Polynucleotide constructs and cell lines for recombinant AAV production

Abstract

The inventions provide mammalian cell lines integrated with recombinant adeno-associated virus (rAAV) plasmids, cell genomes and methods of making the cell genomes for production of rAAV. The inventions also provide regulatory fusion proteins (RFPs) (which can act as activators or repressors) and repressor proteins (which act as repressors), such as antibiotic repressors, and arranged operators to control transcription of at least one polynucleotide of interest, such as AAV Rep genes and / or non-AAV helper genes.

Description

135975-75120 REGN 11751 POLYNUCLEOTIDE CONSTRUCTS AND CELL LINES FOR RECOMBINANT AAV PRODUCTION This application claims priority to U.S. Serial No.63 / 688,635, filed August 29, 2024, which is hereby incorporated by reference. Field of the Inventions

[0001] The inventions relate to recombinant adeno-associated virus (rAAV) plasmids, cell genomes and methods of the making the cell genomes for production of rAAV. The present inventions described herein provide, among other things, cells, cell cultures, polynucleotide (typically DNA) and polypeptide constructs, systems and methods for controlling the transcription of one or more polynucleotide sequences of interest. The inventions described herein further provide regulatory fusion proteins (RFPs) and repressor proteins, such as antibiotic repressors, and tandemly arranged operators to control transcription of at least one polynucleotide of interest, such as AAV Rep genes. Reference to Electronic Sequence Listing

[0002] This application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on August 26, 2025, is named “11751.xml” and is 394,859 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety. Background

[0003] The wild type AAV genome includes a capsid gene referred to as “Cap” or “cap”. Cap in nature is translated to produce, via alternative start codons and transcript splicing, three size-variant structural proteins referred to as VP1 (about135975-75120 REGN 11751 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). The wild-type AAV genome also includes a Rep gene, which is responsible for DNA replication and capsid packing, The Rep protein, however, is known to be toxic to cells in cell culture.

[0004] Various methods for controlled transcription of a polynucleotide sequence of interest in a cell are known to the art and described in U.S. Patent No.9,469,856. For example, No et al., Proc. Natl. Acad. Sci. USA 93:3346-3351 (1996) describe a controllable gene expression system utilizing a chimeric transactivator consisting of the ecdysone nuclear receptor fused to the VP16 transactivation domain from herpes simplex virus. Gossen et al., Proc. Natl. Acad. Sci. USA 89:5547-5551 (1992) describe a single system for controlling transcription of a polynucleotide sequence of interest based on a chimeric protein consisting of the tetracycline repressor protein fused with the VP16 transactivation domain. Gossen et al., Science 268: 1766-69 (1995) describes the fusion of a VP16 activation domain with a mutated ‘reverse’ Tet repressor that requires tetracycline for induction. Tetracycline-inducible gene expression is discussed in Ortiz and Johnson, Molec. Biochem. Parasitology 128: 43-40 (2003). Other described single control systems are cited in U.S. Patent No. 9,469,856, including Sadowski et al., Nature 335:563-564 (1988); Brent et al., Cell 40:729-736 (1985); Labow et al., Mol. Cell. Biol.10:3342-3356 (1990), for example.

[0005] Problems resulting from leaky transcription related to the sole reliance on minimal promoters have led to systems using fusions of the steroid-binding domains of the glucocorticoid or estrogen nuclear receptors. See, for example, Mattioni et al., Methods Cell Biol.43:335-352 (1994); Louvion et al. Gene 131:129-134 (1993); Iida et al. J. Virol.70: 6054-6059 (1996).

[0006] Further improvements in regulated expression systems are described and claimed in U.S. Patent No.9,469,856. However, ever tighter control of polynucleotide transcription and polypeptide expression is desired, particularly for the Rep gene.135975-75120 REGN 11751

[0007] Recombinant AAV (rAAV) has been produced in HEK 293, HeLa BHK, human amniotic (for example, epithelial cells such as HAEpiC), and CHO lines, for example. First generation rAAV has been 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. The AAV Cap and Rep gene, and viral helper polynucleotides, would be expressed via plasmids using transient expression.

[0008] There have been problems, however, due to expressing VP1, VP2 and VP3 proteins in appropriate ratios, while controlling the timing and expression of the Rep protein to minimize its cytotoxic effects.

[0009] The present inventions provide controlled expression of polynucleotide transcription and polypeptide expression of adeno-associated virus (AAV) rep plasmids, AAV cap plasmids, helper gene plasmids, and mammalian cell genomes. Summary of the Inventions

[0010] The present inventions advantageously include and utilize regulatory fusion proteins (RFPs) (which can act as activators or repressors) and repressor proteins (which act as repressors) and arranged operators to control transcription of at least one polynucleotide of interest, such as AAV Rep genes and / or non-AAV helper genes, including but not limited adenovirus helper genes. The transcriptional control approach utilizes the inventions in International Publication No. WO 2023 / 069929 A1, which is hereby incorporated by reference.

[0011] As employed herein, a first RFP can bind to a first operator, which can be located 5’ of a promoter and a polynucleotide of interest to be transcribed. A second RFP or a repressor protein can bind to a second operator. The second operator can be located 3’ of a promoter but 5’ of a polynucleotide to be transcribed. In some aspects, the second operator can be located 3’ of a promoter but 5’ of a polynucleotide to be transcribed and another second operator optionally can be operably linked to a polynucleotide encoding the first RFP or a repressor protein. The polynucleotide of interest can encode an AAV Rep protein, such as an AAV Rep gene having an active p19 promoter or an AAV Rep gene with reduced p19 promoter activity. The polynucleotides of interest can be one or more non-AAV helper135975-75120 REGN 11751 gene(s), such as one or more of an adenovirus gene, a herpesvirus gene, a human papilloma virus gene, a bocavirus gene and a baculovirus gene. T

[0012] The descriptions of aspects and aspects of the inventions provide methods for controlling the transcription of a polynucleotides of interest in cells, wherein the method comprises (I) maintaining a cell in a medium without an effective amount of a ligand of both an activator and a repressor, wherein a cell comprises: (A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding the activator; (C) a second operator; and (D) a polynucleotide encoding the repressor, wherein transcription of the polynucleotide of interest is inhibited in the absence of the ligand of both the activator and the repressor; and (II) controlling the cell to transcribe the polynucleotide of interest by maintaining the cell in a medium with an effective amount of the ligand of both the activator and the repressor. The second operator can be operably linked and positioned 3’ with respect to the promoter and 5’ with respect to the polynucleotide of interest. The activator can bind to the first operator in the presence of the ligand to permit transcription of the polynucleotide of interest. The polynucleotide of interest can encode an AAV Rep protein, such as an AAV Rep gene having an active p19 promoter or an AAV Rep gene with reduced p19 promoter activity. The polynucleotide of interest can be one or more non-AAV helper gene(s), such as one or more of an adenovirus gene, a herpesvirus gene, a human papilloma virus gene, a bocavirus gene and a baculovirus gene. The repressor can be a repressor protein, such as an antibiotic repressor, wherein transcription of the polynucleotide of interest is inhibited in the absence of the ligand, and wherein transcription is permitted in the presence of the ligand. The repressor protein can bind to the second operator in the absence of the ligand. The activator can be a regulatory fusion protein (RFP). The ligand can be selected from the group consisting of tetracycline and doxycycline. An activator RFP can be a reverse tetracycline transactivator. A repressor protein can be an antibiotic repressor, such as a tetracycline repressor.

[0013] Further provided are methods for controlling the transcription of polynucleotides of interest in a cell, wherein the method comprises (I) maintaining a cell in a medium without an effective amount of a ligand of an activator (activator ligand) and with an effective amount of ligand of a repressor (repressor ligand),135975-75120 REGN 11751 wherein the cell comprises: (A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding the activator; (C) a second operator; and (D) a polynucleotide encoding the repressor, wherein transcription of the polynucleotide of interest is inhibited in the absence of the activator ligand and the presence of the repressor ligand; and (II) controlling the cell to transcribe the polynucleotide of interest by maintaining the cell in a medium with an effective amount of the activator ligand and without an effective amount of the repressor ligand. The second operator can be operably linked and positioned 3’ with respect to the promoter and 5’ with respect to the polynucleotide of interest. The activator can bind to the first operator in the presence of the activator ligand to permit transcription of the polynucleotide of interest. The activator can be a regulatory fusion protein (RFP). The repressor can be a regulatory fusion protein (RFP), wherein transcription of the polynucleotide of interest is inhibited in the presence of the repressor ligand, and transcription is permitted in the absence of the repressor ligand. An activator RFP can be a reverse tetracycline transactivator. The activator ligand can be selected from the group consisting of tetracycline and doxycycline. A repressor RFP can be ArcEr, and the repressor RFP ligand can be selected from the group consisting of estrogen, estradiol (E2), tamoxifen and 4-hydroxytamoxifen (OHT). The polynucleotide of interest can encode an AAV Rep protein, such as an AAV Rep gene having an active p19 promoter an AAV Rep gene with reduced p19 promoter activity. The polynucleotide of interest can be one or more non-AAV helper gene(s), such as one or more of an adenovirus gene, a herpesvirus gene, a human papilloma virus gene, a bocavirus gene and a baculovirus gene.

[0014] There also are provided methods for controlling the transcription of polynucleotides of interest in cell culture, wherein the methods comprise: I. maintaining at least one cell in a medium with or without an effective amount of a first ligand of a first regulatory fusion protein (RFP) and with an effective amount of a second ligand of a second RFP, wherein the cell comprises: (A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding a first RFP, where the first RFP comprises: (1) a transcription activating domain fused to a first DNA binding domain; and (2) a ligand-binding domain; wherein the first ligand is135975-75120 REGN 11751 capable of binding to the ligand-binding domain of the first RFP, and wherein the DNA binding domain of the first RFP is capable of binding to the operator positioned 5’ when in the presence of the first ligand; (C) a second operator; and (D) a polynucleotide encoding the second RFP that differs from the first RFP, wherein the second RFP comprises: (1) a second DNA binding domain; and (2) a ligand-binding domain; wherein the second ligand is capable of binding to the ligand-binding domain of the second RFP, and wherein the second RFP is capable of binding to the second operator in the presence of the second ligand; wherein transcription of the polynucleotide of interest is inhibited in the absence of an effective amount of the first ligand and in the presence of an effective amount of the second ligand; and (II) controlling the cell to transcribe the polynucleotide of interest by maintaining the cell in a medium with an effective amount of the first ligand and without an effective amount of the second ligand. The polynucleotide of interest can encode an AAV Rep protein, such as an AAV Rep gene having an active p19 promoter or an AAV Rep gene with reduced p19 promoter activity. The second operator can be operably linked and positioned 3’ with respect to the promoter and 5’ with respect to the polynucleotide sequence encoding the protein of interest. Another second operator optionally can be operably linked to the polynucleotide sequence encoding the first RFP. The first RFP as an activator can be a reverse tetracycline transactivator (rtTA). The second RFP as a repressor can comprise an Arc repressor binding domain fused to the estrogen receptor ligand binding domain (ArcEr). The first operator can be a Tet Response Element (TRE). The second operator can be an Arc operator (AO). The cells can further comprise a repressor that is altered by the first ligand. The repressor can be a tet repressor protein (TetR). Additionally, the polynucleotide encoding the first RFP can be operably linked to promoter and optionally a second Arc operator. The promoter can be a CMV promoter, such as CMVmin. ArcEr can control the transcription of the polynucleotide encoding rtTA. The polynucleotide of interest can encode an AAV Rep protein, such as an AAV Rep gene having an active p19 promoter or an AAV Rep gene with reduced p19 promoter activity. The polynucleotide of interest can be one or more non-AAV helper gene(s), such as one or more of an adenovirus gene, a herpesvirus gene, a human papilloma virus gene, a bocavirus gene and a baculovirus gene.135975-75120 REGN 11751

[0015] There also are provided methods for controlling the transcription of polynucleotides of interest in cell culture, wherein the methods comprise: (I) maintaining at least one cell in a medium with or without an effective amount of a first ligand of a first regulatory fusion protein (RFP) and with an effective amount of a second ligand of a second RFP, wherein the cell comprises: (A) a promoter operably linked to a polynucleotide of interest and controlled by Tet Response Element (TRE) operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding a first RFP, where the first RFP comprises: (1) a transcription activating domain fused to a DNA binding domain; and (2) a ligand-binding domain, wherein the first ligand is capable of binding to the ligand-binding domain of the first RFP, and wherein the DNA binding domain of the first RFP is capable of binding to the operator positioned 5’ when in the presence of the first ligand; (C) an Arc operator operably linked and positioned 3’ with respect to the promoter and 5’ with respect to the polynucleotide encoding the protein of interest; and (D) a polynucleotide encoding the second RFP, wherein the second RFP comprises: (1) an Arc repressor DNA-binding domain; and (2) a ligand-binding domain; wherein the second ligand is capable of binding to the ligand-binding domain of the second RFP, and wherein the second RFP is capable of binding to the Arc operator in the presence of the second ligand; wherein transcription of the polynucleotide encoding the protein of interest is inhibited in the absence of an effective amount the first ligand and in the presence of an effective amount of the second ligand; and (II) controlling the cell to transcribe the polynucleotide of interest by maintaining the cell in a medium with an effective amount of the first ligand and without an effective amount of the second ligand. The first ligand can be selected from the group consisting of tetracycline, doxycycline and derivatives thereof. The second ligand can be selected from the group consisting of estrogen, estradiol (E2), tamoxifen, 4-hydroxytamoxifen (OHT) and derivatives thereof. The ligand-binding domain of the second RFP can be the ligand binding domain of a steroid receptor. The first regulatory fusion protein (RFP) as an activator can be a reverse tetracycline transactivator (rtTA). The second RFP as a repressor can be ArcER, which has the Arc repressor binding domain fused to the estrogen receptor ligand binding domain (Arc is a repressor from phage P22). The promoter operably linked to the polynucleotide sequence encoding a polypeptide of interest can be a CMV promoter, such as a CMVmin promoter. A CMV promoter and an Arc135975-75120 REGN 11751 operator optionally can be operably linked to the polynucleotide encoding the first RFP. An SV40 E / L promoter, or other constitutive promoter, can be operably linked to the polynucleotide encoding the second RFP. The polynucleotide of interest can encode an AAV Rep protein, such as an AAV Rep gene having an active p19 promoter or an AAV Rep gene with reduced p19 promoter activity. The polynucleotide of interest can be one or more non-AAV helper gene(s), such as one or more of an adenovirus gene, a herpesvirus gene, a human papilloma virus gene, a bocavirus gene and a baculovirus gene.

[0016] Moreover, there are provided methods for controlling the transcription of polynucleotides of interest in a cell, wherein the method comprises (I) maintaining at least one cell in a medium without an effective amount of a ligand of a regulatory fusion protein (RFP) and a repressor protein, wherein the cell comprises: (A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding the RFP, wherein the RFP comprises: (1) a transcription activating domain fused to a DNA binding domain; and (2) a ligand-binding domain; wherein the ligand is capable of binding to the ligand-binding domain of the RFP, and wherein the DNA binding domain of the RFP is capable of binding to the first operator when in the presence of the ligand; (C) a second operator; and (D) a polynucleotide encoding the repressor protein, wherein the repressor protein can bind to the second operator only in the absence of the ligand, wherein transcription of the polynucleotide is inhibited in the absence of an effective amount of the ligand; and (II) controlling the cell to transcribe the polynucleotide of interest by maintaining the cell in a medium with an effective amount of the ligand. The second operator can be operably linked and positioned 3’ with respect to the promoter and 5’ with respect to the polynucleotide of interest. The repressor protein can bind to the second operator in the absence of the ligand to inhibit transcription of the polynucleotide of interest. The RFP can bind to the first operator in the presence of the ligand to permit transcription of the polynucleotide of interest. The ligand can be selected from the group consisting of tetracycline and doxycycline. The activator RFP can be a reverse tetracycline transactivator (rtTA). The repressor protein can be a tetracycline repressor (TetR). The first operator can be a Tet Response Element (TRE). The second operator can be a Tet operator. The polynucleotide of interest135975-75120 REGN 11751 can encode an AAV Rep protein, such as an AAV Rep gene having an active p19 promoter or an AAV Rep gene with reduced p19 promoter activity. The polynucleotide of interest can be one or more non-AAV helper gene(s), such as one or more of an adenovirus gene, a herpesvirus gene, a human papilloma virus gene, a bocavirus gene and a baculovirus gene.

[0017] Additionally, there are provided methods for controlling the transcription of polynucleotides of interest in cell culture, wherein the methods comprise: (I) maintaining at least one cell in a medium with or without an effective amount of a first ligand of a first regulatory fusion protein (RFP) and with an effective amount of a second ligand of a second RFP, wherein the cell comprises: (A) a promoter operably linked to a polynucleotide of interest and controlled by a Tet Response Element (TRE) operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding a first RFP, wherein the first RFP comprises: (1) a transcription activating domain fused to a DNA binding domain; and (2) a ligand- binding domain; wherein the first ligand is capable of binding to the ligand-binding domain of the first RFP, and wherein the DNA binding domain of the first RFP is capable of binding to the TRE positioned 5’ when in the presence of the first ligand; (C) a Tet operator operably linked and positioned 3’ with respect to the promoter and 5’ with respect to the polynucleotide of interest; and (D) a polynucleotide encoding the second RFP, wherein the second RFP comprises: (1) an Arc repressor DNA- binding domain; and (2) a ligand-binding domain; wherein the second ligand is capable of binding to the ligand-binding domain of the second RFP, and wherein the second RFP is capable of binding to the Arc operator in the presence of the second ligand; wherein transcription of the polynucleotide is inhibited in the absence of an effective amount of the first ligand and the presence of an effective amount of the second ligand; and (II) controlling the cell to transcribe the polynucleotide of interest by maintaining the cell in a medium with an effective amount of the first ligand and without an effective amount of the second ligand. The first ligand can be selected from the group consisting of tetracycline, doxycycline and derivatives thereof. The second ligand can be selected from the group consisting of estrogen, estradiol (E2), tamoxifen, 4-hydroxytamoxifen (OHT) and derivatives thereof. The ligand-binding domain of the second RFP can be the ligand binding domain of a steroid receptor. The first regulatory fusion protein (RFP) as an activator can be a reverse tetracycline135975-75120 REGN 11751 transactivator (rtTA). The second RFP as a repressor can be ArcER. The promoter operably linked to the polynucleotide sequence encoding a polypeptide of interest can be a CMV promoter, such as CMVmin. A CMV promoter and an Arc operator optionally can be operably linked to the polynucleotide encoding the first RFP. An SV40 E / L promoter, or other constitutive promoter, can be operably linked to the polynucleotide encoding the second RFP. The cells can further comprise a polynucleotide encoding a repressor that is altered by the first ligand. The repressor can be TetR. The polynucleotide of interest can encode an AAV Rep protein, such as an AAV Rep gene having an active p19 promoter or an AAV Rep gene with reduced p19 promoter activity. The polynucleotide of interest can be one or more non-AAV helper gene(s), such as one or more of an adenovirus gene, a herpesvirus gene, a human papilloma virus gene, a bocavirus gene and a baculovirus gene.

[0018] There also are provided methods for controlling the transcription of polynucleotides of interest in cell culture, wherein the methods comprise: maintaining at least one cell in a medium with or without an effective amount of a first ligand of a first regulatory fusion protein (RFP) and with an effective amount of a second ligand of a second RFP, wherein the cell comprises (A) a promoter; (B) an Arc operator; and (C) a polynucleotide encoding a reverse tetracycline transactivator fusion protein (rtTA), wherein (A), (B) and (C) are operably linked, and wherein transcription of the rtTA polynucleotide is controlled by a fusion protein comprising an Arc repressor binding domain and an estrogen receptor ligand binding domain (ArcEr); wherein rtTA can control the transcription of a polynucleotide of interest. The promoter can be a CMV promoter, such as CMVmin. The first ligand can be selected from the group consisting of tetracycline and doxycycline and derivatives thereof. The second ligand can be selected from the group consisting of estrogen, estradiol (E2), tamoxifen, 4- hydroxytamoxifen (OHT) and derivatives thereof. The polynucleotide of interest can encode an AAV Rep protein, such as an AAV Rep gene having an active p19 promoter or an AAV Rep gene with reduced p19 promoter activity. The polynucleotide of interest can be one or more non-AAV helper gene(s), such as one or more of an adenovirus gene, a herpesvirus gene, a human papilloma virus gene, a bocavirus gene and a baculovirus gene.

[0019] The methods above can be combined. For example, a method wherein the polynucleotide of interest is an AAV Rep gene can be practiced along with a135975-75120 REGN 11751 method wherein the polynucleotide of interest is one or more non-AAV helper gene(s).

[0020] The inventions also provide cells capable of controlled transcription of at least one polynucleotide of interest, wherein a cell comprises: (A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding an activator; (C) a second operator; and (D) a polynucleotide encoding a repressor, wherein transcription of the polynucleotide of interest is inhibited in the absence of a ligand of both the activator and the repressor, and is permitted in the presence of the ligand of both the activator and the repressor. The second operator can be operably linked and positioned 3’ with respect to the promoter and 5’ with respect to the polynucleotide of interest. The activator can bind to the first operator in the presence of the ligand to permit transcription of the polynucleotide of interest. The repressor can be a repressor protein, wherein transcription of the polynucleotide of interest is inhibited in the absence of the ligand, and wherein transcription is permitted in the presence of the ligand. The repressor protein can bind to the second operator in the absence of the ligand. The activator can be a regulatory fusion protein (RFP). The ligand can be selected from the group consisting of tetracycline and doxycycline. The activator RFP can be a reverse tetracycline transactivator. The repressor protein can be a tetracycline repressor. The polynucleotide of interest can encode an AAV Rep protein, such as an AAV Rep gene having an active p19 promoter or an AAV Rep gene with reduced p19 promoter activity. The polynucleotide of interest can be one or more non-AAV helper gene(s), such as one or more of an adenovirus gene, a herpesvirus gene, a human papilloma virus gene, a bocavirus gene and a baculovirus gene.

[0021] Additionally, there are provided cells capable of controlled transcription of at least one polynucleotide of interest, wherein a cell comprises: (A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding a first regulatory fusion protein (RFP), where the first RFP comprises: (1) a transcription activating domain fused to a first DNA binding domain; and (2) a ligand- binding domain; wherein the first ligand is capable of binding to the ligand-binding domain of the first RFP, and wherein the DNA binding domain of the first RFP is135975-75120 REGN 11751 capable of binding to the operator positioned 5’ when in the presence of the first ligand; (C) a second operator; and (D) a polynucleotide encoding the second RFP that differs from the first RFP, wherein the second RFP comprises: (1) a second DNA-binding domain; and (2) a ligand-binding domain; wherein the second ligand is capable of binding to the ligand-binding domain of the second RFP, and wherein the second RFP is capable of binding to the second operator in the presence of the second ligand; wherein transcription of the polynucleotide is inhibited in the absence of the first ligand and in the presence of the second ligand and is permitted in the presence of the first ligand and absence of the second ligand. The second operator can be operably linked and positioned 3’ with respect to the promoter and 5’ with respect to the polynucleotide sequence encoding the protein of interest. The second operator optionally can be operably linked to the polynucleotide sequence encoding the first RFP. The cells can comprise a polynucleotide that encodes the repressor that is altered by the first ligand. The repressor can be TetR. The polynucleotide (B) encoding the first RFP can be operably linked to promoter and a second Arc operator. The promoter can be a CMV promoter, such as CMVmin. The first RFP as an activator can be a reverse tetracycline transactivator fusion protein (rtTA) and the second RFP as a repressor can be a fusion protein comprising an Arc repressor binding domain and an estrogen receptor ligand binding domain (ArcEr). ArcEr can control the transcription of the polynucleotide encoding rtTA. The polynucleotide of interest can encode an AAV Rep protein, such as an AAV Rep gene having an active p19 promoter or an AAV Rep gene with reduced p19 promoter activity. The polynucleotide of interest can be one or more non-AAV helper gene(s), such as one or more of an adenovirus gene, a herpesvirus gene, a human papilloma virus gene, a bocavirus gene and a baculovirus gene.

[0022] Furthermore, there are provided cells capable of controlled transcription of polynucleotides of interest, wherein a cell comprises (A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding an activator; (C) a second operator; and (D) a polynucleotide encoding a repressor; wherein transcription of the polynucleotide of interest is inhibited in the absence of an effective amount if an activator ligand and the presence of an effective amount of a repressor ligand; and permitted in the presence of an effective amount of the135975-75120 REGN 11751 activator ligand and the absence of an effective amount of the repressor ligand. The second operator can be operably linked and positioned 3’ with respect to the promoter and 5’ with respect to the polynucleotide of interest. The activator can bind to the first operator in the presence of the activator ligand to permit transcription of the polynucleotide of interest. The activator can be a regulatory fusion protein (RFP). The repressor can be a regulatory fusion protein (RFP), wherein transcription of the polynucleotide of interest is inhibited in the presence of the repressor ligand, and transcription is permitted in the absence of the repressor ligand. The activator RFP can be a reverse tetracycline transactivator. The activator ligand can be selected from the group consisting of tetracycline and doxycycline. The repressor RFP can be ArcEr. The repressor ligand can be selected from the group consisting of estrogen, estradiol (E2), tamoxifen and 4-hydroxytamoxifen (OHT). The polynucleotide of interest can encode an AAV Rep protein, such as an AAV Rep gene having an active p19 promoter or an AAV Rep gene with reduced p19 promoter activity. The polynucleotide of interest can be one or more non-AAV helper gene(s), such as one or more of an adenovirus gene, a herpesvirus gene, a human papilloma virus gene, a bocavirus gene and a baculovirus gene.

