Use Of Ion Concentrations To Increase The Packaging Efficiency Of Recombinant Adeno-associated Virus

Abstract

The present invention is directed to methods for increasing the efficiencies with which recombinant adeno-associated virus (rAAV) are packaged, so as to increase their production titers. More specifically, the invention relates to a method for increasing the production titer of rAAV by transfected cells by increasing the ionic strength of the cell culture media through the administration of additional ions.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of, and claims priority to, U.S. patent application Ser. No. 16/511,612 (pending), which was filed on Jul. 15, 2019, which application is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention is directed to methods for increasing the efficiencies with which recombinant adeno-associated virus (rAAV) are packaged, so as to increase their production titers. More specifically, the invention relates to a method for increasing the production titer of rAAV by transfected cells by increasing the ionic strength of the cell culture media through the administration of additional ions.

REFERENCE TO SEQUENCE LISTING

[0003] This application includes one or more Sequence Listings pursuant to 37 C.F.R. 1.821 et seq., which are disclosed in computer-readable media (file name: 2650-0002US_ST25.txt, created on Jul. 15, 2019, and having a size of 38,334 bytes), which file is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

I. Adeno-Associated Virus (AAV)

[0004] Adeno-Associated Virus (AAV) is a small, naturally-occurring, non-pathogenic virus belonging to the Dependovirus genus of the Parvoviridae (Balakrishnan, B. et al. (2014) "Basic Biology of Adeno-Associated Virus (AAV) Vectors Used in Gene Therapy," Curr. Gene Ther. 14(2):86-100; Zinn, E. et al. (2014) "Adeno-Associated Virus: Fit To Serve," Curr. Opin. Virol. 0:90-97). Despite not causing disease, AAV is known to be able to infect humans and other primates and is prevalent in human populations (Johnson, F. B. et al. (1972) "Immunological Reactivity of Antisera Prepared Against the Sodium Dodecyl Sulfate-Treated Structural Polypeptides of Adenovirus-Associated Virus," J. Virol. 9(6):1017-1026). AAV infect a broad range of different cell types (e.g., cells of the central nervous system, heart, kidney, liver, lung, pancreas, retinal pigment epithelium or photoreceptor cells, or skeletal muscle cells). Twelve serotypes of the virus (e.g., AAV2, AAV5, AAV6, etc.), exhibiting different tissue infection capabilities ("tropisms"), have been identified (Colella, P. et al. (2018) "Emerging Issues in AAV-Mediated In Vivo Gene Therapy," Molec. Ther. Meth. Clin. Develop. 8:87-104; Hocquemiller, M. et al. (2016) "Adeno-Associated Virus-Based Gene Therapy for CNS Diseases," Hum. Gene Ther. 27(7):478-496; Lisowski, L. et al. (2015) "Adeno-Associated Virus Serotypes For Gene Therapeutics," 24:59-67).

[0005] AAV is a single-stranded DNA virus that is composed of approximately 4,700 nucleotides. The viral genome may be described as having a 5' half and a 3' half which together comprise the genes that encode the virus' proteins (FIG. 1). The 5' half of the AAV genome comprises the AAV rep gene, which, through the use of multiple reading frames, staggered initiating promoters (p5, p19 and p40) and alternate splicing, encodes four non-structural Rep proteins (Rep40, Rep52, Rep68 and Rep78) that are required for viral transcription control and replication and for the packaging of viral genomes into the viral capsule (Lackner, D. F. et al. (2002) "Studies of the Mechanism of Transactivation of the Adeno-Associated Virus p19 Promoter by Rep Protein," J. Virol. 76(16):8225-8235). The 3' half the AAV genome comprises the AAV capsid gene (cap), which encodes three capsid proteins (VP): VP1, VP2 and VP3. The three capsid proteins are translated from a single mRNA transcript that is controlled by a single promoter (p40 in case of AAV2). The 3' half of the AAV genome also comprises the AAP gene, which encodes the AAV assembly-activating protein (AAP). Sixty VP monomers (comprising approximately 5 copies of VP1, 5 copies of VP2, and 50 copies of VP3) self-assemble around the AAV genome to form the icosahedral protein shell (capsid) of the mature viral particle (Buning, H. et al. (2019) "Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors," Mol. Ther. Meth. Clin. Devel. 12:P248-P265; Van Vliet K. M. et al. (2008) The Role of the Adeno-Associated Virus Capsid in Gene Transfer. In: DRUG DELIVERY SYSTEMS, Jain, K. K. (eds.), Meth. Molec. Biol. 437:51-91). The AAV AAP protein is believed to be required for stabilizing and transporting newly produced VP proteins from the cytoplasm into the cell nucleus. The 3' half of the AAV genome also comprises the AAV X gene, which is believed to encode a protein that supports genome replication (Colella, P. et al. (2018) "Emerging Issues in AAV-Mediated In Vivo Gene Therapy," Molec. Ther. Meth. Clin. Develop. 8:87-104; Buning, H. et al. (2019) "Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors," Mol. Ther. Meth. Clin. Devel. 12:P248-P265; Cao, M. et al. (2014) "The X Gene Of Adeno-Associated Virus 2 (AAV2) Is Involved In Viral DNA Replication," PLoS ONE 9, e104596:1-10).

[0006] The above-described AAV gene-coding sequences are flanked by two AAV-specific palindromic inverted terminal repeated sequences (ITR) of 145 nucleotides (Balakrishnan, B. et al. (2014) "Basic Biology of Adeno-Associated Virus (AAV) Vectors Used in Gene Therapy," Curr. Gene Ther. 14(2):86-100; Colella, P. et al. (2018) "Emerging Issues in AAV-Mediated In Vivo Gene Therapy," Molec. Ther. Meth. Clin. Develop. 8:87-104).

[0007] AAV is an inherently defective virus, lacking the capacity to perform at least two critical functions: the ability to initiate the synthesis of viral-specific products and the ability to assemble such products to form the icosahedral protein shell (capsid) of the mature infectious viral particle. It thus requires a co-infecting "helper" virus, such as adenovirus (Ad), herpes simplex virus (HSV), cytomegalovirus (CMV), vaccinia virus or human papillomavirus to provide the viral-associated (VA) RNA that is not encoded by the genes of the AAV genome. Such VA RNA is not translated, but plays a role in regulating the translation of other viral genes. Similarly, the AAV genome does not include genes that encode the viral proteins E1a, E1b, E2a, and E4 of Ad; thus, these proteins must also be provided by a co-infecting "helper" virus. The E1a protein greatly stimulate viral gene transcription during the productive infection. The E1b protein block apoptosis in adenovirus-infected cells, and thus allow productive infection to proceed. The E2a protein plays a role in the elongation phase of viral strand displacement replication by unwinding the template and enhancing the initiation of transcription. The E4 protein has been shown to affect transgene persistence, vector toxicity and immunogenicity (see, Grieger, J. C. et al. (2012) "Adeno-Associated Virus Vectorology, Manufacturing, and Clinical Applications," Meth. Enzymol. 507:229-254; Dyson, N. et al. (1992) "Adenovirus E1A Targets Key Regulators Of Cell Proliferation," Canc. Surv. 12:161-195; Jones N. C. (1990) "Transformation By The Human Adenoviruses," Semin. Cancer Biol. 1(6):425-435; Ben-Israel, H. et al. (2002) "Adenovirus and Cell Cycle Control," Front. Biosci. 7:d1369-d1395; Hoeben, R. C. et al. (2013) "Adenovirus DNA Replication," Cold Spring Harb. Perspect. Biol. 5:a013003 (pages 1-11); Berk, A. J. (2013) "Adenoviridae: The Viruses And Their Replication, In: FIELDS VIROLOGY, 6.sup.th Edition (Knipe, D. M. et al., Eds.), Vol. 2., Lippincott Williams & Wilkins, Philadelphia, pages 1704-1731; Weitzman, M. D. (2005) "Functions Of The Adenovirus E4 Proteins And Their Impact On Viral Vectors," Front. Biosci. 10:1106-1117).

[0008] AAV viruses infect both dividing and non-dividing cells, and persist as circular episomal molecules or can be integrated into the DNA of a host cell at specific chromosomic loci (Adeno-Associated Virus Integration Sites or AAVS) (Duan, D. (2016) "Systemic Delivery Of Adeno-Associated Viral Vectors," Curr. Opin. Virol. 21:16-25; Grieger, J. C. et al. (2012) "Adeno-Associated Virus Vectorology, Manufacturing, and Clinical Applications," Meth. Enzymol. 507:229-254). AAV remains latent in such infected cells unless a helper virus is present to provide the functions needed for AAV replication and maturation.

II. rAAV and Their Use in Gene Therapy

[0009] In light of AAV's properties, recombinantly-modified versions of AAV (rAAV) have found substantial utility as vectors for gene therapy (see, Naso, M. F. et al. (2017) "Adeno-Associated Virus (AAV) as a Vector for Gene Therapy," BioDrugs 31:317-334; Berns, K. I. et al. (2017) "AAV: An Overview of Unanswered Questions," Human Gene Ther. 28(4):308-313; Berry, G. E. et al. (2016) "Cellular Transduction Mechanisms Of Adeno-Associated Viral Vectors," Curr. Opin. Virol. 21:54-60; Blessing, D. et al. (2016) "Adeno-Associated Virus And Lentivirus Vectors: A Refined Toolkit For The Central Nervous System," 21:61-66; Santiago-Ortiz, J. L. (2016) "Adeno-Associated Virus (AAV) Vectors in Cancer Gene Therapy," J. Control Release 240:287-301; Salganik, M. et al. (2015) "Adeno-Associated Virus As A Mammalian DNA Vector," Microbiol. Spectr. 3(4):1-32; Hocquemiller, M. et al. (2016) "Adeno-Associated Virus-Based Gene Therapy for CNS Diseases," Hum. Gene Ther. 27(7):478-496; Lykken, E. A. et al. (2018) "Recent Progress And Considerations For AAV Gene Therapies Targeting The Central Nervous System," J. Neurodevelop. Dis. 10:16:1-10; Buning, H. et al. (2019) "Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors," Mol. Ther. Meth. Clin. Devel. 12:P248-P265; During, M. J. et al. (1998) "In Vivo Expression Of Therapeutic Human Genes For Dopamine Production In The Caudates Of MPTP-Treated Monkeys Using An AAV Vector," Gene Ther. 5:820-827; Grieger, J. C. et al. (2012) "Adeno-Associated Virus Vectorology, Manufacturing, and Clinical Applications," Meth. Enzymol. 507:229-254; Kotterman, M. A. et al. (2014) "Engineering Adeno-Associated Viruses For Clinical Gene Therapy," Nat. Rev. Genet. 15(7):445-451; Kwon, I. et al. (2007) "Designer Gene Delivery Vectors: Molecular Engineering and Evolution of Adeno-Associated Viral Vectors for Enhanced Gene Transfer," Pharm. Res. 25(3):489-499).

[0010] rAAV are typically produced using circular plasmids ("rAAV plasmid vector"). The AAV rep and cap genes are typically deleted from such constructs and replaced with a promoter, a .beta.-globin intron, a cloning site into which a therapeutic gene of choice (transgene) has been inserted, and a poly-adenylation ("polyA") site. The inverted terminal repeated sequences (ITR) of the rAAV are, however, retained, so that the transgene expression cassette of the rAAV plasmid vector is flanked by AAV ITR sequences (Colella, P. et al. (2018) "Emerging Issues in AAV-Mediated In Vivo Gene Therapy," Molec. Ther. Meth. Clin. Develop. 8:87-104; Buning, H. et al. (2019) "Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors," Mol. Ther. Meth. Clin. Devel. 12:P248-P265). Thus, in the 5' to 3' direction, the rAAV comprises a 5' ITR, the transgene expression cassette of the rAAV, and a 3' ITR.

