.1, 1 / 33.2, 1 / 33.3, 1 / 33.4, 1 / 33.5, 1 / 33.6, / 33.7, 1 / 33.8, 1 / 33.9, 1 / 34, 1 / 34.1, 1 / 34.2, 1 / 34.3, 1 / 34.4, 1 / 34.5, 1 / 34.6, 1 / 34.7, / 34.8, 1 / 34.9, 1 / 35, 1 / 35.1, 1 / 35.2, 1 / 35.3, 1 / 35.4, 1 / 35.5, 1 / 35.6, 1 / 35.7, 1 / 35.8, / 35.9, 1 / 36, 1 / 36.1, 1 / 36.2, 1 / 36.3, 1 / 36.4, 1 / 36.5, 1 / 36.6, 1 / 36.7, 1 / 36.8, 1 / 36.9, / 37, 1 / 37.1, 1 / 37.2, 1 / 37.3, 1 / 37.4, 1 / 37.5, 1 / 37.6, 1 / 37.7, 1 / 37.8, 1 / 37.9, 1 / 38,REGN 11547 1 / 38.1, 1 / 38.2, 1 / 38.3, 1 / 38.4, 1 / 38.5, 1 / 38.6, 1 / 38.7, 1 / 38.8, 1 / 38.9, 1 / 39, 1 / 39.1, 1 / 39.2, 1 / 39.3, 1 / 39.4, 1 / 39.5, 1 / 39.6, 1 / 39.7, 1 / 39.8, 1 / 39.9, 1 / 40, 1 / 40.1, 1 / 40.2, 1 / 40.3, 1 / 40.4, 1 / 40.5, 1 / 40.6, 1 / 40.7, 1 / 40.8, 1 / 40.9, 1 / 41, 1 / 41.1, 1 / 41.2, 1 / 41.3, 1 / 41.4, 1 / 41.5, 1 / 41.6, 1 / 41.7, 1 / 41.8, 1 / 41.9, 1 / 42, 1 / 42.1, 1 / 42.2, 1 / 42.3, 1 / 42.4, 1 / 42.5, 1 / 42.6, 1 / 42.7, 1 / 42.8, 1 / 42.9, 1 / 43, 1 / 43.1, 1 / 43.2, 1 / 43.3, 1 / 43.4, 1 / 43.5, 1 / 43.6, 1 / 43.7, 1 / 43.8, 1 / 43.9, 1 / 44, 1 / 44.1, 1 / 44.2, 1 / 44.3, 1 / 44.4, 1 / 44.5, 1 / 44.6, 1 / 44.7, 1 / 44.8, 1 / 44.9, 1 / 45, 1 / 45.1, 1 / 45.2, 1 / 45.3, 1 / 45.4, 1 / 45.5, 1 / 45.6, 1 / 45.7, 1 / 45.8, 1 / 45.9, 1 / 46, 1 / 46.1, 1 / 46.2, 1 / 46.3, 1 / 46.4, 1 / 46.5, 1 / 46.6, 1 / 46.7, 1 / 46.8, 1 / 46.9, 1 / 47, 1 / 47.1, 1 / 47.2, 1 / 47.3, 1 / 47.4, 1 / 47.5, 1 / 47.6, 1 / 47.7, 1 / 47.8, 1 / 47.9, 1 / 48, 1 / 48.1, 1 / 48.2, 1 / 48.3, 1 / 48.4, 1 / 48.5, 1 / 48.6, 1 / 48.7, 1 / 48.8, 1 / 48.9, 1 / 49, 1 / 49.1, 1 / 49.2, 1 / 49.3, 1 / 49.4, 1 / 49.5, 1 / 49.6, 1 / 49.7, 1 / 49.8, 1 / 49.9, 1 / 50, 1 / 50.1, 1 / 50.2, 1 / 50.3, 1 / 50.4, 1 / 50.5, 1 / 50.6, 1 / 50.7, 1 / 50.8, 1 / 50.9, 1 / 51, 1 / 51.1, 1 / 51.2, 1 / 51.3, 1 / 51.4, 1 / 51.5, 1 / 51.6, 1 / 51.7, 1 / 51.8, 1 / 51.9, 1 / 52, 1 / 52.1, 1 / 52.2, 1 / 52.3, 1 / 52.4, 1 / 52.5, 1 / 52.6, 1 / 52.7, 1 / 52.8, 1 / 52.9, 1 / 53, 1 / 53.1, 1 / 53.2, 1 / 53.3, 1 / 53.4, 1 / 53.5, 1 / 53.6, 1 / 53.7, 1 / 53.8, 1 / 53.9, 1 / 54, 1 / 54.1, 1 / 54.2, 1 / 54.3, 1 / 54.4, 1 / 54.5, 1 / 54.6, 1 / 54.7, 1 / 54.8, 1 / 54.9, 1 / 55, 1 / 55.1, 1 / 55.2, 1 / 55.3, 1 / 55.4, 1 / 55.5, 1 / 55.6, 1 / 55.7, 1 / 55.8, 1 / 55.9, 1 / 56, 1 / 56.1, 1 / 56.2, 1 / 56.3, 1 / 56.4, 1 / 56.5, 1 / 56.6, 1 / 56.7, 1 / 56.8, 1 / 56.9, 1 / 57, 1 / 57.1, 1 / 57.2, 1 / 57.3, 1 / 57.4, 1 / 57.5, 1 / 57.6, 1 / 57.7, 1 / 57.8, 1 / 57.9, 1 / 58, 1 / 58.1, 1 / 58.2, 1 / 58.3, 1 / 58.4, 1 / 58.5, 1 / 58.6, 1 / 58.7, 1 / 58.8, 1 / 58.9, 1 / 59, 1 / 59.1, 1 / 59.2, 1 / 59.3, 1 / 59.4, 1 / 59.5, 1 / 59.6, 1 / 59.7, 1 / 59.8, 1 / 59.9, 1 / 60 or ratios thereabout or therebetween. Ranges of ratios of 1 / 1 to 1 / 60 can be employed, and subranges as set forth below. More preferred mosaicisms are:REGN 11547 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, / 11.7, 1 / 11.8, 1 / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, / 12.8, 1 / 12.9 or 1 / 13.

[0255] Further preferred mosaicisms are: / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, or 1 / 12.

[0256] Still more preferred mosaicisms are: / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, / 10.9, or 1 / 11.

[0257] Preferred ranges of mosaicisms are 1 / 4 to 1 / 13; 1 / 5 to 1 / 12; 1 / 6 1 / 11, 1 / 7 to 1 / 10.5. or 1 / 7.5 to 1 / 10.REGN 11547 (6) Preferred molar ratios of AAV-FM to SCM-RM are as follows: 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300, 1:305, 1:310, 1:315, 1:320, 1:325, 1:330, 1:335, 1:340, 1:345, 1:350, 1:355, 1:360, 1:365, 1:370, 1:375, 1:380, 1:385, 1:390, 1:395, 1:400, 1:405, 1:410, 1:415, 1:420, 1:425, 1:430, 1:435, 1:440, 1:445, 1:450, 1:455, 1:460, 1:465, 1:470, 1:475, 1:480, 1:485, 1:490, 1:495, 1:500, 1:505, 1:510, 1:515, 1:520, 1:525, 1:530, 1:535, 1:540, 1:545, 1:550, 1:555, 1:560, 1:565, 1:570, 1:575, 1:580, 1:585, 1:590, 1:595, 1:600, 1:605, 1:610, 1:615, 1:620, 1:625, 1:630, 1:635, 1:640, 1:645, 1:650, 1:655, 1:660, 1:665, 1:670, 1:675, 1:680, 1:685, 1:690, 1:695, 1:700, 1:705, 1:710, 1:715, 1:720, 1:725, 1:730, 1:735, 1:740, 1:745, 1:750, 1:755, 1:760, 1:765, 1:770, 1:775, 1:780, 1:785, 1:790, 1:795, 1:800, 1:805, 1:810, 1:815, 1:820, 1:825, 1:830, 1:835, 1:840, 1:845, 1:850, 1:855, 1:860, 1:865, 1:870, 1:875, 1:880, 1:885, 1:890, 1:895, 1:900, 1:905, 1:910, 1:915, 1:920, 1:925, 1:930, 1:935, 1:940, 1:945, 1:950, 1:955, 1:960, 1:965, 1:970, 1:975, 1:980, 1:985, 1:990, 1:995, 1:1000, 1:>1000 or any ratio thereabout or therebetween.

[0258] More preferred ratios AAV-FM to SCM-RM are: 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300 or any ratio thereabout or therebetween.REGN 11547

[0259] Preferred ranges of AAV-FM to SCM-RM are 1:20 to 1:1000, and more preferred ranges are: 1:30 to 1:100, 1:40 to 1:100, 1:50 to 1:1001:60 to 1:100, 1:70 to 1:100, 1:80 to 1:100, 1:90:1:100, 1:30 to 1:150, 1:40 to 1:150, 1:50 to 1:1501:60 to 1:150, 1:70 to 1:150, 1:80 to 1:150, 1:90 to :150, 1:100 to 1:150, 1:30 to 1:200, 1:40 to 1:200, 1:50 to 1:2001:60 to 1:200, 1:70 to 1:200, 1:80 to 1:200, 1:90:1:200, 1:100 to 1:200, 1:30 to 1:250, 1:40 to 1:250, 1:50 to 1:2501:60 to 1:250, 1:70 to 1:250, 1:80 to 1:250, 1:90 to :250, 1:100 to 1:250, 1:30 to 1:300, 1:40 to 1:300, 1:50 to 1:3001:60 to 1:300, 1:70 to 1:300, 1:80 to 1:300, 1:90:1:300, or 1:100 to 1:300. (7) The inventions provide for a desired conjugation of a level of conjugation of at least 10%, at least 20%, at least 30%, and at least 40%. Preferred levels are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100%, any of which can be considered complete depending on the purpose. More preferably, the desired level of conjugation typically will be about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Ranges of desired conjugation levels can be 10% to 100%, 20% to 99%, 30% to 99%, 40% to 99%, 50% to 99%, 60% to 99%, 70% to 99%, 80% to 99%, or 90% to 99%. Preferred ranges of desired conjugation levels can be 40% to 100%, 40% to 99%, 45% to 95%, 50% to 95%, 55% to 90%, 60% to 90%, 65% to 95%, 65% toREGN 11547 85%, 65% to 90%, 70% to 90%, 75% to 90%, 75% to 85%, 75% to 80%, or 80% to 85%. More preferably, the range is 65% to 95%.

[0260] The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein. Points 1-7 above also apply to the second chart below: :

[0261] Variant AAV27m8: AAV27m8 (also referred to as AAV2.7m8) can be used and is characterized by a 10-amino acid peptide ‘LALGETTRPA’, referred to as ‘7m8’, inserted at position 588 of the AAV2 capsid protein sequence. (Dalkara, D., et al. "In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous. Sci. Transl. Med.2013; 5:REGN 11547 189ra76." Gene therapy restores vision in a canine model of childhood blindness. Nat Genet 28.1 (2001): 92-5).

[0262] Variant AAV2-4Y-F: AAV2 Quad Y-F can be used and is a modified AAV2 comprises a mutated AAV2 VP3 capsid protein comprising phenylalanines (F) at each of the positions corresponding to Y272, Y444, Y500, and Y730 in a wild type AAV2 VP3 capsid protein (Petrs-Silva, Hilda, et al. "High- efficiency transduction of the mouse retina by tyrosine-mutant AAV serotype vectors." Molecular therapy 17.3 (2009): 463-471). EXAMPLE 17 – AAV3

[0263] Covalently surface modified AAV3 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and harvesting the resulting AAV, and then conjugating the resulting AAV with a second cognate member (SCM) that is fused to a retargeting molecule The first chart below uses SpyTag as the first member of a specific binding pair, and Adenovirus Helper Genes. Modifications can be undertaken as disclosed below:REGN 11547(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV3. (2) Plasmids pRC-FM can be modified for other specific binding pairs. The first member (FM) should be complementary to a second cognate member (SCM) that is fused to a retargeting molecule (for example, an antibody, as well derivatives and fragments thereof, and still other molecules). See Definitions and Description sections of the Detailed Description, the Figures and associated text for “retargeting” and “retargeting molecules”. The populations of FMs and SCMs can be homogenous or heterogeneous as long as the selected FMs can come in contact with appropriate SCMs so that binding can occur. (3) Detargeting mutations are optional and can be undertaken as disclosed herein and in publications. See Definitions and Description sections of the Detailed Description, the Figures and associated text for “detargeting” and “detargeting mutations”. (4) To lessen the possibility of encountering neutralizing antibodies when the covalently surface modified AAV is administered to a subject, KTag can be used asREGN 11547 an SCM to bind with SpyTag (FM) (SEQ ID NOS: 54 and 86). KTag and SpyLigase are based upon SpyCatcher. KTag (SEQ ID NO: 85) has only 10 amino acids, which renders it less immunogenic. KTag can be bound to SpyTag in the presence of SpyLigase (SEQ ID NO: 61). The SpyLigase can be removed by subsequent purification, such as by chromatography and / or filtration so that the SpyLigase will not have an appreciable presence in the final drug substance. (5) Preferred mosaicisms are as follows: 1 / 1, 1 / 1.1, 1 / 1.2, 1 / 1.3, 1 / 1.4, 1 / 1.5, 1 / 1.6, 1 / 1.7, 1 / 1.8, 1 / 1.9, 1 / 2, 1 / 2.1, 1 / 2.2, 1 / 2.3, 1 / 2.4, 1 / 2.5, 1 / 2.6, 1 / 2.7, 1 / 2.8, 1 / 2.9, 1 / 3, 1 / 3.1, 1 / 3.2, 1 / 3.3, 1 / 3.4, 1 / 3.5, 1 / 3.6, 1 / 3.7, 1 / 3.8, 1 / 3.9, 1 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, 1 / 12.8, 1 / 12.9, 1 / 13, 1 / 13.1, 1 / 13.2, 1 / 13.3, 1 / 13.4, 1 / 13.5, 1 / 13.6, 1 / 13.7, 1 / 13.8, 1 / 13.9, 1 / 14, 1 / 14.1, 1 / 14.2, 1 / 14.3, 1 / 14.4, 1 / 14.5, 1 / 14.6, 1 / 14.7, 1 / 14.8, 1 / 14.9, 1 / 15, 1 / 15.1, 1 / 15.2, 1 / 15.3, 1 / 15.4, 1 / 15.5, 1 / 15.6, 1 / 15.7, 1 / 15.8, 1 / 15.9, 1 / 16, 1 / 16.1, 1 / 16.2, 1 / 16.3, 1 / 16.4, 1 / 16.5, 1 / 16.6, 1 / 16.7, 1 / 16.8, 1 / 16.9, 1 / 17, 1 / 17.1, 1 / 17.2, 1 / 17.3, 1 / 17.4, 1 / 17.5, 1 / 17.6, 1 / 17.7, 1 / 17.8, 1 / 17.9, 1 / 18, 1 / 18.1, 1 / 18.2, 1 / 18.3, 1 / 18.4, 1 / 18.5, 1 / 18.6, 1 / 18.7, 1 / 18.8, 1 / 18.9, 1 / 19, 11 / 9.1, 1 / 19.2, 1 / 19.3, 1 / 19.4, 1 / 91.5, 1 / 19.6, 1 / 19.7, 1 / 19.8, 1 / 19.9, 1 / 20, 1 / 21, 1 / 21.1, 1 / 21.2, 1 / 21.3, 1 / 21.4, 1 / 21.5, 1 / 21.6, 1 / 21.7, 1 / 21.8, 1 / 21.9,1 / 22, 1 / 22.1, 1 / 22.2, 1 / 22.3, 1 / 22.4, 1 / 22.5, 1 / 22.6,REGN 11547 / 22.7, 1 / 22.8, 1 / 22.9, 1 / 23, 1 / 23.1, 1 / 23.2, 1 / 23.3, 1 / 23.4, 1 / 23.5, 1 / 23.6, 1 / 23.7, / 23.8, 1 / 23.9, 1 / 24, 1 / 24.1, 1 / 24.2, 1 / 24.3, 1 / 24.4, 1 / 24.5, 1 / 24.6, 1 / 24.7, 1 / 24.8, / 24.9, 1 / 25, 1 / 25.1, 1 / 25.2, 1 / 25.3, 1 / 25.4, 1 / 25.5, 1 / 25.6, 1 / 25.7, 1 / 25.8, 1 / 25.9, / 26, 1 / 26.1, 1 / 26.2, 1 / 26.3, 1 / 26.4, 1 / 26.5, 1 / 26.6, 1 / 26.7, 1 / 26.8, 12 / 6.9, 1 / 27, / 27.1, 1 / 27.2, 1 / 27.3, 1 / 27.4, 1 / 27.5, 1 / 27.6, 1 / 27.7, 1 / 27.8, 1 / 27.9, 1 / 28, 1 / 28.1, / 28.2, 1 / 28.3, 1 / 28.4, 1 / 28.5, 1 / 28.6, 1 / 28.7, 1 / 28.8, 1 / 28.9, 1 / 29, 1 / 29.1, 1 / 29.2, / 29.3, 1 / 29.4, 1 / 29.5, 1 / 29.6, 1 / 29.7, 1 / 29.8, 1 / 29.9, 1 / 30, 1 / 30.1, 1 / 30.2, 1 / 30.3, / 30.4, 1 / 30.5, 1 / 30.6, 1 / 30.7, 1 / 30.8, 1 / 30.9, 1 / 31, 1 / 31.1, 1 / 31.2, 1 / 31.3, 1 / 31.4, / 31.5, 1 / 31.6, 1 / 31.7, 1 / 31.8, 1 / 31.9, 1 / 32, 1 / 32.1, 1 / 32.2, 1 / 32.3, 1 / 32.4, 1 / 32.5, / 32.6, 1 / 32.7, 1 / 32.8, 1 / 32.9, 1 / 33, 1 / 33.1, 1 / 33.2, 1 / 33.3, 1 / 33.4, 1 / 33.5, 1 / 33.6, / 33.7, 1 / 33.8, 1 / 33.9, 1 / 34, 1 / 34.1, 1 / 34.2, 1 / 34.3, 1 / 34.4, 1 / 34.5, 1 / 34.6, 1 / 34.7, / 34.8, 1 / 34.9, 1 / 35, 1 / 35.1, 1 / 35.2, 1 / 35.3, 1 / 35.4, 1 / 35.5, 1 / 35.6, 1 / 35.7, 1 / 35.8, / 35.9, 1 / 36, 1 / 36.1, 1 / 36.2, 1 / 36.3, 1 / 36.4, 1 / 36.5, 1 / 36.6, 1 / 36.7, 1 / 36.8, 1 / 36.9, / 37, 1 / 37.1, 1 / 37.2, 1 / 37.3, 1 / 37.4, 1 / 37.5, 1 / 37.6, 1 / 37.7, 1 / 37.8, 1 / 37.9, 1 / 38, / 38.1, 1 / 38.2, 1 / 38.3, 1 / 38.4, 1 / 38.5, 1 / 38.6, 1 / 38.7, 1 / 38.8, 1 / 38.9, 1 / 39, 1 / 39.1, / 39.2, 1 / 39.3, 1 / 39.4, 1 / 39.5, 1 / 39.6, 1 / 39.7, 1 / 39.8, 1 / 39.9, 1 / 40, 1 / 40.1, 1 / 40.2, / 40.3, 1 / 40.4, 1 / 40.5, 1 / 40.6, 1 / 40.7, 1 / 40.8, 1 / 40.9, 1 / 41, 1 / 41.1, 1 / 41.2, 1 / 41.3, / 41.4, 1 / 41.5, 1 / 41.6, 1 / 41.7, 1 / 41.8, 1 / 41.9, 1 / 42, 1 / 42.1, 1 / 42.2, 1 / 42.3, 1 / 42.4, / 42.5, 1 / 42.6, 1 / 42.7, 1 / 42.8, 1 / 42.9, 1 / 43, 1 / 43.1, 1 / 43.2, 1 / 43.3, 1 / 43.4, 1 / 43.5, / 43.6, 1 / 43.7, 1 / 43.8, 1 / 43.9, 1 / 44, 1 / 44.1, 1 / 44.2, 1 / 44.3, 1 / 44.4, 1 / 44.5, 1 / 44.6, / 44.7, 1 / 44.8, 1 / 44.9, 1 / 45, 1 / 45.1, 1 / 45.2, 1 / 45.3, 1 / 45.4, 1 / 45.5, 1 / 45.6, 1 / 45.7, / 45.8, 1 / 45.9, 1 / 46, 1 / 46.1, 1 / 46.2, 1 / 46.3, 1 / 46.4, 1 / 46.5, 1 / 46.6, 1 / 46.7, 1 / 46.8, / 46.9, 1 / 47, 1 / 47.1, 1 / 47.2, 1 / 47.3, 1 / 47.4, 1 / 47.5, 1 / 47.6, 1 / 47.7, 1 / 47.8, 1 / 47.9, / 48, 1 / 48.1, 1 / 48.2, 1 / 48.3, 1 / 48.4, 1 / 48.5, 1 / 48.6, 1 / 48.7, 1 / 48.8, 1 / 48.9, 1 / 49, / 49.1, 1 / 49.2, 1 / 49.3, 1 / 49.4, 1 / 49.5, 1 / 49.6, 1 / 49.7, 1 / 49.8, 1 / 49.9, 1 / 50, 1 / 50.1,REGN 11547 1 / 50.2, 1 / 50.3, 1 / 50.4, 1 / 50.5, 1 / 50.6, 1 / 50.7, 1 / 50.8, 1 / 50.9, 1 / 51, 1 / 51.1, 1 / 51.2, 1 / 51.3, 1 / 51.4, 1 / 51.5, 1 / 51.6, 1 / 51.7, 1 / 51.8, 1 / 51.9, 1 / 52, 1 / 52.1, 1 / 52.2, 1 / 52.3, 1 / 52.4, 1 / 52.5, 1 / 52.6, 1 / 52.7, 1 / 52.8, 1 / 52.9, 1 / 53, 1 / 53.1, 1 / 53.2, 1 / 53.3, 1 / 53.4, 1 / 53.5, 1 / 53.6, 1 / 53.7, 1 / 53.8, 1 / 53.9, 1 / 54, 1 / 54.1, 1 / 54.2, 1 / 54.3, 1 / 54.4, 1 / 54.5, 1 / 54.6, 1 / 54.7, 1 / 54.8, 1 / 54.9, 1 / 55, 1 / 55.1, 1 / 55.2, 1 / 55.3, 1 / 55.4, 1 / 55.5, 1 / 55.6, 1 / 55.7, 1 / 55.8, 1 / 55.9, 1 / 56, 1 / 56.1, 1 / 56.2, 1 / 56.3, 1 / 56.4, 1 / 56.5, 1 / 56.6, 1 / 56.7, 1 / 56.8, 1 / 56.9, 1 / 57, 1 / 57.1, 1 / 57.2, 1 / 57.3, 1 / 57.4, 1 / 57.5, 1 / 57.6, 1 / 57.7, 1 / 57.8, 1 / 57.9, 1 / 58, 1 / 58.1, 1 / 58.2, 1 / 58.3, 1 / 58.4, 1 / 58.5, 1 / 58.6, 1 / 58.7, 1 / 58.8, 1 / 58.9, 1 / 59, 1 / 59.1, 1 / 59.2, 1 / 59.3, 1 / 59.4, 1 / 59.5, 1 / 59.6, 1 / 59.7, 1 / 59.8, 1 / 59.9, 1 / 60 or ratios thereabout or therebetween. Ranges of ratios of 1 / 1 to 1 / 60 can be employed, and subranges as set forth below.