[0023] There also are provided cells capable of controlled transcription of at least one polynucleotide of interest, wherein a cell comprises: (A) a promoter operably linked to a polynucleotide of interest and controlled by Tet Response Element (TRE) operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding a first regulatory fusion protein (RFP), where the first RFP comprises: (1) a transcription activating domain fused to a DNA binding domain; and (2) a ligand- binding domain; wherein the first ligand is capable of binding to the ligand-binding domain of the first RFP, and wherein the DNA binding domain of the first RFP is capable of binding to the operator positioned 5’ when in the presence of the first ligand; (C) an Arc operator operably linked and positioned 3’ with respect to the promoter and 5’ with respect to the polynucleotide encoding the protein of interest; and (D) a polynucleotide encoding the second RFP, wherein the second RFP comprises: (1) an Arc repressor DNA-binding domain; and (2) a ligand-binding domain; wherein the second ligand is capable of binding to the ligand-binding domain of the second RFP, and wherein the second RFP is capable of binding to the Arc operator in the presence of the second ligand; wherein transcription of the135975-75120 REGN 11751 polynucleotide is inhibited in the absence of the first ligand and in the presence of the second ligand and is permitted in the presence of the first ligand and absence of the second ligand. The first ligand can be selected from the group consisting of tetracycline, doxycycline and derivatives thereof. The second ligand can be selected from the group consisting of estrogen, estradiol (E2), tamoxifen, 4-hydroxytamoxifen (OHT) and derivatives thereof. The ligand-binding domain of the second RFP can be the ligand binding domain of a steroid receptor. The first regulatory fusion protein (RFP) as an activator can be a reverse tetracycline transactivator (rtTA). The second RFP as a repressor can be ArcER. The promoter operably linked to the polynucleotide sequence encoding a polypeptide of interest can be a CMV promoter, such as a CMVmin promoter. A CMV promoter and an Arc operator optionally can be operably linked to the polynucleotide encoding the first RFP. An SV40 E / L promoter, or other constitutive promoter, can be operably linked to the polynucleotide encoding the second RFP. The polynucleotide of interest can encode an AAV Rep protein, such as an AAV Rep gene having an active p19 promoter or an AAV Rep gene with reduced p19 promoter activity. The polynucleotide of interest can be one or more non-AAV helper gene(s), such as one or more of an adenovirus gene, a herpesvirus gene, a human papilloma virus gene, a bocavirus gene and a baculovirus gene.

[0024] In addition, there are provided cells capable of controlled transcription of polynucleotides of interest, wherein a cell comprises (A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding a regulatory fusion protein (RFP), wherein the RFP comprises: (1) a transcription activating domain fused to a DNA binding domain; and (2) a ligand-binding domain; wherein the ligand is capable of binding to the ligand-binding domain of the RFP, and wherein the DNA binding domain of the RFP is capable of binding to the first operator when in the presence of the ligand; (C) a second operator; and (D) a polynucleotide encoding a repressor protein, wherein the repressor protein can bind to the second operator only in the absence of the ligand, wherein transcription of the polynucleotide of interest is inhibited in the absence of an effective amount of the ligand of both the activator and the repressor, and is permitted in the presence of an effective amount of the ligand of both the activator and the repressor. The second135975-75120 REGN 11751 operator can be operably linked and positioned 3’ with respect to the promoter and 5’ with respect to the polynucleotide of interest. The repressor protein can bind to the second operator in the absence of the ligand to inhibit transcription of the polynucleotide of interest. The RFP can bind to the first operator in the presence of the ligand to permit transcription of the polynucleotide of interest. The ligand can be selected from the group consisting of tetracycline and doxycycline. The activator RFP can be a reverse tetracycline transactivator (rtTA). The repressor protein can be a tetracycline repressor (TetR). The first operator can be a Tet Response Element (TRE). The second operator can be a Tet operator. The polynucleotide of interest can encode an AAV Rep protein, such as an AAV Rep gene having an active p19 promoter or an AAV Rep gene with reduced p19 promoter activity. The polynucleotide of interest can be one or more non-AAV helper gene(s), such as one or more of an adenovirus gene, a herpesvirus gene, a human papilloma virus gene, a bocavirus gene and a baculovirus gene.

[0025] Additionally, there are provided cells capable of controlled transcription of at least one polynucleotide of interest, wherein a cell comprises: (A) a promoter operably linked to a polynucleotide of interest and controlled by a Tet Response Element (TRE) operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding a first regulatory fusion protein (first RFP), where the first RFP comprises: (1) a transcription activating domain fused to a DNA binding domain; and (2) a ligand-binding domain; wherein the first ligand is capable of binding to the ligand-binding domain of the first RFP, and wherein the DNA binding domain of the first RFP is capable of binding to the TRE positioned 5’ when in the presence of the first ligand; (C) a Tet operator operably linked and positioned 3’ with respect to the promoter and 5’ with respect to the polynucleotide of interest; and (D) a polynucleotide encoding the second RFP, wherein the second RFP comprises: (1) an Arc repressor DNA-binding domain; and (2) a ligand-binding domain; wherein the second ligand is capable of binding to the ligand-binding domain of the second RFP, and wherein the second RFP is capable of binding to the Arc operator in the presence of the second ligand; wherein transcription of the polynucleotide is inhibited in the absence of the first ligand and in the presence of the second ligand and is permitted in the presence of the first ligand and absence of the second ligand. The first ligand can be selected from the group consisting of tetracycline, doxycycline and135975-75120 REGN 11751 derivatives thereof. The second ligand can be selected from the group consisting of estrogen, estradiol (E2), tamoxifen, 4-hydroxytamoxifen (OHT) and derivatives thereof. The first regulatory fusion protein (RFP) as an activator can be a reverse tetracycline transactivator (rtTA). The second RFP as a repressor can be ArcER. The promoter operably linked to the polynucleotide sequence encoding a polypeptide of interest can be a CMV promoter, such as a CMVmin. A CMV promoter and an Arc operator optionally can be operably linked to the polynucleotide encoding the first RFP. An SV40 E / L promoter, or other constitutive promoter, can be operably linked to the polynucleotide encoding the second RFP. The cell can further comprise a polynucleotide encoding a repressor that is altered by the first ligand. The repressor can be TetR. The polynucleotide of interest can encode an AAV Rep protein, such as an AAV Rep gene having an active p19 promoter or an AAV Rep gene with reduced p19 promoter activity. The polynucleotide of interest can be one or more non-AAV helper gene(s), such as one or more of an adenovirus gene, a herpesvirus gene, a human papilloma virus gene, a bocavirus gene and a baculovirus gene.

[0026] Additionally, there are provided cells capable of controlled transcription of at least one polynucleotide of interest when present, wherein a cell comprises: (A) a polynucleotide sequence encoding a first regulatory fusion protein (first RFP), where the first RFP comprises: (1) a transcription activating domain fused to a DNA binding domain; and (2) a ligand-binding domain; wherein the first ligand is capable of binding to the ligand-binding domain of the first RFP, and wherein the DNA binding domain of the first RFP is capable of binding to the operator positioned 5’ when in the presence of the first ligand; (B) a polynucleotide sequence encoding the second regulatory fusion protein (second RFP), wherein the second RFP comprises: (1) a DNA binding domain comprising an Arc repressor DNA-binding domain; and (2) a ligand-binding domain; wherein the second ligand is capable of binding to the ligand- binding domain of the second RFP, and wherein the second RFP is capable of binding to an Arc operator in the presence of the second ligand; (C) one or more insertion sites for a polynucleotide of interest that is operably linked to a promoter and at least one operator. The first ligand can be selected from the group consisting of tetracycline, doxycycline and derivatives thereof. The second ligand can be selected from the group consisting of estrogen, estradiol (E2), tamoxifen, 4-135975-75120 REGN 11751 hydroxytamoxifen (OHT) and derivatives thereof. The first regulatory fusion protein (RFP) as an activator can be a reverse tetracycline transactivator (rtTA). The second RFP as a repressor can be ArcER. The promoter operably linked to the polynucleotide sequence encoding a polypeptide of interest can be a CMV promoter, such as a CMVmin promoter. A CMV promoter and an Arc operator optionally can be operably linked to the polynucleotide encoding the first RFP. An SV40 E / L promoter or other constitutive promoter can be operably linked to the polynucleotide encoding the second RFP. A cell can further comprise a polynucleotide encoding a repressor that is altered by the first ligand. The repressor can be TetR. The polynucleotide of interest can encode an AAV Rep protein, such as an AAV Rep gene having an active p19 promoter or an AAV Rep gene with reduced p19 promoter activity. The polynucleotide of interest can be one or more non-AAV helper gene(s), such as one or more of an adenovirus gene, a herpesvirus gene, a human papilloma virus gene, a bocavirus gene and a baculovirus gene.

[0027] There also are provided cells capable of controlling the transcription of polynucleotides of interest, wherein a cell comprises (A) a promoter; (B) an Arc operator; and (C) a polynucleotide encoding a reverse tetracycline transactivator fusion protein (rtTA), wherein (A), (B) and (C) are operably linked, and wherein transcription of the rtTA polynucleotide is controlled by a fusion protein comprising an Arc repressor binding domain and an estrogen receptor ligand binding domain (ArcEr); wherein rtTA can control the transcription of a polynucleotide of interest. The promoter can be a CMV promoter, such as CMVmin. The first ligand can be selected from the group consisting of tetracycline and doxycycline. The second ligand can be selected from the group consisting of estrogen, estradiol (E2), tamoxifen, 4-hydroxytamoxifen (OHT) and derivatives thereof. The polynucleotide of interest can encode an AAV Rep protein, such as an AAV Rep gene having an active p19 promoter or an AAV Rep gene with reduced p19 promoter activity. The polynucleotide of interest can be one or more non-AAV helper gene(s), such as one or more of an adenovirus gene, a herpesvirus gene, a human papilloma virus gene, a bocavirus gene and a baculovirus gene.

[0028] The cells above can contain constructs allowing for the controlled expression of an AAV Rep gene and one or more non-AAV helper gene(s).135975-75120 REGN 11751

[0029] The inventions provide polynucleotides, wherein each can comprise in the 5’ to 3’ direction (i) a tetracycline response element (TRE), (ii) a first promoter, (iii) an Arc operator (AO) or a tetracycline operator (TO), (iv) an adeno-associated virus (AAV) Rep gene having a p19 promoter, and (v) a portion of an AAV Cap gene. The (i) tetracycline response element (TRE), (ii) the first promoter, (iii) the Arc operator (AO) or tetracycline operator (TO), and (iv) the AAV Rep gene can be operably linked. The p19 promoter can be associated with a non-ATG start codon mutation, such as an ACG, CTG, GTG, TTG mutation and the like. The first promoter can be a CMV promoter, such as a CMV min promoter. A polynucleotide can AO, or a polynucleotide can comprise TO. The polynucleotide can be used to express Rep 78 and 68,

[0030] The inventions further provide cells capable of controlled transcription of AAV Rep genes, wherein a cell comprises a cell genome comprising (A) a promoter operably linked to an AAV Rep gene having an active p19 promoter and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding an activator; (C) a second operator; and (D) a polynucleotide encoding a repressor; wherein transcription of the AAV Rep gene is inhibited in the absence of an effective amount if an activator ligand and the presence of an effective amount of a repressor ligand; and permitted in the presence of an effective amount of the activator ligand and the absence of an effective amount of the repressor ligand, and wherein the cell can express Rep 78 protein and Rep 68 protein. The second operator can be operably linked and positioned 3’ with respect to the promoter and 5’ with respect to the AAV Rep gene. The activator can bind to the first operator in the presence of the activator ligand to permit transcription of the AAV Rep gene. The first operator can be a tetracycline response element (TRE). The second operator can be a tetracycline operator (TO). The cell may not express or may minimally expresses Rep52 and Rep40. The cell genome can comprise a portion of an AAV Cap gene located 3’ of the AAV Rep gene. The activator can be a regulatory fusion protein (RFP). The RFP can be a reverse tetracycline transactivator. The repressor protein can be a tetracycline repressor protein. The activator ligand and the repressor ligand can be selected from the group consisting of tetracycline and doxycycline.135975-75120 REGN 11751

[0031] The inventions also provide cells capable of controlled transcription of AAV Rep genes, wherein a cell comprises a cell genome comprising: (A) a promoter operably linked to an AAV Rep gene having an active p19 promoter and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding a first regulatory fusion protein (RFP), where the first RFP comprises: (1) a transcription activating domain fused to a first DNA binding domain; and (2) a ligand-binding domain; wherein the first ligand is capable of binding to the ligand-binding domain of the first RFP, and wherein the DNA binding domain of the first RFP is capable of binding to the operator positioned 5’ when in the presence of the first ligand; (C) a second operator; and (D) a polynucleotide encoding the second RFP that differs from the first RFP, wherein the second RFP comprises: (1) a DNA- binding domain; and (2) a ligand-binding domain; wherein the second ligand is capable of binding to the ligand-binding domain of the second RFP, and wherein the second RFP is capable of binding to the second operator in the presence of the second ligand; wherein transcription of the polynucleotide is inhibited in the absence of an effective amount of the first ligand and in the presence of an effective amount of the second ligand and is permitted in the presence of an effective amount of the first ligand and absence of an effective amount of the second ligand, and wherein the cell can express Rep 78 protein and Rep 68 protein. The second operator can be operably linked and positioned 3’ with respect to the promoter and 5’ with respect to the AAV Rep polynucleotide. The cell may not express or may minimally express Rep 52 and Rep 40. The first RFP can be a reverse tetracycline transactivator. The second RFP can comprise (1) an Arc repressor DNA-binding domain; and (2) a ligand-binding domain; wherein the second ligand is capable of binding to the ligand- binding domain of the second RFP, and wherein the second RFP is capable of binding to the Arc operator in the presence of the second ligand; wherein transcription of the polynucleotide is inhibited in the absence of the first ligand and the presence of the second ligand. The ligand-binding domain of the second RFP can be a ligand binding domain of a steroid receptor. The ligand-binding domain of the second RFP can be an estrogen receptor ligand binding domain. The second RFP can be ArcEr. The first operator can be a tetracycline response element (TRE). The second operator can be an Arc operator (AO). The first ligand can be selected from the group consisting of tetracycline and doxycycline. The second ligand can be135975-75120 REGN 11751 selected from the group consisting of estrogen, estradiol (E2), tamoxifen, 4- hydroxytamoxifen (OHT) and derivatives thereof. The cell genome can comprise a portion of an AAV Cap gene located 3’ of the AAV Rep gene.

[0032] The inventions also provide methods for controlling the transcription of AAV Rep genes in a cell, wherein the methods comprise: I. maintaining a cell in a medium without an effective amount of a ligand of an activator (activator ligand) and with an effective amount of ligand of a repressor (repressor ligand), wherein the cell is capable of controlled transcription of an AAV Rep gene, wherein the cell comprises a cell genome comprising (A) a promoter operably linked to an AAV Rep gene having an active p19 promoter and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding an activator; (C) a second operator; and (D) a polynucleotide encoding a repressor; wherein transcription of the AAV Rep gene is inhibited in the absence of an effective amount if an activator ligand and the presence of an effective amount of a repressor ligand; and permitted in the presence of an effective amount of the activator ligand and the absence of an effective amount of the repressor ligand; and II. controlling the cell to transcribe the AAV Rep gene by maintaining the cell in a medium with an effective amount of the activator ligand and without an effective amount of the repressor ligand, and wherein the cell can express Rep 78 protein and Rep 68 protein. The second operator can be operably linked and positioned 3’ with respect to the promoter and 5’ with respect to the AAV Rep gene. The activator can bind to the first operator in the presence of the activator ligand to permit transcription of the AAV Rep gene. The cell may not express or may minimally express Rep 52 and Rep 40. The cell genome can comprise a portion of an AAV Cap gene located 3’ of the AAV Rep gene. The activator is a regulatory fusion protein (RFP). The RFP can be a reverse tetracycline transactivator. The repressor protein can be a tetracycline repressor protein. The activator ligand and the repressor ligand can be selected from the group consisting of tetracycline and doxycycline.

[0033] The inventions further provide methods for controlling the transcription of AAV Rep genes in a cell, wherein the methods comprise: I. maintaining a cell in a medium without an effective amount of a ligand of an activator (activator ligand) and with an effective amount of ligand of a repressor (repressor ligand), wherein the cell is capable of controlled transcription of an AAV Rep gene, and wherein the cell135975-75120 REGN 11751 comprises a cell genome comprising: (A) a promoter operably linked to AAV Rep gene having an active p19 promoter and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding a first regulatory fusion protein (RFP), where the first RFP comprises: (1) a transcription activating domain fused to a first DNA binding domain; and (2) a ligand-binding domain; wherein the first ligand is capable of binding to the ligand-binding domain of the first RFP, and wherein the DNA binding domain of the first RFP is capable of binding to the operator positioned 5’ when in the presence of the first ligand; (C) a second operator; and (D) a polynucleotide encoding the second RFP that differs from the first RFP, wherein the second RFP comprises: (1) a DNA-binding domain; and (2) a ligand-binding domain; wherein the second ligand is capable of binding to the ligand-binding domain of the second RFP, and wherein the second RFP is capable of binding to the second operator in the presence of the second ligand; wherein transcription of the polynucleotide is inhibited in the absence of an effective amount of the first ligand and in the presence of an effective amount of the second ligand and is permitted in the presence of an effective amount of the first ligand and absence of an effective amount of the second ligand; and II. controlling the cell to transcribe the AAV Rep gene by maintaining the cell in a medium with an effective amount of the activator ligand and without an effective amount of the repressor ligand, and wherein the cell can express Rep 78 protein and Rep 68 protein. The second operator can be operably linked and positioned 3’ with respect to the promoter and 5’ with respect to the AAV Rep gene. The activator can bind to the first operator in the presence of the activator ligand to permit transcription of the AAV Rep gene. The cells may not express or may minimally express Rep 52 and Rep 40. The first RFP can be a reverse tetracycline transactivator. The second RFP can comprise (1) an Arc repressor DNA-binding domain; and (2) a ligand-binding domain; wherein the second ligand is capable of binding to the ligand-binding domain of the second RFP, and wherein the second RFP is capable of binding to the Arc operator in the presence of the second ligand; wherein transcription of the polynucleotide is inhibited in the absence of the first ligand and the presence of the second ligand. The ligand-binding domain of the second RFP can be the ligand binding domain of a steroid receptor. The ligand-binding domain of the second RFP can be an estrogen receptor ligand binding domain. The second RFP can be ArcEr.135975-75120 REGN 11751 The first operator can be a tetracycline response element (TRE). The second operator can be an Arc operator (AO). The first ligand can be selected from the group consisting of tetracycline and doxycycline. The second ligand can be selected from the group consisting of estrogen, estradiol (E2), tamoxifen, 4-hydroxytamoxifen (OHT) and derivatives thereof. The cell genome comprises a portion of an AAV Cap gene located 3’ of the AAV Rep gene.

[0034] The cells above can contain constructs allowing for the controlled expression of an AAV Rep gene and one or more non-AAV helper gene(s

[0035] The inventions further provide polynucleotides comprising in the 5’ to 3’ direction (i) a tetracycline response element (TRE), (ii) a first promoter, (iii) an Arc operator (AO), (iv) an AAV Rep gene with reduced p19 promoter activity, and (v) a portion of an AAV Cap gene. The (i) tetracycline response element (TRE), (ii) the first promoter, (iii) the Arc operator (AO), (iv) the AAV Rep gene, and (v) a portion of an AAV Cap gene are operably linked. The first promoter can be a CMV promoter, such as a CMV min promoter. The polynucleotide can comprise (iii) AO or the polynucleotide can comprise (iii) TO. The polynucleotide can be used to express Rep 52 and 40.

[0036] The inventions also provide cells capable of controlled transcription of an AAV Rep genes, wherein a cell comprises a cell genome comprising (A) a promoter operably linked to an AAV Rep gene with reduced p19 promoter activity and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding an activator; (C) a second operator; and (D) a polynucleotide encoding a repressor; wherein transcription of the AAV Rep gene is inhibited in the absence of an effective amount if an activator ligand and the presence of an effective amount of a repressor ligand; and permitted in the presence of an effective amount of the activator ligand and the absence of an effective amount of the repressor ligand, and wherein the cell can express Rep 52 protein and Rep 40 protein. The second operator can be operably linked and positioned 3’ with respect to the promoter and 5’ with respect to the AAV Rep gene. The activator can bind to the first operator in the presence of the activator ligand to permit transcription of the AAV Rep gene. The first operator can be a tetracycline response element (TRE). The second operator can be a tetracycline operator (TO). The cell may not express135975-75120 REGN 11751 or may minimally express Rep 78 and Rep 68. The cell genome may comprise a portion of an AAV Cap gene located 3’ of the AAV Rep gene. The activator may a regulatory fusion protein (RFP), such as a reverse tetracycline transactivator. The repressor protein may be a tetracycline repressor protein. The activator ligand and the repressor ligand may be selected from the group consisting of tetracycline and doxycycline.

[0037] The inventions also provide cells capable of controlled transcription of AAV Rep genes, wherein the cell comprises a cell genome comprising: (A) a promoter operably linked to an AAV Rep gene with reduced p19 promoter activity and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding a first regulatory fusion protein (RFP), where the first RFP comprises: (1) a transcription activating domain fused to a first DNA binding domain; and (2) a ligand-binding domain; wherein the first ligand is capable of binding to the ligand-binding domain of the first RFP, and wherein the DNA binding domain of the first RFP is capable of binding to the operator positioned 5’ when in the presence of the first ligand; (C) a second operator; and (D) a polynucleotide encoding the second RFP that differs from the first RFP, wherein the second RFP comprises: (1) a DNA-binding domain; and (2) a ligand-binding domain; wherein the second ligand is capable of binding to the ligand-binding domain of the second RFP, and wherein the second RFP is capable of binding to the second operator in the presence of the second ligand; wherein transcription of the polynucleotide is inhibited in the absence of an effective amount of the first ligand and in the presence of an effective amount of the second ligand and is permitted in the presence of an effective amount of the first ligand and absence of an effective amount of the second ligand, and wherein the cell can express Rep 52 protein and Rep 40 protein. The second operator is operably linked and positioned 3’ with respect to the promoter and 5’ with respect to the AAV Rep polynucleotide. The cell may not express or may minimally express Rep 78 and Rep 68. The first RFP can be a reverse tetracycline transactivator. The second RFP can comprise (1) an Arc repressor DNA-binding domain; and (2) a ligand-binding domain; wherein the second ligand is capable of binding to the ligand-binding domain of the second RFP, and wherein the second RFP is capable of binding to the Arc operator in the presence of the second ligand; wherein transcription of the polynucleotide is inhibited in the135975-75120 REGN 11751 absence of the first ligand and the presence of the second ligand. The ligand-binding domain of the second RFP can be a ligand binding domain of a steroid receptor, such as an estrogen receptor ligand binding domain. The second RFP can be ArcEr. The first operator can be a tetracycline response element (TRE). The second operator can be an Arc operator (AO). The first ligand can be selected from the group consisting of tetracycline and doxycycline. The second ligand can be selected from the group consisting of estrogen, estradiol (E2), tamoxifen, 4-hydroxytamoxifen (OHT) and derivatives thereof. The cell genome can comprise a portion of an AAV Cap gene located 3’ of the AAV Rep gene.

[0038] The cells above can contain constructs allowing for the controlled expression of an AAV Rep gene and one or more non-AAV helper gene(s).

[0039] The inventions further provide methods for controlling the transcription of AAV Rep genes in a cell, wherein the methods comprise: maintaining a cell in a medium without an effective amount of a ligand of an activator (activator ligand) and with an effective amount of ligand of a repressor (repressor ligand), wherein the cell is capable of controlled transcription of an AAV Rep gene, and wherein the cell comprises a cell genome comprising (A) a promoter operably linked to an AAV Rep gene with reduced p19 promoter activity and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding an activator; (C) a second operator; and (D) a polynucleotide encoding a repressor; wherein transcription of the AAV Rep gene is inhibited in the absence of an effective amount if an activator ligand and the presence of an effective amount of a repressor ligand; and permitted in the presence of an effective amount of the activator ligand and the absence of an effective amount of the repressor ligand; and II. controlling the cell to transcribe the AAV Rep gene by maintaining the cell in a medium with an effective amount of the activator ligand and without an effective amount of the repressor ligand, and wherein the cell can express Rep 52 protein and Rep 40 protein. The second operator is operably linked and positioned 3’ with respect to the promoter and 5’ with respect to the AAV Rep gene. The activator binds to the first operator in the presence of the activator ligand to permit transcription of the AAV Rep gene. The cell may not express or may minimally express Rep 78 and Rep 68. The cell genome comprises a portion of an AAV Cap gene located 3’ of the AAV Rep gene. The activator can be a regulatory fusion protein (RFP), such as a reverse135975-75120 REGN 11751 tetracycline transactivator. The repressor protein can be a tetracycline repressor protein. The activator ligand and the repressor ligand can be selected from the group consisting of tetracycline and doxycycline.