[0011] rAAV have been used to deliver a transgene to patients suffering from any of a multitude of genetic diseases (e.g., hereditary lipoprotein lipase deficiency (LPLD), Leber's congenital amaurosis (LCA), aromatic L-amino acid decarboxylase deficiency (AADC), choroideremia and hemophilia), and have utility in new clinical modalities, such as in interfering RNA (RNAi) therapy and gene-modifying strategies such as Crispr/Cas9 (U.S. Pat. Nos. 8,697,359, 10,000,772, 10,113,167, 10,227,611; Lino, C. A. et al. (2018) "Delivering CRISPR: A Review Of The Challenges And Approaches," Drug Deliv. 25(1):1234-1237; Ferreira, V. et al. (2014) "Immune Responses To AAV-Vectors, The Glybera Example From Bench To Bedside" Front. Immunol. 5(82):1-15), Buning, H. et al. (2019) "Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors," Mol. Ther. Meth. Clin. Devel. 12:P248-P265; Rastall, D. P. W. (2017) "Current and Future Treatments for Lysosomal Storage Disorders," Curr. Treat Options Neurol. 19(12):45; Kay, M. et al. (2017) "Future Of rAAV Gene Therapy: Platform For RNA Gene Editing And Beyond," Human Gene Ther. 28:361-372); Berns, K. I. et al. (2017) "AAV: An Overview of Unanswered Questions," Human Gene Ther. 28(4):308-313). More than 150 clinical trials involving rAAV have been instituted (Buning, H. et al. (2019) "Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors," Mol. Ther. Meth. Clin. Devel. 12:P248-P265; Clement, N. et al. (2016) "Manufacturing Of Recombinant Adeno-Associated Viral Vectors For Clinical Trials," Meth. Clin. Develop. 3:16002:1-7). The most commonly used AAV serotype for such recombinantly-modified AAV is AAV2, which is capable of infecting cells of the central nervous system, kidney, retinal pigment epithelium and photoreceptor cells. Another AAV serotype is AAV9, which infects muscle cells, also has been widely used (Duan, D. (2016) "Systemic Delivery Of Adeno-Associated Viral Vectors," Curr. Opin. Virol. 21:16-25). AAV serotypes are described in U.S. Pat. Nos. 10,301,650; 10,266,846; 10,265,417; 10,214,785; 10,214,566; 10,202,657; 10,046,016; 9,884,071; 9,856,539; 9,737,618; 9,677,089; 9,458,517; 9,457,103; 9,441,244; 9,193,956; 8,846,389; 8,507,267; 7,906,111; 7,479,554; 7,186,552; 7,105,345; 6,984,517; 6,962,815; and 6,733,757.

III. Methods of rAAV Production

[0012] rAAV containing a desired transgene expression cassette are typically produced by human cells (such as HEK293) grown in either adhesion or suspension. Since, as described above, rAAV are defective viruses, additional functions must be provided in order to replicate and package rAAV.

[0013] Typically, rAAV are produced by transiently transfecting cells with an rAAV plasmid vector and a second plasmid vector that comprises an AAV helper function-providing polynucleotide that provides the Rep52 and Rep78 genes that are required for vector transcription control and replication, and for the packaging of viral genomes into the viral capsule (Rep40 and Rep68 are not required for rAAV production) and the cap genes that were excised from the AAV in order to produce the rAAV. The second plasmid vector may additionally comprise a non-AAV helper function-providing polynucleotide that encodes the viral transcription and translation factors (E1a, E1b, E2a, VA and E4) required for AAV proliferation, so as to comprise, in concert with the rAAV, a double plasmid transfection system (Grimm, D. et al. (1998) "Novel Tools For Production And Purification Of Recombinant Adeno-Associated Virus Vectors," Hum. Gene Ther. 9:2745-2760; Penaud-Budloo, M. et al. (2018) "Pharmacology of Recombinant Adeno-associated Virus Production," Molec. Ther. Meth. Clin. Develop. 8:166-180).

[0014] However, it has become increasingly common to clone the AAV helper function-providing polynucleotide (which provides the required rep and cap genes) into an "AAV helper plasmid," and to clone the non-AAV helper function-providing polynucleotide (which provides the genes that encode the viral transcription and translation factors) on a different plasmid (i.e., an "Ad helper plasmid"), so that such plasmids, in concert with an rAAV plasmid vector, comprise a triple plasmid transfection system (FIG. 2). Use of the triple plasmid transfection system has the advantage of permitting one to easily switch one cap gene for another, thereby facilitating changes in the rAAV's serotype. The use of helper plasmids, rather than helper viruses, permits rAAV to be produced without additionally producing particles of the helper virus (Francois, A. et al. (2018) "Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls," Molec. Ther. Meth. Clin. Develop. 10:223-236; Matsushita, T. et al. (1998) "Adeno-Associated Virus Vectors Can Be Efficiently Produced Without Helper Virus," Gene Ther. 5:938-945).

[0015] The transient transfection of plasmid DNAs comprising an rAAV plasmid vector, a plasmid vector providing AAV helper functions rep and cap genes, and a plasmid vector providing non-AAV helper functions into HEK293 cells by calcium phosphate coprecipitation has become the standard method to produce rAAV in the research laboratory (Grimm, D. et al. (1998) "Novel Tools For Production And Purification Of Recombinant Adeno-Associated Virus Vectors," Hum. Gene Ther. 9:2745-2760). However, the use of such a calcium phosphate-mediated transfection process with suspension-cultured transfected mammalian cells requires media exchanges, and is thus not considered ideal for the large-scale rAAV production that is required in order to produce therapeutic doses of rAAV (Lock, M. et al. (2010) "Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale," Hum. Gene Ther. 21:1259-1271). For this reason, polyethylenimine (PEI), has been used as a transfection reagent and has been found to provide yields of virus that are similar to those obtained using calcium phosphate-mediated transfection (Durocher, Y. et al. (2007) "Scalable Serum-Free Production Of Recombinant Adeno-Associated Virus Type 2 By Transfection Of 293 Suspension Cells," J. Virol. Meth. 144:32-40).

[0016] rAAV may alternatively be produced in insect cells (e.g., sf9 cells) using baculoviral vectors (see. e.g., U.S. Pat. Nos. 9,879,282; 9,879,279; 8,945,918; 8,163,543; 7,271,002 and 6,723,551), or in HSV-infected baby hamster kidney (BHK) cells (e.g., BHK21) (Francois, A. et al. (2018) "Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls," Molec. Ther. Meth. Clin. Develop. 10:223-236). Methods of rAAV production are reviewed in Grieger, J. C. et al. (2012) "Adeno-Associated Virus Vectorology, Manufacturing, and Clinical Applications," Meth. Enzymol. 507:229-254, and in Penaud-Budloo, M. et al. (2018) "Pharmacology of Recombinant Adeno-associated Virus Production," Molec. Ther. Meth. Clin. Develop. 8:166-180.

IV. Methods of rAAV Purification and Recovery

[0017] After production, rAAV are typically collected and purified by one or more overnight CsCl gradient centrifugations (Zolotukhin, S. et al. (1999) "Recombinant Adeno-Associated Virus Purification Using Novel Methods Improves Infectious Titer And Yield," Gene Ther. 6:973-985), followed by desalting to form a purified rAAV production stock. Titers of 10.sup.12-10.sup.13 infectious rAAV capsids/mL are obtainable.

[0018] Because rAAV infection does not cause a cytopathic effect, plaque assays cannot be used to determine the infectious titer of an rAAV preparation. Infectious titer is thus typically measured as the median tissue culture infective dose (TCID50). In this method, a HeLa-derived AAV2 rep- and cap-expressing cell line is grown in a 96-well plate and infected with replicate 10-fold serial dilutions of the rAAV preparation, in the presence of adenovirus of serotype 5. After infection, vector genome replication is determined by quantitative PCR (qPCR) (Zen, Z. et al. (2004) "Infectious Titer Assay For Adeno-Associated Virus Vectors With Sensitivity Sufficient To Detect Single Infectious Events," Hum. Gene Ther. 15:709-715). Alternatively, the infectious titer of an rAAV preparation can be measured using the infectious center assay (ICA). This assay uses HeLa rep-cap cells and Ad, but, after incubation, involves transferring the cells to a membrane. A labeled probe that is complementary to a portion of the employed transgene is used to detect infectious centers (representing individual infected cells) via hybridization. Although more widely used, the TCID50 assay has been reported to lead to a higher background than the ICA and to overestimate vector infectivity relative to the ICA (Francois, A. et al. (2018) "Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls," Molec. Ther. Meth. Clin. Develop. 10:223-236). Methods of producing and purifying rAAV are described inter alia in U.S. Pat. Nos. 10,294,452; 10,161,011; 10,017,746; 9,598,703; 7,625,570; 7,439,065; 7,419,817; 7,208,315; 6,995,006; 6,989,264; 6,846,665 and 6,841,357.

[0019] As discussed above, multiple rounds of overnight cesium chloride gradient centrifugation are typically employed in order to produce rAAV in the research laboratory. However, prolonged exposure to CsCl has been reported to compromise the potency of rAAV plasmid vectors (Zolotukhin, S. et al. (1999) "Recombinant Adeno-Associated Virus Purification Using Novel Methods Improves Infectious Titer And Yield," Gene Ther. 6:973-985). Additionally, such gradients have a limited loading capacity for cell lysate, and thus limit the amount of rAAV that may be purified. Although an isotonic alternative gradient medium, iodixanol, has been used to purify rAAV plasmid vectors, iodixanol shares the same loading capacity drawback as CsCl for rAAV production.

[0020] In order to overcome such gradient-specific constraints, researchers have developed ion-exchange chromatographic methods, affinity purification methods, and antibody-affinity based methods of rAAV purification (Auricchio, A. et al. (2001) "Isolation Of Highly Infectious And Pure Adeno-Associated Virus Type 2 Vectors With A Single-Step Gravity-Flow Column," Hum. Gene Ther. 12:71-76; Brument, N. et al. (2002) "A Versatile And Scalable Two-Step Ion-Exchange Chromatography Process For The Purification Of Recombinant Adeno-Associated Virus Serotypes-2 And -5," Mol. Ther. 6:678-686; Zolotukhin, S. et al. (2002) "Production And Purification Of Serotype 1, 2, And 5 Recombinant Adeno-Associated Viral Vectors," Methods 28:158-167; Davidoff, A. M. et al. (2004) "Purification Of Recombinant Adeno-Associated Virus Type 8 Vectors By Ion Exchange Chromatography Generates Clinical Grade Vector Stock," J. Virol. Methods 121:209-215; Smith, R. H. et al. (2009) "A Simplified Baculovirus-AAV Expression Vector System Coupled With One-Step Affinity Purification Yields High-Titer rAAV Stocks From Insect Cells," Mol. Ther. 17:1888-1896; Lock, M. et al. (2010) "Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale," Hum. Gene Ther. 21:1259-1271). Unfortunately, however, such chromatography-based purification methods are generally unable to separate vector-related impurities, such as empty capsids from fully functional vector particles. Thus, despite its drawbacks, CsCl gradient centrifugation remains the best characterized method for removing empty particles from rAAV vector preparations.