[0264] More preferred mosaicisms are: 1 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, 1 / 12.8, 1 / 12.9 or 1 / 13.

[0265] Further preferred mosaicisms are: 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9,REGN 11547 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, or 1 / 12.

[0266] Still more preferred mosaicisms are: 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, or 1 / 11.

[0267] Preferred ranges of mosaicisms are 1 / 4 to 1 / 13; 1 / 5 to 1 / 12; 1 / 6 to 1 / 11, 1 / 7 to 1 / 10.5. or 1 / 7.5 to 1 / 10. (6) Preferred molar ratios of AAV-FM to SCM-RM are as follows: 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300, 1:305, 1:310, 1:315, 1:320, 1:325, 1:330, 1:335, 1:340, 1:345, 1:350, 1:355, 1:360, 1:365, 1:370, 1:375, 1:380, 1:385, 1:390, 1:395, 1:400, 1:405, 1:410, 1:415, 1:420, 1:425, 1:430, 1:435, 1:440, 1:445, 1:450, 1:455, 1:460, 1:465, 1:470, 1:475, 1:480, 1:485, 1:490, 1:495, 1:500, 1:505, 1:510, 1:515, 1:520, 1:525, 1:530, 1:535, 1:540, 1:545, 1:550, 1:555, 1:560, 1:565, 1:570, 1:575, 1:580, 1:585, 1:590, 1:595, 1:600, 1:605, 1:610, 1:615, 1:620, 1:625, 1:630, 1:635, 1:640, 1:645, 1:650, 1:655, 1:660, 1:665, 1:670, 1:675, 1:680, 1:685, 1:690, 1:695, 1:700, 1:705, 1:710, 1:715, 1:720, 1:725, 1:730, 1:735, 1:740, 1:745, 1:750, 1:755,REGN 11547 1:760, 1:765, 1:770, 1:775, 1:780, 1:785, 1:790, 1:795, 1:800, 1:805, 1:810, 1:815, 1:820, 1:825, 1:830, 1:835, 1:840, 1:845, 1:850, 1:855, 1:860, 1:865, 1:870, 1:875, 1:880, 1:885, 1:890, 1:895, 1:900, 1:905, 1:910, 1:915, 1:920, 1:925, 1:930, 1:935, 1:940, 1:945, 1:950, 1:955, 1:960, 1:965, 1:970, 1:975, 1:980, 1:985, 1:990, 1:995, 1:1000, 1:>1000 or any ratio thereabout or therebetween.

[0268] More preferred ratios AAV-FM to SCM-RM are: 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300 or any ratio thereabout or therebetween.

[0269] Preferred ranges of AAV-FM to SCM-RM are 1:20 to 1:1000, and more preferred ranges are: 1:30 to 1:100, 1:40 to 1:100, 1:50 to 1:1001:60 to 1:100, 1:70 to 1:100, 1:80 to 1:100, 1:90:1:100, 1:30 to 1:150, 1:40 to 1:150, 1:50 to 1:1501:60 to 1:150, 1:70 to 1:150, 1:80 to 1:150, 1:90 to :150, 1:100 to 1:150, 1:30 to 1:200, 1:40 to 1:200, 1:50 to 1:2001:60 to 1:200, 1:70 to 1:200, 1:80 to 1:200, 1:90:1:200, 1:100 to 1:200, 1:30 to 1:250, 1:40 to 1:250, 1:50 to 1:2501:60 to 1:250, 1:70 to 1:250, 1:80 to 1:250, 1:90 to :250, 1:100 to 1:250, 1:30 to 1:300, 1:40 to 1:300, 1:50 to 1:3001:60 to 1:300, 1:70 to 1:300, 1:80 to 1:300, 1:90:1:300, or 1:100 to 1:300. (7) The inventions provide for a desired conjugation of a level of conjugation of at least 10%, at least 20%, at least 30%, and at least 40%. Preferred levels are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100%, any of which can be considered complete depending on the purpose. More preferably, theREGN 11547 desired level of conjugation typically will be about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0270] Ranges of desired conjugation levels can be 10% to 100%, 20% to 99%, 30% to 99%, 40% to 99%, 50% to 99%, 60% to 99%, 70% to 99%, 80% to 99%, or 90% to 99%. Preferred ranges of desired conjugation levels can be 40% to 100%, 40% to 99%, 45% to 95%, 50% to 95%, 55% to 90%, 60% to 90%, 65% to 95%, 65% to 85%, 65% to 90%, 70% to 90%, 75% to 90%, 75% to 85%, 75% to 80%, or 80% to 85%. More preferably, the range is 65% to 95%.

[0271] The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein. Points 1-7 above also apply to the second chart below:REGN 11547EXAMPLE 18 – AAV4

[0272] Covalently surface modified AAV4 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and harvesting the resulting AAV, and then conjugating the resulting AAV with a second cognate member (SCM) that is fused to a retargeting molecule The first chart below uses SpyTag as the first member of a specific binding pair, and Adenovirus Helper Genes. Modifications can be undertaken as disclosed below:REGN 11547(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV4. (2) Plasmids pRC-FM can be modified for other specific binding pairs. The first member (FM) should be complementary to a second cognate member (SCM) that is fused to a retargeting molecule (for example, an antibody, as well derivatives and fragments thereof, and still other molecules). See Definitions and Description sections of the Detailed Description, the Figures and associated text for “retargeting” and “retargeting molecules”. The populations of FMs and SCMs can be homogenous or heterogeneous as long as the selected FMs can come in contact with appropriate SCMs so that binding can occur. (3) Detargeting mutations are optional and can be undertaken as disclosed herein and in publications. See Definitions and Description sections of the Detailed Description, the Figures and associated text for “detargeting” and “detargeting mutations”. (4) To lessen the possibility of encountering neutralizing antibodies when the covalently surface modified AAV is administered to a subject, KTag can be used as an SCM to bind with SpyTag (FM) (SEQ ID NOS: 54 and 86). KTag and SpyLigase are based upon SpyCatcher. KTag (SEQ ID NO: 85) has only 10 amino acids, which renders it less immunogenic. KTag can be bound to SpyTag in the presence ofREGN 11547 SpyLigase (SEQ ID NO: 61). The SpyLigase can be removed by subsequent purification, such as by chromatography and / or filtration so that the SpyLigase will not have an appreciable presence in the final drug substance. (5) Preferred mosaicisms are as follows: 1 / 1, 1 / 1.1, 1 / 1.2, 1 / 1.3, 1 / 1.4, 1 / 1.5, 1 / 1.6, 1 / 1.7, 1 / 1.8, 1 / 1.9, 1 / 2, 1 / 2.1, 1 / 2.2, 1 / 2.3, 1 / 2.4, 1 / 2.5, 1 / 2.6, 1 / 2.7, 1 / 2.8, 1 / 2.9, 1 / 3, 1 / 3.1, 1 / 3.2, 1 / 3.3, 1 / 3.4, 1 / 3.5, 1 / 3.6, 1 / 3.7, 1 / 3.8, 1 / 3.9, 1 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, 1 / 12.8, 1 / 12.9, 1 / 13, 1 / 13.1, 1 / 13.2, 1 / 13.3, 1 / 13.4, 1 / 13.5, 1 / 13.6, 1 / 13.7, 1 / 13.8, 1 / 13.9, 1 / 14, 1 / 14.1, 1 / 14.2, 1 / 14.3, 1 / 14.4, 1 / 14.5, 1 / 14.6, 1 / 14.7, 1 / 14.8, 1 / 14.9, 1 / 15, 1 / 15.1, 1 / 15.2, 1 / 15.3, 1 / 15.4, 1 / 15.5, 1 / 15.6, 1 / 15.7, 1 / 15.8, 1 / 15.9, 1 / 16, 1 / 16.1, 1 / 16.2, 1 / 16.3, 1 / 16.4, 1 / 16.5, 1 / 16.6, 1 / 16.7, 1 / 16.8, 1 / 16.9, 1 / 17, 1 / 17.1, 1 / 17.2, 1 / 17.3, 1 / 17.4, 1 / 17.5, 1 / 17.6, 1 / 17.7, 1 / 17.8, 1 / 17.9, 1 / 18, 1 / 18.1, 1 / 18.2, 1 / 18.3, 1 / 18.4, 1 / 18.5, 1 / 18.6, 1 / 18.7, 1 / 18.8, 1 / 18.9, 1 / 19, 11 / 9.1, 1 / 19.2, 1 / 19.3, 1 / 19.4, 1 / 91.5, 1 / 19.6, 1 / 19.7, 1 / 19.8, 1 / 19.9, 1 / 20, 1 / 21, 1 / 21.1, 1 / 21.2, 1 / 21.3, 1 / 21.4, 1 / 21.5, 1 / 21.6, 1 / 21.7, 1 / 21.8, 1 / 21.9,1 / 22, 1 / 22.1, 1 / 22.2, 1 / 22.3, 1 / 22.4, 1 / 22.5, 1 / 22.6, 1 / 22.7, 1 / 22.8, 1 / 22.9, 1 / 23, 1 / 23.1, 1 / 23.2, 1 / 23.3, 1 / 23.4, 1 / 23.5, 1 / 23.6, 1 / 23.7, 1 / 23.8, 1 / 23.9, 1 / 24, 1 / 24.1, 1 / 24.2, 1 / 24.3, 1 / 24.4, 1 / 24.5, 1 / 24.6, 1 / 24.7, 1 / 24.8, 1 / 24.9, 1 / 25, 1 / 25.1, 1 / 25.2, 1 / 25.3, 1 / 25.4, 1 / 25.5, 1 / 25.6, 1 / 25.7, 1 / 25.8, 1 / 25.9,REGN 11547 / 26, 1 / 26.1, 1 / 26.2, 1 / 26.3, 1 / 26.4, 1 / 26.5, 1 / 26.6, 1 / 26.7, 1 / 26.8, 12 / 6.9, 1 / 27, / 27.1, 1 / 27.2, 1 / 27.3, 1 / 27.4, 1 / 27.5, 1 / 27.6, 1 / 27.7, 1 / 27.8, 1 / 27.9, 1 / 28, 1 / 28.1, / 28.2, 1 / 28.3, 1 / 28.4, 1 / 28.5, 1 / 28.6, 1 / 28.7, 1 / 28.8, 1 / 28.9, 1 / 29, 1 / 29.1, 1 / 29.2, / 29.3, 1 / 29.4, 1 / 29.5, 1 / 29.6, 1 / 29.7, 1 / 29.8, 1 / 29.9, 1 / 30, 1 / 30.1, 1 / 30.2, 1 / 30.3, / 30.4, 1 / 30.5, 1 / 30.6, 1 / 30.7, 1 / 30.8, 1 / 30.9, 1 / 31, 1 / 31.1, 1 / 31.2, 1 / 31.3, 1 / 31.4, / 31.5, 1 / 31.6, 1 / 31.7, 1 / 31.8, 1 / 31.9, 1 / 32, 1 / 32.1, 1 / 32.2, 1 / 32.3, 1 / 32.4, 1 / 32.5, / 32.6, 1 / 32.7, 1 / 32.8, 1 / 32.9, 1 / 33, 1 / 33.1, 1 / 33.2, 1 / 33.3, 1 / 33.4, 1 / 33.5, 1 / 33.6, / 33.7, 1 / 33.8, 1 / 33.9, 1 / 34, 1 / 34.1, 1 / 34.2, 1 / 34.3, 1 / 34.4, 1 / 34.5, 1 / 34.6, 1 / 34.7, / 34.8, 1 / 34.9, 1 / 35, 1 / 35.1, 1 / 35.2, 1 / 35.3, 1 / 35.4, 1 / 35.5, 1 / 35.6, 1 / 35.7, 1 / 35.8, / 35.9, 1 / 36, 1 / 36.1, 1 / 36.2, 1 / 36.3, 1 / 36.4, 1 / 36.5, 1 / 36.6, 1 / 36.7, 1 / 36.8, 1 / 36.9, / 37, 1 / 37.1, 1 / 37.2, 1 / 37.3, 1 / 37.4, 1 / 37.5, 1 / 37.6, 1 / 37.7, 1 / 37.8, 1 / 37.9, 1 / 38, / 38.1, 1 / 38.2, 1 / 38.3, 1 / 38.4, 1 / 38.5, 1 / 38.6, 1 / 38.7, 1 / 38.8, 1 / 38.9, 1 / 39, 1 / 39.1, / 39.2, 1 / 39.3, 1 / 39.4, 1 / 39.5, 1 / 39.6, 1 / 39.7, 1 / 39.8, 1 / 39.9, 1 / 40, 1 / 40.1, 1 / 40.2, / 40.3, 1 / 40.4, 1 / 40.5, 1 / 40.6, 1 / 40.7, 1 / 40.8, 1 / 40.9, 1 / 41, 1 / 41.1, 1 / 41.2, 1 / 41.3, / 41.4, 1 / 41.5, 1 / 41.6, 1 / 41.7, 1 / 41.8, 1 / 41.9, 1 / 42, 1 / 42.1, 1 / 42.2, 1 / 42.3, 1 / 42.4, / 42.5, 1 / 42.6, 1 / 42.7, 1 / 42.8, 1 / 42.9, 1 / 43, 1 / 43.1, 1 / 43.2, 1 / 43.3, 1 / 43.4, 1 / 43.5, / 43.6, 1 / 43.7, 1 / 43.8, 1 / 43.9, 1 / 44, 1 / 44.1, 1 / 44.2, 1 / 44.3, 1 / 44.4, 1 / 44.5, 1 / 44.6, / 44.7, 1 / 44.8, 1 / 44.9, 1 / 45, 1 / 45.1, 1 / 45.2, 1 / 45.3, 1 / 45.4, 1 / 45.5, 1 / 45.6, 1 / 45.7, / 45.8, 1 / 45.9, 1 / 46, 1 / 46.1, 1 / 46.2, 1 / 46.3, 1 / 46.4, 1 / 46.5, 1 / 46.6, 1 / 46.7, 1 / 46.8, / 46.9, 1 / 47, 1 / 47.1, 1 / 47.2, 1 / 47.3, 1 / 47.4, 1 / 47.5, 1 / 47.6, 1 / 47.7, 1 / 47.8, 1 / 47.9, / 48, 1 / 48.1, 1 / 48.2, 1 / 48.3, 1 / 48.4, 1 / 48.5, 1 / 48.6, 1 / 48.7, 1 / 48.8, 1 / 48.9, 1 / 49, / 49.1, 1 / 49.2, 1 / 49.3, 1 / 49.4, 1 / 49.5, 1 / 49.6, 1 / 49.7, 1 / 49.8, 1 / 49.9, 1 / 50, 1 / 50.1, / 50.2, 1 / 50.3, 1 / 50.4, 1 / 50.5, 1 / 50.6, 1 / 50.7, 1 / 50.8, 1 / 50.9, 1 / 51, 1 / 51.1, 1 / 51.2, / 51.3, 1 / 51.4, 1 / 51.5, 1 / 51.6, 1 / 51.7, 1 / 51.8, 1 / 51.9, 1 / 52, 1 / 52.1, 1 / 52.2, 1 / 52.3, / 52.4, 1 / 52.5, 1 / 52.6, 1 / 52.7, 1 / 52.8, 1 / 52.9, 1 / 53, 1 / 53.1, 1 / 53.2, 1 / 53.3, 1 / 53.4,REGN 11547 1 / 53.5, 1 / 53.6, 1 / 53.7, 1 / 53.8, 1 / 53.9, 1 / 54, 1 / 54.1, 1 / 54.2, 1 / 54.3, 1 / 54.4, 1 / 54.5, 1 / 54.6, 1 / 54.7, 1 / 54.8, 1 / 54.9, 1 / 55, 1 / 55.1, 1 / 55.2, 1 / 55.3, 1 / 55.4, 1 / 55.5, 1 / 55.6, 1 / 55.7, 1 / 55.8, 1 / 55.9, 1 / 56, 1 / 56.1, 1 / 56.2, 1 / 56.3, 1 / 56.4, 1 / 56.5, 1 / 56.6, 1 / 56.7, 1 / 56.8, 1 / 56.9, 1 / 57, 1 / 57.1, 1 / 57.2, 1 / 57.3, 1 / 57.4, 1 / 57.5, 1 / 57.6, 1 / 57.7, 1 / 57.8, 1 / 57.9, 1 / 58, 1 / 58.1, 1 / 58.2, 1 / 58.3, 1 / 58.4, 1 / 58.5, 1 / 58.6, 1 / 58.7, 1 / 58.8, 1 / 58.9, 1 / 59, 1 / 59.1, 1 / 59.2, 1 / 59.3, 1 / 59.4, 1 / 59.5, 1 / 59.6, 1 / 59.7, 1 / 59.8, 1 / 59.9, 1 / 60 or ratios thereabout or therebetween. Ranges of ratios of 1 / 1 to 1 / 60 can be employed, and subranges as set forth below.

