Production and purification of covalently surface modified adeno-associated virus

Abstract

The present inventions provide covalently surface modified adeno-associated viruses can comprise gene of interest (GOIs) and advantageously can be targeted to certain cell and tissue types for preventative and therapeutic purposes. 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

135975-54620 (REGN 11546) PRODUCTION AND PURIFICATION OF COVALENTLY SURFACE MODIFIED ADENO-ASSOCIATED VIRUS This application claims priority to U.S. Application Ser. No.63 / 657,304, filed June 7, 2024; U.S. Application Ser. No.63 / 654,328, filed May 31, 2024; U.S. Application Ser. No.63 / 620,585, filed January 12, 2024; U.S. Application Ser. No. 63 / 620,002, filed January 11, 2024; and U.S. Application Ser. No.63 / 537,170, filed September 7, 2023. These applications are incorporated by reference in their entirety. FIELD OF THE INVENTIONS

[0001] Recombinant AAV of any serotype (rAAV) containing gene(s) of interest (GOI) are increasingly being used in preventative and therapeutic capacities, such as in vaccines and in gene therapy. The present inventions provide systems and methods for engineering adeno-associated virus (AAV) to produce 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, and improved approaches for purification. Covalently surface modified adeno-associated viruses can comprise a gene of interest (GOI) and advantageously can be targeted to certain cell and tissue types for preventative and therapeutic purposes, according to the inventions described herein. REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0002] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in135975-54620 (REGN 11546) its entirety. This .XML copy, created on August 27, 2024, is named “11546.xml” and is 400,620 bytes in size. The sequence listing contained in this .XML file is part of the specification and is incorporated by reference 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 and 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). Recombinant AAVs (rAAVs) containing genes of interest (GOIs) are increasingly being used in preventative and therapeutic capacities, such as in vaccines and in gene therapy. 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 2 ME149651883v.1135975-54620 (REGN 11546) codons and transcript splicing, three size-variant structural proteins referred to as VP1 (about 90kDa), VP2 (about 72kDa) and VP3 (about 60kDa). 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 VP2 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, BHK, human amniotic (for example, epithelial cells such as HAEpiC), CHO, HeLa and Sf9 lines. 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] Different AAV serotypes are known to preferentially transduce different tissues. Tissue specificity is limited, and AAV is known to preferentially transduce the liver, which can be a safety and efficacy concern. In view of these natural limitations, there exist needs to vary the targeting of rAAVs to control target specificity, including at least lessening the targeting of the liver. These goals were not met until the present inventions.

[0008] The production of covalently surface modified AAV requires the production of both the AAV vector and the retargeting surface modification. Producing the two entities separately requires separate production vessels and purification trains, which can be resource demanding and labor intensive. The present invention provides methods to simultaneously produce all necessary covalently surface modified AAV components within the same producing cell line, for example, in a suspension cultured fed-batch production bioreactor. A serum free suspension platform has been widely adopted in traditional biologics, and is 3 ME149651883v.1135975-54620 (REGN 11546) increasing in popularity for cell and gene therapy industry. It allows increased safety and consistency from batch to batch, and process scale up to 1 kL scale or greater.

[0009] Depth filtration is used for the purification of AAVs. In current manufacturing processes for rAAV, expensive endonuclease treatment is used prior to depth filtration to break down large amounts of chromatin post-cellular lysis. Without this treatment, traditional depth filters tend to foul rapidly, making clarification impractical without the use of excessive filter area. The present inventions provide for improvements in depth filtration to enhance the efficiency and lower costs of rAAV purification.

[0010] Purification of surface-modified AAVs also requires effective methods to capture surface-modified AAVs from the bioreactor harvest mixture. Challenges in affinity capture include difficulty in binding to the AAV surface epitopes in case of heavy conjugation, and difficulty in eluting the surface-modified AAV without destabilizing the molecule leading to aggregation, precipitation, and / or fragmentation. These difficulties often lead to an imbalance in the level of conjugation in the affinity capture load material relative to the eluate stream; that is, a loss of the some or all of the conjugated species during affinity capture due to preferential purification of the less conjugated / unconjugated AAVs. The goal of high-yield affinity capture of conjugated AAV species was not met until the present inventions. SUMMARY OF THE INVENTIONS

[0011] Many potential gene therapy applications would benefit from cell / tissue-specific retargeting and liver detargeting that is beyond current AAV vector control capabilities. The current inventions advantageously employ 4 ME149651883v.1135975-54620 (REGN 11546) covalently surface modified AAV for retargeting. Additionally, according to the inventions, mutations can be introduced into the AAV Cap proteins to detarget the liver.

[0012] Genetic components encoding for the covalently surface modified AAV can be introduced via transient transfection of six plasmids into cells, referred to as a “hexad transfection.” The AAV vector and surface modification components interact and form covalent bonds at the end of production after cell lysis. The plasmid concentration and ratios used during the hexad transfection are optimized using multivariate design of experiment (DOE) approaches to achieve high AAV titer and fast conjugation reaction rates. The hexad transfection provides all the necessary components, namely AAV rep and cap functional and structural proteins, helper polynucleotide sequences, retargeting molecule components (such as antibody heavy chain and light chains), sequences encoding specific binding pairs, genes of interest, and detargeting mutations (if desired). One or more retargeting molecules can bind to one or more targets.

[0013] 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, improved affinity capture and improved ionic exchange chromatography. Depth filtration can be employed with lowered or without endonuclease treatment. Affinity capture with optimized loading and elution conditions to achieve high yield without loss of heavily conjugated species.

[0014] Modulation of the level of surface modification is demonstrated through the introduction of different plasmid ratios during the transient hexad 5 ME149651883v.1135975-54620 (REGN 11546) transfection. Optimal level of AAV surface modification is identified to improve tissue specificity and transduction efficiency.

[0015] The inventions provide methods of producing a covalently surface modified adeno-associated viruses (AAV), wherein the methods comprise the steps of: (A) transfecting a cell with: (i) a plasmid comprising a gene of interest flanked by AAV inverted terminal repeats; (ii) a plasmid comprising an AAV rep gene and an AAV cap gene; (iii) a plasmid AAV rep and cap genes and a polynucleotide sequence encoding a first member of a specific binding pair (“first member”); (iv) a plasmid comprising one or more helper polynucleotide sequences; (v) a plasmid comprising a polynucleotide sequence encoding a first portion of a retargeting molecule and a polynucleotide encoding a second cognate member of the specific binding pair (“second member”); and (vi) a plasmid comprising a polynucleotide encoding a second portion of a retargeting molecule; (B) culturing the transfected cell to allow expression of plasmids (i) to (vi) and assembly of components to form covalently surface modified adeno-associated virus; and (C) harvesting the covalently surface modified adeno-associated virus. The cells can be mammalian cells, such as human and rodent cells. Insect cells also can be used. The cells can be a mammalian cell, such as a human cell, for example HEK293 (both adherent and suspension), HeLa and amniotic cells.

[0016] The retargeting molecules can be Fc-containing proteins having, such as monoclonal antibodies, which have portions. For example, the first portion of the retargeting molecule can be an antibody heavy chain, and the second portion of the retargeting molecule is an antibody light chain. Optionally, the first 6 ME149651883v.1135975-54620 (REGN 11546) portion of the retargeting molecule can be an antibody light chain, and the second portion of the retargeting molecule can be an antibody heavy chain.

[0017] The specific binding pair can be SpyTag-SpyCatcher, for example. In such a case, the first member of a specific binding pair can be a SpyTag peptide For example, the polynucleotide encoding the SpyTag peptide can be inserted into the AAV cap gene to encode recombinant capsid proteins. The second cognate member of the specific binding pair can be a SpyCatcher protein, for example.

[0018] The methods also can include having the cells express at least one recombinant capsid protein selected from the group consisting of a recombinant VP1 protein comprising a SpyTag amino acid sequence, a recombinant VP2 protein comprising a SpyTag amino acid sequence, and a recombinant VP3 protein comprising a SpyTag amino acid sequence. Additionally, the VP1 protein can be mutated in a galactose binding domain to detarget liver cells, such as a detargeting mutation is at least one selected from the group consisting of N272A and W503A, for example. Other detargeting mutations can be employed as well, including but not limited to those disclosed herein.

[0019] The methods also provide for a covalently surface modified adeno-associated virus comprising a plurality of first members bound to second cognate members, wherein the second cognate members also are bound to retargeting molecules; and a gene of interest. The first members can be SpyTag peptides and second cognate members at SpyCatcher proteins. The retargeting molecules can be antibodies, antibody fragments or antibody derivatives.

[0020] Covalently surface modified adeno-associated viruses can be provided according to the inventions by having the cells express at least one 7 ME149651883v.1135975-54620 (REGN 11546) recombinant capsid protein selected from the group consisting of a recombinant VP1 protein comprising a SpyTag amino acid sequence, a recombinant VP2 protein comprising a SpyTag amino acid sequence, and a recombinant VP3 protein comprising a SpyTag amino acid sequence. Additionally, the VP1 protein can be mutated in a galactose binding domain to detarget liver cells, such as a detargeting mutation is at least one selected from the group consisting of N272A and W503A, including but not limited to those disclosed herein.

[0021] The inventions further provide covalently surface modified adeno-associated virus comprising a plurality of first members bound to second cognate members, wherein the second cognate members also are bound to retargeting molecules, and a gene of interest, wherein the covalently surface modified adeno-associated virus is made by a method comprising the steps of: A) transfecting a cell with: (i) a plasmid comprising a gene of interest flanked by AAV inverted terminal repeats; (ii) a plasmid comprising an AAV rep gene and an AAV cap gene; (iii) a plasmid comprising AAV rep and cap genes and a polynucleotide sequence encoding a first member of a specific binding pair; (iv) a plasmid comprising one or more helper polynucleotide sequences; (v) a plasmid comprising a polynucleotide sequence encoding a first portion of a retargeting molecule and a polynucleotide encoding a second cognate member of the specific binding pair; and (vi) a plasmid comprising a polynucleotide encoding a second portion of a retargeting molecule; (B) culturing the transfected cell to allow expression of plasmids (i) to (vi) and assembly of proteins to form covalently surface modified adeno-associated virus; and (C) harvesting the covalently surface modified adeno- associated virus. 8 ME149651883v.1135975-54620 (REGN 11546)

[0022] The retargeting molecules can be Fc-containing proteins having, such as monoclonal antibodies, which have portions. For example, the first portion of the retargeting molecule can be an antibody heavy chain, and the second portion of the retargeting molecule is an antibody light chain. Optionally, the first portion of the retargeting molecule can be an antibody light chain, and the second portion of the retargeting molecule can be an antibody heavy chain.

[0023] The specific binding pair can be SpyTag-SpyCatcher, for example. In such a case, the first member of a specific binding pair can be a SpyTag peptide For example, the polynucleotide encoding the SpyTag peptide can be inserted into the AAV cap gene to encode recombinant capsid proteins. The second cognate member of the specific binding pair can be a SpyCatcher protein, for example.

[0024] The cells can be a mammalian cell, such as a human cell, for example HEK293 (both adherent and suspension), HeLa and amniotic cells.

[0025] The covalently surface modified adeno-associated viruses can comprise a plurality of first members bound to second cognate members, wherein the second cognate members also are bound to retargeting molecules; and a gene of interest. The first members can be SpyTag peptides and second cognate members at SpyCatcher proteins. The retargeting molecules can be antibodies, antibody fragments or antibody derivatives. During production, covalently surface modified adeno-associated viruses can be subjected to the step of (D) purifying the covalently surface modified adeno-associated virus using depth filtration. The depth filtration can be performed without an endonuclease. During production, covalently surface modified adeno-associated viruses can be subjected to the step of comprise 9 ME149651883v.1135975-54620 (REGN 11546) the step of (E) purifying the covalently surface modified adeno-associated viruses using affinity chromatography.

[0026] The covalently surface modified adeno-associated virus can comprise a plurality of first members bound to second cognate members, wherein the second cognate members also are bound to retargeting molecules; and a gene of interest. The first members can be SpyTag peptides and second cognate members can be SpyCatcher proteins. The retargeting molecules can be antibodies, antibody fragments or antibody derivatives. The helper polynucleotide sequences can encode adenovirus E4, adenovirus E2, and VA RNA, for example, 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.

[0027] The inventions also provide methods of screening retargeting molecules for production of a covalently surface modified adeno-associated virus species, wherein the method comprises the steps of: (I) providing (A) a first plurality of nucleic acids encoding retargeting molecules that are different from one another and (B) a second plurality of DNA barcodes that are different from one another, wherein each individual DNA barcode of the second plurality is assigned to an individual covalently surface modified adeno-associated virus comprising a retargeting molecule of the first plurality for creating a covalently surface modified adeno-associated virus species; (II) producing covalently surface modified adeno- associated virus species by (A) transfecting a cell with: (i) a plasmid comprising a polynucleotide that comprises a DNA barcode, wherein the polynucleotide that comprises the DNA barcode is flanked by AAV inverted terminal repeats; (ii) a plasmid comprising an AAV rep gene and an AAV cap gene; (iii) a plasmid 10 ME149651883v.1135975-54620 (REGN 11546) comprising AAV rep and cap genes and a polynucleotide sequence encoding a first member of a specific binding pair; (iv) a plasmid comprising one or more helper polynucleotide sequences; (v) a plasmid comprising a polynucleotide sequence encoding a first portion of a retargeting molecule of the first plurality and a polynucleotide encoding a second cognate member of the specific binding pair; and (vi) a plasmid comprising a polynucleotide encoding a second portion of a retargeting molecule of the first plurality; (B) culturing the transfected cell to allow expression of plasmids (i) to (vi) and assembly of components to form a covalently surface modified adeno-associated virus species; (C) harvesting the covalently surface modified adeno-associated virus species, wherein the covalently surface modified adeno-associated virus species will become part of a covalently surface modified adeno-associated virus library; (D) repeating steps (A) to (C) to produce each covalently surface modified adeno-associated virus species to form the covalently surface modified adeno-associated viruses library; and (E) detecting each DNA barcode of the second plurality in order to screen each species to determine a property.

[0028] The AAV library produced can be used to dose model animals (for example, rodents or non-human primates), the target tissue is then extracted and sequenced to quantify enrichment of the DNA barcodes. The distribution of barcodes in different body parts / tissues can be assessed to see which covalently surface modified adeno-associated virus species are best at delivering GOI payloads (containing barcodes) to a specific body part, preventing the delivery of excess GOI payloads to unwanted body parts such as the liver and / or characterizing genomic titer. 11 ME149651883v.1135975-54620 (REGN 11546)

[0029] The retargeting molecule can be an Fc-containing protein, such as a monoclonal antibody, including multispecific antibodies, such as bispecific and trispecific antibodies. The Fc-containing protein can be an Fc-fusion protein, such as a receptor- Fc-fusion protein, which includes trap proteins. The retargeting molecule also can be selected from the group consisting of an fab, f(ab’), f(Ab’)2, a single chain antibody and a mini-trap protein, for example. The helper polynucleotide sequences can encode adenovirus E4, adenovirus E2, and VA RNA, for example. 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.

[0030] The inventions further provide methods of screening covalently surface modified adeno-associated virus species, wherein the method comprises the steps of: (I) providing (A) a first plurality of nucleic acids encoding a component of a covalently surface modified adeno-associated virus species (component), wherein each component of the first plurality are different from one another and (B) a second plurality of DNA barcodes that are different from one another, wherein each individual DNA barcode of the second plurality is assigned to an individual component of the first plurality for creating a covalently surface modified adeno- associated virus species; (II) producing covalently surface modified adeno- associated virus species by (A) transfecting a cell with: (i) a plasmid comprising a polynucleotide that comprises a DNA barcode, wherein the polynucleotide that comprises the DNA barcode is flanked by AAV inverted terminal repeats; (ii) a plasmid comprising an AAV rep gene and an AAV cap gene; (iii) a plasmid comprising AAV rep and cap genes and a polynucleotide sequence encoding a first 12 ME149651883v.1135975-54620 (REGN 11546) member of a specific binding pair; (iv) a plasmid comprising one or more helper polynucleotide sequences; (v) a plasmid comprising a polynucleotide sequence encoding a first portion of a retargeting molecule and a polynucleotide encoding a second cognate member of the specific binding pair; and (vi) a plasmid comprising a polynucleotide encoding a second portion of a retargeting molecule; (B) culturing the transfected cell to allow expression of plasmids (i) to (vi) and assembly of components to form a covalently surface modified adeno-associated virus species; (C) harvesting the covalently surface modified adeno-associated virus species, wherein the covalently surface modified adeno-associated virus species will become part of a covalently surface modified adeno-associated virus library; (D) repeating steps (A) to (C) to produce each covalently surface modified adeno- associated virus species to form the covalently surface modified adeno-associated viruses library; and (E) detecting each DNA barcode of the second plurality in order to screen each species to determine a property. The component can be selected from the group consisting of a retargeting molecule, an AAV cap gene, an AAV rep gene, an ITR, a helper polynucleotide sequence(s), a first member of a specific binding pair, and a second cognate member of a specific binding pair. The helper sequences can encode adenovirus E4, adenovirus E2, and VA RNA, for example. 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.

[0031] The inventions further comprise covalently surface modified adeno-associated viruses (AAV) produced and / or purified by the methods. 13 ME149651883v.1135975-54620 (REGN 11546)

[0032] The inventions also provide AAV preparations produced by any of the above the methods, and drug products made from the AAV preparations.

[0033] 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, rh10, rh39, rh43, rh74,

[0034] 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

[0035] Figure 1 schematically depicts a covalently surface modified AAV bound to a target cells. In Figure 1, the covalently surface modified AAV comprises a plurality of SpyTag (SpyT) peptide sequences on the capsid surface. The SpyTag amino acid sequences can bind to the SpyCatcher (SpyC) protein. The SpyCatcher protein and retargeting molecule are expressed as a fusion protein, and are later allowed to conjugate with the SpyTag peptide in the AAV capsid. For example, where the retargeting molecule is a monoclonal antibody (mAb), the SpyCatcher peptide sequences a preferably fused at the Fc portion of the mAb. The mAb at the Fv portion recognizes a target on the surface of a target cell, such as a tissue specific surface antigen, to be transduced by the AAV. The AAV can further comprise a GOI (not shown).

[0036] Figures 2A and 2B schematically depict and describe hexad transfection systems and methods for producing covalently surface modified AAV in a eukaryotic cell, such as HEK 293F. As described in Figure 2A, the hexad 14 ME149651883v.1135975-54620 (REGN 11546) transfection system utilizes six plasmids (schematically depicted) 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 is used as an example) 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 if 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); (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); (v) pHC-SpyC - a plasmid comprising polynucleotides encoding a mAb heavy chain sequence and a SpyCatcher protein sequence (a second cognate member if a specific binding pair), and optionally a first selection marker gene, such as an antibiotic resistance gene (for example, ampicillin resistance). This is an exemplary plasmid pRC-second cognate member (pRC-SCM); and (vi) pLC - a plasmid comprising a polynucleotide encoding a mAB light chain sequence, and optionally a first selection marker gene, such as an antibiotic resistance gene (for example, ampicillin resistance). Promoters that are functional in eukaryotic cells, such as AAV P5, CMV, lac, CAG, CAGG, and SV40 promoters can be used to initiate transcription, but are not shown. Additionally, internal 15 ME149651883v.1135975-54620 (REGN 11546) ribosome entry site (IRES), recombinase recognition sites (RRS), ehancers and operators optionally can be included, but are not shown.

[0037] Figure 2B 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 a first member of a specific binding pair, here the SpyTag 13 amino acid peptide sequence as an example, 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.

[0038] Figure 3 schematically depicts a first Design of Experiment for initial optimization of three plasmid transfection prior to the introduction of pRC- SpyT and mAb HC and LC plasmids

[0039] Figure 4 schematically depicts a second Design of Experiment to investigate the impact of bioreactor pH and agitation speed at three different phases of production and the transition timings between phases on vector titer and quality

[0040] Figures 5A-5C schematically depict a comparison of covalently surface modified AAVs with different detargeting mutations and varying levels of antibody conjugation. Figure 5A schematically describes and depicts AAVs as follows: Lane 1 – AAV without a detargeting mutation, a SpyTag insertion nor mAb; Lane 2 – AAV without a SpyTag insertion nor mAb conjugation, but has a detargeting mutation (N272A); Lane 3 – AAV without an mAb, but has a SpyTag insertion at some VP proteins and detargeting mutations (N272A or W503A). The fraction of the pRC-SpyT plasmid 16 ME149651883v.1135975-54620 (REGN 11546) used during transfection is 1 / 8 of the total RC plasmids. Lanes 5 to 10 – AAV with a SpyTag insertion at some VP proteins, a detargeting mutation (N272A) and an mAb. The fraction of the pRC-SpyT plasmid used during transfection is 1 / 4, 1 / 5, 1 / 6.7, 1 / 8, 1 / 10 and 1 / 20 of the total RC plasmid in the direction of Lane 5 to Lane 10; and Lanes 11 and 12 - AAV with a SpyTag insertion at some VP proteins, a detargeting mutation (W503A) and an mAb. The fraction of the pRC-SpyT plasmid used during transfection is 1 / 4 of the total RC plasmid in Lane 11 and 1 / 8 in Lane 12.

[0041] Figure 5B shows antibody conjugated capsids only in lanes 5 to 12, and unconjugated capsids in lanes 1 to 12.

[0042] Figure 5C uses green fluorescent protein labeled calcium voltage-gated channel auxiliary subunit gamma 1 (CACNG1) in a transduction assay. CACNG1 was bound by the mAb of the covalently surface modified AAV. Figure 5C shows the infectivity results of the covalently surface modified (in this case anti-CACNG1 mAb conjugated) AAV with varying levels of antibody conjugation. CACNG1 positive HEK293 cells was dosed with the respective AAV virus preps in each lane, and the intensity of the fluorescent signal indicates infectivity of the AAV virus prep. Lower SpyTag plasmid fractions corresponded to higher infectivity.

[0043] Figures 6A-6C schematically depict the effect of the SpyCatcher-mAb titer on the conjugation efficiency of the AAV. Fractions at 1 / 5 and 1 / 8 of SpyT-RepCap plasmid (pRC-SpyT) were tested. Figure 6A is a bar graph depicting mAb titers affected by the concentration mAb plasmid DNA used during hexad transfection. Figure 6B is a bar graph of genomic titers showing that SpyTag AAV titer was not affected by SpyCatcher mAb titer within the range studied. Figure 17 ME149651883v.1135975-54620 (REGN 11546) 6C shows gel electrophoresis of capsids. VP1, VP2 and VP3 of unmodified capsids ran further on the gel than antibody-modified capsids. The data show that increasing the SpyCatcher mAb plasmid DNA led to a more efficient conjugation of AAV.

[0044] Figure 7 is a bar graph schematically depicting data relating to the selection of SpyTag fraction and SpyCatcher mAb DNA concentration. Spy Tag fractions ranged from 1 / 5 to 1 / 30. The percent change in mAb DNA concentration ranged 0% to 200% Lane 5, which had a 1 / 10 SpyTag fraction and a 100% increase in mAb DNA (corresponding to mAb plasmid DNA concentration of 0.56 µg / mL of cell culture during hexad transfection) showed the highest transduction efficiency. The signal was generated using a green fluorescent reporter gene.

[0045] Figure 8 schematically depicts data from a low pH hold used to increase the efficiency of the conjugation reaction.

[0046] Figures 9A and 9B schematically depict data on filtrate quality analytics for Harvest RC (HRC) and C0SP filtration of rAAV8 (3 x 106cells / ml) and rAAV9 (1.5 x 106cells / ml) . Figure 9A is a bar graph for AAV8 depicting in order bars for bioreactor lysate (white bars), filtrate pool where transmembrane pressure (TMP) is 5 psi (light gray bars), and filtrate pool where TMP is 15 psi (black bars). HRC (solid bars) and C0SP (a standard filter used as a control) (hatched and labeled bars) are filtration trains with different endonuclease and salt conditions in the load. Figure 9B is a bar graph for AAV9 depicting in order bars for bioreactor lysate (white bars), filtrate pool where TMP is 5 psi (light gray bars), and filtrate pool where TMP is 15 psi (black bars). HRC (solid bars) are filtration trains with different endonuclease and salt conditions in the load. In Figure 9A, significant breakthrough of HCDNA into the filtrate was observed for C0SP at the “C0SP Negative Control”, 18 ME149651883v.1135975-54620 (REGN 11546) and for HRC at 250 mM NaCl and 0 U / ml endonuclease (dark gray bars). In Figure 9B, significant breakthrough of HCDNA into the filtrate was observed for HRC at 250 mM NaCl and 0 U / ml endonuclease (dark gray bars) and 250 mM NaCl and 10 U / ml endonuclease (dark gray bars).

[0047] Figures 10A-10F schematically depict data for depth filtration treatment at various salt and endonuclease conditions. Figures 10A-10C provide data from testing of AAV8 and Figures 10D-10F provide data from testing of AAV9.

[0048] Figure 11 schematically depicts data set forth in Figures 10A- 10F. Throughputs for endonuclease at the 10 U / ml condition were midway between the 0 U / ml and 100 U / ml endonuclease conditions.

[0049] Figure 12 schematically depicts data set forth in Figures 10A- 10F regarding the effects of salt.

[0050] Figure 13 schematically depicts data showing that salt addition reduces load turbidity and increases throughput at higher pressures.

[0051] Figure 14 schematically depicts data obtained from Figures 10A- 10F showing that a combined cake-fiber fouling model is a good representation for the mechanism of Harvest RC filtration (depth filtration).

[0052] Figures 15A-15C schematically depict data obtained from Figures 10A-10F. Cake formation parameters (KC) vs NaCl (mM) added with 0 U / ml endonuclease (Figure 15A), 10 U / ml endonuclease (Figure 15B), and 100 U / ml endonuclease (Figure 15C).

[0053] Figure 16 schematically depicts three productions performed using the suspension miniature bioreactor system, and the expected transduction assay luciferase signal for the three productions alone and the pooled material with or without soluble SpyTag quenching. Triangles attached to an AAV represent 19 ME149651883v.1135975-54620 (REGN 11546) SpyTag peptide inserted into the VP proteins. Triangles not attached to an AAV represent soluble SpyTag peptide.

[0054] Figure 17 schematically depicts the luciferase signal from the transduction assay when cells are dosed with the respective AAV products at a series of multiplicity of infection (MOI) levels. The 8D3 is a particular mouse-TfR Fab construct.

[0055] Figure 18 schematically depicts data using a POROS CaptureSelect AAV9 resin to capture AAV9-SpyT-SpyC-mAb. Monoclonal antibodies against CACNG1, ASGR1 and Fel d1 were used as retargeting molecules. Less conjugated AAV9 (1 / 20 and 1 / 30) achieved higher yields.

[0056] Figure 19 schematically depicts and compares various buffers used for affinity capture of an AAV-SpyT-SpyC-Tfr Fab. A Poros CaptureSelect AAV9 column loaded with 1013to 1015capsids per milliliter was employed. The yield of the affinity capture step was heavily dependent on the choice of elution buffer. Low yields were achieved in many elution buffers along with loss of the heaviest conjugated species, whereas certain buffers were able to achieve yield > 90% along with successful capture of the heavily-conjugated AAV species.

[0057] Figure 20 schematically illustrates a two-step affinity capture protocol utilizing (i) affinity to AAV surface epitopes and (ii) affinity to the antibody surface epitopes. The two affinity capture steps can be employed in any order. Resins used for affinity capture via AAV surface epitopes can comprise POROS CaptureSelect AAVX, POROS CaptureSelect AAV8, POROS CaptureSelect AAV9, Capto AVB, AVB Sepharose, Avipure AAV2, Avipure AAV8, Avipure AAV9, among others. Resins used for affinity capture via antibody surface epitopes can comprise mAbSelectSuRe, mAbSelect PrimaA, Capto L, mAbSelect VL, KappaSelect, among 20 ME149651883v.1135975-54620 (REGN 11546) others. The two-step capture protocol allows effective removal of unconjugated antibody as well as unconjugated AAV, resulting in a pool with only conjugated AAV. Pools include eluents.

[0058] Figure 21 schematically depicts a purification train.

[0059] Figure 22 schematically depicts data showing that using the hexad transfection production process to create a library comprising unique covalently surface modified AA species. The average vector genome titer obtained from the bioreactor lysate was 1.8 x 1011vg / ml. Unique DNA barcodes were employed as a biomarker encoded inside the AAV vector, and was matched to a Fab candidate (a type of retargeting molecule) conjugated to the outside of the AAV vector. The library contained species that exhibited various genomic titers, which is a measurement of production.

[0060] Figure 23 schematically 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.

[0061] Figure 24A 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 24B is a graph schematically comparing 21 ME149651883v.1135975-54620 (REGN 11546) 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.

[0062] Figure 25 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.

[0063] Figure 26 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.

[0064] Figure 27 is a graph schematically 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 26 with a feed comprising an exemplary AAV, here AAV9 comprising a SpyTag insert.

[0065] Figure 28 schematically depicts data from a 5-in-series configuration according to Figure 26 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 22 ME149651883v.1135975-54620 (REGN 11546) 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 26 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.

[0066] Figure 29A schematically depicts a design space model based on Figures 27 and 28 using the 5-in-series configuration of Figure 26. Here, the process target was 35 LMH, and the intersecting lines indicate a VCF of 8 and a TMP of 10psi. An exemplary acceptable zone would be a VCF of 6-10 and a TMP of 7.5 to 12.5 psi. Figure 29B 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

[0067] Figure 30 schematically 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 exemplary AAV9 contained an average of 6 SpyTag peptids per capsid. Capsid titer in retenate (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.

[0068] Figure 31 is a graph schematically 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 23 ME149651883v.1135975-54620 (REGN 11546) 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 of 8x.

[0069] Figure 32 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.

[0070] Figure 33 is a graph schematically 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.

[0071] Figure 34 is a graph schematically 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.

[0072] Figure 35 schematically depicts and 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 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. 24 ME149651883v.1135975-54620 (REGN 11546)

[0073] Figure 36 schematically depicts the overall pilot scale process, and is similar to parts of the production process of Figure 23.

[0074] Figure 37 schematically depicts and 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 (2.7 to 5.0 minutes).

[0075] Figure 38 schematically 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. DETAILED DESCRIPTION OF THE INVENTIONS Definitions

[0076] 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 this invention belongs.

[0077] 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 25 ME149651883v.1135975-54620 (REGN 11546) 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.

[0078] “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, and 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).

[0079] 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 26 ME149651883v.1135975-54620 (REGN 11546) 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.

[0080] The phrase "bispecific antibody" includes an antibody capable of selectively binding two or more epitopes. Bispecific antibodies generally comprise two different heavy chains, with each heavy chain specifically binding a different epitope -- 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, or 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. A typical 27 ME149651883v.1135975-54620 (REGN 11546) 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.

[0081] The term “components” refers to any constituent molecules needed to produce a covalently surface modified adeno-associated viruses and include, but are not limited to promoters, polyadenylation signals, transgenes, genes encoding retargeting molecules, AAV cap genes, AAV rep genes, ITRs, helper polynucleotide sequence(s), genes encoding a first member of a specific binding pair and a second cognate member of a specific binding pair, as well as peptides encoded by the genes and sequences. Optional sequences include detargeting mutation sequences, IRESs, RRSs, introns, operators and enhancers.

[0082] The phrase “assembly of components” refers to peptide 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.

[0083] The phrase “DNA barcode” refers to types of unique nucleotide sequences that can be used for identification purposes. DNA barcodes typically 28 ME149651883v.1135975-54620 (REGN 11546) contain the same amount of base pairs (for example, 32 base pair pairs), but each type will have an unique sequence, and are commercially available. The DNA barcodes can include terminal single stranded hairpins. Exemplary DNA barcode sequences are disclosed in Example 34, which sets forth one strand of the unique basepairs.

[0084] 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, and 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.

[0085] 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 29 ME149651883v.1135975-54620 (REGN 11546) 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.

[0086] “Recombinase recognition sites” (RRS), also known as “heterospecific recombination sites,” are used in recombinase mediated cassette exchange (RMCE). Cre / Lox Dre / Rox, VCre / 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, lox 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.

[0087] 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).

[0088] “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 30 ME149651883v.1135975-54620 (REGN 11546) 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.

[0089] “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).

[0090] 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 proteins 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.

[0091] “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 31 ME149651883v.1135975-54620 (REGN 11546) 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 create charge variation.

[0092] "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 proteins 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 embodiments comprises a hinge region followed by a CH2 and CH3 domain of an immunoglobulin. In some embodiments, 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, 32 ME149651883v.1135975-54620 (REGN 11546) 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.

[0093] “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”. The 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.

[0094] “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.

[0095] 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.” 33 ME149651883v.1135975-54620 (REGN 11546)

[0096] A “nucleotide of interest” includes GOIs and sequences encoding non-translated RNAs / non-coding RNAs (such as, but not limited to, antisense RNA, small interfering RNA, micro RNA, catalytic RNA and ribozymes). NOIs and GOIs also can be referred to as “payloads.”

[0097] “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), 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.

[0098] “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.

[0099] 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 34 ME149651883v.1135975-54620 (REGN 11546) 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.

[0100] “Detargeting” refers to reducing or abolishing AAV natural preferential transduction by mutating Cap proteins. For example, mutations in the galactose binding domain of VP1 assist in detargeting the liver. These mutations are optional and can be referred to as “detargeting mutations,” and are discussed herein in greater detail.

[0101] 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 other AAVs are as follows: AAV1 – N500E; AAV2 – R585A and R588A; 35 ME149651883v.1135975-54620 (REGN 11546) AAV5 – T571S; AAV6 – N500E, K531A and K531E. These and others are set forth in the chart below:Other mutations are available in publications and otherwise available, and can be used according to the inventions. 36 ME149651883v.1135975-54620 (REGN 11546)

[0102] "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.

[0103] 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 is bound to a polypeptide that is part of a specific binding pair. For example, a retargeting molecule could be bound to SpyCatcher 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, IgG-ScFv, IgG-dab, ScFV-Fc-ScFV, trispecifics) , Fc-fusion proteins, receptor-Fc fusion proteins, trap proteins can be useful as retargeting molecules. Mini-trap proteins also can be useful as retargeting molecules.

[0104] All human and non-human antibody classes can be used as retargeting molecules. IgA, IgD IgE, IgG and IgM 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 37 ME149651883v.1135975-54620 (REGN 11546) 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.

[0105] "Specific binding pair," "protein:protein binding pair" and the like includes 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.

[0106] 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 occur 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 38 ME149651883v.1135975-54620 (REGN 11546) 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.

[0107] “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 an 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 39 ME149651883v.1135975-54620 (REGN 11546) 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.

[0108] “Selectable” or “selection” marker proteins include proteins conferring certain traits, including but not limited to drug resistance or other selective 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 40 ME149651883v.1135975-54620 (REGN 11546) 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.

[0109] 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, 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 41 ME149651883v.1135975-54620 (REGN 11546) 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- 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 42 ME149651883v.1135975-54620 (REGN 11546) ectonucleoside triphosphate diphosphohydrolase 3 (hENTPD3). Other targets can be selected by the person skilled in the art. See WO 2019 / 006046.

[0110] 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. Description

[0111] The inventions advantageously employ one or more "specific binding pairs," also referred to as "protein:protein binding pairs." An exemplary system 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 is about 15 kD in size. 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.

[0112] Figure 1 contains a schematic depiction of a covalently surface modified AAV bound to a target cells. In Figure 1, the AAV comprises a plurality of 43 ME149651883v.1135975-54620 (REGN 11546) SpyTag amino acid sequences on the capsid surface. The SpyTag sequences can bind to the SpyCatcher protein. The SpyCatcher protein is expressed as a fusion protein with a retargeting molecule, here a monoclonal antibody (mAb) in Figure 1. Preferably the SpyCatcher protein is fused to the heavy chain at the Fc portion of the mAb. The mAb at the Fv portion recognizes a target on the surface of a target cell to be transduced by the AAV. The AAV can further comprise a GOI (not shown) to be expressed once the covalently surface modified AAV.

[0113] Figures 2A and 2B depict and describe hexad transfection systems and methods for producing covalently surface modified AAV in a eukaryotic cell, such as HEK 293. As described in Figure 2A, the hexad transfection system utilizes six plasmids (schematically depicted) as follows: (i) pGOI – a plasmid comprising a GOI (for example, a transgene) flanked by two AAV inverted terminal repeats (ITRs), and optionally a first selection marker gene, such as an antibiotic resistance gene (for example, ampicillin resistance); (ii) pRC - a plasmid comprising AAV (here AAV9 for example) rep and cap genes, and optionally a first 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 first selection marker gene, such as an antibiotic resistance gene (for example, ampicillin resistance). This is an exemplary plasmid pRC-first member (pRC-FM); (iv) pHELP - a plasmid comprising adenovirus helper genes E4 and E2, and VA RNA , (exemplary helper genes), and optionally a first selection marker gene, such as an antibiotic resistance gene (for example, ampicillin resistance); 44 ME149651883v.1135975-54620 (REGN 11546) (v) pHC-SpyC - a plasmid comprising polynucleotides encoding a mAb heavy chain sequence and a SpyCatcher protein sequence (a second cognate member of a specific binding pair), and optionally a first selection marker gene, such as an antibiotic resistance gene (for example, ampicillin resistance). This is an exemplary plasmid pRC-second cognate member (pRC-SCM); and (vi) pLC - a plasmid comprising a polynucleotide encoding a mAB light chain sequence, and optionally a first selection marker gene, such as an antibiotic resistance gene (for example, ampicillin resistance). The following chart provides a helpful summation of this example:45 ME149651883v.1135975-54620 (REGN 11546)

[0114] Transfection is preferably undertaken in mammalian cells, preferably human cell lines. Human cell lines include amniotic cells (such as Human Amniotic Epithelial cells), Hela cells, Per.C6 cells and HEK 293 cells. Examples of HEK 293 cells include, but are not limited, to HEK 293, HEK 293A, HEK 293E, HEK 293F, HEK 293FT, HEK 293FTM, HEK 293H, HEK 293MSR, HEK 293S, HEK 293SG, HEK 293SGGD, HEK 293T and mutants and variants thereof. Rodent cells and insect cells also can be used.

[0115] 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 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.

[0116] Figure 2B schematically depicts at the top a pRC-SpyT plasmid comprising the rep and cap genes, 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. 46 ME149651883v.1135975-54620 (REGN 11546)

[0117] Covalently surface modified recombinant AAV can be produced using any AAV serotype, for example, AAV1, AAV2,), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV rh10, AAV rh39, AAV rh43, and AAV rh74, and any other variants AAVs (for example, AAV27m8 or AAV2quad(Y-F), can be modified with genes of interest. Recombinant AAV capsids with modified viral capsid proteins to permit retargeting of AAV are disclosed in WO 2019 / 006046.

[0118] Modified capsid protein approaches according to the inventions utilize a first member and a second cognate member of a specific binding pair, which first member and second cognate member specifically interact to form a chemical, preferably covalent, bond. The first member, when displayed on a capsid protein, acts as a scaffold for any retargeting molecule (often referred to as a targeting ligand, see Yan et al., Pharmaceutics 202416, 248) fused to the second cognate member, but upon binding of the first member and second cognate member, an isopeptide bond forms, and the recombinant viral particle acts as a targeting vector. The covalently surface modified AAVs also can comprise GOIs and ITRs.

[0119] The second cognate member can be operably linked to a retargeting molecule. The first member can be flanked by a first and / or second linker that link(s) the first member to the capsid protein, and wherein the first and / or second linker is each independently at least one amino acid in length. The first and second linker can be identical or non-identical.

[0120] 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 47 ME149651883v.1135975-54620 (REGN 11546) 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, 48 ME149651883v.1135975-54620 (REGN 11546) 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.

[0121] 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.

[0122] SpyTag002:SpyCatcher002 system is described in Keeble et al (2017) Angew Chem Int Ed Engl 56: 16521-25. See WO 2019 / 006046.

[0123] SpyTag003:Spay Catcher003 also has been created. Spy Tag002:SpyCatcher002 and SpyTag003:SpyCatcher003 are different iterations of Spy Tag:Spy Catcher.

[0124] 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 49 ME149651883v.1135975-54620 (REGN 11546) between the complementary proteins. Veggiani (2016)), supra. See WO 2019 / 006046.

[0125] 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.

[0126] 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.

[0127] The present inventions are amenable for production in mammalian cell culture. Exemplary rodent cell lines are CHO, Per.C6 cells, Sp2 / 0 cells, 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, a preferred human cell line, include, but are not limited, to HEK293, HEK293A, HEK293E, HEK293F, HEK293FT, HEK293FTM, HEK293H, HEK293MSR, HEK293S, HEK293SG, HEK293SGGD, HEK293T and mutants and variants thereof. 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.