[0021] It has been observed that rAAV of various serotypes is released to the supernatant in both calcium phosphate- and PEI-transfected cultures (Lock, M. et al. (2010) "Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale," Hum. Gene Ther. 21:1259-1271; U.S. Pat. Nos. 6,566,118 and 6,989,264, and US Patent Publication US 2005/0266567). U.S. Pat. Nos. 6,566,118 and 6,989,264, and US Patent Publication US 2005/0266567 disclose that high titers of recombinant AAV vectors are released into the supernatant of cell suspensions if the culture medium had been formulated to initially comprise an osmolarity of between about 100 mOsM to about 650 mOsM using NaCl (i.e., 50-325 mM NaCl) and other, but unspecified, salts, mannitol or glucose, or by manipulating the conductivity of the culture medium to be at least about 5 mS, using an ionic solute such as Na.sup.+ or K.sup.+. An initial osmolarity of 300 mOsM (150 mM) NaCl was found to be optimal. Adamson-Small, L. et al. (2017) similarly demonstrated that 60-90 mM sodium chloride in the production medium resulted in a significant increase in rAAV9 transducing units and capsid proteins under infection conditions in which increased sodium chloride was present 4-6 hr post-transduction (WO 2017/112948; Adamson-Small, L. et al. (2017) "Sodium Chloride Enhances Recombinant Adeno-Associated Virus Production in a Serum-Free Suspension Manufacturing Platform Using the Herpes Simplex Virus System," Hum. Gene Ther. Meth. 28(1):1-14).

[0022] Lock, M. et al. (2010) disclose a PEI transfection-based- and supernatant harvest-based-technique for facilitating the recovery of rAAV particles (Lock, M. et al. (2010) "Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale," Hum. Gene Ther. 21:1259-1271). The method is based on the observation that rAAV belonging to AAV serotypes other than AAV2 were released primarily into the culture medium of calcium phosphate-transfected cells and were not retained in the cell lysate. As such, Lock, M. et al. (2010) discloses that for such rAAV serotypes, the production culture medium represents a relatively pure source of rAAV plasmid vector that possesses a lower level of cellular contaminants and that these factors improve the loading capacity and resolution of purification gradients. In the disclosed method, rAAV, including rAAV belonging to AAV2 serotype, were transfected into HEK293 cells using calcium phosphate. Seventy-two hours (or 120 hours) post-transfection, serum-free media was added and the incubation was continued for an additional 28 hours. Benzonase.RTM., a genetically engineered endonuclease that degrades all forms for DNA and RNA, was then added to the culture supernatant. After 2 hours, NaCl was added to 500 mM and the incubation was resumed for an additional 2 hr before harvesting the culture medium. The clarified medium was then concentrated 125-fold by tangential flow filtration (TFF), and the rAAV was purified using iodixanol step gradients. The method could be employed with AAV of serotypes AAV1, AAV6, AAV7, AAV8, and AAV9. Use of the high-salt incubation of Lock et al. (2010) is disclosed to lead to a further 20% release of rAAV6 and rAAV9 plasmid vectors to the culture medium (relative to the methods of U.S. Pat. Nos. 6,566,118 and 6,989,264 and US Patent Publication US 2005/0266567), but was seen to have elicited little change with respect to other serotypes. Although the average overall yields of rAAV8 and rAAV9 were high (2.2.times.10.sup.14 genome copies), yields of other rAAV serotypes were significantly lower (e.g., 6.7.times.10.sup.13 genome copies for rAAV6). Although the estimated purity of the produced rAAV exceeded 90%, between 35% and 50% of the produced rAAV8 and rAAV9 were lost in the processing steps, and 80-85% of the produced rAAV6 were lost in processing, and rAAV2 were mostly retained within the cells and not released into the culture medium.

[0023] Provision of salt has also been used to permeabilize cells in order to more easily measure transgene-associated gene expression. Thus, for example, During, M. J. et al. (1998) used a "release buffer" containing 135 mm NaCl, 3 mm KCl, 1.2 mm CaCl.sub.2, 1.0 mm MgCl.sub.2, 10 mm glucose, 200 mm ascorbate and 2 mm sodium mono- and dibasic phosphate buffered to pH 7.4 to promote the release of dopamine from HEK 293 cells that had been transfected with an rAAV expressing human tyrosine hydroxylase (TH) and aromatic amino decarboxylase (AADC) (During, M. J. et al. (1998) "In Vivo Expression Of Therapeutic Human Genes For Dopamine Production In The Caudates Of MPTP-Treated Monkeys Using An AAV Vector," Gene Ther. 5:820-827).

[0024] However, despite all such prior successes, a need remains to develop methods capable of addressing problems that presently limit the applicability of rAAV to gene therapy (Grieger, J. C. et al. (2012) "Adeno-Associated Virus Vectorology, Manufacturing, and Clinical Applications," Meth. Enzymol. 507:229-254; Kotterman, M. A. et al. (2014) "Engineering Adeno-Associated Viruses For Clinical Gene Therapy," Nat. Rev. Genet. 15(7):445-451; Kwon, I. et al. (2007) "Designer Gene Delivery Vectors: Molecular Engineering and Evolution of Adeno-Associated Viral Vectors for Enhanced Gene Transfer," Pharm. Res. 25(3):489-499; Naso, M. F. et al. (2017) "Adeno-Associated Virus (AAV) as a Vector for Gene Therapy," BioDrugs 31:317-334). Such problems include: [0025] (1) The Limited Tissue-Specific Tropism of rAAV: One such problem reflects the limited tissue-specific tropisms of AAV and rAAV. The use of multiple helper plasmids, encoding capsid proteins of differing serotypes (i.e., "mosaic" capsids) has been exploited as a way to increase the range of tissue types that can be infected by rAAV (Hauck, B. et al. (2003) "Generation And Characterization Of Chimeric Recombinant AAV Vectors," Mol. Ther. 7:419-425; Rabinowitz, J. E. et al. (2004) "Crossdressing The Virion: The Transcapsidation Of Adeno-Associated Virus Serotypes Functionally Defines Subgroups," J. Virol. 78:4421-4432; Lisowski, L. et al. (2015) "Adeno-Associated Virus Serotypes For Gene Therapeutics," 24:59-67). [0026] (2) The Prevalence of anti-rAAV Immune Responses: A second such problem reflects the fact that 30-80% of humans have been naturally exposed to AAV infection (mainly AAV2) and 20-67% of humans harbor titers of neutralizing anti-AAV capsid antibodies in their blood and other bodily fluids (Liu, Q. et al. (2014) "Neutralizing Antibodies Against AAV2, AAV5 And AAV8 In Healthy And HIV-1-Infected Subjects In China: Implications For Gene Therapy Using AAV Vectors," Gene Ther. 21:732-738; Vandamme, C. et al. (2017) "Unraveling the Complex Story of Immune Responses to AAV Vectors Trial After Trial," Hum. Gene. Ther. 28(11):1061-1074). The presence of these antibodies attenuates the effectiveness of rAAV therapy by preventing transgene expression. Synthetic polymer conjugates (e.g., polyethylene glycol (PEG)) have been used as a means for shielding rAAV from neutralizing antibodies (Le, H. T. et al. (2005) "Utility Of Pegylated Recombinant Adeno-Associated Viruses For Gene Transfer," J. Control. Release 108:161-177; Lee, G. K. et al. (2005) "PEG Conjugation Moderately Protects Adeno-Associated Viral Vectors Against Antibody Neutralization," Biotechnol. Bioeng. 92:24-34). The use of rAAV having alternative serotypes or mutated non-immunogenic capsids has also been pursued (Smith, J. K. et al. (2018) "Creating An Arsenal Of Adeno-Associated Virus (AAV) Gene Delivery Stealth Vehicles," PLoS Pathog. 14(5):1-6). [0027] (3) The Limitation of rAAV Packaging Capacity: The packaging efficiency of rAAV has been found to significantly decrease beyond 5 kb, with lager genomes being encapsidated with 5' truncations (Wu, Z. et al. (2010) "Effect Of Genome Size On AAV Vector Packaging," Molec. Ther. 18:80-86; Ghosh, A. et al. (2007) "Expanding Adeno-Associated Viral Vector Capacity: A Tale Of Two Vectors," Biotechnol. Genet. Eng. Rev. 24:165-177; McClements, M. E. et al. (2017) "Adeno-associated Virus (AAV) Dual Vector Strategies for Gene Therapy Encoding Large Transgenes," Yale J. Biol. Med. 90:611-623). [0028] (4) The Limitations of Large-Scale Manufacturing Technologies: The ability to manufacture rAAV in amounts sufficient for use in large-scale therapy has been a barrier to the successful application of the technology, with process yields ranging from below 5% to below 30% (Lock, M. et al. (2010) "Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale," Hum. Gene Ther. 21:1259-1271).

[0029] These problems are, in some cases, inter-related. For example, the presence of empty particles in the final product exposes the recipient of the vector to a large source of AAV antigen that can lead to unwanted immune responses and toxicity. Thus, improved methods for increasing packaging efficiency and obtaining high production titers are of great importance.

[0030] The present invention is directed to improved methods for increasing the efficiency of rAAV packaging by altering the concentration of ions in a culturing medium during the production of rAAV.

SUMMARY OF THE INVENTION

[0031] The present invention is directed to methods for increasing the efficiency with which recombinant adeno-associated virus (rAAV) are packaged, so as to increase their production titers. More specifically, the invention relates to a method for increasing the production titer of rAAV by transfected cells by increasing the ionic strength of the cell culture media through the administration of additional ions.

[0032] In detail, the invention provides a method for increasing the production titer of recombinantly-modified adeno-associated virus (rAAV), wherein the method comprises the steps: [0033] (A) culturing cells that have been transfected with the rAAV in an initial culture medium for an initial period under conditions sufficient to permit the production of rAAV, wherein the cells additionally contain an AAV helper function-providing polynucleotide and a non-AAV helper function-providing polynucleotide; [0034] (B) changing the ionic strength of the culture medium after the initial period by adding one or more ions other than Na.sup.+ to the culture medium; and [0035] (C) continuing the culturing of the cells to thereby produce a production titer of with the rAAV that is greater than a titer obtained in the absence of step (B).

[0036] The invention additionally provides the embodiment of such method wherein each of the added ion(s) is provided in an amount sufficient to increase the concentration of such ion in the initial culture medium by from about 10 mM to about 80 mM.

[0037] The invention additionally provides the embodiment of such methods wherein the production titer is at least 50% greater than the titer obtained from a similarly conducted cell culturing in the absence of the step (B).

[0038] The invention additionally provides the embodiment of such methods wherein the rAAV comprises a transgene cassette that encodes a protein, or comprises a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition.

[0039] The invention additionally provides the embodiment of such methods wherein the rAAV belongs to the rAAV1, rAAV2, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9 or rAAV10 serotype, or to a hybrid of such serotypes.

[0040] The invention additionally provides the embodiment of such methods wherein the rAAV belongs to the rAAV2, rAAV5, or rAAV9 serotype, or to a hybrid of the serotypes.

[0041] The invention additionally provides the embodiment of such methods wherein the added ions comprise one or more of K.sup.+, Ca.sup.++, or Mg.sup.++.

[0042] The invention additionally provides the embodiment of such methods wherein the added ions comprise one or more of CO.sub.3.sup..dbd., HCO.sub.3.sup.-, HPO.sub.4.sup.-, PO.sub.4.sup..dbd., SCN.sup.-, SO.sub.4.sup..dbd., HSO.sub.4.sup.-, and Cl.sup.-.

[0043] The invention additionally provides the embodiment of such methods wherein the added ions comprise one or more of acetate, aspartate, biphthalate, bitartrate, butoxyethoxy acetate, caprylate, citrate, dehydroacetate, diacetate, dihydroxy glycinate, d-saccharate, gluconate, glutamate, glycinate, glycosulfate, hydroxymethane sulfonate, lactate, methionate, oxalate, phenate, phenosulfonate, propionate, propionate, saccharin, salicylate, sarcosinate, sorbate, thioglycolate, and toluene sulfonate.