[0273] More preferred mosaicisms are: 1 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, 1 / 12.8, 1 / 12.9 or 1 / 13.

[0274] Further preferred mosaicisms are: 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, or 1 / 12.REGN 11547

[0275] Still more preferred mosaicisms are: 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, or 1 / 11.

[0276] Preferred ranges of mosaicisms are 1 / 4 to 1 / 13; 1 / 5 to 1 / 12; 1 / 6 to 1 / 11, 1 / 7 to 1 / 10.5. or 1 / 7.5 to 1 / 10. (6) Preferred molar ratios of AAV-FM to SCM-RM are as follows: 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300, 1:305, 1:310, 1:315, 1:320, 1:325, 1:330, 1:335, 1:340, 1:345, 1:350, 1:355, 1:360, 1:365, 1:370, 1:375, 1:380, 1:385, 1:390, 1:395, 1:400, 1:405, 1:410, 1:415, 1:420, 1:425, 1:430, 1:435, 1:440, 1:445, 1:450, 1:455, 1:460, 1:465, 1:470, 1:475, 1:480, 1:485, 1:490, 1:495, 1:500, 1:505, 1:510, 1:515, 1:520, 1:525, 1:530, 1:535, 1:540, 1:545, 1:550, 1:555, 1:560, 1:565, 1:570, 1:575, 1:580, 1:585, 1:590, 1:595, 1:600, 1:605, 1:610, 1:615, 1:620, 1:625, 1:630, 1:635, 1:640, 1:645, 1:650, 1:655, 1:660, 1:665, 1:670, 1:675, 1:680, 1:685, 1:690, 1:695, 1:700, 1:705, 1:710, 1:715, 1:720, 1:725, 1:730, 1:735, 1:740, 1:745, 1:750, 1:755, 1:760, 1:765, 1:770, 1:775, 1:780, 1:785, 1:790, 1:795, 1:800, 1:805, 1:810, 1:815, 1:820, 1:825, 1:830, 1:835, 1:840, 1:845, 1:850, 1:855, 1:860, 1:865, 1:870, 1:875, 1:880, 1:885, 1:890, 1:895, 1:900, 1:905, 1:910, 1:915, 1:920, 1:925, 1:930, 1:935,REGN 11547 1:940, 1:945, 1:950, 1:955, 1:960, 1:965, 1:970, 1:975, 1:980, 1:985, 1:990, 1:995, 1:1000, 1:>1000 or any ratio thereabout or therebetween.

[0277] More preferred ratios AAV-FM to SCM-RM are: 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300 or any ratio thereabout or therebetween. Preferred ranges of AAV-FM to SCM-RM are 1:20 to 1:1000, and more preferred ranges are: 1:30 to 1:100, 1:40 to 1:100, 1:50 to 1:1001:60 to 1:100, 1:70 to 1:100, 1:80 to 1:100, 1:90:1:100, 1:30 to 1:150, 1:40 to 1:150, 1:50 to 1:1501:60 to 1:150, 1:70 to 1:150, 1:80 to 1:150, 1:90 to :150, 1:100 to 1:150, 1:30 to 1:200, 1:40 to 1:200, 1:50 to 1:2001:60 to 1:200, 1:70 to 1:200, 1:80 to 1:200, 1:90:1:200, 1:100 to 1:200, 1:30 to 1:250, 1:40 to 1:250, 1:50 to 1:2501:60 to 1:250, 1:70 to 1:250, 1:80 to 1:250, 1:90 to :250, 1:100 to 1:250, 1:30 to 1:300, 1:40 to 1:300, 1:50 to 1:300 1:60 to 1:300, 1:70 to 1:300, 1:80 to 1:300, 1:90:1:300, or 1:100 to 1:300. (7) The inventions provide for a desired conjugation of a level of conjugation of at least 10%, at least 20%, at least 30%, and at least 40%. Preferred levels are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100%, any of which can be considered complete depending on the purpose. More preferably, the desired level of conjugation typically will be about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,REGN 11547 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0278] Ranges of desired conjugation levels can be 10% to 100%, 20% to 99%, 30% to 99%, 40% to 99%, 50% to 99%, 60% to 99%, 70% to 99%, 80% to 99%, or 90% to 99%. Preferred ranges of desired conjugation levels can be 40% to 100%, 40% to 99%, 45% to 95%, 50% to 95%, 55% to 90%, 60% to 90%, 65% to 95%, 65% to 85%, 65% to 90%, 70% to 90%, 75% to 90%, 75% to 85%, 75% to 80%, or 80% to 85%. More preferably, the range is 65% to 95%.

[0279] The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein. Points 1-7 above also apply to the second chart below:REGN 11547 EXAMPLE 19 – AAV5

[0280] Covalently surface modified AAV5 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and harvesting the resulting AAV, and then conjugating the resulting AAV with a second cognate member (SCM) that is fused to a retargeting molecule The first chart below uses SpyTag as the first member of a specific binding pair, and Adenovirus Helper Genes. Modifications can be undertaken as disclosed below:(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV5. (2) Plasmids pRC-FM can be modified for other specific binding pairs. The first member (FM) should be complementary to a second cognate member (SCM) that isREGN 11547 fused to a retargeting molecule (for example, an antibody, as well derivatives and fragments thereof, and still other molecules). See Definitions and Description sections of the Detailed Description, the Figures and associated text for “retargeting” and “retargeting molecules”. The populations of FMs and SCMs can be homogenous or heterogeneous as long as the selected FMs can come in contact with appropriate SCMs so that binding can occur. (3) Detargeting mutations are optional and can be undertaken as disclosed herein and in publications. See Definitions and Description sections of the Detailed Description, the Figures and associated text for “detargeting” and “detargeting mutations”. (4) To lessen the possibility of encountering neutralizing antibodies when the covalently surface modified AAV is administered to a subject, KTag can be used as an SCM to bind with SpyTag (FM) (SEQ ID NOS: 54 and 86). KTag and SpyLigase are based upon SpyCatcher. KTag (SEQ ID NO: 85) has only 10 amino acids, which renders it less immunogenic. KTag can be bound to SpyTag in the presence of SpyLigase (SEQ ID NO: 61). The SpyLigase can be removed by subsequent purification, such as by chromatography and / or filtration so that the SpyLigase will not have an appreciable presence in the final drug substance. (5) Preferred mosaicisms are as follows: 1 / 1, 1 / 1.1, 1 / 1.2, 1 / 1.3, 1 / 1.4, 1 / 1.5, 1 / 1.6, 1 / 1.7, 1 / 1.8, 1 / 1.9, 1 / 2, 1 / 2.1, 1 / 2.2, 1 / 2.3, 1 / 2.4, 1 / 2.5, 1 / 2.6, 1 / 2.7, 1 / 2.8, 1 / 2.9, 1 / 3, 1 / 3.1, 1 / 3.2, 1 / 3.3, 1 / 3.4, 1 / 3.5, 1 / 3.6, 1 / 3.7, 1 / 3.8, 1 / 3.9, 1 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2,REGN 11547 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, 1 / 12.8, 1 / 12.9, / 13, 1 / 13.1, 1 / 13.2, 1 / 13.3, 1 / 13.4, 1 / 13.5, 1 / 13.6, 1 / 13.7, 1 / 13.8, 1 / 13.9, 1 / 14, / 14.1, 1 / 14.2, 1 / 14.3, 1 / 14.4, 1 / 14.5, 1 / 14.6, 1 / 14.7, 1 / 14.8, 1 / 14.9, 1 / 15, 1 / 15.1, / 15.2, 1 / 15.3, 1 / 15.4, 1 / 15.5, 1 / 15.6, 1 / 15.7, 1 / 15.8, 1 / 15.9, 1 / 16, 1 / 16.1, 1 / 16.2, / 16.3, 1 / 16.4, 1 / 16.5, 1 / 16.6, 1 / 16.7, 1 / 16.8, 1 / 16.9, 1 / 17, 1 / 17.1, 1 / 17.2, 1 / 17.3, / 17.4, 1 / 17.5, 1 / 17.6, 1 / 17.7, 1 / 17.8, 1 / 17.9, 1 / 18, 1 / 18.1, 1 / 18.2, 1 / 18.3, 1 / 18.4, / 18.5, 1 / 18.6, 1 / 18.7, 1 / 18.8, 1 / 18.9, 1 / 19, 11 / 9.1, 1 / 19.2, 1 / 19.3, 1 / 19.4, 1 / 91.5, / 19.6, 1 / 19.7, 1 / 19.8, 1 / 19.9, 1 / 20, 1 / 21, 1 / 21.1, 1 / 21.2, 1 / 21.3, 1 / 21.4, 1 / 21.5, / 21.6, 1 / 21.7, 1 / 21.8, 1 / 21.9,1 / 22, 1 / 22.1, 1 / 22.2, 1 / 22.3, 1 / 22.4, 1 / 22.5, 1 / 22.6, / 22.7, 1 / 22.8, 1 / 22.9, 1 / 23, 1 / 23.1, 1 / 23.2, 1 / 23.3, 1 / 23.4, 1 / 23.5, 1 / 23.6, 1 / 23.7, / 23.8, 1 / 23.9, 1 / 24, 1 / 24.1, 1 / 24.2, 1 / 24.3, 1 / 24.4, 1 / 24.5, 1 / 24.6, 1 / 24.7, 1 / 24.8, / 24.9, 1 / 25, 1 / 25.1, 1 / 25.2, 1 / 25.3, 1 / 25.4, 1 / 25.5, 1 / 25.6, 1 / 25.7, 1 / 25.8, 1 / 25.9, / 26, 1 / 26.1, 1 / 26.2, 1 / 26.3, 1 / 26.4, 1 / 26.5, 1 / 26.6, 1 / 26.7, 1 / 26.8, 12 / 6.9, 1 / 27, / 27.1, 1 / 27.2, 1 / 27.3, 1 / 27.4, 1 / 27.5, 1 / 27.6, 1 / 27.7, 1 / 27.8, 1 / 27.9, 1 / 28, 1 / 28.1, / 28.2, 1 / 28.3, 1 / 28.4, 1 / 28.5, 1 / 28.6, 1 / 28.7, 1 / 28.8, 1 / 28.9, 1 / 29, 1 / 29.1, 1 / 29.2, / 29.3, 1 / 29.4, 1 / 29.5, 1 / 29.6, 1 / 29.7, 1 / 29.8, 1 / 29.9, 1 / 30, 1 / 30.1, 1 / 30.2, 1 / 30.3, / 30.4, 1 / 30.5, 1 / 30.6, 1 / 30.7, 1 / 30.8, 1 / 30.9, 1 / 31, 1 / 31.1, 1 / 31.2, 1 / 31.3, 1 / 31.4, / 31.5, 1 / 31.6, 1 / 31.7, 1 / 31.8, 1 / 31.9, 1 / 32, 1 / 32.1, 1 / 32.2, 1 / 32.3, 1 / 32.4, 1 / 32.5, / 32.6, 1 / 32.7, 1 / 32.8, 1 / 32.9, 1 / 33, 1 / 33.1, 1 / 33.2, 1 / 33.3, 1 / 33.4, 1 / 33.5, 1 / 33.6, / 33.7, 1 / 33.8, 1 / 33.9, 1 / 34, 1 / 34.1, 1 / 34.2, 1 / 34.3, 1 / 34.4, 1 / 34.5, 1 / 34.6, 1 / 34.7, / 34.8, 1 / 34.9, 1 / 35, 1 / 35.1, 1 / 35.2, 1 / 35.3, 1 / 35.4, 1 / 35.5, 1 / 35.6, 1 / 35.7, 1 / 35.8, / 35.9, 1 / 36, 1 / 36.1, 1 / 36.2, 1 / 36.3, 1 / 36.4, 1 / 36.5, 1 / 36.6, 1 / 36.7, 1 / 36.8, 1 / 36.9,REGN 11547 1 / 37, 1 / 37.1, 1 / 37.2, 1 / 37.3, 1 / 37.4, 1 / 37.5, 1 / 37.6, 1 / 37.7, 1 / 37.8, 1 / 37.9, 1 / 38, 1 / 38.1, 1 / 38.2, 1 / 38.3, 1 / 38.4, 1 / 38.5, 1 / 38.6, 1 / 38.7, 1 / 38.8, 1 / 38.9, 1 / 39, 1 / 39.1, 1 / 39.2, 1 / 39.3, 1 / 39.4, 1 / 39.5, 1 / 39.6, 1 / 39.7, 1 / 39.8, 1 / 39.9, 1 / 40, 1 / 40.1, 1 / 40.2, 1 / 40.3, 1 / 40.4, 1 / 40.5, 1 / 40.6, 1 / 40.7, 1 / 40.8, 1 / 40.9, 1 / 41, 1 / 41.1, 1 / 41.2, 1 / 41.3, 1 / 41.4, 1 / 41.5, 1 / 41.6, 1 / 41.7, 1 / 41.8, 1 / 41.9, 1 / 42, 1 / 42.1, 1 / 42.2, 1 / 42.3, 1 / 42.4, 1 / 42.5, 1 / 42.6, 1 / 42.7, 1 / 42.8, 1 / 42.9, 1 / 43, 1 / 43.1, 1 / 43.2, 1 / 43.3, 1 / 43.4, 1 / 43.5, 1 / 43.6, 1 / 43.7, 1 / 43.8, 1 / 43.9, 1 / 44, 1 / 44.1, 1 / 44.2, 1 / 44.3, 1 / 44.4, 1 / 44.5, 1 / 44.6, 1 / 44.7, 1 / 44.8, 1 / 44.9, 1 / 45, 1 / 45.1, 1 / 45.2, 1 / 45.3, 1 / 45.4, 1 / 45.5, 1 / 45.6, 1 / 45.7, 1 / 45.8, 1 / 45.9, 1 / 46, 1 / 46.1, 1 / 46.2, 1 / 46.3, 1 / 46.4, 1 / 46.5, 1 / 46.6, 1 / 46.7, 1 / 46.8, 1 / 46.9, 1 / 47, 1 / 47.1, 1 / 47.2, 1 / 47.3, 1 / 47.4, 1 / 47.5, 1 / 47.6, 1 / 47.7, 1 / 47.8, 1 / 47.9, 1 / 48, 1 / 48.1, 1 / 48.2, 1 / 48.3, 1 / 48.4, 1 / 48.5, 1 / 48.6, 1 / 48.7, 1 / 48.8, 1 / 48.9, 1 / 49, 1 / 49.1, 1 / 49.2, 1 / 49.3, 1 / 49.4, 1 / 49.5, 1 / 49.6, 1 / 49.7, 1 / 49.8, 1 / 49.9, 1 / 50, 1 / 50.1, 1 / 50.2, 1 / 50.3, 1 / 50.4, 1 / 50.5, 1 / 50.6, 1 / 50.7, 1 / 50.8, 1 / 50.9, 1 / 51, 1 / 51.1, 1 / 51.2, 1 / 51.3, 1 / 51.4, 1 / 51.5, 1 / 51.6, 1 / 51.7, 1 / 51.8, 1 / 51.9, 1 / 52, 1 / 52.1, 1 / 52.2, 1 / 52.3, 1 / 52.4, 1 / 52.5, 1 / 52.6, 1 / 52.7, 1 / 52.8, 1 / 52.9, 1 / 53, 1 / 53.1, 1 / 53.2, 1 / 53.3, 1 / 53.4, 1 / 53.5, 1 / 53.6, 1 / 53.7, 1 / 53.8, 1 / 53.9, 1 / 54, 1 / 54.1, 1 / 54.2, 1 / 54.3, 1 / 54.4, 1 / 54.5, 1 / 54.6, 1 / 54.7, 1 / 54.8, 1 / 54.9, 1 / 55, 1 / 55.1, 1 / 55.2, 1 / 55.3, 1 / 55.4, 1 / 55.5, 1 / 55.6, 1 / 55.7, 1 / 55.8, 1 / 55.9, 1 / 56, 1 / 56.1, 1 / 56.2, 1 / 56.3, 1 / 56.4, 1 / 56.5, 1 / 56.6, 1 / 56.7, 1 / 56.8, 1 / 56.9, 1 / 57, 1 / 57.1, 1 / 57.2, 1 / 57.3, 1 / 57.4, 1 / 57.5, 1 / 57.6, 1 / 57.7, 1 / 57.8, 1 / 57.9, 1 / 58, 1 / 58.1, 1 / 58.2, 1 / 58.3, 1 / 58.4, 1 / 58.5, 1 / 58.6, 1 / 58.7, 1 / 58.8, 1 / 58.9, 1 / 59, 1 / 59.1, 1 / 59.2, 1 / 59.3, 1 / 59.4, 1 / 59.5, 1 / 59.6, 1 / 59.7, 1 / 59.8, 1 / 59.9, 1 / 60 or ratios thereabout or therebetween. Ranges of ratios of 1 / 1 to 1 / 60 can be employed, and subranges as set forth below. More preferred mosaicisms are:REGN 11547 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, / 11.7, 1 / 11.8, 1 / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, / 12.8, 1 / 12.9 or 1 / 13.

[0281] Further preferred mosaicisms are: / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, or 1 / 12.

[0282] Still more preferred mosaicisms are: / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, / 10.9, or 1 / 11.

[0283] Preferred ranges of mosaicisms are 1 / 4 to 1 / 13; 1 / 5 to 1 / 12; 1 / 6 1 / 11, 1 / 7 to 1 / 10.5. or 1 / 7.5 to 1 / 10.REGN 11547 (6) Preferred molar ratios of AAV-FM to SCM-RM are as follows: 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300, 1:305, 1:310, 1:315, 1:320, 1:325, 1:330, 1:335, 1:340, 1:345, 1:350, 1:355, 1:360, 1:365, 1:370, 1:375, 1:380, 1:385, 1:390, 1:395, 1:400, 1:405, 1:410, 1:415, 1:420, 1:425, 1:430, 1:435, 1:440, 1:445, 1:450, 1:455, 1:460, 1:465, 1:470, 1:475, 1:480, 1:485, 1:490, 1:495, 1:500, 1:505, 1:510, 1:515, 1:520, 1:525, 1:530, 1:535, 1:540, 1:545, 1:550, 1:555, 1:560, 1:565, 1:570, 1:575, 1:580, 1:585, 1:590, 1:595, 1:600, 1:605, 1:610, 1:615, 1:620, 1:625, 1:630, 1:635, 1:640, 1:645, 1:650, 1:655, 1:660, 1:665, 1:670, 1:675, 1:680, 1:685, 1:690, 1:695, 1:700, 1:705, 1:710, 1:715, 1:720, 1:725, 1:730, 1:735, 1:740, 1:745, 1:750, 1:755, 1:760, 1:765, 1:770, 1:775, 1:780, 1:785, 1:790, 1:795, 1:800, 1:805, 1:810, 1:815, 1:820, 1:825, 1:830, 1:835, 1:840, 1:845, 1:850, 1:855, 1:860, 1:865, 1:870, 1:875, 1:880, 1:885, 1:890, 1:895, 1:900, 1:905, 1:910, 1:915, 1:920, 1:925, 1:930, 1:935, 1:940, 1:945, 1:950, 1:955, 1:960, 1:965, 1:970, 1:975, 1:980, 1:985, 1:990, 1:995, 1:1000, 1:>1000 or any ratio thereabout or therebetween.