[0040] The inventions further include methods for controlling the transcription of AAV Rep genes in a cell, wherein the methods comprise: I. maintaining a cell in a medium without an effective amount of a ligand of an activator (activator ligand) and with an effective amount of ligand of a repressor (repressor ligand), wherein the cell is capable of controlled transcription of an AAV Rep gene, wherein the cell comprises a cell genome comprising: (A) a promoter operably linked to AAV Rep gene with reduced p19 promoter activity and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding a first regulatory fusion protein (RFP), where the first RFP comprises: (1) a transcription activating domain fused to a first DNA binding domain; and (2) a ligand- binding domain; wherein the first ligand is capable of binding to the ligand-binding domain of the first RFP, and wherein the DNA binding domain of the first RFP is capable of binding to the operator positioned 5’ when in the presence of the first ligand; (C) a second operator; and (D) a polynucleotide encoding the second RFP that differs from the first RFP, wherein the second RFP comprises: (1) a DNA- binding domain; and (2) a ligand-binding domain; wherein the second ligand is capable of binding to the ligand-binding domain of the second RFP, and wherein the second RFP is capable of binding to the second operator in the presence of the second ligand; wherein transcription of the polynucleotide is inhibited in the absence of an effective amount of the first ligand and in the presence of an effective amount of the second ligand and is permitted in the presence of an effective amount of the first ligand and absence of an effective amount of the second ligand; and II. controlling the cell to transcribe the AAV Rep gene by maintaining the cell in a medium with an effective amount of the activator ligand and without an effective amount of the repressor ligand, and wherein the cell can express Rep 52 protein and Rep 40 protein. The second operator can be operably linked and positioned 3’ with respect to the promoter and 5’ with respect to the AAV Rep gene. The activator can bind to the first operator in the presence of the activator ligand to permit transcription of the AAV Rep gene. The cell may not express or may minimally express Rep 78 and Rep 68. The first RFP can a reverse tetracycline transactivator. The second135975-75120 REGN 11751 RFP may comprise (1) an Arc repressor DNA-binding domain; and (2) a ligand- binding domain; wherein the second ligand is capable of binding to the ligand- binding domain of the second RFP, and wherein the second RFP is capable of binding to the Arc operator in the presence of the second ligand; wherein transcription of the polynucleotide is inhibited in the absence of the first ligand and the presence of the second ligand. The ligand-binding domain of the second RFP may be the ligand binding domain of a steroid receptor, such as an estrogen receptor ligand binding domain. The second RFP can be ArcEr. The first operator can be a tetracycline response element (TRE). The second operator can be an Arc operator (AO). The first ligand can be selected from the group consisting of tetracycline and doxycycline. The second ligand can be selected from the group consisting of estrogen, estradiol (E2), tamoxifen, 4-hydroxytamoxifen (OHT) and derivatives thereof. The cell genome can comprise a portion of an AAV Cap gene located 3’ of the AAV Rep gene.

[0041] The methods above can be combined. For example, a method wherein the polynucleotide of interest is an AAV Rep gene can be practiced along with a method wherein the polynucleotide of interest is one or more non-AAV helper gene(s).

[0042] The inventions further provide polynucleotides comprising in the 5’ to 3’ direction (a) a tetracycline response element (TRE), (b) a first promoter, (c) an Arc operator (AO) or a tetracycline operator (TO), (d) a polynucleotide of interest, wherein the polynucleotide of interest is selected from the group consisting of: (i) a Rep gene fused to a portion of an AAV Cap gene to provide alternative forms Rep68 and Rep40, and (ii) a non-AAV helper gene. The (a) tetracycline response element (TRE), (b) the first promoter, (c) the Arc operator (AO) or tetracycline operator (TO), and (d) the polynucleotide of interest can be operably linked. The polynucleotide can comprise (iii) AO. The polynucleotide can comprise (iii) TO. The inventions also provide cells capable of controlled transcription of polynucleotides of interest, wherein the cell comprises a cell genome comprising (A) a promoter operably linked to the polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding an activator; (C) a second operator; and (D) a135975-75120 REGN 11751 polynucleotide encoding a repressor, wherein transcription of the polynucleotide of interest is inhibited in the absence of an effective amount if an activator ligand and the presence of an effective amount of a repressor ligand; and permitted in the presence of an effective amount of the activator ligand and the absence of an effective amount of the repressor ligand, and wherein the polynucleotide of interest is selected from the group consisting of: (i) a Rep gene fused to a portion of an AAV Cap gene to provide alternative forms Rep68 and Rep40, and (ii) a non-AAV helper genes. The first operator can be a tetracycline response element (TRE). The second operator can be a tetracycline operator (TO).

[0043] The invention further comprises cell capable of controlled transcription of polynucleotides of interest, wherein the cell comprises a cell genome comprising: (A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding a first regulatory fusion protein (RFP), where the first RFP comprises: (1) a transcription activating domain fused to a first DNA binding domain; and (2) a ligand-binding domain; wherein the first ligand is capable of binding to the ligand-binding domain of the first RFP, and wherein the DNA binding domain of the first RFP is capable of binding to the operator positioned 5’ when in the presence of the first ligand; (C) a second operator; and (D) a polynucleotide encoding the second RFP that differs from the first RFP, wherein the second RFP comprises: (1) a DNA- binding domain; and (2) a ligand-binding domain; wherein the second ligand is capable of binding to the ligand-binding domain of the second RFP, and wherein the second RFP is capable of binding to the second operator in the presence of the second ligand; wherein transcription of the polynucleotide of interest is inhibited in the absence of an effective amount of the first ligand and in the presence of an effective amount of the second ligand and is permitted in the presence of an effective amount of the first ligand and absence of an effective amount of the second ligand, wherein the polynucleotide of interest is selected from the group consisting of : (i) a Rep gene fused to a portion of an AAV Cap gene to provide alternative forms Rep68 and Rep40, and (ii) a non-AAV helper genes. The first operator can be a tetracycline response element (TRE). The second operator can be an Arc operator (AO).

[0044] The inventions further comprise methods for controlling the transcription of polynucleotides of interest, wherein the method comprises: I. maintaining a cell in a135975-75120 REGN 11751 medium without an effective amount of a ligand of an activator (activator ligand) and with an effective amount of ligand of a repressor (repressor ligand), wherein the cell is capable of controlled transcription of an AAV Rep gene, and wherein the cell comprises a cell genome comprising: (A) a promoter operably linked to the polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding an activator; (C) a second operator; and (D) a polynucleotide encoding a repressor, wherein transcription of the polynucleotide of interest is inhibited in the absence of an effective amount if an activator ligand and the presence of an effective amount of a repressor ligand; and permitted in the presence of an effective amount of the activator ligand and the absence of an effective amount of the repressor ligand, and wherein the polynucleotide of interest is selected from the group consisting of : (i) a Rep gene fused to a portion of an AAV Cap gene to provide alternative forms Rep68 and Rep40, and (ii) one non-AAV helper genes. The methods can further comprise the step of II. controlling the cell to transcribe the polynucleotide of interest by maintaining the cell in a medium with an effective amount of the activator ligand and without an effective amount of the repressor ligand. The first operator can be a tetracycline response element (TRE). The second operator can be a tetracycline operator (TO).

[0045] The inventions also comprise methods for controlling the transcription of polynucleotides of interest in a cell, wherein the method comprises: I. maintaining a cell in a medium without an effective amount of a ligand of an activator (activator ligand) and with an effective amount of ligand of a repressor (repressor ligand), wherein the cell is capable of controlled transcription of an AAV Rep gene, and wherein the cell comprises a cell genome comprising: (A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding a first regulatory fusion protein (RFP), where the first RFP comprises: (1) a transcription activating domain fused to a first DNA binding domain; and (2) a ligand-binding domain; wherein the first ligand is capable of binding to the ligand-binding domain of the first RFP, and wherein the DNA binding domain of the first RFP is capable of binding to the operator positioned 5’ when in the presence of the first ligand; (C) a second operator; and (D) a polynucleotide encoding the second RFP that differs135975-75120 REGN 11751 from the first RFP, wherein the second RFP comprises: (1) a DNA-binding domain; and (2) a ligand-binding domain; wherein the second ligand is capable of binding to the ligand-binding domain of the second RFP, and wherein the second RFP is capable of binding to the second operator in the presence of the second ligand; wherein transcription of the polynucleotide of interest is inhibited in the absence of an effective amount of the first ligand and in the presence of an effective amount of the second ligand and is permitted in the presence of an effective amount of the first ligand and absence of an effective amount of the second ligand, wherein the polynucleotide of interest is selected from the group consisting of :(i) a Rep gene fused to a portion of an AAV Cap gene to provide alternative forms Rep68 and Rep40, and (ii) a non-AAV helper gene. The methods can further comprise the step of II. controlling the cell to transcribe the AAV Rep gene by maintaining the cell in a medium with an effective amount of the activator ligand and without an effective amount of the repressor ligand. The first operator can be a tetracycline response element (TRE). The second operator can be an Arc operator (AO).

[0046] The inventions further comprise cells capable of controlled transcription of a polynucleotides of interest, wherein the cell comprises a cell genome comprising I. a first DNA construct comprising (A) a promoter operably linked to the polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding an activator; (C) a second operator; and (D) a polynucleotide encoding a repressor, wherein transcription of the polynucleotide of interest is inhibited in the absence of an effective amount if an activator ligand and the presence of an effective amount of a repressor ligand; and permitted in the presence of an effective amount of the activator ligand and the absence of an effective amount of the repressor ligand, and wherein the polynucleotide of interest is a Rep gene fused to a portion of an AAV Cap gene to provide alternative forms Rep68 and Rep40; and II. a second DNA construct comprising (A) a promoter operably linked to the polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding an activator; (C) a second operator; and (D) a polynucleotide encoding a repressor; wherein transcription of the polynucleotide of interest is inhibited in the absence of an effective amount if an activator ligand and the presence of an effective amount of a repressor ligand; and permitted in the135975-75120 REGN 11751 presence of an effective amount of the activator ligand and the absence of an effective amount of the repressor ligand, and wherein the polynucleotide of interest comprises a non-AAV helper gene. The first operator can be a tetracycline response element (TRE). The second operator can be a tetracycline operator (TO). The second DNA construct comprises more than one non-AAV helper gene. The non- AAV helper gene can be selected from the group consisting of an adenovirus gene, a herpesvirus gene, a human papilloma virus gene, a bocavirus gene and a baculovirus gene. An adenovirus gene can be selected from the group consisting of E4 orf6-VA, E2A ORF 2, E2 ORF4, E2A DBP, E2A 22K, E2A 33K, and E2A 33K native. The polynucleotide of interest of the second DNA construct comprises in the 5’ to 3’ direction one or more selected from the group consisting of: (A) adenovirus E2A DBP, a 2A sequence, adenovirus E2A ORF2, a 2A sequence, and adenovirus E2A ORF4; (B) adenovirus E2A DBP, a 2A sequence, adenovirus E2A ORF2, a 2A sequence, adenovirus E2A ORF4, a 2A sequence, adenovirus ORF6 and VA; and (C) adenovirus E4 orf6, a 2A sequence, adenovirus E2A DBP, a 2A sequence, and adenovirus E2A 33K. Adenovirus 22K and 33K proteins are discussed in Adesero et al., Human Gene Ther.35, nos.1 and 2 (2024); Su et al., Scientific Reps.13: 21670 (2023).

[0047] The inventions further provide cells capable of controlled transcription of polynucleotides of interest, wherein the cell comprises a cell genome comprising I. a first DNA construct comprising (A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding a first regulatory fusion protein (RFP), where the first RFP comprises: (1) a transcription activating domain fused to a first DNA binding domain; and (2) a ligand-binding domain; wherein the first ligand is capable of binding to the ligand-binding domain of the first RFP, and wherein the DNA binding domain of the first RFP is capable of binding to the operator positioned 5’ when in the presence of the first ligand; (C) a second operator; and (D) a polynucleotide encoding the second RFP that differs from the first RFP, wherein the second RFP comprises: (1) a DNA-binding domain; and (2) a ligand- binding domain; wherein the second ligand is capable of binding to the ligand- binding domain of the second RFP, and wherein the second RFP is capable of binding to the second operator in the presence of the second ligand; wherein135975-75120 REGN 11751 transcription of the polynucleotide of interest is inhibited in the absence of an effective amount of the first ligand and in the presence of an effective amount of the second ligand and is permitted in the presence of an effective amount of the first ligand and absence of an effective amount of the second ligand, wherein the polynucleotide of interest is a Rep gene fused to a portion of an AAV Cap gene to provide alternative forms Rep68 and Rep40; and II. a second DNA construct comprising (A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding a first regulatory fusion protein (RFP), where the first RFP comprises: (1) a transcription activating domain fused to a first DNA binding domain; and (2) a ligand-binding domain; wherein the first ligand is capable of binding to the ligand-binding domain of the first RFP, and wherein the DNA binding domain of the first RFP is capable of binding to the operator positioned 5’ when in the presence of the first ligand; (C) a second operator; and (D) a polynucleotide encoding the second RFP that differs from the first RFP, wherein the second RFP comprises: (1) a DNA-binding domain; and (2) a ligand-binding domain; wherein the second ligand is capable of binding to the ligand-binding domain of the second RFP, and wherein the second RFP is capable of binding to the second operator in the presence of the second ligand; wherein transcription of the polynucleotide of interest is inhibited in the absence of an effective amount of the first ligand and in the presence of an effective amount of the second ligand and is permitted in the presence of an effective amount of the first ligand and absence of an effective amount of the second ligand, wherein the polynucleotide of interest comprises a non-AAV helper gene. The first operator can be a tetracycline response element (TRE). The second operator can be an Arc tetracycline operator (AO). The second DNA construct can comprise more than one non-AAV helper gene.

[0048] The non-AAV helper gene can be selected from the group consisting of an adenovirus gene, a herpesvirus gene, a human papilloma virus gene, a bocavirus gene and a baculovirus gene. An adenovirus gene can be selected from the group consisting of E4 orf6-VA, E2A ORF 2, E2 ORF4, E2A DBP, E2A 22K, E2A 33K, and E2A 33K native. The polynucleotide of interest of the second DNA construct can comprise in the 5’ to 3’ direction one or more selected from the group consisting of: (A) adenovirus E2A DBP, a 2A sequence, adenovirus E2A ORF2, a 2A sequence,135975-75120 REGN 11751 and adenovirus E2A ORF4; (B) adenovirus E2A DBP, a 2A sequence, adenovirus E2A ORF2, a 2A sequence, adenovirus E2A ORF4, a 2A sequence, adenovirus ORF6 and VA.

[0049] The inventions further provide polynucleotides comprising a Cap gene, wherein the polynucleotide is selected from the group consisting of A. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box and one or more operators, such as 2 to 5 Lac operators (LacO x 2-5) to control an AAV Cap gene, and (iii) a Lac repressor (LacI), wherein the LacI is under the control of an mCMV promoter, and wherein LacI is operably linked to LacO x 2-5; B. a polynucleotide comprising in the 5’ to 3’ direction (i) one or more operators, such as 2 to 5 Lac operators (LacO x 2-5), (ii) an hCMV promoter, (iii) a TATA box to control an AAV Cap gene, (iv) an IRES, (v) a polynucleotide encoding AAV rep52, (vi) a first polyadenylation signal (pA), (vii) a Lac repressor (LacI), and (viii) a second pA , wherein the LacI is under the control of an mCMV promoter, and wherein LacI is operably linked to LacO x 2-5; C. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) 1 to 5 operators, (iv) an AAV Cap gene, iv) a 2A polynucleotide sequence, (vi) a polynucleotide encoding AAV rep52, (viii) and a first polyadenylation signal (pA), (ix) a Lac repressor (LacI), and ix) a second pA, wherein the LacI is under the control of an mCMV promoter, and optionally comprise 1 to 2 or more ArcO, 1 to 2 or more TO or 2 to 5 or more LacO: D. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a first TATA box, (iii) 1 to 5 operators, (iv) an AAV Cap gene, (v) a first polyadenylation signal (pA), (vi) a second hCMV promoter, (vii) a second TATA box, (viii) a polynucleotide encoding AAV rep52, (ix) a second pA, (x) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and (x) a second pA., and optionally comprise 1 to 2 or more ArcO, 1 to 2 or more TO or 2 to 5 or more LacO: E. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) 2 to 5 Lac operators (LacO x 2-5), (iv) an AAV Cap gene, (v) an IRES, (vi) a polynucleotide encoding an AAV rep52, (vii) a 2A polynucleotide sequence, (viii) a polynucleotide encoding VP3-SpyTag fusion protein, (ix) a first pA, (x) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and135975-75120 REGN 11751 (xi) a second pA, and optionally comprise 1 to 2 or more ArcO or 1 to 2 or more TO instead of 2 to 5 LacO; F. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) 2 to 5 Lac operators (LacO x 2-5), (iv) an AAV Cap gene, (v) an IRES, (vi) a polynucleotide encoding VP3-SpyTag fusion protein, (vii) a 2A polynucleotide sequence, (viii) a polynucleotide encoding an AAV rep52, (ix) a first pA, (x) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and (xi) a second pA, and optionally comprise 1 to 2 or more ArcO or 1 to 2 or more TO instead of 2 to 5 LacO; and G. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) a first set of LacO x 2-5, (iv) an AAV Cap gene, (v) an IRES, (vi) a polynucleotide encoding VP3-SpyTag fusion protein, (vii) a first pA, (viii) a second hCMV promoter, (ix) a second TATA box, (x) a second set of LacO x 2-5, (xi) a polynucleotide encoding an AAV rep52, (xii) a second pA, (xiii) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and (xiv) and a third pA, and optionally comprise 1 to 2 or more ArcO or 1 to 2 or more TO instead of 2 to 5 LacO.

[0050] The inventions also provide cells comprising a polynucleotide comprising a Cap gene, wherein the polynucleotide is selected from the group consisting of A. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box and one or more operators, such as 2 to 5 Lac operators (LacO x 2-5) to control an AAV Cap gene, and (iii) a Lac repressor (LacI), wherein the LacI is under the control of an mCMV promoter, and wherein LacI is operably linked to LacO x 2-5; B. a polynucleotide comprising in the 5’ to 3’ direction (i) one or more operators, such as 2 to 5 Lac operators (LacO x 2-5), (ii) an hCMV promoter, (iii) a TATA box to control an AAV Cap gene, (iv) an IRES, (v) a polynucleotide encoding AAV rep52, (vi) a first polyadenylation signal (pA), (vii) a Lac repressor (LacI), and (viii) a second pA , wherein the LacI is under the control of an mCMV promoter, and wherein LacI is operably linked to LacO x 2-5; C. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) 1 to 5 operators, (iv) an AAV Cap gene, iv) a 2A polynucleotide sequence, (vi) a polynucleotide encoding AAV rep52, (viii) and a first polyadenylation135975-75120 REGN 11751 signal (pA), (ix) a Lac repressor (LacI), and ix) a second pA, wherein the LacI is under the control of an mCMV promoter, and optionally comprise 1 to 2 or more ArcO, 1 to 2 or more TO or 2 to 5 or more LacO: D. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a first TATA box, (iii) 1 to 5 operators, (iv) an AAV Cap gene, (v) a first polyadenylation signal (pA), (vi) a second hCMV promoter, (vii) a second TATA box, (viii) a polynucleotide encoding AAV rep52, (ix) a second pA, (x) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and (x) a second pA., and optionally comprise 1 to 2 or more ArcO, 1 to 2 pr more TO or 2 to 5 or more LacO: E. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) 2 to 5 Lac operators (LacO x 2-5), (iv) an AAV Cap gene, (v) an IRES, (vi) a polynucleotide encoding an AAV rep52, (vii) a 2A polynucleotide sequence, (viii) a polynucleotide encoding VP3-SpyTag fusion protein, (ix) a first pA, (x) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and (xi) a second pA, and optionally comprise 1 to 2 or more ArcO or 1 to 2 or more TO instead of 2 to 5 LacO; F. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) 2 to 5 Lac operators (LacO x 2-5), (iv) an AAV Cap gene, (v) an IRES, (vi) a polynucleotide encoding VP3-SpyTag fusion protein, (vii) a 2A polynucleotide sequence, (viii) a polynucleotide encoding an AAV rep52, (ix) a first pA, (x) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and (xi) a second pA, and optionally comprise 1 to 2 or more ArcO or 1 to 2 or more TO instead of 2 to 5 LacO; and G. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) a first set of LacO x 2-5, (iv) an AAV Cap gene, (v) an IRES, (vi) a polynucleotide encoding VP3-SpyTag fusion protein, (vii) a first pA, (viii) a second hCMV promoter, (ix) a second TATA box, (x) a second set of LacO x 2-5, (xi) a polynucleotide encoding an AAV rep52, (xii) a second pA, (xiii) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and (xiv) and a third pA, and optionally comprise 1 to 2 or more ArcO or 1 to 2 or more TO instead of 2 to 5 LacO.135975-75120 REGN 11751 These Cap gene constructs can be used with any of the Rep gene constructs and non-AAV helper gene constructs disclosed herein. The inventions also provide cell genomes as set forth in this summary

[0051] The inventions further provide methods for controlling the transcription of a polynucleotide in a cell using any of the cells and cell genomes as set forth in this summary.

[0052] The inventions also provide polynucleotides comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box and one or more operators, such as 2 to 5 Lac operators (LacO x 2-5) to control an AAV Cap gene, and (iii) a Lac repressor (LacI), wherein the LacI is under the control of an mCMV promoter, and wherein LacI is operably linked to LacO x 2-5.

[0053] The inventions also provide polynucleotides comprising in the 5’ to 3’ direction (i) one or more operators, such as 2 to 5 Lac operators (LacO x 2-5), (ii) an hCMV promoter, (iii) a TATA box to control an AAV Cap gene, (iv) an IRES, (v) a polynucleotide encoding AAV rep52, (vi) a first polyadenylation signal (pA), (vii) a Lac repressor (LacI), and (viii) a second pA , wherein the LacI is under the control of an mCMV promoter, and wherein LacI is operably linked to LacO x 2-5.

[0054] The inventions also provide polynucleotides comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) 1 to 5 operators, (iv) an AAV Cap gene, iv) a 2A polynucleotide sequence, (vi) a polynucleotide encoding AAV rep52, (viii) and a first polyadenylation signal (pA), (ix) a Lac repressor (LacI), and ix) a second pA, wherein the LacI is under the control of an mCMV promoter, and wherein LacI is operably linked to LacO x 2-5. For example, it is possible to use 1 to 2 or more ArcO, 1 to 2 or more TO or 2 to 5 or more LacO.

[0055] The inventions also provide polynucleotides comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a first TATA box, (iii) 1 to 5 operators, (iv) an AAV Cap gene, (v) a first polyadenylation signal (pA), (vi) a second hCMV promoter, (vii) a second TATA box, (viii) a polynucleotide encoding AAV rep52, (ix) a second pA, (x) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, wherein LacI is operably linked to LacO x 2-5, and (x) a second pA. For135975-75120 REGN 11751 example, it is possible to use 1 to 2 or more ArcO, 1 to 2 TO ore more or 2 to 5 or more LacO.

[0056] The inventions also provide polynucleotides comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) 2 to 5 Lac operators (LacO x 2- 5), (iv) an AAV Cap gene, (v) an IRES, (vi) a polynucleotide encoding an AAV rep52, (vii) a 2A polynucleotide sequence, (viii) a polynucleotide encoding VP3-SpyTag fusion protein (a type of recombinant capsid protein), (ix) a first pA, (x) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and (xi) a second pA. For example, it is also possible to use 1 to 2 or more ArcO or 1 to 2 or more TO instead of 2 to 5 LacO. SpyTag is a peptide tag and member of a specific binding pair. SpyTag can be covalently bound to SpyCatcher by an isopeptide bond.

[0057] The inventions also provide polynucleotides comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) 2 to 5 Lac operators (LacO x 2- 5), (iv) an AAV Cap gene, (v) an IRES, (vi) a polynucleotide encoding VP3-SpyTag fusion protein (a type of recombinant capsid protein), (vii) a 2A polynucleotide sequence, (viii) a polynucleotide encoding an AAV rep52, (ix) a first pA, (x) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and (xi) a second pA. For example, it is also possible to use 1 to 2 or more ArcO or 1 to 2 or more TO instead of 2 to 5 LacO.

[0058] The inventions also provide polynucleotides comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) a first set of LacO x 2-5, (iv) an AAV Cap gene, (v) an IRES, (vi) a polynucleotide encoding VP3-SpyTag fusion protein (a type of recombinant capsid protein), (vii) a first pA, (viii) a second hCMV promoter, (ix) a second TATA box, (x) a second set of LacO x 2-5, (xi) a polynucleotide encoding an AAV rep52, (xii) a second pA, (xiii) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and (xiv) and a third pA. For example, it is also possible to use 1 to 2 or more ArcO or 1 to 2 or more TO instead of 2 to 5 LacO.

[0059] The inventions provide adeno-associated virus (AAV) plasmids including, in a 5’ to 3’ orientation: (a) a tetracycline response element (TRE), (b) a first promoter, (c) an Arc operator (AO) or a tetracycline operator (TO), (d) a135975-75120 REGN 11751 polynucleotide encoding an AAV rep protein, and (e) a polynucleotide encoding a portion of an AAV cap protein.

[0060] The inventions also provide adeno-associated virus (AAV) plasmids comprising, in a 5’ to 3’ orientation: (a) a tetracycline response element (TRE), (b) a first promoter, (c) an Arc operator (AO) or a tetracycline operator (TO), (d) a polynucleotide encoding a AAV rep polypeptide, and (e) a polynucleotide encoding a portion of an AAV cap polypeptide. The AAV promoter can be an AAV p19 promoter.

[0061] The inventions also provide adeno-associated virus (AAV) plasmids comprising, in a 5’ to 3’ orientation: (a) a promoter, (b) a lactose operator, (c) a Kozak consensus sequence, and (d) a Cap gene.

[0062] The inventions also provide adeno-associated virus (AAV) plasmids comprising, in a 5’ to 3’ orientation: (a) a promoter, (b) lactose operator (LacO), (b) a Kozak consensus sequence, (d) a polynucleotide encoding a portion of an AAV Cap polypeptide, (e) a cap gene, (f) an internal ribosome entry site (IRES), and (g) a polynucleotide sequence encoding rep52 polypeptide.