[0044] The invention additionally provides the embodiment of such methods wherein the added ions comprise K.sup.+ and CO.sub.3.sup..dbd..

[0045] The invention additionally provides the embodiment of such methods wherein the cells are human embryonic kidney cells, baby hamster kidney cells or sf9 insect cells.

[0046] The invention additionally provides the embodiment of such methods wherein the cells are HEK293 human embryonic kidney cells.

[0047] The invention additionally provides the embodiment of such methods wherein the cells are BHK21 baby hamster kidney cells.

[0048] The invention additionally provides the embodiment of such methods wherein the initial culture medium is Dulbecco's Modified Eagle's Medium or Dulbecco's Modified Eagle's Medium supplemented with serum.

[0049] The invention additionally provides a pharmaceutical composition that comprises: [0050] (A) a preparation of recombinantly-modified adeno-associated virus (rAAV) produced by any of the above-described methods, wherein the rAAV comprises a transgene cassette that encodes a protein, or a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition, and wherein the pharmaceutical composition contains an effective amount of the rAAV preparation; and [0051] (B) a pharmaceutically acceptable carrier.

[0052] The invention additionally provides a preparation of recombinantly-modified adeno-associated virus (rAAV) produced by any of the above-described methods, wherein the rAAV comprises a transgene cassette that encodes a protein, or a transcribed nucleic acid, or the above-described pharmaceutical composition for use in the treatment of a genetic or heritable disease or condition.

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG. 1 provides a schematic genetic map of the wild-type (Wt) AAV genome.

[0054] FIG. 2 provides a schematic of the structural domain of the wild-type AAV2 genome (1), a recombinant AAV (rAAV) (2), complementing "AAV helper plasmid" (3) and an adenovirus helper plasmid ("Ad helper plasmid") (4). The wild-type (Wt) AAV2 (1) is composed of AAV-specific palindromic inverted terminal repeated sequences (ITR), a 5' half containing genes that encode the Rep proteins and a 3' half containing genes that encode the Cap proteins. The rAAV (2) is formed by replacing the Rep- and Cap-encoding genes of the wild-type (Wt) AAV2 (1) with a transgene cassette that comprises a promoter (Pro), the exogenous transgene of interest, and a polyadenylation site (pA). In order to produce the rAAV (2), a complementing "AAV helper" plasmid vector (3) and an adenovirus helper plasmid vector (Ad helper plasmid) (4) are provided. The complementing AAV helper plasmid (3) provides Rep and Cap proteins. The Ad helper plasmid (4) provides adenovirus proteins E1a, E1b, E2a, VA and E4.

[0055] FIG. 3 shows a map of the AAV helper plasmid vector pAAV-RC2.

[0056] FIG. 4 shows a map of the non-AAV helper plasmid vector pHelper-Kan.

[0057] FIG. 5 shows a map of the rAAV plasmid vector pAV-CMV-EGFP.

[0058] FIG. 6 shows a map of the rAAV plasmid vector pAV-TBG-EGFP.

[0059] FIGS. 7A-7C show the effect of cation and cation concentration on the production of rAAV by transfected cells. FIG. 7A shows the extent of expression of the enhanced green fluorescent protein (EGFP) in the transfected cells and the titering of the rAAV stocks using the infectious center assay. Stocks were produced by growing transfected HEK293 cells in Dulbecco's Modified Eagle's Medium in the presence of additionally added NaCl, KCl, CaCl.sub.2 or MgCl.sub.2. The additional concentration of such provided salt is 0, 20, 40, 60, 80 or 100 mM. FIG. 7A shows the infectious center assay. FIG. 7B is a graph of the fold-change in the titers of AAV vectors and salt concentration. FIG. 7C is a graph of the fold-change in Total Genomes (TG) of AAV as a function of cation and cation concentration. The concentration shown in the Figure is the concentration increase in the culturing medium provided by the addition of such salts.

[0060] FIGS. 8A-8B show the effect of cation and cation concentration on the production of rAAV stocks. FIG. 8A shows the extent of expression of the enhanced green fluorescent protein (EGFP) in the transfected cells and the titering of the rAAV stocks using the infectious center assay. Stocks were produced by growing transfected HEK293 cells in Dulbecco's Modified Eagle's Medium in the presence of additionally added 12 salts. The additional concentration of such provided salt is 40, 50, 60 or 70 mM. FIG. 8A shows the infectious center assay. FIG. 8B is a graph of the fold-change in the titers of AAV vectors and salt concentration. The Figure shows the fold-change in rAAV titer for rAAV that were produced in the presence of different anions and differing additionally provided concentrations of such anions. The concentration shown in the Figure is the concentration increase in the culturing medium provided by the addition of such anions.

[0061] FIGS. 9A-9B demonstrate that the provision of KHCO.sub.3 caused unexpectedly higher titers of rAAV, relative to other ions (FIG. 9A: fold-change in AAV titer in culture medium; FIG. 9B: fold-change in Total Genomes). The concentration shown in the Figure (40, 50, 60 or 70 mM) is the concentration increase in the culturing medium provided by the addition of such KHCO.sub.3.

[0062] FIG. 10 shows the fold-change in the total amount of rAAV produced, and in the amount of rAAV released into the medium by cells that had been co-transfected with an Ad helper plasmid, a plasmid that provides the AAV ITRs, an enhanced green fluorescent protein-encoding transgene cassette and either an AAV2 helper plasmid or an AAV8 helper plasmid in order to provide the AAV rep and cap gene functions. At 2, 4, 6, 8, and 10 hours post-transfection, KHCO.sub.3 was added to produce an additional concentration of 30 mM in the culturing medium and the fold-change of rAAV that had been released into the medium (AAV2-medium and AAV8-medium) and the total genomes in the cell lysis (AAV2-total and AAV8-total) were assessed at 72 hours post-transfection.

[0063] FIGS. 11A-11B show the effect of providing KHCO.sub.3 on the enhancement of the production of rAAV of different serotypes. FIG. 11A: shows the fold-change of rAAV released into the medium after 24 hours; FIG. 11B shows the fold-change of total genomes of rAAV; KHCO.sub.3-30 denotes that KHCO.sub.3 was added to produce an additional concentration of 30 mM in the culturing medium; KHCO.sub.3-55 denotes that KHCO.sub.3 was added to produce an additional concentration of 55 mM in the culturing medium.

[0064] FIG. 12 shows the ability of cells cultured in suspension to produce enhanced levels of rAAV in response to the provision of KHCO.sub.3. Provision of KHCO.sub.3 sufficient to increase the concentration of KHCO.sub.3 in the culturing medium by greater than about 20 mM enhanced production of rAAV5 and rAAV6 after 20 hours.

DETAILED DESCRIPTION OF THE INVENTION

I. The Methods of the Present Invention

[0065] The present invention is directed to methods for increasing the efficiencies with which recombinant adeno-associated virus (rAAV) are packaged, so as to increase their production titers. More specifically, the invention relates to a method for increasing the production titer of rAAV by transfected cells by increasing the ionic strength of the cell culture media through the administration of additional ions.

[0066] As used herein, the term "AAV" is intended to denote adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally-occurring and recombinant forms. As used herein, the term "rAAV" is intended to denote a recombinantly-modified version of AAV that comprises a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV). The rAAV may be single-stranded or double-stranded, and may be composed of deoxyribonucleotides or ribonucleotides.

[0067] As used herein, the term "AAV helper functions" denotes AAV proteins (e.g., Rep and Cap) and/or polynucleotides of AAV that are required for the replication and packaging of an rAAV. Such AAV helper functions are provided by an "AAV helper function-providing polynucleotide," which as such term is used herein is a virus, plasmid vector, a non-plasmid vector, or a polynucleotide that has been integrated into a cellular chromosome, that provides AAV helper functions. AAV helper plasmids that may be used in accordance with the present invention to provide AAV helper functions, such as pAAV-RC (Agilent; Addgene; Cell Biolabs), pAAV-RC2 (Cell Biolabs), etc., are commercially available. Plasmid pAAV-RC2 (SEQ ID NO:1; FIG. 3) is an AAV helper plasmid that may be used in accordance with the present invention to provide AAV helper functions.