[0284] More preferred ratios AAV-FM to SCM-RM are: 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300 or any ratio thereabout or therebetween.REGN 11547 Preferred ranges of AAV-FM to SCM-RM are 1:20 to 1:1000, and more preferred ranges are: 1:30 to 1:100, 1:40 to 1:100, 1:50 to 1:1001:60 to 1:100, 1:70 to 1:100, 1:80 to 1:100, 1:90:1:100, 1:30 to 1:150, 1:40 to 1:150, 1:50 to 1:1501:60 to 1:150, 1:70 to 1:150, 1:80 to 1:150, 1:90 to :150, 1:100 to 1:150, 1:30 to 1:200, 1:40 to 1:200, 1:50 to 1:2001:60 to 1:200, 1:70 to 1:200, 1:80 to 1:200, 1:90:1:200, 1:100 to 1:200, 1:30 to 1:250, 1:40 to 1:250, 1:50 to 1:2501:60 to 1:250, 1:70 to 1:250, 1:80 to 1:250, 1:90 to :250, 1:100 to 1:250, 1:30 to 1:300, 1:40 to 1:300, 1:50 to 1:3001:60 to 1:300, 1:70 to 1:300, 1:80 to 1:300, 1:90:1:300, or 1:100 to 1:300. (7) The inventions provide for a desired conjugation of a level of conjugation of at least 10%, at least 20%, at least 30%, and at least 40%. Preferred levels are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100%, any of which can be considered complete depending on the purpose. More preferably, the desired level of conjugation typically will be about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0285] Ranges of desired conjugation levels can be 10% to 100%, 20% to 99%, 30% to 99%, 40% to 99%, 50% to 99%, 60% to 99%, 70% to 99%, 80% to 99%, or 90% to 99%. Preferred ranges of desired conjugation levels can be 40% to 100%, 40% to 99%, 45% to 95%, 50% to 95%, 55% to 90%, 60% to 90%, 65% to 95%, 65% to 85%, 65% to 90%, 70% to 90%, 75% to 90%, 75% to 85%, 75% to 80%, or 80% to 85%. More preferably, the range is 65% to 95%.REGN 11547

[0286] The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein. Points 1-7 above also apply to the second chart below:EXAMPLE 20 – AAV6

[0287] Covalently surface modified AAV6 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and harvesting the resulting AAV, and then conjugating the resulting AAV with a second cognate member (SCM) that is fused to a retargeting molecule The first chart belowREGN 11547 uses SpyTag as the first member of a specific binding pair, and Adenovirus Helper Genes. Modifications can be undertaken as disclosed below:(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV6. (2) Plasmids pRC-FM can be modified for other specific binding pairs. The first member (FM) should be complementary to a second cognate member (SCM) that is fused to a retargeting molecule (for example, an antibody, as well derivatives and fragments thereof, and still other molecules). See Definitions and Description sections of the Detailed Description, the Figures and associated text for “retargeting” and “retargeting molecules”. The populations of FMs and SCMs can be homogenousREGN 11547 or heterogeneous as long as the selected FMs can come in contact with appropriate SCMs so that binding can occur. (3) Detargeting mutations are optional and can be undertaken as disclosed herein and in publications. See, e.g., Definitions and Description sections of the Detailed Description, the Figures and associated text for “detargeting” and “detargeting mutations”. (4) To lessen the possibility of encountering neutralizing antibodies when the covalently surface modified AAV is administered to a subject, KTag can be used as an SCM to bind with SpyTag (FM) (SEQ ID NOS: 54 and 86). KTag and SpyLigase are based upon SpyCatcher. KTag (SEQ ID NO: 85) has only 10 amino acids, which renders it less immunogenic. KTag can be bound to SpyTag in the presence of SpyLigase (SEQ ID NO: 61). The SpyLigase can be removed by subsequent purification, such as by chromatography and / or filtration so that the SpyLigase will not have an appreciable presence in the final drug substance. (5) Preferred mosaicisms are as follows: 1 / 1, 1 / 1.1, 1 / 1.2, 1 / 1.3, 1 / 1.4, 1 / 1.5, 1 / 1.6, 1 / 1.7, 1 / 1.8, 1 / 1.9, 1 / 2, 1 / 2.1, 1 / 2.2, 1 / 2.3, 1 / 2.4, 1 / 2.5, 1 / 2.6, 1 / 2.7, 1 / 2.8, 1 / 2.9, 1 / 3, 1 / 3.1, 1 / 3.2, 1 / 3.3, 1 / 3.4, 1 / 3.5, 1 / 3.6, 1 / 3.7, 1 / 3.8, 1 / 3.9, 1 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, 1 / 12.8, 1 / 12.9,REGN 11547 / 13, 1 / 13.1, 1 / 13.2, 1 / 13.3, 1 / 13.4, 1 / 13.5, 1 / 13.6, 1 / 13.7, 1 / 13.8, 1 / 13.9, 1 / 14, / 14.1, 1 / 14.2, 1 / 14.3, 1 / 14.4, 1 / 14.5, 1 / 14.6, 1 / 14.7, 1 / 14.8, 1 / 14.9, 1 / 15, 1 / 15.1, / 15.2, 1 / 15.3, 1 / 15.4, 1 / 15.5, 1 / 15.6, 1 / 15.7, 1 / 15.8, 1 / 15.9, 1 / 16, 1 / 16.1, 1 / 16.2, / 16.3, 1 / 16.4, 1 / 16.5, 1 / 16.6, 1 / 16.7, 1 / 16.8, 1 / 16.9, 1 / 17, 1 / 17.1, 1 / 17.2, 1 / 17.3, / 17.4, 1 / 17.5, 1 / 17.6, 1 / 17.7, 1 / 17.8, 1 / 17.9, 1 / 18, 1 / 18.1, 1 / 18.2, 1 / 18.3, 1 / 18.4, / 18.5, 1 / 18.6, 1 / 18.7, 1 / 18.8, 1 / 18.9, 1 / 19, 11 / 9.1, 1 / 19.2, 1 / 19.3, 1 / 19.4, 1 / 91.5, / 19.6, 1 / 19.7, 1 / 19.8, 1 / 19.9, 1 / 20, 1 / 21, 1 / 21.1, 1 / 21.2, 1 / 21.3, 1 / 21.4, 1 / 21.5, / 21.6, 1 / 21.7, 1 / 21.8, 1 / 21.9,1 / 22, 1 / 22.1, 1 / 22.2, 1 / 22.3, 1 / 22.4, 1 / 22.5, 1 / 22.6, / 22.7, 1 / 22.8, 1 / 22.9, 1 / 23, 1 / 23.1, 1 / 23.2, 1 / 23.3, 1 / 23.4, 1 / 23.5, 1 / 23.6, 1 / 23.7, / 23.8, 1 / 23.9, 1 / 24, 1 / 24.1, 1 / 24.2, 1 / 24.3, 1 / 24.4, 1 / 24.5, 1 / 24.6, 1 / 24.7, 1 / 24.8, / 24.9, 1 / 25, 1 / 25.1, 1 / 25.2, 1 / 25.3, 1 / 25.4, 1 / 25.5, 1 / 25.6, 1 / 25.7, 1 / 25.8, 1 / 25.9, / 26, 1 / 26.1, 1 / 26.2, 1 / 26.3, 1 / 26.4, 1 / 26.5, 1 / 26.6, 1 / 26.7, 1 / 26.8, 12 / 6.9, 1 / 27, / 27.1, 1 / 27.2, 1 / 27.3, 1 / 27.4, 1 / 27.5, 1 / 27.6, 1 / 27.7, 1 / 27.8, 1 / 27.9, 1 / 28, 1 / 28.1, / 28.2, 1 / 28.3, 1 / 28.4, 1 / 28.5, 1 / 28.6, 1 / 28.7, 1 / 28.8, 1 / 28.9, 1 / 29, 1 / 29.1, 1 / 29.2, / 29.3, 1 / 29.4, 1 / 29.5, 1 / 29.6, 1 / 29.7, 1 / 29.8, 1 / 29.9, 1 / 30, 1 / 30.1, 1 / 30.2, 1 / 30.3, / 30.4, 1 / 30.5, 1 / 30.6, 1 / 30.7, 1 / 30.8, 1 / 30.9, 1 / 31, 1 / 31.1, 1 / 31.2, 1 / 31.3, 1 / 31.4, / 31.5, 1 / 31.6, 1 / 31.7, 1 / 31.8, 1 / 31.9, 1 / 32, 1 / 32.1, 1 / 32.2, 1 / 32.3, 1 / 32.4, 1 / 32.5, / 32.6, 1 / 32.7, 1 / 32.8, 1 / 32.9, 1 / 33, 1 / 33.1, 1 / 33.2, 1 / 33.3, 1 / 33.4, 1 / 33.5, 1 / 33.6, / 33.7, 1 / 33.8, 1 / 33.9, 1 / 34, 1 / 34.1, 1 / 34.2, 1 / 34.3, 1 / 34.4, 1 / 34.5, 1 / 34.6, 1 / 34.7, / 34.8, 1 / 34.9, 1 / 35, 1 / 35.1, 1 / 35.2, 1 / 35.3, 1 / 35.4, 1 / 35.5, 1 / 35.6, 1 / 35.7, 1 / 35.8, / 35.9, 1 / 36, 1 / 36.1, 1 / 36.2, 1 / 36.3, 1 / 36.4, 1 / 36.5, 1 / 36.6, 1 / 36.7, 1 / 36.8, 1 / 36.9, / 37, 1 / 37.1, 1 / 37.2, 1 / 37.3, 1 / 37.4, 1 / 37.5, 1 / 37.6, 1 / 37.7, 1 / 37.8, 1 / 37.9, 1 / 38, / 38.1, 1 / 38.2, 1 / 38.3, 1 / 38.4, 1 / 38.5, 1 / 38.6, 1 / 38.7, 1 / 38.8, 1 / 38.9, 1 / 39, 1 / 39.1, / 39.2, 1 / 39.3, 1 / 39.4, 1 / 39.5, 1 / 39.6, 1 / 39.7, 1 / 39.8, 1 / 39.9, 1 / 40, 1 / 40.1, 1 / 40.2, / 40.3, 1 / 40.4, 1 / 40.5, 1 / 40.6, 1 / 40.7, 1 / 40.8, 1 / 40.9, 1 / 41, 1 / 41.1, 1 / 41.2, 1 / 41.3,REGN 11547 1 / 41.4, 1 / 41.5, 1 / 41.6, 1 / 41.7, 1 / 41.8, 1 / 41.9, 1 / 42, 1 / 42.1, 1 / 42.2, 1 / 42.3, 1 / 42.4, 1 / 42.5, 1 / 42.6, 1 / 42.7, 1 / 42.8, 1 / 42.9, 1 / 43, 1 / 43.1, 1 / 43.2, 1 / 43.3, 1 / 43.4, 1 / 43.5, 1 / 43.6, 1 / 43.7, 1 / 43.8, 1 / 43.9, 1 / 44, 1 / 44.1, 1 / 44.2, 1 / 44.3, 1 / 44.4, 1 / 44.5, 1 / 44.6, 1 / 44.7, 1 / 44.8, 1 / 44.9, 1 / 45, 1 / 45.1, 1 / 45.2, 1 / 45.3, 1 / 45.4, 1 / 45.5, 1 / 45.6, 1 / 45.7, 1 / 45.8, 1 / 45.9, 1 / 46, 1 / 46.1, 1 / 46.2, 1 / 46.3, 1 / 46.4, 1 / 46.5, 1 / 46.6, 1 / 46.7, 1 / 46.8, 1 / 46.9, 1 / 47, 1 / 47.1, 1 / 47.2, 1 / 47.3, 1 / 47.4, 1 / 47.5, 1 / 47.6, 1 / 47.7, 1 / 47.8, 1 / 47.9, 1 / 48, 1 / 48.1, 1 / 48.2, 1 / 48.3, 1 / 48.4, 1 / 48.5, 1 / 48.6, 1 / 48.7, 1 / 48.8, 1 / 48.9, 1 / 49, 1 / 49.1, 1 / 49.2, 1 / 49.3, 1 / 49.4, 1 / 49.5, 1 / 49.6, 1 / 49.7, 1 / 49.8, 1 / 49.9, 1 / 50, 1 / 50.1, 1 / 50.2, 1 / 50.3, 1 / 50.4, 1 / 50.5, 1 / 50.6, 1 / 50.7, 1 / 50.8, 1 / 50.9, 1 / 51, 1 / 51.1, 1 / 51.2, 1 / 51.3, 1 / 51.4, 1 / 51.5, 1 / 51.6, 1 / 51.7, 1 / 51.8, 1 / 51.9, 1 / 52, 1 / 52.1, 1 / 52.2, 1 / 52.3, 1 / 52.4, 1 / 52.5, 1 / 52.6, 1 / 52.7, 1 / 52.8, 1 / 52.9, 1 / 53, 1 / 53.1, 1 / 53.2, 1 / 53.3, 1 / 53.4, 1 / 53.5, 1 / 53.6, 1 / 53.7, 1 / 53.8, 1 / 53.9, 1 / 54, 1 / 54.1, 1 / 54.2, 1 / 54.3, 1 / 54.4, 1 / 54.5, 1 / 54.6, 1 / 54.7, 1 / 54.8, 1 / 54.9, 1 / 55, 1 / 55.1, 1 / 55.2, 1 / 55.3, 1 / 55.4, 1 / 55.5, 1 / 55.6, 1 / 55.7, 1 / 55.8, 1 / 55.9, 1 / 56, 1 / 56.1, 1 / 56.2, 1 / 56.3, 1 / 56.4, 1 / 56.5, 1 / 56.6, 1 / 56.7, 1 / 56.8, 1 / 56.9, 1 / 57, 1 / 57.1, 1 / 57.2, 1 / 57.3, 1 / 57.4, 1 / 57.5, 1 / 57.6, 1 / 57.7, 1 / 57.8, 1 / 57.9, 1 / 58, 1 / 58.1, 1 / 58.2, 1 / 58.3, 1 / 58.4, 1 / 58.5, 1 / 58.6, 1 / 58.7, 1 / 58.8, 1 / 58.9, 1 / 59, 1 / 59.1, 1 / 59.2, 1 / 59.3, 1 / 59.4, 1 / 59.5, 1 / 59.6, 1 / 59.7, 1 / 59.8, 1 / 59.9, 1 / 60 or ratios thereabout or therebetween. Ranges of ratios of 1 / 1 to 1 / 60 can be employed, and subranges as set forth below.

[0288] More preferred mosaicisms are: 1 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3,REGN 11547 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, 1 / 12.8, 1 / 12.9 or 1 / 13.

[0289] Further preferred mosaicisms are: 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, or 1 / 12.

[0290] Still more preferred mosaicisms are: 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, or 1 / 11.

[0291] Preferred ranges of mosaicisms are 1 / 4 to 1 / 13; 1 / 5 to 1 / 12; 1 / 6 to 1 / 11, 1 / 7 to 1 / 10.5. or 1 / 7.5 to 1 / 10. (6) Preferred molar ratios of AAV-FM to SCM-RM are as follows: 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275,REGN 11547 1:280, 1:285, 1:290, 1:295, 1:300, 1:305, 1:310, 1:315, 1:320, 1:325, 1:330, 1:335, 1:340, 1:345, 1:350, 1:355, 1:360, 1:365, 1:370, 1:375, 1:380, 1:385, 1:390, 1:395, 1:400, 1:405, 1:410, 1:415, 1:420, 1:425, 1:430, 1:435, 1:440, 1:445, 1:450, 1:455, 1:460, 1:465, 1:470, 1:475, 1:480, 1:485, 1:490, 1:495, 1:500, 1:505, 1:510, 1:515, 1:520, 1:525, 1:530, 1:535, 1:540, 1:545, 1:550, 1:555, 1:560, 1:565, 1:570, 1:575, 1:580, 1:585, 1:590, 1:595, 1:600, 1:605, 1:610, 1:615, 1:620, 1:625, 1:630, 1:635, 1:640, 1:645, 1:650, 1:655, 1:660, 1:665, 1:670, 1:675, 1:680, 1:685, 1:690, 1:695, 1:700, 1:705, 1:710, 1:715, 1:720, 1:725, 1:730, 1:735, 1:740, 1:745, 1:750, 1:755, 1:760, 1:765, 1:770, 1:775, 1:780, 1:785, 1:790, 1:795, 1:800, 1:805, 1:810, 1:815, 1:820, 1:825, 1:830, 1:835, 1:840, 1:845, 1:850, 1:855, 1:860, 1:865, 1:870, 1:875, 1:880, 1:885, 1:890, 1:895, 1:900, 1:905, 1:910, 1:915, 1:920, 1:925, 1:930, 1:935, 1:940, 1:945, 1:950, 1:955, 1:960, 1:965, 1:970, 1:975, 1:980, 1:985, 1:990, 1:995, 1:1000, 1:>1000 or any ratio thereabout or therebetween.

[0292] More preferred ratios AAV-FM to SCM-RM are: 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300 or any ratio thereabout or therebetween. Preferred ranges of AAV-FM to SCM-RM are 1:20 to 1:1000, and more preferred ranges are: 1:30 to 1:100, 1:40 to 1:100, 1:50 to 1:1001:60 to 1:100, 1:70 to 1:100, 1:80 to 1:100, 1:90:1:100, 1:30 to 1:150, 1:40 to 1:150, 1:50 to 1:1501:60 to 1:150, 1:70 to 1:150, 1:80 to 1:150, 1:90 to :150, 1:100 to 1:150, 1:30 to 1:200, 1:40 to 1:200, 1:50 to 1:2001:60 to 1:200, 1:70 to 1:200, 1:80 to 1:200, 1:90:1:200,REGN 11547 1:100 to 1:200, 1:30 to 1:250, 1:40 to 1:250, 1:50 to 1:2501:60 to 1:250, 1:70 to 1:250, 1:80 to 1:250, 1:90 to :250, 1:100 to 1:250, 1:30 to 1:300, 1:40 to 1:300, 1:50 to 1:3001:60 to 1:300, 1:70 to 1:300, 1:80 to 1:300, 1:90:1:300, or 1:100 to 1:300. (7) The inventions provide for a desired conjugation of a level of conjugation of at least 10%, at least 20%, at least 30%, and at least 40%. Preferred levels are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100%, any of which can be considered complete depending on the purpose. More preferably, the desired level of conjugation typically will be about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0293] Ranges of desired conjugation levels can be 10% to 100%, 20% to 99%, 30% to 99%, 40% to 99%, 50% to 99%, 60% to 99%, 70% to 99%, 80% to 99%, or 90% to 99%. Preferred ranges of desired conjugation levels can be 40% to 100%, 40% to 99%, 45% to 95%, 50% to 95%, 55% to 90%, 60% to 90%, 65% to 95%, 65% to 85%, 65% to 90%, 70% to 90%, 75% to 90%, 75% to 85%, 75% to 80%, or 80% to 85%. More preferably, the range is 65% to 95%. The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein. Points 1-7 above also apply to the second chart below:REGN 11547EXAMPLE 21 – AAV 7