[0063] The AAV rep protein can be an AAV2 rep protein. The AAV promoter is an AAV p19 promoter, wherein the p19 promoter can have a p19 promoter can be associated with a non-ATG start mutation, such as an ACG, CTG, GTG, TTG mutation and the like. The (a) tetracycline response element (TRE), (b) the first promoter, (c) Arc operator (AO) or a tetracycline operator (TO), and (d) the AAV rep gene can be operably linked. The first promoter can be a CMV promoter, and the polynucleotide can be encoding an AAV rep protein such as rep78 or rep52. The rep78 can have the sequence located in SEQ ID NOS: 2, 3, 9, 11, 13, 15, 17, 19, 22, 24, 26, or 28, for example, and the rep52 can have the sequence of SEQ ID NO: 4, for example.

[0064] The inventions also provide mammalian cell genomes comprising any of the rep and cap plasmid disclosed herein. The mammalian cell genome can be selected from the group consisting of HEK-293, Hela, CHO, BHK, or human amniotic cell. The mammalian cell genome can further comprise: a polynucleotide encoding AAV Rep; a polynucleotide encoding Ad E1A; a polynucleotide encoding Ad E1B; a polynucleotide encoding Ad E2A, E2A orf, E2A ORF4, E2A 22K, E2A 33K, E2A 33K135975-75120 REGN 11751 native, or E2A DBP; a polynucleotide encoding Ad E4 or E4 orf 6; a polynucleotide encoding VA RNA; and a polynucleotide encoding AAV ITRs and a protein of interest.

[0065] The inventions also provide cell cultures comprising any of the mammalian cell genome disclosed herein. The cell culture can be cultured in a suitable culture medium, which allows the cell culture to produce AAV VP1, VP2 and VP3 proteins.

[0066] The inventions also provide methods of manufacturing a recombinant adeno-associated virus comprising the steps of: (I) introducing into a mammalian cell genome integrated with adenovirus (Ad) helper genes E1A and E1B: (a) a plasmid of claim comprising, in a 5’ to 3’ orientation: (i) a tetracycline response element (TRE), (ii) a first promoter, (iii) an Arc operator (AO) or a tetracycline operator (TO), (iv) a polynucleotide encoding an AAV rep protein, and (v) a polynucleotide encoding a portion of an AAV cap protein, (b) a helper plasmid comprising one or more helper genes selected from Ad E4 orf6, E2A and VA RNA, and (c) a transgene plasmid comprising a transgene of interest flanked by inverse terminal repeats (ITRs),wherein the introducing step is under conditions that permit formation of the recombinant adeno-associated virus; and (II) collecting the recombinant adeno- associated virus.

[0067] The introducing step can be under conditions that permit formation of the recombinant adeno-associated virus. The methods further can include the step of collecting the recombinant adeno-associated virus. The transgene plasmid further can include a promoter operably linked to the transgene of interest. The mammalian cell can be a HEK-293, Hela, CHO, BHK, or human amniotic cells. A transgene of interest includes any gene that is desired to be expressed and can fit within an AAV capsid. More than one transgene of interest is possible if the totality of genes is of a combined size that can fit within an AAV capsid.

[0068] The viral yield can be at least 1 x 1010viral genomes (vg) / ml (for example, 1 x 1010vg / mL, 2 x 1010vg / mL, 3 x 1010vg / mL, 4 x 1010vg / mL, 5 x 1010vg / mL, or greater, such as such as 1 x 1010vg / mL to 5 x 1010vg / mL, 1 x 1010vg / mL to 1 x 1011vg / mL, 1 x 1010vg / mL to 5 x 1011vg / mL, or 1 x 1010vg / mL to 1.5 x 1012vg / mL, or greater, as measured using ddPCR of clarified lysate. An example set forth below showed a viral yield of 1.2 x 1012vg / ml.135975-75120 REGN 11751

[0069] The inventions also provide methods of manufacturing a recombinant adeno-associated virus comprising the steps of: (I) introducing into a mammalian cell genome integrated with adenovirus (Ad) helper genes E1A and E1B: (a) a plasmid of claim comprising, in a 5’ to 3’ orientation: (i) a tetracycline response element (TRE), (ii) a first promoter, (iii) an Arc operator (AO) or a tetracycline operator (TO), (iv) a polynucleotide encoding an AAV rep protein or a AAV rep polypeptide, and (v) a polynucleotide encoding a portion of an AAV cap protein.,(b) a helper plasmid comprising one or more helper genes selected from Ad E4orf6, E2A and VA RNA, and (c) a transgene plasmid comprising a transgene of interest flanked by inverse terminal repeats (ITRs), wherein the introducing step is under conditions that permit formation of the recombinant adeno-associated virus; and (II) collecting the recombinant adeno-associated virus.

[0070] The inventions also provide methods of manufacturing a recombinant adeno-associated virus comprising the steps of: (I) introducing into a mammalian cell genome integrated with adenovirus (Ad) helper genes E1A and E1B:(a) a plasmid of claim comprising, in a 5’ to 3’ orientation: (i) a tetracycline response element (TRE), (ii) a first promoter, (iii) lactose operator (LacO), (iv) a Kozak consensus sequence (v) a cap gene, (vi) an internal ribosome entry site (IRES), and (vii) a polynucleotide sequence encoding rep52 polypeptide, (b) a helper plasmid comprising one or more helper genes selected from Ad E4orf6, E2A and VA RNA, and (c) a transgene plasmid comprising a transgene of interest flanked by inverse terminal repeats (ITRs), wherein the introducing step is under conditions that permit formation of the recombinant adeno-associated virus; and (II) collecting the recombinant adeno- associated virus.

[0071] The inventions also provide methods of manufacturing recombinant adeno-associated virus by introducing into a mammalian cell: (a) a plasmid including, in a 5’ to 3’ orientation: (i) a polynucleotide encoding an AAV rep protein; (ii) a tetracycline response element (TRE); (iii) an AAV promoter (such as p40 or p41); and (iv) a polynucleotide encoding an AAV capsid protein; (b) a helper plasmid containing one or more helper genes selected from E4, E2a and VA; and (c) a transgene plasmid including a transgene of interest flanked by inverse terminal repeats. The introducing step can be under conditions that permit formation of the recombinant adeno-associated virus. The methods further can include the step of135975-75120 REGN 11751 collecting the recombinant adeno-associated virus. The polynucleotide encoding an AAV capsid protein can be a polynucleotide encoding an AAV1, AAV2, AAV2quad(Y-F), AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, rh10, rh39, rh43, rh74, Anc80, Anc80L65, DJ, DJ / 8, DJ / 9, 7m8, PHP.B, PHP.eB, or PHP.S capsid protein. The tetracycline response element (TRE), as described herein. The transgene plasmid of the inventions can further include a promoter operably linked to the transgene of interest. The mammalian cell can be a HEK-293, Hela, CHO, BHK, or human amniotic cells. A transgene of interest includes any gene / polynucleotide that is desired to be expressed and can fit within an AAV capsid. More than one transgene of interest flanked by ITRs is possible if the totality of genes is of a combined size that can fit within an AAV capsid.

[0072] The inventions further provide cell genomes comprising the plasmids according to the inventions, and cell cultures comprising the cells according to the inventions.

[0073] The recombinant AAVs can have detargeting mutations, and / or can be retargeted using covalent approaches, such as the use of specific binding pairs, for example Spy Tag and Spy Catcher. Alternatively, non-covalent approaches for retargeting can be employed, such as the use of bispecific antibodies that can bind to the AAV capsid and a target antigen, receptor and / or ligand on a cell.

[0074] There also are provided master cell banks, working cell banks, developmental cell banks, cell cultures, seed cultures, and production cultures comprising cells according to the inventions, as well as bioreactors and fermenters containing cell cultures comprising cells according to the inventions described herein and cells comprising cell genomes containing polynucleotides of interest. Brief Description of the Figures

[0075] FIGURE 1 depicts induction of the transcription of a polynucleotide of interest) in the presence of dox and absence of OHT. The left side depicts the repressed state where dox is absent and OHT is present. The right side depicts the induced state where dox is present and OHT is absent. The figure shows an example of a tandem arrangement of a Tet Response Element (TRE) and an Arc135975-75120 REGN 11751 operator (AO). A TATA box is schematically depicted 5’ to AO and 3’ to the CMVmin promoter.

[0076] FIGURE 2 depicts induction of the transcription of a polynucleotide of interest under the control of rtTA and TetR. A Tet Response Element (TRE) and a Tetracycline operator (TetO or TO) can be in a tandem arrangement. Induction of the transcription of the polynucleotide of interest occurs in the presence of dox. A TATA box is schematically depicted 5’ to TO and 3’ to the CMVmin promoter.

[0077] FIGURE 3 depicts representation of polynucleotide constructs for the systems of FIGURES 1 and 2. The top construct show the ArcER 5’ of a selection marker gene, and IRES and a color reporter gene. CMV and AO control the expression of rtTA polynucleotide. The bottom construct depicts a TRE and AO or TO controlling expression of a rep polynucleotide, which is an example of a polynucleotide of interest.

[0078] FIGURE 4 is representation of FIGURES 1 and 2 as depicted in FIGURES 3, 5 and 6.

[0079] FIGURE 5 depicts a recombinant Rep and partial Cap expression cassette. Large Reps (Rep 78, Rep 68) are expressed. Small Reps (Rep 52, Rep 40) are not expressed or only minimally expressed due to ATG to ACG mutation near the p19 promoter of the Rep gene, preferably full length, which is a polynucleotide of interest.

[0080] FIGURE 6 depicts another recombinant Rep and partial Cap expression cassette. Small Reps (Rep 52, Rep 40) are expressed. The Rep gene has reduced p19 promoter activity (due to a non-ATG start codon) and preferably does not express or only minimally express Rep 78 and 68 (these also are a polynucleotides of interest).

[0081] FIGURES 7A and 7B concern VP1, VP2 and VP3 protein expression. Figure 7A is a schematic depiction of recombinant Cap expression cassette showing a polynucleotide in the 5’ to 3’ direction with (i) an hCMV promoter, (ii) a lactose operator or Arc operator, (iii) a Kozak consensus sequence, and (iv) a representative Cap gene (here, Cap is from AAV5). Figure 7B depicts HEK293 expression of Cap gene and achieves a desired protein VP ratio of about 1:1:10 for VP1:VP2:VP3. VP1135975-75120 REGN 11751 is lower due to ACG initiation in combination with Kozak sequence, VP2 is lower due to ACG initiations, and VP3 is normal due to use of ATG initiation.

[0082] FIGURE 8 depicts a recombinant Rep and Cap expression cassette that adds an IRES and a Rep gene to the construct of FIGURE 7A. Cap is expressed from an hCMV LacO promoter; regulated by LacR (+IPTG induces expression). An approximated correct ratio is achieved by ACG initiation of VP1 in combination with Kozak sequence, and native ACG of VP2 and ATG of VP3. Since Rep 52 is engaged in packaging and can be low in the initial rep expressing cells, IRES-Rep52 can be added downstream of cap expression cassette.

[0083] FIGURE 9A depicts a cap expression cassette containing a Kozak-ACG- Cap system a representative Cap gene (here, Cap is from AAV5). Low VP1 is achieved by alternative start codons and not by splicing. FIGURE 9B depicts a cap expression cassette containing 2-5 Lac operators (LacO x 2-5) or Arc Operator (ArcO or AO), a Kozak-ACG-Cap system having a representative Cap gene (here, Cap is from AAV5). Low VP1 is achieved by alternative start codons and not by splicing. ACG is a weaker alternative to ATG initiation codon. Translation can start from ACG, but this event is less efficient, so the amount of protein generated from ACG start can be lower than from the ATG. Kozak sequence enhances translation initiation and can be included to help ACG initiation.

[0084] FIGURE 10 illustrates a cap expression cassette containing an IRES-Cap- IRES-Cap (ICIC). The schematic diagram of a polynucleotide comprising a promoter, an intron, two internal ribosome entry sites, two polynucleotides encoding AAV and a representative Cap protein (here, Cap is from AAV5). See WO 2023 / 069926 A1.

[0085] FIGURE 11 illustrates a cap expression cassette containing an IRES-Cap- IRES-Cap (ICIC). The schematic diagram of a polynucleotide comprising a CMV promoter, a Lac operator (LacO) or an Arc Operator (ArcO), a CMV intron, two internal ribosome entry sites, two polynucleotides encoding a representative Cap protein (here, Cap is from AAV5).

[0086] FIGURE 12A is a schematic depiction in a 5’ to 3’ direction of an hCMV promoter, a TATA box and 2 to 5 Lac operators (LacO x 2-5) to control a Cap gene followed by an mCMV promoter controlling a Lac repressor (LacI). Lac repressor135975-75120 REGN 11751 protein is schematically depicted interacting with LacO x 2-5. In the presence (+) of IPTG (isopropyl β-D-1 thiogalactopyranoside), LacI will not bind to LacO. FIGURE 12B is a schematic depiction in a 5’ to 3’ direction of an hCMV promoter, a TATA box and an Arc operator to control a Cap gene followed by a mCMV promoter controlling an ArcER repressor (ArcER). ArcER repressor protein is schematically depicted interacting with an ArcO. In the absence (-) of OHT, ArcER no longer binds to AO.

[0087] FIGURE 13A is a schematic depiction in a 5’ to 3’ direction of an hCMV promoter, a TATA box, 2 to 5 Lac operators (LacO x 2-5), a Cap polynucleotide followed by an IRES, a polynucleotide encoding rep52, a polyadenylation signal (pA), a LacI polynucleotide and a pA under control of an mCMV promoter. FIGURE 13B is a schematic depiction in a 5’ to 3’ direction of an hCMV promoter, a TATA box, 2 to 5 Lac operators (LacO x 2-5), a Cap polynucleotide followed by an IRES, a polynucleotide encoding rep52, a polyadenylation signal (pA), and a LacI polynucleotide under control of an mCMV promoter. FIGURE 13C is a schematic depiction in a 5’ to 3’ direction of an hCMV promoter, a TATA box, an Arc operator, a Cap polynucleotide followed by an IRES, and a polynucleotide encoding rep52, a pA and an ArcEr polynucleotide under control of an mCMV promoter.

[0088] FIGURE 14A is a schematic depiction in a 5’ to 3’ direction of an hCMV promoter, TATA box, 2 to 5 Lac operators (LacO x 2-5), to control a Cap gene, followed by 2A (a small peptide allowing multiple proteins to be expressed from one open reading frame), a polynucleotide encoding rep52 and a polyadenylation signal (pA), followed by a LacI polynucleotide and a pA under control of an mCMV promoter. FIGURE 14B is a schematic depiction in a 5’ to 3’ direction an hCMV promoter, TATA box, and an AO to control a Cap gene, followed by 2A, a polynucleotide encoding rep52 and a polyadenylation signal (pA), followed by a ArcEr polynucleotide and a pA under control of an mCMV promoter.

[0089] FIGURE 15A is a schematic depiction in a 5’ to 3’ direction of an hCMV promoter, TATA box, 2 to 5 Lac operators (LacO x 2-5), to control a Cap gene, followed by a polyadenylation signal (pA). Next, in a 5’ to 3’ direction there is an hCMV promoter, TATA box, 2 to 5 Lac operators (LacO x 2-5), to control a polynucleotide encoding rep52 and a pA, followed by a LacI polynucleotide and a pA under control of an mCMV promoter. FIGURE 15B is a schematic depiction in a 5’135975-75120 REGN 11751 to 3’ direction of an hCMV promoter, TATA box and an AO, to control a Cap gene, followed by a polyadenylation signal (pA). Next, in a 5’ to 3’ direction of an hCMV promoter, TATA box, and an AO to control a polynucleotide encoding rep52 and a pA., followed by an mCMV promoter controlling ArcEr and a pA.

[0090] FIGURE 16A is a schematic depiction in a 5’ to 3’ direction of an hCMV promoter, a TATA box and 2 to 5 Lac operators (LacO x 2-5) to control a Cap polynucleotide, followed by an IRES, a polynucleotide encoding rep52, a 2A polynucleotide sequence and a polynucleotide encoding VP3-SpyTag fusion protein and a pA, followed by a LacI polynucleotide and a pA under control of an mCMV promoter. FIGURE 16B is a schematic depiction in a 5’ to 3’ direction of an hCMV promoter, a TATA box and 2 to 5 Lac operators (LacO x 2-5) to control a Cap polynucleotide, followed by an IRES, a polynucleotide encoding rep52, a 2A polynucleotide sequence and a polynucleotide encoding VP3-SpyTag fusion protein and pA, followed by a LacI polynucleotide under control of an mCMV promoter . FIGURE 16C is a schematic depiction in a 5’ to 3’ direction of an hCMV promoter, a TATA box and an AO to control a Cap polynucleotide, followed by an IRES, a polynucleotide encoding rep52, a 2A polynucleotide sequence and a polynucleotide encoding VP3-SpyTag fusion protein and pA, followed by an ArcEr polynucleotide under control of an mCMV promoter.

[0091] FIGURE 17A is a schematic depiction in a 5’ to 3’ direction of an hCMV promoter, a TATA box and 2 to 5 Lac operators (LacO x 2-5) to control a Cap polynucleotide, followed by an IRES, a polynucleotide encoding VP3-SpyTag fusion protein, a 2A polynucleotide sequence and a polynucleotide encoding rep52 and a pA, followed by a LacI polynucleotide and a pA under control of an mCMV promoter. FIGURE 17B is a schematic depiction in a 5’ to 3’ direction of an hCMV promoter, a TATA box and AO to control a Cap polynucleotide, followed by an IRES, a polynucleotide encoding VP3-SpyTag fusion protein, a 2A polynucleotide sequence and a polynucleotide encoding rep52 and a pA, followed by an ArcEr polynucleotide under control of an mCMV promoter. FIGURE 17C is a schematic depiction in a 5’ to 3’ direction of an hCMV promoter, a TATA box and AO to control a Cap polynucleotide, followed by an IRES, a polynucleotide encoding VP3-SpyTag fusion protein, a 2A polynucleotide sequence and a polynucleotide encoding rep52 and a135975-75120 REGN 11751 pA, followed by an ArcEr polynucleotide and a pA under control of an mCMV promoter.

[0092] FIGURE 18A is a schematic depiction in a 5’ to 3’ direction of an hCMV promoter, a TATA box and 2 to 5 Lac operators (LacO x 2-5) to control a Cap polynucleotide, followed by an IRES, a polynucleotide encoding VP3-SpyTag fusion protein, and a pA, followed by a second hCMV promoter, second TATA box and a second LacO x 2-5 to control expression of a polynucleotide encoding Rep52 and a pA, followed by a LacI polynucleotide and a pA under control of an mCMV promoter. FIGURE 18B is a schematic depiction in a 5’ to 3’ direction of an hCMV promoter, a TATA box and an AO to control a Cap polynucleotide, followed by an IRES, a polynucleotide encoding VP3-SpyTag fusion protein and a pA, followed by a second hCMV promoter, second TATA box and a second AO to control a polynucleotide encoding Rep52, followed by an mCMV promoter controlling ArcEr and a pA.

[0093] FIGURE 19 is a schematic showing that the lac repressor (LacI) regulation works for Cap in a transient transfection. IPTG addition induced CAP expression by removal of LacI.

[0094] FIGURE 20 illustrating data showing delaying Cap induction results in better infectivity (Higher Full:Empty ratio). Replication of ITR-GOI was induced by adding Dox (-OHT) on day 0 and allowing it to accumulate for 24 hours, then IPTG was added to induce Cap expression. Less empty capsids were generated; cap is made when enough ITR is replicated to be packaged. If cap is made in the absence of sufficient amount of ITR-GOI, it results in formation of empty capsids.

[0095] FIGURE 21 is a representative of FIGURES 22-31. pAd helper individual genes can be put together in expression cassettes where individual ORFs can be joined by 2A peptides (see FIGURES 29-31), and can be controlled by a TRE, promoter TATA box and AO or TO.

[0096] FIGURE 22 is a schematic depiction in a 5’ to 3’ direction of a pAd helper gene E4orf6 with VA RNA in an expression cassette.

[0097] FIGURE 23 is a schematic depiction in a 5’ to 3’ direction of a pAd helper gene E2A ORF2 in an expression cassette.135975-75120 REGN 11751

[0098] FIGURE 24 is a schematic depiction in a 5’ to 3’ direction of a pAd helper gene E2A ORF4 in an expression cassette.

[0099] FIGURE 25 is a schematic depiction in a 5’ to 3’ direction of a pAd helper gene E2A DBP in an expression cassette.

[0100] FIGURE 26 is a schematic depiction in a 5’ to 3’ direction of a pAd helper gene E2A 22K in an expression cassette.

[0101] FIGURE 27 is a schematic depiction in a 5’ to 3’ direction of a pAd helper l gene E2A 33K in an expression cassette.

[0102] FIGURE 28 is a schematic depiction in a 5’ to 3’ direction of a pAd helper gene E2A 33K native in an expression cassette.

[0103] FIGURE 29 is a schematic depiction in a 5’ to 3’ direction of pAd helper genes E2A-DBP, E2A-ORF2 and E2A-ORF4 joined in consolidated expression cassettes where individual ORFs are connected by 2A peptides.

[0104] FIGURE 30 is a schematic depiction in a 5’ to 3’ direction of pAd helper genes E2A-DBP, E2A-ORF2, E2A-ORF4 and E4ORF6-VA joined in consolidated expression cassettes where individual ORFs are connected by 2A peptides.

[0105] FIGURE 31 is a schematic depiction in a 5’ to 3’ direction of pAd helper genes E4ORF6, E2A-DBP, and E2A 33K native joined in consolidated expression cassettes where individual ORFs are connected by 2A peptides.

[0106] FIGURE 32 is a schematic depiction of a polynucleotide showing Splice Acceptor and Donor locations and sequences.

[0107] FIGURES 33A to 33N is a schematic depiction of an AAV Rep polynucleotide joined to an AAV Cap polynucleotide. Detailed Description of the Inventions Definitions

[0108] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the inventions belong.135975-75120 REGN 11751

[0109] 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, number amount, density, degree, increase, decrease, percentage, ratio, value, purity, pH, concentration, presence of a form or variant, temperature or amount of time, as is apparent from the teachings contained herein. For example, “about” can signify values either above or below the stated value in a range of approx. + / - 10% or more or less depending on the ability to perform. Thus, this term encompasses values beyond those simply resulting from systematic error.

[0110] An “effective amount” of a compound refers to the amount of compound needed to cause the intended result and is typically defined in terms of molar or weight concentration of the compound when present in a medium. Ligands are an example of compounds.

[0111] “Capable of binding” refers to the ability of a molecule, such a regulatory fusion protein or portion thereof to bind to another molecule or portion thereof, such ligand binding domains, nucleic acid binding domains, operators, response elements and the like. Typically, binding can permit an action or function or block an action or function.

[0112] A “nucleic acid moiety” includes any arrangement of single stranded or double stranded nucleotide sequences. Nucleic acid moieties can include, but are not limited to, polynucleotides, promoters, enhancers, operators, repressors, transcription termination signals, ribosomal entry sites and polyadenylation signals.

[0113] A “DNA cassette” or “cassette” is a type of nucleic acid moiety that typically comprises at least a promoter, at least one open reading frame and optionally a polyadenylation signal. One or more operators also are optional. A DNA cassette thus is a polynucleotide that comprises two or more shorter polynucleotides. A cassette can comprise one or more gene and promoters, enhancers, operators, repressors, transcription termination signals, ribosomal entry sites, introns and polyadenylation signals.

[0114] A ”DNA construct” can comprise one or more shorter DNA sequences.135975-75120 REGN 11751

[0115] As used herein, the terms “AAV” and “adeno-associated virus” refer to a Dependoparvovirus within the Parvoviridae genus of viruses. AAV can refer to an AAV derived from a naturally occurring “wild-type” virus, an AAV derived from a rAAV genome packaged into a capsid derived from capsid proteins encoded by a naturally occurring cap gene and / or a rAAV genome packaged into a capsid derived from capsid proteins encoded by a non-natural capsid cap gene.

[0116] As used herein, the term “recombinant AAV” refers to a recombinant AAV. Recombinant AAV includes an AAV genome in which part or all of the native rep and cap genes have been replaced with heterologous sequences, such as genes of interest (GOI), which are flanked by AAV inverted terminal repeats (ITRs). Recombinant AAV also include AAV with insertions and deletions to the AAV genome. An example of an insertion is the addition of heterologous sequences to the Cap gene.

[0117] As used herein, the term “polynucleotide encoding an AAV capsid protein” refers to the nucleic acid sequences that encode capsid proteins that form, or contribute to the formation of, the capsid, or protein shell, of the virus. In the case of AAV, the polynucleotide encoding an AAV capsid protein encodes capsid proteins VP1, VP2, and VP3.

[0118] As used herein, “administration” refers to providing or giving a subject a therapeutic agent (for example, an rAAV vector produced using a modified plasmid described herein), by any effective route. Exemplary routes of administration are described herein below.

[0119] As used herein, a “cell genome” refers to polynucleotide sequences in a cell, such as chromosomal DNA, mitochondrial DNA. episomal DNA and plasmid DNA. A cell genome can include native sequences, non-native sequences and sequences that are synthesized in whole or in part. The polynucleotide sequences can be transcribed or not transcribed. Transcribed sequences can be translated or not translated.

[0120] As used herein, the term “cell type” refers to a group of cells sharing a phenotype that is statistically separable based on gene expression data. For instance, cells of a common cell type may share similar structural and / or functional characteristics, such as similar gene activation patterns and antigen presentation135975-75120 REGN 11751 profiles. Cells of a common cell type may include those that are isolated from a common tissue (for example, epithelial tissue, neural tissue, connective tissue, or muscle tissue) and / or those that are isolated from a common organ, tissue system, blood vessel, or other structure and / or region in an organism.