[0128] Covalently surface modified AAV vectors can be produced using a suspension cultured HEK293 derived cell line, such as HEK293F, using a 50 ME149651883v.1135975-54620 (REGN 11546) commercial media CTS LV-MAX (Thermo Fisher Scientific). These viral vectors have been successfully produced in fed-batch bioreactors at various scales from 200mL to 50L with temperature controlled at about 37°C, pH controlled between about 6.7 – 7.5, agitation power input of about 22 W / m3, dissolved oxygen level of about 30%. Cells are typically transfected at about 24 hours post inoculation of the production bioreactor, and harvested at 3-5 days post transfection.

[0129] 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).

[0130] All major antibody classes, namely IgG, IgA, IgM, IgD and IgE, can be used as targeting 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, F(ab’) or a F(ab')2, 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. Derivatives, components, domains, chains and fragments of the above also are included as types of targeting molecules.

[0131] As stated above, 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 to lower the AAV preferential transduction of the liver. The 51 ME149651883v.1135975-54620 (REGN 11546) 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. The inventions further provide mutations for other AAVs are as follows: AAV1 – N500E AAV2 – R585A and R588A AAV5 – T571S AAV6 – N500E, K531A and K531E. These and others are set forth in the chart below:52 ME149651883v.1135975-54620 (REGN 11546)

[0132] Still other mutations for all AAV serotypes are available to the person skilled in the art.

[0133] The AAVs were evaluated using affinity chromatography. Available affinity capture resins include, but are not limited to: POROS CaptureSelect AAVX, POROS CaptureSelect AAV8, POROS CaptureSelect AAV9, Capto AVB, AVB Sepharose, Avipure AAV2, Avipure AAV8, Avipure AAV9, among others. Resins used for affinity capture via antibody surface epitopes can comprise mAbSelectSuRe, mAbSelect PrimaA, Capto L, mAbSelect VL, and KappaSelect.

[0134] The inventions provide the skilled person with the ability to modulate the percent of SpyTag inserted capsid and subsequently the level of antibody conjugation through changing the fraction of the pRC-SpyT plasmid during hexad transfection. Level of antibody conjugation correlates with in-vitro infectivity.

[0135] A low pH hold in a bioreactor harvest has been found to be beneficial in order to drive the conjugation reaction to completion; that is, to ensure that any available SpyT peptides on AAV capsids are conjugated to SpyC- Antibodies, which are expressed in excess, as described herein.

[0136] The inventions are further described by the following Examples, which do not limit the inventions in any manner and are applicable to all sections of the descriptions of the inventions and the aspects of the inventions. 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.

[0137] The AAV serotype sequences and component sequence types that are used or can be used according to the inventions, and are discussed in the Description, Summary, Examples and Figures are representative and not limiting. Any AAV serotype sequence and component sequence can be used according to 53 ME149651883v.1135975-54620 (REGN 11546) 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 – Transfection Parameters – Design of Experiment 1

[0138] At 24 hours post inoculation of the production bioreactor, cells can be transiently transfected with 6 plasmids to produce the rAAV and additional components for covalent surface modification. The FectoVIR-AAV (Polyplus) is selected as the transfection reagent to deliver the plasmids into the cells. To achieve high yield production of rAAV, parameters defining the transfection procedure should to be optimized, including the cell density at the time of transfection, plasmid and transfection reagent concentration and ratios, and transfection complex incubation time.

[0139] A triple transfection using three plasmids (pGOI, pRC, pHELP) was used as a simplified platform to optimize the relative amounts of AAV transgene, functional and structural gene Rep and Cap, and helper polynucleotide sequences as a first step, prior to the introduction of plasmids encoding for the various components for covalent surface modification. A total of 6 factors were evaluated in a 24-run D-optimal custom Design of Experiment (DOE) study, referred to as DOE 1. Additional 10 bioreactor runs were added to augment the study design to further characterize the effects on the edge of the studied factor ranges. Table 1 summarizes the range studied and the optimal value identified for each factor to maximize the rAAV vector genome titer and percent of full rAAV capsids. Figure 3 depicts the prediction profiler from Design of Experiment study 1 on the effect of transfection parameters on rAAV titer, percent full capsids, and residual host cell 54 ME149651883v.1135975-54620 (REGN 11546) DNA (hcDNA). Optimal amount of RC plasmid identified from this study was used for subsequent hexad transfection but split at a fixed ratio between pRC and pRC- SpyT. The parameters are set forth below in Table 1: Table 1* The basis of the ratios are the GOI plasmid (pGOI).

[0140] Significant models were obtained for viral genomic titer (Radj2= 0.791, RMSE = 7.1 x 1010vector genomes (vg) / mL, p<0.0001), capsid titer (Radj2= 0.815, RMSE = 1.3 x 1012cp / mL, p<0.0001) and percentage of full capsids (Radj2= 0.842, RMSE = 4.4%, p<0.0001) using a standard least squares fit algorithm.

[0141] The data indicate that increasing the total DNA concentration and pRC / pGOI ratio and lowering the transfection reagent to DNA ratio increased the vector genome titer, but lowered the percentage of filled capsids. Reducing the transfection complex incubation time had no impact on vector genome titer, but improved the percent of full capsids. Increasing the cell density at transfection and the pHELP / pGOI ratio to a medium range improved both titer and percent of full capsids. Total RC plasmid to pGOI ratio should be about between 3.5 – 6.5, and 55 ME149651883v.1135975-54620 (REGN 11546) pHELP to pGOI ratio should be about 0.4 – 2.0. Optimal parameter values are identified to achieve a balance in the optimization objectives. The predicted titer is 4.3 x 1011vg / mL and percent full capsids of 25% EXAMPLE 2 – Bioreactor Operating Parameters-Design of Experiment 2

[0142] Controlling the cell culture environment at optimal state in respect to temperature, aeration, and pH can significantly enhance viral production. Specifically, pH of the bioreactor can be optimized to meet the demands of various phases of the viral production process, with the pH suited for cell growth potentially being different from the optimal pH for transfection complex internalization and viral vector production. Similarly, the agitation speed of the bioreactor can be studied to identify the optimal agitation speed for cell growth with minimal cell aggregation, and the agitation speed suitable for transfection without significant shear force to the transfection complex. To evaluate the impact of these two bioreactor parameters in detail, a total of 8 factors were studied in a 46-run D-optimal custom Design of Experiment study (DOE 2) to investigate the impact of the bioreactor pH and agitation speed at three different phases of production and the transition timings between phases on vector titer and quality. Table 2 summarizes the range studied and the optimal value identified for each factor to maximize the rAAV vector genome titer and percent of full rAAV capsids.

[0143] Figure 4 depicts the prediction profiler from DOE 2 on the effect of the bioreactor parameters on rAAV titer and percent full capsids. The SpyTag AAV (here, AAV9) was produced in this experiment without the introduction of any covalent surface modification. Optimal bioreactor setpoints identified from this study is validated on the production of conjugated AAV9. RC plasmid ratio used in this study is identified from the previous transfection multivariate study, but split 56 ME149651883v.1135975-54620 (REGN 11546) between the pRC and pRC-SpyT at a fixed ratio of 9:1 (1 / 10 SpyTag fraction). Table 2HPreT, hours pre-transfection; HPostT, hours post-transfection. Agitation speed is expressed in power input per unit volume in W / m3unit.

[0144] The data indicate that controlling the bioreactor pH at a high level of 7.3 during the transfection complex update phase and production of AAV improves the vector titer, while a modest pH of 7.1 is ideal for the cell growth phase prior to transfection. Shifting the pH from the growth pH up to transfection / production pH at an earlier time point is beneficial to improve percent of full capsids. Agitation speed, on the other hand, has minimal impact on viral production within the range studied. Optimal bioreactor pH and agitation set points are identified to achieve a balance between improving vector genome titer and percent of full capsids. Using these optimal parameter values, the model predicted a vector genome titer of 2.9 x 1011vg / mL and a percent of full capsids of 17.7%. EXAMPLE 3 – The Effect of Varying pRC-SpyT to pRC Plasmid Ratios 57 ME149651883v.1135975-54620 (REGN 11546)

[0145] The average number of covalent surface modifications on a single AAV is dominated by the number of SpyTag insertions on the VP proteins that make up the AAV capsid. These SpyTag insertions provide available docking sites for the free SpyCatcher antibodies to bind and form covalent bonds. The percentage of VP proteins that contain the SpyTag insertion is dictated at the genetic level by the relative amount of the RC plasmids with or without the SpyTag sequence that are introduced to the cells.

[0146] This study demonstrated for the first time the production of antibody conjugated AAV9 using a suspension cultured HEK293 cell line in a controlled fed-batch bioreactor system. The SpyTag AAV (here, AAV9) and SpyCatcher-Rm (anti-CACNG1) are produced simultaneously following the hexad transfection of plasmids encoding for all necessary components. A range of SpyTag fractions were used to demonstrate the ability to tune the percentage of SpyTag insertion to the AAV capsid and subsequently the level of antibody conjugation through the introduction of the pRC-SpyT and pRC plasmids at different ratios during hexad transfection. The AAV9 capsids with two point mutations (N272A and W503A) were produced in separate productions to support the investigation of AAV9 detargeting from the liver tissue. Various control conditions were included in the production study design to progressively introduce the detargeting and retargeting components and facilitate understanding of the impact of these components on viral production titer.

[0147] As shown in Figure 2A, there are two plasmids encoding AAV Rep and Cap. Plasmid RC (pRC) encodes Rep and Cap, but not SpyT, whereas pRC-SpyT encodes Rep and Cap (with Spy-T inserted into Cap ) (see Figure 2B). 58 ME149651883v.1135975-54620 (REGN 11546)

[0148] Figure 5A shows AAV with no SpyTag insertion (lanes 1-2); AAV with 1 / 8 SpyT insertion and a N272A detargeting mutation (lane 3) or a W503A detargeting mutation (lane 4); AAV with 1 / 4 to 1 / 20 SpyT insertion, antibody sequences and a N272A detargeting mutation (lanes 5-10); AAV with 1 / 4 to 1 / 8 SpyT insertion, antibody sequences and a W503A detargeting mutation (lanes 11 and 12).

[0149] The SpyTag fraction refers to the part of pRC-SpyT plasmid of the total RC plasmids at plasmid level. SpyTag Insertion refers to the addition of SpyTag to the VP proteins. The SpyTag insertion is on a percentage of VP proteins and is largely dependent on the fraction of the pRC-SpyT plasmid used during transfection. “Conjugation” refers to the linking of SpyCatcher-Rm (for example, a mAb or Fab) to the SpyTag AAV.

[0150] The illustrations at the bottom of Figure 5A depict the concept of capsid modification. Samples were stored at 4°C for 10 days to permit conjugation to complete.

[0151] Figure 5B shows that unmodified capsids are produced with each construct (bottom set of bands). SpyTag AAV capsids are produced in lanes 3-4 reflected by the additional VP3 band with slightly increased molecular weight. Antibody modified capsids are produced in lanes 5-12 when antibody plasmids are introduced during the hexad transfection. The antibody conjugation to the AAV capsid proteins results in a shift in molecular weight (top set of bands). The concurrent lack of the bands corresponding to the SpyTag AAV capsids indicate that antibody conjugation is close to completion and few un-conjugated SpyTag AAV is remaining following the extended hold for 10 days at 4°C. 59 ME149651883v.1135975-54620 (REGN 11546)

[0152] Figure 5C shows the infectivity results of the covalently surface modified (in this case anti-CACNG1 mAb conjugated) AAV with varying levels of antibody conjugation. In this cell-based assay, HEK293 cells that express CACNG1 surface antigen are dosed with the respective AAV vector preps in each lane. Two different spiking volumes of the vector preps are tested to screen for appropriate multiplicity of infection for this assay. The AAV vectors produced in this study carry a recombinant viral payload containing a CAGG promoter and a transgene encoding for green fluorescent protein. Cells that are transduced with the AAV vectors will receive the recombinant viral payload and express GFP, and the intensity of the fluorescent signal indicates infectivity of the AAV virus prep.

[0153] Antibody conjugation led to greater infectivity than control AAV (lanes 1 and 2) and AAV with only detargeting mutations (lanes 3 and 4). Infectivity of the vector preps negatively correlates with level of antibody conjugation. Lower SpyTag plasmid fractions, resulting in less antibody conjugation, corresponded to higher infectivity. EXAMPLE 4 - The Effect of Increasing Rm and SpyCatcher-Rm Plasmids

[0154] The conjugation of the SpyCatcher-Rm (for example, a mAb or Fab) to SpyTag AAV occurs predominantly at the end of the fed-batch production when cells are lysed apart using chemical detergent. SpyTag AAV that primarily retained inside the cells comes together with the SpyCatcher Rm that is secreted outside of the cells, and the spontaneous conjugation reaction occurs. The rate of the conjugation reaction is dependent on the temperature, pH and detergent concentration of the reaction mixture (Zakeri B, Howarth M, et al., 2012, PNAS), as well as the concentrations of the SpyTag AAV and SpyCatcher-Rm as reaction 60 ME149651883v.1135975-54620 (REGN 11546) substrates. A faster conjugation kinetic is beneficial to allow completion of reaction well within the time frame of the processing step and reduce production batch to batch variability in case the process intermediate hold time is varied.

[0155] Using a total DNA concentration of 2 µg / mL for the four plasmids encoding for SpyTag AAV, and a total DNA concentration of 0.28 µg / mL for the pHC-SpyC (heavy chain and SpyCatcher-Rm) and pLC (light chain) plasmids encoding for the SpyCatcher-Rm during transfection, the cells produce on average 30-fold SpyCatcher-Rm in excess in molar concentration relative to SpyTag AAV. At this ratio of SpyCatcher-Rm over SpyTag AAV in the clarified cell lysate, the conjugation reaction progresses at a suboptimal rate, showing close to complete conjugation only after extended incubation for 10 days at 4°C.

[0156] To study the effect of SpyCatcher-Rm level on rate of the conjugation reaction in the clarified lysis, the total DNA concentration of the pHC-pSpyC and pLC antibody plasmids used during transfection were changed. The mass ratio of pHC:pLC was kept the same at 1:2. A total of six different concentration conditions are studied in addition to the control condition, by lowering the total DNA concentration (represented by negative sign) by 75%, 50%, 25% or by increasing the concentration by 50%, 100% or 200%.

[0157] Figure 6A is a bar graph depicting the impact of mAb plasmid DNA concentration used in transfection on SpyCatcher-Rm (here, SpyCatcher- mAb) production titer. Increasing the mAb plasmid DNA concentration across the entire studied range improved the mAb titer by 45 – 100%. Figure 6B is a bar graph depicting SpyTag AAV titer was not affected by SpyCatcher mAb titer within the range studied. Figure 6C shows that Increasing the SpyCatcher mAb plasmid DNA (pHC-SpyC) led to more efficient conjugation of AAV in the 1 / 8 SpyTag fraction 61 ME149651883v.1135975-54620 (REGN 11546) condition. In Figure 6C, lanes 1 to 6 are for the 1 / 5 fraction of SpyT (pRC-SpyT plasmids to pRC plasmids is 1:4), and lanes 7 to 12 are for the 1 / 8 ratio of SpyT (pRC-SpyT plasmids to pRC plasmids is 1:7). To allow faster conjugation kinetics while maintaining the AAV production titer, the total concentration of the antibody plasmids is doubled (+100% condition) to 0.56 ug / mL during transfection in the final production process. EXAMPLE 5 – Transduction Efficiency

[0158] The plasmid concentration and ratios used during transient transfection were further evaluated for their indirect impact on transduction efficiency of the vector product. See Figure 7. The range of the SpyTag fraction was extended to 1 / 30, and the range of mAb DNA concentration was kept above control level (positive sign) to enhance rate of conjugation. Spy Tag fractions ranged from 1 / 5 (pRC-SpyT to pRC is 1:4) to 1 / 30 (pRC-SpyT to pRC is 1:29). The percent change in mAB DNA concentration ranged 0% to 200%, meaning 0% = 0.28 µg / mL, 100% = 0.56 µg / mL, 200% = 1.12 µg / mL. Lane 5, which had a 1 / 10 SpyTag fraction (pRC-Spy T to pRC is 1:9) and a 100% increase in mAb DNA showed the highest transduction efficiency. The signal was generated using a green fluorescent reporter gene. EXAMPLE 6 – Low pH Hold

[0159] The hexad transfection process involves supplying AAV, AAV- SpyT, and SpyC-Antibody plasmids simultaneously to mammalian cells, such as HEK293 cells, and therefore results in expression of AAV capsids, AAV-SpyT capsids, and SpyC-Antibodies in the bioreactor. While some of the AAV-SpyT capsids are conjugated to SpyC-Antibodies naturally in the bioreactor, a proportion 62 ME149651883v.1135975-54620 (REGN 11546) remain unconjugated. This is disadvantageous as the objective is to achieve maximum production of conjugated AAV-SpyT-SpyC-Antibody species.

[0160] A low pH hold in the clarified bioreactor harvest has been found to be beneficial in order to drive the conjugation reaction to completion; that is, to ensure that any available SpyT peptides on AAV capsids are conjugated to SpyC- Antibodies, which are expressed in excess. Figure 8 sets forth data from an experiment showing the usefulness of low pH hold to drive the conjugation reaction to completion for material from four different 2 liter bioreactors (Bioreactor A, B, C, D) cultivated using the hexad transfection approach with different SpyC-Antibody plasmids in each bioreactor. Experiments were conducted at pH values of 6, 7 and 8, and pH 6 performed the best.

[0161] Lanes 1, 3, 5 and 7 show the bioreactor lysate material directly after clarification for Bioreactors A, B, C and D, respectively. It can be seen that there is a faint band above the "VP3" band, indicating the presence of VP3-SpyT; that is, a denatured VP3 capsid protein with the SpyT peptide included in its sequence. There is also a faint band in the upper half of the gel indicating the presence of VP3-SpyT-SpyC-Ab; that is, the VP3 capsid protein conjugated to a SpyC-antibody.

[0162] Lanes 2, 4, 6, and 8 show the material from Bioreactors A, B, C, and D, respectively, after clarification followed by an overnight hold at pH 6. In these lanes, the VP3-SpyT band no longer appears, and the bands in the upper half of the gel are darker. This indicates that the unconjugated VP1 / 2 / 3-SpyT proteins have been completely conjugated to SpyC-Ab, causing the higher molecular weight bands to become darker in the upper half of the gel. Thus, the low pH hold can be seen to be a part of the production process for conjugated AAV-SpyT-SpyC-Ab 63 ME149651883v.1135975-54620 (REGN 11546) species using the hexad transfection approach, and is useful for driving the conjugation reaction to completion and resulting in maximum yield of conjugated species. EXAMPLE 7 – Depth Filtration

[0163] Cell lysis is an early part of AAV 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), Tritons (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.

[0164] Depth filtration typically follows lysis. Single-use chromatographic clarification uses a next-generation synthetic fibrous anion- exchange chromatographic clarification media to simultaneously conduct depth filtration, removal of cellular debris, soluble negatively charged impurities, and sterilize filter cell harvest material. Although traditionally used for mAb processing, these filters offer a unique benefit for rAAV production as they can process chromatin-containing material without excessive reduction in capacity, presenting an opportunity to eliminate expensive endonuclease (for example, Benzoase) treatment.

[0165] Harvest RC (3M) is a single-use chromatographic clarification unit consisting of synthetic fibrous anion exchange (AEX) media and a 0.2 µm polyethersulfone (PES) membrane. Cells are bound inside the fibrous AEX media by, allowing efficient retention of large and small particles without caking that rapidly fouls the filter. This structure also captures large strands of DNA without rapid 64 ME149651883v.1135975-54620 (REGN 11546) fouling, unlike conventional depth filters which require the DNA to be digested by endonuclease prior to clarification.

[0166] Tests with the Harvest RC (HRC) filters were conducted under different conditions of endonuclease addition and salt treatment. Comparison runs were also conducted with traditional C0SP depth filtration media made of polypropylene fibers. HRC data and C0SP data are set forth in Figure 9A (rAAV8 at 3 x 106cells / ml) and Figure 9B (rAAV9 at 1.5 x 106cells / ml). The top figures are for Total Host Cell DNA (ng / ml) and the bottom figures are for Total Host Cell Protein (ng / ml).

[0167] Figure 9A is a bar graph for AAV8 depicting in order bars for bioreactor lysate (white bars), filtrate pool where transmembrane pressure (TMP) is 5 psi (light gray bars), and filtrate pool where TMP is 15 psi (black bars). HRC (solid bars) and C0SP (a standard filter used as a control) (hatched and labeled bars) are filtration trains with different endonuclease and salt conditions in the load. Figure 9B is a bar graph for AAV9 depicting in order bars for bioreactor lysate (white bars), filtrate pool where TMP is 5 psi (light gray bars), and filtrate pool where TMP is 15 psi (black bars). HRC (solid bars) are filtration trains with different endonuclease and salt conditions in the load. In Figure 9A, significant breakthrough of HCDNA into the filtrate was observed for C0SP at the “C0SP Negative Control”, and for HRC at 250 mM NaCl and 0 U / ml endonuclease (dark gray bars). In Figure 9B, significant breakthrough of HCDNA into the filtrate was observed for HRC at 250 mM NaCl and 0 U / ml endonuclease (dark gray bars) and 250 mM NaCl and 10 U / ml endonuclease (dark gray bars).

[0168] Notably, the C0SP filter was unable to achieve significant removal of Host Cell DNA in the endonuclease-free train, with greater than 10465 ME149651883v.1135975-54620 (REGN 11546) ng / mL present in both the 5 and 15 psi fractions. In contrast, the HRC filters successfully removed Host Cell DNA to less than 102ng / mL in all filtration trains in low-salt conditions for rAAV8 (Figure 9A). In particular, for the endonuclease-free- trains a 100-fold reduction in host cell DNA (“HCDNA”) was achieved, from greater than 104ng / mL in the load material to less than 102 ng / mL in the filtrate fractions.

[0169] Additionally, capsid binding was not observed in any of the experiments on bioreactor harvest material, likely due the presence of sufficient HCDNA and other negatively charged impurities to compete with the capsids for the charged binding sites on the Harvest RC filter.

[0170] However, in the high salt condition when 250 mM NaCl was added to the load material, the HRC filter was unable to achieve effective Host Cell DNA (HCDNA) removal for the medium-endonuclease case, though the high salt level did not affect the filtrate quality in the high-endonuclease case. This is likely due to the salt competing with the Host Cell DNA for binding onto the anion exchange sites on the HRC filter, lowering the binding capacity. Finally, no significant reduction or impact on Host Cell Protein was observed between the load material and the filtrate fractions for any of the tested conditions.

[0171] For rAAV9, the low-salt and endonuclease-free conditions once again performed remarkably well, with 1000-fold reduction of Host Cell DNA from greater than 104ng / mL in the load material to 101ng / mL in both the 5 and 15 psi filtrate fractions for 0-100 mM NaCl. See Figure 9B. In all runs, it was observed that high salt conditions of 250 mM NaCl led to significant Host Cell DNA breakthrough into the filtrate for both the 0 U / mL and 10 U / mL endonuclease filtration trains, showing that limiting salt addition is critical for endonuclease-free clarification to be successful. The data with AAV8 and AAV9 show that an endonuclease-free 66 ME149651883v.1135975-54620 (REGN 11546) clarification process can provide reduction of Host Cell DNA to comparable levels as conventional processes that use 100 U / mL of endonuclease in the bioreactor during lysis.

[0172] Figures 10A-10F provide data for depth filtration treatment at various salt and endonuclease conditions. Figures 10A-10C provide data from HRC depth filtration of AAV8 and Figures 10D-10F provide data from HRC depth filtration of AAV9. Figures 10A and 10D have no endonuclease added. Figures 10B and 10E have 10 units / ml of an endonuclease added. Figures 10C and 10F have 100 units / ml of an endonuclease added. Each of Figures 10B-10F contain data with concentrations of 0 mM NaCl, 100 mM NaCl and 250 mM NaCl. Figure 10A contains data with concentrations of 0 mM NaCl and 100 mM NaCl with HRC and 0 mM NaCl with C0SP. Figure 10C data with concentrations of 0 mM NaCl, 100 mM NaCl and 250 mM NaCl for HRC and 250 mM NaCl for C0SP.

[0173] Data from HRC and C0SP with AAV8 and AAV9 indicate Host Cell DNA breakthrough in Figure 10A (0 mM NaCl with 0 U / m endonuclease) (C0SP) (AAV8), Figure 10B (250 mM NaCl with 10 U / m endonuclease) (HRC) (AAV8), Figure 10D (250 mM NaCl with 0 U / m endonuclease) (HRC) (AAV9), and Figure 10E (250 mM NaCl with 10 U / m endonuclease) (HRC) (AAV9). Experimental characterization of the effect of salt shows that it is important to limit salt addition to prevent Host Cell DNA breakthrough into the filtrate. However, an amount of salt (for example, less than 100 mM) can be beneficial to increase overall process throughput and prevent the differential pressure across the filter from increasing too steeply.

[0174] Overall, this approach enables endonuclease-free clarification of rAAV. Thus, using salt conditions of 0-100 mM, for example, clarification can be 67 ME149651883v.1135975-54620 (REGN 11546) undertaken with low endonuclease or without an endonuclease altogether, and thereby significantly reduce purification costs.

[0175] Figure 11 depicts data set forth in Figures 10A-10F. A three-fold in throughput (liters filtered per meters2of filter area) at 5 pounds per square inch (psi) differential pressure at different endonuclease conditions, specifically at 0 U / ml, 10 U / ml and 100 U / ml of endonuclease, using AAV8 and AAV9. The 10 U / ml condition was midway between the 0 U / ml and 100 U / ml endonuclease conditions. Capsid and genomic yields in the filtrate pool of all runs were greater than 90% and comparable throughputs were observed at scales of 3.2 cm2and 25 cm2.

[0176] Figure 12 depicts data set forth in Figures 10A-10F regarding the effects of salt (NaCl). The pressure range from 5-20 psi is a safe zone for flush and filter blowdown operations with depth filtration. If the pressure increases too sharply from 5-20 psi, there can be a loss of hold-up material because the modules should not be operated above a limit of 20 psi. Salt conditions, such as about 1 mM to 110 mM or more, but less than 250 mM. Preferably 1 mM to 100 mM, more preferably 1mM to 75 mM, still more preferably 1 mM to 50 mM, and yet more preferably 1 mM to 25 mM. For example, preferred salt conditions such as 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, and 110 mM, and ranges formed between any of these values (for example, 25 mM to 100 mM, 50 mM to 100 mM, 75 mM to 100 mM, 90 mM to 110 mM) can be selected.

[0177] Figure 13 depicts data showing that salt (NaCl) addition (See Figure 12) reduces load turbidity and increases throughput at higher pressures. For example, salt conditions, such as about 1 mM to 110 mM or more, but less than 250 mM. Preferably 1 mM to 100 mM, more preferably 1 mM to 75 mM, still more 68 ME149651883v.1135975-54620 (REGN 11546) preferably 1 mM to 50 mM, and yet more preferably 1 mM to 25 mM. For example, preferred salt conditions such as about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 105 mM, and 110 mM, and ranges formed between any of these values (for example, 25 mM to 100 mM, 50 mM to 100 mM, 75 mM to 100 mM, 90 mM to 110 mM) can be selected to reduce turbidity and increase throughput at higher pressures.

[0178] Treatment with 250 mM NaCl resulted in host cell DNA breakthrough into the filtrate for low and zero-endonuclease conditions.

[0179] Figure 14 depicts data set forth in Figures 10A-10F showing that a combined cake-fiber fouling model is a good representation for the mechanism of Harvest RC filtration (depth filtration). In Figure 14, a filtration train with 0 U / ml endonuclease and 100 mM NaCl with AAV9 was analyzed.

[0180] Harvest RC filters contain fibrous anion exchange media layered above a flat sheet sterile filter. Low to zero endonuclease results in long strands of host cell DNA that are expected to bind to the AEX fibers, thereby causing fiber coating. Cake formation is expected at the flat-sheet filter and between tightly packed fibers. A cake-fiber fouling model resulted in excellent fit with R2> 0.95 and was better than cake formation or fiber coating models individually.

[0181] The following model can be used:Kc = cake filtration constant. s / m269 ME149651883v.1135975-54620 (REGN 11546) Kf= fiber coating constant, 1 / m V = volume filtered, rn3 / m2P = pressure, kg / ms2J = solvent flux, m / s ∅ = filter solid or fiber volume fraction

[0182] See G. R. Bolton, D. LaCasse, M. J. Lazzara, and R. Kuriyel, “The fiber-coating model of biopharmaceutical depth filtration,” AIChE Journal, vol. 51, no.11. Wiley, pp.2978–2987 ( 2005).

[0183] Figures 15A-15C depict data set forth in Figures 10A-10F. Cake formation parameters (KC) vs NaCl (mM) added with 0 U / ml endonuclease (Figure 15A), 10 U / ml endonuclease (Figure 15B), and 100 U / ml endonuclease (Figure 15C).

[0184] Empirical parameters extracted from the best-fit model curves were plotted for the different load conditions. The cake-fiber fouling model uses two parameters, namely cake formation parameter Kcand fiber coating parameter Kf. Kc increased with increasing salt for all endonuclease conditions. This is expected as Cl- competes with HCDNA for AEX binding sites and causes more of a cake filtration than fiber coating mechanism. Kfdecreased two-fold between the 0 U / mL and the 10 U / mL endonuclease conditions, from an average of 6 x 10-6to 3 x 10-6. This suggests that the fiber coating effect is stronger in the endonuclease-free condition. That is, the long strands of undigested HCDNA are binding strongly onto the AEX fibers and creating a thickening effect.

[0185] The methods of the inventions provided a reduction of HCDNA from 5x104ng / mL to 50 ng / mL for rAAV8 and 2x104ng / mL to 10 ng / mL for rAAV9 in endonuclease-free conditions. This is comparable to traditional depth filtration media with 100 U / mL endonuclease treatment in the load. 70 ME149651883v.1135975-54620 (REGN 11546)

[0186] Overall, this approach enables endonuclease-free clarification of rAAV. Thus, using salt conditions of 0-100 mM, for example, clarification can be undertaken with low endonuclease or without an endonuclease altogether, and thereby significantly reduce purification costs.

[0187] Following depth filtration, the pool can be subjected to tangential flow filtration (TFF) for concentration and buffer exchange before further chromatography, such as affinity chromatography. Conventional TFF can be employed, which requires 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 EXAMPLE 8 – Removal of Excess SpyCatcher-Rm

[0188] The hexad transfection production process represents an efficient method for the production of covalently modified AAV for retargeting. A population of retargeted AAVs with different covalent modifications (that is, covalently surface modified AAV species) can be produced using this method, and combined together (pooling) to form a mixture for in vivo screening of best retargeting candidate. The pooling step ideally would occur prior to purification unit operations so the subsequent purification process can be completed in a single train. To enable pooling of different production material prior to purification, excess SpyCatcher-Rm (non-conjugated) within each production should be 71 ME149651883v.1135975-54620 (REGN 11546) inactivated / quenched to prevent cross conjugation to unintended, unconjugated SpyTag-AAV after pooling.

[0189] The present inventions can utilize a 13 amino acid SpyTag peptide in soluble form (Example 33) to bind to the excess SpyCatcher-Rm (here SpyCatcher Fabs) and achieve a two log reduction in unintended cross conjugation between the SpyTag AAV and SpyCatcher Fab. The concentration of the soluble SpyTag was screened and the effect was demonstrated as discussed below. See Figures 16-17.

[0190] In this study, three suspension cultured fed-batch productions were completed in miniature 250 ml bioreactors, producing 1) an unconjugated SpyTag AAV species carrying a nano luciferase (nLuc) reporter gene, 2) a mouse- TfR Fab (8D3) conjugated SpyTag AAV carrying an alternative transgene (in this case gene encoding for green fluorescent protein), 3) a mouse-TfR Fab (8D3) conjugated SpyTag AAV carrying a nano luciferase reporter gene (nLuc). The production material was assessed in a transduction assay using HEK293 cells expressing the mouse-TfR cell surface receptor (mTfR+ HEK293) and luciferase as the reporter signal. Production 1 and 2 material alone showed low luciferase signal due to the absence of mTfR Fab conjugation or absence of relevant reporter gene, while production 3 material showed intense luciferase signal as a positive control. The soluble SpyTag at a concentration of 0, 33, 100, 300, and 900 ug / ml was co- incubated with the production 1 and 2 material separately at room temperature, pH 6.0 for 24 hours prior to pooling of the two production material together. The combined material was then assayed in the transduction assay. Comparing with no soluble SpyTag quenching condition (yellow), the soluble SpyTag at concentrations of 33 – 900 ug / mL (green) resulted in two log reduction in cross conjugation signal. 72 ME149651883v.1135975-54620 (REGN 11546)

[0191] Figure 16 and 17 schematically depict a proof of concept study demonstrating the use of soluble SpyTag peptide to quench excess SpyCatcher Fab. Figure 16 illustrates the three productions performed using the suspension miniature bioreactor system, and the anticipated transduction assay luciferase signal (nLuc) for the three productions alone and the pooled material with or without soluble SpyTag quenching. Triangles attached to an AAV represent SpyTag peptide inserted into the VP proteins. Triangles not attached to an AAV represent soluble SpyTag peptide. Figure 17 depicts the luciferase signal from the transduction assay when cells are dosed with the respective AAV products at a series of multiplicity of infection (MOI) levels. The 8D3 is a particular mouse-TfR Fab candidate.

[0192] Parameters and ranges: Concentration ranges for the soluble SpyTag include 2 – 1000 ug / mL pH ranges include 5.0 – 8.0 Duration of quenching 1 – 48 hours Temperature for quenching 2 – 37°C Higher soluble SpyTag concentration (100 – 900 ug / mL) and longer duration of quenching (12 – 48 hours) are preferred under unfavorable conjugation conditions (pH >= 7.0). Lower soluble SpyTag concentrations (2 – 100 ug / mL) and shorter duration of quenching (1 – 24 hours) may be sufficient under favorable conjugation conditions (pH 5 – 7). This approach is useful with the Hexad transfection and other transfection regimens. EXAMPLE 9 – Genomic Yield with Affinity Capture

[0193] Purification of covalently surface modified AAVs (also referred to as “retargeted AAVs”) requires effective methods to capture surface-modified AAVs from the bioreactor harvest mixture. Challenges in affinity capture include difficulty 73 ME149651883v.1135975-54620 (REGN 11546) in binding to the AAV surface epitopes in case of heavy conjugation, or difficulty in eluting the surface-modified AAV without destabilizing the molecule, thereby leading to aggregation, precipitation, and / or fragmentation. These difficulties often lead to an imbalance in the level of conjugation in the affinity capture load material relative to the eluate stream; that is, a loss of the some to all of the conjugated species during affinity capture due to preferential purification of the less conjugated and unconjugated AAVs. The goal of high-yield affinity capture of conjugated AAV species was not met until the present inventions.

[0194] Several screening runs were carried out on different resins and with different elution buffers. Poros CaptureSelect AAVX did not demonstrate good binding affinity for conjugated rAAV species, for example, AAV9-SpyT-SpyC-mAb. In contrast, Poros CaptureSelect AAV9 resin did exhibit good binding with conjugated rAAV species, for example, AAV9-SpyT-SpyC-mAb, but the yield was dependent on the choice of elution buffers.

[0195] Low yields were achieved in many elution buffers, such as yields of approximately 10% for glycine buffers along with loss of the heaviest conjugated species. A buffer comprising about: 50 mM acetate, 50 mM histidine, 0.001% P188, pH 3.0 was able to achieve yield of greater than 90% along with successful capture of the heavily conjugated AAV species. Notably, this buffer also resulted in highest yield for unconjugated AAV9-SpyTag species as produced in the post-processing conjugation system instead of the hexad transfection system.

[0196] Figure 18 depicts data using a POROS CaptureSelect AAV9 resin to capture AAV9-SpyT-SpyC-mAb. Monoclonal antibodies against CACNG1, ASGR1 and Fel d 1 were used as retargeting molecules. Average column loading was 2.5 x 1012viral genomes / ml resin and 9.2 x1013capsids / ml resin. 74 ME149651883v.1135975-54620 (REGN 11546)

[0197] Less conjugated AAV9 (1 / 20 and 1 / 30) achieved higher yields. It is believed that affinity chromatography favored less conjugated AAV9 because such AAV9 would have more available epitopes.

[0198] Figure 19 compares various buffers used for affinity capture of an AAV-SpyT-SpyC-Tfr Fab. The buffers left to right comprised (i) glycine, (ii) acetate, (iii) acetate and arginine, (iv) acetate and histidine, and (v) acetate, histidine and arginine. Buffer (iv) acetate and histidine provided the highest yield. A POROS CaptureSelect AAV9 column loaded with 1013to 1015capsids per milliliter was employed.

[0199] Figure 20 compares the level of conjugation in the eluate of affinity capture using (i) a single affinity capture step specific to AAV surface epitopes, and (ii) after a second affinity capture step specific to antibody surface epitopes. POROS CaptureSelect AAV9 was useful for removing free antibody (fAb). The two-step affinity capture (POROS CaptureSelect AAV9 and Capto L) strategy resulted in removal of unconjugated AAV species. See Figure 20 and Table 3. Table 375 ME149651883v.1135975-54620 (REGN 11546)

[0200] Following affinity capture, the pool optionally can be subjected to tangential flow filtration for concentration. Thereafter, the pool optionally can be subjected to centrifugation, such as Iodixanol Gradient Centrifugation to enrich for full capsids and then subjected again to Tangential Flow Filtration (TFF) to concentrate and a buffer change to a storage buffer, or other chromatographic methods, such as AEX. 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). EXAMPLE 10 – Production of Fab conjugated barcoded AAV library

[0201] The hexad transfection methodology allows simultaneous production of SpyTag AAV and SpyCatcher-Rm within the same production bioreactor, and is an efficient way to produce a library of covalently surface modified AAV species for screening studies when the AAV and / or conjugated retargeting molecules (Rm) are uniquely different.

[0202] Using the hexad transfection method, a library of 48 Fab conjugated AAV vectors (which are covalently surface modified AAV species) were produced for a non-human primate study to screen for top Fab candidates to improve tissue specificity and transduction efficiency. Each of these conjugated AAV vectors carries a polynucleotide, such as transgene comprising a unique 32 base pair (bp) sequence as a “DNA barcode,” which are commercially available from companies such as PacBio.

[0203] Each unique DNA barcode serves as a biomarker encoded inside the AAV vector, and is matched to a Fab candidate conjugated on the outside of the AAV vector. The library of Fab conjugated AAV vectors were pooled and injected into mice and non-human primates. At the end of the study, the tissue of 76 ME149651883v.1135975-54620 (REGN 11546) interest was harvested, and the enrichment of DNA barcodes in the target tissue, representing the preferential transduction of the AAV vector retargeted by the corresponding Fab candidate, was evaluated using next generation sequencing technologies.

[0204] Production of the library of Fab conjugated AAV vectors starts with thawing of a vial of the suspension cultured HEK293 cell line followed by a series of cell expansion steps to gradually increase the cell culture volume. Cells were then inoculated into the production bioreactors, with each conjugated species produced separately in a fed-batch production bioreactor using the hexad transfection method. At the end of the productions, cells were lysed and clarified via depth filtration. The material was held at pH 6.0 at room temperature for 24 hours to allow conjugation reaction to reach completion.