[0294] Covalently surface modified AAV 7 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and harvesting the resulting AAV, and then conjugating the resulting AAV with a second cognate member (SCM) that is fused to a retargeting molecule The first chart below uses SpyTag as the first member of a specific binding pair, and Adenovirus Helper Genes. Modifications can be undertaken as disclosed below:REGN 11547(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV7. (2) Plasmids pRC-FM can be modified for other specific binding pairs. The first member (FM) should be complementary to a second cognate member (SCM) that is fused to a retargeting molecule (for example, an antibody, as well derivatives and fragments thereof, and still other molecules). See Definitions and Description sections of the Detailed Description, the Figures and associated text for “retargeting” and “retargeting molecules”. The populations of FMs and SCMs can be homogenous or heterogeneous as long as the selected FMs can come in contact with appropriate SCMs so that binding can occur. (3) Detargeting mutations are optional and can be undertaken as disclosed herein and in publications. See Definitions and Description sections of the Detailed Description, the Figures and associated text for “detargeting” and “detargeting mutations”.REGN 11547 (4) To lessen the possibility of encountering neutralizing antibodies when the covalently surface modified AAV is administered to a subject, KTag can be used as an SCM to bind with SpyTag (FM) (SEQ ID NOS: 54 and 86). KTag and SpyLigase are based upon SpyCatcher. KTag (SEQ ID NO: 85) has only 10 amino acids, which renders it less immunogenic. KTag can be bound to SpyTag in the presence of SpyLigase (SEQ ID NO: 61). The SpyLigase can be removed by subsequent purification, such as by chromatography and / or filtration so that the SpyLigase will not have an appreciable presence in the final drug substance. (5) Preferred mosaicisms are as follows: 1 / 1, 1 / 1.1, 1 / 1.2, 1 / 1.3, 1 / 1.4, 1 / 1.5, 1 / 1.6, 1 / 1.7, 1 / 1.8, 1 / 1.9, 1 / 2, 1 / 2.1, 1 / 2.2, 1 / 2.3, 1 / 2.4, 1 / 2.5, 1 / 2.6, 1 / 2.7, 1 / 2.8, 1 / 2.9, 1 / 3, 1 / 3.1, 1 / 3.2, 1 / 3.3, 1 / 3.4, 1 / 3.5, 1 / 3.6, 1 / 3.7, 1 / 3.8, 1 / 3.9, 1 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, 1 / 12.8, 1 / 12.9, 1 / 13, 1 / 13.1, 1 / 13.2, 1 / 13.3, 1 / 13.4, 1 / 13.5, 1 / 13.6, 1 / 13.7, 1 / 13.8, 1 / 13.9, 1 / 14, 1 / 14.1, 1 / 14.2, 1 / 14.3, 1 / 14.4, 1 / 14.5, 1 / 14.6, 1 / 14.7, 1 / 14.8, 1 / 14.9, 1 / 15, 1 / 15.1, 1 / 15.2, 1 / 15.3, 1 / 15.4, 1 / 15.5, 1 / 15.6, 1 / 15.7, 1 / 15.8, 1 / 15.9, 1 / 16, 1 / 16.1, 1 / 16.2, 1 / 16.3, 1 / 16.4, 1 / 16.5, 1 / 16.6, 1 / 16.7, 1 / 16.8, 1 / 16.9, 1 / 17, 1 / 17.1, 1 / 17.2, 1 / 17.3, 1 / 17.4, 1 / 17.5, 1 / 17.6, 1 / 17.7, 1 / 17.8, 1 / 17.9, 1 / 18, 1 / 18.1, 1 / 18.2, 1 / 18.3, 1 / 18.4, 1 / 18.5, 1 / 18.6, 1 / 18.7, 1 / 18.8, 1 / 18.9, 1 / 19, 11 / 9.1, 1 / 19.2, 1 / 19.3, 1 / 19.4, 1 / 91.5,REGN 11547 / 19.6, 1 / 19.7, 1 / 19.8, 1 / 19.9, 1 / 20, 1 / 21, 1 / 21.1, 1 / 21.2, 1 / 21.3, 1 / 21.4, 1 / 21.5, / 21.6, 1 / 21.7, 1 / 21.8, 1 / 21.9,1 / 22, 1 / 22.1, 1 / 22.2, 1 / 22.3, 1 / 22.4, 1 / 22.5, 1 / 22.6, / 22.7, 1 / 22.8, 1 / 22.9, 1 / 23, 1 / 23.1, 1 / 23.2, 1 / 23.3, 1 / 23.4, 1 / 23.5, 1 / 23.6, 1 / 23.7, / 23.8, 1 / 23.9, 1 / 24, 1 / 24.1, 1 / 24.2, 1 / 24.3, 1 / 24.4, 1 / 24.5, 1 / 24.6, 1 / 24.7, 1 / 24.8, / 24.9, 1 / 25, 1 / 25.1, 1 / 25.2, 1 / 25.3, 1 / 25.4, 1 / 25.5, 1 / 25.6, 1 / 25.7, 1 / 25.8, 1 / 25.9, / 26, 1 / 26.1, 1 / 26.2, 1 / 26.3, 1 / 26.4, 1 / 26.5, 1 / 26.6, 1 / 26.7, 1 / 26.8, 12 / 6.9, 1 / 27, / 27.1, 1 / 27.2, 1 / 27.3, 1 / 27.4, 1 / 27.5, 1 / 27.6, 1 / 27.7, 1 / 27.8, 1 / 27.9, 1 / 28, 1 / 28.1, / 28.2, 1 / 28.3, 1 / 28.4, 1 / 28.5, 1 / 28.6, 1 / 28.7, 1 / 28.8, 1 / 28.9, 1 / 29, 1 / 29.1, 1 / 29.2, / 29.3, 1 / 29.4, 1 / 29.5, 1 / 29.6, 1 / 29.7, 1 / 29.8, 1 / 29.9, 1 / 30, 1 / 30.1, 1 / 30.2, 1 / 30.3, / 30.4, 1 / 30.5, 1 / 30.6, 1 / 30.7, 1 / 30.8, 1 / 30.9, 1 / 31, 1 / 31.1, 1 / 31.2, 1 / 31.3, 1 / 31.4, / 31.5, 1 / 31.6, 1 / 31.7, 1 / 31.8, 1 / 31.9, 1 / 32, 1 / 32.1, 1 / 32.2, 1 / 32.3, 1 / 32.4, 1 / 32.5, / 32.6, 1 / 32.7, 1 / 32.8, 1 / 32.9, 1 / 33, 1 / 33.1, 1 / 33.2, 1 / 33.3, 1 / 33.4, 1 / 33.5, 1 / 33.6, / 33.7, 1 / 33.8, 1 / 33.9, 1 / 34, 1 / 34.1, 1 / 34.2, 1 / 34.3, 1 / 34.4, 1 / 34.5, 1 / 34.6, 1 / 34.7, / 34.8, 1 / 34.9, 1 / 35, 1 / 35.1, 1 / 35.2, 1 / 35.3, 1 / 35.4, 1 / 35.5, 1 / 35.6, 1 / 35.7, 1 / 35.8, / 35.9, 1 / 36, 1 / 36.1, 1 / 36.2, 1 / 36.3, 1 / 36.4, 1 / 36.5, 1 / 36.6, 1 / 36.7, 1 / 36.8, 1 / 36.9, / 37, 1 / 37.1, 1 / 37.2, 1 / 37.3, 1 / 37.4, 1 / 37.5, 1 / 37.6, 1 / 37.7, 1 / 37.8, 1 / 37.9, 1 / 38, / 38.1, 1 / 38.2, 1 / 38.3, 1 / 38.4, 1 / 38.5, 1 / 38.6, 1 / 38.7, 1 / 38.8, 1 / 38.9, 1 / 39, 1 / 39.1, / 39.2, 1 / 39.3, 1 / 39.4, 1 / 39.5, 1 / 39.6, 1 / 39.7, 1 / 39.8, 1 / 39.9, 1 / 40, 1 / 40.1, 1 / 40.2, / 40.3, 1 / 40.4, 1 / 40.5, 1 / 40.6, 1 / 40.7, 1 / 40.8, 1 / 40.9, 1 / 41, 1 / 41.1, 1 / 41.2, 1 / 41.3, / 41.4, 1 / 41.5, 1 / 41.6, 1 / 41.7, 1 / 41.8, 1 / 41.9, 1 / 42, 1 / 42.1, 1 / 42.2, 1 / 42.3, 1 / 42.4, / 42.5, 1 / 42.6, 1 / 42.7, 1 / 42.8, 1 / 42.9, 1 / 43, 1 / 43.1, 1 / 43.2, 1 / 43.3, 1 / 43.4, 1 / 43.5, / 43.6, 1 / 43.7, 1 / 43.8, 1 / 43.9, 1 / 44, 1 / 44.1, 1 / 44.2, 1 / 44.3, 1 / 44.4, 1 / 44.5, 1 / 44.6, / 44.7, 1 / 44.8, 1 / 44.9, 1 / 45, 1 / 45.1, 1 / 45.2, 1 / 45.3, 1 / 45.4, 1 / 45.5, 1 / 45.6, 1 / 45.7, / 45.8, 1 / 45.9, 1 / 46, 1 / 46.1, 1 / 46.2, 1 / 46.3, 1 / 46.4, 1 / 46.5, 1 / 46.6, 1 / 46.7, 1 / 46.8, / 46.9, 1 / 47, 1 / 47.1, 1 / 47.2, 1 / 47.3, 1 / 47.4, 1 / 47.5, 1 / 47.6, 1 / 47.7, 1 / 47.8, 1 / 47.9,REGN 11547 1 / 48, 1 / 48.1, 1 / 48.2, 1 / 48.3, 1 / 48.4, 1 / 48.5, 1 / 48.6, 1 / 48.7, 1 / 48.8, 1 / 48.9, 1 / 49, 1 / 49.1, 1 / 49.2, 1 / 49.3, 1 / 49.4, 1 / 49.5, 1 / 49.6, 1 / 49.7, 1 / 49.8, 1 / 49.9, 1 / 50, 1 / 50.1, 1 / 50.2, 1 / 50.3, 1 / 50.4, 1 / 50.5, 1 / 50.6, 1 / 50.7, 1 / 50.8, 1 / 50.9, 1 / 51, 1 / 51.1, 1 / 51.2, 1 / 51.3, 1 / 51.4, 1 / 51.5, 1 / 51.6, 1 / 51.7, 1 / 51.8, 1 / 51.9, 1 / 52, 1 / 52.1, 1 / 52.2, 1 / 52.3, 1 / 52.4, 1 / 52.5, 1 / 52.6, 1 / 52.7, 1 / 52.8, 1 / 52.9, 1 / 53, 1 / 53.1, 1 / 53.2, 1 / 53.3, 1 / 53.4, 1 / 53.5, 1 / 53.6, 1 / 53.7, 1 / 53.8, 1 / 53.9, 1 / 54, 1 / 54.1, 1 / 54.2, 1 / 54.3, 1 / 54.4, 1 / 54.5, 1 / 54.6, 1 / 54.7, 1 / 54.8, 1 / 54.9, 1 / 55, 1 / 55.1, 1 / 55.2, 1 / 55.3, 1 / 55.4, 1 / 55.5, 1 / 55.6, 1 / 55.7, 1 / 55.8, 1 / 55.9, 1 / 56, 1 / 56.1, 1 / 56.2, 1 / 56.3, 1 / 56.4, 1 / 56.5, 1 / 56.6, 1 / 56.7, 1 / 56.8, 1 / 56.9, 1 / 57, 1 / 57.1, 1 / 57.2, 1 / 57.3, 1 / 57.4, 1 / 57.5, 1 / 57.6, 1 / 57.7, 1 / 57.8, 1 / 57.9, 1 / 58, 1 / 58.1, 1 / 58.2, 1 / 58.3, 1 / 58.4, 1 / 58.5, 1 / 58.6, 1 / 58.7, 1 / 58.8, 1 / 58.9, 1 / 59, 1 / 59.1, 1 / 59.2, 1 / 59.3, 1 / 59.4, 1 / 59.5, 1 / 59.6, 1 / 59.7, 1 / 59.8, 1 / 59.9, 1 / 60 or ratios thereabout or therebetween. Ranges of ratios of 1 / 1 to 1 / 60 can be employed, and subranges as set forth below.

[0295] More preferred mosaicisms are: 1 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, 1 / 12.8, 1 / 12.9 or 1 / 13.

[0296] Further preferred mosaicisms are: 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3,REGN 11547 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, or 1 / 12.

[0297] Still more preferred mosaicisms are: 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, or 1 / 11.

[0298] Preferred ranges of mosaicisms are 1 / 4 to 1 / 13; 1 / 5 to 1 / 12; 1 / 6 to 1 / 11, 1 / 7 to 1 / 10.5. or 1 / 7.5 to 1 / 10. (6) Preferred molar ratios of AAV-FM to SCM-RM are as follows: 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300, 1:305, 1:310, 1:315, 1:320, 1:325, 1:330, 1:335, 1:340, 1:345, 1:350, 1:355, 1:360, 1:365, 1:370, 1:375, 1:380, 1:385, 1:390, 1:395, 1:400, 1:405, 1:410, 1:415, 1:420, 1:425, 1:430, 1:435, 1:440, 1:445, 1:450, 1:455, 1:460, 1:465, 1:470, 1:475, 1:480, 1:485, 1:490, 1:495, 1:500, 1:505, 1:510, 1:515, 1:520, 1:525, 1:530, 1:535, 1:540, 1:545, 1:550, 1:555, 1:560, 1:565, 1:570, 1:575, 1:580, 1:585, 1:590, 1:595, 1:600, 1:605, 1:610, 1:615, 1:620, 1:625, 1:630, 1:635,REGN 11547 1:640, 1:645, 1:650, 1:655, 1:660, 1:665, 1:670, 1:675, 1:680, 1:685, 1:690, 1:695, 1:700, 1:705, 1:710, 1:715, 1:720, 1:725, 1:730, 1:735, 1:740, 1:745, 1:750, 1:755, 1:760, 1:765, 1:770, 1:775, 1:780, 1:785, 1:790, 1:795, 1:800, 1:805, 1:810, 1:815, 1:820, 1:825, 1:830, 1:835, 1:840, 1:845, 1:850, 1:855, 1:860, 1:865, 1:870, 1:875, 1:880, 1:885, 1:890, 1:895, 1:900, 1:905, 1:910, 1:915, 1:920, 1:925, 1:930, 1:935, 1:940, 1:945, 1:950, 1:955, 1:960, 1:965, 1:970, 1:975, 1:980, 1:985, 1:990, 1:995, 1:1000, 1:>1000 or any ratio thereabout or therebetween.

[0299] More preferred ratios AAV-FM to SCM-RM are: 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300 or any ratio thereabout or therebetween

[0300] Preferred ranges of AAV-FM to SCM-RM are 1:20 to 1:1000, and more preferred ranges are: 1:30 to 1:100, 1:40 to 1:100, 1:50 to 1:1001:60 to 1:100, 1:70 to 1:100, 1:80 to 1:100, 1:90:1:100, 1:30 to 1:150, 1:40 to 1:150, 1:50 to 1:1501:60 to 1:150, 1:70 to 1:150, 1:80 to 1:150, 1:90 to :150, 1:100 to 1:150, 1:30 to 1:200, 1:40 to 1:200, 1:50 to 1:2001:60 to 1:200, 1:70 to 1:200, 1:80 to 1:200, 1:90:1:200, 1:100 to 1:200, 1:30 to 1:250, 1:40 to 1:250, 1:50 to 1:2501:60 to 1:250, 1:70 to 1:250, 1:80 to 1:250, 1:90 to :250, 1:100 to 1:250, 1:30 to 1:300, 1:40 to 1:300, 1:50 to 1:3001:60 to 1:300, 1:70 to 1:300, 1:80 to 1:300, 1:90:1:300, or 1:100 to 1:300. (7) The inventions provide for a desired conjugation of a level of conjugation of atREGN 11547 least 10%, at least 20%, at least 30%, and at least 40%. Preferred levels are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100%, any of which can be considered complete depending on the purpose. More preferably, the desired level of conjugation typically will be about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0301] Ranges of desired conjugation levels can be 10% to 100%, 20% to 99%, 30% to 99%, 40% to 99%, 50% to 99%, 60% to 99%, 70% to 99%, 80% to 99%, or 90% to 99%. Preferred ranges of desired conjugation levels can be 40% to 100%, 40% to 99%, 45% to 95%, 50% to 95%, 55% to 90%, 60% to 90%, 65% to 95%, 65% to 85%, 65% to 90%, 70% to 90%, 75% to 90%, 75% to 85%, 75% to 80%, or 80% to 85%. More preferably, the range is 65% to 95%.

[0302] The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein. Points 1-7 above also apply to the second chart below:REGN 11547EXAMPLE 22 – AAV8