TABLE-US-00001 Coding Strand of Plasmid pAAV-RC2 (SEQ ID NO: 1): ccgggccccc cctcgaggtc gacggtatcg ggggagctcg cagggtctcc attttgaagc gggaggtttg aacgcgcagc cgccatgccg gggttttacg agattgtgat taaggtcccc agcgaccttg acgagcatct gcccggcatt tctgacagct ttgtgaactg ggtggccgag aaggaatggg agttgccgcc agattctgac atggatctga atctgattga gcaggcaccc ctgaccgtgg ccgagaagct gcagcgcgac tttctgacgg aatggcgccg tgtgagtaag gccccggagg ctcttttctt tgtgcaattt gagaagggag agagctactt ccacatgcac gtgctcgtgg aaaccaccgg ggtgaaatcc atggttttgg gacgtttcct gagtcagatt cgcgaaaaac tgattcagag aatttaccgc gggatcgagc cgactttgcc aaactggttc gcggtcacaa agaccagaaa tggcgccgga ggcgggaaca aggtggtgga tgagtgctac atccccaatt acttgctccc caaaacccag cctgagctcc agtgggcgtg gactaatatg gaacagtatt taagcgcctg tttgaatctc acggagcgta aacggttggt ggcgcagcat ctgacgcacg tgtcgcagac gcaggagcag aacaaagaga atcagaatcc caattctgat gcgccggtga tcagatcaaa aacttcagcc aggtacatgg agctggtcgg gtggctcgtg gacaagggga ttacctcgga gaagcagtgg atccaggagg accaggcctc atacatctcc ttcaatgcgg cctccaactc gcggtcccaa atcaaggctg ccttggacaa tgcgggaaag attatgagcc tgactaaaac cgcccccgac tacctggtgg gccagcagcc cgtggaggac atttccagca atcggattta taaaattttg gaactaaacg ggtacgatcc ccaatatgcg gcttccgtct ttctgggatg ggccacgaaa aagttcggca agaggaacac catctggctg tttgggcctg caactaccgg gaagaccaac atcgcggagg ccatagccca cactgtgccc ttctacgggt gcgtaaactg gaccaatgag aactttccct tcaacgactg tgtcgacaag atggtgatct ggtgggagga ggggaagatg accgccaagg tcgtggagtc ggccaaagcc attctcggag gaagcaaggt gcgcgtggac cagaaatgca agtcctcggc ccagatagac ccgactcccg tgatcgtcac ctccaacacc aacatgtgcg ccgtgattga cgggaactca acgaccttcg aacaccagca gccgttgcaa gaccggatgt tcaaatttga actcacccgc cgtctggatc atgactttgg gaaggtcacc aagcaggaag tcaaagactt tttccggtgg gcaaaggatc acgtggttga ggtggagcat gaattctacg tcaaaaaggg tggagccaag aaaagacccg cccccagtga cgcagatata agtgagccca aacgggtgcg cgagtcagtt gcgcagccat cgacgtcaga cgcggaagct tcgatcaact acgcagacag gtaccaaaac aaatgttctc gtcacgtggg catgaatctg atgctgtttc cctgcagaca atgcgagaga atgaatcaga attcaaatat ctgcttcact cacggacaga aagactgttt agagtgcttt cccgtgtcag aatctcaacc cgtttctgtc gtcaaaaagg cgtatcagaa actgtgctac attcatcata tcatgggaaa ggtgccagac gcttgcactg cctgcgatct ggtcaatgtg gatttggatg actgcatctt tgaacaataa atgatttaaa tcaggtatgg ctgccgatgg ttatcttcca gattggctcg aggacactct ctctgaagga ataagacagt ggtggaagct caaacctggc ccaccaccac caaagcccgc agagcggcat aaggacgaca gcaggggtct tgtgcttcct gggtacaagt acctcggacc cttcaacgga ctcgacaagg gagagccggt caacgaggca gacgccgcgg ccctcgagca cgacaaagcc tacgaccggc agctcgacag cggagacaac ccgtacctca agtacaacca cgccgacgcg gagtttcagg agcgccttaa agaagatacg tcttttgggg gcaacctcgg acgagcagtc ttccaggcga aaaagagggt tcttgaacct ctgggcctgg ttgaggaacc tgttaagacg gctccgggaa aaaagaggcc ggtagagcac tctcctgtgg agccagactc ctcctcggga accggaaagg cgggccagca gcctgcaaga aaaagattga attttggtca gactggagac gcagactcag tacctgaccc ccagcctctc ggacagccac cagcagcccc ctctggtctg ggaactaata cgatggctac aggcagtggc gcaccaatgg cagacaataa cgagggcgcc gacggagtgg gtaattcctc gggaaattgg cattgcgatt ccacatggat gggcgacaga gtcatcacca ccagcacccg aacctgggcc ctgcccacct acaacaacca cctctacaaa caaatttcca gccaatcagg agcctcgaac gacaatcact actttggcta cagcacccct tgggggtatt ttgacttcaa cagattccac tgccactttt caccacgtga ctggcaaaga ctcatcaaca acaactgggg attccgaccc aagagactca acttcaagct ctttaacatt caagtcaaag aggtcacgca gaatgacggt acgacgacga ttgccaataa ccttaccagc acggttcagg tgtttactga ctcggagtac cagctcccgt acgtcctcgg ctcggcgcat caaggatgcc tcccgccgtt cccagcagac gtcttcatgg tgccacagta tggatacctc accctgaaca acgggagtca ggcagtagga cgctcttcat tttactgcct ggagtacttt ccttctcaga tgctgcgtac cggaaacaac tttaccttca gctacacttt tgaggacgtt cctttccaca gcagctacgc tcacagccag agtctggacc gtctcatgaa tcctctcatc gaccagtacc tgtattactt gagcagaaca aacactccaa gtggaaccac cacgcagtca aggcttcagt tttctcaggc cggagcgagt gacattcggg accagtctag gaactggctt cctggaccct gttaccgcca gcagcgagta tcaaagacat ctgcggataa caacaacagt gaatactcgt ggactggagc taccaagtac cacctcaatg gcagagactc tctggtgaat ccgggcccgg ccatggcaag ccacaaggac gatgaagaaa agttttttcc tcagagcggg gttctcatct ttgggaagca aggctcagag aaaacaaatg tggacattga aaaggtcatg attacagacg aagaggaaat caggacaacc aatcccgtgg ctacggagca gtatggttct gtatctacca acctccagag aggcaacaga caagcagcta ccgcagatgt caacacacaa ggcgttcttc caggcatggt ctggcaggac agagatgtgt accttcaggg gcccatctgg gcaaagattc cacacacgga cggacatttt cacccctctc ccctcatggg tggattcgga cttaaacacc ctcctccaca gattctcatc aagaacaccc cggtacctgc gaatccttcg accaccttca gtgcggcaaa gtttgcttcc ttcatcacac agtactccac gggacaggtc agcgtggaga tcgagtggga gctgcagaag gaaaacagca aacgctggaa tcccgaaatt cagtacactt ccaactacaa caagtctgtt aatgtggact ttactgtgga cactaatggc gtgtattcag agcctcgccc cattggcacc agatacctga ctcgtaatct gtaattgctt gttaatcaat aaaccgttta attcgtttca gttgaacttt ggtctctgcg tatttctttc ttatctagtt tccatgctct aggatccact agtaacggcc gccagtgtgc tggaattcgg ctttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc tctagaggtc ctgtattaga ggtcacgtga gtgttttgcg acattttgcg acaccatgtg gtcacgctgg gtatttaagc ccgagtgagc acgcagggtc tccattttga agcgggaggt ttgaacgcgc agccgccaag ccgaattctg cagatatcca aacactggcg gccgctcgac tagagcggcc gccaccgcgg tggagctcca gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc

aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac gactcactat agggcgaatt gggta

[0068] In SEQ ID NO:1, residues 85-1950 of pAAV-RC2 encode the Rep protein, Rep78 (with residues 484-663 corresponding to the P19 promoter, residues 1464-1643 corresponding to the P40 promoter and residues 1668-1676 being a donor site); residues 1967-4174 encode the capsid protein, VP1; residues 1992-2016 encodes a portion of the Rep68 protein; residues 4175-4256 encode a polyA sequence; residues 4610-4626 are M13 Rev sequences; residues 4634-4650 are Lac operator sequences; 4658-4688 are Lac promoter sequences; residues 4951-5675 correspond to pMB ori sequences, residues 5771-6631 encode an ampicillin resistance determinant; and residues 6632-6730 are bla promoter sequences (FIG. 3).

[0069] As used herein, the term "non-AAV helper functions" denotes proteins of Ad, CMV, HSV or other non-AAD viruses (e.g., E1a, E1b, E2a, VA and E4) and/or polynucleotides of Ad, CMV, HSV or other non-AAD viruses that are required for the replication and packaging of an rAAV. Such non-AAV helper functions are provided by a "non-AAV helper function-providing polynucleotide," which as such term is used herein is a virus, plasmid vector, a non-plasmid vector, or a polynucleotide that has been integrated into a cellular chromosome, that provides non-AAV helper functions. The vector, pHelper and derivatives thereof (commercially available from Cell Biolabs, Inc., Invitrogen and Stratagene) are suitable non-AAV helper function-providing polynucleotide (see, e.g., Matsushita, T. et al. (1998) "Adeno-Associated Virus Vectors Can Be Efficiently Produced Without Helper Virus," Gene Ther. 5:938-945; Sharma, A. et al. (2010) "Transduction Efficiency Of AAV 2/6, 2/8 And 2/9 Vectors For Delivering Genes In Human Corneal Fibroblasts," Brain Res. Bull. 81(2-3):273-278). Plasmid pHelper-Kan (SEQ ID NO:2; FIG. 4) is a non-AAV helper function-providing polynucleotide that may be used in accordance with the present invention to provide non-AAV helper functions.

TABLE-US-00002 Coding Strand of Plasmid pHelper-Kan (SEQ ID NO: 2): ggtacccaac tccatgctta acagtcccca ggtacagccc accctgcgtc gcaaccagga acagctctac agcttcctgg agcgccactc gccctacttc cgcagccaca gtgcgcagat taggagcgcc acttcttttt gtcacttgaa aaacatgtaa aaataatgta ctaggagaca ctttcaataa aggcaaatgt ttttatttgt acactctcgg gtgattattt accccccacc cttgccgtct gcgccgttta aaaatcaaag gggttctgcc gcgcatcgct atgcgccact ggcagggaca cgttgcgata ctggtgttta gtgctccact taaactcagg cacaaccatc cgcggcagct cggtgaagtt ttcactccac aggctgcgca ccatcaccaa cgcgtttagc aggtcgggcg ccgatatctt gaagtcgcag ttggggcctc cgccctgcgc gcgcgagttg cgatacacag ggttgcagca ctggaacact atcagcgccg ggtggtgcac gctggccagc acgctcttgt cggagatcag atccgcgtcc aggtcctccg cgttgctcag ggcgaacgga gtcaactttg gtagctgcct tcccaaaaag ggtgcatgcc caggctttga gttgcactcg caccgtagtg gcatcagaag gtgaccgtgc ccggtctggg cgttaggata cagcgcctgc atgaaagcct tgatctgctt aaaagccacc tgagcctttg cgccttcaga gaagaacatg ccgcaagact tgccggaaaa ctgattggcc ggacaggccg cgtcatgcac gcagcacctt gcgtcggtgt tggagatctg caccacattt cggccccacc ggttcttcac gatcttggcc ttgctagact gctccttcag cgcgcgctgc ccgttttcgc tcgtcacatc catttcaatc acgtgctcct tatttatcat aatgctcccg tgtagacact taagctcgcc ttcgatctca gcgcagcggt gcagccacaa cgcgcagccc gtgggctcgt ggtgcttgta ggttacctct gcaaacgact gcaggtacgc ctgcaggaat cgccccatca tcgtcacaaa ggtcttgttg ctggtgaagg tcagctgcaa cccgcggtgc tcctcgttta gccaggtctt gcatacggcc gccagagctt ccacttggtc aggcagtagc ttgaagtttg cctttagatc gttatccacg tggtacttgt ccatcaacgc gcgcgcagcc tccatgccct tctcccacgc agacacgatc ggcaggctca gcgggtttat caccgtgctt tcactttccg cttcactgga ctcttccttt tcctcttgcg tccgcatacc ccgcgccact gggtcgtctt cattcagccg ccgcaccgtg cgcttacctc ccttgccgtg cttgattagc accggtgggt tgctgaaacc caccatttgt agcgccacat cttctctttc ttcctcgctg tccacgatca cctctgggga tggcgggcgc tcgggcttgg gagaggggcg cttctttttc tttttggacg caatggccaa atccgccgtc gaggtcgatg gccgcgggct gggtgtgcgc ggcaccagcg catcttgtga cgagtcttct tcgtcctcgg actcgagacg ccgcctcagc cgcttttttg ggggcgcgcg gggaggcggc ggcgacggcg acggggacga cacgtcctcc atggttggtg gacgtcgcgc cgcaccgcgt ccgcgctcgg gggtggtttc gcgctgctcc tcttcccgac tggccatttc cttctcctat aggcagaaaa agatcatgga gtcagtcgag aaggaggaca gcctaaccgc cccctttgag ttcgccacca ccgcctccac cgatgccgcc aacgcgccta ccaccttccc cgtcgaggca cccccgcttg aggaggagga agtgattatc gagcaggacc caggttttgt aagcgaagac gacgaggatc gctcagtacc aacagaggat aaaaagcaag accaggacga cgcagaggca aacgaggaac aagtcgggcg gggggaccaa aggcatggcg actacctaga tgtgggagac gacgtgctgt tgaagcatct gcagcgccag tgcgccatta tctgcgacgc gttgcaagag cgcagcgatg tgcccctcgc catagcggat gtcagccttg cctacgaacg ccacctgttc tcaccgcgcg taccccccaa acgccaagaa aacggcacat gcgagcccaa cccgcgcctc aacttctacc ccgtatttgc cgtgccagag gtgcttgcca cctatcacat ctttttccaa aactgcaaga tacccctatc ctgccgtgcc aaccgcagcc gagcggacaa gcagctggcc ttgcggcagg gcgctgtcat acctgatatc gcctcgctcg acgaagtgcc aaaaatcttt gagggtcttg gacgcgacga gaaacgcgcg gcaaacgctc tgcaacaaga aaacagcgaa aatgaaagtc actgtggagt gctggtggaa cttgagggtg acaacgcgcg cctagccgtg ctgaaacgca gcatcgaggt cacccacttt gcctacccgg cacttaacct accccccaag gttatgagca cagtcatgag cgagctgatc gtgcgccgtg cacgacccct ggagagggat gcaaacttgc aagaacaaac cgaggagggc ctacccgcag ttggcgatga gcagctggcg cgctggcttg agacgcgcga gcctgccgac ttggaggagc gacgcaagct aatgatggcc gcagtgcttg ttaccgtgga gcttgagtgc atgcagcggt tctttgctga cccggagatg cagcgcaagc tagaggaaac gttgcactac acctttcgcc agggctacgt gcgccaggcc tgcaaaattt ccaacgtgga gctctgcaac ctggtctcct accttggaat tttgcacgaa aaccgcctcg ggcaaaacgt gcttcattcc acgctcaagg gcgaggcgcg ccgcgactac gtccgcgact gcgtttactt atttctgtgc tacacctggc aaacggccat gggcgtgtgg cagcaatgcc tggaggagcg caacctaaag gagctgcaga agctgctaaa gcaaaacttg aaggacctat ggacggcctt caacgagcgc tccgtggccg cgcacctggc ggacattatc ttccccgaac gcctgcttaa aaccctgcaa cagggtctgc cagacttcac cagtcaaagc atgttgcaaa actttaggaa ctttatccta gagcgttcag gaattctgcc cgccacctgc tgtgcgcttc ctagcgactt tgtgcccatt aagtaccgtg aatgccctcc gccgctttgg ggtcactgct accttctgca gctagccaac taccttgcct accactccga catcatggaa gacgtgagcg gtgacggcct actggagtgt cactgtcgct gcaacctatg caccccgcac cgctccctgg tctgcaattc gcaactgctt agcgaaagtc aaattatcgg tacctttgag ctgcagggtc cctcgcctga cgaaaagtcc gcggctccgg ggttgaaact cactccgggg ctgtggacgt cggcttacct tcgcaaattt gtacctgagg actaccacgc ccacgagatt aggttctacg aagaccaatc ccgcccgcca aatgcggagc ttaccgcctg cgtcattacc cagggccaca tccttggcca attgcaagcc atcaacaaag cccgccaaga gtttctgcta cgaaagggac ggggggttta cctggacccc cagtccggcg aggagctcaa cccaatcccc ccgccgccgc agccctatca gcagccgcgg gcccttgctt cccaggatgg cacccaaaaa gaagctgcag ctgccgccgc cgccacccac ggacgaggag gaatactggg acagtcaggc agaggaggtt ttggacgagg aggaggagat gatggaagac tgggacagcc tagacgaagc ttccgaggcc gaagaggtgt cagacgaaac accgtcaccc tcggtcgcat tcccctcgcc ggcgccccag aaattggcaa ccgttcccag catcgctaca acctccgctc ctcaggcgcc gccggcactg cctgttcgcc gacccaaccg tagatgggac accactggaa ccagggccgg taagtctaag cagccgccgc cgttagccca agagcaacaa cagcgccaag gctaccgctc gtggcgcggg cacaagaacg ccatagttgc ttgcttgcaa gactgtgggg gcaacatctc cttcgcccgc cgctttcttc tctaccatca cggcgtggcc ttcccccgta acatcctgca ttactaccgt catctctaca gcccctactg caccggcggc agcggcagcg gcagcaacag cagcggtcac acagaagcaa aggcgaccgg atagcaagac tctgacaaag cccaagaaat ccacagcggc ggcagcagca ggaggaggag cgctgcgtct ggcgcccaac gaacccgtat cgacccgcga gcttagaaat aggatttttc ccactctgta tgctatattt caacaaagca ggggccaaga acaagagctg aaaataaaaa acaggtctct gcgctccctc acccgcagct gcctgtatca caaaagcgaa gatcagcttc ggcgcacgct ggaagacgcg gaggctctct tcagcaaata ctgcgcgctg actcttaagg actagtttcg cgccctttct caaatttaag cgcgaaaact acgtcatctc cagcggccac acccggcgcc