[0205] Prior to the pooling of the conjugated AAV vectors from different productions, the excess free SpyCatcher Fabs were quenched by the addition of soluble SpyTag followed by another 24-hour room temperature hold at pH 6.0. The small soluble peptide bound to the excess SpyCatcher Fab and prevented it from cross conjugating to unintended AAV vectors. The soluble SpyTag quenched material was then pooled and further purified through a purification train comprising tangential flow filtration (TFF), affinity capture, iodixanol ultracentrifugation and a final TFF. The process flow diagram is shown in Figure 21. Using the hexad transfection production process, the average vector genome titer obtained from the bioreactor lysate was 1.8 x 1011vg / mL (Figure 22). The 48 different DNA Bar codes used to identify each Fab conjugated AAV vector are set forth in Example 34. 77 ME149651883v.1135975-54620 (REGN 11546) EXAMPLE 11 – Production of Covalently Surface Modified AAV using Adherent HEK 293T cells

[0206] Adherent HEK 293T cells were used to produce covalently surface modified AAVs: Splitting & plating of Adherent 293T cells (at Day 3 or Day 4 of growth):

[0207] Lifting & Passaging 1. Prepare & warm fresh formulated media a. 500 ml DMEM, 50 ml FBS, 5 ml NEAA, 5 ml PenStrep i. Final concentrations of: 10%-FBS, 1% NEAA, 1% PenStrep, good for one month; 2. Remove confluent HEK293T T225s from incubator, check cells under microscope, and aspirate spent media w / vacuum line; 3. Add 5 ml of TrypLE Select / T225, evenly disperse over cells, and wait 2-5 minutes for cells to dissociate from flask surface (cells do not need to be rinsed with phosphate buffered saline (PBS) prior to addition of TrypLE Select); 4. Lightly tap flasks to loosen cells and add 5 ml of formulated DMEM per T225; 5. Repeatedly rinse / wash cells off w / pipette controller and transfer cells to appropriately sized vessel (50 ml-200 ml conical tube); 6. Count cells w / Countess and seed 4-6 new T225 flasks for next passaging cycle a. 5 x 106cells for 4 days, 1 x 107cells for 3 days (in 30 ml formulated DMEM / T225); 7. Return cells to 37°C incubator for 3-4 days (based on seeding density).

[0208] Seeding (10-Layer Cell-Stack) 78 ME149651883v.1135975-54620 (REGN 11546) Seed HEK293T cells for AAV production according to below table and incubate at 37°C: a. For cell-stacks, mark off cap used for adding media & cells. Make sure to use same side for transfection; b. For 10-layer cell-stacks, evenly divide cells between the 2 media bottles.Note: each layer in a cell-stack is ~415cm plates

[0209] Transfection of 293T Cells (Day 0): 10-Layer Cell-Stack 1. For each cell-stack, add about 15 ml of unformulated DMEM to 2x 50 ml conical tubes a. 1x 15 ml (PEI tube), 1x 15 ml (DNA tube;) 2. To DNA tube & PEI tube, add appropriate amounts of DNA and PEI (see table below) & vortex each for 10 seconds a. 1 ug DNA: 4 µg polyethylenimine (PEI) ratio; 79 ME149651883v.1135975-54620 (REGN 11546) 3. pRC-SpyT to pRC Ratio 1 / 84. Combine DNA and PEI solutions and vortex for 10 seconds. Incubate mixture for 10 minutes at room temperature; 5. During the 10-minute incubation: Label cell-stacks appropriately and bring multi-Liter bucket into hood for waste; 6. 1 minute left in incubation: Unscrew marked cap. Pour spent media into the plastic bucket ; 7. When 10 minutes has elapsed, gently swirl DNA-PEI complexes, and add to formulated media. Swirl formulated media + complexes gently to mix a. For 10-layer cell-stacks, evenly divide complexes between the 2 media bottles; 80 ME149651883v.1135975-54620 (REGN 11546) 8. Slowly add formulated media + complexes into the cell stack through labeled cap side; 9. Tilt the cell stack on the long side (unmarked cap side). Wait for media levels to equalize. Slowly turn to the short side (side without caps). Finally, slowly lay cell stack flat and gently rock to cover cells with media and complexes; 10. Check that the layers appear similar in volume and return to 37°C incubator for 3-4 days.

[0210] Collections (Day 3 or 4): 10-Layer Cell-Stack (lysate collection & processing) 1. Sterile-filter portion of antibody-containing supernatant (~100 ml) and pour remainder into appropriately sized waste bucket for disposal; 2. Add appropriate amount (see below) of PBS + 4mm EDTA to cell-stack, evenly distribute over cells, and incubate at 37°C for ~5 minutes a. 10-Layer (~800 ml); 3. Shake cell-stack side-to-side and up / down to dislodge cells. Ensure that cells are lifted and pour PBS / EDTA+cells into appropriate number of 200-225ml conical tubes; 4. Pellet cells and debris at 4000rpm for 25 minutes; 5. Carefully pour off supernate and re-suspend in appropriate volume of lysis buffer (see below) a. 10-Layer (35 ml); 6. Transfer lysate to appropriate # of 50 ml conical tubes a. Use 2x 50 ml tube (17.5 ml of resuspended lysate in each); 7. Vortex and freeze in dry ice + 100% EtOH bath; 81 ME149651883v.1135975-54620 (REGN 11546) 8. Thaw cells in 37°C water bath; 9. Repeat for a total of 3 freeze / thaw cycles a. Cell lysates can be kept frozen at -80°C at any point for processing later; 10. Add 35 ml total of supernatant back to lysate (17.5 ml per 50 ml conical) a. total volume now about 70 ml, split into 2 x 50 ml conicals containing ~35 ml each); 11. Add total 25 µL of Denarase total to complexing AAVs (about 12.5 µL per 50 ml conical); 12. Vortex to mix and incubate at 37°C for 1 hour with occasional vortexing; 13. Pellet at 10,000 x g for 10 minutes; 14. Filter through 0.2 µm PES filter or 50 ml MDI filter and discard pellet.

[0211] Iodixanol Gradient Ultracentrifugation (for isolation of full AAV capsids, 80-90% full) 1. Using 10 ml syringe + 16 or 19-gauge stainless steel canula needle, prepare iodixanol (IDX) gradients by underlaying iodixanol solutions in ascending order of density: a. 7 mL 15% Iodixanol b. 6 mL 25% Iodixanol c. 7 mL 40% Iodixanol d. 5 mL 60% Iodixanol Note: dispense IDX solutions slowly to avoid mixing / disturbing the different layers, and mark interface between layers at conclusion of gradient laying; 82 ME149651883v.1135975-54620 (REGN 11546) 2. Gently load concentrated medium or cell lysate onto gradient with pipette controller and fill remaining volume as necessary with 1x PBS + 0.001% Pluronic (for media) or lysis buffer (for lysate) a. Capacity for each IDX column is ~12-13 ml (~10 plates worth of volume) b. Weigh and balance tubes in buckets with caps to minimize centrifugation error; 3. Insert into Beckman Coulter Type SW 32 Ti Swinging-Bucket Rotor; 4. Ultracentrifuge virus for 16-18 hours overnight at 29,600 rpm at 10°C. Use max acceleration and deceleration of “5”; 5. After centrifugation, carefully remove tubes from the rotor with disposable forceps a. Spray buckets with Envirocide, and then 70% ethyl alcohol to clean; 6. Gently insert sterile beveled 1.5” 19G needle with 3 mL syringe roughly 0.5 mL below interface of 40% and 60% iodixanol solutions. Draw 3 mL of volume a. Use 3D-printed AAV IDX extractor for support; . Remove needle, block puncture, and discard tube in a waste bottle.

[0212] Buffer Exchange and Concentration (for lower titer serotypes) (Reagent Consideration: 1x PBS + 0.1% Pluronic, 1x PBS + 0.01% Pluronic, 1x PBS + 0.001% Pluronic, 100 kDa Amicon concentrator (15 ml)) 1. Add 1x PBS + 0.1% Pluronic to 100 kDa Amicon concentrator, allow concentration filter to incubate for 5 minutes, and centrifuge at 4000 x g for 2 minutes. Discard flow-through; 2. Add 1x PBS + 0.01% Pluronic to 100 kDa Amicon and centrifuge at 4000 x g for 2 minutes. Discard flow-through; 83 ME149651883v.1135975-54620 (REGN 11546) 3. Add 1x PBS + 0.001% Pluronic to 100 kDa Amicon and centrifuge at 4000 x g for 2 minutes. Discard flow-through; 4. Dilute 3 ml iodixanol fraction to 15mL with 1x PBS + 0.001% Pluronic; 5. Centrifuge sample at 4000 x g until volume is reduced (<1.5mL) and discard flow-through a. 2-3 fractions of the same AAV can be combined for use in a single Amicon (dilute appropriately). Additional fractions will increase spin times dramatically b. Likely no more than 5-10 minutes needed for initial spin with a single gradient; 6. Resuspend to top of Amicon with additional quantity of sample or 1x PBS + 0.001% Pluronic and repeat centrifugation; 7. Continue to refill and centrifuge an additional 3x; 8. Reduce to desired volume (refer to final concentration table) and filter using 0.2 µm Spin-X tube (10,000 RPM, 1 minute); 9. Store purified AAV at 4°C for short term use (<2 weeks) or aliquot and store at -80°C. The yield of this protocol for AAV9 was 1 to 2.5 x 1013vg after Iodixanol (IDX) purification, with greater than 50% full capsids. EXAMPLE 12 – PROCESS ANALYTICAL TECHNOLOGY AND IMPROVED PURIFICATION

[0213] Figure 23 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, 84 ME149651883v.1135975-54620 (REGN 11546) which contains the desired biological material, such as AAV. See Adams et al., Biotech. Bioeng.117: 3199-3211 (2020).

[0214] The bottom section of Figure 23 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,

[0215] 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.

[0216] For further comparison, Figure 24A 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 biological 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.

[0217] Figure 24B 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. 85 ME149651883v.1135975-54620 (REGN 11546)

[0218] Figure 25 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 completed in less than a day, whereas the batch process can be multi-day.

[0219] 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.

[0220] 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.

[0221] 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.

[0222] Figure 26 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 86 ME149651883v.1135975-54620 (REGN 11546) 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.

[0223] Figure 27 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 26 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.

[0224] Figure 28 depicts data from a 5-in-series configuration according to Figure 26 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 26 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.

[0225] Figure 29A is a design space model based on Figures 27 and 28 using the 5-in-series configuration of Figure 26. Here, the process target was 35 LMH, and the intersecting lines indicate a VCF of 8 and a TMP of 10psi. An exemplary acceptable zone would be a VCF of 6-10 and a TMP of 7.5 to 12.5 psi. Figure 29B is an exemplary comparison of process parameters between SPTFF and Batch TFF. With Batch TFF, typically there would be one batch before the 87 ME149651883v.1135975-54620 (REGN 11546) 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.

[0226] Figure 30 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. 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.

[0227] Figure 31 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 demonstrated 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.

[0228] Figure 32 depicts a tween micelle build-up on the TFF membrane. Without being bound by any theory or hypothesis, it is believed that 88 ME149651883v.1135975-54620 (REGN 11546) 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.

[0229] Figure 33 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.

[0230] Figure 34 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.

[0231] With Batch TFF, flux decline can be address by increasing processing time. However, with SPTFF immediate control is desired.

[0232] Figure 35 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 of the figure, Option 1 (SPTFF 89 ME149651883v.1135975-54620 (REGN 11546) 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 29A

[0233] Figure 36 depicts the overall pilot scale process, and is similar to parts of the production process of Figure 23.

[0234] Figure 37 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 (2.7 to 5.0 minutes). 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.

[0235] Figure 38 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 90 ME149651883v.1135975-54620 (REGN 11546) 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.

[0236] 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 maximizes 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.

[0237] EXAMPLES OF COVALENTLY SURFACE MODIFIED AAV

[0238] 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. 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. 91 ME149651883v.1135975-54620 (REGN 11546)

[0239] EXAMPLE 13 – AAV1 Covalently surface modified AAV1 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and then harvesting the covalently surface modified AAV1. The first chart below uses SpyTag-SpyCatcher as specific binding pairs, and Adenovirus Helper Genes. Modifications are possible and the considerations include:92 ME149651883v.1135975-54620 (REGN 11546) (1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV1; (2) Plasmids pRC-FM and pHC-SCM can be modified to use other specific binding pairs. The first member (FM) and the second cognate member (SCM) should be complementary in order to bond. (3) Plasmids pHC-SCM and pLC can be modified to use other Retargeting Molecules. For example, the heavy chain (HC) and the light chain (LC) should be complementary in order form a functional Retargeting Molecule. (4) Detargeting mutations can be undertaken as disclosed herein and in publications. The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein:93 ME149651883v.1135975-54620 (REGN 11546)

[0240] EXAMPLE 14 – AAV2 Covalently surface modified AAV2 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and then harvesting the covalently surface modified AAV2. The first chart below uses SpyTag-SpyCatcher as specific binding pairs, and Adenovirus Helper Genes. Modifications are possible and the considerations include:94 ME149651883v.1135975-54620 (REGN 11546)(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV2; (2) Plasmids pRC-FM and pHC-SCM can be modified to use other specific binding pairs. The first member (FM) and the second cognate member (SCM) should be complementary in order to bond. (3) Plasmids pHC-SCM and pLC can be modified to use other Retargeting Molecules. For example, the heavy chain (HC) and the light chain (LC) should be complementary in order form a functional Retargeting Molecule. (4) Detargeting mutations can be undertaken as disclosed herein and in publications. The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein:95 ME149651883v.1135975-54620 (REGN 11546)Serotype 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: 189ra76." Gene therapy restores vision in a canine model of childhood blindness. Nat Genet 28.1 (2001): 92-5). Serotype AAV2-4Y-F: AAV2 Quad Y-F can be used and is referred to a modified AAV2 comprising 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).

[0241] EXAMPLE 15 – AAV3 Covalently surface modified AAV3 can be made by transfecting 96 ME149651883v.1135975-54620 (REGN 11546) a cell with plasmids as set forth below, followed by culturing the transfected cells and then harvesting the covalently surface modified AAV3. The first chart below uses SpyTag-SpyCatcher as specific binding pairs, and Adenovirus Helper Genes. Modifications are possible and the considerations include:(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV1; 97 ME149651883v.1135975-54620 (REGN 11546) (2) Plasmids pRC-FM and pHC-SCM can be modified to use other specific binding pairs. The first member (FM) and the second cognate member (SCM) should be complementary in order to bond. (3) Plasmids pHC-SCM and pLC can be modified to use other Retargeting Molecules. For example, the heavy chain (HC) and the light chain (LC) should be complementary in order form a functional Retargeting Molecule. (4) Detargeting mutations can be undertaken as disclosed herein and in publications. The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein:98 ME149651883v.1135975-54620 (REGN 11546)

[0242] EXAMPLE 16 – AAV4 Covalently surface modified AAV4 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and then harvesting the covalently surface modified AAV4. The first chart below uses SpyTag-SpyCatcher as specific binding pairs, and Adenovirus Helper Genes. Modifications are possible and the considerations include:99 ME149651883v.1135975-54620 (REGN 11546)(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV4; (2) Plasmids pRC-FM and pHC-SCM can be modified to use other specific binding pairs. The first member (FM) and the second cognate member (SCM) should be complementary in order to bond. (3) Plasmids pHC-SCM and pLC can be modified to use other Retargeting Molecules. For example, the heavy chain (HC) and the light chain (LC) should be complementary in order form a functional Retargeting Molecule. (4) Detargeting mutations can be undertaken as disclosed herein and in publications. The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein:100 ME149651883v.1135975-54620 (REGN 11546)

[0243] EXAMPLE 17 – AAV5 Covalently surface modified AAV5 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and then harvesting the covalently surface modified AAV5. The first chart below uses SpyTag-SpyCatcher as specific binding pairs, and Adenovirus Helper Genes. Modifications are possible and the considerations include:101 ME149651883v.1135975-54620 (REGN 11546)(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV1; (2) Plasmids pRC-FM and pHC-SCM can be modified to use other specific binding pairs. The first member (FM) and the second cognate member (SCM) should be complementary in order to bond. (3) Plasmids pHC-SCM and pLC can be modified to use other Retargeting Molecules. For example, the heavy chain (HC) and the light chain (LC) should be complementary in order form a functional Retargeting Molecule. (4) Detargeting mutations can be undertaken as disclosed herein and in publications. The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein:102 ME149651883v.1135975-54620 (REGN 11546)

[0244] EXAMPLE 18 – AAV6 Covalently surface modified AAV6 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and then harvesting the covalently surface modified AAV6. The first chart below uses SpyTag-SpyCatcher as specific binding pairs, and Adenovirus Helper Genes. Modifications are possible and the considerations include:103 ME149651883v.1135975-54620 (REGN 11546)(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV1; (2) Plasmids pRC-FM and pHC-SCM can be modified to use other specific binding pairs. The first member (FM) and the second cognate member (SCM) should be complementary in order to bond. (3) Plasmids pHC-SCM and pLC can be modified to use other Retargeting Molecules. For example, the heavy chain (HC) and the light chain (LC) should be complementary in order form a functional Retargeting Molecule. (4) Detargeting mutations can be undertaken as disclosed herein and in publications. The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein: 104 ME149651883v.1135975-54620 (REGN 11546)

[0245] EXAMPLE 19 – AAV7 Covalently surface modified AAV7 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and then harvesting the covalently surface modified AAV7. The first chart below uses SpyTag-SpyCatcher as specific binding pairs, and Adenovirus Helper Genes. Modifications are possible and the considerations include:105 ME149651883v.1135975-54620 (REGN 11546)(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV7; (2) Plasmids pRC-FM and pHC-SCM can be modified to use other specific binding pairs. The first member (FM) and the second cognate member (SCM) should be complementary in order to bond. (3) Plasmids pHC-SCM and pLC can be modified to use other Retargeting Molecules. For example, the heavy chain (HC) and the light chain (LC) should be complementary in order form a functional Retargeting Molecule. 106 ME149651883v.1135975-54620 (REGN 11546) (4) Detargeting mutations can be undertaken as disclosed herein and in publications. The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein:

[0246] EXAMPLE 20 – AAV8 Covalently surface modified AAV8 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and then harvesting the covalently surface modified AAV8. The first chart below uses SpyTag-SpyCatcher as specific binding pairs, and Adenovirus Helper Genes. 107 ME149651883v.1135975-54620 (REGN 11546) Modifications are possible and the considerations include:(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV8; (2) Plasmids pRC-FM and pHC-SCM can be modified to use other specific binding pairs. The first member (FM) and the second cognate member (SCM) should be complementary in order to bond. 108 ME149651883v.1135975-54620 (REGN 11546) (3) Plasmids pHC-SCM and pLC can be modified to use other Retargeting Molecules. For example, the heavy chain (HC) and the light chain (LC) should be complementary in order form a functional Retargeting Molecule. (4) Detargeting mutations can be undertaken as disclosed herein and in publications. The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein:

[0247] EXAMPLE 21 – AAV9 Covalently surface modified AAV9 can be made by transfecting 109 ME149651883v.1135975-54620 (REGN 11546) a cell with plasmids as set forth below, followed by culturing the transfected cells and then harvesting the covalently surface modified AAV9. The first chart below uses SpyTag-SpyCatcher as specific binding pairs, and Adenovirus Helper Genes. Modifications are possible and the considerations include:(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV9; 110 ME149651883v.1135975-54620 (REGN 11546) (2) Plasmids pRC-FM and pHC-SCM can be modified to use other specific binding pairs. The first member (FM) and the second cognate member (SCM) should be complementary in order to bond. (3) Plasmids pHC-SCM and pLC can be modified to use other Retargeting Molecules. For example, the heavy chain (HC) and the light chain (LC) should be complementary in order form a functional Retargeting Molecule. (4) Detargeting mutations can be undertaken as disclosed herein and in publications. The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein:111 ME149651883v.1135975-54620 (REGN 11546)

[0248] EXAMPLE 22 – AAV10 Covalently surface modified AAV10 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and then harvesting the covalently surface modified AAV10. The first chart below uses SpyTag-SpyCatcher as specific binding pairs, and Adenovirus Helper Genes. Modifications are possible and the considerations include:112 ME149651883v.1135975-54620 (REGN 11546)(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV10; (2) Plasmids pRC-FM and pHC-SCM can be modified to use other specific binding pairs. The first member (FM) and the second cognate member (SCM) should be complementary in order to bond. (3) Plasmids pHC-SCM and pLC can be modified to use other Retargeting Molecules. For example, the heavy chain (HC) and the light chain (LC) should be complementary in order form a functional Retargeting Molecule. (4) Detargeting mutations can be undertaken as disclosed herein and in publications. The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein:113 ME149651883v.1135975-54620 (REGN 11546)

[0249] EXAMPLE 23 – AAV11 Covalently surface modified AAV11 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and then harvesting the covalently surface modified AAV11. The first chart below uses SpyTag-SpyCatcher as specific binding pairs, and Adenovirus Helper Genes. Modifications are possible and the considerations include:114 ME149651883v.1135975-54620 (REGN 11546)(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV7; (2) Plasmids pRC-FM and pHC-SCM can be modified to use other specific binding pairs. The first member (FM) and the second cognate member (SCM) should be complementary in order to bond. (3) Plasmids pHC-SCM and pLC can be modified to use other Retargeting Molecules. For example, the heavy chain (HC) and the light chain (LC) should be complementary in order form a functional Retargeting Molecule. (4) Detargeting mutations can be undertaken as disclosed herein and in publications. The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein:115 ME149651883v.1135975-54620 (REGN 11546)

[0250] EXAMPLE 24 – AAV12 Covalently surface modified AAV12 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and then harvesting the covalently surface modified AAV12. The first chart below uses SpyTag-SpyCatcher as specific binding pairs, and Adenovirus Helper Genes. Modifications are possible and the considerations include:116 ME149651883v.1135975-54620 (REGN 11546)(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV12; (2) Plasmids pRC-FM and pHC-SCM can be modified to use other specific binding pairs. The first member (FM) and the second cognate member (SCM) should be complementary in order to bond. (3) Plasmids pHC-SCM and pLC can be modified to use other Retargeting Molecules. For example, the heavy chain (HC) and the light chain (LC) should be complementary in order form a functional Retargeting Molecule. (4) Detargeting mutations can be undertaken as disclosed herein and in publications. The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein: 117 ME149651883v.1135975-54620 (REGN 11546)

[0251] EXAMPLE 25 – AAV13 Covalently surface modified AAV13 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and then harvesting the covalently surface modified AAV13. The first chart below uses SpyTag-SpyCatcher as specific binding pairs, and Adenovirus Helper Genes. Modifications are possible and the considerations include:118 ME149651883v.1135975-54620 (REGN 11546)(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV13; (2) Plasmids pRC-FM and pHC-SCM can be modified to use other specific binding pairs. The first member (FM) and the second cognate member (SCM) should be complementary in order to bond. (3) Plasmids pHC-SCM and pLC can be modified to use other Retargeting Molecules. For example, the heavy chain (HC) and the light chain (LC) should be complementary in order form a functional Retargeting Molecule. 119 ME149651883v.1135975-54620 (REGN 11546) (4) Detargeting mutations can be undertaken as disclosed herein and in publications. The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein:

[0252] EXAMPLE 26 – AAV rh10 Covalently surface modified AAV rh10 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and then harvesting the covalently surface modified AAV rh10. 120 ME149651883v.1135975-54620 (REGN 11546) The first chart below uses SpyTag-SpyCatcher as specific binding pairs, and Adenovirus Helper Genes. Modifications are possible and the considerations include:(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV rh10; 121 ME149651883v.1135975-54620 (REGN 11546) (2) Plasmids pRC-FM and pHC-SCM can be modified to use other specific binding pairs. The first member (FM) and the second cognate member (SCM) should be complementary in order to bond. (3) Plasmids pHC-SCM and pLC can be modified to use other Retargeting Molecules. For example, the heavy chain (HC) and the light chain (LC) should be complementary in order form a functional Retargeting Molecule. (4) Detargeting mutations can be undertaken as disclosed herein and in publications. The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein:122 ME149651883v.1135975-54620 (REGN 11546)

[0253] EXAMPLE 27 – AAV rh39 Covalently surface modified AAV rh39 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and then harvesting the covalently surface modified AAV rh39. The first chart below uses SpyTag-SpyCatcher as specific binding pairs, and Adenovirus Helper Genes. Modifications are possible and the considerations include:123 ME149651883v.1135975-54620 (REGN 11546)(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV rh39; (2) Plasmids pRC-FM and pHC-SCM can be modified to use other specific binding pairs. The first member (FM) and the second cognate member (SCM) should be complementary in order to bond. (3) Plasmids pHC-SCM and pLC can be modified to use other Retargeting Molecules. For example, the heavy chain (HC) and the light chain (LC) should be complementary in order form a functional Retargeting Molecule. (4) Detargeting mutations can be undertaken as disclosed herein and in publications. The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein:124 ME149651883v.1135975-54620 (REGN 11546)

[0254] EXAMPLE 28 – AAV rh43 Covalently surface modified AAV rh43 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and then harvesting the covalently surface modified AAV rh43. The first chart below uses SpyTag-SpyCatcher as specific binding pairs, and Adenovirus Helper Genes. Modifications are possible and the considerations include:125 ME149651883v.1135975-54620 (REGN 11546)(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV rh43; (2) Plasmids pRC-FM and pHC-SCM can be modified to use other specific binding pairs. The first member (FM) and the second cognate member (SCM) should be complementary in order to bond. (3) Plasmids pHC-SCM and pLC can be modified to use other Retargeting Molecules. For example, the heavy chain (HC) and the light chain (LC) should be complementary in order form a functional Retargeting Molecule. (4) Detargeting mutations can be undertaken as disclosed herein and in publications. The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein:126 ME149651883v.1135975-54620 (REGN 11546)

[0255] EXAMPLE 29 – AAV rh74 Covalently surface modified AAV rh74 can be made by transfecting a cell with plasmids as set forth below, followed by culturing the transfected cells and then harvesting the covalently surface modified AAV rh74. The first chart below uses SpyTag-SpyCatcher as specific binding pairs, and Adenovirus Helper Genes. Modifications are possible and the considerations include:127 ME149651883v.1135975-54620 (REGN 11546)(1) Plasmid pGOI can comprise one or more genes that are desired to be expressed in covalently surface modified AAV rh74; (2) Plasmids pRC-FM and pHC-SCM can be modified to use other specific binding pairs. The first member (FM) and the second cognate member (SCM) should be complementary in order to bond. (3) Plasmids pHC-SCM and pLC can be modified to use other Retargeting Molecules. For example, the heavy chain (HC) and the light chain (LC) should be complementary in order form a functional Retargeting Molecule. (4) Detargeting mutations can be undertaken as disclosed herein and in publications. The second chart below cites to exemplary modifications to the above chart based upon the descriptions and sequence examples contained herein: 128 ME149651883v.1135975-54620 (REGN 11546)SEQUENCE EXAMPLES

[0256] The following examples set forth exemplary sequences, which are optional for use and do not limit the inventions in any manner. Other AAV sequences, helper sequences, Specific Binding Pair sequences, and DNA Barcode sequences are available and accessible. Gene of interest sequences and retargeting molecule sequences can be selected based upon the purpose of the covalently surface modified AAV and the cell / tissue to be targeted. 129 ME149651883v.1135975-54620 (REGN 11546) EXAMPLE 30 – AAV Polynucleotide Sequences