[0121] As used herein, the term “endogenous” refers to a molecule (for example, a polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (for example, a human) or in a particular location within an organism (for example, an organ, a tissue, or a cell, such as a human cell, for example, a human cochlear supporting cell).

[0122] As used herein, the term “express” refers to one or more of the following events, depending on context: (1) production of an RNA template from a DNA sequence (for example, by transcription); (2) processing of an RNA transcript (for example, by splicing, editing, 5′ cap formation, and / or 3′ end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein. The term “expression product” refers to a protein or RNA molecule produced by any of these events.

[0123] As used herein, the term “exogenous” describes a molecule (for example, a polypeptide, nucleic acid, or cofactor) that is not found naturally in a particular organism (for example, a human) or in a particular location within an organism (for example, an organ, a tissue, or a cell, such as a human cell, for example, a human cochlear supporting cell). Exogenous materials include those that are provided from an external source to an organism or to cultured matter extracted therefrom.

[0124] As used herein, the term “heterologous” refers to a combination of elements that is not naturally occurring. For example, a heterologous transgene refers to a transgene that is not naturally expressed by the promoter to which it is operably linked.

[0125] As used herein, the term “target host cell” refers to a cell that has been transformed, or is capable of being transformed, with a nucleic acid sequence and thereby expresses a gene of interest or recombinant virus, and preferably will be from a cell line. The term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent cell, so long as the gene of interest is present.135975-75120 REGN 11751

[0126] Target host cells that are suitable for use with the inventions can be readily selected by those of skill in the art. The cell line can be a eukaryotic cell line such as a yeast cell line, insect cell line (for example, Sf9 and Sf21 cells) or mammalian cell lines. Preferred mammalian cell genomes include primate cells (including human), canine cells and rodent cells. Cells can be primary cells or immortalized cells. Suitable cell genomes can be selected from Vero cells, COS cells, HEK 293 cells, HeLa cells, CHO cells, BHK cells, MDCK cells, amniotic cells (human), embryonic cells, cell lines transfected with viral genes, for example, AD5 E1, including but not limited to an immortalized human retinal cell transfected with an adenovirus gene, for example, a PER.C6 cell, or an NSO cell. The cell genome can be a Chinese hamster ovary (CHO) cell line. Some examples of 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. The cell genome can be a HEK293 cell. Some examples of HEK293 cells include, but are not limited, to HEK293, HEK293A, HEK293E, HEK293F, HEK293FT, HEK293FTM, HEK293H, HEK293MSR, HEK293S, HEK293SG, HEK293SGGD, HEK293T and mutants and variants thereof.

[0127] As used herein, the term “naturally occurring” refers to materials which are found in nature or a form of the material that is found in nature.

[0128] As used herein, “2A peptide(s)” refer to peptides are often used to generate two peptides from one RNA transcript. There are four widely used 2A peptides (T2A, P2A, E2A, F2A), ranging from 18-22 amino acids, which all end in GP. While the exact mechanism is under study, the translating ribosome skips and fails to generate a peptide bond between Gly and Pro resulting in two separate peptide chains. The resultant N-terminal chain contains 17-21 amino acids of the 2A peptide, and the C-terminal chain contains a leading Proline. This event is widely described (and accepted) as a cleavage event even though no cleavage occurs. The four peptides have differing levels of ‘cleavage’ efficiency and in general P2A and T2A peptides are used as they offer close to 100% efficiency. In principle, 2A peptides are like IRES elements, which also generate two peptides from a single transcript. A difference is that when using 2A peptides the two chains are expressed at similar levels. With IRES elements, the second chain is often expressed at a lower level than the first. 2A peptides are encoded by 2A genes (2A polynucleotides).135975-75120 REGN 11751

[0129] One widely used modification of the 2A peptide is to include a furin cleavage site upstream of the 2A peptide. After skipping, the N-terminal protein is cleaved at the furin cleavage site (by Furin) leaving some nucleotides which are cleaved off by cellular carboxypeptidases. Lin et al., Biotechnol. J.12: 1700268 (2017); Liu et al., Scientifc Reports 7:2193 (2017).

[0130] "Operably linked" refers to one or more nucleotide sequences in functional relationships with one or more other nucleotide sequences. Such functional relationships can directly or indirectly control, which refers to inducing, causing, regulating, enhancing, facilitating, permitting, influencing, attenuating, stopping, preventing, repressing and / or blocking one or more actions or activities in accordance with the selected design for a selected purpose. Exemplars include single-stranded or double-stranded nucleic acid moieties and can comprise two or more nucleotide sequences arranged within a given moiety in such a way that sequence(s) can exert at least one functional effect on other(s). For example, a promoter operably linked to the coding region of a DNA polynucleotide sequence can facilitate transcription of the coding region. Other elements, such as enhancers, operators, repressors, transcription termination signals, ribosomal entry sites and polyadenylation signals also can be operably linked with a polynucleotide(s) of interest to control its transcription. Arrangements and spacing to achieve operable linkages can be ascertained by approaches available to the person skilled in the art, such as screening using western blots and RT-PCR. Operable linkage can occur with sequences on the same cassette or different cassettes.

[0131] As used herein, the term “plasmid” refers to a to an extrachromosomal circular double stranded DNA molecule into which additional DNA segments may be ligated. A plasmid is a type of vector, a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Certain plasmids are capable of autonomous replication in a host cell into which they are introduced (for example, bacterial plasmids having a bacterial origin of replication and episomal mammalian plasmids). Other vectors (for example, non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Certain plasmids are capable of directing the expression of genes to which they are operably linked.135975-75120 REGN 11751

[0132] “Protein”, “Polypeptide” or “peptide” includes a sequence 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.

[0133] A “homologous sequence” in the context of nucleic acid sequences refers to a sequence that is substantially homologous to a reference nucleic acid sequence. Two sequences can be considered to be substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding nucleotides are identical over a relevant stretch of residues. The relevant stretch can be a complete (i.e., full) sequence.

[0134] "Polynucleotide sequence" as used herein can refer to the polynucleotide material itself and / or to the sequence information (that is, the succession of letters used as abbreviations for bases) that biochemically characterizes a specific nucleic acid. A polynucleotide sequence presented herein is presented in a 5' to 3' direction unless otherwise indicated. A gene can be referred to as a polynucleotide,

[0135] “Protein of interest” or “polypeptide of interest” (POI) for expression in a recombinant AAV can have any amino acid sequence, and includes any protein, polypeptide, or peptide, and derivatives, components, domains, chains and fragments thereof. Included are, but not limited to, viral proteins, bacterial proteins, fungal proteins, plant proteins and animal (including human) proteins. Protein types can include, but are not limited to, antibodies, bi-specific antibodies, multi-specific antibodies, antibody chains (including heavy and light), antibody fragments, Fv fragments, Fc fragments, Fc-containing proteins, Fc-fusion proteins, receptor Fc- fusion proteins, receptors, receptor domains, trap and mini-trap proteins, enzymes, factors, repressors, activators, ligands, reporter proteins, selection proteins, protein hormones, protein toxins, structural proteins, storage proteins, transport proteins, signal proteins, neurotransmitters and contractile proteins. POIs include viral proteins used as non-AAV helper proteins, such as adenovirus helper proteins, and AAV proteins, such as Rep proteins and Cap proteins. Derivatives, components, chains and fragments of the above also are included. The sequences can be natural, semi- synthetic or synthetic.135975-75120 REGN 11751

[0136] Protein(s) of interest and polypeptide(s) of interest are encoded by “gene(s) of interest,” which also can be referred to as “polynucleotide(s) of interest.” Where multiple genes (same or different) are integrated, they can be referred to as “first,” “second”, “third,” “fourth,” “fifth,” “sixth,” “seventh,” “eighth,” “ninth,” “tenth,” etc. as is apparent from the context of use. As used herein, proteins of interest and polypeptides of interest include but are not limited to (i) AAV Rep proteins in their various forms, Cap proteins (VP1 protein, VP2 protein, VPs protein, including fusion VP proteins that comprise a member of a specific binding pair, such as SpyTag), and non-AAV helper proteins, and are encoded by genes of interest / polynucleotides of interest.

[0137] A “Rep gene type” refer to any polynucleotide sequence that encode one or more of Rep78, Rep68, Rep52, Rep40 and any mutants and derivative thereof. The Rep gene type can include a p19 promoter, lack p19 promoter activity or have an altered p19 promoter. Other codons can be altered, including start codons, such as changing ATG to ACG,CTG, GTG, TTG or the like, in order to reduce transcription from the p19 promoter (referred to as reduced p19 promoter activity). The Rep gene type can include genes of the native length, longer versions, and truncated versions. The Rep gene type also includes genes that engineered to include non-coding sequences, such as introns, and include split versions of the gene that reside on one or more cassettes.

[0138] A “polynucleotide of interest” refers to any polynucleotide or gene sequence that encode, for example, one or more of Rep78, Rep68, Rep52, Rep40, helper genes, other genes of interest, such as therapeutic proteins and enzymes, and any mutants and derivative thereof. The polynucleotide of interest can include a p19 promoter, have reduced p19 promoter activity, lack a p19 promoter or have an altered p19 promoter. Other codons can be altered, including start codons, such as changing ATG to ACG,CTG, GTG, TTG or the like, in order to reduce transcription from the p19 promoter (referred to as reduced p19 promoter activity”). The polynucleotide of interest includes genes of the native length, longer versions, and truncated versions. The polynucleotide of interest also includes genes that engineered to include non-coding sequences, such as introns, and include split versions of the gene that reside on one or more cassettes.135975-75120 REGN 11751

[0139] As used herein, the term "promoter" refers to a recognition site on DNA that is bound by an RNA polymerase. The polymerase drives transcription of the transgene. “Promoter” also indicates a DNA sequence that cause transcription of a DNA sequence to which it is operably linked, i.e., linked in such a way as to permit transcription of the nucleotide sequence of interest when the appropriate signals are present and / or repressors are absent. The transcription of a polynucleotide of interest may be placed under control of any promoter or enhancer element known in the art. A eukaryotic promoter can be operably linked to a TATA Box, and most eukaryotic promoters have TATA boxes. The TATA Box is typically located upstream of the transcription start site.

[0140] Promoters include, but are not limited to, promoters such as p19, p40, and p41. Other useful promoters that also can be used include, but are not limited to, the SV40 early promoter region, SV40 E / L (early late) promoter, the promoter contained in the 3' long terminal repeat of Rous sarcoma virus, the regulatory sequences of the metallothionein gene, mouse or human cytomegalovirus major immediate early (CMV-MIE) promoter and other CMV promoters, including CMVmin promoters. Plant expression vectors comprising the nopaline synthetase promoter region, the cauliflower mosaic virus 35S RNA promoter, and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase; promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I; insulin; immunoglobulin; mouse mammary tumor virus; albumin; C.-feto protein; C.1-antitrypsin; 3-globin, and myosin light chain-2. Various forms of the CMV promoter are preferred and the CMVmin promoter is exemplified here. Promoters depicted in the Figures and mentioned in the Examples are exemplary, and other promoters can be used by the person skilled in the art.

[0141] Minimal promoters, such as CMVmin promoters, tend to be truncated promoters or core promoters and can be used in controlled expression systems. Minimal promoters are more amenable to control. Minimal promoters and development approaches are widely known and disclosed in, for example, Saxena et al., Methods Molec. Biol.1651:263-73 (2017); Ede et al., ACS Synth Biol.5:395-404135975-75120 REGN 11751 (2016); Brown et al., Biotech Bioeng.111:1638-47 (2014); Morita et al., Biotechniques 0:1-5 (2012); Lagrange et al., Genes Dev.12:34-44 (1998). There are many CMVmin promoters described in the field. It also is possible to use TATA box sequences to perform the role of a promoter. “Reduced promoter activity” can be brought about by altering a promotor or selecting a weak promoter. Activity also can be reduced by the use of a non-ATG start codon, such as ACG, CTG, GTG, TTG or the like, when referring to the DNA level. Non- ATG (DNA level) and non-AUG (RNA level) start codons are discussed in Cao et al. , Non-AUG start codons: expanding and regulating the small and alternative ORFeome, Exp. Cell Res.391(1):11973 (June 1, 2020).

[0142] “Operator” indicates a DNA sequence that is introduced in or near a polynucleotide sequence in such a way that the polynucleotide sequence may be regulated by the interaction of a molecule capable of binding to the operator and, as a result, prevent or allow transcription of the polynucleotide sequence, as the case may be. One skilled in the art will recognize that the operator must be located sufficiently in proximity to the promoter such that it is capable of controlling transcription by the promoter, which can be considered a type of operable linkage. The operator may be placed either downstream or upstream of the promoter. These include, but are not limited to, the operator region of the Lex A gene of E. coli, which binds the Lex A peptide and the lactose and 45 tryptophan operators, which bind the repressor proteins encoded by the Lad and trpR genes of E. coli. The bacteriophage operators from the lambda Pi and the phage P22 Mnt and Arc. Alternatively, when the transcription blocking domain of the RFP is a restriction enzyme, the operator is the recognition sequence for that enzyme. Preferred operators are the Tet operator, and the Arc operator exemplified herein. Operators can have a native sequence or a mutant sequence (for example, synthetic or semi-synthetic). For example, mutant sequences of the Tet operator are disclosed in Wissmann et al., Nucleic Acids Res. 14: 4253-66 (1986). TRE also functions as an operator and can comprise native operator sequences, mutant operator sequences or combinations of native and mutant operator sequences.

[0143] The phrases “percent identity” or “% identical,” in their various grammatical forms, when describing a sequence is meant to include homologous sequences that135975-75120 REGN 11751 display the recited identity along regions of contiguous homology, but the presence of gaps, deletions, or insertions that have no homolog in the compared sequence are not taken into account in calculating percent identity. As used herein, a “percent identity” or “% identical” determination between homologs would not include a comparison of sequences where the homolog has no homologous sequence to compare in an alignment. Thus, “percent identity” and “% identical” do not include penalties for gaps, deletions, and insertions.

[0144] A “homologous sequence” in the context of nucleic acid sequences refers to a sequence that is substantially homologous to a reference nucleic acid sequence. Two sequences can be considered to be substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding nucleotides are identical over a relevant stretch of residues. The relevant stretch can be a complete (i.e., full) sequence.

[0145] “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.

[0146] A “regulatory fusion protein” or “RFP” is a protein that comprises a ligand binding domain and a DNA binding domain that originate from different proteins. Steroid-binding domains of the glucocorticoid or estrogen nuclear receptors can be employed as ligand binding domains. The reverse Tet DNA binding domain (rTet) also is useful as a ligand binding domain and can bind DNA as well. Exemplary RFPs for use according to the inventions described herein are the reverse tetracycline transactivator (rtTA) and the fusion protein comprising the Arc repressor binding domain (Arc) and the estrogen receptor ligand binding domain (ArcER). Other components for RFPs include the DNA-binding domain of yeast activator GAL4 fused to HSV VP16; the KRAB domain of human Kox1 fused to a prokaryotic135975-75120 REGN 11751 Tet repressor (TetR-KRAB); ligand-binding domain of the estrogen receptor (ER) to the carboxyl end of the tTA transactivator (TetR-VP16); and a catalytically inactive form of Cas9 fused to repeats of the minimal activation domain of VP16 (dCas9- VP64). Other fusion proteins include LexA-VP16 and LacI-VP16. Polynucleotides encoding regulatory fusion proteins (for example, rtTA and ArcEr) can be integrated into the cellular genome as described herein.

[0147] The term "recombinant capsid protein" includes a capsid protein that has at least one mutation in comparison to the corresponding capsid protein of the wild- type virus, which wild-type may be a reference and / or control virus for comparative study. A recombinant capsid protein includes a capsid protein that comprises a heterologous amino acid sequence, which may be inserted into and / or displayed by the capsid protein. "Heterologous" in a general context means heterologous as compared to the virus, from which the capsid protein is derived. The inserted amino acids can simply be inserted between two given amino acids of the capsid protein. An insertion of amino acids can also go along with a deletion of given amino acids of the capsid protein at the site of insertion, for example, 1 or more capsid protein amino acids are substituted by 5 or more heterologous amino acids). An example of a heterologous amino acid sequence that can be inserted is a member of a specific binding pair, such SpyTag.

[0148] "Retargeting" or "redirecting" may include scenarios 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 to abolished by insertion of the heterologous epitope, and which retargeting to more a specific cell in the tissue or a specific organ in the organism can be achieved with the retargeting molecule that binds a marker expressed by the specific cell. Such retargeting or redirecting may also include a scenario in which the wildtype vector targets a tissue, which targeting of the tissue is reduced to abolished by insertion of the heterologous epitope, and which retargeting to a completely different tissue is achieved with the retargeting molecule. Covalent and non-covalent approaches can be employed.

[0149] “Detargeting” refers to reducing or abolishing AAV natural preferential transduction by mutating Cap proteins. For example, mutations in the galactose135975-75120 REGN 11751 binding domain of VP1 assist in detargeting the liver. These mutations can be referred to as “detargeting mutations,” and are discussed herein in greater detail.

[0150] 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; AAV5 – T571S; and AAV6 – N500E, K531A and K531E. These and others are set forth in the chart below:135975-75120 REGN 11751

[0151] Still other mutations for all AAV serotypes are available to the person skilled in the art.

[0152] The term “retargeting molecule” is a molecule useful for targeting an antigen, receptor and / or ligand found on the surface of a cell. The retargeting molecule is bound to a polypeptide that is part of a specific binding pair, and is a covalent approach. See WO 2019 / 006046, WO 2025 / 054526 and WO 2025 / 111473 publications. For example, a targeting 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 and mini-trap proteins, are useful as retargeting molecules. In the literature, the phrase “targeting ligand” has been used for molecules that are useful for targeting. See Yan et al., Pharmaceutics 202416, 248.

[0153] Non-covalent approaches for retargeting also can be employed, such as the use of bispecific antibodies that can bind to the AAV capsid and a target antigen, receptor and / or ligand on a cell. See U.S. Patent 12,258,597.

[0154] All antibody classes, namely IgG, IgA, IgM, IgD and IgE, can be used as retargeting molecules. IgG is a preferred class and includes subclasses IgG1 (including IgG1λ and IgG1κ), IgG2, IgG3, and IgG4. Further antibody types include a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecifϊc antibody, a bispecific antibody, a trispecific antibody, an antigen binding antibody fragment, a single chain antibody, a diabody, triabody or tetrabody, a Fab fragment or a F(ab')2 fragment, an IgD antibody, an IgE antibody,135975-75120 REGN 11751 an IgM antibody, an IgG antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

[0155] "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 Spy Tag:Spy Catcher, 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.

[0156] 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 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.135975-75120 REGN 11751

[0157] The term "peptide tag" or “tag” includes polypeptides that are (1) heterologous to the protein, which is tagged with the peptide tag, (2) a member of a specific protein-protein binding pair capable of forming an isopeptide bond, and (3) no more than 50 amino acids in length.

[0158] The term "target cells" includes any cells in which expression of a nucleotide of interest is desired. Preferably, target cells exhibit a target, such as a receptor, ligand and / or antigen 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. 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, TRP2-135975-75120 REGN 11751 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 human ectonucleoside triphosphate diphosphohydrolase 3 (hENTPD3). Other targets can be selected by the person skilled in the art. See WO 2019 / 006046.

[0159] A “repressor protein”, also referred to as a “repressor,” is a protein that can bind to DNA in order to repressor transcription. Repressors are of eukaryotic and prokaryotic origin. Prokaryotic repressors are preferred. Examples of repressor families include: TetR, LysR, LacI, ArsR, IcIR, MerR, AsnC, MarR, DeoR, GntR and Crp families. Repressor proteins in the TetR family include: ArcR, ActII, AmeR, AmrR, ArpR, BpeR, EnvR, EthR, HemR, HydR, IfeR, LanK, LfrR, LmrA, MtrR, Pip, PqrA, QacR, RifQ, RmrR, SimReg2, SmeT, SrpR, TcmR, TetR, TtgR, TrgW, UrdK, VarR YdeS, ArpA, BarA, Aur1B, CalR1, CprB, FarA, JadR*, JadR2, MphB, NonG, PhlF, TylQ, VanT, TarA, TylP, BM1P1, Bm3R1, ButR, CampR, CamR, DhaR, KstR, LexA-like, AcnR, PaaRR, PsbI, Th1R, UidR, YDH1, BetI, McbR, MphR, PhaD, Q9ZF45, TtK, Yhgd, YixD, CasR, IcaR, LitR, LuxR, LuxT, OpaR, Orf2, SmcR, HapR, Ef0113, HlyIIR, BarB, ScbR, MmfR, AmtR, PsrA andYjdC proteins See Ramos et al., Microbiol. Mol. Biol. Rev., 69: 326-56 (2005). Still other repressors include PurR, LacR, MetJ and PadR, Repressor proteins are encoded by genes referred to as “repressor genes” or “repressor protein genes.”

[0160] “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 or fluorescent reaction, and often are referred to as “fluorescent proteins” or “color proteins.” However, a reporter protein also can be non-enzymatic and non-fluorescent as long as it can be detected by another protein135975-75120 REGN 11751 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 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.

[0161] “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 cause135975-75120 REGN 11751 a cell to stop propagating, stop protein production and / or are lethal to the cell in the presence of the negative selectable marker proteins. Thymidine kinase and certain fusion proteins can serve as negative selectable markers, including but not limited to GyrB-PKR. See White et al., Biotechniques, 50: 303-309 (May 2011). Selectable marker proteins and corresponding genes can be referred to generically as first (1), second (2), third (3), fourth (4), fifth (5), sixth (6), seventh (7), eighth (8), ninth (9), tenth (10), etc., as is apparent from the context of usage.

[0162] A “Stable Integration Site” or “SIS” is a region for site-specific integration of DNA polynucleotides, including cassettes that comprise genes and / or other open reading frames, promoters and optionally other elements. DNA constructs can be inserted into an SIS by a variety of approaches. Multiple Stable Integration Sites can be created and located on different chromosomes, different regions of the same chromosome or different positions in a same region of a chromosome. See WO 2023 / 069931 A1; US 2019 / 0233544 A1; U.S. Patent No.9,816,110; and U.S. Patent No.7,771,997 describe stable integration sites, including those located at enhanced expression and stability regions.

[0163] A “Tetracycline Response Element” or “TRE” comprises seven copies of the 19 nucleotide TetO spaced apart by spacers comprising 17-18 nucleotides and are commercially available. TetO sequences can vary, and nucleotide substitutions are known. For example, altered sequences based on the Tet operator are disclosed in Wissmann et al., Nucleic Acids Res.14: 4253-66 (1986). The spacers are not sequence specific. The spacers can be similar, but all should not be identical. A TRE is considered a type of operator as used herein, for example, sequences are disclosed in International Publication No. WO 2023 / 069929 A1.

[0164] As used herein, the term “pharmaceutical composition” refers to a mixture containing a therapeutic agent, optionally in combination with one or more pharmaceutically acceptable excipients, diluents, and / or carriers, to be administered to a subject, such as a mammal, for example, a human, in order to prevent, treat or control a particular disease or condition affecting or that may affect the subject.

[0165] As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions and / or dosage forms, which are suitable for contact with the tissues of a subject, such as a mammal (for example, a human)135975-75120 REGN 11751 without excessive toxicity, irritation, allergic response, and other problem complications commensurate with a reasonable benefit / risk ratio.

[0166] As used herein, the term “polynucleotide encoding an AAV rep protein” refers to the nucleic acid sequences that encode Rep proteins (that is, rep78, rep68, rep52, and / or rep40) related to the replication and production of AAV.

[0167] As used herein, the term “plasmid” refers to a plasmid that provides the viral rep and cap gene functions. This plasmid can be useful for the production of AAVs from rAAV genomes lacking fully functional rep and / or the capsid gene sequences.

[0168] As used herein, the term “subject” refers to any organism to which a composition in accordance with the invention may be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include any animal (for example, mammals such as mice, rats, rabbits, non-human primates, and humans). A subject may seek and be in need of treatment, require treatment, be receiving treatment, be receiving treatment in the future, and be a human and animal who is under care by a trained professional for a particular disease and condition. Preferably, the subject is a human.

[0169] As used herein, the terms “transcription regulatory element” and “regulatory sequence” refer to a polynucleotide that controls, at least in part, the transcription of a gene of interest. Transcription regulatory elements may include promoters, enhancers, and other polynucleotides (for example, polyadenylation signals) that control or help to control gene transcription. Examples of transcription regulatory elements are described, for example, in Lorence, Recombinant Gene Expression: Reviews and Protocols (Humana Press, New York, NY, 2012).

[0170] As used herein, the term “transfection” refers to any of a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, for example, electroporation, lipofection, calcium phosphate precipitation, DEAE-dextran transfection, Nucleofection, squeeze-poration, sonoporation, optical transfection, magnetofection, impalefection and the like.

[0171] As used herein, the terms "transduction" and “transduce” refer to a method of introducing a vector DNA construct or a part thereof into a cell. Wherein the vector135975-75120 REGN 11751 DNA construct is contained in a viral vector such as for example an AAV vector, transduction refers to viral infection of the cell and subsequent transfer and integration of the vector DNA construct or part thereof into the cell genome.

[0172] As used herein, the term "transgene" refers to a recombinant nucleic acid (for example, DNA) encoding a gene product, such as a peptide, protein, or RNA (for example, a protein-encoding mRNA or an inhibitory RNA, such as a miRNA, shRNA, or the like). Transgenes also can be referred to as genes of interest or polynucleotides of interest. In addition to the coding region for the gene product, the transgene may include, or be operably linked to, one or more elements to facilitate or enhance expression, such as a promoter, enhancer(s), destabilizing domain(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s), and / or other functional elements. According to the current inventions, any known suitable promoter, enhancer(s), destabilizing domain(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s), and / or other functional elements which can be utilized. Examples of transgenes are identified herein. Transgenes can be genes / polynucleotides of interest.