agcacctgtc gtcagcgcca ttatgagcaa ggaaattccc acgccctaca tgtggagtta ccagccacaa atgggacttg cggctggagc tgcccaagac tactcaaccc gaataaacta catgagcgcg ggaccccaca tgatatcccg ggtcaacgga atccgcgccc accgaaaccg aattctcctc gaacaggcgg ctattaccac cacacctcgt aataacctta atccccgtag ttggcccgct gccctggtgt accaggaaag tcccgctccc accactgtgg tacttcccag agacgcccag gccgaagttc agatgactaa ctcaggggcg cagcttgcgg gcggctttcg tcacagggtg cggtcgcccg ggcgttttag ggcggagtaa cttgcatgta ttgggaattg tagttttttt aaaatgggaa gtgacgtatc gtgggaaaac ggaagtgaag atttgaggaa gttgtgggtt ttttggcttt cgtttctggg cgtaggttcg cgtgcggttt tctgggtgtt ttttgtggac tttaaccgtt acgtcatttt ttagtcctat atatactcgc tctgtacttg gcccttttta cactgtgact gattgagctg gtgccgtgtc gagtggtgtt ttttaatagg tttttttact ggtaaggctg actgttatgg ctgccgctgt ggaagcgctg tatgttgttc tggagcggga gggtgctatt ttgcctaggc aggagggttt ttcaggtgtt tatgtgtttt tctctcctat taattttgtt atacctccta tgggggctgt aatgttgtct ctacgcctgc gggtatgtat tcccccgggc tatttcggtc gctttttagc actgaccgat gttaaccaac ctgatgtgtt taccgagtct tacattatga ctccggacat gaccgaggaa ctgtcggtgg tgctttttaa tcacggtgac cagttttttt acggtcacgc cggcatggcc gtagtccgtc ttatgcttat aagggttgtt tttcctgttg taagacaggc ttctaatgtt taaatgtttt tttttttgtt attttatttt gtgtttaatg caggaacccg cagacatgtt tgagagaaaa atggtgtctt tttctgtggt ggttccggaa cttacctgcc tttatctgca tgagcatgac tacgatgtgc ttgctttttt gcgcgaggct ttgcctgatt ttttgagcag caccttgcat tttatatcgc cgcccatgca acaagcttac ataggggcta cgctggttag catagctccg agtatgcgtg tcataatcag tgtgggttct tttgtcatgg ttcctggcgg ggaagtggcc gcgctggtcc gtgcagacct gcacgattat gttcagctgg ccctgcgaag ggacctacgg gatcgcggta tttttgttaa tgttccgctt ttgaatctta tacaggtctg tgaggaacct gaatttttgc aatcatgatt cgctgcttga ggctgaaggt ggagggcgct ctggagcaga tttttacaat ggccggactt aatattcggg atttgcttag agacatattg ataaggtggc gagatgaaaa ttatttgggc atggttgaag gtgctggaat gtttatagag gagattcacc ctgaagggtt tagcctttac gtccacttgg acgtgagggc agtttgcctt ttggaagcca ttgtgcaaca tcttacaaat gccattatct gttctttggc tgtagagttt gaccacgcca ccggagggga gcgcgttcac ttaatagatc ttcattttga ggttttggat aatcttttgg aataaaaaaa aaaaaacatg gttcttccag ctcttcccgc tcctcccgtg tgtgactcgc agaacgaatg tgtaggttgg ctgggtgtgg cttattctgc ggtggtggat gttatcaggg cagcggcgca tgaaggagtt tacatagaac ccgaagccag ggggcgcctg gatgctttga gagagtggat atactacaac tactacacag agcgagctaa gcgacgagac cggagacgca gatctgtttg tcacgcccgc acctggtttt gcttcaggaa atatgactac gtccggcgtt ccatttggca tgacactacg accaacacga tctcggttgt ctcggcgcac tccgtacagt agggatcgcc tacctccttt tgagacagag acccgcgcta ccatactgga ggatcatccg ctgctgcccg aatgtaacac tttgacaatg cacaacgtga gttacgtgcg aggtcttccc tgcagtgtgg gatttacgct gattcaggaa tgggttgttc cctgggatat ggttctgacg cgggaggagc ttgtaatcct gaggaagtgt atgcacgtgt gcctgtgttg tgccaacatt gatatcatga cgagcatgat gatccatggt tacgagtcct gggctctcca ctgtcattgt tccagtcccg gttccctgca gtgcatagcc ggcgggcagg ttttggccag ctggtttagg atggtggtgg atggcgccat gtttaatcag aggtttatat ggtaccggga ggtggtgaat tacaacatgc caaaagaggt aatgtttatg tccagcgtgt ttatgagggg tcgccactta atctacctgc gcttgtggta tgatggccac gtgggttctg tggtccccgc catgagcttt ggatacagcg ccttgcactg tgggattttg aacaatattg tggtgctgtg ctgcagttac tgtgctgatt taagtgagat cagggtgcgc tgctgtgccc ggaggacaag gcgtctcatg ctgcgggcgg tgcgaatcat cgctgaggag accactgcca tgttgtattc ctgcaggacg gagcggcggc ggcagcagtt tattcgcgcg ctgctgcagc accaccgccc tatcctgatg cacgattatg actctacccc catgtaggcg tggacttccc cttcgccgcc cgttgagcaa ccgcaagttg gacagcagcc tgtggctcag cagctggaca gcgacatgaa cttaagcgag ctgcccgggg agtttattaa tatcactgat gagcgtttgg ctcgacagga aaccgtgtgg aatataacac ctaagaatat gtctgttacc catgatatga tgctttttaa ggccagccgg ggagaaagga ctgtgtactc tgtgtgttgg gagggaggtg gcaggttgaa tactagggtt ctgtgagttt gattaaggta cggtgatcaa tataagctat gtggtggtgg ggctatacta ctgaatgaaa aatgacttga aattttctgc aattgaaaaa taaacacgtt gaaacataac atgcaacagg ttcacgattc tttattcctg ggcaatgtag gagaaggtgt aagagttggt agcaaaagtt tcagtggtgt attttccact ttcccaggac catgtaaaag acatagagta agtgcttacc tcgctagttt ctgtggattc actagaatcg atgtaggatg ttgcccctcc tgacgcggta ggagaagggg agggtgccct gcatgtctgc cgctgctctt gctcttgccg ctgctgagga ggggggcgca tctgccgcag caccggatgc atctgggaaa agcaaaaaag gggctcgtcc ctgtttccgg aggaatttgc aagcggggtc ttgcatgacg gggaggcaaa cccccgttcg ccgcagtccg gccggcccga gactcgaacc gggggtcctg cgactcaacc cttggaaaat aaccctccgg ctacagggag cgagccactt aatgctttcg ctttccagcc taaccgctta cgccgcgcgc ggccagtggc caaaaaagct agcgcagcag ccgccgcgcc tggaaggaag ccaaaaggag cgctcccccg ttgtctgacg tcgcacacct gggttcgaca cgcgggcggt aaccgcatgg atcacggcgg acggccggat ccggggttcg aaccccggtc gtccgccatg atacccttgc gaatttatcc accagaccac ggaagagtgc ccgcttacag gctctccttt tgcacggtct agagcgtcaa cgactgcgca cgcctcaccg gccagagcgt cccgaccatg gagcactttt tgccgctgcg caacatctgg aaccgcgtcc gcgactttcc gcgcgcctcc accaccgccg ccggcatcac ctggatgtcc aggtacatct acggattacg tcgacgttta aaccatatga tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa

aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gtcagaagaa ctcgtcaaga aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt tccaccatga tattcggcaa gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg ggcatgctcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg tccagatcat cctgatcgac aagaccggct tccatccgag tacgtgctcg ctcgatgcga tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt gcatcagcca tgatggatac tttctcggca ggagcaaggt gagatgacag gagatcctgc cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagtaca gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcttgcagt tcattcaggg caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac agccggaaca cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat agcctctcca cccaagcggc cggagaacct gcgtgcaatc catcttgttc aatcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gatggatcc

[0070] In SEQ ID NO:2, residues 1-5343 of pHelper-Kan are derived from adenovirus, and include a polynucleotide encoding the E2A protein (residues 258-1847); residues 5344-8535 are derived from adenovirus, and include a polynucleotide encoding the E4orf6 protein; residues 9423-10011 correspond to ori sequences; residues 10182-10976 encode a kanamycin resistance determinant expressed by a bla promoter sequence (residues 10977-11081); residues 11107-11561 correspond to f1 ori sequences (FIG. 4).