[0257] AAV Rep, Cap and ITR sequences are known in the art. The present inventions are amenable to all AAV serotypes. AAV sequences from various AAV serotypes are set forth below. Many of these sequences are available from the National Center for Biotechnology Information (NCBI). AAV-1 Full Genome: NC_002077 CapVP1: (SEQ ID NO: 1) ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACTTG AAACCTGGAGCCCCGAAGCCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTAC AAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCAC GACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTT CAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTT CTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAGACGGCTCCTGGAAAGAAACGTCCGGTAGAGCAGTCGCCA CAAGAGCCAGACTCCTCCTCGGGCATCGGCAAGACAGGCCAGCAGCCCGCTAAAAAGAGACTCAATTTTGGTCAG ACTGGCGACTCAGAGTCAGTCCCCGATCCACAACCTCTCGGAGAACCTCCAGCAACCCCCGCTGCTGTGGGACCT ACTACAATGGCTTCAGGCGGTGGCGCACCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAATGCCTCA GGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGCACCTGGGCCTTGCCC ACCTACAATAACCACCTCTACAAGCAAATCTCCAGTGCTTCAACGGGGGCCAGCAACGACAACCACTACTTCGGC TACAGCACCCCCTGGGGGTATTTTGATTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAGCGACTC ATCAACAACAATTGGGGATTCCGGCCCAAGAGACTCAACTTCAAACTCTTCAACATCCAAGTCAAGGAGGTCACG ACGAATGATGGCGTCACAACCATCGCTAATAACCTTACCAGCACGGTTCAAGTCTTCTCGGACTCGGAGTACCAG CTTCCGTACGTCCTCGGCTCTGCGCACCAGGGCTGCCTCCCTCCGTTCCCGGCGGACGTGTTCATGATTCCGCAA TACGGCTACCTGACGCTCAACAATGGCAGCCAAGCCGTGGGACGTTCATCCTTTTACTGCCTGGAATATTTCCCT TCTCAGATGCTGAGAACGGGCAACAACTTTACCTTCAGCTACACCTTTGAGGAAGTGCCTTTCCACAGCAGCTAC GCGCACAGCCAGAGCCTGGACCGGCTGATGAATCCTCTCATCGACCAATACCTGTATTACCTGAACAGAACTCAA AATCAGTCCGGAAGTGCCCAAAACAAGGACTTGCTGTTTAGCCGTGGGTCTCCAGCTGGCATGTCTGTTCAGCCC AAAAACTGGCTACCTGGACCCTGTTATCGGCAGCAGCGCGTTTCTAAAACAAAAACAGACAACAACAACAGCAAT TTTACCTGGACTGGTGCTTCAAAATATAACCTCAATGGGCGTGAATCCATCATCAACCCTGGCACTGCTATGGCC TCACACAAAGACGACGAAGACAAGTTCTTTCCCATGAGCGGTGTCATGATTTTTGGAAAAGAGAGCGCCGGAGCT TCAAACACTGCATTGGACAATGTCATGATTACAGACGAAGAGGAAATTAAAGCCACTAACCCTGTGGCCACCGAA AGATTTGGGACCGTGGCAGTCAATTTCCAGAGCAGCAGCACAGACCCTGCGACCGGAGATGTGCATGCTATGGGA GCATTACCTGGCATGGTGTGGCAAGATAGAGACGTGTACCTGCAGGGTCCCATTTGGGCCAAAATTCCTCACACA GATGGACACTTTCACCCGTCTCCTCTTATGGGCGGCTTTGGACTCAAGAACCCGCCTCCTCAGATCCTCATCAAA AACACGCCTGTTCCTGCGAATCCTCCGGCGGAGTTTTCAGCTACAAAGTTTGCTTCATTCATCACCCAATACTCC ACAGGACAAGTGAGTGTGGAAATTGAATGGGAGCTGCAGAAAGAAAACAGCAAGCGCTGGAATCCCGAAGTGCAG TACACATCCAATTATGCAAAATCTGCCAACGTTGATTTTACTGTGGACAACAATGGACTTTATACTGAGCCTCGC CCCATTGGCACCCGTTACCTTACCCGTCCCCTGTAA Rep78: (SEQ ID NO: 2) ATGCCGGGCTTCTACGAGATCGTGATCAAGGTGCCGAGCGACCTGGACGAGCACCTGCCGGGCATTTCTGACTCG TTTGTGAGCTGGGTGGCCGAGAAGGAATGGGAGCTGCCCCCGGATTCTGACATGGATCTGAATCTGATTGAGCAG GCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTCCTGGTCCAATGGCGCCGCGTGAGTAAGGCCCCGGAG GCCCTCTTCTTTGTTCAGTTCGAGAAGGGCGAGTCCTACTTCCACCTCCATATTCTGGTGGAGACCACGGGGGTC AAATCCATGGTGCTGGGCCGCTTCCTGAGTCAGATTAGGGACAAGCTGGTGCAGACCATCTACCGCGGGATCGAG CCGACCCTGCCCAACTGGTTCGCGGTGACCAAGACGCGTAATGGCGCCGGAGGGGGGAACAAGGTGGTGGACGAG TGCTACATCCCCAACTACCTCCTGCCCAAGACTCAGCCCGAGCTGCAGTGGGCGTGGACTAACATGGAGGAGTAT ATAAGCGCCTGTTTGAACCTGGCCGAGCGCAAACGGCTCGTGGCGCAGCACCTGACCCACGTCAGCCAGACCCAG GAGCAGAACAAGGAGAATCTGAACCCCAATTCTGACGCGCCTGTCATCCGGTCAAAAACCTCCGCGCGCTACATG 130 ME149651883v.1135975-54620 (REGN 11546) GAGCTGGTCGGGTGGCTGGTGGACCGGGGCATCACCTCCGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCGTAC ATCTCCTTCAACGCCGCTTCCAACTCGCGGTCCCAGATCAAGGCCGCTCTGGACAATGCCGGCAAGATCATGGCG CTGACCAAATCCGCGCCCGACTACCTGGTAGGCCCCGCTCCGCCCGCGGACATTAAAACCAACCGCATCTACCGC ATCCTGGAGCTGAACGGCTACGAACCTGCCTACGCCGGCTCCGTCTTTCTCGGCTGGGCCCAGAAAAGGTTCGGG AAGCGCAACACCATCTGGCTGTTTGGGCCGGCCACCACGGGCAAGACCAACATCGCGGAAGCCATCGCCCACGCC GTGCCCTTCTACGGCTGCGTCAACTGGACCAATGAGAACTTTCCCTTCAATGATTGCGTCGACAAGATGGTGATC TGGTGGGAGGAGGGCAAGATGACGGCCAAGGTCGTGGAGTCCGCCAAGGCCATTCTCGGCGGCAGCAAGGTGCGC GTGGACCAAAAGTGCAAGTCGTCCGCCCAGATCGACCCCACCCCCGTGATCGTCACCTCCAACACCAACATGTGC GCCGTGATTGACGGGAACAGCACCACCTTCGAGCACCAGCAGCCGTTGCAGGACCGGATGTTCAAATTTGAACTC ACCCGCCGTCTGGAGCATGACTTTGGCAAGGTGACAAAGCAGGAAGTCAAAGAGTTCTTCCGCTGGGCGCAGGAT CACGTGACCGAGGTGGCGCATGAGTTCTACGTCAGAAAGGGTGGAGCCAACAAAAGACCCGCCCCCGATGACGCG GATAAAAGCGAGCCCAAGCGGGCCTGCCCCTCAGTCGCGGATCCATCGACGTCAGACGCGGAAGGAGCTCCGGTG GACTTTGCCGACAGGTACCAAAACAAATGTTCTCGTCACGCGGGCATGCTTCAGATGCTGTTTCCCTGCAAGACA TGCGAGAGAATGAATCAGAATTTCAACATTTGCTTCACGCACGGGACGAGAGACTGTTCAGAGTGCTTCCCCGGC GTGTCAGAATCTCAACCGGTCGTCAGAAAGAGGACGTATCGGAAACTCTGTGCCATTCATCATCTGCTGGGGCGG GCTCCCGAGATTGCTTGCTCGGCCTGCGATCTGGTCAACGTGGACCTGGATGACTGTGTTTCTGAGCAATAA AAV-2 Full Genome: NC_001401 Rep78: (SEQ ID NO: 3) ATGCCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCCGGCATTTCTGACAGC TTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGATTGAGCAG GCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGGAG GCCCTTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCACATGCACGTGCTCGTGGAAACCACCGGGGTG AAATCCATGGTTTTGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGAG CCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGAACAAGGTGGTGGATGAG TGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGGCGTGGACTAATATGGAACAGTAT TTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAG GAGCAGAACAAAGAGAATCAGAATCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTACATG GAGCTGGTCGGGTGGCTCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATAC ATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGC CTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTATAAA ATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTTCGGC AAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACT GTGCCCTTCTACGGGTGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATC TGGTGGGAGGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTGCGC GTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCTCCAACACCAACATGTGC GCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTTGAACTC ACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGAT CACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCA GATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAAC TACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGC GAGAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCA GAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGTG CCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAATAA Rep52: (SEQ ID NO: 4) ATGGAGCTGGTCGGGTGGCTCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCA TACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAAGATTATG AGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTAT AAAATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTTC GGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCAC ACTGTGCCCTTCTACGGGTGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTG ATCTGGTGGGAGGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTG CGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCTCCAACACCAACATG TGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTTGAA 131 ME149651883v.1135975-54620 (REGN 11546) CTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAG GATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGAC GCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATC AACTACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAA TGCGAGAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTG TCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAG GTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAATAA CapVP1: (SEQ ID NO: 5) ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTC AAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTAC AAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCAC GACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTT CAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT CTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCT GTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAG ACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACT AATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCG GGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCC ACCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTAC AGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATC AACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAG AATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTC CCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTAT GGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCT CAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCT CACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACT CCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGG AACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATAC TCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGC CACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACA AATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAG TATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTT CTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGAC GGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAAC ACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACG GGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTAC ACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCC ATTGGCACCAGATACCTGACTCGTAATCTGTAA CapVP2: (SEQ ID NO: 6) ACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCG GGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCT CTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCA GACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGAC AGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAACAAATTTCCAGC CAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTC CACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAAC TTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAACCTTACC AGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTC CCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTA GGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGC TACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTC ATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTT TCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGA GTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGC AGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGC GGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAA 132 ME149651883v.1135975-54620 (REGN 11546) GAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAAC AGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTAC CTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTC GGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGT GCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAG AAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTT ACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA CapVP3: (SEQ ID NO:7) ATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAAT TGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTAC AACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACC CCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAAC AACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGAC GGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTAC GTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATAC CTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATG CTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGC CAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGT GGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGG CTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGG ACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAG GACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTG GACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGT TCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCA GGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACAT TTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCG GTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAG GTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCC AACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGC ACCAGATACCTGACTCGTAATCTGTAA CapAAP: (SEQ ID NO:8) CTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTA ATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGG GAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCA CCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACA GCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCA ACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGA ATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCC CGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATG GATACCTCACCCTGA AAV-3 Full Genome: NC_001729 Rep78: (SEQ ID NO:9) ATGCCGGGGTTCTACGAGATTGTCCTGAAGGTCCCGAGTGACCTGGACGAGCGCCTGCCGGGCATTTCTAACTCG TTTGTTAACTGGGTGGCCGAGAAGGAATGGGACGTGCCGCCGGATTCTGACATGGATCCGAATCTGATTGAGCAG GCACCCCTGACCGTGGCCGAAAAGCTTCAGCGCGAGTTCCTGGTGGAGTGGCGCCGCGTGAGTAAGGCCCCGGAG GCCCTCTTTTTTGTCCAGTTCGAAAAGGGGGAGACCTACTTCCACCTGCACGTGCTGATTGAGACCATCGGGGTC AAATCCATGGTGGTCGGCCGCTACGTGAGCCAGATTAAAGAGAAGCTGGTGACCCGCATCTACCGCGGGGTCGAG CCGCAGCTTCCGAACTGGTTCGCGGTGACCAAAACGCGAAATGGCGCCGGGGGCGGGAACAAGGTGGTGGACGAC TGCTACATCCCCAACTACCTGCTCCCCAAGACCCAGCCCGAGCTCCAGTGGGCGTGGACTAACATGGACCAGTAT TTAAGCGCCTGTTTGAATCTCGCGGAGCGTAAACGGCTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAG GAGCAGAACAAAGAGAATCAGAACCCCAATTCTGACGCGCCGGTCATCAGGTCAAAAACCTCAGCCAGGTACATG GAGCTGGTCGGGTGGCTGGTGGACCGCGGGATCACGTCAGAAAAGCAATGGATTCAGGAGGACCAGGCCTCGTAC 133 ME149651883v.1135975-54620 (REGN 11546) ATCTCCTTCAACGCCGCCTCCAACTCGCGGTCCCAGATCAAGGCCGCGCTGGACAATGCCTCCAAGATCATGAGC CTGACAAAGACGGCTCCGGACTACCTGGTGGGCAGCAACCCGCCGGAGGACATTACCAAAAATCGGATCTACCAA ATCCTGGAGCTGAACGGGTACGATCCGCAGTACGCGGCCTCCGTCTTCCTGGGCTGGGCGCAAAAGAAGTTCGGG AAGAGGAACACCATCTGGCTCTTTGGGCCGGCCACGACGGGTAAAACCAACATCGCGGAAGCCATCGCCCACGCC GTGCCCTTCTACGGCTGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGATTGCGTCGACAAGATGGTGATC TGGTGGGAGGAGGGCAAGATGACGGCCAAGGTCGTGGAGAGCGCCAAGGCCATTCTGGGCGGAAGCAAGGTGCGC GTGGACCAAAAGTGCAAGTCATCGGCCCAGATCGAACCCACTCCCGTGATCGTCACCTCCAACACCAACATGTGC GCCGTGATTGACGGGAACAGCACCACCTTCGAGCATCAGCAGCCGCTGCAGGACCGGATGTTTGAATTTGAACTT ACCCGCCGTTTGGACCATGACTTTGGGAAGGTCACCAAACAGGAAGTAAAGGACTTTTTCCGGTGGGCTTCCGAT CACGTGACTGACGTGGCTCATGAGTTCTACGTCAGAAAGGGTGGAGCTAAGAAACGCCCCGCCTCCAATGACGCG GATGTAAGCGAGCCAAAACGGGAGTGCACGTCACTTGCGCAGCCGACAACGTCAGACGCGGAAGCACCGGCGGAC TACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTTTTTCCCTGTAAAACATGC GAGAGAATGAATCAAATTTCCAATGTCTGTTTTACGCATGGTCAAAGAGACTGTGGGGAATGCTTCCCTGGAATG TCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGAAGACTTATCAGAAACTGTGTCCAATTCATCATATCCTGGGA AGGGCACCCGAGATTGCCTGTTCGGCCTGCGATTTGGCCAATGTGGACTTGGATGACTGTGTTTCTGAGCAATAA CapVP1: (SEQ ID NO:10) ATGGCTGCTGACGGTTATCTTCCAGATTGGCTCGAGGACAACCTTTCTGAAGGCATTCGTGAGTGGTGGGCTCTG AAACCTGGAGTCCCTCAACCCAAAGCGAACCAACAACACCAGGACAACCGTCGGGGTCTTGTGCTTCCGGGTTAC AAATACCTCGGACCCGGTAACGGACTCGACAAAGGAGAGCCGGTCAACGAGGCGGACGCGGCAGCCCTCGAACAC GACAAAGCTTACGACCAGCAGCTCAAGGCCGGTGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTT CAGGAGCGTCTTCAAGAAGATACGTCTTTTGGGGGCAACCTTGGCAGAGCAGTCTTCCAGGCCAAAAAGAGGATC CTTGAGCCTCTTGGTCTGGTTGAGGAAGCAGCTAAAACGGCTCCTGGAAAGAAGGGGGCTGTAGATCAGTCTCCT CAGGAACCGGACTCATCATCTGGTGTTGGCAAATCGGGCAAACAGCCTGCCAGAAAAAGACTAAATTTCGGTCAG ACTGGAGACTCAGAGTCAGTCCCAGACCCTCAACCTCTCGGAGAACCACCAGCAGCCCCCACAAGTTTGGGATCT AATACAATGGCTTCAGGCGGTGGCGCACCAATGGCAGACAATAACGAGGGTGCCGATGGAGTGGGTAATTCCTCA GGAAATTGGCATTGCGATTCCCAATGGCTGGGCGACAGAGTCATCACCACCAGCACCAGAACCTGGGCCCTGCCC ACTTACAACAACCATCTCTACAAGCAAATCTCCAGCCAATCAGGAGCTTCAAACGACAACCACTACTTTGGCTAC AGCACCCCTTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATT AACAACAACTGGGGATTCCGGCCCAAGAAACTCAGCTTCAAGCTCTTCAACATCCAAGTTAGAGGGGTCACGCAG AACGATGGCACGACGACTATTGCCAATAACCTTACCAGCACGGTTCAAGTGTTTACGGACTCGGAGTATCAGCTC CCGTACGTGCTCGGGTCGGCGCACCAAGGCTGTCTCCCGCCGTTTCCAGCGGACGTCTTCATGGTCCCTCAGTAT GGATACCTCACCCTGAACAACGGAAGTCAAGCGGTGGGACGCTCATCCTTTTACTGCCTGGAGTACTTCCCTTCG CAGATGCTAAGGACTGGAAATAACTTCCAATTCAGCTATACCTTCGAGGATGTACCTTTTCACAGCAGCTACGCT CACAGCCAGAGTTTGGATCGCTTGATGAATCCTCTTATTGATCAGTATCTGTACTACCTGAACAGAACGCAAGGA ACAACCTCTGGAACAACCAACCAATCACGGCTGCTTTTTAGCCAGGCTGGGCCTCAGTCTATGTCTTTGCAGGCC AGAAATTGGCTACCTGGGCCCTGCTACCGGCAACAGAGACTTTCAAAGACTGCTAACGACAACAACAACAGTAAC TTTCCTTGGACAGCGGCCAGCAAATATCATCTCAATGGCCGCGACTCGCTGGTGAATCCAGGACCAGCTATGGCC AGTCACAAGGACGATGAAGAAAAATTTTTCCCTATGCACGGCAATCTAATATTTGGCAAAGAAGGGACAACGGCA AGTAACGCAGAATTAGATAATGTAATGATTACGGATGAAGAAGAGATTCGTACCACCAATCCTGTGGCAACAGAG CAGTATGGAACTGTGGCAAATAACTTGCAGAGCTCAAATACAGCTCCCACGACTGGAACTGTCAATCATCAGGGG GCCTTACCTGGCATGGTGTGGCAAGATCGTGACGTGTACCTTCAAGGACCTATCTGGGCAAAGATTCCTCACACG GATGGACACTTTCATCCTTCTCCTCTGATGGGAGGCTTTGGACTGAAACATCCGCCTCCTCAAATCATGATCAAA AATACTCCGGTACCGGCAAATCCTCCGACGACTTTCAGCCCGGCCAAGTTTGCTTCATTTATCACTCAGTACTCC ACTGGACAGGTCAGCGTGGAAATTGAGTGGGAGCTACAGAAAGAAAACAGCAAACGTTGGAATCCAGAGATTCAG TACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTAGACACTAATGGTGTTTATAGTGAACCTCGC CCTATTGGAACCCGGTATCTCACACGAAACTTGTGA AAV-4 Full Genome: NC_001829 Rep78: (SEQ ID NO:11) ATGCCGGGGTTCTACGAGATCGTGCTGAAGGTGCCCAGCGACCTGGACGAGCACCTGCCCGGCATTTCTGACTCT TTTGTGAGCTGGGTGGCCGAGAAGGAATGGGAGCTGCCGCCGGATTCTGACATGGACTTGAATCTGATTGAGCAG GCACCCCTGACCGTGGCCGAAAAGCTGCAACGCGAGTTCCTGGTCGAGTGGCGCCGCGTGAGTAAGGCCCCGGAG GCCCTCTTCTTTGTCCAGTTCGAGAAGGGGGACAGCTACTTCCACCTGCACATCCTGGTGGAGACCGTGGGCGTC AAATCCATGGTGGTGGGCCGCTACGTGAGCCAGATTAAAGAGAAGCTGGTGACCCGCATCTACCGCGGGGTCGAG 134 ME149651883v.1135975-54620 (REGN 11546) CCGCAGCTTCCGAACTGGTTCGCGGTGACCAAGACGCGTAATGGCGCCGGAGGCGGGAACAAGGTGGTGGACGAC TGCTACATCCCCAACTACCTGCTCCCCAAGACCCAGCCCGAGCTCCAGTGGGCGTGGACTAACATGGACCAGTAT ATAAGCGCCTGTTTGAATCTCGCGGAGCGTAAACGGCTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAG GAGCAGAACAAGGAAAACCAGAACCCCAATTCTGACGCGCCGGTCATCAGGTCAAAAACCTCCGCCAGGTACATG GAGCTGGTCGGGTGGCTGGTGGACCGCGGGATCACGTCAGAAAAGCAATGGATCCAGGAGGACCAGGCGTCCTAC ATCTCCTTCAACGCCGCCTCCAACTCGCGGTCACAAATCAAGGCCGCGCTGGACAATGCCTCCAAAATCATGAGC CTGACAAAGACGGCTCCGGACTACCTGGTGGGCCAGAACCCGCCGGAGGACATTTCCAGCAACCGCATCTACCGA ATCCTCGAGATGAACGGGTACGATCCGCAGTACGCGGCCTCCGTCTTCCTGGGCTGGGCGCAAAAGAAGTTCGGG AAGAGGAACACCATCTGGCTCTTTGGGCCGGCCACGACGGGTAAAACCAACATCGCGGAAGCCATCGCCCACGCC GTGCCCTTCTACGGCTGCGTGAACTGGACCAATGAGAACTTTCCGTTCAACGATTGCGTCGACAAGATGGTGATC TGGTGGGAGGAGGGCAAGATGACGGCCAAGGTCGTAGAGAGCGCCAAGGCCATCCTGGGCGGAAGCAAGGTGCGC GTGGACCAAAAGTGCAAGTCATCGGCCCAGATCGACCCAACTCCCGTGATCGTCACCTCCAACACCAACATGTGC GCGGTCATCGACGGAAACTCGACCACCTTCGAGCACCAACAACCACTCCAGGACCGGATGTTCAAGTTCGAGCTC ACCAAGCGCCTGGAGCACGACTTTGGCAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCGTCAGAT CACGTGACCGAGGTGACTCACGAGTTTTACGTCAGAAAGGGTGGAGCTAGAAAGAGGCCCGCCCCCAATGACGCA GATATAAGTGAGCCCAAGCGGGCCTGTCCGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTCCGGTGGAC TACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGTATGAATCTGATGCTTTTTCCCTGCCGGCAATGC GAGAGAATGAATCAGAATGTGGACATTTGCTTCACGCACGGGGTCATGGACTGTGCCGAGTGCTTCCCCGTGTCA GAATCTCAACCCGTGTCTGTCGTCAGAAAGCGGACGTATCAGAAACTGTGTCCGATTCATCACATCATGGGGAGG GCGCCCGAGGTGGCCTGCTCGGCCTGCGAACTGGCCAATGTGGACTTGGATGACTGTGACATGGAACAATAA CapVP1: (SEQ ID NO:12) ATGACTGACGGTTACCTTCCAGATTGGCTAGAGGACAACCTCTCTGAAGGCGTTCGAGAGTGGTGGGCGCTGCAA CCTGGAGCCCCTAAACCCAAGGCAAATCAACAACATCAGGACAACGCTCGGGGTCTTGTGCTTCCGGGTTACAAA TACCTCGGACCCGGCAACGGACTCGACAAGGGGGAACCCGTCAACGCAGCGGACGCGGCAGCCCTCGAGCACGAC AAGGCCTACGACCAGCAGCTCAAGGCCGGTGACAACCCCTACCTCAAGTACAACCACGCCGACGCGGAGTTCCAG CAGCGGCTTCAGGGCGACACATCGTTTGGGGGCAACCTCGGCAGAGCAGTCTTCCAGGCCAAAAAGAGGGTTCTT GAACCTCTTGGTCTGGTTGAGCAAGCGGGTGAGACGGCTCCTGGAAAGAAGAGACCGTTGATTGAATCCCCCCAG CAGCCCGACTCCTCCACGGGTATCGGCAAAAAAGGCAAGCAGCCGGCTAAAAAGAAGCTCGTTTTCGAAGACGAA ACTGGAGCAGGCGACGGACCCCCTGAGGGATCAACTTCCGGAGCCATGTCTGATGACAGTGAGATGCGTGCAGCA GCTGGCGGAGCTGCAGTCGAGGGCGGACAAGGTGCCGATGGAGTGGGTAATGCCTCGGGTGATTGGCATTGCGAT TCCACCTGGTCTGAGGGCCACGTCACGACCACCAGCACCAGAACCTGGGTCTTGCCCACCTACAACAACCACCTC TACAAGCGACTCGGAGAGAGCCTGCAGTCCAACACCTACAACGGATTCTCCACCCCCTGGGGATACTTTGACTTC AACCGCTTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGCATGCGACCCAAA GCCATGCGGGTCAAAATCTTCAACATCCAGGTCAAGGAGGTCACGACGTCGAACGGCGAGACAACGGTGGCTAAT AACCTTACCAGCACGGTTCAGATCTTTGCGGACTCGTCGTACGAACTGCCGTACGTGATGGATGCGGGTCAAGAG GGCAGCCTGCCTCCTTTTCCCAACGACGTCTTTATGGTGCCCCAGTACGGCTACTGTGGACTGGTGACCGGCAAC ACTTCGCAGCAACAGACTGACAGAAATGCCTTCTACTGCCTGGAGTACTTTCCTTCGCAGATGCTGCGGACTGGC AACAACTTTGAAATTACGTACAGTTTTGAGAAGGTGCCTTTCCACTCGATGTACGCGCACAGCCAGAGCCTGGAC CGGCTGATGAACCCTCTCATCGACCAGTACCTGTGGGGACTGCAATCGACCACCACCGGAACCACCCTGAATGCC GGGACTGCCACCACCAACTTTACCAAGCTGCGGCCTACCAACTTTTCCAACTTTAAAAAGAACTGGCTGCCCGGG CCTTCAATCAAGCAGCAGGGCTTCTCAAAGACTGCCAATCAAAACTACAAGATCCCTGCCACCGGGTCAGACAGT CTCATCAAATACGAGACGCACAGCACTCTGGACGGAAGATGGAGTGCCCTGACCCCCGGACCTCCAATGGCCACG GCTGGACCTGCGGACAGCAAGTTCAGCAACAGCCAGCTCATCTTTGCGGGGCCTAAACAGAACGGCAACACGGCC ACCGTACCCGGGACTCTGATCTTCACCTCTGAGGAGGAGCTGGCAGCCACCAACGCCACCGATACGGACATGTGG GGCAACCTACCTGGCGGTGACCAGAGCAACAGCAACCTGCCGACCGTGGACAGACTGACAGCCTTGGGAGCCGTG CCTGGAATGGTCTGGCAAAACAGAGACATTTACTACCAGGGTCCCATTTGGGCCAAGATTCCTCATACCGATGGA CACTTTCACCCCTCACCGCTGATTGGTGGGTTTGGGCTGAAACACCCGCCTCCTCAAATTTTTATCAAGAACACC CCGGTACCTGCGAATCCTGCAACGACCTTCAGCTCTACTCCGGTAAACTCCTTCATTACTCAGTACAGCACTGGC CAGGTGTCGGTGCAGATTGACTGGGAGATCCAGAAGGAGCGGTCCAAACGCTGGAACCCCGAGGTCCAGTTTACC TCCAACTACGGACAGCAAAACTCTCTGTTGTGGGCTCCCGATGCGGCTGGGAAATACACTGAGCCTAGGGCTATC GGTACCCGCTACCTCACCCACCACCTGTAA AAV-5 Full Genome: NC_006152 Rep78: (SEQ ID NO:13) 135 ME149651883v.1135975-54620 (REGN 11546) ATGGCTACCTTCTATGAAGTCATTGTTCGCGTCCCATTTGACGTGGAGGAACATCTGCCTGGAATTTCTGACAGC TTTGTGGACTGGGTAACTGGTCAAATTTGGGAGCTGCCTCCAGAGTCAGATTTAAATTTGACTCTGGTTGAACAG CCTCAGTTGACGGTGGCTGATAGAATTCGCCGCGTGTTCCTGTACGAGTGGAACAAATTTTCCAAGCAGGAGTCC AAATTCTTTGTGCAGTTTGAAAAGGGATCTGAATATTTTCATCTGCACACGCTTGTGGAGACCTCCGGCATCTCT TCCATGGTCCTCGGCCGCTACGTGAGTCAGATTCGCGCCCAGCTGGTGAAAGTGGTCTTCCAGGGAATTGAACCC CAGATCAACGACTGGGTCGCCATCACCAAGGTAAAGAAGGGCGGAGCCAATAAGGTGGTGGATTCTGGGTATATT CCCGCCTACCTGCTGCCGAAGGTCCAACCGGAGCTTCAGTGGGCGTGGACAAACCTGGACGAGTATAAATTGGCC GCCCTGAATCTGGAGGAGCGCAAACGGCTCGTCGCGCAGTTTCTGGCAGAATCCTCGCAGCGCTCGCAGGAGGCG GCTTCGCAGCGTGAGTTCTCGGCTGACCCGGTCATCAAAAGCAAGACTTCCCAGAAATACATGGCGCTCGTCAAC TGGCTCGTGGAGCACGGCATCACTTCCGAGAAGCAGTGGATCCAGGAAAATCAGGAGAGCTACCTCTCCTTCAAC TCCACCGGCAACTCTCGGAGCCAGATCAAGGCCGCGCTCGACAACGCGACCAAAATTATGAGTCTGACAAAAAGC GCGGTGGACTACCTCGTGGGGAGCTCCGTTCCCGAGGACATTTCAAAAAACAGAATCTGGCAAATTTTTGAGATG AATGGCTACGACCCGGCCTACGCGGGATCCATCCTCTACGGCTGGTGTCAGCGCTCCTTCAACAAGAGGAACACC GTCTGGCTCTACGGACCCGCCACGACCGGCAAGACCAACATCGCGGAGGCCATCGCCCACACTGTGCCCTTTTAC GGCTGCGTGAACTGGACCAATGAAAACTTTCCCTTTAATGACTGTGTGGACAAAATGCTCATTTGGTGGGAGGAG GGAAAGATGACCAACAAGGTGGTTGAATCCGCCAAGGCCATCCTGGGGGGCTCAAAGGTGCGGGTCGATCAGAAA TGTAAATCCTCTGTTCAAATTGATTCTACCCCTGTCATTGTAACTTCCAATACAAACATGTGTGTGGTGGTGGAT GGGAATTCCACGACCTTTGAACACCAGCAGCCGCTGGAGGACCGCATGTTCAAATTTGAACTGACTAAGCGGCTC CCGCCAGATTTTGGCAAGATTACTAAGCAGGAAGTCAAGGACTTTTTTGCTTGGGCAAAGGTCAATCAGGTGCCG GTGACTCACGAGTTTAAAGTTCCCAGGGAATTGGCGGGAACTAAAGGGGCGGAGAAATCTCTAAAACGCCCACTG GGTGACGTCACCAATACTAGCTATAAAAGTCTGGAGAAGCGGGCCAGGCTCTCATTTGTTCCCGAGACGCCTCGC AGTTCAGACGTGACTGTTGATCCCGCTCCTCTGCGACCGCTCAATTGGAATTCAAGGTATGATTGCAAATGTGAC TATCATGCTCAATTTGACAACATTTCTAACAAATGTGATGAATGTGAATATTTGAATCGGGGCAAAAATGGATGT ATCTGTCACAATGTAACTCACTGTCAAATTTGTCATGGGATTCCCCCCTGGGAAAAGGAAAACTTGTCAGATTTT GGGGATTTTGACGATGCCAATAAAGAACAGTAA CapVP1: (SEQ ID NO:14) ATGTCTTTTGTTGATCACCCTCCAGATTGGTTGGAAGAAGTTGGTGAAGGTCTTCGCGAGTTTTTGGGCCTTGAA GCGGGCCCACCGAAACCAAAACCCAATCAGCAGCATCAAGATCAAGCCCGTGGTCTTGTGCTGCCTGGTTATAAC TATCTCGGACCCGGAAACGGTCTCGATCGAGGAGAGCCTGTCAACAGGGCAGACGAGGTCGCGCGAGAGCACGAC ATCTCGTACAACGAGCAGCTTGAGGCGGGAGACAACCCCTACCTCAAGTACAACCACGCGGACGCCGAGTTTCAG GAGAAGCTCGCCGACGACACATCCTTCGGGGGAAACCTCGGAAAGGCAGTCTTTCAGGCCAAGAAAAGGGTTCTC GAACCTTTTGGCCTGGTTGAAGAGGGTGCTAAGACGGCCCCTACCGGAAAGCGGATAGACGACCACTTTCCAAAA AGAAAGAAGGCTCGGACCGAAGAGGACTCCAAGCCTTCCACCTCGTCAGACGCCGAAGCTGGACCCAGCGGATCC CAGCAGCTGCAAATCCCAGCCCAACCAGCCTCAAGTTTGGGAGCTGATACAATGTCTGCGGGAGGTGGCGGCCCA TTGGGCGACAATAACCAAGGTGCCGATGGAGTGGGCAATGCCTCGGGAGATTGGCATTGCGATTCCACGTGGATG GGGGACAGAGTCGTCACCAAGTCCACCCGAACCTGGGTGCTGCCCAGCTACAACAACCACCAGTACCGAGAGATC AAAAGCGGCTCCGTCGACGGAAGCAACGCCAACGCCTACTTTGGATACAGCACCCCCTGGGGGTACTTTGACTTT AACCGCTTCCACAGCCACTGGAGCCCCCGAGACTGGCAAAGACTCATCAACAACTACTGGGGCTTCAGACCCCGG TCCCTCAGAGTCAAAATCTTCAACATTCAAGTCAAAGAGGTCACGGTGCAGGACTCCACCACCACCATCGCCAAC AACCTCACCTCCACCGTCCAAGTGTTTACGGACGACGACTACCAGCTGCCCTACGTCGTCGGCAACGGGACCGAG GGATGCCTGCCGGCCTTCCCTCCGCAGGTCTTTACGCTGCCGCAGTACGGTTACGCGACGCTGAACCGCGACAAC ACAGAAAATCCCACCGAGAGGAGCAGCTTCTTCTGCCTAGAGTACTTTCCCAGCAAGATGCTGAGAACGGGCAAC AACTTTGAGTTTACCTACAACTTTGAGGAGGTGCCCTTCCACTCCAGCTTCGCTCCCAGTCAGAACCTGTTCAAG CTGGCCAACCCGCTGGTGGACCAGTACTTGTACCGCTTCGTGAGCACAAATAACACTGGCGGAGTCCAGTTCAAC AAGAACCTGGCCGGGAGATACGCCAACACCTACAAAAACTGGTTCCCGGGGCCCATGGGCCGAACCCAGGGCTGG AACCTGGGCTCCGGGGTCAACCGCGCCAGTGTCAGCGCCTTCGCCACGACCAATAGGATGGAGCTCGAGGGCGCG AGTTACCAGGTGCCCCCGCAGCCGAACGGCATGACCAACAACCTCCAGGGCAGCAACACCTATGCCCTGGAGAAC ACTATGATCTTCAACAGCCAGCCGGCGAACCCGGGCACCACCGCCACGTACCTCGAGGGCAACATGCTCATCACC AGCGAGAGCGAGACGCAGCCGGTGAACCGCGTGGCGTACAACGTCGGCGGGCAGATGGCCACCAACAACCAGAGC TCCACCACTGCCCCCGCGACCGGCACGTACAACCTCCAGGAAATCGTGCCCGGCAGCGTGTGGATGGAGAGGGAC GTGTACCTCCAAGGACCCATCTGGGCCAAGATCCCAGAGACGGGGGCGCACTTTCACCCCTCTCCGGCCATGGGC GGATTCGGACTCAAACACCCACCGCCCATGATGCTCATCAAGAACACGCCTGTGCCCGGAAATATCACCAGCTTC TCGGACGTGCCCGTCAGCAGCTTCATCACCCAGTACAGCACCGGGCAGGTCACCGTGGAGATGGAGTGGGAGCTC AAGAAGGAAAACTCCAAGAGGTGGAACCCAGAGATCCAGTACACAAACAACTACAACGACCCCCAGTTTGTGGAC TTTGCCCCGGACAGCACCGGGGAATACAGAACCACCAGACCTATCGGAACCCGATACCTTACCCGACCCCTTTAA AAV-6 Full Genome: AF028704 136 ME149651883v.1135975-54620 (REGN 11546) Rep78: (SEQ ID NO:15) ATGCCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCCGGCATTTCTGACAGC TTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGATTGAGCAG GCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTCCTGGTCCAGTGGCGCCGCGTGAGTAAGGCCCCGGAG GCCCTCTTCTTTGTTCAGTTCGAGAAGGGCGAGTCCTACTTCCACCTCCATATTCTGGTGGAGACCACGGGGGTC AAATCCATGGTGCTGGGCCGCTTCCTGAGTCAGATTAGGGACAAGCTGGTGCAGACCATCTACCGCGGGATCGAG CCGACCCTGCCCAACTGGTTCGCGGTGACCAAGACGCGTAATGGCGCCGGAGGGGGGAACAAGGTGGTGGACGAG TGCTACATCCCCAACTACCTCCTGCCCAAGACTCAGCCCGAGCTGCAGTGGGCGTGGACTAACATGGAGGAGTAT ATAAGCGCGTGTTTAAACCTGGCCGAGCGCAAACGGCTCGTGGCGCACGACCTGACCCACGTCAGCCAGACCCAG GAGCAGAACAAGGAGAATCTGAACCCCAATTCTGACGCGCCTGTCATCCGGTCAAAAACCTCCGCACGCTACATG GAGCTGGTCGGGTGGCTGGTGGACCGGGGCATCACCTCCGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCGTAC ATCTCCTTCAACGCCGCCTCCAACTCGCGGTCCCAGATCAAGGCCGCTCTGGACAATGCCGGCAAGATCATGGCG CTGACCAAATCCGCGCCCGACTACCTGGTAGGCCCCGCTCCGCCCGCCGACATTAAAACCAACCGCATTTACCGC ATCCTGGAGCTGAACGGCTACGACCCTGCCTACGCCGGCTCCGTCTTTCTCGGCTGGGCCCAGAAAAGGTTCGGA AAACGCAACACCATCTGGCTGTTTGGGCCGGCCACCACGGGCAAGACCAACATCGCGGAAGCCATCGCCCACGCC GTGCCCTTCTACGGCTGCGTCAACTGGACCAATGAGAACTTTCCCTTCAACGATTGCGTCGACAAGATGGTGATC TGGTGGGAGGAGGGCAAGATGACGGCCAAGGTCGTGGAGTCCGCCAAGGCCATTCTCGGCGGCAGCAAGGTGCGC GTGGACCAAAAGTGCAAGTCGTCCGCCCAGATCGATCCCACCCCCGTGATCGTCACCTCCAACACCAACATGTGC GCCGTGATTGACGGGAACAGCACCACCTTCGAGCACCAGCAGCCGTTGCAGGACCGGATGTTCAAATTTGAACTC ACCCGCCGTCTGGAGCATGACTTTGGCAAGGTGACAAAGCAGGAAGTCAAAGAGTTCTTCCGCTGGGCGCAGGAT CACGTGACCGAGGTGGCGCATGAGTTCTACGTCAGAAAGGGTGGAGCCAACAAGAGACCCGCCCCCGATGACGCG GATAAAAGCGAGCCCAAGCGGGCCTGCCCCTCAGTCGCGGATCCATCGACGTCAGACGCGGAAGGAGCTCCGGTG GACTTTGCCGACAGGTACCAAAACAAATGTTCTCGTCACGCGGGCATGCTTCAGATGCTGTTTCCCTGCAAAACA TGCGAGAGAATGAATCAGAATTTCAACATTTGCTTCACGCACGGGACCAGAGACTGTTCAGAATGTTTCCCCGGC GTGTCAGAATCTCAACCGGTCGTCAGAAAGAGGACGTATCGGAAACTCTGTGCCATTCATCATCTGCTGGGGCGG GCTCCCGAGATTGCTTGCTCGGCCTGCGATCTGGTCAACGTGGATCTGGATGACTGTGTTTCTGAGCAATAA 137 ME149651883v.1135975-54620 (REGN 11546) CapVP1: (SEQ ID NO:16) ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACTTG AAACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTAC AAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGATGCAGCGGCCCTCGAGCAC GACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTT CAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGAGGGTT CTCGAACCTTTTGGTCTGGTTGAGGAAGGTGCTAAGACGGCTCCTGGAAAGAAACGTCCGGTAGAGCAGTCGCCA CAAGAGCCAGACTCCTCCTCGGGCATTGGCAAGACAGGCCAGCAGCCCGCTAAAAAGAGACTCAATTTTGGTCAG ACTGGCGACTCAGAGTCAGTCCCCGACCCACAACCTCTCGGAGAACCTCCAGCAACCCCCGCTGCTGTGGGACCT ACTACAATGGCTTCAGGCGGTGGCGCACCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAATGCCTCA GGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGAACATGGGCCTTGCCC ACCTATAACAACCACCTCTACAAGCAAATCTCCAGTGCTTCAACGGGGGCCAGCAACGACAACCACTACTTCGGC TACAGCACCCCCTGGGGGTATTTTGATTTCAACAGATTCCACTGCCATTTCTCACCACGTGACTGGCAGCGACTC ATCAACAACAATTGGGGATTCCGGCCCAAGAGACTCAACTTCAAGCTCTTCAACATCCAAGTCAAGGAGGTCACG ACGAATGATGGCGTCACGACCATCGCTAATAACCTTACCAGCACGGTTCAAGTCTTCTCGGACTCGGAGTACCAG TTGCCGTACGTCCTCGGCTCTGCGCACCAGGGCTGCCTCCCTCCGTTCCCGGCGGACGTGTTCATGATTCCGCAG TACGGCTACCTAACGCTCAACAATGGCAGCCAGGCAGTGGGACGGTCATCCTTTTACTGCCTGGAATATTTCCCA TCGCAGATGCTGAGAACGGGCAATAACTTTACCTTCAGCTACACCTTCGAGGACGTGCCTTTCCACAGCAGCTAC GCGCACAGCCAGAGCCTGGACCGGCTGATGAATCCTCTCATCGACCAGTACCTGTATTACCTGAACAGAACTCAG AATCAGTCCGGAAGTGCCCAAAACAAGGACTTGCTGTTTAGCCGGGGGTCTCCAGCTGGCATGTCTGTTCAGCCC AAAAACTGGCTACCTGGACCCTGTTACCGGCAGCAGCGCGTTTCTAAAACAAAAACAGACAACAACAACAGCAAC TTTACCTGGACTGGTGCTTCAAAATATAACCTTAATGGGCGTGAATCTATAATCAACCCTGGCACTGCTATGGCC TCACACAAAGACGACAAAGACAAGTTCTTTCCCATGAGCGGTGTCATGATTTTTGGAAAGGAGAGCGCCGGAGCT TCAAACACTGCATTGGACAATGTCATGATCACAGACGAAGAGGAAATCAAAGCCACTAACCCCGTGGCCACCGAA AGATTTGGGACTGTGGCAGTCAATCTCCAGAGCAGCAGCACAGACCCTGCGACCGGAGATGTGCATGTTATGGGA GCCTTACCTGGAATGGTGTGGCAAGACAGAGACGTATACCTGCAGGGTCCTATTTGGGCCAAAATTCCTCACACG GATGGACACTTTCACCCGTCTCCTCTCATGGGCGGCTTTGGACTTAAGCACCCGCCTCCTCAGATCCTCATCAAA AACACGCCTGTTCCTGCGAATCCTCCGGCAGAGTTTTCGGCTACAAAGTTTGCTTCATTCATCACCCAGTATTCC ACAGGACAAGTGAGCGTGGAGATTGAATGGGAGCTGCAGAAAGAAAACAGCAAACGCTGGAATCCCGAAGTGCAG TATACATCTAACTATGCAAAATCTGCCAACGTTGATTTCACTGTGGACAACAATGGACTTTATACTGAGCCTCGC CCCATTGGCACCCGTTACCTCACCCGTCCCCTGTAA AAV-7 Full Genome: NC_006260 Rep78: (SEQ ID NO:17) ATGCCGGGTTTCTACGAGATCGTGATCAAGGTGCCGAGCGACCTGGACGAGCACCTGCCGGGCATTTCTGACTCG TTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGCTGCCCCCGGATTCTGACATGGATCTGAATCTGATCGAGCAG GCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTCCTGGTCCAATGGCGCCGCGTGAGTAAGGCCCCGGAG