[0303] Covalently surface modified AAV8 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and harvesting the resulting AAV, and then conjugating the resulting AAV with a second cognate member (SCM) that is fused to a retargeting molecule The first chart below uses SpyTag as the first member of a specific binding pair, and Adenovirus Helper Genes. Modifications can be undertaken as disclosed below:REGN 11547(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV8. (2) Plasmids pRC-FM can be modified for other specific binding pairs. The first member (FM) should be complementary to a second cognate member (SCM) that is fused to a retargeting molecule (for example, an antibody, as well derivatives and fragments thereof, and still other molecules). See Definitions and Description sections of the Detailed Description, the Figures and associated text for “retargeting” and “retargeting molecules”. The populations of FMs and SCMs can be homogenous or heterogeneous as long as the selected FMs can come in contact with appropriate SCMs so that binding can occur. (3) Detargeting mutations are optional and can be undertaken as disclosed herein and in publications. See Definitions and Description sections of the Detailed Description, the Figures and associated text for “detargeting” and “detargeting mutations”.REGN 11547 (4) To lessen the possibility of encountering neutralizing antibodies when the covalently surface modified AAV is administered to a subject, KTag can be used as an SCM to bind with SpyTag (FM) (SEQ ID NOS: 54 and 86). KTag and SpyLigase are based upon SpyCatcher. KTag (SEQ ID NO: 85) has only 10 amino acids, which renders it less immunogenic. KTag can be bound to SpyTag in the presence of SpyLigase (SEQ ID NO: 61). The SpyLigase can be removed by subsequent purification, such as by chromatography and / or filtration so that the SpyLigase will not have an appreciable presence in the final drug substance. (5) Preferred mosaicisms are as follows: 1 / 1, 1 / 1.1, 1 / 1.2, 1 / 1.3, 1 / 1.4, 1 / 1.5, 1 / 1.6, 1 / 1.7, 1 / 1.8, 1 / 1.9, 1 / 2, 1 / 2.1, 1 / 2.2, 1 / 2.3, 1 / 2.4, 1 / 2.5, 1 / 2.6, 1 / 2.7, 1 / 2.8, 1 / 2.9, 1 / 3, 1 / 3.1, 1 / 3.2, 1 / 3.3, 1 / 3.4, 1 / 3.5, 1 / 3.6, 1 / 3.7, 1 / 3.8, 1 / 3.9, 1 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, 1 / 12.8, 1 / 12.9, 1 / 13, 1 / 13.1, 1 / 13.2, 1 / 13.3, 1 / 13.4, 1 / 13.5, 1 / 13.6, 1 / 13.7, 1 / 13.8, 1 / 13.9, 1 / 14, 1 / 14.1, 1 / 14.2, 1 / 14.3, 1 / 14.4, 1 / 14.5, 1 / 14.6, 1 / 14.7, 1 / 14.8, 1 / 14.9, 1 / 15, 1 / 15.1, 1 / 15.2, 1 / 15.3, 1 / 15.4, 1 / 15.5, 1 / 15.6, 1 / 15.7, 1 / 15.8, 1 / 15.9, 1 / 16, 1 / 16.1, 1 / 16.2, 1 / 16.3, 1 / 16.4, 1 / 16.5, 1 / 16.6, 1 / 16.7, 1 / 16.8, 1 / 16.9, 1 / 17, 1 / 17.1, 1 / 17.2, 1 / 17.3, 1 / 17.4, 1 / 17.5, 1 / 17.6, 1 / 17.7, 1 / 17.8, 1 / 17.9, 1 / 18, 1 / 18.1, 1 / 18.2, 1 / 18.3, 1 / 18.4, 1 / 18.5, 1 / 18.6, 1 / 18.7, 1 / 18.8, 1 / 18.9, 1 / 19, 11 / 9.1, 1 / 19.2, 1 / 19.3, 1 / 19.4, 1 / 91.5,REGN 11547 / 19.6, 1 / 19.7, 1 / 19.8, 1 / 19.9, 1 / 20, 1 / 21, 1 / 21.1, 1 / 21.2, 1 / 21.3, 1 / 21.4, 1 / 21.5, / 21.6, 1 / 21.7, 1 / 21.8, 1 / 21.9,1 / 22, 1 / 22.1, 1 / 22.2, 1 / 22.3, 1 / 22.4, 1 / 22.5, 1 / 22.6, / 22.7, 1 / 22.8, 1 / 22.9, 1 / 23, 1 / 23.1, 1 / 23.2, 1 / 23.3, 1 / 23.4, 1 / 23.5, 1 / 23.6, 1 / 23.7, / 23.8, 1 / 23.9, 1 / 24, 1 / 24.1, 1 / 24.2, 1 / 24.3, 1 / 24.4, 1 / 24.5, 1 / 24.6, 1 / 24.7, 1 / 24.8, / 24.9, 1 / 25, 1 / 25.1, 1 / 25.2, 1 / 25.3, 1 / 25.4, 1 / 25.5, 1 / 25.6, 1 / 25.7, 1 / 25.8, 1 / 25.9, / 26, 1 / 26.1, 1 / 26.2, 1 / 26.3, 1 / 26.4, 1 / 26.5, 1 / 26.6, 1 / 26.7, 1 / 26.8, 12 / 6.9, 1 / 27, / 27.1, 1 / 27.2, 1 / 27.3, 1 / 27.4, 1 / 27.5, 1 / 27.6, 1 / 27.7, 1 / 27.8, 1 / 27.9, 1 / 28, 1 / 28.1, / 28.2, 1 / 28.3, 1 / 28.4, 1 / 28.5, 1 / 28.6, 1 / 28.7, 1 / 28.8, 1 / 28.9, 1 / 29, 1 / 29.1, 1 / 29.2, / 29.3, 1 / 29.4, 1 / 29.5, 1 / 29.6, 1 / 29.7, 1 / 29.8, 1 / 29.9, 1 / 30, 1 / 30.1, 1 / 30.2, 1 / 30.3, / 30.4, 1 / 30.5, 1 / 30.6, 1 / 30.7, 1 / 30.8, 1 / 30.9, 1 / 31, 1 / 31.1, 1 / 31.2, 1 / 31.3, 1 / 31.4, / 31.5, 1 / 31.6, 1 / 31.7, 1 / 31.8, 1 / 31.9, 1 / 32, 1 / 32.1, 1 / 32.2, 1 / 32.3, 1 / 32.4, 1 / 32.5, / 32.6, 1 / 32.7, 1 / 32.8, 1 / 32.9, 1 / 33, 1 / 33.1, 1 / 33.2, 1 / 33.3, 1 / 33.4, 1 / 33.5, 1 / 33.6, / 33.7, 1 / 33.8, 1 / 33.9, 1 / 34, 1 / 34.1, 1 / 34.2, 1 / 34.3, 1 / 34.4, 1 / 34.5, 1 / 34.6, 1 / 34.7, / 34.8, 1 / 34.9, 1 / 35, 1 / 35.1, 1 / 35.2, 1 / 35.3, 1 / 35.4, 1 / 35.5, 1 / 35.6, 1 / 35.7, 1 / 35.8, / 35.9, 1 / 36, 1 / 36.1, 1 / 36.2, 1 / 36.3, 1 / 36.4, 1 / 36.5, 1 / 36.6, 1 / 36.7, 1 / 36.8, 1 / 36.9, / 37, 1 / 37.1, 1 / 37.2, 1 / 37.3, 1 / 37.4, 1 / 37.5, 1 / 37.6, 1 / 37.7, 1 / 37.8, 1 / 37.9, 1 / 38, / 38.1, 1 / 38.2, 1 / 38.3, 1 / 38.4, 1 / 38.5, 1 / 38.6, 1 / 38.7, 1 / 38.8, 1 / 38.9, 1 / 39, 1 / 39.1, / 39.2, 1 / 39.3, 1 / 39.4, 1 / 39.5, 1 / 39.6, 1 / 39.7, 1 / 39.8, 1 / 39.9, 1 / 40, 1 / 40.1, 1 / 40.2, / 40.3, 1 / 40.4, 1 / 40.5, 1 / 40.6, 1 / 40.7, 1 / 40.8, 1 / 40.9, 1 / 41, 1 / 41.1, 1 / 41.2, 1 / 41.3, / 41.4, 1 / 41.5, 1 / 41.6, 1 / 41.7, 1 / 41.8, 1 / 41.9, 1 / 42, 1 / 42.1, 1 / 42.2, 1 / 42.3, 1 / 42.4, / 42.5, 1 / 42.6, 1 / 42.7, 1 / 42.8, 1 / 42.9, 1 / 43, 1 / 43.1, 1 / 43.2, 1 / 43.3, 1 / 43.4, 1 / 43.5, / 43.6, 1 / 43.7, 1 / 43.8, 1 / 43.9, 1 / 44, 1 / 44.1, 1 / 44.2, 1 / 44.3, 1 / 44.4, 1 / 44.5, 1 / 44.6, / 44.7, 1 / 44.8, 1 / 44.9, 1 / 45, 1 / 45.1, 1 / 45.2, 1 / 45.3, 1 / 45.4, 1 / 45.5, 1 / 45.6, 1 / 45.7, / 45.8, 1 / 45.9, 1 / 46, 1 / 46.1, 1 / 46.2, 1 / 46.3, 1 / 46.4, 1 / 46.5, 1 / 46.6, 1 / 46.7, 1 / 46.8, / 46.9, 1 / 47, 1 / 47.1, 1 / 47.2, 1 / 47.3, 1 / 47.4, 1 / 47.5, 1 / 47.6, 1 / 47.7, 1 / 47.8, 1 / 47.9,REGN 11547 1 / 48, 1 / 48.1, 1 / 48.2, 1 / 48.3, 1 / 48.4, 1 / 48.5, 1 / 48.6, 1 / 48.7, 1 / 48.8, 1 / 48.9, 1 / 49, 1 / 49.1, 1 / 49.2, 1 / 49.3, 1 / 49.4, 1 / 49.5, 1 / 49.6, 1 / 49.7, 1 / 49.8, 1 / 49.9, 1 / 50, 1 / 50.1, 1 / 50.2, 1 / 50.3, 1 / 50.4, 1 / 50.5, 1 / 50.6, 1 / 50.7, 1 / 50.8, 1 / 50.9, 1 / 51, 1 / 51.1, 1 / 51.2, 1 / 51.3, 1 / 51.4, 1 / 51.5, 1 / 51.6, 1 / 51.7, 1 / 51.8, 1 / 51.9, 1 / 52, 1 / 52.1, 1 / 52.2, 1 / 52.3, 1 / 52.4, 1 / 52.5, 1 / 52.6, 1 / 52.7, 1 / 52.8, 1 / 52.9, 1 / 53, 1 / 53.1, 1 / 53.2, 1 / 53.3, 1 / 53.4, 1 / 53.5, 1 / 53.6, 1 / 53.7, 1 / 53.8, 1 / 53.9, 1 / 54, 1 / 54.1, 1 / 54.2, 1 / 54.3, 1 / 54.4, 1 / 54.5, 1 / 54.6, 1 / 54.7, 1 / 54.8, 1 / 54.9, 1 / 55, 1 / 55.1, 1 / 55.2, 1 / 55.3, 1 / 55.4, 1 / 55.5, 1 / 55.6, 1 / 55.7, 1 / 55.8, 1 / 55.9, 1 / 56, 1 / 56.1, 1 / 56.2, 1 / 56.3, 1 / 56.4, 1 / 56.5, 1 / 56.6, 1 / 56.7, 1 / 56.8, 1 / 56.9, 1 / 57, 1 / 57.1, 1 / 57.2, 1 / 57.3, 1 / 57.4, 1 / 57.5, 1 / 57.6, 1 / 57.7, 1 / 57.8, 1 / 57.9, 1 / 58, 1 / 58.1, 1 / 58.2, 1 / 58.3, 1 / 58.4, 1 / 58.5, 1 / 58.6, 1 / 58.7, 1 / 58.8, 1 / 58.9, 1 / 59, 1 / 59.1, 1 / 59.2, 1 / 59.3, 1 / 59.4, 1 / 59.5, 1 / 59.6, 1 / 59.7, 1 / 59.8, 1 / 59.9, 1 / 60 or ratios thereabout or therebetween. Ranges of ratios of 1 / 1 to 1 / 60 can be employed, and subranges as set forth below.

[0304] More preferred mosaicisms are: 1 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, 1 / 12.8, 1 / 12.9 or 1 / 13.

[0305] Further preferred mosaicisms are: 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3,REGN 11547 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, or 1 / 12.

[0306] Still more preferred mosaicisms are: 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, or 1 / 11.

[0307] Preferred ranges of mosaicisms are 1 / 4 to 1 / 13; 1 / 5 to 1 / 12; 1 / 6 to 1 / 11, 1 / 7 to 1 / 10.5. or 1 / 7.5 to 1 / 10. (6) Preferred molar ratios of AAV-FM to SCM-RM are as follows: 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300, 1:305, 1:310, 1:315, 1:320, 1:325, 1:330, 1:335, 1:340, 1:345, 1:350, 1:355, 1:360, 1:365, 1:370, 1:375, 1:380, 1:385, 1:390, 1:395, 1:400, 1:405, 1:410, 1:415, 1:420, 1:425, 1:430, 1:435, 1:440, 1:445, 1:450, 1:455, 1:460, 1:465, 1:470, 1:475, 1:480, 1:485, 1:490, 1:495, 1:500, 1:505, 1:510, 1:515, 1:520, 1:525, 1:530, 1:535, 1:540, 1:545, 1:550, 1:555, 1:560, 1:565, 1:570, 1:575, 1:580, 1:585, 1:590, 1:595, 1:600, 1:605, 1:610, 1:615, 1:620, 1:625, 1:630, 1:635,REGN 11547 1:640, 1:645, 1:650, 1:655, 1:660, 1:665, 1:670, 1:675, 1:680, 1:685, 1:690, 1:695, 1:700, 1:705, 1:710, 1:715, 1:720, 1:725, 1:730, 1:735, 1:740, 1:745, 1:750, 1:755, 1:760, 1:765, 1:770, 1:775, 1:780, 1:785, 1:790, 1:795, 1:800, 1:805, 1:810, 1:815, 1:820, 1:825, 1:830, 1:835, 1:840, 1:845, 1:850, 1:855, 1:860, 1:865, 1:870, 1:875, 1:880, 1:885, 1:890, 1:895, 1:900, 1:905, 1:910, 1:915, 1:920, 1:925, 1:930, 1:935, 1:940, 1:945, 1:950, 1:955, 1:960, 1:965, 1:970, 1:975, 1:980, 1:985, 1:990, 1:995, 1:1000, 1:>1000 or any ratio thereabout or therebetween.

[0308] More preferred ratios AAV-FM to SCM-RM are: 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300 or any ratio thereabout or therebetween.

[0309] Preferred ranges of AAV-FM to SCM-RM are 1:20 to 1:1000, and more preferred ranges are: 1:30 to 1:100, 1:40 to 1:100, 1:50 to 1:1001:60 to 1:100, 1:70 to 1:100, 1:80 to 1:100, 1:90:1:100, 1:30 to 1:150, 1:40 to 1:150, 1:50 to 1:1501:60 to 1:150, 1:70 to 1:150, 1:80 to 1:150, 1:90 to :150, 1:100 to 1:150, 1:30 to 1:200, 1:40 to 1:200, 1:50 to 1:2001:60 to 1:200, 1:70 to 1:200, 1:80 to 1:200, 1:90:1:200, 1:100 to 1:200, 1:30 to 1:250, 1:40 to 1:250, 1:50 to 1:2501:60 to 1:250, 1:70 to 1:250, 1:80 to 1:250, 1:90 to :250, 1:100 to 1:250, 1:30 to 1:300, 1:40 to 1:300, 1:50 to 1:3001:60 to 1:300, 1:70 to 1:300, 1:80 to 1:300, 1:90:1:300, or 1:100 to 1:300.REGN 11547 . (7) The inventions provide for a desired conjugation of a level of conjugation of at least 10%, at least 20%, at least 30%, and at least 40%. Preferred levels are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100%, any of which can be considered complete depending on the purpose. More preferably, the desired level of conjugation typically will be about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Ranges of desired conjugation levels can be 10% to 100%, 20% to 99%, 30% to 99%, 40% to 99%, 50% to 99%, 60% to 99%, 70% to 99%, 80% to 99%, or 90% to 99%. Preferred ranges of desired conjugation levels can be 40% to 100%, 40% to 99%, 45% to 95%, 50% to 95%, 55% to 90%, 60% to 90%, 65% to 95%, 65% to 85%, 65% to 90%, 70% to 90%, 75% to 90%, 75% to 85%, 75% to 80%, or 80% to 85%. More preferably, the range is 65% to 95%. The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein. Points 1-7 above also apply to the second chart below:REGN 11547EXAMPLE 23 – AAV9

[0310] Covalently surface modified AAV9 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and harvesting the resulting AAV, and then conjugating the resulting AAV with a second cognate member (SCM) that is fused to a retargeting molecule The first chart below uses SpyTag as the first member of a specific binding pair, and Adenovirus Helper Genes. Modifications can be undertaken as disclosed below:REGN 11547(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV9. (2) Plasmids pRC-FM can be modified for other specific binding pairs. The first member (FM) should be complementary to a second cognate member (SCM) that is fused to a retargeting molecule (for example, an antibody, as well derivatives and fragments thereof, and still other molecules). See Definitions and Description sections of the Detailed Description, the Figures and associated text for “retargeting” and “retargeting molecules”. The populations of FMs and SCMs can be homogenous or heterogeneous as long as the selected FMs can come in contact with appropriate SCMs so that binding can occur. (3) Detargeting mutations are optional and can be undertaken as disclosed herein and in publications. See Definitions and Description sections of the Detailed Description, the Figures and associated text for “detargeting” and “detargeting mutations”. (4) To lessen the possibility of encountering neutralizing antibodies when the covalently surface modified AAV is administered to a subject, KTag can be used as an SCM to bind with SpyTag (FM) (SEQ ID NOS: 54 and 86). KTag and SpyLigaseREGN 11547 are based upon SpyCatcher. KTag (SEQ ID NO: 85) has only 10 amino acids, which renders it less immunogenic. KTag can be bound to SpyTag in the presence of SpyLigase (SEQ ID NO: 61). The SpyLigase can be removed by subsequent purification, such as by chromatography and / or filtration so that the SpyLigase will not have an appreciable presence in the final drug substance. (5) Preferred mosaicisms are as follows: 1 / 1, 1 / 1.1, 1 / 1.2, 1 / 1.3, 1 / 1.4, 1 / 1.5, 1 / 1.6, 1 / 1.7, 1 / 1.8, 1 / 1.9, 1 / 2, 1 / 2.1, 1 / 2.2, 1 / 2.3, 1 / 2.4, 1 / 2.5, 1 / 2.6, 1 / 2.7, 1 / 2.8, 1 / 2.9, 1 / 3, 1 / 3.1, 1 / 3.2, 1 / 3.3, 1 / 3.4, 1 / 3.5, 1 / 3.6, 1 / 3.7, 1 / 3.8, 1 / 3.9, 1 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, 1 / 12.8, 1 / 12.9, 1 / 13, 1 / 13.1, 1 / 13.2, 1 / 13.3, 1 / 13.4, 1 / 13.5, 1 / 13.6, 1 / 13.7, 1 / 13.8, 1 / 13.9, 1 / 14, 1 / 14.1, 1 / 14.2, 1 / 14.3, 1 / 14.4, 1 / 14.5, 1 / 14.6, 1 / 14.7, 1 / 14.8, 1 / 14.9, 1 / 15, 1 / 15.1, 1 / 15.2, 1 / 15.3, 1 / 15.4, 1 / 15.5, 1 / 15.6, 1 / 15.7, 1 / 15.8, 1 / 15.9, 1 / 16, 1 / 16.1, 1 / 16.2, 1 / 16.3, 1 / 16.4, 1 / 16.5, 1 / 16.6, 1 / 16.7, 1 / 16.8, 1 / 16.9, 1 / 17, 1 / 17.1, 1 / 17.2, 1 / 17.3, 1 / 17.4, 1 / 17.5, 1 / 17.6, 1 / 17.7, 1 / 17.8, 1 / 17.9, 1 / 18, 1 / 18.1, 1 / 18.2, 1 / 18.3, 1 / 18.4, 1 / 18.5, 1 / 18.6, 1 / 18.7, 1 / 18.8, 1 / 18.9, 1 / 19, 11 / 9.1, 1 / 19.2, 1 / 19.3, 1 / 19.4, 1 / 91.5, 1 / 19.6, 1 / 19.7, 1 / 19.8, 1 / 19.9, 1 / 20, 1 / 21, 1 / 21.1, 1 / 21.2, 1 / 21.3, 1 / 21.4, 1 / 21.5, 1 / 21.6, 1 / 21.7, 1 / 21.8, 1 / 21.9,1 / 22, 1 / 22.1, 1 / 22.2, 1 / 22.3, 1 / 22.4, 1 / 22.5, 1 / 22.6, 1 / 22.7, 1 / 22.8, 1 / 22.9, 1 / 23, 1 / 23.1, 1 / 23.2, 1 / 23.3, 1 / 23.4, 1 / 23.5, 1 / 23.6, 1 / 23.7,REGN 11547 / 23.8, 1 / 23.9, 1 / 24, 1 / 24.1, 1 / 24.2, 1 / 24.3, 1 / 24.4, 1 / 24.5, 1 / 24.6, 1 / 24.7, 1 / 24.8, / 24.9, 1 / 25, 1 / 25.1, 1 / 25.2, 1 / 25.3, 1 / 25.4, 1 / 25.5, 1 / 25.6, 1 / 25.7, 1 / 25.8, 1 / 25.9, / 26, 1 / 26.1, 1 / 26.2, 1 / 26.3, 1 / 26.4, 1 / 26.5, 1 / 26.6, 1 / 26.7, 1 / 26.8, 12 / 6.9, 1 / 27, / 27.1, 1 / 27.2, 1 / 27.3, 1 / 27.4, 1 / 27.5, 1 / 27.6, 1 / 27.7, 1 / 27.8, 1 / 27.9, 1 / 28, 1 / 28.1, / 28.2, 1 / 28.3, 1 / 28.4, 1 / 28.5, 1 / 28.6, 1 / 28.7, 1 / 28.8, 1 / 28.9, 1 / 29, 1 / 29.1, 1 / 29.2, / 29.3, 1 / 29.4, 1 / 29.5, 1 / 29.6, 1 / 29.7, 1 / 29.8, 1 / 29.9, 1 / 30, 1 / 30.1, 1 / 30.2, 1 / 30.3, / 30.4, 1 / 30.5, 1 / 30.6, 1 / 30.7, 1 / 30.8, 1 / 30.9, 1 / 31, 1 / 31.1, 1 / 31.2, 1 / 31.3, 1 / 31.4, / 31.5, 1 / 31.6, 1 / 31.7, 1 / 31.8, 1 / 31.9, 1 / 32, 1 / 32.1, 1 / 32.2, 1 / 32.3, 1 / 32.4, 1 / 32.5, / 32.6, 1 / 32.7, 1 / 32.8, 1 / 32.9, 1 / 33, 1 / 33.1, 1 / 33.2, 1 / 33.3, 1 / 33.4, 1 / 33.5, 1 / 33.6, / 33.7, 1 / 33.8, 1 / 33.9, 1 / 34, 1 / 34.1, 1 / 34.2, 1 / 34.3, 1 / 34.4, 1 / 34.5, 1 / 34.6, 1 / 34.7, / 34.8, 1 / 34.9, 1 / 35, 1 / 35.1, 1 / 35.2, 1 / 35.3, 1 / 35.4, 1 / 35.5, 1 / 35.6, 1 / 35.7, 1 / 35.8, / 35.9, 1 / 36, 1 / 36.1, 1 / 36.2, 1 / 36.3, 1 / 36.4, 1 / 36.5, 1 / 36.6, 1 / 36.7, 1 / 36.8, 1 / 36.9, / 37, 1 / 37.1, 1 / 37.2, 1 / 37.3, 1 / 37.4, 1 / 37.5, 1 / 37.6, 1 / 37.7, 1 / 37.8, 1 / 37.9, 1 / 38, / 38.1, 1 / 38.2, 1 / 38.3, 1 / 38.4, 1 / 38.5, 1 / 38.6, 1 / 38.7, 1 / 38.8, 1 / 38.9, 1 / 39, 1 / 39.1, / 39.2, 1 / 39.3, 1 / 39.4, 1 / 39.5, 1 / 39.6, 1 / 39.7, 1 / 39.8, 1 / 39.9, 1 / 40, 1 / 40.1, 1 / 40.2, / 40.3, 1 / 40.4, 1 / 40.5, 1 / 40.6, 1 / 40.7, 1 / 40.8, 1 / 40.9, 1 / 41, 1 / 41.1, 1 / 41.2, 1 / 41.3, / 41.4, 1 / 41.5, 1 / 41.6, 1 / 41.7, 1 / 41.8, 1 / 41.9, 1 / 42, 1 / 42.1, 1 / 42.2, 1 / 42.3, 1 / 42.4, / 42.5, 1 / 42.6, 1 / 42.7, 1 / 42.8, 1 / 42.9, 1 / 43, 1 / 43.1, 1 / 43.2, 1 / 43.3, 1 / 43.4, 1 / 43.5, / 43.6, 1 / 43.7, 1 / 43.8, 1 / 43.9, 1 / 44, 1 / 44.1, 1 / 44.2, 1 / 44.3, 1 / 44.4, 1 / 44.5, 1 / 44.6, / 44.7, 1 / 44.8, 1 / 44.9, 1 / 45, 1 / 45.1, 1 / 45.2, 1 / 45.3, 1 / 45.4, 1 / 45.5, 1 / 45.6, 1 / 45.7, / 45.8, 1 / 45.9, 1 / 46, 1 / 46.1, 1 / 46.2, 1 / 46.3, 1 / 46.4, 1 / 46.5, 1 / 46.6, 1 / 46.7, 1 / 46.8, / 46.9, 1 / 47, 1 / 47.1, 1 / 47.2, 1 / 47.3, 1 / 47.4, 1 / 47.5, 1 / 47.6, 1 / 47.7, 1 / 47.8, 1 / 47.9, / 48, 1 / 48.1, 1 / 48.2, 1 / 48.3, 1 / 48.4, 1 / 48.5, 1 / 48.6, 1 / 48.7, 1 / 48.8, 1 / 48.9, 1 / 49, / 49.1, 1 / 49.2, 1 / 49.3, 1 / 49.4, 1 / 49.5, 1 / 49.6, 1 / 49.7, 1 / 49.8, 1 / 49.9, 1 / 50, 1 / 50.1, / 50.2, 1 / 50.3, 1 / 50.4, 1 / 50.5, 1 / 50.6, 1 / 50.7, 1 / 50.8, 1 / 50.9, 1 / 51, 1 / 51.1, 1 / 51.2,REGN 11547 1 / 51.3, 1 / 51.4, 1 / 51.5, 1 / 51.6, 1 / 51.7, 1 / 51.8, 1 / 51.9, 1 / 52, 1 / 52.1, 1 / 52.2, 1 / 52.3, 1 / 52.4, 1 / 52.5, 1 / 52.6, 1 / 52.7, 1 / 52.8, 1 / 52.9, 1 / 53, 1 / 53.1, 1 / 53.2, 1 / 53.3, 1 / 53.4, 1 / 53.5, 1 / 53.6, 1 / 53.7, 1 / 53.8, 1 / 53.9, 1 / 54, 1 / 54.1, 1 / 54.2, 1 / 54.3, 1 / 54.4, 1 / 54.5, 1 / 54.6, 1 / 54.7, 1 / 54.8, 1 / 54.9, 1 / 55, 1 / 55.1, 1 / 55.2, 1 / 55.3, 1 / 55.4, 1 / 55.5, 1 / 55.6, 1 / 55.7, 1 / 55.8, 1 / 55.9, 1 / 56, 1 / 56.1, 1 / 56.2, 1 / 56.3, 1 / 56.4, 1 / 56.5, 1 / 56.6, 1 / 56.7, 1 / 56.8, 1 / 56.9, 1 / 57, 1 / 57.1, 1 / 57.2, 1 / 57.3, 1 / 57.4, 1 / 57.5, 1 / 57.6, 1 / 57.7, 1 / 57.8, 1 / 57.9, 1 / 58, 1 / 58.1, 1 / 58.2, 1 / 58.3, 1 / 58.4, 1 / 58.5, 1 / 58.6, 1 / 58.7, 1 / 58.8, 1 / 58.9, 1 / 59, 1 / 59.1, 1 / 59.2, 1 / 59.3, 1 / 59.4, 1 / 59.5, 1 / 59.6, 1 / 59.7, 1 / 59.8, 1 / 59.9, 1 / 60 or ratios thereabout or therebetween. Ranges of ratios of 1 / 1 to 1 / 60 can be employed, and subranges as set forth below.