[0071] As discussed above, AAV helper function-providing polynucleotides and non-AAV helper function-providing polynucleotides are typically employed in concert with an rAAV plasmid vector to comprise a triple plasmid transfection system. Multiple commercially available rAAV plasmid vectors (e.g., pAV-CMV-EGFP, pGOI, etc. (Cell Biolabs, Inc., Invitrogen and Stratagene)) may be used in accordance with the present invention. An illustrative rAAV plasmid vector that may be used in accordance with the present invention is pAV-CMV-EGFP (SEQ ID NO:3; FIG. 5) which comprises a 5' ITR, a U6 promoter, CMV enhancer and promoter sequences, a polynucleotide encoding the enhanced green fluorescent protein (EGFP) (Gambotto, A. et al. (2000) "Immunogenicity Of Enhanced Green Fluorescent Protein (EGFP) In BALB/C Mice: Identification Of An H2-Kd-Restricted CTL Epitope," Gene Ther. 7(23):2036-2040; Tsien, R. Y. (1998) "The Green Fluorescent Protein," Annu. Rev. Biochem. 67:509-544; Cinelli, R. A. et al. (2000) "The Enhanced Green Fluorescent Protein As A Tool For The Analysis Of Protein Dynamics And Localization: Local Fluorescence Study At The Single-Molecule Level," Photochem. Photobiol. 71(6):771-776; Chopra A. (2008) "Recombinant Adenovirus With Enhanced Green Fluorescent Protein," In: MOLECULAR IMAGING AND CONTRAST AGENT DATABASE (MICAD), National Center for Biotechnology Information, Bethesda Md.), FLAG-tag and 6.times.His-tag sites for facilitating recovery or localization of expressed proteins, an SV40 poly(A) site and a 3' ITR.

TABLE-US-00003 Coding Strand of Plasmid pAV-CMV-EGFP (SEQ ID NO: 3): cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg ccctccagtg agcgagcgcg cagagaggga gtggccaact ccatcactag gggttcctgc ggccgcacgc gtctagttat taatagtaat cgaattcgtg ttactcataa ctagtaaggt cgggcaggaa gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttgggtt tatatatctt gtggaaagga cgcgggatcc actggaccag gcagcagcgt cagaagactt ttttggaaaa gcttgactag taatactgta atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt gttttgcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagagatc cggtaccgag gagatctgcc gccgcgatcg ccggcgcgcc agatctcacg cttaactagc tagcggaccg acgcgtacgc ggccgctcga gatggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta agtcgaggat tataaggatg acgacgataa attcgtcgag caccaccacc accaccacta ataaggttta tccgatccac cggatctaga taagatatcc gatccaccgg atctagataa ctgatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta acgcggtaac cacgtgcgga ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacctgccag cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc

ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt

[0072] In SEQ ID NO:3, residues 1-128 of pAV-CMV-EGFP correspond to the 5' ITR; residues 201-441 are U6 promoter sequences; residues 562-865 are human cytomegalovirus (CMV) immediate early enhancer sequences; residues 866-1068 comprise the CMV immediate early promoter; residues 1192-1911 comprise a mammalian codon-optimized polynucleotide that encodes the EGFP; residues 1918-1941 encode the FLAG-tag; residues 1951-1968 encode the 6.times.His-tag; residues 2139-2260 encode the SV40 poly(A) sequence; residues 2293-2433 correspond to the 3' ITR; residues 2508-22963 correspond to F1 ori sequences; residues 3350-4210 encode an ampicillin resistance determinant and its signal sequence (residues 3350-3418) expressed by a bla promoter sequence (residues 3245-3349); residues 4381-4969 correspond to an ori sequence (FIG. 5).

[0073] A second illustrative rAAV plasmid vector that may be used in accordance with the present invention is pAV-TBG-EGFP (SEQ ID NO:4; FIG. 6) which comprises a 5' ITR, a thyroid hormone-binding globulin (TBG) promoter, a polynucleotide encoding the enhanced green fluorescent protein (EGFP), FLAG-tag and 6.times.His-tag sites for facilitating recovery or localization of expressed proteins, an SV40 poly(A) site and a 3' ITR.

TABLE-US-00004 Coding Strand of Plasmid pAV-TBG-EGFP (SEQ ID NO: 4): cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct gcggccggtc gcgtctagta ctagtaggtt aatttttaaa aagcagtcaa aagtccaagt ggcccttggc agcatttact ctctctgttt gctctggtta ataatctcag gagcacaaac attccagatc caggttaatt tttaaaaagc agtcaaaagt ccaagtggcc cttggcagca tttactctct ctgtttgctc tggttaataa tctcaggagc acaaacattc cagatccggc gcgccagggc tggaagctac ctttgacatc atttcctctg cgaatgcatg tataatttct acagaaccta ttagaaagga tcacccagcc tctgcttttg tacaactttc ccttaaaaaa ctgccaattc cactgctgtt tggcccaata gtgagaactt tttcctgctg cctcttggtg cttttgccta tggcccctat tctgcctgct gaagacactc ttgccagcat ggacttaaac ccctccagct ctgacaatcc tctttctctt ttgttttaca tgaagggtct ggcagccaaa gcaatcactc aaagttcaaa ccttatcatt ttttgctttg ttcctcttgg ccttggtttt gtacatcagc tttgaaaata ccatcccagg gttaatgctg gggttaattt ataactaaga gtgctctagt tttgcaatac aggacatgct ataaaaatgg aaagatgttg ctttctgaga gacaggtacc gaggagatct gccgccgcga tcgccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacttacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtagac gcgtacgcgg ccgctcgagg attataagga tgacgacgat aaattcgtcg agcaccacca ccaccaccac taataaggtt tatccgatcc accggatcta gataagatat ccgatccacc ggatctagat aactgatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct taacgcggta accacgtgcg gacccaacgg ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcacat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacctgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gt

[0074] In SEQ ID NO:4, residues 1-130 of pAV-TBG-EGFP correspond to the 5' ITR; residues 150-854 are TBG promoter sequences, with residues 415-824 comprising the TBG promoter; residues 886-1608 encode the EGFP; residues 1630-1653 encode the FLAG-tag; residues 1663-1680 encode the 6.times.His-tag; residues 1851-1972 encode the poly(A) sequence; residues 2005-2145 corresponds to the 3' ITR; residues 2220-2675 correspond to F1 ori sequences; residues 3062-3922 encode an ampicillin resistance determinant and its signal sequence (residues 3062-3130) expressed by a bla promoter sequence (residues 2957-3061); residues 4093-4681 correspond to an ori sequence (FIG. 6).

[0075] As used herein, the term "production titer" is intended to denote the amount of concentration of infectious rAAV in a preparation. Such amounts or concentrations are preferably determined by titering the AAV or rAAV in such preparation. The production titers of the rAAV preparations of the present invention are preferably titered after subjecting producing cells (e.g., HEK293 transformed with an rAAV plasmid vector, an AAV helper vector providing Rep and Cap proteins, and an Ad helper vector providing required adenovirus transcription and translation factors) to three rounds of freeze/thawing, followed by sonication to release the rAAV particles. The preparation is then centrifuged. The employed AAV vector is localized to the supernatant. An aliquot of the preparation is treated with proteinase K, and the number of AAV genomes is determined. An aliquot of the preparation is infected into HeLa-32C2 cells (which express AAV2 Rep and Cap proteins), and infectious titer is measured using the infectious center assay (ICA) (Francois, A. et al. (2018) "Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls," Molec. Ther. Meth. Clin. Develop. 10:223-236) or more preferably, as the median tissue culture infective dose (TCID50) (Zen, Z. et al. (2004) "Infectious Titer Assay For Adeno-Associated Virus Vectors With Sensitivity Sufficient To Detect Single Infectious Events," Hum. Gene Ther. 15:709-715).

[0076] As used herein, an rAAV production titer is said to be "increased" by the methods of the present invention if the production titer obtained from the use of the methods of the present invention is at least 10% greater, more preferably at least 20% greater, still more preferably at least 30% greater, still more preferably at least 40% greater, still more preferably at least 50% greater, still more preferably at least 60% greater, still more preferably at least 70% greater, still more preferably at least 80% greater, still more preferably at least 90% greater, still more preferably at least 2-fold greater, still more preferably at least 110% greater, still more preferably at least 120% greater, still more preferably at least 130% greater, still more preferably at least 140% greater, still more preferably at least 2.5-fold greater, still more preferably at least 160% greater, still more preferably at least 170% greater, still more preferably at least 180% greater, still more preferably at least 190% greater, and still more preferably at least 3-fold greater than the titer obtained from a similarly conducted production in which the additionally provided ions were not provided.

[0077] The rAAV whose production titer may be increased using the methods of the present invention may comprise any transgene cassette that permits the rAAV to be packaged into an rAAV plasmid vector that may be encapsidated within an AAV capsid particle. Without limitation, such transgene cassette(s) may be of human, primate (including chimpanzee, gibbon, gorilla, orangutan, etc.), cercopithecine (including baboon, cynomolgus monkey, velvet monkey, etc.), canine, glirine (including rat, mouse, hamster, guinea pig, etc.), feline, ovine, caprine, or equine origin.

[0078] In preferred embodiments, such an rAAV or rAAV plasmid vector will encode a protein (e.g., an enzyme, hormone, antibody, receptor, ligand, etc.), or comprise a transcribed nucleic acid, that is relevant to a genetic or heritable disease or condition, such that it may be used in gene therapy to treat such disease or condition.

[0079] The methods of the present invention may be used to increase the production titer of rAAV and rAAV plasmid vectors in cells that have been transfected with a desired rAAV or rAAV plasmid vector, and with such one or more viruses and/or helper plasmids that can provide proteins or RNA molecules that are not provided by such rAAV or rAAV plasmid vectors, but are required for their production. As discussed above, such proteins or RNA molecules include the genes encoding the Rep52 and Rep78 proteins that are required for vector transcription control and replication, and for the packaging of viral genomes into the viral capsule, and, in the case of rAAV, cap genes that encode VP capsid proteins required to form infectious particles. Such proteins or RNA molecules also include the viral transcription and translation factors (E1a, E1b, E2a, VA and E4) required for AAV proliferation. In one embodiment for producing the rAAV of the present invention, all of these genes and RNA molecules are provided on the same helper virus (or more preferably, helper vector) so as to comprise, in concert with an rAAV, a double plasmid transfection system. More preferably, however, for producing the rAAV of the present invention, the required rep and cap genes are provided by one plasmid, and the genes that encode the viral transcription and translation factors are provided on a second plasmid, so that such plasmids, in concert with the rAAV, comprise a triple plasmid transfection system.

[0080] The methods of the present invention may be employed to increase the production titer of rAAV belonging to any serotype, including the AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10 serotypes and the rAAV1, rAAV2, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, and rAAV10 serotypes, and including hybrid serotypes (e.g., AAV2/5 and rAAV2/5, which is a hybrid of serotypes 2 and 5 and thus has the trophism of both such serotypes).

[0081] The methods of the present invention may be employed to enhance the production titers of rAAV that are to be produced using "helper" RNA or proteins provided by an adenovirus, a herpes simplex virus, a cytomegalovirus, a vaccinia virus or a papillomavirus.