GCCCTGTTCTTTGTTCAGTTCGAGAAGGGCGAGAGCTACTTCCACCTTCACGTTCTGGTGGAGACCACGGGGGTC AAGTCCATGGTGCTAGGCCGCTTCCTGAGTCAGATTCGGGAGAAGCTGGTCCAGACCATCTACCGCGGGGTCGAG CCCACGCTGCCCAACTGGTTCGCGGTGACCAAGACGCGTAATGGCGCCGGCGGGGGGAACAAGGTGGTGGACGAG TGCTACATCCCCAACTACCTCCTGCCCAAGACCCAGCCCGAGCTGCAGTGGGCGTGGACTAACATGGAGGAGTAT ATAAGCGCGTGTTTGAACCTGGCCGAACGCAAACGGCTCGTGGCGCAGCACCTGACCCACGTCAGCCAGACGCAG GAGCAGAACAAGGAGAATCTGAACCCCAATTCTGACGCGCCCGTGATCAGGTCAAAAACCTCCGCGCGCTACATG GAGCTGGTCGGGTGGCTGGTGGACCGGGGCATCACCTCCGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCGTAC ATCTCCTTCAACGCCGCCTCCAACTCGCGGTCCCAGATCAAGGCCGCGCTGGACAATGCCGGCAAGATCATGGCG CTGACCAAATCCGCGCCCGACTACCTGGTGGGGCCCTCGCTGCCCGCGGACATTAAAACCAACCGCATCTACCGC ATCCTGGAGCTGAACGGGTACGATCCTGCCTACGCCGGCTCCGTCTTTCTCGGCTGGGCCCAGAAAAAGTTCGGG AAGCGCAACACCATCTGGCTGTTTGGGCCCGCCACCACCGGCAAGACCAACATTGCGGAAGCCATCGCCCACGCC GTGCCCTTCTACGGCTGCGTCAACTGGACCAATGAGAACTTTCCCTTCAACGATTGCGTCGACAAGATGGTGATC TGGTGGGAGGAGGGCAAGATGACGGCCAAGGTCGTGGAGTCCGCCAAGGCCATTCTCGGCGGCAGCAAGGTGCGC GTGGACCAAAAGTGCAAGTCGTCCGCCCAGATCGACCCCACCCCCGTGATCGTCACCTCCAACACCAACATGTGC GCCGTGATTGACGGGAACAGCACCACCTTCGAGCACCAGCAGCCGTTGCAGGACCGGATGTTCAAATTTGAACTC ACCCGCCGTCTGGAGCACGACTTTGGCAAGGTGACGAAGCAGGAAGTCAAAGAGTTCTTCCGCTGGGCCAGTGAT CACGTGACCGAGGTGGCGCATGAGTTCTACGTCAGAAAGGGCGGAGCCAGCAAAAGACCCGCCCCCGATGACGCG GATATAAGCGAGCCCAAGCGGGCCTGCCCCTCAGTCGCGGATCCATCGACGTCAGACGCGGAAGGAGCTCCGGTG GACTTTGCCGACAGGTACCAAAACAAATGTTCTCGTCACGCGGGCATGATTCAGATGCTGTTTCCCTGCAAAACG 138 ME149651883v.1135975-54620 (REGN 11546) TGCGAGAGAATGAATCAGAATTTCAACATTTGCTTCACACACGGGGTCAGAGACTGTTTAGAGTGTTTCCCCGGC GTGTCAGAATCTCAACCGGTCGTCAGAAAAAAGACGTATCGGAAACTCTGCGCGATTCATCATCTGCTGGGGCGG GCGCCCGAGATTGCTTGCTCGGCCTGCGACCTGGTCAACGTGGACCTGGACGACTGCGTTTCTGAGCAATAA CapVP1: (SEQ ID NO:18) ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACCTG AAACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACAACGGCCGGGGTCTGGTGCTTCCTGGCTAC AAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCAC GACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTT CAGGAGCGTCTGCAAGAAGATACGTCATTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTT CTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAGACGGCTCCTGCAAAGAAGAGACCGGTAGAGCCGTCACCT CAGCGTTCCCCCGACTCCTCCACGGGCATCGGCAAGAAAGGCCAGCAGCCCGCCAGAAAGAGACTCAATTTCGGT CAGACTGGCGACTCAGAGTCAGTCCCCGACCCTCAACCTCTCGGAGAACCTCCAGCAGCGCCCTCTAGTGTGGGA TCTGGTACAGTGGCTGCAGGCGGTGGCGCACCAATGGCAGACAATAACGAAGGTGCCGACGGAGTGGGTAATGCC TCAGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATTACCACCAGCACCCGAACCTGGGCCCTG CCCACCTACAACAACCACCTCTACAAGCAAATCTCCAGTGAAACTGCAGGTAGTACCAACGACAACACCTACTTC GGCTACAGCACCCCCTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGA CTCATCAACAACAACTGGGGATTCCGGCCCAAGAAGCTGCGGTTCAAGCTCTTCAACATCCAGGTCAAGGAGGTC ACGACGAATGACGGCGTTACGACCATCGCTAATAACCTTACCAGCACGATTCAGGTATTCTCGGACTCGGAATAC CAGCTGCCGTACGTCCTCGGCTCTGCGCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGACGTCTTCATGATTCCT CAGTACGGCTACCTGACTCTCAACAATGGCAGTCAGTCTGTGGGACGTTCCTCCTTCTACTGCCTGGAGTACTTC CCCTCTCAGATGCTGAGAACGGGCAACAACTTTGAGTTCAGCTACAGCTTCGAGGACGTGCCTTTCCACAGCAGC TACGCACACAGCCAGAGCCTGGACCGGCTGATGAATCCCCTCATCGACCAGTACTTGTACTACCTGGCCAGAACA CAGAGTAACCCAGGAGGCACAGCTGGCAATCGGGAACTGCAGTTTTACCAGGGCGGGCCTTCAACTATGGCCGAA CAAGCCAAGAATTGGTTACCTGGACCTTGCTTCCGGCAACAAAGAGTCTCCAAAACGCTGGATCAAAACAACAAC AGCAACTTTGCTTGGACTGGTGCCACCAAATATCACCTGAACGGCAGAAACTCGTTGGTTAATCCCGGCGTCGCC ATGGCAACTCACAAGGACGACGAGGACCGCTTTTTCCCATCCAGCGGAGTCCTGATTTTTGGAAAAACTGGAGCA ACTAACAAAACTACATTGGAAAATGTGTTAATGACAAATGAAGAAGAAATTCGTCCTACTAATCCTGTAGCCACG GAAGAATACGGGATAGTCAGCAGCAACTTACAAGCGGCTAATACTGCAGCCCAGACACAAGTTGTCAACAACCAG GGAGCCTTACCTGGCATGGTCTGGCAGAACCGGGACGTGTACCTGCAGGGTCCCATCTGGGCCAAGATTCCTCAC ACGGATGGCAACTTTCACCCGTCTCCTTTGATGGGCGGCTTTGGACTTAAACATCCGCCTCCTCAGATCCTGATC AAGAACACTCCCGTTCCCGCTAATCCTCCGGAGGTGTTTACTCCTGCCAAGTTTGCTTCGTTCATCACACAGTAC AGCACCGGACAAGTCAGCGTGGAAATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATT CAGTACACCTCCAACTTTGAAAAGCAGACTGGTGTGGACTTTGCCGTTGACAGCCAGGGTGTTTACTCTGAGCCT CGCCCTATTGGCACTCGTTACCTCACCCGTAATCTGTAA AAV-8 Full Genome: NC_006261 Rep78: (SEQ ID NO:19) ATGCCGGGCTTCTACGAGATCGTGATCAAGGTGCCGAGCGACCTGGACGAGCACCTGCCGGGCATTTCTGACTCG TTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGCTGCCCCCGGATTCTGACATGGATCGGAATCTGATCGAGCAG GCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTCCTGGTCCAATGGCGCCGCGTGAGTAAGGCCCCGGAG GCCCTCTTCTTTGTTCAGTTCGAGAAGGGCGAGAGCTACTTTCACCTGCACGTTCTGGTCGAGACCACGGGGGTC AAGTCCATGGTGCTAGGCCGCTTCCTGAGTCAGATTCGGGAAAAGCTTGGTCCAGACCATCTACCCGCGGGGTCG AGCCCCACCTTGCCCAACTGGTTCGCGGTGACCAAAGACGCGGTAATGGCGCCGGCGGGGGGGAACAAGGTGGTG GACGAGTGCTACATCCCCAACTACCTCCTGCCCAAGACTCAGCCCGAGCTGCAGTGGGCGTGGACTAACATGGAG GAGTATATAAGCGCGTGCTTGAACCTGGCCGAGCGCAAACGGCTCGTGGCGCAGCACCTGACCCACGTCAGCCAG ACGCAGGAGCAGAACAAGGAGAATCTGAACCCCAATTCTGACGCGCCCGTGATCAGGTCAAAAACCTCCGCGCGC TATATGGAGCTGGTCGGGTGGCTGGTGGACCGGGGCATCACCTCCGAGAAGCAGTGGATCCAGGAGGACCAGGCC TCGTACATCTCCTTCAACGCCGCCTCCAACTCGCGGTCCCAGATCAAGGCCGCGCTGGACAATGCCGGCAAGATC ATGGCGCTGACCAAATCCGCGCCCGACTACCTGGTGGGGCCCTCGCTGCCCGCGGACATTACCCAGAACCGCATC TACCGCATCCTCGCTCTCAACGGCTACGACCCTGCCTACGCCGGCTCCGTCTTTCTCGGCTGGGCTCAGAAAAAG TTCGGGAAACGCAACACCATCTGGCTGTTTGGACCCGCCACCACCGGCAAGACCAACATTGCGGAAGCCATCGCC CACGCCGTGCCCTTCTACGGCTGCGTCAACTGGACCAATGAGAACTTTCCCTTCAATGATTGCGTCGACAAGATG GTGATCTGGTGGGAGGAGGGCAAGATGACGGCCAAGGTCGTGGAGTCCGCCAAGGCCATTCTCGGCGGCAGCAAG GTGCGCGTGGACCAAAAGTGCAAGTCGTCCGCCCAGATCGACCCCACCCCCGTGATCGTCACCTCCAACACCAAC ATGTGCGCCGTGATTGACGGGAACAGCACCACCTTCGAGCACCAGCAGCCTCTCCAGGACCGGATGTTTAAGTTC 139 ME149651883v.1135975-54620 (REGN 11546) GAACTCACCCGCCGTCTGGAGCACGACTTTGGCAAGGTGACAAAGCAGGAAGTCAAAGAGTTCTTCCGCTGGGCC AGTGATCACGTGACCGAGGTGGCGCATGAGTTTTACGTCAGAAAGGGCGGAGCCAGCAAAAGACCCGCCCCCGAT GACGCGGATAAAAGCGAGCCCAAGCGGGCCTGCCCCTCAGTCGCGGATCCATCGACGTCAGACGCGGAAGGAGCT CCGGTGGACTTTGCCGACAGGTACCAAAACAAATGTTCTCGTCACGCGGGCATGCTTCAGATGCTGTTTCCCTGC AAAACGTGCGAGAGAATGAATCAGAATTTCAACATTTGCTTCACACACGGGGTCAGAGACTGCTCAGAGTGTTTC CCCGGCGTGTCAGAATCTCAACCGGTCGTCAGAAAGAGGACGTATCGGAAACTCTGTGCGATTCATCATCTGCTG GGGCGGGCTCCCGAGATTGCTTGCTCGGCCTGCGATCTGGTCAACGTGGACCTGGATGACTGTGTTTCTGAGCAA TAA CapVP1: (SEQ ID NO:20) ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGCGCTG AAACCTGGAGCCCCGAAGCCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTAC AAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCAC GACAAGGCCTACGACCAGCAGCTGCAGGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTT CAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTT CTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAGACGGCTCCTGGAAAGAAGAGACCGGTAGAGCCATCACCC CAGCGTTCTCCAGACTCCTCTACGGGCATCGGCAAGAAAGGCCAACAGCCCGCCAGAAAAAGACTCAATTTTGGT CAGACTGGCGACTCAGAGTCAGTTCCAGACCCTCAACCTCTCGGAGAACCTCCAGCAGCGCCCTCTGGTGTGGGA CCTAATACAATGGCTGCAGGCGGTGGCGCACCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAGTTCC TCGGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTG CCCACCTACAACAACCACCTCTACAAGCAAATCTCCAACGGGACATCGGGAGGAGCCACCAACGACAACACCTAC TTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTTAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAG CGACTCATCAACAACAACTGGGGATTCCGGCCCAAGAGACTCAGCTTCAAGCTCTTCAACATCCAGGTCAAGGAG GTCACGCAGAATGAAGGCACCAAGACCATCGCCAATAACCTCACCAGCACCATCCAGGTGTTTACGGACTCGGAG TACCAGCTGCCGTACGTTCTCGGCTCTGCCCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGACGTGTTCATGATT CCCCAGTACGGCTACCTAACACTCAACAACGGTAGTCAGGCCGTGGGACGCTCCTCCTTCTACTGCCTGGAATAC TTTCCTTCGCAGATGCTGAGAACCGGCAACAACTTCCAGTTTACTTACACCTTCGAGGACGTGCCTTTCCACAGC AGCTACGCCCACAGCCAGAGCTTGGACCGGCTGATGAATCCTCTGATTGACCAGTACCTGTACTACTTGTCTCGG ACTCAAACAACAGGAGGCACGGCAAATACGCAGACTCTGGGCTTCAGCCAAGGTGGGCCTAATACAATGGCCAAT CAGGCAAAGAACTGGCTGCCAGGACCCTGTTACCGCCAACAACGCGTCTCAACGACAACCGGGCAAAACAACAAT AGCAACTTTGCCTGGACTGCTGGGACCAAATACCATCTGAATGGAAGAAATTCATTGGCTAATCCTGGCATCGCT ATGGCAACACACAAAGACGACGAGGAGCGTTTTTTTCCCAGTAACGGGATCCTGATTTTTGGCAAACAAAATGCT GCCAGAGACAATGCGGATTACAGCGATGTCATGCTCACCAGCGAGGAAGAAATCAAAACCACTAACCCTGTGGCT ACAGAGGAATACGGTATCGTGGCAGATAACTTGCAGCAGCAAAACACGGCTCCTCAAATTGGAACTGTCAACAGC CAGGGGGCCTTACCCGGTATGGTCTGGCAGAACCGGGACGTGTACCTGCAGGGTCCCATCTGGGCCAAGATTCCT CACACGGACGGCAACTTCCACCCGTCTCCGCTGATGGGCGGCTTTGGCCTGAAACATCCTCCGCCTCAGATCCTG ATCAAGAACACGCCTGTACCTGCGGATCCTCCGACCACCTTCAACCAGTCAAAGCTGAACTCTTTCATCACGCAA TACAGCACCGGACAGGTCAGCGTGGAAATTGAATGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCCGAG ATCCAGTACACCTCCAACTACTACAAATCTACAAGTGTGGACTTTGCTGTTAATACAGAAGGCGTGTACTCTGAA CCCCGCCCCATTGGCACCCGTTACCTCACCCGTAATCTGTAA AAV-9 Cap only: AY530579 CapVP1: (SEQ ID NO:21) ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATTCGCGAGTGGTGGGCTTTG AAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACATCAAGACAACGCTCGAGGTCTTGTGCTTCCGGGTTAC AAATACCTTGGACCCGGCAACGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCAC GACAAGGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCCGAGTTC CAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTT CTTGAACCTCTTGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCT CAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAAGAGACTCAATTTCGGTCAG ACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAATCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCT CTTACAATGGCTTCAGGTGGTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCG GGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCC ACCTACAACAATCACCTCTACAAGCAAATCTCCAACAGCACATCTGGAGGATCTTCAAATGACAACGCCTACTTC GGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGA CTCATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTCAACATTCAGGTCAAAGAGGTT ACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTACCAGCACGGTCCAGGTCTTCACGGACTCAGACTAT 140 ME149651883v.1135975-54620 (REGN 11546) CAGCTCCCGTACGTGCTCGGGTCGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCT CAGTACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACTGCCTGGAATATTTC CCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCTACGAGTTTGAGAACGTACCTTTCCATAGCAGC TACGCTCACAGCCAAAGCCTGGACCGACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCAAAGACT ATTAACGGTTCTGGACAGAATCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATGGCTGTCCAGGGA AGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTCAACCACTGTGACTCAAAACAACAACAGCGAA TTTGCTTGGCCTGGAGCTTCTTCTTGGGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCC AGCCACAAAGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGGAACTGGAAGA GACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAAATTAAAACTACTAACCCGGTAGCAACGGAG TCCTATGGACAAGTGGCCACAAACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGGGTTCAAAACCAAGGA ATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGCAAGGACCCATTTGGGCCAAAATTCCTCACACG GACGGCAACTTTCACCCTTCTCCGCTGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAA AACACACCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATCACCCAGTATTCT ACTGGCCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATCCAG TACACTTCCAACTATTACAAGTCTAATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGC CCCATTGGCACCAGATACCTGACTCGTAATCTGTAA AAV-10 Partial Genome: AY631965 Rep78: (SEQ ID NO:22) ATGCCGGGCTTCTACGAGATCGTGATCAAGGTGCCGAGCGACCTGGACGAGCACCTGCCGGGCATTTCTGACTCG TTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGCTGCCCCCGGATTCTGACATGGATCGGAATCTGATCGAGCAG GCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTCCTGGTCCACTGGCGCCGCGTGAGTAAGGCCCCGGAG GCCCTCTTCTTTGTTCAGTTCGAGAAGGGCGAGTCCTACTTTCACCTGCACGTTCTGGTCGAGACCACGGGGGTC AAGTCCATGGTCCTGGGCCGCTTCCTGAGTCAGATCAGAGACAGGCTGGTGCAGACCATCTACCGCGGGGTAGAG CCCACGCTGCCCAACTGGTTCGCGGTGACCAAGACGCGAAATGGCGCCGGCGGGGGGAACAAGGTGGTGGACGAG TGCTACATCCCCAACTACCTCCTGCCCAAGACGCAGCCCGAGCTGCAGTGGGCGTGGACTAACATGGAGGAGTAT ATAAGCGCGTGTCTGAACCTCGCGGAGCGTAAACGGCTCGTGGCGCAGCACCTGACCCACGTCAGCCAGACGCAG GAGCAGAACAAGGAGAATCTGAACCCGAATTCTGACGCGCCCGTGATCAGGTCAAAAACCTCCGCGCGCTACATG GAGCTGGTCGGGTGGCTGGTGGACCGGGGCATCACCTCCGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCGTAC ATCTCCTTCAACGCCGCCTCCAACTCGCGGTCCCAGATCAAGGCCGCGCTGGACAATGCCGGAAAGATCATGGCG CTGACCAAATCCGCGCCCGACTACCTGGTAGGCCCGTCCTTACCCGCGGACATTAAGGCCAACCGCATCTACCGC ATCCTGGAGCTCAACGGCTACGACCCCGCCTACGCCGGCTCCGTCTTCCTGGGCTGGGCGCAGAAAAAGTTCGGT AAAAGGAATACAATTTGGCTGTTCGGGCCCGCCACCACCGGCAAGACCAACATCGCGGAAGCCATCGCCCACGCC GTGCCCTTCTACGGCTGCGTCAACTGGACCAATGAGAACTTTCCCTTCAACGATTGCGTCGACAAGATGGTGATC TGGTGGGAGGAGGGCAAGATGACCGCCAAGGTCGTGGAGTCCGCCAAGGCCATTCTGGGCGGAAGCAAGGTGCGC GTCGACCAAAAGTGCAAGTCCTCGGCCCAGATCGACCCCACGCCCGTGATCGTCACCTCCAACACCAACATGTGC GCCGTGATCGACGGGAACAGCACCACCTTCGAGCACCAGCAGCCCCTGCAGGACCGCATGTTCAAGTTCGAGCTC ACCCGCCGTCTGGAGCACGACTTTGGCAAGGTGACCAAGCAGGAAGTCAAAGAGTTCTTCCGCTGGGCTCAGGAT CACGTGACTGAGGTGACGCATGAGTTCTACGTCAGAAAGGGCGGAGCCACCAAAAGACCCGCCCCCAGTGACGCG GATATAAGCGAGCCCAAGCGGGCCTGCCCCTCAGTTGCGGAGCCATCGACGTCAGACGCGGAAGCACCGGTGGAC TTTGCGGACAGGTACCAAAACAAATGTTCTCGTCACGCGGGCATGCTTCAGATGCTGTTTCCCTGCAAGACATGC GAGAGAATGAATCAGAATTTCAACGTCTGCTTCACGCACGGGGTCAGAGACTGCTCAGAGTGCTTCCCCGGCGCG TCAGAATCTCAACCTGTCGTCAGAAAAAAGACGTATCAGAAACTGTGCGCGATTCATCATCTGCTGGGGCGGGCA CCCGAGATTGCGTGTTCGGCCTGCGATCTCGTCAACGTGGACTTGGATGACTGTGTTTCTGAGCAATAA CapVP1: (SEQ ID NO:23) ATGGCTGCTGACGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACCTG AAACCTGGAGCCCCCAAGCCCAAGGCCAACCAGCAGAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTAC AAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCAC GACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTT CAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCGGGTT CTCGAACCTCTCGGTCTGGTTGAGGAAGCTGCTAAGACGGCTCCTGGAAAGAAGAGACCGGTAGAACCGTCACCT CAGCGTTCCCCCGACTCCTCCACGGGCATCGGCAAGAAAGGCCAGCAGCCCGCTAAAAAGAGACTGAACTTTGGG CAGACTGGCGAGTCAGAGTCAGTCCCCGACCCTCAACCAATCGGAGAACCACCAGCAGGCCCCTCTGGTCTGGGA TCTGGTACAATGGCTGCAGGCGGTGGCGCTCCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAGTTCC TCAGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTG 141 ME149651883v.1135975-54620 (REGN 11546) CCCACCTACAACAACCACCTCTACAAGCAAATCTCCAACGGGACATCGGGAGGAAGCACCAACGACAACACCTAC TTCGGCTACAGCACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAG CGACTCATCAACAACAACTGGGGATTCCGGCCAAAAAGACTCAGCTTCAAGCTCTTCAACATCCAGGTCAAGGAG GTCACGCAGAATGAAGGCACCAAGACCATCGCCAATAACCTTACCAGCACGATTCAGGTATTTACGGACTCGGAA TACCAGCTGCCGTACGTCCTCGGCTCCGCGCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGATGTCTTCATGATT CCCCAGTACGGCTACCTGACACTGAACAATGGAAGTCAAGCCGTAGGCCGTTCCTCCTTCTACTGCCTGGAATAT TTTCCATCTCAAATGCTGCGAACTGGAAACAATTTTGAATTCAGCTACACCTTCGAGGACGTGCCTTTCCACAGC AGCTACGCACACAGCCAGAGCTTGGACCGACTGATGAATCCTCTCATTGACCAGTACCTGTACTACTTATCCAGA ACTCAGTCCACAGGAGGAACTCAAGGTACCCAGCAATTGTTATTTTCTCAAGCTGGGCCTGCAAACATGTCGGCT CAGGCCAAGAACTGGCTGCCTGGACCTTGCTACCGGCAGCAGCGAGTCTCCACGACACTGTCGCAAAACAACAAC AGCAACTTTGCTTGGACTGGTGCCACCAAATATCACCTGAACGGAAGAGACTCTCTGGTGAATCCCGGTGTCGCC ATGGCAACCCACAAGGACGACGAGGAACGCTTCTTCCCGTCGAGCGGAGTCCTGATGTTTGGAAAACAGGGTGCT GGAAGAGACAATGTGGACTACAGCAGCGTTATGCTAACAAGCGAAGAAGAAATTAAAACCACTAACCCTGTAGCC ACAGAACAATACGGCGTGGTGGCTGACAACTTGCAGCAAGCCAATACAGGGCCTATTGTGGGAAATGTCAACAGC CAAGGAGCCTTACCTGGCATGGTCTGGCAGAACCGAGACGTGTACCTGCAGGGTCCCATCTGGGCCAAGATTCCT CACACGGACGGCAACTTTCACCCGTCTCCTCTGATGGGCGGCTTTGGACTTAAACACCCGCCTCCACAGATCCTG ATCAAGAACACGCCGGTACCTGCGGATCCTCCAACAACGTTCAGCCAGGCGAAATTGGCTTCCTTCATCACGCAG TACAGCACCGGACAGGTCAGCGTGGAAATCGAGTGGGAGCTGCAGAAGGAGAACAGCAAACGCTGGAACCCAGAG ATTCAGTACACTTCAAACTACTACAAATCTACAAATGTGGACTTTGCTGTCAATACAGAGGGAACTTATTCTGAG CCTCGCCCCATTGGTACTCGTTATCTGACACGTAATCTGTAA AAV-11 Partial Genome: AY631966 Rep78: (SEQ ID NO:24) ATGCCGGGCTTCTACGAGATCGTGATCAAGGTGCCGAGCGACCTGGACGAGCACCTGCCGGGCATTTCTGACTCG TTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGCTGCCCCCGGATTCTGACATGGATCGGAATCTGATCGAGCAG GCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTCCTGGTCCACTGGCGCCGCGTGAGTAAGGCCCCGGAG GCCCTCTTCTTTGTTCAGTTCGAGAAGGGCGAGTCCTACTTCCACCTCCACGTTCTCGTCGAGACCACGGGGGTC AAGTCCATGGTCCTGGGCCGCTTCCTGAGTCAGATCAGAGACAGGCTGGTGCAGACCATCTACCGCGGGGTCGAG CCCACGCTGCCCAACTGGTTCGCGGTGACCAAGACGCGAAATGGCGCCGGCGGGGGGAACAAGGTGGTGGACGAG TGCTACATCCCCAACTACCTCCTGCCCAAGACCCAGCCCGAGCTGCAGTGGGCGTGGACTAACATGGAGGAGTAT ATAAGCGCGTGTCTAAACCTCGCGGAGCGTAAACGGCTCGTGGCGCAGCACCTGACCCACGTCAGCCAGACGCAG GAGCAGAACAAGGAGAATCTGAACCCGAATTCTGACGCGCCCGTGATCAGGTCAAAAACCTCCGCGCGCTACATG GAGCTGGTCGGGTGGCTGGTGGACCGGGGCATCACCTCCGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCGTAC ATCTCCTTCAACGCCGCCTCCAACTCGCGGTCCCAGATCAAGGCCGCGCTGGACAATGCCGGAAAGATCATGGCG CTGACCAAATCCGCGCCCGACTACCTGGTAGGCCCGTCCTTACCCGCGGACATTAAGGCCAACCGCATCTACCGC ATCCTGGAGCTCAACGGCTACGACCCCGCCTACGCCGGCTCCGTCTTCCTGGGCTGGGCGCAGAAAAAGTTCGGT AAACGCAACACCATCTGGCTGTTTGGGCCCGCCACCACCGGCAAGACCAACATCGCGGAAGCCATAGCCCACGCC GTGCCCTTCTACGGCTGCGTGAACTGGACCAATGAGAACTTTCCCTTCAACGATTGCGTCGACAAGATGGTGATC TGGTGGGAGGAGGGCAAGATGACCGCCAAGGTCGTGGAGTCCGCCAAGGCCATTCTGGGCGGAAGCAAGGTGCGC GTGGACCAAAAGTGCAAGTCCTCGGCCCAGATCGACCCCACGCCCGTGATCGTCACCTCCAACACCAACATGTGC GCCGTGATCGACGGGAACAGCACCACCTTCGAGCACCAGCAGCCGCTGCAGGACCGCATGTTCAAGTTCGAGCTC ACCCGCCGTCTGGAGCACGACTTTGGCAAGGTGACCAAGCAGGAAGTCAAAGAGTTCTTCCGCTGGGCTCAGGAT CACGTGACTGAGGTGGCGCATGAGTTCTACGTCAGAAAGGGCGGAGCCACCAAAAGACCCGCCCCCAGTGACGCG GATATAAGCGAGCCCAAGCGGGCCTGCCCCTCAGTTCCGGAGCCATCGACGTCAGACGCGGAAGCACCGGTGGAC TTTGCGGACAGGTACCAAAACAAATGTTCTCGTCACGCGGGCATGCTTCAGATGCTGTTTCCCTGCAAGACATGC GAGAGAATGAATCAGAATTTCAACGTCTGCTTCACGCACGGGGTCAGAGACTGCTCAGAGTGCTTCCCCGGCGCG TCAGAATCTCAACCCGTCGTCAGAAAAAAGACGTATCAGAAACTGTGCGCGATTCATCATCTGCTGGGGCGGGCA CCCGAGATTGCGTGTTCGGCCTGCGATCTCGTCAACGTGGACTTGGATGACTGTGTTTCTGAGCAATAA CapVP1: (SEQ ID NO:25) ATGGCTGCTGACGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACCTG AAACCTGGAGCCCCGAAGCCCAAGGCCAACCAGCAGAAGCAGGACGACGGCCGGGGTCTGGTGCTTCCTGGCTAC AAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCAC GACAAGGCCTACGACCAGCAGCTCAAAGCGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTT CAGGAGCGTCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGAGGGTA CTCGAACCTCTGGGCCTGGTTGAAGAAGGTGCTAAAACGGCTCCTGGAAAGAAGAGACCGTTAGAGTCACCACAA 142 ME149651883v.1135975-54620 (REGN 11546) GAGCCCGACTCCTCCTCGGGCATCGGCAAAAAAGGCAAACAACCAGCCAGAAAGAGGCTCAACTTTGAAGAGGAC ACTGGAGCCGGAGACGGACCCCCTGAAGGATCAGATACCAGCGCCATGTCTTCAGACATTGAAATGCGTGCAGCA CCGGGCGGAAATGCTGTCGATGCGGGACAAGGTTCCGATGGAGTGGGTAATGCCTCGGGTGATTGGCATTGCGAT TCCACCTGGTCTGAGGGCAAGGTCACAACAACCTCGACCAGAACCTGGGTCTTGCCCACCTACAACAACCACTTG TACCTGCGTCTCGGAACAACATCAAGCAGCAACACCTACAACGGATTCTCCACCCCCTGGGGATATTTTGACTTC AACAGATTCCACTGTCACTTCTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGACTACGACCAAAA GCCATGCGCGTTAAAATCTTCAATATCCAAGTTAAGGAGGTCACAACGTCGAACGGCGAGACTACGGTCGCTAAT AACCTTACCAGCACGGTTCAGATATTTGCGGACTCGTCGTATGAGCTCCCGTACGTGATGGACGCTGGACAAGAG GGGAGCCTGCCTCCTTTCCCCAATGACGTGTTCATGGTGCCTCAATATGGCTACTGTGGCATCGTGACTGGCGAG AATCAGAACCAAACGGACAGAAACGCTTTCTACTGCCTGGAGTATTTTCCTTCGCAAATGTTGAGAACTGGCAAC AACTTTGAAATGGCTTACAACTTTGAGAAGGTGCCGTTCCACTCAATGTATGCTCACAGCCAGAGCCTGGACAGA CTGATGAATCCCCTCCTGGACCAGTACCTGTGGCACTTACAGTCGACTACCTCTGGAGAGACTCTGAATCAAGGC AATGCAGCAACCACATTTGGAAAAATCAGGAGTGGAGACTTTGCCTTTTACAGAAAGAACTGGCTGCCTGGGCCT TGTGTTAAACAGCAGAGATTCTCAAAAACTGCCAGTCAAAATTACAAGATTCCTGCCAGCGGGGGCAACGCTCTG TTAAAGTATGACACCCACTATACCTTAAACAACCGCTGGAGCAACATCGCGCCCGGACCTCCAATGGCCACAGCC GGACCTTCGGATGGGGACTTCAGTAACGCCCAGCTTATATTCCCTGGACCATCTGTTACCGGAAATACAACAACT TCAGCCAACAATCTGTTGTTTACATCAGAAGAAGAAATTGCTGCCACCAACCCAAGAGACACGGACATGTTTGGC CAGATTGCTGACAATAATCAGAATGCTACAACTGCTCCCATAACCGGCAACGTGACTGCTATGGGAGTGCTGCCT GGCATGGTGTGGCAAAACAGAGACATTTACTACCAAGGGCCAATTTGGGCCAAGATCCCACACGCGGACGGACAT TTTCATCCTTCACCGCTGATTGGTGGGTTTGGACTGAAACACCCGCCTCCCCAGATATTCATCAAGAACACTCCC GTACCTGCCAATCCTGCGACAACCTTCACTGCAGCCAGAGTGGACTCTTTCATCACACAATACAGCACCGGCCAG GTCGCTGTTCAGATTGAATGGGAAATTGAAAAGGAACGCTCCAAACGCTGGAATCCTGAAGTGCAGTTTACTTCA AACTATGGGAACCAGTCTTCTATGTTGTGGGCTCCTGATACAACTGGGAAGTATACAGAGCCGCGGGTTATTGGC TCTCGTTATTTGACTAATCATTTGTAA AAV-12 Partial Genome: DQ813647 Rep78: (SEQ ID NO:26) ATGCCGGGGTTCTACGAGGTGGTGATCAAGGTGCCCAGCGACCTGGACGAGCACCTGCCCGGCATTTCTGACTCC TTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCCCCGGATTCTGACATGGATCAGAATCTGATTGAGCAG GCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGAGTTCCTGGTGGAATGGCGCCGAGTGAGTAAATTTCTGGAG GCCAAGTTTTTTGTGCAGTTTGAAAAGGGGGACTCGTACTTTCATTTGCATATTCTGATTGAAATTACCGGCGTG AAATCCATGGTGGTGGGCCGCTACGTGAGTCAGATTAGGGATAAACTGATCCAGCGCATCTACCGCGGGGTCGAG CCCCAGCTGCCCAACTGGTTCGCGGTCACAAAGACCCGAAATGGCGCCGGAGGCGGGAACAAGGTGGTGGACGAG TGCTACATCCCCAACTACCTGCTCCCCAAGGTCCAGCCCGAGCTTCAGTGGGCGTGGACTAACATGGAGGAGTAT ATAAGCGCCTGTTTGAACCTCGCGGAGCGTAAACGGCTCGTGGCGCAGCACCTGACGCACGTCTCCCAGACCCAG GAGGGCGACAAGGAGAATCTGAACCCGAATTCTGACGCGCCGGTGATCCGGTCAAAAACCTCCGCCAGGTACATG GAGCTGGTCGGGTGGCTGGTGGACAAGGGCATCACGTCCGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCGTAC ATCTCCTTCAACGCGGCCTCCAACTCCCGGTCGCAGATCAAGGCGGCCCTGGACAATGCCTCCAAAATCATGAGC CTCACCAAAACGGCTCCGGACTATCTCATCGGGCAGCAGCCCGTGGGGGACATTACCACCAACCGGATCTACAAA ATCCTGGAACTGAACGGGTACGACCCCCAGTACGCCGCCTCCGTCTTTCTCGGCTGGGCCCAGAAAAAGTTTGGA AAGCGCAACACCATCTGGCTGTTTGGGCCCGCCACCACCGGCAAGACCAACATCGCGGAAGCCATCGCCCACGCG GTCCCCTTCTACGGCTGCGTCAACTGGACCAATGAGAACTTTCCCTTCAACGACTGCGTCGACAAAATGGTGATT TGGTGGGAGGAGGGCAAGATGACCGCCAAGGTCGTAGAGTCCGCCAAGGCCATTCTGGGCGGCAGCAAGGTGCGC GTGGACCAAAAATGCAAGGCCTCTGCGCAGATCGACCCCACCCCCGTGATCGTCACCTCCAACACCAACATGTGC GCCGTGATTGACGGGAACAGCACCACCTTCGAGCACCAGCAGCCCCTGCAGGACCGGATGTTCAAGTTTGAACTC ACCCGCCGCCTCGACCACGACTTTGGCAAGGTCACCAAGCAGGAAGTCAAGGACTTTTTCCGGTGGGCGGCTGAT CACGTGACTGACGTGGCTCATGAGTTTTACGTCACAAAGGGTGGAGCTAAGAAAAGGCCCGCCCCCTCTGACGAG GATATAAGCGAGCCCAAGCGGCCGCGCGTGTCATTTGCGCAGCCGGAGACGTCAGACGCGGAAGCTCCCGGAGAC TTCGCCGACAGGTACCAAAACAAATGTTCTCGTCACGCGGGTATGCTGCAGATGCTCTTTCCCTGCAAGACGTGC GAGAGAATGAATCAGAATTCCAACGTCTGCTTCACGCACGGTCAGAAAGATTGCGGGGAGTGCTTTCCCGGGTCA GAATCTCAACCGGTTTCTGTCGTCAGAAAAACGTATCAGAAACTGTGCATCCTTCATCAGCTCCGGGGGGCACCC GAGATCGCCTGCTCTGCTTGCGACCAACTCAACCCCGATTTGGACGATTGCCAATTTGAGCAATAA CapVP1: (SEQ ID NO:27) ATGGCTGCTGACGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAAGGCATTCGCGAGTGGTGGGCGCTG AAACCTGGAGCTCCACAACCCAAGGCCAACCAACAGCATCAGGACAACGGCAGGGGTCTTGTGCTTCCTGGGTAC 143 ME149651883v.1135975-54620 (REGN 11546) AAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCAC GACAAGGCCTACGACAAGCAGCTCGAGCAGGGGGACAACCCGTATCTCAAGTACAACCACGCCGACGCCGAGTTC CAGCAGCGCTTGGCGACCGACACCTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGATT CTCGAGCCTCTGGGTCTGGTTGAAGAGGGCGTTAAAACGGCTCCTGGAAAGAAACGCCCATTAGAAAAGACTCCA AATCGGCCGACCAACCCGGACTCTGGGAAGGCCCCGGCCAAGAAAAAGCAAAAAGACGGCGAACCAGCCGACTCT GCTAGAAGGACACTCGACTTTGAAGACTCTGGAGCAGGAGACGGACCCCCTGAGGGATCATCTTCCGGAGAAATG TCTCATGATGCTGAGATGCGTGCGGCGCCAGGCGGAAATGCTGTCGAGGCGGGACAAGGTGCCGATGGAGTGGGT AATGCCTCCGGTGATTGGCATTGCGATTCCACCTGGTCAGAGGGCCGAGTCACCACCACCAGCACCCGAACCTGG GTCCTACCCACGTACAACAACCACCTGTACCTGCGAATCGGAACAACGGCCAACAGCAACACCTACAACGGATTC TCCACCCCCTGGGGATACTTTGACTTTAACCGCTTCCACTGCCACTTTTCCCCACGCGACTGGCAGCGACTCATC AACAACAACTGGGGACTCAGGCCGAAATCGATGCGTGTTAAAATCTTCAACATACAGGTCAAGGAGGTCACGACG TCAAACGGCGAGACTACGGTCGCTAATAACCTTACCAGCACGGTTCAGATCTTTGCGGATTCGACGTATGAACTC CCATACGTGATGGACGCCGGTCAGGAGGGGAGCTTTCCTCCGTTTCCCAACGACGTCTTTATGGTTCCCCAATAC GGATACTGCGGAGTTGTCACTGGAAAAAACCAGAACCAGACAGACAGAAATGCCTTTTACTGCCTGGAATACTTT CCATCCCAAATGCTAAGAACTGGCAACAATTTTGAAGTCAGTTACCAATTTGAAAAAGTTCCTTTCCATTCAATG TACGCGCACAGCCAGAGCCTGGACAGAATGATGAATCCTTTACTGGATCAGTACCTGTGGCATCTGCAATCGACC ACTACCGGAAATTCCCTTAATCAAGGAACAGCTACCACCACGTACGGGAAAATTACCACTGGAGACTTTGCCTAC TACAGGAAAAACTGGTTGCCTGGAGCCTGCATTAAACAACAAAAATTTTCAAAGAATGCCAATCAAAACTACAAG ATTCCCGCCAGCGGGGGAGACGCCCTTTTAAAGTATGACACGCATACCACTCTAAATGGGCGATGGAGTAACATG GCTCCTGGACCTCCAATGGCAACCGCAGGTGCCGGGGACTCGGATTTTAGCAACAGCCAGCTGATCTTTGCCGGA CCCAATCCGAGCGGTAACACGACCACATCTTCAAACAATTTGTTGTTTACCTCAGAAGAGGAGATTGCCACAACA AACCCACGAGACACGGACATGTTTGGACAGATTGCAGATAATAATCAAAATGCCACCACCGCCCCTCACATCGCT AACCTGGACGCTATGGGAATTGTTCCCGGAATGGTCTGGCAAAACAGAGACATCTACTACCAGGGCCCTATTTGG GCCAAGGTCCCTCACACGGACGGACACTTTCACCCTTCGCCGCTGATGGGAGGATTTGGACTGAAACACCCGCCT CCACAGATTTTCATCAAAAACACCCCCGTACCCGCCAATCCCAATACTACCTTTAGCGCTGCAAGGATTAATTCT TTTCTGACGCAGTACAGCACCGGACAAGTTGCCGTTCAGATCGACTGGGAAATTCAGAAGGAGCATTCCAAACGC TGGAATCCCGAAGTTCAATTTACTTCAAACTACGGCACTCAAAATTCTATGCTGTGGGCTCCCGACAATGCTGGC AACTACCACGAACTCCGGGCTATTGGGTCCCGTTTCCTCACCCACCACTTGTAA AAV-13 Partial Genome: EU285562 Rep78: (SEQ ID NO:28) ATGCCGGGATTCTACGAGATTGTCCTGAAGGTGCCCAGCGACCTGGACGAGCACCTGCCTGGCATTTCTGACTCT TTTGTAAACTGGGTGGCGGAGAAGGAATGGGAGCTGCCGCCGGATTCTGACATGGATCTGAATCTGATTGAGCAG GCACCCCTAACCGTGGCCGAAAAGCTGCAACGCGAATTCCTGGTCGAGTGGCGCCGCGTGAGTAAGGCCCCGGAG GCCCTCTTCTTTGTTCAGTTCGAGAAGGGGGACAGCTACTTCCACCTACACATTCTGGTGGAGACCGTGGGCGTG AAATCCATGGTGGTGGGCCGCTACGTGAGCCAGATTAAAGAGAAGCTGGTGACCCGCATCTACCGCGGGGTCGAG CCGCAGCTTCCGAACTGGTTCGCGGTGACCAAGACGCGTAATGGCGCCGGAGGCGGGAACAAGGTGGTGGACGAC TGCTACATCCCCAACTACCTGCTCCCCAAGACCCAGCCCGAGCTCCAGTGGGCGTGGACTAATATGGACCAGTAT TTAAGCGCCTGTTTGAATCTCGCGGAGCGTAAACGGCTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAG GAGCAGAACAAAGAGAACCAGAATCCCAATTCTGACGCGCCGGTGATCAGATCAAAAACCTCCGCGAGGTACATG GAGCTGGTCGGGTGGCTGGTGGACCGCGGGATCACGTCAGAAAAGCAATGGATCCAGGAGGACCAGGCCTCTTAC ATCTCCTTCAACGCCGCCTCCAACTCGCGGTCACAAATCAAGGCCGCACTGGACAATGCCTCCAAATTTATGAGC CTGACAAAAACGGCTCCGGACTACCTGGTGGGAAACAACCCGCCGGAGGACATTACCAGCAACCGGATCTACAAA ATCCTCGAGATGAACGGGTACGATCCGCAGTACGCGGCCTCCGTCTTCCTGGGCTGGGCGCAAAAGAAGTTCGGG AAGAGGAACACCATCTGGCTCTTTGGGCCGGCCACGACGGGTAAAACCAACATCGCTGAAGCTATCGCCCACGCC GTGCCCTTTTACGGCTGCGTGAACTGGACCAATGAGAACTTTCCGTTCAACGATTGCGTCGACAAGATGGTGATC TGGTGGGAGGAGGGCAAGATGACGGCCAAGGTCGTGGAGTCCGCCAAGGCCATTCTGGGCGGAAGCAAGGTGCGC GTGGACCAAAAGTGCAAGTCATCGGCCCAGATCGACCCAACTCCCGTCATCGTCACCTCCAACACCAACATGTGC GCGGTCATCGACGGAAATTCCACCACCTTCGAGCACCAACAACCACTCCAAGACCGGATGTTCAAGTTCGAGCTC ACCAAGCGCCTGGAGCACGACTTTGGCAAGGTCACCAAGCAGGAAGTCAAGGACTTTTTCCGGTGGGCGTCAGAT CACGTGACTGAGGTGTCTCACGAGTTTTACGTCAGAAAGGGTGGAGCTAGAAAGAGGCCCGCCCCCAATGACGCA GATATAAGTGAGCCCAAGCGGGCCTGTCCGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTCCGGTGGAC TACGCGGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTTTTTCCCTGCCGGCAATGC GAGAGAATGAATCAGAATGTGGACATTTGCTTCACGCACGGGGTCATGGACTGTGCCGAGTGCTTCCCCGTGTCA GAATCTCAACCCGTGTCTGTCGTCAGAAAGCGGACATATCAGAAACTGTGTCCGATTCATCACATCATGGGGAGG GCGCCCGAGGTGGCTTGTTCGGCCTGCGATCTGGCCAATGTGGACTTGGATGACTGTGACATGGAGCAATAA 144 ME149651883v.1135975-54620 (REGN 11546) CapVP1: (SEQ ID NO:29) ATGACTGACGGTTACCTTCCAGATTGGCTAGAGGACAACCTCTCTGAAGGCGTTCGAGAGTGGTGGGCGCTGCAA CCTGGAGCCCCTAAACCCAAGGCAAATCAACAACATCAGGACAACGCTCGGGGTCTTGTGCTTCCGGGTTACAAA TACCTCGGACCCGGCAACGGACTTGACAAGGGGGAACCCGTCAACGCAGCGGACGCGGCAGCCCTCGAACACGAC AAGGCCTACGACCAGCAGCTCAAGGCCGGTGACAACCCCTACCTCAAGTACAACCACGCCGACGCCGAGTTTCAG GAGCGTCTTCAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCCAAAAAGAGGATCCTT GAGCCTCTGGGTCTGGTTGAGGAAGCGGCTAAGACGGCTCCTGGAAAAAAGAGACCTGTAGAGCAATCTCCAGCA GAACCGGACTCCTCTTCGGGCATCGGCAAATCAGGCCAGCAGCCCGCTAGAAAAAGACTGAATTTTGGTCAGACT GGCGACACAGAGTCAGTCCCAGACCCTCAACCACTCGGACAACCTCCCGCAGCCCCCTCTGGTGTGGGATCTACT ACAATGGCTTCAGGCGGTGGCGCACCAATGGCAGACAATAACGAGGGTGCCGATGGAGTGGGTAATTCCTCAGGA AATTGGCATTGCGATTCCCAATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGCACCTGGGCCCTGCCCACC TACAACAATCACCTCTACAAGCAAATCTCCAGCCAATCAGGAGCCACCAACGACAACCACTACTTTGGCTACAGC ACCCCCTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAAC AACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAAT GACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCCGAGTACCAGCTCCCG TACGTCCTCGGCTCGGCGCATCAGGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTCCCACAGTATGGA TACCTCACCCTGAACAACGGGAGTCAGGCGGTAGGACGCTCTTCCTTTTACTGCCTGGAGTACTTTCCTTCTCAG ATGCTGCGTACTGGAAACAACTTTCAGTTTAGCTACACTTTTGAAGACGTGCCTTTCCACAGCAGCTACGCTCAC AGCCAAAGTCTGGACCGTCTCATGAATCCTCTGATCGACCAGTACCTGTACTATCTGAACAGGACACAAACAGCC AGTGGAACTCAGCAGTCTCGGCTACTGTTTAGCCAAGCTGGACCCACCAGTATGTCTCTTCAAGCTAAAAACTGG CTGCCTGGACCTTGCTACAGACAGCAGCGTCTGTCAAAGCAGGCAAACGACAACAACAACAGCAACTTTCCCTGG ACTGGTGCCACCAAATATCATCTGAATGGCCGGGACTCATTGGTGAACCCGGGCCCTGCTATGGCCAGTCACAAG GATGACAAAGAAAAGTTTTTCCCCATGCATGGAACCCTGATATTTGGTAAAGAAGGAACAAATGCCAACAACGCG GATTTGGAAAATGTCATGATTACAGATGAAGAAGAAATCCGCACCACCAATCCCGTGGCTACGGAGCAGTACGGG ACTGTGTCAAATAATTTGCAAAACTCAAACGCTGGTCCAACTACTGGAACTGTCAATCACCAAGGAGCGTTACCT GGTATGGTGTGGCAGGATCGAGACGTGTACCTGCAGGGACCCATTTGGGCCAAGATTCCTCACACCGATGGACAC TTTCATCCTTCTCCACTGATGGGAGGTTTTGGGCTCAAACACCCGCCTCCTCAGATCATGATCAAAAACACTCCC GTTCCAGCCAATCCTCCCACAAACTTTAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGGCAG GTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAGAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCC AACTACAACAAATCTGTTAATGTGGACTTTACTGTGGACACTAATGGTGTGTATTCAGAGCCTCGCCCCATTGGC ACCAGATACCTGACTCGTAATCTGTAA AAV rh10 (SEQ ID NO:30) GenBank: AY243015.1 - Non-human primate Adeno-associated virus isolate AAVrh.10 capsid protein (VP1) gene, complete cds 1 ATGGCTGCCG ATGGTTATCT TCCAGATTGG CTCGAGGACA ACCTCTCTGA GGGCATTCGC 61 GAGTGGTGGG ACTTGAAACC TGGAGCCCCG AAACCCAAAG CCAACCAGCA AAAGCAGGAC 121 GACGGCCGGG GTCTGGTGCT TCCTGGCTAC AAGTACCTCG GACCCTTCAA CGGACTCGAC 181 AAGGGGGAGC CCGTCAACGC GGCGGACGCA GCGGCCCTCG AGCACGACAA GGCCTACGAC 241 CAGCAGCTCA AAGCGGGTGA CAATCCGTAC CTGCGGTATA ACCACGCCGA CGCCGAGTTT 301 CAGGAGCGTC TGCAAGAAGA TACGTCTTTT GGGGGCAACC TCGGGCGAGC AGTCTTCCAG 361 GCCAAGAAGC GGGTTCTCGA ACCTCTCGGT CTGGTTGAGG AAGGCGCTAA GACGGCTCCT 421 GGAAAGAAGA GACCGGTAGA GCCATCACCC CAGCGTTCTC CAGACTCCTC TACGGGCATC 481 GGCAAGAAAG GCCAGCAGCC CGCGAAAAAG AGACTCAACT TTGGGCAGAC TGGCGACTCA 541 GAGTCAGTGC CCGACCCTCA ACCAATCGGA GAACCCCCCG CAGGCCCCTC TGGTCTGGGA 601 TCTGGTACAA TGGCTGCAGG CGGTGGCGCT CCAATGGCAG ACAATAACGA AGGCGCCGAC 661 GGAGTGGGTA GTTCCTCAGG AAATTGGCAT TGCGATTCCA CATGGCTGGG CGACAGAGTC 721 ATCACCACCA GCACCCGAAC CTGGGCCCTC CCCACCTACA ACAACCACCT CTACAAGCAA 781 ATCTCCAACG GGACTTCGGG AGGAAGCACC AACGACAACA CCTACTTCGG CTACAGCACC 841 CCCTGGGGGT ATTTTGACTT TAACAGATTC CACTGCCACT TCTCACCACG TGACTGGCAG 901 CGACTCATCA ACAACAACTG GGGATTCCGG CCCAAGAGAC TCAACTTCAA GCTCTTCAAC 961 ATCCAGGTCA AGGAGGTCAC GCAGAATGAA GGCACCAAGA CCATCGCCAA TAACCTTACC 1021 AGCACGATTC AGGTCTTTAC GGACTCGGAA TACCAGCTCC CGTACGTCCT CGGCTCTGCG 1081 CACCAGGGCT GCCTGCCTCC GTTCCCGGCG GACGTCTTCA TGATTCCTCA GTACGGGTAC 1141 CTGACTCTGA ACAATGGCAG TCAGGCCGTG GGCCGTTCCT CCTTCTACTG CCTGGAGTAC 1201 TTTCCTTCTC AAATGCTGAG AACGGGCAAC AACTTTGAGT TCAGCTACCA