[0173] As used herein, “treatment” and “treating,” in reference to a disease or condition, refer to an approach for obtaining beneficial or desired results, for example, clinical results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease or condition; stabilized (that is, not worsening) state of disease, disorder, or condition; preventing spread of disease or condition; delay or slowing the progress of the disease or condition; amelioration or palliation of the disease or condition; and remission (whether partial or total), whether detectable or undetectable. “Ameliorating” or “palliating” a disease or condition means that the extent and / or undesirable clinical manifestations of the disease, disorder, or condition are lessened and / or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.135975-75120 REGN 11751

[0174] As used herein, the term “vector” refers to a nucleic acid vector, for example, a DNA vector, such as a plasmid, cosmid, or artificial chromosome, an RNA vector, a virus, or any other suitable replicon (for example, viral vector). A variety of vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into a prokaryotic or eukaryotic cell. Examples of such expression vectors are described in, for example, Gellissen, Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems (John Wiley & Sons, Marblehead, MA, 2006). Expression vectors suitable for use with the compositions and methods described herein contain a polynucleotide sequence as well as, for example, additional sequence elements used for the expression of proteins. Certain vectors that can be used for the expression of a viral capsid protein as described herein include vectors that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription. Other useful vectors for expression of a transgene contain polynucleotide sequences that enhance the rate of translation of the transgene or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, for example, 5’ and 3’ untranslated regions and a polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector. The expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.

[0175] As used herein, the term “viral capsid protein” refers to a capsid protein composing a proteinaceous shell. Such a proteinaceous shell is generally composed of one or more viral capsid proteins and when assembled is capable of being loaded with one or more polynucleotide molecules. A viral capsid protein described herein may, for example, be a viral protein (VP) 1, VP2, and VP3. Further, a viral capsid protein described herein may refer to a viral capsid protein from any AAV.

[0176] As used herein, the term “VP1” refers to a capsid protein that is a component of an AAV capsid and is encoded by the Cap gene. As used herein, a VP1 may possess a surface binding site that interacts with one or more molecules on the surface of a cell to initiate the process of cell entry (for example, endocytic135975-75120 REGN 11751 entry and receptor-mediated fusion). As used herein, a VP1 molecule may self- assemble into a structure composed of VP1, VP2, and / or VP3 molecules.

[0177] As used herein, the term “VP2” refers to a capsid protein that is a component of an AAV capsid and is encoded by the Cap gene. As used herein, a VP2 protein may facilitate capsid entry into a host cell, for example, by mediating associations with and exit from the endoplasmic reticulum of a host cell and by facilitating the entry of a nucleic acid molecule into a host cell nucleus. As used herein, a VP2 molecule may self-assemble into a structure composed of VP1, VP2, and / or VP3 molecules.

[0178] As used herein, the term “VP3” refers to a capsid protein that is a component of an AAV capsid and is encoded by the Cap gene. As used herein, a VP3 may facilitate capsid entry into a host cell, for example, by mediating associations with and exit from the endoplasmic reticulum of a host cell and by facilitating the entry of a nucleic acid molecule into a host cell nucleus. As used herein, a VP3 molecule may self-assemble into a structure composed of VP1, VP2, and / or VP3 molecules.

[0179] As used herein, the term “wild-type” refers to a genotype with high frequency for a particular gene in a given organism.

[0180] 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 merely 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

[0181] Described herein are compositions and methods for producing AAV vectors (for example, AAV5 vectors). The present inventions provide plasmids containing a tetracycline response element (TRE), a polynucleotide encoding an135975-75120 REGN 11751 AAV rep protein and a polynucleotide encoding an AAV capsid protein (for example, an AAV5 capsid protein). The polynucleotide encoding an AAV capsid protein (for example, AAV5 capsid protein) can encode AAV capsid proteins VP1, VP2, and VP3. In addition, the inventions provide methods of producing AAV vectors (for example, AAV5 vectors) using the modified plasmid (for example, by transducing a cell with the modified plasmid, a helper plasmid, and a transgene plasmid). The compositions and methods described herein can be used to increase AAV production (for example, AAV5 production) compared to approaches using unmodified plasmids (for example, plasmids containing the same polynucleotides encoding rep and capsid proteins but lacking the TRE.) In addition, the compositions and methods described herein can be used to increase the percent of full AAV vectors produced (for example, AAV vectors containing a transgene or polynucleotide to be expressed). AAV vectors (for example, AAV5 vectors) produced using the methods described herein can contain a transgene of interest and can be used in gene therapy applications.

[0182] Additionally, polynucleotide (typically, DNA) constructs described herein provide an advantage that the transcription of polynucleotide containing a polynucleotide of interest is tightly controlled so that the cells are able to survive to a stage permitting large scale expression of a Rep protein. By “tightly controlled” it is meant that transcription of the polynucleotide of interest can be substantially reduced. For example, in the repressed state at least a 10-fold decrease in transcription of polynucleotide of interest can be achieved relative to the level of transcription as seen in the induced state. In the repressed state, at least a 20-fold decrease in transcription of polynucleotide of interest can be achieved relative to the level of transcription in the induced state. In the repressed state, at least a 50-fold decrease in transcription of polynucleotide of interest can be achieved relative to the level of transcription as seen in the induced state. In the repressed state, at least a 100-fold decrease in transcription of polynucleotide of interest can be achieved relative to the level of transcription as seen in the induced state. In the repressed state, at least a 500-fold decrease in transcription of polynucleotide of interest can be achieved relative to the level of transcription as seen in the induced state.

[0183] As a corollary to the repressed state, the degree of induction of transcription of the polynucleotide of interest can be at least 10-fold greater than in135975-75120 REGN 11751 the repressed state. The degree of induction of transcription of the polynucleotide of interest can be least 20-fold greater than in the repressed state. The degree of induction of transcription of the polynucleotide of interest can be at least 50-fold greater than in the repressed state. The degree of induction of transcription of the polynucleotide of interest can be at least 100-fold greater than in the repressed state. The degree of induction of transcription of the polynucleotide of interest can be at least 500-fold greater than in the repressed state.

[0184] The degree or amount of transcription of the polynucleotide of interest may be determined by methods known to those of skill in the art. For example, the level of expression of Rep protein in a host cell can be determined based on the amount of the corresponding mRNA that is present in the cell. Messenger RNA transcribed from a polynucleotide sequence can be quantified by various methods known by those of skill in the art, including but not limited to, Northern blot hybridization, ribonuclease RNA protection, in situ hybridization to cellular RNA or by PCR.

[0185] By way of a further example, the level of expression of Rep protein in a host cell may also be determined based on the amount of polypeptide of interest encoded by the selected sequence. Polypeptides encoded by a polynucleotide sequence can be quantified by various methods known by those of skill in the art, including but not limited to, ELISA, Western blotting, radioimmunoassays, immunoprecipitation, assay of the biological activity of the polypeptide, immunostaining of the polypeptide followed by FACS analysis or by homogeneous time resolved fluorescence assays (HTRF). Controllable Transcription and Expression Systems

[0186] The present inventions relate to a controllable transcription and expression system that may be used to control the transcription of any polynucleotide sequence of interest. The described controllable transcription and expression system comprises at least two controllable operator systems. One of the operator systems can be located 5’ to a promoter that is operably linked to the polynucleotide sequence of interest and the second operator system can be located 3’ of the promoter. The operator systems may comprise operators that are operably linked to a promoter that drives transcription of the polynucleotide sequence of interest. The135975-75120 REGN 11751 polynucleotide of interest may encode a polypeptide and / or product (for example, RNA) of interest.

[0187] In one aspect, controllable transcription as described herein allows for transcription of the polynucleotide of interest in the presence of a first ligand and the absence of a second ligand. See Figure 1. Briefly, when present, the first ligand binds to a ligand binding site on a first regulatory fusion protein (RFP) which comprises a (1) a transcription activating domain fused to a DNA binding domain; and (2) a ligand-binding domain. Upon binding of the first ligand to the ligand-binding domain of the first RFP, the DNA binding domain of the first RFP binds to a first operator, allowing for transcription from the promoter, but only if transcription is not inhibited by the second operator system. The second operator system is controlled by a second ligand. Briefly, when present, the second ligand binds to a ligand binding site on a second regulatory fusion protein (RFP) which comprises a (1) a transcription blocking domain fused to a DNA binding domain; and (2) a ligand- binding domain. Upon binding of the second ligand to the ligand-binding domain of the second RFP, the DNA binding domain of the second RFP binds to a second operator, blocking transcription from the promoter. Thus, in the presence of the second ligand and absence of the first ligand, transcription is repressed; whereas in the absence of the second ligand and the presence of the first ligand, transcription is permitted. Figure 1 illustrates examples of control of transcription utilizing this system.

[0188] The first operator system may comprise at least one operator that is operably linked to a promoter that drives transcription of the polynucleotide sequence of interest. The operator of the first operator system may be located 5’ to a promoter that is operably linked to the polynucleotide sequence of interest. Examples of such configurations are shown in Figures 1 and 2 where the first operator system comprises a TRE.

[0189] The first operator system also may comprise a regulatory fusion protein (RFP) which comprises a (1) a transcription activating domain fused to a DNA binding domain; and (2) a ligand-binding domain. The first operator system may further comprise a ligand that binds to the ligand-binding domain of the first RFP. Upon binding of the ligand to the ligand-binding domain of the first RFP, the DNA135975-75120 REGN 11751 binding domain of the RFP binds to the operator, for example TRE, thereby allowing for transcription from the promoter, but only if transcription is not inhibited by the second operator system as discussed herein. Other system components are known to those of skill in the art, and include for example, the tetracycline on systems (Tet- On), tetracycline on advanced (Tet-On Advanced), tetracycline on 3G systems (Tet- On 3G), cumate-inducible systems, lactose-inducible systems, and variations thereof.

[0190] The second operator system can be located 3’ to a promoter that is operably linked to the polynucleotide sequence encoding the polypeptide of interest. Examples of such configurations are shown in Figures 1 and 2. The second regulatory element comprises at least one operator that is operably linked to a promoter that drives transcription of the polynucleotide sequence of interest. Examples of such configurations are shown in Figures 1 and 2 where the second operator system can comprise either a tetracycline operator (TetO or TO) or an Arc operator (ArcO or AO). The second operator can be ArcO (AO) (Figure 1) or TetO (TO) (Figure 2).

[0191] By way of a non-limiting example, suitable controllable operator components for the second operator system are described in U.S. Patent No. 9,469,856.

[0192] Suitable promoters for use with the described system are known and can be determined by those of skill in the art in combinations of choice. The promoter operably linked to the polynucleotide sequences can be selected from, but is not limited to, the SV40 early promoter region, SV40 E / L promoter, the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus, the regulatory sequences of the metallothionein gene, mouse or human cytomegalovirus major immediate early (MIE) promoter; CMVmin promoters, plant expression vectors comprising the nopaline synthetase promoter region, the cauliflower mosaic virus 35S RNA promoter, and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase; promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized135975-75120 REGN 11751 in transgenic animals: elastase I; insulin; immunoglobulin; mouse mammary tumor virus; albumin; α-fetoprotein; α1-antitrypsin; β-globin; and myosin light chain-2. The promoter can be the human CMV-MIEmin or other CMVmin promoters. Approaches for developing minimal promoters are described in Saxena et al., Methods Molec. Biol.1651:263-73 (2017); Ede et al., ACS Synth Biol.5:395-404 (2016); Brown et al., Biotech Bioeng.111:1638-47 (2014); Morita et al., Biotechniques 0:1-5 (2012); Lagrange et al. Genes Dev.12:34-44 (1998).

[0193] The polynucleotide sequence of interest can encode a polypeptide of interest. The polynucleotide of interest can be a native gene, including variants thereof, or a synthetic, semi-synthetic or optimized sequence. The polynucleotide sequence of interest also can encode a product (for example, RNA) of interest. More specifically, products of interest may be non-coding RNAs.

[0194] “Protein of interest” or “polypeptide of interest” (POI) for expression in a recombinant AAV can have any amino acid sequence, and includes any protein, polypeptide, or peptide, and derivatives, components, domains, chains and fragments thereof. Included are, but not limited to, viral proteins, bacterial proteins, fungal proteins, plant proteins and animal (including human) proteins. Protein types can include, but are not limited to, antibodies, bi-specific antibodies, multi-specific antibodies, antibody chains (including heavy and light), antibody fragments, Fv fragments, Fc fragments, Fc-containing proteins, Fc-fusion proteins, receptor Fc- fusion proteins, receptors, receptor domains, trap and mini-trap proteins, enzymes, factors, repressors, activators, ligands, reporter proteins, selection proteins, protein hormones, protein toxins, structural proteins, storage proteins, transport proteins, neurotransmitters and contractile proteins. Derivatives, components, chains and fragments of the above also are included. The sequences can be natural, semi- synthetic or synthetic. Proteins of interest and polypeptides of interest are encoded by “genes of interest,” which also can be referred to as “polynucleotides of interest.” Where multiple genes (same or different) are integrated, they can be referred to as “first,” “second”, “third,” “fourth,” “fifth,” “sixth,” “seventh,” “eighth,” “ninth,” “tenth,” etc. as is apparent from the context of use.

[0195] A polypeptide of interest also can include cytotoxic proteins, such as viral proteins. For example, adenovirus E1A, E1B, E2A and E4 are used to perform135975-75120 REGN 11751 functions for production of adeno-associated virus (AAV) but have been reported to be toxic effects in certain cell types. AAV Rep also has been reported to by cytotoxic in certain cell types. Additionally, proteins used in genetic alterations, such as Cre recombinase, Flp recombinase, Zinc finger (ZFN) proteins and dimers, TALEN, bxb 1 integrase, CRISPR associated proteins (Types I-VI; including Cas1, Cas2, Cas3, Cas4, Cas, Cas6, Cas7, Cas8, Cas9, Cas10, Cas11, Cas12 and Cas13) and other nucleases and integrases, can be POIs, and thereby controlled according to the present inventions. Cells Capable of Controlled Transcription and Expression

[0196] A cell comprising a promoter operably linked to a polynucleotide sequence of interest, wherein the promoter can be controlled by at least two operators operably linked to the promoter is provided. The promoter operably linked to the polynucleotide sequence of interest, the operators operably linked to the promoter can be integrated into the cell genome. Transcription of the polynucleotide sequence of interest is controlled by the operators, allowing the transcription of the polynucleotide of interest to be permitted or repressed as preferred.

[0197] Cells that are suitable for use with these inventions can be readily selected by those of skill in the art. The cell line can be a eukaryotic cell line such as a yeast cell line, insect cell line (for example, Sf9 and Sf21 cells) or a mammalian cell line. Preferred mammalian cells include primate cells (including human), canine cells and rodent cells. Cells can be primary cells or immortalized cells. Suitable cells can be selected from Vero cells, COS cells, HEK293 cells, HeLa cells, CHO cells, BHK cells, Sp2 / 0 cells, MDCK cells, amniotic cells (including human), embryonic cells, cell lines transfected with viral genes, for example, AD5 E1, including but not limited to an immortalized human retinal cell transfected with an adenovirus gene, for example, a PER.C6 cell, or an NSO cell. The cell can be a Chinese hamster ovary (CHO) cell line. Some examples of CHO cells include, but are not limited to, CHO-ori, CHO-K1, CHO-s, CHO-DHB11, CHO-DXB11, CHO-K1SV, and mutants / variants thereof. The CHO cell can be the CHO cell line designated K1. Examples of HEK293 cells include, but are not limited, to HEK293, HEK293A, HEK293E, HEK293F,135975-75120 REGN 11751 HEK293FT, HEK293FTM, HEK293H, HEK293MSR, HEK293S, HEK293SG, HEK293SGGD, HEK293T and mutants and variants thereof. Transgenes of interest for recombinant AAVs and Gene Therapy

[0198] A transgene of interest includes any gene that is desired to be expressed and can fit within an AAV capsid. More than one transgene of interest is possible if the totality of genes are of a combined size that can fit within an AAV capsid.

[0199] Such rAAV vectors can also contain marker or reporter genes. Useful rAAV vectors can have one or more of the AAV WT genes deleted in whole or in part but retain functional flanking ITR sequences. The AAV ITRs can be of any serotype suitable for a particular application. Methods for using rAAV vectors are described, for example, in Pupo et al., Mol Ther.7:3515-3541 (2022), and Wang et al., Nat Rev Drug Discov.18:358-378 (2019).

[0200] Production of rAAV vectors for gene therapy can be carried out in vitro using suitable producer cell lines. Producer cells can be any cell type possessing the genes necessary to promote AAV genome replication, capsid assembly, and packaging. Exemplary producer cells can include human embryonic kidney 293 (HEK-293) cells or derivatives thereof, HeLa cells, human amniotic cells, CHO cells, BHK cells, insect cells and others. EXAMPLES

[0201] The following examples are put forth so as to provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated, and are intended to be purely exemplary of the inventions and are not intended to limit the scope of what the inventors regard as their inventions.

[0202] The inventions provide HEK293T and other HEK293 cells engineered to express rtTA transcriptional activator and ArcER transcriptional repressor. Cells were then transfected with expression plasmids that encode Rep78 and / or Rep52 under the control of rtTA / ArcER regulated promoter. The promoter can be comprised of minimal CMV promoter sequence flanked by TRE repeat elements on the 5’ and 2 Arc operator sequences on the 3’ of the TATA box. Other cell types can be employed as well, for example Hela, CHO, BHK, epithelial cells or amniotic cells.135975-75120 REGN 11751 Example 1 Two Regulatory Fusion Proteins (rtTA and ArcER) Allow for Tight Control

[0203] HEK293 cells were constructed that stably express the polynucleotide of interest (a gene of interest (GOI)) under the control of both the TRE and ArcO (AO). As shown in Figure 1, transcription of polynucleotide of interest is from the CMVmin promoter with a TATA box. The CMVmin promoter is flanked 5’ by a TRE and 3’ by an AO. The reverse tetracycline transcriptional activator (rtTA) is an RFP composed of a reverse Tet DNA binding domain (rTet) and the VP16 transactivation domain (VP16 AD). The rTet moiety can bind the ligand tetracycline, doxycycline (dox) and derivatives thereof. ArcER is an RFP where the Arc repressor binding domain (Arc) is fused to the estrogen receptor ligand binding domain (ER). The ER moiety can bind estrogen, estradiol (E2), tamoxifen, 4-hydroxytamoxifen (OHT) and other derivatives thereof. For example, the polynucleotide can be a Rep-type gene or a non-AAV helper gene (that is, a non-AAV gene that helps produce AAV).

[0204] Figure 1 depicts a repressed GOI and induced GOI. In the repressed state, the first ligand, here dox, is absent, which means that rtTA is unable to bind TRE to allow transcription (trx) to proceed. Additionally, the second ligand, here OHT, is present and binds to the ArcER regulatory fusion protein (RFP), thereby resulting in inhibition of transcription of polynucleotide of interest.

[0205] In the induced state, the first ligand, here dox, binds to rtTA and enables it to bind TRE, which is permissive for transcription (trx). When the second ligand is absent, here OHT, ArcER does not inhibit transcription of Polynucleotide of interest. It is believed that ArcER is not transported into the nucleus in the absence of an estrogen receptor ligand like OHT or E2. Thus, transcription can proceed where the first ligand is present, and the second ligand is absent.

[0206] For example, Rep gene expressed in HEK293 cells (TRE-AO) can be made by inserting a DNA cassette encoding selectable markers and reporter proteins, as well as polynucleotides encoding rtTA and ArcER into the genome of the cell at the Stable Site 1 according to U.S. Patent No.7,771,997. A cassette containing a polynucleotide encoding Polynucleotide of interest with a CMVmin135975-75120 REGN 11751 promoter can be inserted into the cellular genome at Stable Site 2 according to the teachings of U.S. Patent No.9,816,110, for example. The reporter protein was included to confirm integration of the expression cassette into the cellular genome. See Figure 3.

[0207] Stable integration of the two expression cassettes was confirmed utilizing the included selectable markers.

[0208] The ability to tightly control transcription of polynucleotide of interest in the TRE-AO HEK293 cells was then tested and compared to a negative control (unmodified cell). As expected, when the ligand for ArcER, here E2, was present and the ligand for rtTA, here Dox, was absent, expression of polynucleotide of interest was highly suppressed. The levels of Polynucleotide of interest expression were close to those of the negative control. Additionally, levels of polynucleotide of interest expression in the TRE-TO HEK293 cells (See Figure 2 and Example 2) were lower than those in a control cell (standard CMV-TO) that was able to express polynucleotide of interest in the presence of Dox. This demonstrates that the TRE- AO system provided tighter control of transcription as compared to a TetO alone. Further, with +dox and -E2, high levels of polynucleotide of interest expression were observed (TRE-TO induced).

[0209] Thus, the TRE-AO system provides a means of tightly controlling the transcription of polynucleotides of interest. Example 2 Regulatory Fusion Protein (rtTA) with Repressor Protein (TetR) Allows for Tight Control

[0210] Another approach to express the Polynucleotide of interest (a gene of interest (GOI)) under the control of TRE and a separate TetO (TO). Transcription of a polynucleotide of interest is from the CMVmin promoter with a TATA box. The CMVmin promoter is flanked 5’ by a TRE and 3’ by a TetO (TO). In the absence of the ligand dox, rtTA cannot bind to TRE, which prevents transcription. Additionally, the Tet Repressor protein (TetR) binds to the tetracycline operator (TO) in the absence of the ligand dox, which also blocks transcription (FIGURE 2). For135975-75120 REGN 11751 example, the polynucleotide can be a Rep-type gene or a non-AAV helper gene (that is, a non-AAV gene that helps produce AAV).

[0211] When the TetR ligand is present, here dox, it binds to the rtTA, and thereby is permissive for transcription. Additionally, the dox ligand binds to TetR, which lessens the affinity of the Tet repressor for TO and is permissive for transcription. The polynucleotide encoding the repressor protein, such as TetR, can be inserted randomly into the genome or site-specifically into the genome. Example 3 HEK 293 Cells

[0212] One or more Cap-containing polynucleotides according to Figures 5-18B are transfected into the HEK293 cells. Also, transfected into the HEK293 cell genome a polynucleotide containing Polynucleotide of interests according to Figures 1 and 2. A preferred promoter is the hCMV-IE promoter, and optionally a tet operator can be operably linked to the promoter for expression control. Optionally, an intron can be located 3’ of the promoter. A preferred intron is an hCMV-IE intron. AAV Cap, Rep and ITRs can be obtained from any AAV serotype. AAV polynucleotide sequences are set forth in Example 11.

[0213] AAV ITRs and Rep and Ad E1A, E1B, E2A (or E2A partial sequence (E2A orf)), E4 (or E4 partial sequence (E4 orf 6)) and VA RNA can be randomly integrated, site-specifically integrated or remain on a plasmid. Adenovirus polynucleotide sequences are available and are exemplified in Example 12. Example 4 Cap Gene expression and Desired Protein VP Ratios

[0214] Cap gene containing polynucleotide (such as shown in Figure 7A) is transfected into the HEK293 cell genome. The polynucleotide is a Cap expression cassette in the 5’ to 3’ direction with (i) an hCMV promoter, (ii) a lactose operator, (iii) a Kozak consensus sequence, and (iv) a representative Cap gene from AAV5. A Western blot analysis revealed expression of the desired protein VP ratio of approximately 1:1:10 for VP1:VP2:VP3, respectively, as depicted in Figure 7B. The135975-75120 REGN 11751 expression of protein VP1 is low due to ACG initiation in combination with Kozak sequence, expression of protein VP2 is low due to ACG initiations, and the expression of protein VP3 is with typical ATG initiation.

[0215] The Cap constructs, as depicted in Figures 7A, 8 and 9A-B, contain a modified Kozak consensus sequence which replaces the ATG start codon with the non-canonical ACG start codon. Expression of Cap without the Kozak and with an ATG start codon results in primarily VP1 expression. In that format, changing the start codon to ACG results VP3 greater than VP2 much greater than VP1 expression. Adding the Kozak-like element results in expression of VP1:VP2:VP3 at ratios more closely resembling the natural AAV production of 1:1:10. As a translation initiation factor it is believed that the Kozak-like element employed is signaling that the VP1 ACG is a non-canonical start codon, albeit its expression pales to the actually native ATG present in the downstream VP3. Example 5 Cap Gene expression and Desired Protein VP Ratios

[0216] Cap gene containing polynucleotide (such as shown in Figures 12A and 12B) is transfected into the HEK293 cell genome. The polynucleotide is a Cap expression cassette in the 5’ to 3’ direction with an hCMV promoter, a TATA box and 2 to 5 Lac operators (LacO x 2-5) or Arc Operator (AO) to control a Cap gene followed by a mCMV promoter controlling a Lac repressor (LacI) or ArcER. The LacI protein interacts with LacO x 2-5 and the ArcER is operably linked to the AO. A Western blot analysis revealed expression of the desired protein VP ratio of approximately 1:1:10 for VP1:VP2:VP3, respectively, as indicated in Figure 19. The VP expression data shows that the LacI regulation works for Cap gene. Example 6 Delaying Cap Induction Results in Better Infectivity

[0217] Cap gene containing polynucleotide (such as shown in Figures 12A and 12B) is transfected into the HEK293 cell genome. The polynucleotide is a Cap expression cassette in the 5’ to 3’ direction with an hCMV promoter, a TATA box and 2 to 5 Lac operators (LacO x 2-5) or Arc Operator (AO) to control a Cap gene135975-75120 REGN 11751 followed by a mCMV promoter controlling a Lac repressor (LacI) or ArcER. The LacI protein interacts with LacO x 2-5 and the ArcER interacts with AO. Example 7 Gene expression Constructs

[0218] Cap gene expression constructs are exemplified in Figures 9A through 20. FIGURE 9A shows a cap expression cassette containing a Kozak-ACG-Cap system a representative Cap gene (here, Cap is from AAV5). Low VP1 is achieved by alternative start codons and not by splicing. FIGURE 9B shows a cap expression cassette containing 2-5 Lac operators (LacO x 2-5) or Arc Operator (ArcO or AO), a Kozak-ACG-Cap system having a representative Cap gene (here, Cap is from AAV5). Low VP1 is achieved by alternative start codons and not by splicing. ACG is a weaker alternative to ATG initiation codon. Translation can start from ACG, but this event is less efficient, so the amount of protein generated from ACG start can be lower than from the ATG. Kozak sequence enhances translation initiation and can be included to help ACG initiation.