[0311] More preferred mosaicisms are: 1 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, 1 / 12.8, 1 / 12.9 or 1 / 13.

[0312] Further preferred mosaicisms are: 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3,REGN 11547 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, or 1 / 12.

[0313] Still more preferred mosaicisms are: 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, or 1 / 11.

[0314] Preferred ranges of mosaicisms are 1 / 4 to 1 / 13; 1 / 5 to 1 / 12; 1 / 6 to 1 / 11, 1 / 7 to 1 / 10.5. or 1 / 7.5 to 1 / 10. (6) Preferred molar ratios of AAV-FM to SCM-RM are as follows: 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300, 1:305, 1:310, 1:315, 1:320, 1:325, 1:330, 1:335, 1:340, 1:345, 1:350, 1:355, 1:360, 1:365, 1:370, 1:375, 1:380, 1:385, 1:390, 1:395, 1:400, 1:405, 1:410, 1:415, 1:420, 1:425, 1:430, 1:435, 1:440, 1:445, 1:450, 1:455, 1:460, 1:465, 1:470, 1:475, 1:480, 1:485, 1:490, 1:495, 1:500, 1:505, 1:510, 1:515, 1:520, 1:525, 1:530, 1:535, 1:540, 1:545, 1:550, 1:555, 1:560, 1:565, 1:570, 1:575, 1:580, 1:585, 1:590, 1:595, 1:600, 1:605, 1:610, 1:615, 1:620, 1:625, 1:630, 1:635, 1:640, 1:645, 1:650, 1:655, 1:660, 1:665, 1:670, 1:675, 1:680, 1:685, 1:690, 1:695, 1:700, 1:705, 1:710, 1:715, 1:720, 1:725, 1:730, 1:735, 1:740, 1:745, 1:750, 1:755, 1:760, 1:765, 1:770, 1:775, 1:780, 1:785, 1:790, 1:795, 1:800, 1:805, 1:810, 1:815,REGN 11547 1:820, 1:825, 1:830, 1:835, 1:840, 1:845, 1:850, 1:855, 1:860, 1:865, 1:870, 1:875, 1:880, 1:885, 1:890, 1:895, 1:900, 1:905, 1:910, 1:915, 1:920, 1:925, 1:930, 1:935, 1:940, 1:945, 1:950, 1:955, 1:960, 1:965, 1:970, 1:975, 1:980, 1:985, 1:990, 1:995, 1:1000, 1:>1000 or any ratio thereabout or therebetween.

[0315] More preferred ratios AAV-FM to SCM-RM are: 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300 or any ratio thereabout or therebetween.

[0316] Preferred ranges of AAV-FM to SCM-RM are 1:20 to 1:1000, and more preferred ranges are: 1:30 to 1:100, 1:40 to 1:100, 1:50 to 1:1001:60 to 1:100, 1:70 to 1:100, 1:80 to 1:100, 1:90:1:100, 1:30 to 1:150, 1:40 to 1:150, 1:50 to 1:1501:60 to 1:150, 1:70 to 1:150, 1:80 to 1:150, 1:90 to :150, 1:100 to 1:150, 1:30 to 1:200, 1:40 to 1:200, 1:50 to 1:2001:60 to 1:200, 1:70 to 1:200, 1:80 to 1:200, 1:90:1:200, 1:100 to 1:200, 1:30 to 1:250, 1:40 to 1:250, 1:50 to 1:2501:60 to 1:250, 1:70 to 1:250, 1:80 to 1:250, 1:90 to :250, 1:100 to 1:250, 1:30 to 1:300, 1:40 to 1:300, 1:50 to 1:3001:60 to 1:300, 1:70 to 1:300, 1:80 to 1:300, 1:90:1:300, or 1:100 to 1:300. (7) The inventions provide for a desired conjugation of a level of conjugation of at least 10%, at least 20%, at least 30%, and at least 40%. Preferred levels are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100%, any of which can be considered complete depending on the purpose. More preferably, theREGN 11547 desired level of conjugation typically will be about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0317] Ranges of desired conjugation levels can be 10% to 100%, 20% to 99%, 30% to 99%, 40% to 99%, 50% to 99%, 60% to 99%, 70% to 99%, 80% to 99%, or 90% to 99%. Preferred ranges of desired conjugation levels can be 40% to 100%, 40% to 99%, 45% to 95%, 50% to 95%, 55% to 90%, 60% to 90%, 65% to 95%, 65% to 85%, 65% to 90%, 70% to 90%, 75% to 90%, 75% to 85%, 75% to 80%, or 80% to 85%. More preferably, the range is 65% to 95%.

[0318] The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein. Points 1-7 above also apply to the second chart below:REGN 11547EXAMPLE 24 – AAV10

[0319] Covalently surface modified AAV10 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and harvesting the resulting AAV, and then conjugating the resulting AAV with a second cognate member (SCM) that is fused to a retargeting molecule The first chart below uses SpyTag as the first member of a specific binding pair, and Adenovirus Helper Genes. Modifications can be undertaken as disclosed below:REGN 11547(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV10. (2) Plasmids pRC-FM can be modified for other specific binding pairs. The first member (FM) should be complementary to a second cognate member (SCM) that is fused to a retargeting molecule (for example, an antibody, as well derivatives and fragments thereof, and still other molecules). See Definitions and Description sections of the Detailed Description, the Figures and associated text for “retargeting” and “retargeting molecules”. The populations of FMs and SCMs can be homogenous or heterogeneous as long as the selected FMs can come in contact with appropriate SCMs so that binding can occur. (3) Detargeting mutations are optional and can be undertaken as disclosed herein and in publications. See Definitions and Description sections of the Detailed Description, the Figures and associated text for “detargeting” and “detargeting mutations”. (4) To lessen the possibility of encountering neutralizing antibodies when the covalently surface modified AAV is administered to a subject, KTag can be used as an SCM to bind with SpyTag (FM) (SEQ ID NOS: 54 and 86). KTag and SpyLigase are based upon SpyCatcher. KTag (SEQ ID NO: 85) has only 10 amino acids, which renders it less immunogenic. KTag can be bound to SpyTag in the presence of SpyLigase (SEQ ID NO: 61). The SpyLigase can be removed by subsequentREGN 11547 purification, such as by chromatography and / or filtration so that the SpyLigase will not have an appreciable presence in the final drug substance. (5) Preferred mosaicisms are as follows: 1 / 1, 1 / 1.1, 1 / 1.2, 1 / 1.3, 1 / 1.4, 1 / 1.5, 1 / 1.6, 1 / 1.7, 1 / 1.8, 1 / 1.9, 1 / 2, 1 / 2.1, 1 / 2.2, 1 / 2.3, 1 / 2.4, 1 / 2.5, 1 / 2.6, 1 / 2.7, 1 / 2.8, 1 / 2.9, 1 / 3, 1 / 3.1, 1 / 3.2, 1 / 3.3, 1 / 3.4, 1 / 3.5, 1 / 3.6, 1 / 3.7, 1 / 3.8, 1 / 3.9, 1 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, 1 / 12.8, 1 / 12.9, 1 / 13, 1 / 13.1, 1 / 13.2, 1 / 13.3, 1 / 13.4, 1 / 13.5, 1 / 13.6, 1 / 13.7, 1 / 13.8, 1 / 13.9, 1 / 14, 1 / 14.1, 1 / 14.2, 1 / 14.3, 1 / 14.4, 1 / 14.5, 1 / 14.6, 1 / 14.7, 1 / 14.8, 1 / 14.9, 1 / 15, 1 / 15.1, 1 / 15.2, 1 / 15.3, 1 / 15.4, 1 / 15.5, 1 / 15.6, 1 / 15.7, 1 / 15.8, 1 / 15.9, 1 / 16, 1 / 16.1, 1 / 16.2, 1 / 16.3, 1 / 16.4, 1 / 16.5, 1 / 16.6, 1 / 16.7, 1 / 16.8, 1 / 16.9, 1 / 17, 1 / 17.1, 1 / 17.2, 1 / 17.3, 1 / 17.4, 1 / 17.5, 1 / 17.6, 1 / 17.7, 1 / 17.8, 1 / 17.9, 1 / 18, 1 / 18.1, 1 / 18.2, 1 / 18.3, 1 / 18.4, 1 / 18.5, 1 / 18.6, 1 / 18.7, 1 / 18.8, 1 / 18.9, 1 / 19, 11 / 9.1, 1 / 19.2, 1 / 19.3, 1 / 19.4, 1 / 91.5, 1 / 19.6, 1 / 19.7, 1 / 19.8, 1 / 19.9, 1 / 20, 1 / 21, 1 / 21.1, 1 / 21.2, 1 / 21.3, 1 / 21.4, 1 / 21.5, 1 / 21.6, 1 / 21.7, 1 / 21.8, 1 / 21.9,1 / 22, 1 / 22.1, 1 / 22.2, 1 / 22.3, 1 / 22.4, 1 / 22.5, 1 / 22.6, 1 / 22.7, 1 / 22.8, 1 / 22.9, 1 / 23, 1 / 23.1, 1 / 23.2, 1 / 23.3, 1 / 23.4, 1 / 23.5, 1 / 23.6, 1 / 23.7, 1 / 23.8, 1 / 23.9, 1 / 24, 1 / 24.1, 1 / 24.2, 1 / 24.3, 1 / 24.4, 1 / 24.5, 1 / 24.6, 1 / 24.7, 1 / 24.8, 1 / 24.9, 1 / 25, 1 / 25.1, 1 / 25.2, 1 / 25.3, 1 / 25.4, 1 / 25.5, 1 / 25.6, 1 / 25.7, 1 / 25.8, 1 / 25.9, 1 / 26, 1 / 26.1, 1 / 26.2, 1 / 26.3, 1 / 26.4, 1 / 26.5, 1 / 26.6, 1 / 26.7, 1 / 26.8, 12 / 6.9, 1 / 27,REGN 11547 / 27.1, 1 / 27.2, 1 / 27.3, 1 / 27.4, 1 / 27.5, 1 / 27.6, 1 / 27.7, 1 / 27.8, 1 / 27.9, 1 / 28, 1 / 28.1, / 28.2, 1 / 28.3, 1 / 28.4, 1 / 28.5, 1 / 28.6, 1 / 28.7, 1 / 28.8, 1 / 28.9, 1 / 29, 1 / 29.1, 1 / 29.2, / 29.3, 1 / 29.4, 1 / 29.5, 1 / 29.6, 1 / 29.7, 1 / 29.8, 1 / 29.9, 1 / 30, 1 / 30.1, 1 / 30.2, 1 / 30.3, / 30.4, 1 / 30.5, 1 / 30.6, 1 / 30.7, 1 / 30.8, 1 / 30.9, 1 / 31, 1 / 31.1, 1 / 31.2, 1 / 31.3, 1 / 31.4, / 31.5, 1 / 31.6, 1 / 31.7, 1 / 31.8, 1 / 31.9, 1 / 32, 1 / 32.1, 1 / 32.2, 1 / 32.3, 1 / 32.4, 1 / 32.5, / 32.6, 1 / 32.7, 1 / 32.8, 1 / 32.9, 1 / 33, 1 / 33.1, 1 / 33.2, 1 / 33.3, 1 / 33.4, 1 / 33.5, 1 / 33.6, / 33.7, 1 / 33.8, 1 / 33.9, 1 / 34, 1 / 34.1, 1 / 34.2, 1 / 34.3, 1 / 34.4, 1 / 34.5, 1 / 34.6, 1 / 34.7, / 34.8, 1 / 34.9, 1 / 35, 1 / 35.1, 1 / 35.2, 1 / 35.3, 1 / 35.4, 1 / 35.5, 1 / 35.6, 1 / 35.7, 1 / 35.8, / 35.9, 1 / 36, 1 / 36.1, 1 / 36.2, 1 / 36.3, 1 / 36.4, 1 / 36.5, 1 / 36.6, 1 / 36.7, 1 / 36.8, 1 / 36.9, / 37, 1 / 37.1, 1 / 37.2, 1 / 37.3, 1 / 37.4, 1 / 37.5, 1 / 37.6, 1 / 37.7, 1 / 37.8, 1 / 37.9, 1 / 38, / 38.1, 1 / 38.2, 1 / 38.3, 1 / 38.4, 1 / 38.5, 1 / 38.6, 1 / 38.7, 1 / 38.8, 1 / 38.9, 1 / 39, 1 / 39.1, / 39.2, 1 / 39.3, 1 / 39.4, 1 / 39.5, 1 / 39.6, 1 / 39.7, 1 / 39.8, 1 / 39.9, 1 / 40, 1 / 40.1, 1 / 40.2, / 40.3, 1 / 40.4, 1 / 40.5, 1 / 40.6, 1 / 40.7, 1 / 40.8, 1 / 40.9, 1 / 41, 1 / 41.1, 1 / 41.2, 1 / 41.3, / 41.4, 1 / 41.5, 1 / 41.6, 1 / 41.7, 1 / 41.8, 1 / 41.9, 1 / 42, 1 / 42.1, 1 / 42.2, 1 / 42.3, 1 / 42.4, / 42.5, 1 / 42.6, 1 / 42.7, 1 / 42.8, 1 / 42.9, 1 / 43, 1 / 43.1, 1 / 43.2, 1 / 43.3, 1 / 43.4, 1 / 43.5, / 43.6, 1 / 43.7, 1 / 43.8, 1 / 43.9, 1 / 44, 1 / 44.1, 1 / 44.2, 1 / 44.3, 1 / 44.4, 1 / 44.5, 1 / 44.6, / 44.7, 1 / 44.8, 1 / 44.9, 1 / 45, 1 / 45.1, 1 / 45.2, 1 / 45.3, 1 / 45.4, 1 / 45.5, 1 / 45.6, 1 / 45.7, / 45.8, 1 / 45.9, 1 / 46, 1 / 46.1, 1 / 46.2, 1 / 46.3, 1 / 46.4, 1 / 46.5, 1 / 46.6, 1 / 46.7, 1 / 46.8, / 46.9, 1 / 47, 1 / 47.1, 1 / 47.2, 1 / 47.3, 1 / 47.4, 1 / 47.5, 1 / 47.6, 1 / 47.7, 1 / 47.8, 1 / 47.9, / 48, 1 / 48.1, 1 / 48.2, 1 / 48.3, 1 / 48.4, 1 / 48.5, 1 / 48.6, 1 / 48.7, 1 / 48.8, 1 / 48.9, 1 / 49, / 49.1, 1 / 49.2, 1 / 49.3, 1 / 49.4, 1 / 49.5, 1 / 49.6, 1 / 49.7, 1 / 49.8, 1 / 49.9, 1 / 50, 1 / 50.1, / 50.2, 1 / 50.3, 1 / 50.4, 1 / 50.5, 1 / 50.6, 1 / 50.7, 1 / 50.8, 1 / 50.9, 1 / 51, 1 / 51.1, 1 / 51.2, / 51.3, 1 / 51.4, 1 / 51.5, 1 / 51.6, 1 / 51.7, 1 / 51.8, 1 / 51.9, 1 / 52, 1 / 52.1, 1 / 52.2, 1 / 52.3, / 52.4, 1 / 52.5, 1 / 52.6, 1 / 52.7, 1 / 52.8, 1 / 52.9, 1 / 53, 1 / 53.1, 1 / 53.2, 1 / 53.3, 1 / 53.4, / 53.5, 1 / 53.6, 1 / 53.7, 1 / 53.8, 1 / 53.9, 1 / 54, 1 / 54.1, 1 / 54.2, 1 / 54.3, 1 / 54.4, 1 / 54.5,REGN 11547 1 / 54.6, 1 / 54.7, 1 / 54.8, 1 / 54.9, 1 / 55, 1 / 55.1, 1 / 55.2, 1 / 55.3, 1 / 55.4, 1 / 55.5, 1 / 55.6, 1 / 55.7, 1 / 55.8, 1 / 55.9, 1 / 56, 1 / 56.1, 1 / 56.2, 1 / 56.3, 1 / 56.4, 1 / 56.5, 1 / 56.6, 1 / 56.7, 1 / 56.8, 1 / 56.9, 1 / 57, 1 / 57.1, 1 / 57.2, 1 / 57.3, 1 / 57.4, 1 / 57.5, 1 / 57.6, 1 / 57.7, 1 / 57.8, 1 / 57.9, 1 / 58, 1 / 58.1, 1 / 58.2, 1 / 58.3, 1 / 58.4, 1 / 58.5, 1 / 58.6, 1 / 58.7, 1 / 58.8, 1 / 58.9, 1 / 59, 1 / 59.1, 1 / 59.2, 1 / 59.3, 1 / 59.4, 1 / 59.5, 1 / 59.6, 1 / 59.7, 1 / 59.8, 1 / 59.9, 1 / 60 or ratios thereabout or therebetween. Ranges of ratios of 1 / 1 to 1 / 60 can be employed, and subranges as set forth below.