GTTTGAGGAC 145 ME149651883v.1135975-54620 (REGN 11546) 1261 GTGCCTTTTC ACAGCAGCTA CGCGCACAGC CAAAGCCTGG ACCGGCTGAT GAACCCCCTC 1321 ATCGACCAGT ACCTGTACTA CCTGTCTCGG ACTCAGTCCA CGGGAGGTAC CGCAGGAACT 1381 CAGCAGTTGC TATTTTCTCA GGCCGGGCCT AATAACATGT CGGCTCAGGC CAAAAACTGG 1441 CTACCCGGGC CCTGCTACCG GCAGCAACGC GTCTCCACGA CACTGTCGCA AAATAACAAC 1501 AGCAACTTTG CCTGGACCGG TGCCACCAAG TATCATCTGA ATGGCAGAGA CTCTCTGGTA 1561 AATCCCGGTG TCGCTATGGC AACCCACAAG GACGACGAAG AGCGATTTTT TCCGTCCAGC 1621 GGAGTCTTAA TGTTTGGGAA ACAGGGAGCT GGAAAAGACA ACGTGGACTA TAGCAGCGTT 1681 ATGCTAACCA GTGAGGAAGA AATTAAAACC ACCAACCCAG TGGCCACAGA ACAGTACGGC 1741 GTGGTGGCCG ATAACCTGCA ACAGCAAAAC GCCGCTCCTA TTGTAGGGGC CGTCAACAGT 1801 CAAGGAGCCT TACCTGGCAT GGTCTGGCAG AACCGGGACG TGTACCTGCA GGGTCCTATC 1861 TGGGCCAAGA TTCCTCACAC GGACGGAAAC TTTCATCCCT CGCCGCTGAT GGGAGGCTTT 1921 GGACTGAAAC ACCCGCCTCC TCAGATCCTG ATTAAGAATA CACCTGTTCC CGCGGATCCT 1981 CCAACTACCT TCAGTCAAGC TAAGCTGGCG TCGTTCATCA CGCAGTACAG CACCGGACAG 2041 GTCAGCGTGG AAATTGAATG GGAGCTGCAG AAAGAAAACA GCAAACGCTG GAACCCAGAG 2101 ATTCAATACA CTTCCAACTA CTACAAATCT ACAAATGTGG ACTTTGCTGT TAACACAGAT 2161 GGCACTTATT CTGAGCCTCG CCCCATCGGC ACCCGTTACC TCACCCGTAA TCTGTAA AAV rh39: (SEQ ID NO:31) GENBANK: EU368921.1 ADENO-ASSOCIATED VIRUS ISOLATE RH.39 CAPSID PROTEIN VP1 GENE, PARTIAL CDS 1 ATGGCTGCCG ATGGTTATCT TCCAGATTGG CTCGAGGACA ACCTCTCTGA GGGCATTCGC 61 GAGTGGTGGG CGCTGAAACC TGGAGCCCCG AAGCCCAAAG CCAACCAGCA AAAGCAGGAC 121 GACGGCCGGG GTCTGGTGCT TCCTGGCTAC AAGTACCTCG GACCCTTCAA CGGACTCGAC 181 AAGGGGGAGC CCGTCAACGC GGCGGACGCA GCGGCCCTCG AGCACGACAA GGCCTACGAC 241 CAGCAGCTCA AAGCGGGTGA CAATCCGTAC CTGCGGTATA ACCACGCCGA CGCCGAGTTT 301 CAGGAGCGTC TGCAAGAAGA TACGTCTTTT GGGGGCAACC TCGGGCGAGC AGTCTTCCAG 361 GCCAAGAAGC GGGTTCTCGA ACCTCTCGGT CTGGTTGAGG AAGCTGCTAA GACGGCTCCT 421 GGAAAGAAGA GACCGGTAGA ACCGTCACCT CAGCGTTCCC CCGACTCCTC CACGGGCATC 481 GGCAAGAAAG GCCAGCAGCC CGCTAAAAAG AGACTGAACT TTGGTCAGAC TGGCGACTCA 541 GAGTCAGTCC CCGACCCTCA ACCAATCGGA GAACCACCAG CAGGCCCCTC TGGTCTGGGA 601 TCTGGTACAA TGGCTGCAGG CGGTGGCGCT CCAATGGCAG ACAATAACGA AGGCGCCGAC 661 GGAGTGGGTA GTTCCTCAGG AAATTGGCAT TGCGATTCCA CATGGCTGGG CGACAGAGTC 721 ATCACCACCA GCACCCGAAC CTGGGCCCTG CCCACCTACA ACAACCACCT CTACAAGCAA 781 ATATCCAATG GGACATCGGG AGGAAGCACC AACGACAACA CCTACTTCGG CTACAGCACC 841 CCCTGGGGGT ATTTTGACTT CAACAGATTC CACTGCCACT TCTCACCACG TGACTGGCAG 901 CGACTCATCA ACAACAACTG GGGATTCCGG CCAAAAAGAC TCAGCTTCAA GCTCTTCAAC 961 ATCCAGGTCA AGGAGGTCAC GCAGAATGAA GGCACCAAGA CCATCGCCAA TAACCTTACC 1021 AGCACGATTC AGGTATTTAC GGACTCGGAA TACCAGCTGC CGTACGTCCT CGGCTCCGCG 1081 CACCAGGGCT GCCTGCCTCC GTTCCCGGCG GACGTCTTCA TGATTCCCCA GTACGGCTAC 1141 CTTACACTGA ACAATGGAAG TCAAGCCGTA GGCCGTTCCT CCTTCTACTG CCTGGAATAT 1201 TTTCCATCTC AAATGCTGCG AACTGGAAAC AATTTTGAAT TCAGCTACAC CTTCGAGGAC 1261 GTGCCTTTCC ACAGCAGCTA CGCACACAGC CAGAGCTTGG ACCGACTGAT GAATCCTCTC 1321 ATCGACCAGT ACCTGTACTA CTTATCCAGA ACTCAGTCCA CAGGAGGAAC TCAAGGTACC 1381 CAGCAATTGT TATTTTCTCA AGCTGGGCCT GCAAACATGT CGGCTCAGGC TAAGAACTGG 1441 CTACCTGGAC CTTGCTACCG GCAGCAGCGA GTCTCTACGA CACTGTCGCA AAACAACAAC 1501 AGCAACTTTG CTTGGACTGG TGCCACCAAA TATCACCTGA ACGGAAGAGA CTCTTTGGTA 1561 AATCCCGGTG TCGCCATGGC AACCCACAAG GACGACGAGG AACGCTTCTT CCCGTCGAGT 1621 GGAGTCCTGA TGTTTGGAAA ACAGGGTGCT GGAAGAGACA ATGTGGACTA CAGCAGCGTT 1681 ATGCTAACCA GCGAAGAAGA AATTAAAACC ACTAACCCTG TAGCCACAGA ACAATACGGT 1741 GTGGTGGCTG ATAACTTGCA GCAAACCAAT ACGGGGCCTA TTGTGGGAAA TGTCAACAGC 1801 CAAGGAGCCT TACCTGGCAT GGTCTGGCAG AACCGAGACG TGTACCTGCA GGGTCCCATC 1861 TGGGCCAAGA TTCCTCACAC GGACGGCAAC TTCCACCCTT CACCGCTAAT GGGAGGATTT 1921 GGACTGAAGC ACCCACCTCC TCAGATCCTG ATCAAGAACA CGCCGGTACC TGCGGATCCT 1981 CCAACAACGT TCAGCCAGGC GAAATTGGCT TCCTTCATTA CGCAGTACAG CACCGGACAG 2041 GTCAGCGTGG AAATCGAGTG GGAGCTGCAG AAGGAGAACA GCAAACGCTG GAACCCAGAG 2101 ATTCAGTACA CTTCAAACTA CTACAAATCT ACAAATGTGG ACTTTGCTGT CAATACAGAG 2161 GGAACTTATT CTGAGCCTCG CCCCATTGGT ACTCGTTACC TCACCCGTAA TCTG 146 ME149651883v.1135975-54620 (REGN 11546) AAV rh43: (SEQ ID NO:32) GENBANK: JA400153.1 AAV serotype, clone rh.43 1 ATGGCTGCCG ATGGTTATCT TCCAGATTGG CTCGAGGACA ACCTCTCTGA GGGCATTCGC 61 GAGTGGTGGG ACTTGAAACC TGGAGCCCCG AAACCCAAAG CCAACCAGCA AAAGCAGGAC 121 GACGGCCGGG GCCTGGTGCT TCCTGGCTAC AAGTACCTCG GACCCTTCAA CGGACTCGAC 181 AAGGGGGAGC CCGTCAACGC GGCGGACGCA GCGGCCCTCG AGCACGACAA GGCCTACGAC 241 CAGCAGCTCG AAGCGGGTGA CAATCCGTAC CTGCGGTATA ACCACGCCGA CGCCGAGTTT 301 CAGGAGCGTC TGCAAGAAGA TACGTCTTTT GGGGGCAACC TCGGGCGAGC AGTCTTCCAG 361 GCCAAGAAGC GGGTTCTCGA ACCTCTCGGT CTGGTTGAGG AAGGCGCTAA GACGGCTCCT 421 GGAAAGAAGA GACCAGTAGA GCAGTCACCC CAAGAACCAG ACTCCTCCTC GGGCATCGGC 481 AAGAAAGGCC AACAGCCCGC CAGAAAAAGA CTCAATTTTG GCCAGACTGG CGACTCAGAG 541 TCAGTTCCAG ACCCTCAACC TCTCGGAGAA CCTCCAGCAG CGCCCTCTGG TGTGGGACCT 601 AATACAATGG CTGCAGGCGG TGGCGCACCA ATGGCAGACA ATAACGAAGG CGCCGACGGA 661 GTGGGTAGTT CCTCGGGAAA TTGGCATTGC GATTCCACAT GGCTGGGCGA CAGAGTCATC 721 ACCACCAGCA CCCGAACCTG GGCCCTGCCC ACCTACAACA ACCACCTCTA CAAGCAAATC 781 TCCAACGGGA CATCGGGAGG AGCCACCAAC GACAACACCT ACTTCGGCTA CAGCACCCCC 841 TGGGGGTATT TTGACTTTAA CAGATTCCAC TGCCACTTTT CACCACGTGA CTGGCAGCGA 901 CTCATCAACA ACAACTGGGG ATTCCGGCCC AAGAGACTCA GCTTCAAGCT CTTCAACATC 961 CAGGTCAAGG AGGTCACGCA GAATGAAGGC ACCAAGACCA TCGCCAATAA CCTCACCAGC 1021 ACCATCCAGG TGTTTACGGA CTCGGAGTAC CAGCTGCCGT ACGTTCTCGG CTCTGCCCAC 1081 CAGGGCTGCC TGCCTCCGTT CCCGGCGGAC GTGTTCATGA TTCCCCAGTA CGGCTACCTA 1141 ACACTCAACA ACGGTAGTCA GGCCGTGGGA CGCTCCTCCT TCTACTGCCT GGAATACTTT 1201 CCTTCGCAGA TGCTGAGAAC CGGCAACAAC TTCCAGTTTA CTTACACCTT CGAGGACGTG 1261 CCTTTCCACA GCAGCTACGC CCACAGCCAG AGCTTGGACC GGCTGATGAA TCCTCTGATT 1321 GACCAGTACC TGTACTACTT GTCTCGGACT CAAACAACAG GAGGCACGGC AAATACGCAG 1381 ACTCTGGGCT TCAGCCAAGG TGGGCCTAAT ACAATGGCCA ATCAGGCAAA GAACTGGCTG 1441 CCAGGACCCT GTTACCGCCA ACAACGCGTC TCAACGACAA CCGGGCAAAA CAACAATAGC 1501 AACTTTGCCT GGACTGCTGG GACCAAATAC CATCTGAATG GAAGAAATTC ATTGGCTAAT 1561 CCTGGCATCG CTATGGCAAC ACACAAAGAC GACGAGGAGC GTTTTTTCCC AGTAACGGGA 1621 TCCTGTTTTT GGCAACAAAA TGCTGCCAGA GACAATGCGG ATTACAGCGA TGTCATGCTC 1681 ACCAGCGAGG AAGAAATCAA AACCACTAAC CCTGTGGCTA CAGAGGAATA CGGTATCGTG 1741 GCAGATAACT TGCAGCAGCA AAACACGGCT CCTCAAATTG GAACTGTCAA CAGCCAGGGG 1801 GCCTTACCCG GTATGGTCTG GCAGAACCGG GACGTGTACC TGCAGGGTCC CATCTGGGCC 1861 AAGATTCCTC ACACGGACGG CAACTTCCAC CCGTCTCCGC TGATGGGCGG CTTTGGCCTG 1921 AAACATCCTC CGCCTCAGAT CCTGATCAAG AACACGCCTG TACCTGCGGA TCCTCCGACC 1981 ACCTTCAACC AGTCAAAGCT GAACTCTTTC ATCACGCAAT ACAGCACCGG ACAGGTCAGC 2041 GTGGAAATTG AATGGGAGCT ACAGAAGGAA AACAGCAAGC GCTGGAACCC CGAGATCCAG 2101 TACACCTCCA ACTACTACAA ATCTACAAGT GTGGACTTTG CTGTTAATAC AGAAGGCGTG 2161 TACTCTGAAC CCCGCCCCAT TGGCACCCGT TACCTCACCC GTAATCTGTA A AAVrh.74: (SEQ ID NO:33) Nucleotide sequence encoding AAV rh74 capsid protein: ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGACCTGAA ACCTGGAGCCCCGAAACCCAAAGCCAACCAGCAAAAGCAGGACAACGGCCGGGGTCTGGTGCTTCCTGGCTACAAGT ACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTCAACGCGGCGGACGCAGCGGCCCTCGAGCACGACAAG GCCTACGACCAGCAGCTCCAAGCGGGTGACAATCCGTACCTGCGGTATAATCACGCCGACGCCGAGTTTCAGGAGCG TCTGCAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGCGCAGTCTTCCAGGCCAAAAAGCGGGTTCTCGAACCTC TGGGCCTGGTTGAATCGCCGGTTAAGACGGCTCCTGGAAAGAAGAGACCGGTAGAGCCATCACCCCAGCGCTCTCCA GACTCCTCTACGGGCATCGGCAAGAAAGGCCAGCAGCCCGCAAAAAAGAGACTCAATTTTGGGCAGACTGGCGACTC AGAGTCAGTCCCCGACCCTCAACCAATCGGAGAACCACCAGCAGGCCCCTCTGGTCTGGGATCTGGTACAATGGCTG CAGGCGGTGGCGCTCCAATGGCAGACAATAACGAAGGCGCCGACGGAGTGGGTAGTTCCTCAGGAAATTGGCATTGC GATTCCACATGGCTGGGCGACAGAGTCATCACCACCAGCACCCGCACCTGGGCCCTGCCCACCTACAACAACCACCT CTACAAGCAAATCTCCAACGGGACCTCGGGAGGAAGCACCAACGACAACACCTACTTCGGCTACAGCACCCCCTGGG GGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAGCGACTCATCAACAACAACTGGGGA TTCCGGCCCAAGAGGCTCAACTTCAAGCTCTTCAACATCCAAGTCAAGGAGGTCACGCAGAATGAAGGCACCAAGAC 147 ME149651883v.1135975-54620 (REGN 11546) CATCGCCAATAACCTTACCAGCACGATTCAGGTCTTTACGGACTCGGAATACCAGCTCCCGTACGTGCTCGGCTCGG CGCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGACGTCTTCATGATTCCTCAGTACGGGTACCTGACTCTGAACAAT GGCAGTCAGGCTGTGGGCCGGTCGTCCTTCTACTGCCTGGAGTACTTTCCTTCTCAAATGCTGAGAACGGGCAACAA CTTTGAATTCAGCTACAACTTCGAGGACGTGCCCTTCCACAGCAGCTACGCGCACAGCCAGAGCCTGGACCGGCTGA TGAACCCTCTCATCGACCAGTACTTGTACTACCTGTCCCGGACTCAAAGCACGGGCGGTACTGCAGGAACTCAGCAG TTGCTATTTTCTCAGGCCGGGCCTAACAACATGTCGGCTCAGGCCAAGAACTGGCTACCCGGTCCCTGCTACCGGCA GCAACGCGTCTCCACGACACTGTCGCAGAACAACAACAGCAACTTTGCCTGGACGGGTGCCACCAAGTATCATCTGA ATGGCAGAGACTCTCTGGTGAATCCTGGCGTTGCCATGGCTACCCACAAGGACGACGAAGAGCGATTTTTTCCATCC AGCGGAGTCTTAATGTTTGGGAAACAGGGAGCTGGAAAAGACAACGTGGACTATAGCAGCGTGATGCTAACCAGCGA GGAAGAAATAAAGACCACCAACCCAGTGGCCACAGAACAGTACGGCGTGGTGGCCGATAACCTGCAACAGCAAAACG CCGCTCCTATTGTAGGGGCCGTCAATAGTCAAGGAGCCTTACCTGGCATGGTGTGGCAGAACCGGGACGTGTACCTG CAGGGTCCCATCTGGGCCAAGATTCCTCATACGGACGGCAACTTTCATCCCTCGCCGCTGATGGGAGGCTTTGGACT GAAGCATCCGCCTCCTCAGATCCTGATTAAAAACACACCTGTTCCCGCCGATCCTCCGACCACCTTCAATCAGGCCA AGCTGGCTTCTTTCATCACGCAGTACAGTACCGGTCAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAGAAC AGCAAACGCTGGAACCCAGAGATTCAGTACACTTCCAACTACTACAAATCTACAAATGTGGACTTTGCTGTCAATAC TGAGGGTACTTATTCCGAGCCTCGCCCCATTGGCACCCGTTACCTCACCCGTAATCTGTAA AAV27M8: AAV27m8 is characterized by a 10-amino acid peptide (SEQ ID NO:34) LALGETTRPA ITR Sequence(SEQ ID NO:35) CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTC GCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT Rep2 Sequence – Contains Rep78 and Rep52 (start codon underlined) (SEQ ID NO:36) ATGCCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCCGGCATTTCTGACAGC TTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGATTGAGCAG GCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGGAG GCCCTTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCACATGCACGTGCTCGTGGAAACCACCGGGGTG AAATCCATGGTTTTGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGAG CCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGAACAAGGTGGTGGATGAG TGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGGCGTGGACTAATATGGAACAGTAT TTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAG GAGCAGAACAAAGAGAATCAGAATCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTACATG GAGCTGGTCGGGTGGCTCGTGGACAAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATAC ATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGC CTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTATAAA ATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTTCGGC AAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACT GTGCCCTTCTACGGGTGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATC TGGTGGGAGGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTGCGC GTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCTCCAACACCAACATGTGC GCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTTGAACTC ACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGAT CACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCA GATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAAC TACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGC GAGAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCA GAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGTG CCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAATAA Cap2 Sequence – contains sequentially VP1, VP2, AAP, VP3 (start codons underlined) (SEQ ID NO:37) 148 ME149651883v.1135975-54620 (REGN 11546) ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTC AAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTAC AAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCAC GACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTT CAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTT CTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCT GTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAG ACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACT AATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCG GGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCC ACCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTAC AGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATC AACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAG AATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTC CCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTAT GGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCT CAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCT CACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACT CCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGG AACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATAC TCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGC CACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACA AATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAG TATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTT CTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGAC GGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAAC ACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACG GGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTAC ACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCC ATTGGCACCAGATACCTGACTCGTAATCTGTAA Cap5 Sequence – contains sequentially VP1, VP2, AAP, VP3 (start codons underlined) (SEQ ID NO:38) ATGGCTTTTGTTGATCACCCTCCAGATTGGTTGGAAGAAGTTGGTGAAGGTCTTCGCGAGTTTTTGGGCCTTGAA GCGGGCCCACCGAAACCAAAACCCAATCAGCAGCATCAAGATCAAGCCCGTGGTCTTGTGCTGCCTGGTTATAAC TATCTCGGACCCGGAAACGGTCTCGATCGAGGAGAGCCTGTCAACAGGGCAGACGAGGTCGCGCGAGAGCACGAC ATCTCGTACAACGAGCAGCTTGAGGCGGGAGACAACCCCTACCTCAAGTACAACCACGCGGACGCCGAGTTTCAG GAGAAGCTCGCCGACGACACATCCTTCGGGGGAAACCTCGGAAAGGCAGTCTTTCAGGCCAAGAAAAGGGTTCTC GAACCTTTTGGCCTGGTTGAAGAGGGTGCTAAGACGGCCCCTACCGGAAAGCGGATAGACGACCACTTTCCAAAA AGAAAGAAGGCTCGGACCGAAGAGGACTCCAAGCCTTCCACCTCGTCAGACGCCGAAGCTGGACCCAGCGGATCC CAGCAGCTGCAAATCCCAGCCCAACCAGCCTCAAGTTTGGGAGCTGATACAATGTCTGCGGGAGGTGGCGGCCCA TTGGGCGACAATAACCAAGGTGCCGATGGAGTGGGCAATGCCTCGGGAGATTGGCATTGCGATTCCACGTGGATG GGGGACAGAGTCGTCACCAAGTCCACCCGAACCTGGGTGCTGCCCAGCTACAACAACCACCAGTACCGAGAGATC AAAAGCGGCTCCGTCGACGGAAGCAACGCCAACGCCTACTTTGGATACAGCACCCCCTGGGGGTACTTTGACTTT AACCGCTTCCACAGCCACTGGAGCCCCCGAGACTGGCAAAGACTCATCAACAACTACTGGGGCTTCAGACCCCGG TCCCTCAGAGTCAAAATCTTCAACATTCAAGTCAAAGAGGTCACGGTGCAGGACTCCACCACCACCATCGCCAAC AACCTCACCTCCACCGTCCAAGTGTTTACGGACGACGACTACCAGCTGCCCTACGTCGTCGGCAACGGGACCGAG GGATGCCTGCCGGCCTTCCCTCCGCAGGTCTTTACGCTGCCGCAGTACGGTTACGCGACGCTGAACCGCGACAAC ACAGAAAATCCCACCGAGAGGAGCAGCTTCTTCTGCCTAGAGTACTTTCCCAGCAAGATGCTGAGAACGGGCAAC AACTTTGAGTTTACCTACAACTTTGAGGAGGTGCCCTTCCACTCCAGCTTCGCTCCCAGTCAGAACCTCTTCAAG CTGGCCAACCCGCTGGTGGACCAGTACTTGTACCGCTTCGTGAGCACAAATAACACTGGCGGAGTCCAGTTCAAC AAGAACCTGGCCGGGAGATACGCCAACACCTACAAAAACTGGTTCCCGGGGCCCATGGGCCGAACCCAGGGCTGG AACCTGGGCTCCGGGGTCAACCGCGCCAGTGTCAGCGCCTTCGCCACGACCAATAGGATGGAGCTCGAGGGCGCG AGTTACCAGGTGCCCCCGCAGCCGAACGGCATGACCAACAACCTCCAGGGCAGCAACACCTATGCCCTGGAGAAC ACTATGATCTTCAACAGCCAGCCGGCGAACCCGGGCACCACCGCCACGTACCTCGAGGGCAACATGCTCATCACC AGCGAGAGCGAGACGCAGCCGGTGAACCGCGTGGCGTACAACGTCGGCGGGCAGATGGCCACCAACAACCAGAGC TCCACCACTGCCCCCGCGACCGGCACGTACAACCTCCAGGAAATCGTGCCCGGCAGCGTGTGGATGGAGAGGGAC GTGTACCTCCAAGGACCCATCTGGGCCAAGATCCCAGAGACGGGGGCGCACTTTCACCCCTCTCCGGCCATGGGC GGATTCGGACTCAAACACCCACCGCCCATGATGCTCATCAAGAACACGCCTGTGCCCGGAAATATCACCAGCTTC 149 ME149651883v.1135975-54620 (REGN 11546) TCGGACGTGCCCGTCAGCAGCTTCATCACCCAGTACAGCACCGGGCAGGTCACCGTGGAGATGGAGTGGGAGCTC AAGAAGGAAAACTCCAAGAGGTGGAACCCAGAGATCCAGTACACAAACAACTACAACGACCCCCAGTTTGTGGAC TTTGCCCCGGACAGCACCGGGGAATACAGAAGCACCAGACCTATCGGAACCCGATACCTTACCCGACCCCTTTAA EXAMPLE 31- Adenovirus Polynucleotide Sequences Adenovirus (Ad) polynucleotides can be selected from any serotype, and representative polynucleotides are exemplified below. E2A Full Sequence (SEQ ID NO:39) CGACCGCACCCTGTGACGAAAGCCGCCCGCAAGCTGCGCCCCTGAGTTAGTCATCTGAACTTCGGCCTGGGCGT CTCTGGGAAGTACCACAGTGGTGGGAGCGGGACTTTCCTGGTACACCAGGGCAGCGGGCCAACTACGGGGATTAA GGTTATTACGAGGTGTGGTGGTAATAGCCGCCTGTTCGAGGAGAATTCGGTTTCGGTGGGCGCGGATTCCGTTGA CCCGGGATATCATGTGGGGTCCCGCGCTCATGTAGTTTATTCGGGTTGAGTAGTCTTGGGCAGCTCCAGCCGCAA GTCCCATTTGTGGCTGGTAACTCCACATGTAGGGCGTGGGAATTTCCTTGCTCATAATGGCGCTGACGACAGGTG CTGGCGCCGGGTGTGGCCGCTGGAGATGACGTAGTTTTCGCGCTTAAATTTGAGAAAGGGCGCGAAACTAGTCCT TAAGAGTCAGCGCGCAGTATTTGCTGAAGAGAGCCTCCGCGTCTTCCAGCGTGCGCCGAAGCTGATCTTCGCTTT TGTGATACAGGCAGCTGCGGGTGAGGGAGCGCAGAGACCTGTTTTTTATTTTCAGCTCTTGTTCTTGGCCCCTGC TTTGTTGAAATATAGCATACAGAGTGGGAAAAATCCTATTTCTAAGCTCGCGGGTCGATACGGGTTCGTTGGGCG CCAGACGCAGCGCTCCTCCTCCTGCTGCTGCCGCCGCTGTGGATTTCTTGGGCTTTGTCAGAGTCTTGCTATCCG GTCGCCTTTGCTTCTGTGTGACCGCTGCTGTTGCTGCCGCTGCCGCTGCCGCCGGTGCAGTAGGGGCTGTAGAGA TGACGGTAGTAATGCAGGATGTTACGGGGGAAGGCCACGCCGTGATGGTAGAGAAGAAAGCGGCGGGCGAAGGAG ATGTTGCCCCCACAGTCTTGCAAGCAAGCAACTATGGCGTTCTTGTGCCCGCGCCACGAGCGGTAGCCTTGGCGC TGTTGTTGCTCTTGGGCTAACGGCGGCGGCTGCTTAGACTTACCGGCCCTGGTTCCAGTGGTGTCCCATCTACGG TTGGGTCGGCGAACAGGCAGTGCCGGCGGCGCCTGAGGAGCGGAGGTTGTAGCGATGCTGGGAACGGTTGCCAAT TTCTGGGGCGCCGGCGAGGGGAATGCGACCGAGGGTGACGGTGTTTCGTCTGACACCTCTTCGGCCTCGGAAGCT TCGTCTAGGCTGTCCCAGTCTTCCATCATCTCCTCCTCCTCGTCCAAAACCTCCTCTGCCTGACTGTCCCAGTAT TCCTCCTCGTCCGTGGGTGGCGGCGGCGGCAGCTGCAGCTTCTTTTTGGGTGCCATCCTGGGAAGCAAGGGCCCG CGGCTGCTGATAGGGCTGCGGCGGCGGGGGGATTGGGTTGAGCTCCTCGCCGGACTGGGGGTCCAGGTAAACCCC CCGTCCCTTTCGTAGCAGAAACTCTTGGCGGGCTTTGTTGATGGCTTGCAATTGGCCAAGGATGTGGCCCTGGGT AATGACGCAGGCGGTAAGCTCCGCATTTGGCGGGCGGGATTGGTCTTCGTAGAACCTAATCTCGTGGGCGTGGTA GTCCTCAGGTACAAATTTGCGAAGGTAAGCCGACGTCCACAGCCCCGGAGTGAGTTTCAACCCCGGAGCCGCGGA CTTTTCGTCAGGCGAGGGACCCTGCAGCTCAAAGGTACCGATAATTTGACTTTCGCTAAGCAGTTGCGAATTGCA GACCAGGGAGCGGTGCGGGGTGCATAGGTTGCAGCGACAGTGACACTCCAGTAGGCCGTCACCGCTCACGTCTTC CATGATGTCGGAGTGGTAGGCAAGGTAGTTGGCTAGCTGCAGAAGGTAGCAGTGACCCCAAAGCGGCGGAGGGCA TTCACGGTACTTAATGGGCACAAAGTCGCTAGGAAGCGCACAGCAGGTGGCGGGCAGAATTCCTGAACGCTCTAG GATAAAGTTCCTAAAGTTTTGCAACATGCTTTGACTGGTGAAGTCTGGCAGACCCTGTTGCAGGGTTTTAAGCAG GCGTTCGGGGAAGATAATGTCCGCCAGGTGCGCGGCCACGGAGCGCTCGTTGAAGGCCGTCCATAGGTCCTTCAA GTTTTGCTTTAGCAGCTTCTGCAGCTCCTTTAGGTTGCGCTCCTCCAGGCATTGCTGCCACACGCCCATGGCCGT TTGCCAGGTGTAGCACAGAAATAAGTAAACGCAGTCGCGGACGTAGTCGCGGCGCGCCTCGCCCTTGAGCGTGGA ATGAAGCACGTTTTGCCCGAGGCGGTTTTCGTGCAAAATTCCAAGGTAGGAGACCAGGTTGCAGAGCTCCACGTT GGAAATTTTGCAGGCCTGGCGCACGTAGCCCTGGCGAAAGGTGTAGTGCAACGTTTCCTCTAGCTTGCGCTGCAT CTCCGGGTCAGCAAAGAACCGCTGCATGCACTCAAGCTCCACGGTAACAAGCACTGCGGCCATCATTAGCTTGCG TCGCTCCTCCAAGTCGGCAGGCTCGCGCGTCTCAAGCCAGCGCGCCAGCTGCTCATCGCCAACTGCGGGTAGGCC CTCCTCGGTTTGTTCTTGCAAGTTTGCATCCCTCTCCAGGGGTCGTGCACGGCGCACGATCAGCTCGCTCATGAC TGTGCTCATAACCTTGGGGGGTAGGTTAAGTGCCGGGTAGGCAAAGTGGGTGACCTCGATGCTGCGTTTCAGCAC GGCTAGGCGCGCGTTGTCACCCTCAAGTTCCACCAGCACTCCACAGTGACTTTCATTTTCGCTGTTTTCTTGTTG CAGAGCGTTTGCCGCGCGTTTCTCGTCGCGTCCAAGACCCTCAAAGATTTTTGGCACTTCGTCGAGCGAGGCGAT ATCAGGTATGACAGCGCCCTGCCGCAAGGCCAGCTGCTTGTCCGCTCGGCTGCGGTTGGCACGGCAGGATAGGGG TATCTTGCAGTTTTGGAAAAAGATGTGATAGGTGGCAAGCACCTCTGGCACGGCAAATACGGGGTAGAAGTTGAG GCGCGGGTTGGGCTCGCATGTGCCGTTTTCTTGGCGTTTGGGGGGTACGCGCGGTGAGAACAGGTGGCGTTCGTA GGCAAGGCTGACATCCGCTATGGCGAGGGGCACATCGCTGCGCTCTTGCAACGCGTCGCAGATAATGGCGCACTG GCGCTGCAGATGCTTCAACAGCACGTCGTCTCCCACATCTAGGTAGTCGCCATGCCTTTGGTCCCCCCGCCCGAC TTGTTCCTCGTTTGCCTCTGCGTCGTCCTGGTCTTGCTTTTTATCCTCTGTTGGTACTGAGCGATCCTCGTCGTC TTCGCTTACAAAACCTGGGTCCTGCTCGATAATCACTTCCTCCTCCTCAAGCGGGGGTGCCTCGACGGGGAAGGT GGTAGGCGCGTTGGCGGCATCGGTGGAGGCGGTGGTGGCGAACTCAAAGGGGGCGGTTAGGCTGTCCTCCTTCTC GACTGACTCCATGATCTTTTTCTGCCTATAGGAGAAGGAAATGGCCAGTCGGGAAGAGGAGCAGCGCGAAACCAC CCCCGAGCGCGGACGCGGTGCGGCGCGACGTCCACCAACCATGGAGGACGTGTCGTCCCCGTCGCCGTCGCCGCC 150 ME149651883v.1135975-54620 (REGN 11546) GCCTCCCCGCGCGCCCCCAAAAAAGCGGCTGAGGCGGCGTCTCGAGTCCGAGGACGAAGAAGACTCGTCACAAGA TGCGCTGGTGCCGCGCACACCCAGCCCGCGGCCATCGACCTCGACGGCGGATTTGGCCATTGCGTCCAAAAAGAA AAAGAAGCGCCCCTCTCCCAAGCCCGAGCGCCCGCCATCCCCAGAGGTGATCGTGGACAGCGAGGAAGAAAGAGA AGATGTGGCGCTACAAATGGTGGGTTTCAGCAACCCACCGGTGCTAATCAAGCACGGCAAGGGAGGTAAGCGCAC GGTGCGGCGGCTGAATGAAGACGACCCAGTGGCGCGGGGTATGCGGACGCAAGAGGAAAAGGAAGAGTCCAGTGA AGCGGAAAGTGAAAGCACGGTGATAAACCCGCTGAGCCTGCCGATCGTGTCTGCGTGGGAGAAGGGCATGGAGGC TGCGCGCGCGTTGATGGACAAGTACCACGTGGATAACGATCTAAAGGCAAACTTCAAGCTACTGCCTGACCAAGT GGAAGCTCTGGCGGCCGTATGCAAGACCTGGCTAAACGAGGAGCACCGCGGGTTGCAGCTGACCTTCACCAGCAA CAAGACCTTTGTGACGATGATGGGGCGATTCCTGCAGGCGTACCTGCAGTCGTTTGCAGAGGTAACCTACAAGCA CCACGAGCCCACGGGCTGCGCGTTGTGGCTGCACCGCTGCGCTGAGATCGAAGGCGAGCTTAAGTGTCTACACGG GAGCATTATGATAAATAAGGAGCACGTGATTGAAATGGATGTGACGAGCGAAAACGGGCAGCGCGCGCTGAAGGA GCAGTCTAGCAAGGCCAAGATCGTGAAGAACCGGTGGGGCCGAAATGTGGTGCAGATCTCCAACACCGACGCAAG GTGCTGCGTGCATGACGCGGCCTGTCCGGCCAATCAGTTTTCCGGCAAGTCTTGCGGCATGTTCTTCTCTGAAGG CGCAAAGGCTCAGGTGGCTTTTAAGCAGATCAAGGCTTTCATGCAGGCGCTGTATCCTAACGCCCAGACCGGGCA CGGTCACCTTCTGATGCCACTACGGTGCGAGTGCAACTCAAAGCCTGGGCATGCACCCTTTTTGGGAAGGCAGCT ACCAAAGTTGACTCCGTTCGCCCTGAGCAACGCGGAGGACCTGGACGCGGATCTGATCTCCGACAAGAGCGTGCT GGCCAGCGTGCACCACCCGGCGCTGATAGTGTTCCAGTGCTGCAACCCTGTGTATCGCAACTCGCGCGCGCAGGG CGGAGGCCCCAACTGCGACTTCAAGATATCGGCGCCCGACCTGCTAAACGCGTTGGTGATGGTGCGCAGCCTGTG GAGTGAAAACTTCACCGAGCTGCCGCGGATGGTTGTGCCTGAGTTTAAGTGGAGCACTAAACACCAGTATCGCAA CGTGTCCCTGCCAGTGGCGCATAGCGATGCGCGGCAGAACCCCTTTGATTTTTAAACGGCGCAGACGGCAAGGGT GGGGGGTAAATAATCACCCGAGAGTGTACAAATAAAAACATTTGCCTTTATTGAAAGTGTCTCCTAGTACATTAT TTTTACATGTTTTTCAAGTGACAAAAAGAAGTGGCGCTCCTAATCTGCGCACTGTGGCTGCGGAAGTAGGGCGAG TGGCGCTCCAGGAAGCTGTAGAGCTGTTCCTGGTTGCGACGCAGGGTGGGCTGTACCTGGGGACTGTTAAGCATG GAGTTGGGTACC E2A ORF Sequence (SEQ ID NO:40) ATGGCCAGTCGGGAAGAGGAGCAGCGCGAAACCACCCCCGAGCGCGGACGCGGTGCGGCGCGACGTCCACCAACC ATGGAGGACGTGTCGTCCCCGTCGCCGTCGCCGCCGCCTCCCCGCGCGCCCCCAAAAAAGCGGCTGAGGCGGCGT CTCGAGTCCGAGGACGAAGAAGACTCGTCACAAGATGCGCTGGTGCCGCGCACACCCAGCCCGCGGCCATCGACC TCGACGGCGGATTTGGCCATTGCGTCCAAAAAGAAAAAGAAGCGCCCCTCTCCCAAGCCCGAGCGCCCGCCATCC CCAGAGGTGATCGTGGACAGCGAGGAAGAAAGAGAAGATGTGGCGCTACAAATGGTGGGTTTCAGCAACCCACCG GTGCTAATCAAGCACGGCAAGGGAGGTAAGCGCACGGTGCGGCGGCTGAATGAAGACGACCCAGTGGCGCGGGGT ATGCGGACGCAAGAGGAAAAGGAAGAGTCCAGTGAAGCGGAAAGTGAAAGCACGGTGATAAACCCGCTGAGCCTG CCGATCGTGTCTGCGTGGGAGAAGGGCATGGAGGCTGCGCGCGCGTTGATGGACAAGTACCACGTGGATAACGAT CTAAAGGCAAACTTCAAGCTACTGCCTGACCAAGTGGAAGCTCTGGCGGCCGTATGCAAGACCTGGCTAAACGAG GAGCACCGCGGGTTGCAGCTGACCTTCACCAGCAACAAGACCTTTGTGACGATGATGGGGCGATTCCTGCAGGCG TACCTGCAGTCGTTTGCAGAGGTAACCTACAAGCACCACGAGCCCACGGGCTGCGCGTTGTGGCTGCACCGCTGC GCTGAGATCGAAGGCGAGCTTAAGTGTCTACACGGGAGCATTATGATAAATAAGGAGCACGTGATTGAAATGGAT GTGACGAGCGAAAACGGGCAGCGCGCGCTGAAGGAGCAGTCTAGCAAGGCCAAGATCGTGAAGAACCGGTGGGGC CGAAATGTGGTGCAGATCTCCAACACCGACGCAAGGTGCTGCGTGCATGACGCGGCCTGTCCGGCCAATCAGTTT TCCGGCAAGTCTTGCGGCATGTTCTTCTCTGAAGGCGCAAAGGCTCAGGTGGCTTTTAAGCAGATCAAGGCTTTC ATGCAGGCGCTGTATCCTAACGCCCAGACCGGGCACGGTCACCTTCTGATGCCACTACGGTGCGAGTGCAACTCA AAGCCTGGGCATGCACCCTTTTTGGGAAGGCAGCTACCAAAGTTGACTCCGTTCGCCCTGAGCAACGCGGAGGAC CTGGACGCGGATCTGATCTCCGACAAGAGCGTGCTGGCCAGCGTGCACCACCCGGCGCTGATAGTGTTCCAGTGC TGCAACCCTGTGTATCGCAACTCGCGCGCGCAGGGCGGAGGCCCCAACTGCGACTTCAAGATATCGGCGCCCGAC CTGCTAAACGCGTTGGTGATGGTGCGCAGCCTGTGGAGTGAAAACTTCACCGAGCTGCCGCGGATGGTTGTGCCT GAGTTTAAGTGGAGCACTAAACACCAGTATCGCAACGTGTCCCTGCCAGTGGCGCATAGCGATGCGCGGCAGAAC CCCTTTGATTTTTAA E4 Full Sequence (SEQ ID NO:41) CCCGGGCGTTTTAGGGCGGAGTAACTTGCATGTATTGGGAATTGTAGTTTTTTTAAAATGGGAAGTGACGTATCG TGGGAAAACGGAAGTGAAGATTTGAGGAAGTTGTGGGTTTTTTGGCTTTCGTTTCTGGGCGTAGGTTCGCGTGCG GTTTTCTGGGTGTTTTTTGTGGACTTTAACCGTTACGTCATTTTTTAGTCCTATATATACTCGCTCTGTACTTGG CCCTTTTTACACTGTGACTGATTGAGCTGGTGCCGTGTCGAGTGGTGTTTTTTAATAGGTTTTTTTACTGGTAAG GCTGACTGTTATGGCTGCCGCTGTGGAAGCGCTGTATGTTGTTCTGGAGCGGGAGGGTGCTATTTTGCCTAGGCA GGAGGGTTTTTCAGGTGTTTATGTGTTTTTCTCTCCTATTAATTTTGTTATACCTCCTATGGGGGCTGTAATGTT GTCTCTACGCCTGCGGGTATGTATTCCCCCGGGCTATTTCGGTCGCTTTTTAGCACTGACCGATGTTAACCAACC TGATGTGTTTACCGAGTCTTACATTATGACTCCGGACATGACCGAGGAACTGTCGGTGGTGCTTTTTAATCACGG 151 ME149651883v.1135975-54620 (REGN 11546) TGACCAGTTTTTTTACGGTCACGCCGGCATGGCCGTAGTCCGTCTTATGCTTATAAGGGTTGTTTTTCCTGTTGT AAGACAGGCTTCTAATGTTTAAATGTTTTTTTTTTTGTTATTTTATTTTGTGTTTAATGCAGGAACCCGCAGACA TGTTTGAGAGAAAAATGGTGTCTTTTTCTGTGGTGGTTCCGGAACTTACCTGCCTTTATCTGCATGAGCATGACT ACGATGTGCTTGCTTTTTTGCGCGAGGCTTTGCCTGATTTTTTGAGCAGCACCTTGCATTTTATATCGCCGCCCA TGCAACAAGCTTACATAGGGGCTACGCTGGTTAGCATAGCTCCGAGTATGCGTGTCATAATCAGTGTGGGTTCTT TTGTCATGGTTCCTGGCGGGGAAGTGGCCGCGCTGGTCCGTGCAGACCTGCACGATTATGTTCAGCTGGCCCTGC GAAGGGACCTACGGGATCGCGGTATTTTTGTTAATGTTCCGCTTTTGAATCTTATACAGGTCTGTGAGGAACCTG AATTTTTGCAATCATGATTCGCTGCTTGAGGCTGAAGGTGGAGGGCGCTCTGGAGCAGATTTTTACAATGGCCGG ACTTAATATTCGGGATTTGCTTAGAGACATATTGATAAGGTGGCGAGATGAAAATTATTTGGGCATGGTTGAAGG TGCTGGAATGTTTATAGAGGAGATTCACCCTGAAGGGTTTAGCCTTTACGTCCACTTGGACGTGAGGGCAGTTTG CCTTTTGGAAGCCATTGTGCAACATCTTACAAATGCCATTATCTGTTCTTTGGCTGTAGAGTTTGACCACGCCAC CGGAGGGGAGCGCGTTCACTTAATAGATCTTCATTTTGAGGTTTTGGATAATCTTTTGGAATAAAAAAAAAAAAA CATGGTTCTTCCAGCTCTTCCCGCTCCTCCCGTGTGTGACTCGCAGAACGAATGTGTAGGTTGGCTGGGTGTGGC TTATTCTGCGGTGGTGGATGTTATCAGGGCAGCGGCGCATGAAGGAGTTTACATAGAACCCGAAGCCAGGGGGCG CCTGGATGCTTTGAGAGAGTGGATATACTACAACTACTACACAGAGCGAGCTAAGCGACGAGACCGGAGACGCAG ATCTGTTTGTCACGCCCGCACCTGGTTTTGCTTCAGGAAATATGACTACGTCCGGCGTTCCATTTGGCATGACAC TACGACCAACACGATCTCGGTTGTCTCGGCGCACTCCGTACAGTAGGGATCGCCTACCTCCTTTTGAGACAGAGA CCCGCGCTACCATACTGGAGGATCATCCGCTGCTGCCCGAATGTAACACTTTGACAATGCACAACGTGAGTTACG TGCGAGGTCTTCCCTGCAGTGTGGGATTTACGCTGATTCAGGAATGGGTTGTTCCCTGGGATATGGTTCTGACGC GGGAGGAGCTTGTAATCCTGAGGAAGTGTATGCACGTGTGCCTGTGTTGTGCCAACATTGATATCATGACGAGCA TGATGATCCATGGTTACGAGTCCTGGGCTCTCCACTGTCATTGTTCCAGTCCCGGTTCCCTGCAGTGCATAGCCG GCGGGCAGGTTTTGGCCAGCTGGTTTAGGATGGTGGTGGATGGCGCCATGTTTAATCAGAGGTTTATATGGTACC GGGAGGTGGTGAATTACAACATGCCAAAAGAGGTAATGTTTATGTCCAGCGTGTTTATGAGGGGTCGCCACTTAA TCTACCTGCGCTTGTGGTATGATGGCCACGTGGGTTCTGTGGTCCCCGCCATGAGCTTTGGATACAGCGCCTTGC ACTGTGGGATTTTGAACAATATTGTGGTGCTGTGCTGCAGTTACTGTGCTGATTTAAGTGAGATCAGGGTGCGCT GCTGTGCCCGGAGGACAAGGCGTCTCATGCTGCGGGCGGTGCGAATCATCGCTGAGGAGACCACTGCCATGTTGT ATTCCTGCAGGACGGAGCGGCGGCGGCAGCAGTTTATTCGCGCGCTGCTGCAGCACCACCGCCCTATCCTGATGC ACGATTATGACTCTACCCCCATGTAGGCGTGGACTTCCCCTTCGCCGCCCGTTGAGCAACCGCAAGTTGGACAGC AGCCTGTGGCTCAGCAGCTGGACAGCGACATGAACTTAAGCGAGCTGCCCGGGGAGTTTATTAATATCACTGATG AGCGTTTGGCTCGACAGGAAACCGTGTGGAATATAACACCTAAGAATATGTCTGTTACCCATGATATGATGCTTT TTAAGGCCAGCCGGGGAGAAAGGACTGTGTACTCTGTGTGTTGGGAGGGAGGTGGCAGGTTGAATACTAGGGTTC TGTGAGTTTGATTAAGGTACGGTGATCAATATAAGCTATGTGGTGGTGGGGCTATACTACTGAATGAAAAATGAC TTGAAATTTTCTGCAATTGAAAAATAAACACGTTGAAACATAACATGCAACAGGTTCACGATTCTTTATTCCTGG GCAATGTAGGAGAAGGTGTAAGAGTTGGTAGCAAAAGTTTCAGTGGTGTATTTTCCACTTTCCCAGGACCATGTA AAAGACATAGAGTAAGTGCTTACCTCGCTAGTTTCTGTGGATTCACTAGAA E4 Orf6 Sequence(SEQ ID NO:42) ATGACTACGTCCGGCGTTCCATTTGGCATGACACTACGACCAACACGATCTCGGTTGTCTCGGCGCACTCCGTAC AGTAGGGATCGCCTACCTCCTTTTGAGACAGAGACCCGCGCTACCATACTGGAGGATCATCCGCTGCTGCCCGAA TGTAACACTTTGACAATGCACAACGTGAGTTACGTGCGAGGTCTTCCCTGCAGTGTGGGATTTACGCTGATTCAG GAATGGGTTGTTCCCTGGGATATGGTTCTGACGCGGGAGGAGCTTGTAATCCTGAGGAAGTGTATGCACGTGTGC CTGTGTTGTGCCAACATTGATATCATGACGAGCATGATGATCCATGGTTACGAGTCCTGGGCTCTCCACTGTCAT TGTTCCAGTCCCGGTTCCCTGCAGTGCATAGCCGGCGGGCAGGTTTTGGCCAGCTGGTTTAGGATGGTGGTGGAT GGCGCCATGTTTAATCAGAGGTTTATATGGTACCGGGAGGTGGTGAATTACAACATGCCAAAAGAGGTAATGTTT ATGTCCAGCGTGTTTATGAGGGGTCGCCACTTAATCTACCTGCGCTTGTGGTATGATGGCCACGTGGGTTCTGTG GTCCCCGCCATGAGCTTTGGATACAGCGCCTTGCACTGTGGGATTTTGAACAATATTGTGGTGCTGTGCTGCAGT TACTGTGCTGATTTAAGTGAGATCAGGGTGCGCTGCTGTGCCCGGAGGACAAGGCGTCTCATGCTGCGGGCGGTG CGAATCATCGCTGAGGAGACCACTGCCATGTTGTATTCCTGCAGGACGGAGCGGCGGCGGCAGCAGTTTATTCGC GCGCTGCTGCAGCACCACCGCCCTATCCTGATGCACGATTATGACTCTACCCCCATGTAG VA Sequence (VA transcripts I and II are underlined) (SEQ ID NO:43) CGTAATCCGTAGATGTACCTGGACATCCAGGTGATGCCGGCGGCGGTGGTGGAGGCGCGCGGAAAGTCGCGGAC GCGGTTCCAGATGTTGCGCAGCGGCAAAAAGTGCTCCATGGTCGGGACGCTCTGGCCGGTGAGGCGTGCGCAGTC GTTGACGCTCTAGACCGTGCAAAAGGAGAGCCTGTAAGCGGGCACTCTTCCGTGGTCTGGTGGATAAATTCGCAA GGGTATCATGGCGGACGACCGGGGTTCGAACCCCGGATCCGGCCGTCCGCCGTGATCCATGCGGTTACCGCCCGC GTGTCGAACCCAGGTGTGCGACGTCAGACAACGGGGGAGCGCTCCTTTTGGCTTCCTTCCAGGCGCGGCGGCTGC TGCGCTAGCTTTTTTGGCCACTGGCCGCGCGCGGCGTAAGCGGTTAGGCTGGAAAGCGAAAGCATTAAGTGGCTC GCTCCCTGTAGCCGGAGGGTTATTTTCCAAGGGTTGAGTCGCAGGACCCCCGGTTCGAGTCTCGGGCCGGCCGGA 152 ME149651883v.1135975-54620 (REGN 11546) CTGCGGCGAACGGGGGTTTGCCTCCCCGTCATGCAAGACCCCGCTTGCAAATTCCTCCGGAAACAGGGACGAGCC CCTTTTTTGCTTTTCCCAGATGCATCCGGTGCTGCGGCAGATGCGCCCCCCTCCTCAGCAGCGGCAAGAGCAAGA GCAGCGGCAGACATGCAGGGCACCCTCCCCTTCTCCTACCGCGTCAGGAGGGGCAACATCCTACATCGA Sequences for E1A and E1B are both contained within Accession AY339865.1 Ad5 E1A Two proteins can be transcribed, a 32 kDa protein (first accession number) and a 27 kDa protein (second accession number). These are both splice variants from the transcript: Accession 1: AAQ19284.1 Accession 2: AAQ19285.1 (SEQ ID NO:44) ATGAGACATATTATCTGCCACGGAGGTGTTATTACCGAAGAAATGGCCGCCAGTCTTTTGGACCAGCTGATCGAA GAGGTACTGGCTGATAATCTTCCACCTCCTAGCCATTTTGAACCACCTACCCTTCACGAACTGTATGATTTAGAC GTGACGGCCCCCGAAGATCCCAACGAGGAGGCGGTTTCGCAGATTTTTCCCGACTCTGTAATGTTGGCGGTGCAG GAAGGGATTGACTTACTCACTTTTCCGCCGGCGCCCGGTTCTCCGGAGCCGCCTCACCTTTCCCGGCAGCCCGAG CAGCCGGAGCAGAGAGCCTTGGGTCCGGTTTCTATGCCAAACCTTGTACCGGAGGTGATCGATCTTACCTGCCAC GAGGCTGGCTTTCCACCCAGTGACGACGAGGATGAAGAGGGTGAGGAGTTTGTGTTAGATTATGTGGAGCACCCC GGGCACGGTTGCAGGTCTTGTCATTATCACCGGAGGAATACGGGGGACCCAGATATTATGTGTTCGCTTTGCTAT ATGAGGACCTGTGGCATGTTTGTCTACAGTCCTGTGTCTGAACCTGAGCCTGAGCCCGAGCCAGAACCGGAGCCT GCAAGACCTACCCGCCGTCCTAAAATGGCGCCTGCTATCCTGAGACGCCCGACATCACCTGTGTCTAGAGAATGC AATAGTAGTACGGATAGCTGTGACTCCGGTCCTTCTAACACACCTCCTGAGATACACCCGGTGGTCCCGCTGTGC CCCATTAAACCAGTTGCCGTGAGAGTTGGTGGGCGTCGCCAGGCTGTGGAATGTATCGAGGACTTGCTTAACGAG CCTGGGCAACCTTTGGACTTGAGCTGTAAACGCCCCAGGCCATAA (SEQ ID NO:45) ATGAGACATATTATCTGCCACGGAGGTGTTATTACCGAAGAAATGGCCGCCAGTCTTTTGGACCAGCTGATCGAA GAGGTACTGGCTGATAATCTTCCACCTCCTAGCCATTTTGAACCACCTACCCTTCACGAACTGTATGATTTAGAC GTGACGGCCCCCGAAGATCCCAACGAGGAGGCGGTTTCGCAGATTTTTCCCGACTCTGTAATGTTGGCGGTGCAG GAAGGGATTGACTTACTCACTTTTCCGCCGGCGCCCGGTTCTCCGGAGCCGCCTCACCTTTCCCGGCAGCCCGAG CAGCCGGAGCAGAGAGCCTTGGGTCCGGTTTCTATGCCAAACCTTGTACCGGAGGTGATCGATCTTACCTGCCAC GAGGCTGGCTTTCCACCCAGTGACGACGAGGATGAAGAGGGTCCTGTGTCTGAACCTGAGCCTGAGCCCGAGCCA GAACCGGAGCCTGCAAGACCTACCCGCCGTCCTAAAATGGCGCCTGCTATCCTGAGACGCCCGACATCACCTGTG TCTAGAGAATGCAATAGTAGTACGGATAGCTGTGACTCCGGTCCTTCTAACACACCTCCTGAGATACACCCGGTG GTCCCGCTGTGCCCCATTAAACCAGTTGCCGTGAGAGTTGGTGGGCGTCGCCAGGCTGTGGAATGTATCGAGGAC TTGCTTAACGAGCCTGGGCAACCTTTGGACTTGAGCTGTAAACGCCCCAGGCCATAA Ad5 E1B_19K Accession: AAQ19286.1 (SEQ ID NO:46) ATGGAGGCTTGGGAGTGTTTGGAAGATTTTTCTGCTGTGCGTAACTTGCTGGAACAGAGCTCTAACAGTACCTCT