[0219] FIGURE 10 depicts a cap expression cassette containing an IRES-Cap- IRES-Cap (ICIC). The schematic diagram of a polynucleotide comprising a promoter, an intron, two internal ribosome entry sites, two polynucleotides encoding AAV and a representative AAV5 Cap protein.

[0220] FIGURE 11 depicts a cap expression cassette containing an IRES-Cap- IRES-Cap (ICIC). The schematic diagram of a polynucleotide comprising a CMV promoter, a Lac operator (LacO) or an Arc Operator (ArcO), a CMV intron, two internal ribosome entry sites, two polynucleotides encoding a representative AAV5 Cap protein.

[0221] FIGURE 12A is a schematic illustration in a 5’ to 3’ direction of an hCMV promoter, a TATA box and 2 to 5 Lac operators (LacO x 2-5) to control a Cap gene followed by an mCMV promoter controlling a Lac repressor (LacI). Lac repressor protein is schematically depicted interacting with LacO x 2-5. In the presence (+) of IPTG (isopropyl β-D-1 thiogalactopyranoside), LacI will not bind to LacO. FIGURE 12B is a schematic depiction in a 5’ to 3’ direction of an hCMV promoter, a TATA box and an Arc operator to control a Cap gene followed by a mCMV promoter controlling135975-75120 REGN 11751 an ArcER repressor (ArcER). ArcER repressor protein is schematically depicted interacting with an ArcO. In the absence (-) of OHT, ArcER no longer binds to AO.

[0222] FIGURE 13A is a schematic illustration in a 5’ to 3’ direction of an hCMV promoter, a TATA box, 2 to 5 Lac operators (LacO x 2-5), a Cap polynucleotide followed by an IRES, a polynucleotide encoding rep52, a polyadenylation signal (pA), a LacI polynucleotide and a pA under control of an mCMV promoter. FIGURE 13B is a schematic depiction in a 5’ to 3’ direction of an hCMV promoter, a TATA box, 2 to 5 Lac operators (LacO x 2-5), a Cap polynucleotide followed by an IRES, a polynucleotide encoding rep52, a polyadenylation signal (pA), and a LacI polynucleotide under control of an mCMV promoter. FIGURE 13C is a schematic depiction in a 5’ to 3’ direction of an hCMV promoter, a TATA box, an Arc operator, a Cap polynucleotide followed by an IRES, and a polynucleotide encoding rep52, a pA and an ArcEr polynucleotide under control of an mCMV promoter.

[0223] FIGURE 14A is a schematic illustration in a 5’ to 3’ direction of an hCMV promoter, TATA box, 2 to 5 Lac operators (LacO x 2-5), to control a Cap gene, followed by 2A (a small peptide allowing multiple proteins to be expressed from one open reading frame), a polynucleotide encoding rep52 and a polyadenylation signal (pA), followed by a LacI polynucleotide and a pA under control of an mCMV promoter. FIGURE 14B is a schematic illustration in a 5’ to 3’ direction an hCMV promoter, TATA box, and an AO to control a Cap gene, followed by 2A, a polynucleotide encoding rep52 and a polyadenylation signal (pA), followed by a ArcEr polynucleotide and a pA under control of an mCMV promoter.

[0224] FIGURE 15A is a schematic illustration in a 5’ to 3’ direction of an hCMV promoter, TATA box, 2 to 5 Lac operators (LacO x 2-5), to control a Cap gene, followed by a polyadenylation signal (pA). Next, in a 5’ to 3’ direction there is an hCMV promoter, TATA box, 2 to 5 Lac operators (LacO x 2-5), to control a polynucleotide encoding rep52 and a pA, followed by a LacI polynucleotide and a pA under control of an mCMV promoter. FIGURE 15B is a schematic illustration in a 5’ to 3’ direction of an hCMV promoter, TATA box and an AO, to control a Cap gene, followed by a polyadenylation signal (pA). Next, in a 5’ to 3’ direction of an hCMV promoter, TATA box, and an AO to control a polynucleotide encoding rep52 and a pA., followed by an mCMV promoter controlling ArcEr and a pA.135975-75120 REGN 11751

[0225] FIGURE 16A is a schematic illustration in a 5’ to 3’ direction of an hCMV promoter, a TATA box and 2 to 5 Lac operators (LacO x 2-5) to control a Cap polynucleotide, followed by an IRES, a polynucleotide encoding rep52, a 2A polynucleotide sequence and a polynucleotide encoding VP3-SpyTag fusion protein and a pA, followed by a LacI polynucleotide and a pA under control of an mCMV promoter. FIGURE 16B is a schematic depiction in a 5’ to 3’ direction of an hCMV promoter, a TATA box and 2 to 5 Lac operators (LacO x 2-5) to control a Cap polynucleotide, followed by an IRES, a polynucleotide encoding rep52, a 2A polynucleotide sequence and a polynucleotide encoding VP3-SpyTag fusion protein and pA, followed by a LacI polynucleotide under control of an mCMV promoter . FIGURE 16C is a schematic illustration in a 5’ to 3’ direction of an hCMV promoter, a TATA box and an AO to control a Cap polynucleotide, followed by an IRES, a polynucleotide encoding rep52, a 2A polynucleotide sequence and a polynucleotide encoding VP3-SpyTag fusion protein and pA, followed by an ArcEr polynucleotide under control of an mCMV promoter.

[0226] FIGURE 17A is a schematic illustration in a 5’ to 3’ direction of an hCMV promoter, a TATA box and 2 to 5 Lac operators (LacO x 2-5) to control a Cap polynucleotide, followed by an IRES, a polynucleotide encoding VP3-SpyTag fusion protein, a 2A polynucleotide sequence and a polynucleotide encoding rep52 and a pA, followed by a LacI polynucleotide and a pA under control of an mCMV promoter. FIGURE 17B is a schematic illustration in a 5’ to 3’ direction of an hCMV promoter, a TATA box and AO to control a Cap polynucleotide, followed by an IRES, a polynucleotide encoding VP3-SpyTag fusion protein, a 2A polynucleotide sequence and a polynucleotide encoding rep52 and a pA, followed by an ArcEr polynucleotide under control of an mCMV promoter. FIGURE 17C is a schematic illustration in a 5’ to 3’ direction of an hCMV promoter, a TATA box and AO to control a Cap polynucleotide, followed by an IRES, a polynucleotide encoding VP3-SpyTag fusion protein, a 2A polynucleotide sequence and a polynucleotide encoding rep52 and a pA, followed by an ArcEr polynucleotide and a pA under control of an mCMV promoter.

[0227] FIGURE 18A is a schematic illustration in a 5’ to 3’ direction of an hCMV promoter, a TATA box and 2 to 5 Lac operators (LacO x 2-5) to control a Cap polynucleotide, followed by an IRES, a polynucleotide encoding VP3-SpyTag fusion135975-75120 REGN 11751 protein, and a pA, followed by a second hCMV promoter, second TATA box and a second LacO x 2-5 to control expression of a polynucleotide encoding Rep52 and a pA, followed by a LacI polynucleotide and a pA under control of an mCMV promoter. FIGURE 18B is a schematic illustration in a 5’ to 3’ direction of an hCMV promoter, a TATA box and an AO to control a Cap polynucleotide, followed by an IRES, a polynucleotide encoding VP3-SpyTag fusion protein and a pA, followed by a second hCMV promoter, second TATA box and a second AO to control a polynucleotide encoding Rep52, followed by an mCMV promoter controlling ArcEr and a pA.

[0228] FIGURE 19 is a schematic presentation showing that the lac repressor (LacI) regulation works for Cap in a transient transfection. IPTG addition induced CAP expression by removal of LacI.

[0229] FIGURE 20 showing data showing delaying Cap induction results in better infectivity (Higher Full:Empty ratio). Replication of ITR-GOI was induced by adding Dox (-OHT) on day 0 and allowing it to accumulate for 24 hours, then IPTG was added to induce Cap expression. Less empty capsids were generated when delayed for two days; cap is made when enough ITR is replicated to be packaged. If cap is made in the absence of sufficient amount of ITR-GOI, it results in formation of empty capsids. Example 8 Helper Gene expression

[0230] FIGURE 21 is a representative of FIGURES 22-31. pAd helper individual genes can be put together in expression cassettes where individual ORFs can be joined by 2A peptides (see FIGURES 29-31), and can be controlled by a TRE, promoter TATA box and AO or TO. AAV ITRs and Rep and Ad E1A, E1B, E2A (or E2A partial sequence (E2A orf)), E4 (or E4 partial sequence (E4 orf 6)) and VA RNA can be randomly integrated, site-specifically integrated or remain on a plasmid. Adenovirus helper polynucleotide sequences are available and are exemplified in Example 13. Helper polynucleotide sequences from other viruses, such as Herpes simplex virus (HSV) helper polynucleotide sequences, Human Papilloma Virus (HPV) Helper Polynucleotide Sequences, and Bocavirus 1 Helper Polynucleotide135975-75120 REGN 11751 Sequence also can be used and are available and exemplified in Examples 20, 21 and 22, respectively.

[0231] FIGURE 22 is a schematic illustration of a pAd helper gene E4orf6 with VA RNA in an expression cassette in a 5’ to 3’ direction .

[0232] FIGURE 23 is a schematic illustration of a pAd helper gene E2A ORF2 in an expression cassette in a 5’ to 3’ direction .

[0233] FIGURE 24 is a schematic illustration of a pAd helper gene E2A ORF4 in an expression cassette in a 5’ to 3’ direction.

[0234] FIGURE 25 is a schematic illustration of a pAd helper gene E2A DBP in an expression cassette in a 5’ to 3’ direction.

[0235] FIGURE 26 is a schematic illustration of a pAd helper gene E2A 22K in an expression cassette in a 5’ to 3’ direction.

[0236] FIGURE 27 is a schematic illustration of a pAd helper l gene E2A 33K in an expression cassette in a 5’ to 3’ direction.

[0237] FIGURE 28 is a schematic illustration of a pAd helper gene E2A 33K native in an expression cassette in a 5’ to 3’ direction.

[0238] FIGURE 29 is a schematic illustration of pAd helper genes E2A-DBP, E2A-ORF2 and E2A-ORF4 joined in consolidated expression cassettes where individual ORFs are connected by 2A peptides in a 5’ to 3’ direction.

[0239] FIGURE 30 is a schematic illustration of pAd helper genes E2A-DBP, E2A-ORF2, E2A-ORF4 and E4ORF6-VA joined in consolidated expression cassettes where individual ORFs are connected by 2A peptides in a 5’ to 3’ direction.

[0240] FIGURE 31 is a schematic illustration of pAd helper genes E4ORF6, E2A- DBP, and E2A 33K native joined in consolidated expression cassettes where individual ORFs are connected by 2A peptides in a 5’ to 3’ direction. Example 9135975-75120 REGN 11751

[0241] FIGURE 32 is schematic map of a polynucleotide showing Splice Donor and Splice Acceptor locations FIGURES 33A to 33N depict Rep2 and Cap5 sequences in a polynucleotide, with sequence joinder depicted in FIGURE 33H. Example 10 Control of the Production of Regulatory Fusion Protein or a Repressor Protein

[0242] Optionally, production of a regulatory fusion protein, such as rtTA, or a repressor protein can be controlled by another regulatory fusion protein and ligand, such as ArcER and AO. An exemplary DNA construct has a CMV promoter, and a TATA box and AO downstream followed by the gene encoding rtTA. ArcER in the presence of the ligand OHT can bind to AO and block transcription (trx). In the absence of a ligand like OHT, ArcER is no longer available to bind AO, which is permissive for transcription (trx) of the gene encoding rtTA.

[0243] The use of an RFP, such as ArcER, to control the level of expression of another RFP, such as rtTA, is another optional approach for controlling transcription of a polynucleotide of interest that is under control of that RFP (rtTA in this instance) according to the inventions described herein. Example 11 Production of eGFP

[0244] HEK293 cells were stably transfected with transcriptional regulators: rtTA and ArcER, Rep (schematically depicted in FIGURE 5), adenovirus helper genes (as depicted in FIGURES 22-29), Cap5 (as depicted in FIGURES 9A-9B) and ITR- flanked eGFP (enhanced green fluorescent protein). Clonal producer cell lines were established and evaluated for AAV production upon induction, using media without repressor molecule (estradiol or 4-hydroxy-tamoxifen), but supplemented with inducer molecules (doxycycline and IPTG). After 3 days, AAV titers were measured to be 1.2 x 1012viral genomes / ml. Example 12 AAV Polynucleotide Sequences135975-75120 REGN 11751

[0245] 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 GAGCTGGTCGGGTGGCTGGTGGACCGGGGCATCACCTCCGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCGTAC ATCTCCTTCAACGCCGCTTCCAACTCGCGGTCCCAGATCAAGGCCGCTCTGGACAATGCCGGCAAGATCATGGCG CTGACCAAATCCGCGCCCGACTACCTGGTAGGCCCCGCTCCGCCCGCGGACATTAAAACCAACCGCATCTACCGC135975-75120 REGN 11751 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 CTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAG GATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGAC GCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATC135975-75120 REGN 11751 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 GAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAAC AGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTAC CTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTC135975-75120 REGN 11751 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 ATCTCCTTCAACGCCGCCTCCAACTCGCGGTCCCAGATCAAGGCCGCGCTGGACAATGCCTCCAAGATCATGAGC CTGACAAAGACGGCTCCGGACTACCTGGTGGGCAGCAACCCGCCGGAGGACATTACCAAAAATCGGATCTACCAA ATCCTGGAGCTGAACGGGTACGATCCGCAGTACGCGGCCTCCGTCTTCCTGGGCTGGGCGCAAAAGAAGTTCGGG135975-75120 REGN 11751 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 CCGCAGCTTCCGAACTGGTTCGCGGTGACCAAGACGCGTAATGGCGCCGGAGGCGGGAACAAGGTGGTGGACGAC TGCTACATCCCCAACTACCTGCTCCCCAAGACCCAGCCCGAGCTCCAGTGGGCGTGGACTAACATGGACCAGTAT ATAAGCGCCTGTTTGAATCTCGCGGAGCGTAAACGGCTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAG135975-75120 REGN 11751 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) ATGGCTACCTTCTATGAAGTCATTGTTCGCGTCCCATTTGACGTGGAGGAACATCTGCCTGGAATTTCTGACAGC TTTGTGGACTGGGTAACTGGTCAAATTTGGGAGCTGCCTCCAGAGTCAGATTTAAATTTGACTCTGGTTGAACAG CCTCAGTTGACGGTGGCTGATAGAATTCGCCGCGTGTTCCTGTACGAGTGGAACAAATTTTCCAAGCAGGAGTCC135975-75120 REGN 11751 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 Rep78: (SEQ ID NO:15)135975-75120 REGN 11751 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 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-7135975-75120 REGN 11751 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 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 AGCACCGGACAAGTCAGCGTGGAAATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACCCGGAGATT135975-75120 REGN 11751 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 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 ACAGAGGAATACGGTATCGTGGCAGATAACTTGCAGCAGCAAAACACGGCTCCTCAAATTGGAACTGTCAACAGC135975-75120 REGN 11751 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 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 CTGACCAAATCCGCGCCCGACTACCTGGTAGGCCCGTCCTTACCCGCGGACATTAAGGCCAACCGCATCTACCGC135975-75120 REGN 11751 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 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 ATAAGCGCGTGTCTAAACCTCGCGGAGCGTAAACGGCTCGTGGCGCAGCACCTGACCCACGTCAGCCAGACGCAG135975-75120 REGN 11751 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 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 GCCAAGTTTTTTGTGCAGTTTGAAAAGGGGGACTCGTACTTTCATTTGCATATTCTGATTGAAATTACCGGCGTG135975-75120 REGN 11751 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 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)135975-75120 REGN 11751 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 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 ACCAGATACCTGACTCGTAATCTGTAA135975-75120 REGN 11751 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 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 CGTCAACAGT135975-75120 REGN 11751 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 GAATCCTCTC135975-75120 REGN 11751 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 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 CTTCAACATC135975-75120 REGN 11751 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 CATCGCCAATAACCTTACCAGCACGATTCAGGTCTTTACGGACTCGGAATACCAGCTCCCGTACGTGCTCGGCTCGG CGCACCAGGGCTGCCTGCCTCCGTTCCCGGCGGACGTCTTCATGATTCCTCAGTACGGGTACCTGACTCTGAACAAT GGCAGTCAGGCTGTGGGCCGGTCGTCCTTCTACTGCCTGGAGTACTTTCCTTCTCAAATGCTGAGAACGGGCAACAA135975-75120 REGN 11751 CTTTGAATTCAGCTACAACTTCGAGGACGTGCCCTTCCACAGCAGCTACGCGCACAGCCAGAGCCTGGACCGGCTGA TGAACCCTCTCATCGACCAGTACTTGTACTACCTGTCCCGGACTCAAAGCACGGGCGGTACTGCAGGAACTCAGCAG TTGCTATTTTCTCAGGCCGGGCCTAACAACATGTCGGCTCAGGCCAAGAACTGGCTACCCGGTCCCTGCTACCGGCA GCAACGCGTCTCCACGACACTGTCGCAGAACAACAACAGCAACTTTGCCTGGACGGGTGCCACCAAGTATCATCTGA ATGGCAGAGACTCTCTGGTGAATCCTGGCGTTGCCATGGCTACCCACAAGGACGACGAAGAGCGATTTTTTCCATCC AGCGGAGTCTTAATGTTTGGGAAACAGGGAGCTGGAAAAGACAACGTGGACTATAGCAGCGTGATGCTAACCAGCGA GGAAGAAATAAAGACCACCAACCCAGTGGCCACAGAACAGTACGGCGTGGTGGCCGATAACCTGCAACAGCAAAACG CCGCTCCTATTGTAGGGGCCGTCAATAGTCAAGGAGCCTTACCTGGCATGGTGTGGCAGAACCGGGACGTGTACCTG CAGGGTCCCATCTGGGCCAAGATTCCTCATACGGACGGCAACTTTCATCCCTCGCCGCTGATGGGAGGCTTTGGACT GAAGCATCCGCCTCCTCAGATCCTGATTAAAAACACACCTGTTCCCGCCGATCCTCCGACCACCTTCAATCAGGCCA AGCTGGCTTCTTTCATCACGCAGTACAGTACCGGTCAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAGAAC AGCAAACGCTGGAACCCAGAGATTCAGTACACTTCCAACTACTACAAATCTACAAATGTGGACTTTGCTGTCAATAC TGAGGGTACTTATTCCGAGCCTCGCCCCATTGGCACCCGTTACCTCACCCGTAATCTGTAA AAV27M8: AAV2 / 7m8 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)135975-75120 REGN 11751 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 TCCACCACTGCCCCCGCGACCGGCACGTACAACCTCCAGGAAATCGTGCCCGGCAGCGTGTGGATGGAGAGGGAC135975-75120 REGN 11751 GTGTACCTCCAAGGACCCATCTGGGCCAAGATCCCAGAGACGGGGGCGCACTTTCACCCCTCTCCGGCCATGGGC GGATTCGGACTCAAACACCCACCGCCCATGATGCTCATCAAGAACACGCCTGTGCCCGGAAATATCACCAGCTTC TCGGACGTGCCCGTCAGCAGCTTCATCACCCAGTACAGCACCGGGCAGGTCACCGTGGAGATGGAGTGGGAGCTC AAGAAGGAAAACTCCAAGAGGTGGAACCCAGAGATCCAGTACACAAACAACTACAACGACCCCCAGTTTGTGGAC TTTGCCCCGGACAGCACCGGGGAATACAGAAGCACCAGACCTATCGGAACCCGATACCTTACCCGACCCCTTTAA Example 13 Adenovirus Polynucleotide Sequences

[0246] 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 CAGAGCGTTTGCCGCGCGTTTCTCGTCGCGTCCAAGACCCTCAAAGATTTTTGGCACTTCGTCGAGCGAGGCGAT135975-75120 REGN 11751 ATCAGGTATGACAGCGCCCTGCCGCAAGGCCAGCTGCTTGTCCGCTCGGCTGCGGTTGGCACGGCAGGATAGGGG TATCTTGCAGTTTTGGAAAAAGATGTGATAGGTGGCAAGCACCTCTGGCACGGCAAATACGGGGTAGAAGTTGAG GCGCGGGTTGGGCTCGCATGTGCCGTTTTCTTGGCGTTTGGGGGGTACGCGCGGTGAGAACAGGTGGCGTTCGTA GGCAAGGCTGACATCCGCTATGGCGAGGGGCACATCGCTGCGCTCTTGCAACGCGTCGCAGATAATGGCGCACTG GCGCTGCAGATGCTTCAACAGCACGTCGTCTCCCACATCTAGGTAGTCGCCATGCCTTTGGTCCCCCCGCCCGAC TTGTTCCTCGTTTGCCTCTGCGTCGTCCTGGTCTTGCTTTTTATCCTCTGTTGGTACTGAGCGATCCTCGTCGTC TTCGCTTACAAAACCTGGGTCCTGCTCGATAATCACTTCCTCCTCCTCAAGCGGGGGTGCCTCGACGGGGAAGGT GGTAGGCGCGTTGGCGGCATCGGTGGAGGCGGTGGTGGCGAACTCAAAGGGGGCGGTTAGGCTGTCCTCCTTCTC GACTGACTCCATGATCTTTTTCTGCCTATAGGAGAAGGAAATGGCCAGTCGGGAAGAGGAGCAGCGCGAAACCAC CCCCGAGCGCGGACGCGGTGCGGCGCGACGTCCACCAACCATGGAGGACGTGTCGTCCCCGTCGCCGTCGCCGCC 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 CCCTTTGATTTTTAA135975-75120 REGN 11751 E4 Full Sequence (SEQ ID NO:41) CCCGGGCGTTTTAGGGCGGAGTAACTTGCATGTATTGGGAATTGTAGTTTTTTTAAAATGGGAAGTGACGTATCG TGGGAAAACGGAAGTGAAGATTTGAGGAAGTTGTGGGTTTTTTGGCTTTCGTTTCTGGGCGTAGGTTCGCGTGCG GTTTTCTGGGTGTTTTTTGTGGACTTTAACCGTTACGTCATTTTTTAGTCCTATATATACTCGCTCTGTACTTGG CCCTTTTTACACTGTGACTGATTGAGCTGGTGCCGTGTCGAGTGGTGTTTTTTAATAGGTTTTTTTACTGGTAAG GCTGACTGTTATGGCTGCCGCTGTGGAAGCGCTGTATGTTGTTCTGGAGCGGGAGGGTGCTATTTTGCCTAGGCA GGAGGGTTTTTCAGGTGTTTATGTGTTTTTCTCTCCTATTAATTTTGTTATACCTCCTATGGGGGCTGTAATGTT GTCTCTACGCCTGCGGGTATGTATTCCCCCGGGCTATTTCGGTCGCTTTTTAGCACTGACCGATGTTAACCAACC TGATGTGTTTACCGAGTCTTACATTATGACTCCGGACATGACCGAGGAACTGTCGGTGGTGCTTTTTAATCACGG 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 GCGCTGCTGCAGCACCACCGCCCTATCCTGATGCACGATTATGACTCTACCCCCATGTAG135975-75120 REGN 11751 VA Sequence (VA transcripts I and II are underlined) (SEQ ID NO:43) CGTAATCCGTAGATGTACCTGGACATCCAGGTGATGCCGGCGGCGGTGGTGGAGGCGCGCGGAAAGTCGCGGAC GCGGTTCCAGATGTTGCGCAGCGGCAAAAAGTGCTCCATGGTCGGGACGCTCTGGCCGGTGAGGCGTGCGCAGTC GTTGACGCTCTAGACCGTGCAAAAGGAGAGCCTGTAAGCGGGCACTCTTCCGTGGTCTGGTGGATAAATTCGCAA GGGTATCATGGCGGACGACCGGGGTTCGAACCCCGGATCCGGCCGTCCGCCGTGATCCATGCGGTTACCGCCCGC GTGTCGAACCCAGGTGTGCGACGTCAGACAACGGGGGAGCGCTCCTTTTGGCTTCCTTCCAGGCGCGGCGGCTGC TGCGCTAGCTTTTTTGGCCACTGGCCGCGCGCGGCGTAAGCGGTTAGGCTGGAAAGCGAAAGCATTAAGTGGCTC GCTCCCTGTAGCCGGAGGGTTATTTTCCAAGGGTTGAGTCGCAGGACCCCCGGTTCGAGTCTCGGGCCGGCCGGA 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 GTTGTGAGACACAAGAATCGCCTGCTACTGTTGTCTTCCGTCCGCCCGGCGATAATACCGACGGAGGAGCAGCAG135975-75120 REGN 11751 CAGCAGCAGGAGGAAGCCAGGCGGCGGCGGCAGGAGCAGAGCCCATGGAACCCGAGAGCCGGCCTGGACCCTCGG GAATGA Ad5 E1B_55K Accession: AAQ19287.1 (SEQ ID NO:47) ATGGAGCGAAGAAACCCATCTGAGCGGGGGGTACCTGCTGGATTTTCTGGCCATGCATCTGTGGAGAGCGGTTGT GAGACACAAGAATCGCCTGCTACTGTTGTCTTCCGTCCGCCCGGCGATAATACCGACGGAGGAGCAGCAGCAGCA GCAGGAGGAAGCCAGGCGGCGGCGGCAGGAGCAGAGCCCATGGAACCCGAGAGCCGGCCTGGACCCTCGGGAATG AATGTTGTACAGGTGGCTGAACTGTATCCAGAACTGAGACGCATTTTGACAATTACAGAGGATGGGCAGGGGCTA AAGGGGGTAAAGAGGGAGCGGGGGGCTTGTGAGGCTACAGAGGAGGCTAGGAATCTAGCTTTTAGCTTAATGACC AGACACCGTCCTGAGTGTATTACTTTTCAACAGATCAAGGATAATTGCGCTAATGAGCTTGATCTGCTGGCGCAG AAGTATTCCATAGAGCAGCTGACCACTTACTGGCTGCAGCCAGGGGATGATTTTGAGGAGGCTATTAGGGTATAT GCAAAGGTGGCACTTAGGCCAGATTGCAAGTACAAGATCAGCAAACTTGTAAATATCAGGAATTGTTGCTACATT TCTGGGAACGGGGCCGAGGTGGAGATAGATACGGAGGATAGGGTGGCCTTTAGATGTAGCATGATAAATATGTGG CCGGGGGTGCTTGGCATGGACGGGGTGGTTATTATGAATGTAAGGTTTACTGGCCCCAATTTTAGCGGTACGGTT TTCCTGGCCAATACCAACCTTATCCTACACGGTGTAAGCTTCTATGGGTTTAACAATACCTGTGTGGAAGCCTGG ACCGATGTAAGGGTTCGGGGCTGTGCCTTTTACTGCTGCTGGAAGGGGGTGGTGTGTCGCCCCAAAAGCAGGGCT TCAATTAAGAAATGCCTCTTTGAAAGGTGTACCTTGGGTATCCTGTCTGAGGGTAACTCCAGGGTGCGCCACAAT GTGGCCTCCGACTGTGGTTGCTTCATGCTAGTGAAAAGCGTGGCTGTGATTAAGCATAACATGGTATGTGGCAAC TGCGAGGACAGGGCCTCTCAGATGCTGACCTGCTCGGACGGCAACTGTCACCTGCTGAAGACCATTCACGTAGCC AGCCACTCTCGCAAGGCCTGGCCAGTGTTTGAGCATAACATACTGACCCGCTGTTCCTTGCATTTGGGTAACAGG AGGGGGGTGTTCCTACCTTACCAATGCAATTTGAGTCACACTAAGATAT...