[0320] More preferred mosaicisms are: 1 / 4, 1 / 4.1, 1 / 4.2, 1 / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, 1 / 12, 1 / 12.1, 1 / 12.2, 1 / 12.3, 1 / 12.4, 1 / 12.5, 1 / 12.6, 1 / 12.7, 1 / 12.8, 1 / 12.9 or 1 / 13.

[0321] Further preferred mosaicisms are: 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, 1 / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1 / 6.3, 1 / 6.4, 1 / 6.5, 1 / 6.6, 1 / 6.7, 1 / 6.8, 1 / 6.9, 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, 1 / 11, 1 / 11.1, 1 / 11.2, 1 / 11.3, 1 / 11.4, 1 / 11.5, 1 / 11.6, 1 / 11.7, 1 / 11.8, 1 / 11.9, or 1 / 12.REGN 11547

[0322] Still more preferred mosaicisms are: 1 / 7, 1 / 7.1, 1 / 7.2, 1 / 7.3, 1 / 7.4, 1 / 7.5, 1 / 7.6, 1 / 7.7, 1 / 7.8, 1 / 7.9, 1 / 8, 1 / 8.1, 1 / 8.2, 1 / 8.3, 1 / 8.4, 1 / 8.5, 1 / 8.6, 1 / 8.7, 1 / 8.8, 1 / 8.9, 1 / 9, 1 / 9.1, 1 / 9.2, 1 / 9.3, 1 / 9.4, 1 / 9.5, 1 / 9.6, 1 / 9.7, 1 / 9.8, 1 / 9.9, 1 / 10, 1 / 10.1, 1 / 10.2, 1 / 10.3, 1 / 10.4, 1 / 10.5, 1 / 10.6, 1 / 10.7, 1 / 10.8, 1 / 10.9, or 1 / 11.

[0323] Preferred ranges of mosaicisms are 1 / 4 to 1 / 13; 1 / 5 to 1 / 12; 1 / 6 to 1 / 11, 1 / 7 to 1 / 10.5. or 1 / 7.5 to 1 / 10. (6) Preferred molar ratios of AAV-FM to SCM-RM are as follows: 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300, 1:305, 1:310, 1:315, 1:320, 1:325, 1:330, 1:335, 1:340, 1:345, 1:350, 1:355, 1:360, 1:365, 1:370, 1:375, 1:380, 1:385, 1:390, 1:395, 1:400, 1:405, 1:410, 1:415, 1:420, 1:425, 1:430, 1:435, 1:440, 1:445, 1:450, 1:455, 1:460, 1:465, 1:470, 1:475, 1:480, 1:485, 1:490, 1:495, 1:500, 1:505, 1:510, 1:515, 1:520, 1:525, 1:530, 1:535, 1:540, 1:545, 1:550, 1:555, 1:560, 1:565, 1:570, 1:575, 1:580, 1:585, 1:590, 1:595, 1:600, 1:605, 1:610, 1:615, 1:620, 1:625, 1:630, 1:635, 1:640, 1:645, 1:650, 1:655, 1:660, 1:665, 1:670, 1:675, 1:680, 1:685, 1:690, 1:695, 1:700, 1:705, 1:710, 1:715, 1:720, 1:725, 1:730, 1:735, 1:740, 1:745, 1:750, 1:755, 1:760, 1:765, 1:770, 1:775, 1:780, 1:785, 1:790, 1:795, 1:800, 1:805, 1:810, 1:815, 1:820, 1:825, 1:830, 1:835, 1:840, 1:845, 1:850, 1:855, 1:860, 1:865, 1:870, 1:875, 1:880, 1:885, 1:890, 1:895, 1:900, 1:905, 1:910, 1:915, 1:920, 1:925, 1:930, 1:935,REGN 11547 1:940, 1:945, 1:950, 1:955, 1:960, 1:965, 1:970, 1:975, 1:980, 1:985, 1:990, 1:995, 1:1000, 1:>1000 or any ratio thereabout or therebetween.

[0324] More preferred ratios AAV-FM to SCM-RM are: 1:30, 1:35, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, 1:80, 1:85, 1:90, 1:95, 1:100, 1:105, 1:110, 1:115, 1:120, 1:125, 1:130, 1:135, 1:140, 1:145, 1:150, 1:155, 1:160, 1:165, 1:170, 1:175, 1:180, 1:185, 1:190, 1:195, 1:200, 1:205, 1:210, 1:215, 1:220, 1:225, 1:230, 1:235, 1:240, 1:245, 1:250, 1:255, 1:260, 1:265, 1:270, 1:275, 1:280, 1:285, 1:290, 1:295, 1:300 or any ratio thereabout or therebetween.

[0325] Preferred ranges of AAV-FM to SCM-RM are 1:20 to 1:1000, and more preferred ranges are: 1:30 to 1:100, 1:40 to 1:100, 1:50 to 1:1001:60 to 1:100, 1:70 to 1:100, 1:80 to 1:100, 1:90:1:100, 1:30 to 1:150, 1:40 to 1:150, 1:50 to 1:1501:60 to 1:150, 1:70 to 1:150, 1:80 to 1:150, 1:90 to :150, 1:100 to 1:150, 1:30 to 1:200, 1:40 to 1:200, 1:50 to 1:2001:60 to 1:200, 1:70 to 1:200, 1:80 to 1:200, 1:90:1:200, 1:100 to 1:200, 1:30 to 1:250, 1:40 to 1:250, 1:50 to 1:2501:60 to 1:250, 1:70 to 1:250, 1:80 to 1:250, 1:90 to :250, 1:100 to 1:250, 1:30 to 1:300, 1:40 to 1:300, 1:50 to 1:3001:60 to 1:300, 1:70 to 1:300, 1:80 to 1:300, 1:90:1:300, or 1:100 to 1:300. (7) The inventions provide for a desired conjugation of a level of conjugation of at least 10%, at least 20%, at least 30%, and at least 40%. Preferred levels are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100%, any of which can be considered complete depending on the purpose. More preferably, the desired level of conjugation typically will be about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,REGN 11547 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0326] Ranges of desired conjugation levels can be 10% to 100%, 20% to 99%, 30% to 99%, 40% to 99%, 50% to 99%, 60% to 99%, 70% to 99%, 80% to 99%, or 90% to 99%. Preferred ranges of desired conjugation levels can be 40% to 100%, 40% to 99%, 45% to 95%, 50% to 95%, 55% to 90%, 60% to 90%, 65% to 95%, 65% to 85%, 65% to 90%, 70% to 90%, 75% to 90%, 75% to 85%, 75% to 80%, or 80% to 85%. More preferably, the range is 65% to 95%.

[0327] The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein. Points 1-7 above also apply to the second chart below:REGN 11547EXAMPLE 25 – AAV11

[0328] Covalently surface modified AAV11 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and harvesting the resulting AAV, and then conjugating the resulting AAV with a second cognate member (SCM) that is fused to a retargeting molecule The first chart below uses SpyTag as the first member of a specific binding pair, and Adenovirus Helper Genes. Modifications can be undertaken as disclosed below:REGN 11547 (1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV11. (2) Plasmids pRC-FM can be modified for other specific binding pairs. The first member (FM) should be complementary to a second cognate member (SCM) that is fused to a retargeting molecule (for example, an antibody, as well derivatives and fragments thereof, and still other molecules). See Definitions and Description sections of the Detailed Description, the Figures and associated text for “retargeting” and “retargeting molecules”. The populations of FMs and SCMs can be homogenous or heterogeneous as long as the selected FMs can come in contact with appropriate SCMs so that binding can occur. (3) Detargeting mutations are optional and can be undertaken as disclosed herein and in publications. See Definitions and Description sections of the Detailed Description, the Figures and associated text for “detargeting” and “detargeting mutations”. (4) To lessen the possibility of encountering neutralizing antibodies when the covalently surface modified AAV is administered to a subject, KTag can be used as an SCM to bind with SpyTag (FM) (SEQ ID NOS: 54 and 86). KTag and SpyLigase are based upon SpyCatcher. KTag (SEQ ID NO: 85) has only 10 amino acids, which renders it less immunogenic. KTag can be bound to SpyTag in the presence of SpyLigase (SEQ ID NO: 61). The SpyLigase can be removed by subsequent purification, such as by chromatography and / or filtration so that the SpyLigase will not have an appreciable presence in the final drug substance. (5) Preferred mosaicisms are as follows: 1 / 1, 1 / 1.1, 1 / 1.2, 1 / 1.3, 1 / 1.4, 1 / 1.5, 1 / 1.6, 1 / 1.7, 1 / 1.8, 1 / 1.9, 1 / 2, 1 / 2.1, 1 / 2.2, 1 / 2.3, 1 / 2.4, 1 / 2.5, 1 / 2.6, 1 / 2.7, 1 / 2.8,REGN 11547 / 2.9, 1 / 3, 1 / 3.1, 1 / 3.2, 1 / 3.3, 1 / 3.4, 1 / 3.5, 1 / 3.6, 1 / 3.7, 1 / 3.8, 1 / 3.9, 1 / 4, 1 / 4.1, 1 / 4.2, / 4.3, 1 / 4.4, 1 / 4.5, 1 / 4.6, 1 / 4.7, 1 / 4.8, 1 / 4.9, 1 / 5, 1 / 5.1, 1 / 5.2, 1 / 5.3, 1 / 5.4, 1 / 5.5, / 5.6, 1 / 5.7, 1 / 5.8, 1 / 5.9, 1 / 6, 1 / 6.1, 1 / 6.2, 1...

Claims

REGN 11547 WHAT IS CLAIMED IS:

1. A method of producing covalently surface modified adeno-associated virus using in vitro conjugation, wherein the method comprises the steps of (I) combining recombinant AAV (rAAV) with retargeting molecules in a ratio selected to achieve a desired level of conjugation; (II) incubating the AAV and retargeting molecules for a period of time and under conditions to achieve the desired level of conjugation.

2. A method of producing a covalently surface modified adeno-associated virus according to claim 1, wherein the rAAV is obtained by (A) transfecting a cell with: (1) a plasmid comprising a gene of interest flanked by adeno-associated virus (AAV) inverted terminal repeats; (2) a plasmid comprising an AAV rep gene and an AAV cap gene; (3) a plasmid comprising AAV rep and cap genes and a polynucleotide sequence encoding a first member of a specific binding pair; and (4) a plasmid comprising one or more helper polynucleotide sequences; (B) culturing the transfected cell from step (A) to allow expression of plasmids (1) to (4) and assembly of proteins formed by the expression of plasmids (1) to (4); and (C) harvesting the recombinant AAV.

3. The method according to claim 2, wherein the cell is a mammalian cell.REGN 11547 4. The method according to claim 3, wherein the mammalian cell is a human cell.

5. The method according to claims 1-4, wherein the recombinant AAV comprises a gene of interest.

6. The method according to claims 1-5, wherein the recombinant AAV comprises a recombinant capsid protein.

7. The method according to claim 6, wherein the recombinant capsid protein comprises an amino acid sequence of a first member of a specific binding pair.

8. The method according to claim 7, wherein the first member of the specific binding pair is SpyTag.

9. The method according to claims 1-8, wherein the retargeting molecule is bound to a second cognate member of a specific binding pair.

10. The method according to claim 9, wherein the second cognate member of the specific binding pair is SpyCatcher.

11. The method according to claims 1-10, wherein the retargeting molecule is an Fc-containing protein.REGN 11547 12. The method according to claim 11, wherein the Fc-containing protein is a monoclonal antibody.

13. The method according to claim 11, wherein the Fc-containing protein is a monoclonal antibody fragment.

14. The method according to claim 11, wherein the Fc-containing protein is an Fc-fusion protein.

15. The method according to claims 1-11, wherein the ratio of AAV to retargeting molecule is 1:20 to 1:1000.

16. The method according to claim 15, wherein the ratio of AAV to retargeting molecule is 1:30 to 1:

300.

17. The method according to claim 16, wherein the ratio of AAV to retargeting molecule is about 1:

300.

18. The method according to claims 1-16, wherein the incubating step is about 4 to 72 hours long.

19. The method according to claims 1-18, wherein the (I) combining step further comprises an additive.

20. The method of claim 19, wherein the additive is isopropyl alcohol.REGN 11547 21. The method of claim 20, wherein the isopropyl alcohol is at a concentration above 5% v / v.

22. The method according to claim 21, wherein isopropyl alcohol is at a concentration of about 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, 20%, 20.5%, 21%, 21.5%, 22%, 22.5%, 23%, 23.5%, 24%, 24.5%, or 25% v / v.

23. The method of claim 22, wherein isopropyl alcohol is at a concentration of about 25% v / v.

24. The method according to claims 1-23, wherein the method achieves optimum conjugation.

25. The method according to claim 24, wherein the method achieves a level of conjugation of at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%.

26. The method according to claims 1-25, wherein the helper polynucleotide sequences encode adenovirus E4, adenovirus E2, and VA RNA.

27. The method of claim 2, wherein the ratio of the plasmids comprising AAV rep and cap genes and a polynucleotide sequence encoding a first member (3) of a specific binding pair to total plasmids comprising AAV rep and cap genes (2 andREGN 11547 3) is selected from the group consisting of 1 / 1 to 1 / 30; 1 / 4 to 1 / 13; 1 / 5 to 1 / 12; 1 / 6 to 1 / 11, 1 / 7 to 1 / 10.5 or 1 / 7.5 to 1 / 10.

28. A method of separating a covalently surface modified adeno- associated virus (AAV) species from unconjugated retargeting molecule species, wherein the method comprises the steps of (a) providing a sample comprising the covalently surface modified adeno- associated virus species produced using in vitro conjugation; (b) subjecting the covalently surface modified adeno-associated virus to anion exchange media using a buffering agent and a salt to separate the covalently surface modified AAV species from retargeting molecule species to provide at least one fraction that is enriched in covalently surface modified AAV species; and (c) harvesting the enriched fraction comprising covalently surface modified AAV species with a defined level of covalent surface modifications.

29. The method according to claim 28, wherein the buffering agent is selected from the group consisting of Bis-Tris-Propane, Bis-Tris, Tris, Glycine, Bicine, Tricine, Acetate, Borate, Citrate, Carbonate, Phosphate, Formate, Sulfate, Succinic acid, Sulfonic acid and variants thereof (for example, MES, PIPES, HEPES, CHES, CAPS, MMS, PBMS), Diethanolamine, and Imidazole, and the salt is selected from the group consisting of NaCl, KCl, MgCl2, CaCl2, NH4Cl, Na2SO4,REGN 11547 CaSO4, K2SO4, MgSO4, (NH4)2SO4, sodium citrate, and tetramethylammonium chloride (TMAC).

30. The method according to claims 28-29, wherein the anion exchange media is selected from the group consisting of CIM QA, CIM DEAE, PRIMA T, Capto Q, Capto Q ImpRes, CAPTO DEAE, POROS HQ, POROS XQ, POROS PI, POROS D, Fractogel EMD TMAE, Fractogel EMD DEAE, Nuvia Q, Nuvia HP-Q, Sartobind STIC PA, Sartobind Q, Natrix Q.

31. A method of separating a covalently surface modified adeno- associated virus (AAV) species from unconjugated retargeting molecule species, wherein the method comprises the steps of (a) providing a sample comprising the covalently surface modified adeno- associated virus species produced using an in vitro conjugation; (b) subjecting the covalently surface modified adeno-associated virus to cation exchange media using a buffering agent and a salt to separate the covalently surface modified AAV species from unconjugated retargeting molecule species to provide at least one fraction that is enriched in covalently surface modified AAV species; and (c) harvesting the enriched fraction comprising covalently surface modified AAV species.REGN 11547 32. The method according to claim 31, wherein the cation exchange media is selected from the group consisting of CIM SO3, Capto S, Capto S ImpRes, Capto S ImpAct, Capto SP, POROS HS, POROS XS, CM Sepharose, Nuvia S, Nuvia HR- Sartobind S, Natrix CH.

33. A method of separating a covalently surface modified adeno- associated virus (AAV) species from unconjugated retargeting molecule species, wherein the method comprises the steps of (a) providing a sample comprising the covalently surface modified adeno- associated virus species produced using an in vitro conjugation; (b) subjecting the covalently surface modified adeno-associated virus to multimodal ion exchange using a buffering agent and a salt and an inorganic salt for loading and a low salt and low pH buffer for elution in order to separate the covalently surface modified AAV species from unconjugated retargeting molecule species to provide a fraction that is enriched in covalently surface modified AAV species; and (c) harvesting at least one enriched fraction comprising covalently surface modified AAV species.

34. A method of separating a covalently surface modified adeno- associated virus (AAV) species from unconjugated retargeting molecule species, wherein the method comprises the steps ofREGN 11547 (a) providing a sample comprising the covalently surface modified adeno- associated virus species produced using an in vitro conjugation; (b) subjecting the covalently surface modified adeno-associated virus to multimodal binding and size separation bead chromatography using a multimodal buffer agent is selected from the group consisting of Bis-Tris-Propane, Bis-Tris, Tris, Glycine, Bicine, Tricine, Acetate, Borate, Citrate, Carbonate, Phosphate, Formate, Sulfate, Succinic acid, Sulfonic acid and variants thereof (for example, MES, PIPES, HEPES, CHES, CAPS, MMS, PBMS), Diethanolamine, and Imidazole, and the salt is selected from the group consisting of NaCl, KCl, MgCl2, CaCl2, NH4Cl, Na2SO4, CaSO4, K2SO4, MgSO4, (NH4)2SO4, sodium citrate, and tetramethylammonium chloride (TMAC) in order to separate the covalently surface modified AAV species from unconjugated retargeting molecule species to provide a fraction that is enriched in covalently surface modified AAV species; and (c) harvesting the enriched fraction comprising covalently surface modified AAV species.

35. The method according to claim 34, wherein beads of the multimodal binding and size separation bead chromatography are functionalized with an octyl amine that is hydrophobic and positively charged.

36. The method according to claim 35 wherein the beads comprise a matrix that provides size exclusion.REGN 11547 37. The method according to claims 34-36, where the beads are CaptoTMCore 400 or CaptoTMCore 700.

38. A method according to any of the above claims, wherein the method further comprises the use of single-pass tangential flow filtration.

39. A covalently surface modified adeno-associated virus (AAV) produced by any of the methods according to claims 1-27. 40 A covalently surface modified adeno-associated virus (AAV) purified by any of the methods according to claims 28-38.

41. A covalently surface modified adeno-associated virus (AAV) made according to an example selected from the group consisting of Example 15, Example 16, Example 17, Example 18, Example 19, Example 20, Example 21, Example 22, Example 23, Example 24, Example 25, Example 26, Example 27, Example 28, Example 29, Example 30 and Example 31.

42. A cell that produces a covalently surface modified adeno-associated virus (AAV) according to claim 41, wherein the cell comprises at least one sequence set forth in a example selected from the group consisting of examples selected from the group consisting of Example 32, Example 33, Example 34, Example 35, Example 36, Example 37, Example 38 and Example 39.REGN 11547 43. A covalently surface modified adeno-associated virus (AAV) produced by a cell according to claim 42.

44. A preparation comprising the covalently surface modified adeno- associated virus (AAV) according to any of the above claims.

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