TGGTTTTGGAGGTTTCTGTGGGGCTCATCCCAGGCAAAGTTAGTCTGCAGAATTAAGGAGGATTACAAGTGGGAA TTTGAAGAGCTTTTGAAATCCTGTGGTGAGCTGTTTGATTCTTTGAATCTGGGTCACCAGGCGCTTTTCCAAGAG AAGGTCATCAAGACTTTGGATTTTTCCACACCGGGGCGCGCTGCGGCTGCTGTTGCTTTTTTGAGTTTTATAAAG GATAAATGGAGCGAAGAAACCCATCTGAGCGGGGGGTACCTGCTGGATTTTCTGGCCATGCATCTGTGGAGAGCG GTTGTGAGACACAAGAATCGCCTGCTACTGTTGTCTTCCGTCCGCCCGGCGATAATACCGACGGAGGAGCAGCAG CAGCAGCAGGAGGAAGCCAGGCGGCGGCGGCAGGAGCAGAGCCCATGGAACCCGAGAGCCGGCCTGGACCCTCGG GAATGA Ad5 E1B_55K Accession: AAQ19287.1 (SEQ ID NO:47) ATGGAGCGAAGAAACCCATCTGAGCGGGGGGTACCTGCTGGATTTTCTGGCCATGCATCTGTGGAGAGCGGTTGT GAGACACAAGAATCGCCTGCTACTGTTGTCTTCCGTCCGCCCGGCGATAATACCGACGGAGGAGCAGCAGCAGCA GCAGGAGGAAGCCAGGCGGCGGCGGCAGGAGCAGAGCCCATGGAACCCGAGAGCCGGCCTGGACCCTCGGGAATG AATGTTGTACAGGTGGCTGAACTGTATCCAGAACTGAGACGCATTTTGACAATTACAGAGGATGGGCAGGGGCTA 153 ME149651883v.1135975-54620 (REGN 11546) AAGGGGGTAAAGAGGGAGCGGGGGGCTTGTGAGGCTACAGAGGAGGCTAGGAATCTAGCTTTTAGCTTAATGACC AGACACCGTCCTGAGTGTATTACTTTTCAACAGATCAAGGATAATTGCGCTAATGAGCTTGATCTGCTGGCGCAG AAGTATTCCATAGAGCAGCTGACCACTTACTGGCTGCAGCCAGGGGATGATTTTGAGGAGGCTATTAGGGTATAT GCAAAGGTGGCACTTAGGCCAGATTGCAAGTACAAGATCAGCAAACTTGTAAATATCAGGAATTGTTGCTACATT TCTGGGAACGGGGCCGAGGTGGAGATAGATACGGAGGATAGGGTGGCCTTTAGATGTAGCATGATAAATATGTGG CCGGGGGTGCTTGGCATGGACGGGGTGGTTATTATGAATGTAAGGTTTACTGGCCCCAATTTTAGCGGTACGGTT TTCCTGGCCAATACCAACCTTATCCTACACGGTGTAAGCTTCTATGGGTTTAACAATACCTGTGTGGAAGCCTGG ACCGATGTAAGGGTTCGGGGCTGTGCCTTTTACTGCTGCTGGAAGGGGGTGGTGTGTCGCCCCAAAAGCAGGGCT TCAATTAAGAAATGCCTCTTTGAAAGGTGTACCTTGGGTATCCTGTCTGAGGGTAACTCCAGGGTGCGCCACAAT GTGGCCTCCGACTGTGGTTGCTTCATGCTAGTGAAAAGCGTGGCTGTGATTAAGCATAACATGGTATGTGGCAAC TGCGAGGACAGGGCCTCTCAGATGCTGACCTGCTCGGACGGCAACTGTCACCTGCTGAAGACCATTCACGTAGCC AGCCACTCTCGCAAGGCCTGGCCAGTGTTTGAGCATAACATACTGACCCGCTGTTCCTTGCATTTGGGTAACAGG AGGGGGGTGTTCCTACCTTACCAATGCAATTTGAGTCACACTAAGATATTGCTTGAGCCCGAGAGCATGTCCAAG GTGAACCTGAACGGGGTGTTTGACATGACCATGAAGATCTGGAAGGTGCTGAGGTACGATGAGACCCGCACCAGG TGCAGACCCTGCGAGTGTGGCGGTAAACATATTAGGAACCAGCCTGTGATGCTGGATGTGACCGAGGAGCTGAGG CCCGATCACTTGGTGCTGGCCTGCACCCGCGCTGAGTTTGGCTCTAGCGATGAAGATACAGATTGA Sequences for E2A and E4A are both contained within Accession MN088492 Ad5 E2A orf: Accession: QHX41645.1 (SEQ ID NO:48) ATGGCCAGTCGGGAAGAGGAGCAGCGCGAAACCACCCCCGAGCGCGGACGCGGTGCGGCGCGACGTCCACCAACC ATGGAGGACGTGTCGTCCCCGTCGCCGTCGCCGCCGCCTCCCCGCGCGCCCCCAAAAAAGCGGCTGAGGCGGCGT CTCGAGTCCGAGGACGAAGAAGACTCGTCACAAGATGCGCTGGTGCCGCGCACACCCAGCCCGCGGCCATCGACC TCGACGGCGGATTTGGCCATTGCGTCCAAAAAGAAAAAGAAGCGCCCCTCTCCCAAGCCCGAGCGCCCGCCATCC CCAGAGGTGATCGTGGACAGCGAGGAAGAAAGAGAAGATGTGGCGCTACAAATGGTGGGTTTCAGCAACCCACCG GTGCTAATCAAGCACGGCAAGGGAGGTAAGCGCACGGTGCGGCGGCTGAATGAAGACGACCCAGTGGCGCGGGGT ATGCGGACGCAAGAGGAAAAGGAAGAGTCCAGTGAAGCGGAAAGTGAAAGCACGGTGATAAACCCGCTGAGCCTG CCGATCGTGTCTGCGTGGGAGAAGGGCATGGAGGCTGCGCGCGCGTTGATGGACAAGTACCACGTGGATAACGAT CTAAAGGCAAACTTCAAGCTACTGCCTGACCAAGTGGAAGCTCTGGCGGCCGTATGCAAGACCTGGCTAAACGAG GAGCACCGCGGGTTGCAGCTGACCTTCACCAGCAACAAGACCTTTGTGACGATGATGGGGCGATTCCTGCAGGCG TACCTGCAGTCGTTTGCAGAGGTAACCTACAAGCACCACGAGCCCACGGGCTGCGCGTTGTGGCTGCACCGCTGC GCTGAGATCGAAGGCGAGCTTAAGTGTCTACACGGGAGCATTATGATAAATAAGGAGCACGTGATTGAAATGGAT GTGACGAGCGAAAACGGGCAGCGCGCGCTGAAGGAGCAGTCTAGCAAGGCCAAGATCGTGAAGAACCGGTGGGGC CGAAATGTGGTGCAGATCTCCAACACCGACGCAAGGTGCTGCGTGCATGACGCGGCCTGTCCGGCCAATCAGTTT TCCGGCAAGTCTTGCGGCATGTTCTTCTCTGAAGGCGCAAAGGCTCAGGTGGCTTTTAAGCAGATCAAGGCTTTC ATGCAGGCGCTGTATCCTAACGCCCAGACCGGGCACGGTCACCTTCTGATGCCACTACGGTGCGAGTGCAACTCA AAGCCTGGGCATGCACCCTTTTTGGGAAGGCAGCTACCAAAGTTGACTCCGTTCGCCCTGAGCAACGCGGAGGAC CTGGACGCGGATCTGATCTCCGACAAGAGCGTGCTGGCCAGCGTGCACCACCCGGCGCTGATAGTGTTCCAGTGC TGCAACCCTGTGTATCGCAACTCGCGCGCGCAGGGCGGAGGCCCCAACTGCGACTTCAAGATATCGGCGCCCGAC CTGCTAAACGCGTTGGTGATGGTGCGCAGCCTGTGGAGTGAAAACTTCACCGAGCTGCCGCGGATGGTTGTGCCT GAGTTTAAGTGGAGCACTAAACACCAGTATCGCAACGTGTCCCTGCCAGTGGCGCATAGCGATGCGCGGCAGAAC CCCTTTGATTTTTAA(SEQ ID NO:49) ATGACTACGTCCGGCGTTCCATTTGGCATGACACTACGACCAACACGATCTCGGTTGTCTCGGCGCACTCCGTAC AGTAGGGATCGCCTACCTCCTTTTGAGACAGAGACCCGCGCTACCATACTGGAGGATCATCCGCTGCTGCCCGAA TGTAACACTTTGACAATGCACAACGCGTGGACTTCCCCTTCGCCGCCCGTTGAGCAACCGCAAGTTGGACAGCAG CCTGTGGCTCAGCAGCTGGACAGCGACATGAACTTAAGCGAGCTGCCCGGGGAGTTTATTAATATCACTGATGAG CGTTTGGCTCGACAGGAAACCGTGTGGAATATAACACCTAAGAATATGTCTGTTACCCATGATATGATGCTTTTT AAGGCCAGCCGGGGAGAAAGGACTGTGTACTCTGTGTGTTGGGAGGGAGGTGGCAGGTTGAATACTAGGGTTCTG TGA 154 ME149651883v.1135975-54620 (REGN 11546) (SEQ ID NO:50) ATGACTACGTCCGGCGTTCCATTTGGCATGACACTACGACCAACACGATCTCGGTTGTCTCGGCGCACTCCGTAC AGTAGGGATCGCCTACCTCCTTTTGAGACAGAGACCCGCGCTACCATACTGGAGGATCATCCGCTGCTGCCCGAA TGTAACACTTTGACAATGCACAACGTGAGTTACGTGCGAGGTCTTCCCTGCAGTGTGGGATTTACGCTGATTCAG GAATGGGTTGTTCCCTGGGATATGGTTCTGACGCGGGAGGAGCTTGTAATCCTGAGGAAGTGTATGCACGTGTGC CTGTGTTGTGCCAACATTGATATCATGACGAGCATGATGATCCATGGTTACGAGTCCTGGGCTCTCCACTGTCAT TGTTCCAGTCCCGGTTCCCTGCAGTGCATAGCCGGCGGGCAGGTTTTGGCCAGCTGGTTTAGGATGGTGGTGGAT GGCGCCATGTTTAATCAGAGGTTTATATGGTACCGGGAGGTGGTGAATTACAACATGCCAAAAGAGGTAATGTTT ATGTCCAGCGTGTTTATGAGGGGTCGCCACTTAATCTACCTGCGCTTGTGGTATGATGGCCACGTGGGTTCTGTG GTCCCCGCCATGAGCTTTGGATACAGCGCCTTGCACTGTGGGATTTTGAACAATATTGTGGTGCTGTGCTGCAGT TACTGTGCTGATTTAAGTGAGATCAGGGTGCGCTGCTGTGCCCGGAGGACAAGGCGTCTCATGCTGCGGGCGGTG CGAATCATCGCTGAGGAGACCACTGCCATGTTGTATTCCTGCAGGACGGAGCGGCGGCGGCAGCAGTTTATTCGC GCGCTGCTGCAGCACCACCGCCCTATCCTGATGCACGATTATGACTCTACCCCCATGTAG Ad5 VA: Accession: AF369965.1 (SEQ ID NO:51) TCGATGTAGGATGTTGCCCCTCCTGACGCGGTAGGAGAAGGGGAGGGTGCCCTGCATGTCTGCCGCTGCTCTTGC TCTTGCCGCTGCTGAGGAGGGGGGCGCATCTGCCGCAGCACCGGATGCATCTGGGAAAAGCAAAAAAGGGGCTCG TCCCTGTTTCCGGAGGAATTTGCAAGCGGGGTCTTGCATGACGGGGAGGCAAACCCCCGTTCGCCGCAGTCCGGC CGGCCCGAGACTCGAACCGGGGGTCCTGCGACTCAACCCTTGGAAAATAACCCTCCGGCTACAGGGAGCGAGCCA CTTAATGCTTTCGCTTTCCAGCCTAACCGCTTACGCCGCGCGCGGCCAGTGGCCAAAAAAGCTAGCGCAGCAGCC GCCGCGCCTGGAAGGAAGCCAAAAGGAGCGCTCCCCCGTTGTCTGACGTCGCACACCTGGGTTCGACACGCGGGC GGTAACCGCATGGATCACGGCGGACGGCCGGATCCGGGGTTCGAACCCCGGTCGTCCGCCATGATACCCTTGCGA ATTTATCCACCAGACCACGGAAGAGTGCCCGCTTACAGGCTCTCCTTTTGCACGGTCTAGAGCGTCAACGACTGC GCACGCCTCACCGGCCAGAGCGTCCCGACCATGGAGCACTTTTTGCCGCTGCGCAACATCTGGAACCGCGTCCGC GACTTTCCGCGCGCCTCCACCACCGCCGCCGGCATCACCTGGATGTCCAGGTACATCTACGGATTACG EXAMPLE 32 – Specific Binding Pair Polynucleotide Sequences Sequences can be found in one or more of these publications, for example, in WO2022 / 234276A1, WO2022129547A1, WO2011 / 098772, WO2016 / 193746, WO2018 / 197854, WO2018 / 189517 or Li et al., J. Mol. Biol.426, 309-317 (2014); or can be designed or obtained by methods known in the art, for example, as described in Zakeri et al., 2012, and in Zakeri et al., 2010. SpyTag Polynucleotide Sequence (SEQ ID NO:52) GCCCACATCGTGATGGTGGACGCCTACAAGCCGACGAAG SpyCatcher Polynucleotide Sequence (SEQ ID NO:53) GTGGATACCCTGTCCGGACTGAGCAGTGAGCAAGGCCAGTCCGGAGATATGACAATTGAAGAAGATAGCGCCACC CATATTAAATTCTCCAAAAGAGATGAGGACGGCAAAGAGCTGGCTGGAGCAACAATGGAGCTGAGAGATTCCTCT GGAAAGACTATTAGTACATGGATCTCTGATGGCCAAGTGAAAGATTTCTATCTGTATCCAGGAAAGTACACATTT 155 ME149651883v.1135975-54620 (REGN 11546) GTCGAAACCGCTGCACCAGACGGATATGAGGTGGCTACAGCTATTACCTTTACAGTGAATGAGCAAGGACAGGTG ACTGTTAATGGCAAAGCTACTAAAGGAGACGCTCATATTTAA EXAMPLE 33– SpyTag Peptide Sequence (SEQ ID NO:54) AHIVMVDAYKPTK EXAMPLE 34 – DNA Barcode Sequences (SEQ ID NO:55) CACATATCAGAGTGCGACACACAGACTGTGAG (SEQ ID NO:56) ACACATCTCGTGAGAGCACGCACACACGCGCG (SEQ ID NO:57) CACTCGACTCTCGCGTCATATATATCAGCTGT (SEQ ID NO:58) TCTGTATCTCTATGTGACAGTCGAGCGCTGCG (SEQ ID NO:59) ACACACGCGAGACAGAACGCGCTATCTCAGAG (SEQ ID NO:60) CTATACGTATATCTATACACTAGATCGCGTGT (SEQ ID NO:61) CTCTCGCATACGCGAGCTCACTACGCGCGCGT (SEQ ID NO:62) CGCATGACACGTGTGTCATAGAGAGATAGTAT (SEQ ID NO:63) CACACGCGCGCTATATTCACGTGCTCACTGTG (SEQ ID NO:64) ACACACTCTATCAGATCACGACACGACGATGT (SEQ ID NO:65) CTATACATAGTGATGTCACTCACGTGTGATAT (SEQ ID NO:66) CAGAGAGATATCTCTGCATGTAGAGCAGAGAG (SEQ ID NO:67) CGCGACACGCTCGCGCCACAGAGACACGCACA (SEQ ID NO:68) CTCACACTCTCTCACACTCTGCTCTGACTCTC (SEQ ID NO:69) TATATATGTCTATAGATCTCTCTATCGCGCTC (SEQ ID NO:70) GATGTCTGAGTGTGTGGAGACTAGAGATAGTG 156 ME149651883v.1135975-54620 (REGN 11546) (SEQ ID NO:71) TCTCGTCGCAGTCTCTATGTGTATATAGATAT (SEQ ID NO:72) GCGCGCGCACTCTCTGGAGACACGTCGCACAC (SEQ ID NO:73) ACACATATCGCACTACGTGTGTCTCGATGCGC (SEQ ID NO:74) CGCACACATAGATACATGTCATATGAGAGTGT (SEQ ID NO:75) TCTCGCGCGTGCACGCCTCGCTCGACGAGCGC (SEQ ID NO:76) TATAGAGCTCTACATAGCTGAGACGACGCGCG (SEQ ID NO:77) ACATATCGTACTCTCTGATATATCGAGTATAT (SEQ ID NO:78) TGTCATGTGTACACACGTGTGCACTCACACTC (SEQ ID NO:79) ACACGTGTGCTCTCTCGATATACGCGAGAGAG (SEQ ID NO:80) CGTGTCTAGCGCGCGCGTGTGAGATATATATC (SEQ ID NO:81) CTCACGTACGTCACACGCGCACGCACTACAGA (SEQ ID NO:82) CACACGAGATCTCATCAGACACACACGCACAT (SEQ ID NO:83) GACGAGCGTCTGAGAGTGTGTCTCTGAGAGTA (SEQ ID NO:84) CACACGCACTGAGATAGATGAGTATAGACACA (SEQ ID NO:85) GCTGTGTGTGCTCGTCTCTCAGATAGTCTATA (SEQ ID NO:86) ACACGCATGACACACTTATATACAGAGTCGAG (SEQ ID NO:87) GCGCTCTCTCACATACTATATGCTCTGTGTGA (SEQ ID NO:88) CTCTATATATCTCGTCAGAGAGCTCTCTCATC (SEQ ID NO:89) GCGAGAGTGAGACGCATGCTCTCGTGTACTGT (SEQ ID NO:90) AGCGCTGCGACACGCGAGACGCGAGCGCGTAG 157 ME149651883v.1135975-54620 (REGN 11546) (SEQ ID NO:91) GCGTGTGTCGAGTGTATGTACGCTCTCTATAT (SEQ ID NO:92) TAGAGAGCGTCGCGTGGTGCACTCGCGCTCTC (SEQ ID NO:93) TATCTCTCGAGTCGCGCTCACACATACACGTC (SEQ ID NO:94) ATAGTACACTCTGTGTTATCTCTGTAGAGTCT (SEQ ID NO:95) GATATATATGTGTGTAGTGACACACAGAGCAC (SEQ ID NO:96) ATATGACATACACGCACGTCTCTCGTCTGTGC (SEQ ID NO:97) ACACAGTAGAGCGAGCGTCGCGCATAGAGCGC (SEQ ID NO:98) CTATCTAGCACTCACACGTGTCACTCTGCGTG (SEQ ID NO:99) CGCGCGAGTATCTCGTAGCACACATATAGCGC (SEQ ID NO:100) GTATATATATACGTCTTCTCACGAGAGCGCAC (SEQ ID NO:101) TAGATGCGAGAGTAGAATAGCGACATCTCTCT (SEQ ID NO:102) GCACGATGTCAGCGCGTGTGCTCTCTACACAG EXAMPLE 35 – Additional Specific Binding Pairs Additional binding pairs are reported. See, for example, WO2022 / 234276A1, WO2011 / 098772, WO2022129547A1, WO2016 / 193746, WO2018 / 197854, WO2018 / 189517 or Li et al., J. Mol. Biol.426, 309-317 (2014).158 ME149651883v.1135975-54620 (REGN 11546)159 ME149651883v.1135975-54620 (REGN 11546)Nucleotide and amino acid sequences for specific binding partners have been published or can be designed or obtained by methods known in the art, for example, as described in Zakeri et al., 2012, and in Zakeri et al., 2010 See, for example:160 ME149651883v.1135975-54620 (REGN 11546)161 ME149651883v.1135975-54620 (REGN 11546)162 ME149651883v.1135975-54620 (REGN 11546)EXAMPLE 36 - Herpes simplex virus (HSV) Polynucleotide Sequences 163 ME149651883v.1135975-54620 (REGN 11546) HSV polynucleotides can be selected from any serotype, and representative polynucleotides are exemplified below. Weindler and Heilbronn 1991 (Weindler, Friedrich W., and R. E. G. I. N. E. Heilbronn. "A subset of herpes simplex virus replication genes provides helper functions for productive adeno-associated virus replication." Journal of virology 65.5 (1991): 2476-2483); Ward et al.2001 (Ward et al. Rep-dependent initiation of adeno-associated virus type 2 DNA replication by a herpes simplex virus type 1 replication complex in a reconstituted system. J. Virol. 2001;75:10250–10258); Herpes Clément et al.2009 (Clément, Nathalie, David R. Knop, and Barry J. Byrne. "Large-scale adeno-associated viral vector production using a herpesvirus-based system enables manufacturing for clinical studies." Human gene therapy 20.8 (2009): 796-806); and Meier et al.2020 (Meier, Anita F., Cornel Fraefel, and Michael Seyffert. "The Interplay between Adeno-Associated Virus and Its Helper Viruses." Viruses 12.6 (2020)) disclose seven HSV replication genes (UL5, UL8, UL9, UL29, UL30, UL42, and UL52) that led to productive AAV replication, of which HSV-1 helicase–primase complex (HP; UL5 / UL8 / UL52) and the single-strand DNA binding protein ICP8 (gene UL29) is sufficient to restore AAV progeny production. HSV replication gene (UL5, UL8, UL52, UL29, UL9, UL30, and UL42) sequences as available at the GenBank are listed below: UL5 helicase-primase helicase subunit [Human alphaherpesvirus 1 (Herpes simplex virus type 1)] Gene ID: 2703420. NCBI Reference Sequence: NC_001806.2. (SEQ ID NO. 202) 1 atggcggcgg ccggcgggga gcgccagcta gacggacaga aacccggccc gccgcacctt 61 cagcaacccg gggaccgacc agccgttcca gggagggccg aggccttttt aaattttacg 121 tctatgcacg gggtgcagcc aatccttaag cgcatccgag agctctcgca acaacagctc 181 gacggagcgc aagtgcccca tctgcagtgg ttccgggacg tggcggcctt agagtccccc 241 gcaggcctgc ccctcaggga gtttccgttc gcggtgtatc ttatcaccgg caacgctggc 301 tccggaaaga gcacgtgcgt gcagacaatc aacgaggtct tggactgtgt ggtgacgggc 361 gccacgcgca ttgcggccca aaacatgtac gccaaactct cgggcgcctt tctcagccga 421 cccatcaaca ccatctttca tgaatttggg tttcgcggga atcacgtcca ggcccaactg 481 ggacagtacc cgtacaccct gaccagcaac cccgcctcgc tggaggacct gcagcgacga 541 gatctgacgt actactggga ggtgattttg gacctcacga agcgcgccct ggccgcctcc 601 gggggcgagg agttgcggaa cgagtttcgc gccctggccg ccctggaacg gaccctgggg 661 ttggccgagg gcgccctgac gcggttggcc ccggccaccc acggggcgct gccggccttt 721 acccgcagca acgtgatcgt catcgacgag gccgggctcc ttgggcgtca cctcctcacg 781 gccgtggtgt attgctggtg gatgattaac gccctgtacc acacccccca gtacgcggcc 841 cgcctgcggc ccgtgttggt gtgtgtgggc tcgccgacgc agacggcgtc cctggagtcg 901 accttcgagc accagaaact gcggtgttcc gtccgccaga gcgagaacgt gctcacgtac 961 ctcatctgca accgcacgct gcgcgagtac gcccgcctct cgtatagctg ggccattttt 1021 attaacaaca aacggtgcgt cgagcacgag ttcggtaacc tcatgaaggt gctggagtac 1081 ggcctgccca tcaccgagga gcacatgcag ttcgtggatc gcttcgtcgt cccggaaaac 1141 tacatcacca accccgccaa cctccccggc tggacgcggc tgttctcctc ccacaaagag 1201 gtgagcgcgt acatggccaa gctccacgcc tacctgaagg tgacccgtga gggggagttc 1261 gtcgtgttca ccctccccgt gcttacgttc gtgtcggtca aggagtttga cgaataccga 1321 cggctgacac accagcccgg cctgacgatt gaaaagtggc tcacggccaa cgccagccgc 1381 atcaccaact actcgcagag ccaggaccag gacgcggggc acatgcgctg cgaggtgcac 1441 agcaaacagc agctggtcgt ggcccgcaac gacgtcactt acgtcctcaa cagccagatc 1501 gcggtgaccg cgcgcctgcg aaaactggtt tttgggttta gtgggacgtt ccgggccttc 1561 gaggcagtgt tgcgtgacga cagctttgta aagactcagg gggagacttc ggtggagttt 1621 gcctacaggt tcctgtcgcg gctcatattt agcgggctta tctcctttta caactttctg 164 ME149651883v.1135975-54620 (REGN 11546) 1681 cagcgcccgg gcctggatgc gacccagagg accctcgcct acgcccgcat gggagaacta 1741 acggcggaga ttctgtctct gcgccccaaa tcttcggggg tgccgacgca ggcgtcggta 1801 atggccgacg caggcgcccc cggcgagcgt gcgtttgatt ttaagcaact ggggccgcgg 1861 gacgggggcc cggacgattt tcccgacgac gacctcgacg ttattttcgc ggggctggac 1921 gaacaacagc tcgacgtgtt ttactgccac tacacccccg gggaaccgga gaccaccgcc 1981 gccgttcaca cccagtttgc gctgctgaag cgggccttcc tcgggagatt ccgaatcctc 2041 caagagctct tcggggaggc atttgaagtc gcccccttta gcacgtacgt ggacaacgtt 2101 atcttccggg gctgcgagat gctgaccggc tcgccgcgcg gggggctgat gtccgtcgcc 2161 ctgcagacgg acaattatac gctcatggga tacacgtacg cacgggtgtt tgcctttgcg 2221 gacgagctgc ggaggcggca cgcgacggcc aacgtggccg agttactgga agaggccccc 2281 ctgccttacg tggtcttgcg ggaccaacac ggcttcatgt ccgtcgtcaa caccaacatc 2341 agcgagtttg tcgagtccat tgactctacg gagctggcca tggccataaa cgccgactac 2401 ggcatcagct ccaagcttgc catgaccatc acgcgctccc agggccttag cctggacaag 2461 gtcgccatct gctttacgcc cggcaacctg cgcctcaaca gcgcgtacgt ggccatgtcc 2521 cgcaccacct cctccgaatt ccttcgcatg aacttaaatc cgctccggga gcgccacgag 2581 cgcgatgacg tcattagtga gcacatacta tcggctctgc gcgatccgaa cgtggtcatt 2641 gtctattaac ccgccgtccc cttacagttc caccgaaccc ggcccggggg actcactacc 2701 caccgcgaga tgtccaatcc acagacgacc atcgcgtata gcctatgcca cgccagggcc 2761 tcgctgacca gcgcactgcc cgacgccgcg caggtggtgc atgtttttga gtacggcacc 2821 cgcgcgatca tggtacgggg ccgggagcgc caggaccgcc tgccgcgcgg aggcgttgtt 2881 atccagcaca cccccattgg gctgttggtg attatcgact gtcgcgccga attttgtgcc 2941 taccgcttta taggccggga cagcaaccag aagctcgaac gcgggtggga cgcccatatg 3001 tacgcgtatc cgttcgactc ctgggtcagc tcctcgcgcg gcgaaagcgc ccggagcgcc 3061 acggccggca ttttgaccgt ggtctggacc gcggacacca tttacatcac tgcaaccatt 3121 tacgggtcgc ccccagagga gacgccaggc gcggcacacg gggtgggcgc cgcgcctcca 3181 cccccgacaa ccgcctgccc cgggacggcc gagtttctcc agcccaccgc ggacctgctg 3241 gtagaggtgc tgcgggagat tcaactgagc cccgccctgg aatacgcaga caaacttttg 3301 gggtcctagg atcccggccg gatcgcgctc gtcacccgac actgaaatgc cccccccccc 3361 ttgcgggcgg tccattaaa UL8 helicase-primase subunit [Human alphaherpesvirus 1 (Herpes simplex virus type 1)] Gene ID: 2703432. NCBI Reference Sequence: NC_001806.2. (SEQ ID NO. 203) 1 atggacaccg cagatatcgt gtgggtggag gagagcgtca gcgccattac cctttacgcg 61 gtatggctgc ccccccgcgc tcgcgagtac ttccacgccc tggtgtattt tgtatgtcgc 121 aacgccgcag gggagggtcg cgcgcgcttt gcggaggtct ccgtcaccgc gacggagctg 181 cgggatttct acggctccgc ggacgtctcc gtccaggccg tcgtggcggc cgcccgcgcc 241 gcgacgacgc cggccgcctc cccgctggag cccctggaga acccgactct gtggcgggcg 301 ctgtacgcgt gcgtcctggc ggccctggag cgccagaccg ggccggtggc cctgttcgcc 361 ccgctgcgta tcggctcgga cccacgcacg ggactggtgg tgaaagttga gagagcgtcg 421 tggggcccgc ccgccgcccc tcgcgccgct ctcctggtcg cggaggccaa cattgacatc 481 gaccctatgg ccctggcggc gcgcgttgcc gagcatcccg acgcgcggct ggcgtgggcg 541 cgcctggcgg ccattcgcga caccccccag tgcgcgtccg ccgcttcgct gaccgttaac 601 atcaccaccg gaaccgcgct atttgcgcgc gaataccaga ctcttgcgtt tccgccgatc 661 aagaaggagg gcgcgttcgg ggacctggtc gaggtgtgcg aggtgggcct gcggccacgc 721 gggcacccgc aacgagtcac ggcacgggtg ctgctgcccc gcgattacga ctactttgta 781 agcgccggcg agaagttctc cgcgccggcg ctcgtcgccc ttttccggca gtggcatacc 841 acggtccacg ccgcccccgg ggccctggcc cccgtctttg cctttctggg gcccgagttt 901 gaggtccggg ggggacccgt cccgtacttt gccgtcctgg ggtttccggg ttggcccacg 961 ttcaccgtgc cggccacggc cgagtcggca cgggacctgg tgcgcggggc cgcggccgct 1021 tacgccgcgc tcctgggggc ctggcccgcg gtgggggcca gggtcgtcct ccccccgcga 1081 gcctggcccg gcgtggcctc ggcggcagcc ggatgcctcc tgcccgcggt gcgggaggcg 1141 gtggcgcggt ggcatcccgc cactaaaatc atccaactgt tagacccgcc cgcggccgtc 1201 gggcccgtct ggacggcgcg gttttgcttc cccggacttc gcgcccagct cctggcggcc 1261 ctggccgacc tcggggggag cgggctggcg gacccccacg gccggacggg cctagcaaga 1321 ctggacgcgc tggtggtggc cgctccctca gagccctggg ccggggccgt cttggagcgc 1381 ctggtcccgg acacgtgcaa cgcctgccct gcgctgcggc agctcctggg tggggtaatg 165 ME149651883v.1135975-54620 (REGN 11546) 1441 gccgccgtct gcctgcagat cgaggagacg gccagctcgg tgaagttcgc ggtctgcggg 1501 ggcgatgggg gtgcgttctg gggtgtcttt aacgtggacc cccaagacgc ggatgcggct 1561 tccggggtga tcgaggacgc ccggcgggcc atcgagacgg ccgtgggagc cgtgcttagg 1621 gccaacggcc tccggctgcg gcacccactg tgcctggccc tcgagggcgt ctacacccac 1681 gcagtcgcct ggagccaggc gggagtgtgg ttctggaact cccgcgacaa cactgaccat 1741 cttgggggat ttcctctccg cgggcccgcg tacaccacgg cggcaggggt cgtacgcgac 1801 acgctgcgac gggtcctggg cctgacaacg gcatgcgtgc cggaggagga cgcactcacg 1861 gcccggggcc ttatggagga cgcctgcgac cgccttatct tggacgcgtt taataaacgg 1921 ttggacgcgg agtactggag cgttcgggtg tccccctttg aggccagcga ccccttgccc 1981 cccactgcct tccgcggcgg cgccttgctg gacgcagagc actactggcg gcgcgtcgtg 2041 cgtgtctgtc ccggaggcgg ggagtcggtc ggcgtccccg tcgatctata cccgcggccc 2101 cttgtgctcc cccccgtgga ctgcgctcat cacctgcgcg aaatcctgcg cgagattgag 2161 ttggtgttta ccggggtgct ggcgggagta tggggcgagg gggggaagtt tgtgtatccc 2221 tttgacgaca agatgtcgtt tctgtttgcc tgagtttgac caataaa UL52 helicase-primase primase subunit [Human alphaherpesvirus 1 (Herpes simplex virus type 1)]. Gene ID: 2703423. NCBI Reference Sequence: NC_001806.2. (SEQ ID NO. 204) 1 atggggcagg aagacgggaa ccgcggggag aggcgggcgg ccgggactcc cgtggaggtg 61 accgcgcttt atgcgaccga cgggtgcgtt attacctctt cgatcgccct cctcacaaac 121 tctctactgg gggccgagcc ggtttatata ttcagctacg acgcatacac gcacgatggc 181 cgtgccgacg ggcccacgga gcaagacagg ttcgaagaga gtcgggcgct ctaccaagcg 241 tcgggcgggc taaatggcga ctccttccga gtaacctttt gtttattggg gacggaagtg 301 ggtgggaccc accaggcccg cgggcgaacc cgacccatgt tcgtctgtcg cttcgagcga 361 gcggacgacg tcgccgcgct acaggacgcc ctggcgcacg ggaccccgct acaaccggac 421 cacatcgccg ccaccctgga cgcggaggcc acgttcgcgc tgcatgcgaa catgatcctg 481 gctctcaccg tggccatcaa caacgccagc ccccgcaccg gacgcgacgc cgccgcggcg 541 cagtatgatc agggcgcgtc cctacgctcg ctcgtggggc gcacgtccct gggacaacgc 601 ggccttacca cgctatacgt ccaccacgag gtgcgcgtgc ttgccgcgta ccgcagggcg 661 tattatggaa gcgcgcagag tcccttctgg tttcttagca aattcgggcc ggacgaaaaa 721 agcctggtgc tcaccactcg gtactacctg cttcaggccc agcgtctggg gggcgcgggg 781 gccacgtacg acctgcaggc catcaaggac atctgcgcca cctacgcgat tccccacgcc 841 ccccgccccg acaccgtcag cgctgcgtcc ctgacctcgt ttgccgccat cacgcggttc 901 tgttgcacga gccagtacgc ccgcggggcc gcggcggccg ggtttccgct ttacgtggag 961 cgccgtattg cggccgacgt ccgcgagacc agtgcgctgg agaagttcat aacccacgat 1021 cgcagttgcc tgcgcgtgtc cgaccgtgaa ttcattacgt acatctacct ggcccatttt 1081 gagtgtttca gccccccgcg cctagccacg catcttcggg ccgtgacgac ccacgacccc 1141 aaccccgcgg ccagcacgga gcagccctcg cccctgggca gggaggccgt ggaacaattt 1201 ttttgtcacg tgcgcgccca actgaatatc ggggagtacg tcaaacacaa cgtgaccccc 1261 cgggagaccg tcctggatgg cgatacggcc aaggcctacc tgcgcgctcg cacgtacgcg 1321 cccggggccc tgacgcccgc ccccgcgtat tgcggggccg tggactccgc caccaaaatg 1381 atggggcgtt tggcggacgc cgaaaagctc ctggtccccc gcgggtggcc cgcgtttgcg 1441 cccgccagtc ccggggagga cacggcgggc ggcacgccgc ccccacagac ctgcggaatt 1501 gtcaagcgcc tcctgagact ggccgccacg gaacagcagg gccccacacc cccggcgatc 1561 gcggcgctta tccgtaatgc ggcggtgcag actcccctgc ccgtctaccg gatatccatg 1621 gtccccacgg gacaggcatt tgccgcgctg gcctgggacg actgggcccg cataacgcgg 166 ME149651883v.1135975-54620 (REGN 11546) 1681 gacgctcgcc tggccgaagc ggtcgtgtcc gccgaagcgg cggcgcaccc cgaccacggc 1741 gcgctgggca ggcggctcac ggatcgcatc cgcgcccagg gccccgtgat gccccctggc 1801 ggcctggatg ccggggggca gatgtacgtg aatcgcaacg agatattcaa cggcgcgctg 1861 gcaatcacaa acatcatcct ggatctcgac atcgccctga aggagcccgt cccctttcgc 1921 cggctccacg aggccctggg ccactttagg cgcggggctc tggctgcggt tcagctcctg 1981 tttcccgcgg cccgcgtgga ccccgacgca tatccctgtt attttttcaa aagcgcatgt 2041 cggcccggcc cggcgtccgt gggttccggc agcggactcg gcaacgacga cgacggggac 2101 tggtttccct gctacgacga cgccggtgat gaggagtggg cggaggaccc gggcgccatg 2161 gacacatccc acgatccccc ggacgacgag gttgcctact ttgacctgtg ccacgaagtc 2221 ggccccacgg cggaacctcg cgaaacggat tcgcccgtgt gttcctgcac cgacaagatc 2281 ggactgcggg tgtgcatgcc cgtccccgcc ccgtacgtcg tccacggttc tctaacgatg 2341 cggggggtgg cacgggtcat ccagcaggcg gtgctgttgg accgagattt tgtggaggcc 2401 atcgggagct acgtaaaaaa cttcctgttg atcgatacgg gggtgtacgc ccacggccac 2461 agcctgcgct tgccgtattt tgccaaaatc gcccccgacg ggcctgcgtg cggaaggctg 2521 ctgccagtgt ttgtgatccc ccccgcctgc aaagacgttc cggcgtttgt cgccgcgcac 2581 gccgacccgc ggcgcttcca ttttcacgcc ccgcccacct atctcgcttc cccccgggag 2641 atccgtgtcc tgcacagcct gggtggggac tatgtgagct tctttgaaag gaaggcgtcc 2701 cgcaacgcgc tggaacactt tgggcgacgc gagaccctga cggaggtcct gggtcggtac 2761 aacgtacagc cggatgcggg ggggaccgtc gaggggttcg catcggaact gctggggcgg 2821 atagtcgcgt gcatcgaaac ccactttccc gaacacgccg gcgaatatca ggccgtatcc 2881 gtccggcggg ccgtcagtaa ggacgactgg gtcctcctac agctagtccc cgttcgcggt 2941 accctgcagc aaagcctgtc gtgtctgcgc tttaagcacg gccgggcgag tcgcgccacg 3001 gcgcggacat tcgtcgcgct gagcgtcggg gccaacaacc gcctgtgcgt gtccttgtgt 3061 cagcagtgct ttgccgccaa atgcgacagc aaccgcctgc acacgctgtt taccattgac 3121 gccggcacgc catgctcgcc gtccgttccc tgcagcacct ctcaaccgtc gtcttgataa 3181 cggcgtacgg cctcgtgctc gtgtggtaca ccgtcttcgg tgccagtccg ctgcaccgat 3241 gtatttacgc ggtacgcccc accggcacca acaacgacac cgccctcgtg tggatgaaaa 3301 tgaaccagac cctattgttt ctgggggccc cgacgcaccc ccccaacggg ggctggcgca 3361 accacgccca tatctgctac gccaatctta tcgcgggtag ggtcgtgccc ttccaggtcc 3421 cacctgacgc catgaatcgt cggatcatga acgtccacga ggcagttaac tgtctggaga 3481 ccctatggta cacacgggtg cgtctggtgg tcgtagggtg gttcctgtat ctggcgttcg 3541 tcgccctcca ccaacgccga tgtatgtttg gcgtcgtgag tcccgcccac aagatggtgg 3601 ccccggccac ctacctcttg aactacgcag gccgcatcgt atcgagcgtg ttcctgcagt 3661 acccctacac gaaaattacc cgcctgctct gcgagctgtc ggtccagcgg caaaacctgg 3721 ttcagttgtt tgagacggac ccggtcacct tcttgtacca ccgccccgcc atcggggtca 3781 tcgtaggctg cgagttgatg ctacgctttg tggccgtggg tctcatcgtc ggcaccgctt 3841 tcatatcccg gggggcatgt gcgatcacat accccctgtt tctgaccatc accacctggt 3901 gttttgtctc caccatcggc ctgacagagc tgtattgtat tctgcggcgg ggcccggccc 3961 ccaagaacgc agacaaggcc gccgccccgg ggcgatccaa ggggctgtcg ggcgtctgcg 4021 ggcgctgctg ttccatcatc ctctcgggca tcgcagtgcg attgtgttat atcgccgtgg 4081 tggccggggt ggtgctcgtg gcgcttcact acgagcagga gatccagagg cgcctgtttg 4141 atgtatgacg tcacatccag gccggcggaa accgtaacgg catatgcaaa ttggaaactg 4201 tcctgtcttg gggcccaccc acccgacgcg tcatatgcaa atgaaaatcg gtcccccgag 167 ME149651883v.1135975-54620 (REGN 11546) 4261 gccacgtgta gcctggatcc caacgacccc gcccatgggt cccaattggc cgtcccgtta 4321 ccaagaccaa cccagccagc gtatccaccc ccgcccgggt ccccgcggaa gcggaacggg 4381 gtatgtgata tgctaattaa a UL29 (ICP8) single-stranded DNA-binding protein [Human alphaherpesvirus 1 (Herpes simplex virus type 1)] Gene ID: 2703458. NCBI Reference Sequence: NC_001806.2. (SEQ ID NO. 205) 1 atggagacaa agcccaagac ggcaaccacc atcaaggtcc cccccgggcc cctgggatac 61 gtgtacgctc gcgcgtgtcc gtccgaaggc atcgagcttc tggcgttact gtcggcacgc 121 agcggcgatt ccgacgtcgc cgtggcgccc ctggtcgtgg gcctgaccgt ggagagcggc 181 tttgaggcca acgtggccgt ggtcgtgggt tctcgcacga cggggctcgg gggtaccgcg 241 gtgtccctga aactgacgcc ctcgcactac agctcgtccg tgtacgtctt tcacggcggc 301 cggcacctgg accccagcac ccaggccccg aacctgacgc gactttgcga gcgggcacgc 361 cgccattttg gcttttcgga ctacaccccc cggcccggcg acctcaaaca cgagacgacg 421 ggggaggcgc tgtgtgagcg cctcggcctg gacccggacc gcgccctcct gtatctggtc 481 gttaccgagg gcttcaagga ggccgtgtgc atcaacaaca cctttctgca cctgggaggc 541 tcggacaagg taaccatagg cggggcggag gtgcaccgca tacccgtgta cccgttgcag 601 ctgttcatgc cggattttag ccgtgtcatc gcagagccgt tcaacgccaa ccaccgatcg 661 atcggggaga attttaccta cccgcttccg ttttttaacc gccccctcaa ccgcctcctg 721 ttcgaggcgg tcgtgggacc cgccgccgtg gcactgcgat gccgaaacgt ggacgccgtg 781 gcccgcgcgg ccgcccacct ggcgtttgac gaaaaccacg agggcgccgc cctccccgcc 841 gacattacgt tcacggcctt cgaagccagc cagggtaaga ccccgcgggg cgggcgcgac 901 ggcggcggca agggcccggc gggcgggttc gaacagcgcc tggcctccgt catggccgga 961 gacgccgccc tggccctcga gtctatcgtg tcgatggccg tctttgacga gccgcccacc 1021 gacatctccg cgtggccgct gttcgagggc caggacacgg ccgcggcccg cgccaacgcc 1081 gtcggggcgt acctggcgcg cgccgcggga ctcgtggggg ccatggtatt tagcaccaac 1141 tcggccctcc atctcaccga ggtggacgac gccggcccgg cggacccaaa ggaccacagc 1201 aaaccctcct tttaccgctt cttcctcgtg cccgggaccc acgtggcggc caacccacag 1261 gtggaccgcg agggacacgt ggtgcccggg ttcgagggtc ggcccaccgc gcccctcgtc 1321 ggcggaaccc aggaatttgc cggcgagcac ctggccatgc tgtgtgggtt ttccccggcg 1381 ctgctggcca agatgctgtt ttacctggag cgctgcgacg gcggcgtgat cgtcgggcgc 1441 caggagatgg acgtgtttcg atacgtcgcg gactccaacc agaccgacgt gccctgtaac 1501 ctatgcacct tcgacacgcg ccacgcctgc gtacacacga cgctcatgcg cctccgggcg 1561 cgccatccaa agttcgccag cgccgcccgc ggagccatcg gcgtcttcgg gaccatgaac 1621 agcatgtaca gcgactgcga cgtgctggga aactacgccg ccttctcggc cctgaagcgc 1681 gcggacggat ccgagaccgc ccggaccatc atgcaggaga cgtaccgcgc ggcgaccgag 1741 cgcgtcatgg ccgaactcga gaccctgcag tacgtggacc aggcggtccc cacggccatg 1801 gggcggctgg agaccatcat caccaaccgc gaggccctgc atacggtggt gaacaacgtc 1861 aggcaggtcg tggaccgcga ggtggagcag ctgatgcgca acctggtgga ggggaggaac 1921 ttcaagtttc gcgacggtct gggcgaggcc aaccacgcca tgtccctgac gctggacccg 1981 tacgcgtgcg ggccgtgccc cctgcttcag cttctcgggc ggcgatccaa cctcgccgtg 2041 taccaggacc tggccctgag tcagtgccac ggggtgttcg ccgggcagtc ggtcgagggg 2101 cgcaactttc gcaatcaatt ccaaccggtg ctgcggcggc gcgtgatgga catgtttaac 2161 aacgggtttc tgtcggccaa aacgctgacg gtcgcgctct cggagggggc ggctatctgc 2221 gcccccagcc taacggccgg ccagacggcc cccgccgaga gcagcttcga gggcgacgtt 2281 gcccgcgtga ccctggggtt tcccaaggag ctgcgcgtca agagccgcgt gttgttcgcg 2341 ggcgcgagcg ccaacgcgtc cgaggccgcc aaggcgcggg tcgccagcct ccagagcgcc 2401 taccagaagc ccgacaagcg cgtggacatc ctcctcggac cgctgggctt tctgctgaag 2461 cagttccacg cggccatctt ccccaacggc aagcccccgg ggtccaacca gccgaacccg 2521 cagtggttct ggacggccct ccaacgcaac cagcttcccg cccggctcct gtcgcgcgag 2581 gacatcgaga ccatcgcgtt cattaaaaag ttttccctgg actacggcgc gataaacttt 2641 attaacctgg cccccaacaa cgtgagcgag ctggcgatgt actacatggc aaaccagatt 2701 ctgcggtact gcgatcactc gacatacttc atcaacaccc ttacggccat catcgcgggg 2761 tcccgccgtc cccccagcgt gcaggctgcg gccgcgtggt ccgcgcaggg cggggcgggc 168 ME149651883v.1135975-54620 (REGN 11546) 2821 ctggaggccg gggcccgcgc gctgatggac gccgtggacg cgcatccggg cgcgtggacg 2881 tccatgttcg ccagctgcaa cctgctgcgg cccgtcatgg cggcgcgccc catggtcgtg 2941 ttggggttga gcatcagcaa gtactacggc atggccggca acgaccgtgt gtttcaggcc 3001 gggaactggg ccagcctgat gggcggcaaa aacgcgtgcc cgctccttat ttttgaccgc 3061 acccgcaagt tcgtcctggc ctgtccccgg gccgggtttg tgtgcgcggc ctcaagcctc 3121 ggcggcggag cgcacgaaag ctcgctgtgc gagcagctcc ggggcattat ctccgagggc 3181 ggggcggccg tcgccagtag cgtgttcgtg gcgaccgtga aaagcctggg gccccgcacc 3241 cagcagctgc agatcgagga ctggctggcg ctcctggagg acgagtacct aagcgaggag 3301 atgatggagc tgaccgcgcg tgccctggag cgcggcaacg gcgagtggtc gacggacgcg 3361 gccctggagg tggcgcacga ggccgaggcc ctagtcagcc aactcggcaa cgccggggag 3421 gtgtttaact ttggggattt tggctgcgag gacgacaacg cgacgccgtt cggcggcccg 3481 ggggccccgg gaccggcatt tgccggccgc aaacgggcgt tccacgggga tgacccgttt 3541 ggggaggggc cccccgacaa aaagggagac ctgacgttgg atatgctgtg aggggttggg 3601 gggtggggga acctagggcg gggcggggaa tgtgtgtaaa ataaa UL9 DNA replication origin-binding helicase [Human alphaherpesvirus 1 (Herpes simplex virus type 1)] Gene ID: 2703434. NCBI Reference Sequence: NC_001806.2. (SEQ ID NO. 206) 1 atgcctttcg tggggggcgc ggagtcggga gatcctctgg gggccgggcg tcccattggg 61 gacgacgagt gcgaacagta cacgtcgagc gtatcgctag cgcggatgtt gtacgggggg 121 gatttggccg aatgggtgcc ccgggttcac ccgaaaacaa cgatcgagcg gcagcagcac 181 ggacccgtca ccttccccaa cgcgagcgcc ccgacggcca ggtgcgtgac tgtggtccgc 241 gcgccaatgg ggtcgggaaa aactaccgcg ctgatccgct ggctgcggga agcgatccac 301 tctccggaca cgagtgtgct cgtcgtctcc tgtcgtcgga gttttaccca gaccctagcg 361 acgcggttcg ctgagtcagg cctggtcgac tttgtcacct acttctcatc caccaattac 421 attatgaacg accgcccctt ccaccgactt atcgtccagg tggaaagcct tcatcgcgtg 481 ggccccaacc ttctgaacaa ctacgacgtc ctcgttctgg acgaggttat gtcgacgctg 541 ggccagctct attcgccaac gatgcagcaa ctgggccgcg tggatgcgtt aatgctacgc 601 ctgctgcgca cctgtcctcg gatcatcgcc atggacgcaa ccgccaacgc gcagttggtg 661 gacttcctgt gcggtctccg gggcgaaaaa aacgtgcatg tggtggtcgg cgagtacgcc 721 atgcccgggt tttcggcgcg ccggtgcctg tttctcccgc gtctggggac cgagctcctg 781 caggctgccc tgcgccc...