Claims

135975-75120 REGN 11751 CLAIMS 1. A polynucleotide comprising in the 5’ to 3’ direction (a) a tetracycline response element (TRE), (b) a first promoter, (c) an Arc operator (AO) or a tetracycline operator (TO), and (d) a polynucleotide of interest, wherein the polynucleotide of interest is selected from the group consisting of: (i) a Rep gene fused to a portion of an AAV Cap gene to provide alternative forms Rep68 and Rep40, and (ii) a non-AAV helper gene.

2. The polynucleotide according to claim 1, wherein the (a) tetracycline response element (TRE), (b) the first promoter, (c) the Arc operator (AO) or tetracycline operator (TO), and (d) the polynucleotide of interest are operably linked.

3. The polynucleotide according to claim 1, wherein the polynucleotide comprises (iii) AO.

4. The polynucleotide according to claim 1, wherein the polynucleotide comprises (iii) TO.

5. A cell capable of controlled transcription of a polynucleotide of interest, wherein the cell comprises a cell genome comprising (A) a promoter operably linked to the polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding an activator; (C) a second operator; and (D) a polynucleotide encoding a repressor; wherein transcription of the polynucleotide of interest is inhibited in the absence of an effective amount if an activator ligand and the presence of an effective amount of a repressor ligand; and permitted in the presence of an effective amount of the activator ligand and the absence of an effective amount of the repressor ligand, and wherein the polynucleotide of interest is selected from the group consisting of : (i) a Rep gene fused to a portion of an AAV Cap gene to provide alternative forms Rep68 and Rep40, and (ii) a non-AAV helper gene.135975-75120 REGN 11751 6. The cell according to claim 5, wherein the first operator is a tetracycline response element (TRE).

7. The cell according to claim 6, wherein the second operator is a tetracycline operator (TO).

8. A cell capable of controlled transcription of a polynucleotide of interest, wherein the cell comprises a cell genome comprising: (A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding a first regulatory fusion protein (RFP), where the first RFP comprises: (1) a transcription activating domain fused to a first DNA binding domain; and (2) a ligand-binding domain; wherein the first ligand is capable of binding to the ligand-binding domain of the first RFP, and wherein the DNA binding domain of the first RFP is capable of binding to the operator positioned 5’ when in the presence of the first ligand; (C) a second operator; and (D) a polynucleotide encoding the second RFP that differs from the first RFP, wherein the second RFP comprises: (1) a DNA-binding domain; and (2) a ligand-binding domain; wherein the second ligand is capable of binding to the ligand-binding domain of the second RFP, and wherein the second RFP is capable of binding to the second operator in the presence of the second ligand; wherein transcription of the polynucleotide of interest is inhibited in the absence of an effective amount of the first ligand and in the presence of an effective amount of the second ligand and is permitted in the presence of an effective amount of the first ligand and absence of an effective amount of the second ligand, wherein the polynucleotide of interest is selected from the group consisting of : (i) a Rep gene fused to a portion of an AAV Cap gene to provide alternative forms Rep68 and Rep40, and135975-75120 REGN 11751 (ii) a non-AAV helper gene.

9. The cell according to claim 8, wherein the first operator is a tetracycline response element (TRE).

10. The cell according to claim 8, wherein the second operator is an Arc operator (AO).

11. A method for controlling the transcription of a polynucleotide in a cell, wherein the method comprises: I. maintaining a cell in a medium without an effective amount of a ligand of an activator (activator ligand) and with an effective amount of ligand of a repressor (repressor ligand), wherein the cell is capable of controlled transcription of an AAV Rep gene, and wherein the cell comprises a cell genome comprising: A) a promoter operably linked to the polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding an activator; (C) a second operator; and (D) a polynucleotide encoding a repressor; wherein transcription of the polynucleotide of interest is inhibited in the absence of an effective amount if an activator ligand and the presence of an effective amount of a repressor ligand; and permitted in the presence of an effective amount of the activator ligand and the absence of an effective amount of the repressor ligand, and wherein the polynucleotide of interest is selected from the group consisting of : (i) a Rep gene fused to a portion of an AAV Cap gene to provide alternative forms Rep68 and Rep40, and (ii) one non-AAV helper genes.

12. The method according to claim 11, further comprising the step of135975-75120 REGN 11751 II. controlling the cell to transcribe the polynucleotide of interest by maintaining the cell in a medium with an effective amount of the activator ligand and without an effective amount of the repressor ligand.

13. The method according to claim 11, wherein the first operator is a tetracycline response element (TRE).

14. The method according to claim 11, wherein the second operator is a tetracycline operator (TO).

15. A method for controlling the transcription of a polynucleotide in a cell, wherein the method comprises: I. maintaining a cell in a medium without an effective amount of a ligand of an activator (activator ligand) and with an effective amount of ligand of a repressor (repressor ligand), wherein the cell is capable of controlled transcription of an AAV Rep gene, and wherein the cell comprises a cell genome comprising: A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding a first regulatory fusion protein (RFP), where the first RFP comprises: (1) a transcription activating domain fused to a first DNA binding domain; and (2) a ligand-binding domain; wherein the first ligand is capable of binding to the ligand-binding domain of the first RFP, and wherein the DNA binding domain of the first RFP is capable of binding to the operator positioned 5’ when in the presence of the first ligand; (C) a second operator; and (D) a polynucleotide encoding the second RFP that differs from the first RFP, wherein the second RFP comprises: (1) a DNA-binding domain; and (2) a ligand-binding domain; wherein the second ligand is capable of binding to the ligand-binding domain of the second RFP, and wherein the second RFP is capable of binding to the second operator in the presence of the second135975-75120 REGN 11751 ligand; wherein transcription of the polynucleotide of interest is inhibited in the absence of an effective amount of the first ligand and in the presence of an effective amount of the second ligand and is permitted in the presence of an effective amount of the first ligand and absence of an effective amount of the second ligand, wherein the polynucleotide of interest is selected from the group consisting of : (i) a Rep gene fused to a portion of an AAV Cap gene t to provide alternative forms Rep68 and Rep40, and (ii) a non-AAV helper gene.

16. The method according to claim 15, further comprising the step of II. controlling the cell to transcribe the AAV Rep gene by maintaining the cell in a medium with an effective amount of the activator ligand and without an effective amount of the repressor ligand.

17. The method according to claim 15, wherein the first operator is a tetracycline response element (TRE).

18. The method according to claim 15, wherein the second operator is an Arc operator (AO).

19. A cell capable of controlled transcription of a polynucleotide of interest, wherein the cell comprises a cell genome comprising I. a first DNA construct comprising (A) a promoter operably linked to the polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding an activator; (C) a second operator; and (D) a polynucleotide encoding a repressor; wherein transcription of the polynucleotide of interest is inhibited in the absence of an effective amount if an activator ligand and the presence of an effective amount of a repressor ligand; and permitted in the presence of an effective amount of the activator ligand and the absence of an effective amount of the repressor ligand, and135975-75120 REGN 11751 wherein the polynucleotide of interest is a Rep gene fused to a portion of an AAV Cap gene to provide alternative forms Rep68 and Rep40; and II. a second DNA construct comprising (A) a promoter operably linked to the polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding an activator; (C) a second operator; and (D) a polynucleotide encoding a repressor; wherein transcription of the polynucleotide of interest is inhibited in the absence of an effective amount if an activator ligand and the presence of an effective amount of a repressor ligand; and permitted in the presence of an effective amount of the activator ligand and the absence of an effective amount of the repressor ligand, and wherein the polynucleotide of interest comprises a non-AAV helper gene.

20. The cell according to claim 19, wherein the first operator is a tetracycline response element (TRE).

21. The cell according to claim 19, wherein the second operator is a tetracycline operator (TO).

22. The cell according to claim 19, wherein the second DNA construct comprises more than one non-AAV helper gene.

23. The cell according to claim 19 or 22, wherein the non-AAV helper gene is selected from the group consisting of an adenovirus gene, a herpesvirus gene, a human papilloma virus gene, a bocavirus gene and a baculovirus gene.

24. The cell according to claim 23, wherein the non-AAV helper gene is an adenovirus gene.135975-75120 REGN 11751 25. The cell according to claim 23, wherein the adenovirus gene is selected from the group consisting of E4 orf6-VA, E2A ORF 2, E2 ORF4, E2A DBP, E2A 22K, E2A 33K, and E2A 33K native.

26. The cell according to claim 22, wherein the polynucleotide of interest of the second DNA construct comprises in the 5’ to 3’ direction one or more selected from the group consisting of: (A) adenovirus E2A DBP, a 2A sequence, adenovirus E2A ORF2, a 2A sequence, and adenovirus E2A ORF4; (B) adenovirus E2A DBP, a 2A sequence, adenovirus E2A ORF2, a 2A sequence, adenovirusE2A ORF4, a 2A sequence, adenovirus ORF6 and VA; and (C) adenovirus E4 orf6, a 2A sequence, adenovirus E2A DBP, a 2A sequence, and adenovirus E2A 33K.

27. A cell capable of controlled transcription of a polynucleotide of interest, wherein the cell comprises a cell genome comprising I. a first DNA construct comprising (A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding a first regulatory fusion protein (RFP), where the first RFP comprises: (1) a transcription activating domain fused to a first DNA binding domain; and (2) a ligand-binding domain; wherein the first ligand is capable of binding to the ligand-binding domain of the first RFP, and wherein the DNA binding domain of the first RFP is capable of binding to the operator positioned 5’ when in the presence of the first ligand; (C) a second operator; and (D) a polynucleotide encoding the second RFP that differs from the first RFP, wherein the second RFP comprises: (1) a DNA-binding domain; and (2) a ligand-binding domain; wherein the second ligand is capable of binding to the ligand-binding domain of the second RFP, and wherein the second135975-75120 REGN 11751 RFP is capable of binding to the second operator in the presence of the second ligand; wherein transcription of the polynucleotide of interest is inhibited in the absence of an effective amount of the first ligand and in the presence of an effective amount of the second ligand and is permitted in the presence of an effective amount of the first ligand and absence of an effective amount of the second ligand, wherein the polynucleotide of interest is a Rep gene fused to a portion of an AAV Cap gene to provide alternative forms Rep68 and Rep40; and II. a second DNA construct comprising (A) a promoter operably linked to a polynucleotide of interest and controlled by a first operator operably linked and positioned 5’ with respect to the promoter; (B) a polynucleotide encoding a first regulatory fusion protein (RFP), where the first RFP comprises: (1) a transcription activating domain fused to a first DNA binding domain; and (2) a ligand-binding domain; wherein the first ligand is capable of binding to the ligand-binding domain of the first RFP, and wherein the DNA binding domain of the first RFP is capable of binding to the operator positioned 5’ when in the presence of the first ligand; (C) a second operator; and (D) a polynucleotide encoding the second RFP that differs from the first RFP, wherein the second RFP comprises: (1) a DNA-binding domain; and (2) a ligand-binding domain; wherein the second ligand is capable of binding to the ligand-binding domain of the second RFP, and wherein the second RFP is capable of binding to the second operator in the presence of the second ligand; wherein transcription of the polynucleotide of interest is inhibited in the absence of an effective amount of the first ligand and in the presence of an effective amount of the second ligand and is permitted in the presence of an effective amount of the first ligand and absence of an effective amount of the second ligand, wherein the polynucleotide of interest comprises a non-AAV helper gene.

28. The cell according to claim 27, wherein the first operator is a tetracycline response element (TRE).135975-75120 REGN 11751 29. The cell according to claim 27, wherein the second operator is an Arc tetracycline operator (AO).

30. The cell according to claim 27, wherein the second DNA construct comprises more than one non-AAV helper gene.

31. The cell according to claim 27 or 30, wherein the non-AAV helper gene is selected from the group consisting of an adenovirus gene, a herpesvirus gene, a human papilloma virus gene, a bocavirus gene and a baculovirus gene.

32. The cell according to claim 27, wherein the non-AAV helper gene is an adenovirus gene.

33. The cell according to claim 32, wherein the adenovirus gene is selected from the group consisting of E4 orf6-VA, E2A ORF 2, E2 ORF4, E2A DBP, E2A 22K, E2A 33K, and E2A 33K native.

34. The cell according to claim 30, wherein the polynucleotide of interest of the second DNA construct comprises in the 5’ to 3’ direction one or more selected from the group consisting of: (A) adenovirus E2A DBP, a 2A sequence, adenovirus E2A ORF2, a 2A sequence, and adenovirus E2A ORF4; (B) adenovirus E2A DBP, a 2A sequence, adenovirus E2A ORF2, a 2A sequence, adenovirusE2A ORF4, a 2A sequence, adenovirus ORF6 and VA; and 35. A polynucleotide comprising a Cap gene, wherein the polynucleotide is selected from the group consisting of A. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box and one or more operators, such as 2 to 5 Lac operators (LacO x 2- 5) to control an AAV Cap gene, and (iii) a Lac repressor (LacI), wherein the LacI is under the control of an mCMV promoter, and wherein LacI is operably linked to LacO x 2-5;135975-75120 REGN 11751 B. a polynucleotide comprising in the 5’ to 3’ direction (i) one or more operators, such as 2 to 5 Lac operators (LacO x 2-5), (ii) an hCMV promoter, (iii) a TATA box to control an AAV Cap gene, (iv) an IRES, (v) a polynucleotide encoding AAV rep52, (vi) a first polydenylation signal (pA), (vii) a Lac repressor (LacI), and (viii) a second pA , wherein the LacI is under the control of an mCMV promoter, and wherein LacI is operably linked to LacO x 2-5; C. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) 1 to 5 operators, (iv) an AAV Cap gene, iv) a 2A polynucleotide sequence, (vi) a polynucleotide encoding AAV rep52, (viii) and a first polydenylation signal (pA), (ix) a Lac repressor (LacI), and ix) a second pA, wherein the LacI is under the control of an mCMV promoter, and optionally comprise 1 to 2 ArcO, 1 to 2 TO or 2 to 5 LacO: D. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a first TATA box, (iii) 1 to 5 operators, (iv) an AAV Cap gene, (v) a first polyadenylation signal (pA), (vi) a second hCMV promoter, (vii) a second TATA box, (viii) a polynucleotide encoding AAV rep52, (ix) a second pA, (x) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and (x) a second pA., and optionally comprise 1 to 2 ArcO, 1 to 2 TO or 2 to 5 LacO: E. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) 2 to 5 Lac operators (LacO x 2-5), (iv) an AAV Cap gene, (v) an IRES, (vi) a polynucleotide encoding an AAV rep52, (vii) a 2A polynucleotide sequence, (viii) a polynucleotide encoding VP3-SpyTag fusion protein, (ix) a first pA, (x) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and (xi) a second pA, and optionally comprise 1 to 2 ArcO or 1 to 2 TO instead of 2 to 5 LacO; F. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) 2 to 5 Lac operators (LacO x 2-5), (iv) an AAV Cap gene, (v) an IRES, (vi) a polynucleotide encoding VP3-SpyTag fusion protein, (vii) a 2A polynucleotide sequence, (viii) a polynucleotide encoding an AAV rep52, (ix) a first pA, (x) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and (xi) a second pA, and optionally comprise 1 to 2 ArcO or 1 to 2 TO instead of 2 to 5 LacO; and135975-75120 REGN 11751 G. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) a first set of LacO x 2-5, (iv) an AAV Cap gene, (v) an IRES, (vi) a polynucleotide encoding VP3-SpyTag fusion protein, (vii) a first pA, (viii) a second hCMV promoter, (ix) a second TATA box, (x) a second set of LacO x 2-5, (xi) a polynucleotide encoding an AAV rep52, (xii) a second pA, (xiii) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and (xiv) and a third pA, and optionally comprise 1 to 2 ArcO or 1 to 2 TO instead of 2 to 5 LacO.

36. A cell according to claim 19, further comprising a polynucleotide comprising a Cap gene, wherein the polynucleotide is selected from the group consisting of A. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box and one or more operators, such as 2 to 5 Lac operators (LacO x 2- 5) to control an AAV Cap gene, and (iii) a Lac repressor (LacI), wherein the LacI is under the control of an mCMV promoter, and wherein LacI is operably linked to LacO x 2-5; B. a polynucleotide comprising in the 5’ to 3’ direction (i) one or more operators, such as 2 to 5 Lac operators (LacO x 2-5), (ii) an hCMV promoter, (iii) a TATA box to control an AAV Cap gene, (iv) an IRES, (v) a polynucleotide encoding AAV rep52, (vi) a first polydenylation signal (pA), (vii) a Lac repressor (LacI), and (viii) a second pA , wherein the LacI is under the control of an mCMV promoter, and wherein LacI is operably linked to LacO x 2-5; C. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) 1 to 5 operators, (iv) an AAV Cap gene, iv) a 2A polynucleotide sequence, (vi) a polynucleotide encoding AAV rep52, (viii) and a first polydenylation signal (pA), (ix) a Lac repressor (LacI), and ix) a second pA, wherein the LacI is under the control of an mCMV promoter, and optionally comprise 1 to 2 ArcO, 1 to 2 TO or 2 to 5 LacO: D. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a first TATA box, (iii) 1 to 5 operators, (iv) an AAV Cap gene, (v) a first polyadenylation signal (pA), (vi) a second hCMV promoter, (vii) a second TATA box, (viii) a polynucleotide encoding AAV rep52, (ix) a second pA, (x) a Lac repressor135975-75120 REGN 11751 (LacI); wherein the LacI is under the control of an mCMV promoter, and (x) a second pA., and optionally comprise 1 to 2 ArcO, 1 to 2 TO or 2 to 5 LacO: E. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) 2 to 5 Lac operators (LacO x 2-5), (iv) an AAV Cap gene, (v) an IRES, (vi) a polynucleotide encoding an AAV rep52, (vii) a 2A polynucleotide sequence, (viii) a polynucleotide encoding VP3-SpyTag fusion protein, (ix) a first pA, (x) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and (xi) a second pA, and optionally comprise 1 to 2 ArcO or 1 to 2 TO instead of 2 to 5 LacO; F. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) 2 to 5 Lac operators (LacO x 2-5), (iv) an AAV Cap gene, (v) an IRES, (vi) a polynucleotide encoding VP3-SpyTag fusion protein, (vii) a 2A polynucleotide sequence, (viii) a polynucleotide encoding an AAV rep52, (ix) a first pA, (x) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and (xi) a second pA, and optionally comprise 1 to 2 ArcO or 1 to 2 TO instead of 2 to 5 LacO; and G. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) a first set of LacO x 2-5, (iv) an AAV Cap gene, (v) an IRES, (vi) a polynucleotide encoding VP3-SpyTag fusion protein, (vii) a first pA, (viii) a second hCMV promoter, (ix) a second TATA box, (x) a second set of LacO x 2-5, (xi) a polynucleotide encoding an AAV rep52, (xii) a second pA, (xiii) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and (xiv) and a third pA, and optionally comprise 1 to 2 ArcO or 1 to 2 TO instead of 2 to 5 LacO.

37. A cell according to claim 27, further comprising a polynucleotide comprising a Cap gene, wherein the polynucleotide is selected from the group consisting of A. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box and one or more operators, such as 2 to 5 Lac operators (LacO x 2- 5) to control an AAV Cap gene, and (iii) a Lac repressor (LacI), wherein the LacI is under the control of an mCMV promoter, and wherein LacI is operably linked to LacO x 2-5;135975-75120 REGN 11751 B. a polynucleotide comprising in the 5’ to 3’ direction (i) one or more operators, such as 2 to 5 Lac operators (LacO x 2-5), (ii) an hCMV promoter, (iii) a TATA box to control an AAV Cap gene, (iv) an IRES, (v) a polynucleotide encoding AAV rep52, (vi) a first polydenylation signal (pA), (vii) a Lac repressor (LacI), and (viii) a second pA , wherein the LacI is under the control of an mCMV promoter, and wherein LacI is operably linked to LacO x 2-5; C. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) 1 to 5 operators, (iv) an AAV Cap gene, iv) a 2A polynucleotide sequence, (vi) a polynucleotide encoding AAV rep52, (viii) and a first polydenylation signal (pA), (ix) a Lac repressor (LacI), and ix) a second pA, wherein the LacI is under the control of an mCMV promoter, and optionally comprise 1 to 2 ArcO, 1 to 2 TO or 2 to 5 LacO: D. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a first TATA box, (iii) 1 to 5 operators, (iv) an AAV Cap gene, (v) a first polyadenylation signal (pA), (vi) a second hCMV promoter, (vii) a second TATA box, (viii) a polynucleotide encoding AAV rep52, (ix) a second pA, (x) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and (x) a second pA., and optionally comprise 1 to 2 ArcO, 1 to 2 TO or 2 to 5 LacO: E. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) 2 to 5 Lac operators (LacO x 2-5), (iv) an AAV Cap gene, (v) an IRES, (vi) a polynucleotide encoding an AAV rep52, (vii) a 2A polynucleotide sequence, (viii) a polynucleotide encoding VP3-SpyTag fusion protein, (ix) a first pA, (x) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and (xi) a second pA, and optionally comprise 1 to 2 ArcO or 1 to 2 TO instead of 2 to 5 LacO; F. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) 2 to 5 Lac operators (LacO x 2-5), (iv) an AAV Cap gene, (v) an IRES, (vi) a polynucleotide encoding VP3-SpyTag fusion protein, (vii) a 2A polynucleotide sequence, (viii) a polynucleotide encoding an AAV rep52, (ix) a first pA, (x) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and (xi) a second pA, and optionally comprise 1 to 2 ArcO or 1 to 2 TO instead of 2 to 5 LacO; and135975-75120 REGN 11751 G. a polynucleotide comprising in the 5’ to 3’ direction (i) an hCMV promoter, (ii) a TATA box, (iii) a first set of LacO x 2-5, (iv) an AAV Cap gene, (v) an IRES, (vi) a polynucleotide encoding VP3-SpyTag fusion protein, (vii) a first pA, (viii) a second hCMV promoter, (ix) a second TATA box, (x) a second set of LacO x 2-5, (xi) a polynucleotide encoding an AAV rep52, (xii) a second pA, (xiii) a Lac repressor (LacI); wherein the LacI is under the control of an mCMV promoter, and (xiv) and a third pA, and optionally comprise 1 to 2 ArcO or 1 to 2 TO instead of 2 to 5 LacO.

38. A method according to claims 1 to 4, wherein alternative forms are produced using a P19 promoter and a non-ATG start codon.

39. A polynucleotide according to claims 11 to 18, wherein alternative forms are produced using a P19 promoter and a non-ATG start codon.

40. A cell according to claims 5 to 10 and 19 to 34, wherein alternative forms are produced using a P19 promoter and a non-ATG start codon.

41. A cell genome as set forth in any of the above claims.

42. A method for controlling the transcription of a polynucleotide in a cell using any of the cells and cell genomes of any of the above claims.