Claims

135975-54620 (REGN 11546) WHAT IS CLAIMED IS:

1. A method of producing a covalently surface modified adeno-associated virus (AAV), wherein the method comprises the steps of (A) transfecting a cell with: (i) a plasmid comprising a gene of interest flanked by AAV inverted terminal repeats; (ii) a plasmid comprising an AAV rep gene and an AAV cap gene; (iii) a plasmid comprising AAV rep and cap genes and a polynucleotide sequence encoding a first member of a specific binding pair; (iv) a plasmid comprising one or more helper polynucleotide sequences; (v) a plasmid comprising a polynucleotide sequence encoding a first portion of a retargeting molecule and a polynucleotide encoding a second cognate member of the specific binding pair; and (vi) a plasmid comprising a polynucleotide encoding a second portion of a retargeting molecule; (B) culturing the transfected cell to allow expression of plasmids (i) to (vi) and assembly of proteins to form covalently surface modified adeno-associated virus; and (C) harvesting the covalently surface modified adeno-associated virus.

2. The method according to claim 1, wherein the cell is a mammalian cell.

3. The method according to claim 2, wherein the mammalian cell is a human cell. 179 ME149651883v.1135975-54620 (REGN 11546) 4. The method according to claim 3, wherein the human cell is a HEK 293 cell.

5. The method according to claim 4, wherein the HEK 293 cell is suspension cultured.

6. The method according to claim 4, wherein the HEK 293 cell is an adherent HEK 293 cell.

7. The method according to claim 1, wherein the retargeting molecule is a monoclonal antibody.

8. The method according to claim 7, wherein the first portion of the retargeting molecule is an antibody heavy chain.

9. The method according to claim 7, wherein the second portion of the retargeting molecule is an antibody light chain.

10. The method according to claim 7, wherein the first portion of the retargeting molecule is an antibody light chain.

11. The method according to claim 7, wherein the second portion of the retargeting molecule is an antibody heavy chain. 180 ME149651883v.1135975-54620 (REGN 11546) 12. The method according to claim 1, wherein the specific binding pair is SpyTag-SpyCatcher.

13. The method according to claim 12, wherein the first member of a specific binding pair is SpyTag peptide.

14. The method according to claim 10, wherein the first member of a specific binding pair is SpyTag peptide, and the polynucleotide encoding the SpyTag peptide is inserted into the AAV cap gene to encode recombinant capsid proteins.

15. The method according to claim 14, wherein the cell expresses at least one recombinant capsid protein selected from the group consisting of a recombinant VP1 protein comprising a SpyTag amino acid sequence, a recombinant VP2 protein comprising a SpyTag amino acid sequence, and a recombinant VP3 protein comprising a SpyTag amino acid sequence.

16. The method according to claim 12, wherein the second cognate member of the specific binding pair is a SpyCatcher protein.

17. The method according to claims 1-3, wherein VP1 protein is mutated in a galactose binding domain to detarget liver cells.

18. The method according to claim 17, wherein a detargeting mutation is at least one selected from the group consisting of N272A and W503A. 181 ME149651883v.1135975-54620 (REGN 11546) 19. The method according to claims 1-3, wherein the covalently surface modified adeno-associated virus comprises a plurality of first members bound to second cognate members, wherein the second cognate members also are bound to retargeting molecules; and a gene of interest.

20. The method according to claim 19, wherein the first members are SpyTag peptides and second cognate members at SpyCatcher proteins.

21. The method according to claims 19-20, wherein the retargeting molecules are antibodies, antibody fragments or antibody derivatives.

22. The method according to claims 1-21, further comprising the step of (D) purifying the covalently surface modified adeno-associated virus using depth filtration followed by single-pass tangential flow filtration.

23. The method according to claim 22, wherein the depth filtration does not require an endonuclease.

24. The method according to claims 22-23, further comprising the step of (E) purifying the covalently surface modified adeno-associated virus using affinity chromatography followed by ionic exchange chromatography.

25. The method according to claims 1-24, wherein the helper polynucleotide sequences encode adenovirus E4, adenovirus E2, and VA RNA. 182 ME149651883v.1135975-54620 (REGN 11546) 26. The method according to claim 1-25, wherein one or more retargeting molecules can bind to one or more targets.

27. A covalently surface modified adeno-associated virus comprising a plurality of first members bound to second cognate members, wherein the second cognate members also are bound to retargeting molecules, and a gene of interest, wherein the covalently surface modified adeno-associated virus is made by a method comprising the steps of: A) transfecting a cell with: (i) a plasmid comprising a gene of interest flanked by AAV inverted terminal repeats; (ii) a plasmid comprising an AAV rep gene and an AAV cap gene; (iii) a plasmid comprising AAV rep and cap genes and a polynucleotide sequence encoding a first member of a specific binding pair; (iv) a plasmid comprising one or more helper polynucleotide sequences; (v) a plasmid comprising a polynucleotide sequence encoding a first portion of a retargeting molecule and a polynucleotide encoding a second cognate member of the specific binding pair; and (vi) a plasmid comprising a polynucleotide encoding a second portion of a retargeting molecule; (B) culturing the transfected cell to allow expression of plasmids (i) to (vi) and assembly of proteins to form covalently surface modified adeno-associated virus; and (C) harvesting the covalently surface modified adeno-associated virus. 183 ME149651883v.1135975-54620 (REGN 11546) 28. The covalently surface modified adeno-associated virus according to claim 27, wherein the cell is a mammalian cell.

29. The covalently surface modified adeno-associated virus according to claim 27, wherein the mammalian cell is a human cell.

30. The covalently surface modified adeno-associated virus according to claim 27, wherein the retargeting molecule is a monoclonal antibody.

31. The covalently surface modified adeno-associated virus according to claim 29, wherein the human cell is a HEK 293 cell.

32. The covalently surface modified adeno-associated virus according to claim 29, wherein the HEK 293 cell is suspension cultured.

33. The covalently surface modified adeno-associated virus according to claim 28, wherein the HEK 293 cell is an adherent HEK 293 cell.

34. The covalently surface modified adeno-associated virus according to claim 30, wherein the first portion of the retargeting molecule is an antibody heavy chain. 184 ME149651883v.1135975-54620 (REGN 11546) 35. The covalently surface modified adeno-associated virus according to claim 309, wherein the second portion of the retargeting molecule is an antibody light chain.

36. The covalently surface modified adeno-associated virus according to claim 30, wherein the first portion of the retargeting molecule is an antibody light chain.

37. The covalently surface modified adeno-associated virus according to claim 30, wherein the second portion of the retargeting molecule is an antibody heavy chain.

38. The covalently surface modified adeno-associated virus according to claim 28, wherein the specific binding pair is SpyTag-SpyCatcher.

39. The covalently surface modified adeno-associated virus according to claim 38, wherein the first member of a specific binding pair is SpyTag peptide.

40. The covalently surface modified adeno-associated virus according to claim 27, wherein the first member of a specific binding pair is SpyTag peptide, and the polynucleotide encoding the SpyTag peptide is inserted into the AAV cap gene to encode recombinant capsid proteins.

41. The covalently surface modified adeno-associated virus according to claim 40, wherein the cell expresses at least one recombinant capsid protein 185 ME149651883v.1135975-54620 (REGN 11546) selected from the group consisting of a recombinant VP1 protein comprising a SpyTag amino acid sequence, a recombinant VP2 protein comprising a SpyTag amino acid sequence, and a recombinant VP3 protein comprising a SpyTag amino acid sequence.

42. The covalently surface modified adeno-associated virus according to claim 38, wherein the second cognate member of the specific binding pair is a SpyCatcher protein.

43. The covalently surface modified adeno-associated virus according to claims 27-29, wherein VP1 protein is mutated in a galactose binding domain to detarget liver cells.

44. The covalently surface modified adeno-associated virus according to claim 43, wherein a detargeting mutation is at least one selected from the group consisting of N272A and W503A.

45. The covalently surface modified adeno-associated virus according to claims 27-29, wherein the covalently surface modified adeno-associated virus comprises a plurality of first members bound to second cognate members, wherein the second cognate members also are bound to retargeting molecules; and a gene of interest. 186 ME149651883v.1135975-54620 (REGN 11546) 46. The covalently surface modified adeno-associated virus according to claim 45, wherein the first members are SpyTag peptides and second cognate members are SpyCatcher proteins.

47. The covalently surface modified adeno-associated virus according to claims 45-46, wherein the retargeting molecules are antibodies, antibody fragments or antibody derivatives.

48. The covalently surface modified adeno-associated virus according to claims 27-47, further comprising the step of (D) purifying the covalently surface modified adeno-associated virus using depth filtration followed by single-pass tangential flow filtration.

49. The covalently surface modified adeno-associated virus according to claim 48, wherein the depth filtration does not require an endonuclease.

50. The covalently surface modified adeno-associated virus according to claims 48-49, further comprising the step of (E) purifying the covalently surface modified adeno-associated virus using affinity chromatography followed by ionic exchange chromatography.

51. The covalently surface modified adeno-associated virus according to claim 27-50, wherein one or more retargeting molecules can bind to one or more targets. 187 ME149651883v.1135975-54620 (REGN 11546) 52. The covalently surface modified adeno-associated virus according to claims 27-51, wherein the helper polynucleotide sequences encode adenovirus E4, adenovirus E2, and VA RNA.

53. An AAV preparation comprising the covalently surface modified adeno- associated virus of any of claims 1-52.

54. A biologic drug product comprising the covalently surface modified adeno-associated virus of any of claims 1-53.

55. A method of screening retargeting molecules for production of a covalently surface modified adeno-associated virus species, wherein the method comprises the steps of: (I) providing (A) a first plurality of nucleic acids encoding retargeting molecules that are different from one another and (B) a second plurality of DNA barcodes that are different from one another, wherein each individual DNA barcode of the second plurality is assigned to an individual covalently surface modified adeno-associated virus comprising a retargeting molecule of the first plurality for creating a covalently surface modified adeno-associated virus species; (II) producing covalently surface modified adeno-associated virus species by (A) transfecting a cell with: (i) a plasmid comprising a polynucleotide that comprises a DNA barcode, wherein the polynucleotide that comprises the DNA barcode is flanked by AAV inverted terminal repeats; 188 ME149651883v.1135975-54620 (REGN 11546) (ii) a plasmid comprising an AAV rep gene and an AAV cap gene; (iii) a plasmid comprising AAV rep and cap genes and a polynucleotide sequence encoding a first member of a specific binding pair; (iv) a plasmid comprising one or more helper sequences; (v) a plasmid comprising a polynucleotide sequence encoding a first portion of a retargeting molecule of the first plurality and a polynucleotide encoding a second cognate member of the specific binding pair; and (vi) a plasmid comprising a polynucleotide encoding a second portion of a retargeting molecule of the first plurality; (B) culturing the transfected cell to allow expression of plasmids (i) to (vi) and assembly of proteins to form a covalently surface modified adeno-associated virus species; (C) harvesting the covalently surface modified adeno-associated virus species, wherein the covalently surface modified adeno-associated virus species will become part of a covalently surface modified adeno-associated virus library; (D) repeating steps (A) to (C) to produce each covalently surface modified adeno-associated virus species to form the covalently surface modified adeno- associated viruses library; and (E) detecting each DNA barcode of the second plurality in order to screen each species to determine a property.

56. The method of claim 55, wherein a species is selected based upon a property, 189 ME149651883v.1135975-54620 (REGN 11546) 57. The method according to claim 55-56, wherein the property is genomic titer.

58. The method according to claims 55-57, wherein the retargeting molecule is an Fc-containing protein.

59. The method according to claim 58, wherein the Fc-containing protein is a monoclonal antibody.

60. The method according to claim 59, wherein the monoclonal antibody is a multispecific antibody, such as a bispecific antibody or a trispecific antibody.

61. The method according to claim 58, wherein the Fc-containing protein is an Fc-fusion protein.

62. The method according to claim 61, wherein the Fc-fusion protein is a receptor-Fc-fusion protein.

63. The method according to claim 61, wherein the receptor-Fc-fusion protein is a trap protein.

64. The method according to claims 55-57, wherein the retargeting molecule is selected from the group consisting of an fab, f(ab’), f(Ab’)2, single chain antibody and a mini-trap protein. 190 ME149651883v.1135975-54620 (REGN 11546) 65. The method according to claims 55-64, wherein the helper polynucleotide sequences encode adenovirus E4, adenovirus E2, and VA RNA.

66. A method of screening covalently surface modified adeno-associated virus species, wherein the method comprises the steps of: (I) providing (A) a first plurality of nucleic acids encoding a component of a covalently surface modified adeno-associated virus species (component), wherein each component of the first plurality are different from one another and (B) a second plurality of DNA barcodes that are different from one another, wherein each individual DNA barcode of the second plurality is assigned to an individual component of the first plurality for creating a covalently surface modified adeno- associated virus species; (II) producing covalently surface modified adeno-associated virus species by (A) transfecting a cell with: (i) a plasmid comprising a polynucleotide that comprises a DNA barcode, wherein the polynucleotide that comprises the DNA barcode is flanked by AAV inverted terminal repeats; (ii) a plasmid comprising an AAV rep gene and an AAV cap gene; (iii) a plasmid comprising AAV rep and cap genes and a polynucleotide sequence encoding a first member of a specific binding pair; (iv) a plasmid comprising one or more helper polynucleotide sequences; (v) a plasmid comprising a polynucleotide sequence encoding a first portion of a retargeting molecule and a polynucleotide encoding a second cognate member of the specific binding pair; and 191 ME149651883v.1135975-54620 (REGN 11546) (vi) a plasmid comprising a polynucleotide encoding a second portion of a retargeting molecule; (B) culturing the transfected cell to allow expression of plasmids (i) to (vi) and assembly of components to form a covalently surface modified adeno- associated virus species; (C) harvesting the covalently surface modified adeno-associated virus species, wherein the covalently surface modified adeno-associated virus species will become part of a covalently surface modified adeno-associated virus library; (D) repeating steps (A) to (C) to produce each covalently surface modified adeno-associated virus species to form the covalently surface modified adeno- associated viruses library; and (E) detecting each DNA barcode of the second plurality in order to screen each species to determine a property.

67. The method according to claim 66, wherein the component is selected from the group consisting of a retargeting molecule, an AAV cap gene, an AAV rep gene, an ITR, a helper polynucleotide sequence(s), a first member of a specific binding pair, and a second cognate member of a specific binding pair.

68. The method according to claims 66-67, wherein the helper sequences encode adenovirus E4, adenovirus E2, and VA RNA. 192 ME149651883v.1

Images