[0082] The methods of the present invention may be employed to enhance the production titers of rAAV produced by cells in adherent monolayer culture or in suspension culture, and may be used with any method capable of producing rAAV. Preferably, however, rAAV is produced by transfecting baby hamster kidney (BHK) cells, or more preferably, human embryonic kidney (HEK) cells grown in tissue culture with the plasmid vectors described above. The BHK cell line BHK-21 (ATCC CCL-10), which lacks endogenous retroviruses is a preferred BHK cell line. The HEK cell line HEK293 (ATCC CRL-1573) and its derivatives, such as HEK293T (ATCC CRL-3216, which is a highly transfectable derivative of the HEK293 cell line into which the temperature-sensitive gene for SV40 T-antigen was inserted) or HEK293T/17 (ATCC.RTM. CRL-11268, which was selected for its ease of transfection) are particularly preferred. The HEK293T/17 SF cell line (ATCC ACS-4500) is a derivative of the 293T/17 cell line (ATCC CRL-11268), adapted to serum-free medium and suspension, and may be employed if desired.

[0083] The preferred base medium of the present invention for culturing such cells is Eagle's Minimum Essential Medium (ATCC Catalog No. 30-2003) or Dulbecco's Modified Eagle's Medium (DMEM; Mediatech, Manassas, Va.). Fetal bovine serum (e.g., FBS; HyClone Laboratories, South Logan, Utah) is added to a final concentration of 10% in order to make the complete growth medium. Eagle's Minimum Essential Medium and Dulbecco's Modified Eagle's Medium are complex media that contain amino acids, vitamins, and optionally glucose, in addition to various inorganic salts. Although different sources differ slightly in the concentrations of such salts, Dulbecco's Modified Eagle's Medium (commercially available from, e.g., ThermoFisher Scientific) additionally contains approximately the inorganic salts shown in Table 1. The media differ in that Dulbecco's modified Eagle's medium contains approximately four times as much of the vitamins and amino acids present in the original formula of Eagle's Minimum Essential Medium, and two to four times as much glucose. Additionally, it contains iron in the form of ferric sulfate and phenol red for pH indication (Yao, T et al. (2017) "Animal-Cell Culture Media: History, Characteristics, And Current Issues," Reproduc. Med. Biol. 16(2): 99-117).

TABLE-US-00005 TABLE 1 Concentration Inorganic Salt Formula mg/L Molarity Calcium Chloride CaCl.sub.2 200 1.80 mM Ferric Nitrate Fe(NO.sub.3).sub.3--9H.sub.2O 0.1 0.25 .mu.M Magnesium Sulfate (Anhyd.) MgSO.sub.4 97.67 0.81 mM Potassium Chloride KCl 400 5.37 mM Sodium Bicarbonate NaHCO.sub.3 3700 44.04 mM Sodium Chloride NaCl 6400 109.5 mM Sodium Phosphate Monobasic NaH.sub.2PO4--H.sub.2O 125 0.78 mM Sodium Phosphate Dibasic Na.sub.2HPO.sub.4--H.sub.2O

[0084] Cells to be used for such transfection are preferably passaged twice weekly to maintain them in exponential growth phase. For small-scale transfections, an aliquot of, for example, 1.times.10.sup.6 HEK293 or BHK cells per well on a multi-well plate, or 1.5.times.10.sup.7 HEK293 cells per 15-cm dish, may be employed. For large-scale production HEK293 or BHK cells may be collected from multiple confluent 15-cm plates, and split into two 10-layer cell stacks (Corning, Corning, N.Y.) containing 1 liter of complete culturing medium. In one embodiment, such cells are grown for 4 days in such medium before transfection. The day before transfection, the two cell stacks may be trypsinized and the cells (e.g., approximately 6.times.10.sup.8 cells) may be resuspended in 200 ml of medium. Preferably, the cells are allowed to attach for 24 hours before transfection. Confluency of the cell stacks may be monitored using a Diaphot inverted microscope (Nikon, Melville, N.Y.) from which the phase-contrast hardware had been removed in order to accommodate the cell stack on the microscope stage.

[0085] As used herein, the phrase "ionic strength" is intended to denote the concentration of ions in a solution. The present invention enhances rAAV production titers by increasing the ionic strength of the culture medium by providing additional ions to the medium used to culture rAAV transfected cells. In one embodiment, the provided ions are cations. Suitable cations include Na.sup.+, K.sup.+, Ca.sup.++, and Mg.sup.++. Such cations may be provided as an inorganic salt or as a salt of organic molecule. In another embodiment, the provided ions are anions. Suitable anions include inorganic anions such as: CO.sub.3.sup..dbd., HCO.sub.3.sup.-, HPO.sub.4.sup.-, PO.sub.4.sup..dbd., SCN.sup.- (thiocyanate), SO.sub.4.sup..dbd., HSO.sub.4.sup.-, and Cl.sup.-, and organic ions, such as: acetate (CH.sub.3COO.sup.-), aspartate, biphthalate, bitartrate, butoxyethoxy acetate, caprylate, citrate (C.sub.6H.sub.5O.sub.7.sup..ident.), dehydroacetate, diacetate, dihydroxy glycinate, d-saccharate, gluconate, glutamate, glycinate, glycosulfate, hydroxymethane sulfonate, lactate, methionate, oxalate, phenate, phenosulfonate, propionate, propionate, saccharin, salicylate, sarcosinate, sorbate, thioglycolate, and toluene sulfonate.

[0086] Such cations or anions may be provided at any concentration sufficient to enhance rAAV production titers over the titers produced in the same culture medium without any such additionally provided cations. Suitable concentrations of such cations or anions include concentrations sufficient to increase the initial concentration of such ion in a culturing medium by from about 30 mM to about 80 mM, by from about 40 mM to about 80 mM, by from about 50 mM to about 80 mM, by from about 60 mM to about 80 mM, by from about 70 mM to about 80 mM, or by about 80 mM, with such concentrations being in addition to any concentration of such ion present in such culture medium prior to such addition. If such culture medium did not initially contain the ions to be administered, then such added ions are preferably provided in an amount sufficient to provide concentrations of the provided ions in such culture medium of from about 30 mM to about 80 mM, by from about 40 mM to about 80 mM, by from about 50 mM to about 80 mM, by from about 60 mM to about 80 mM, by from about 70 mM to about 80 mM or to about 80 mM.

[0087] The ions or salts that are to be added to the initial culture medium may be added at any time prior to the harvesting of produced rAAV. Preferably, such ions or salts will have been added at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 12 hours, at least about 15 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, or at least about 24 hours after the initiation of the culturing.

[0088] As used herein, the term "about" when used with reference to a concentration, amount, or time, is intended to denote such concentration and also a range of concentrations that is within .+-.40% of such concentration, and more preferably within .+-.30% of such concentration, and still more preferably within .+-.20% of such concentration, and still more preferably within .+-.10% of such concentration, and still more preferably within .+-.5% of such concentration. Thus, for example, a recited concentration of 10.0 mM denotes a concentration of 10.0 mM, as well as a concentration between 6-14 mM, and more preferably a concentration between 7-13 mM and still more preferably a concentration between 8-12 mM, and still more preferably a concentration between 9-11 mM, and still more preferably a concentration between 9.5-10.5 mM.

[0089] Thus, for example, since Dulbecco's Modified Eagle's Medium has an initial K.sup.+ concentration of about 5.37 mM, a provision of additional K.sup.+ sufficient to increase the concentration of such cation by about 30 mM would cause the culture medium to have a resultant Na.sup.+ concentration of about 35.4 mM. Likewise, since Dulbecco's Modified Eagle's Medium has an initial HCO.sub.3.sup.- concentration of about 44.04 mM, a provision of additional HCO.sub.3.sup.- sufficient to increase the concentration of such cation by about 30 mM would cause the culture medium to have a resultant HCO.sub.3.sup.- concentration of about 74.04 mM.

[0090] In particular, the present invention thus provides a method for increasing the production titer of recombinantly-modified AAV (rAAV) that comprises the steps: [0091] (A) culturing cells that have been transfected with such rAAV in a culture medium for an initial period under conditions sufficient to permit the production of rAAV; [0092] (B) changing the ionic strength of the culture medium after the initial period by adding one or more ions, and preferably one or more ions other than Na.sup.+, to the culture medium, in an amount sufficient to increase the concentration of such ion in the culture medium by from about 30 mM to about 80 mM; [0093] (C) continuing the culturing of the cells to thereby produce a production titer of rAAV that is greater than a titer obtained in the absence of step (B).

[0094] The invention particularly contemplates the use of KHCO.sub.3 to enhance rAAV production titer. Such KHCO.sub.3 is preferably provided in an amount sufficient to increase the concentrations of K.sup.+ and HCO.sub.3.sup.- in the culture medium by about 20 mM, by about 30 mM, by about 40 mM, or by about 50 mM. Such addition would cause the K.sup.+ concentration in Dulbecco's Modified Eagle's Medium to be about 25 mM, about 35 mM, about 45 mM, or about 55 mM, and would cause the HCO.sub.3.sup.- concentration in such medium to be about 64 mM, about 74 mM, about 84 mM or about 94 mM. If such culture medium did not contain K.sup.+ and HCO.sub.3.sup.- ions, then such KHCO.sub.3 is preferably provided in an amount sufficient to provide concentrations of K.sup.+ and HCO.sub.3.sup.- in such culture medium of about 20 mM, of about 30 mM, or of about 40 mM.

II. Pharmaceutical Compositions of the Present Invention

[0095] The invention additionally includes pharmaceutical compositions that comprise a pharmaceutically acceptable preparation of rAAV produced in accordance with the methods of the present invention, and a pharmaceutically acceptable carrier. The rAAV of such pharmaceutical compositions comprises a transgene cassette that encodes a protein, or comprises a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition, and is present in such pharmaceutical composition in an amount effective to ("effective amount")

[0096] The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the US Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant (e.g., Freund's adjuvant complete and incomplete), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Suitable pharmaceutical excipients are described in U.S. Pat. Nos. 8,852,607; 8,192,975; 6,764,845; 6,759,050; and 7,598,070.

[0097] Generally, the ingredients of compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate, or as an aqueous solution in a hermetically sealed container such as a vial, an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline, or other diluent can be provided so that the ingredients may be mixed prior to administration.

[0098] The invention also provides a pharmaceutical pack or kit comprising one or more containers such pharmaceutical composition. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0099] The rAAV of such pharmaceutical compositions is preferably packaged in a hermetically sealed container, such as a vial, an ampoule or sachette indicating the quantity of the molecule, and optionally including instructions for use. In one embodiment, the rAAV of such kit is supplied as a dry sterilized lyophilized powder or water-free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water, saline, or other diluent to the appropriate concentration for administration to a subject. The lyophilized material should be stored at between 2.degree. C. and 8.degree. C. in their original container and the material should be administered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted. In another embodiment, the rAAV of such kit is supplied as an aqueous solution in a hermetically sealed container and can be diluted, e.g., with water, saline, or other diluent, to the appropriate concentration for administration to a subject. The kit can further comprise one or more other prophylactic and/or therapeutic agents useful for the treatment of the disease or condition, in one or more containers; and/or the kit can further comprise one or more cytotoxic antibodies that bind one or more cancer antigens associated with cancer. In certain embodiments, the other prophylactic or therapeutic agent is a chemotherapeutic. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic.

III. Uses of the Invention

[0100] The methods of the present invention may be used to facilitate the production of rAAV, and may particularly be used to facilitate the production of rAAV that comprise transgene cassettes that encode a protein (e.g., an enzyme, hormone, antibody, receptor, ligand, etc.), or of rAAV that comprise a transcribed nucleic acid, that is relevant to a genetic or heritable disease or condition, such that it may be used in gene therapy to treat such disease or condition. Examples of such diseases and conditions include: achromatopsia (ACHM); alpha-1 antitrypsin (AAT) deficiency; Alzheimer's Disease; aromatic L-amino acid decarboxylase (AADC) deficiency; choroideremia (CHM); cancer; Duchenne muscular dystrophy; dysferlin deficiency; follistatin gene deficiency (BMDSIBM); hemophilia A; hemophilia B; hepatitis A; hepatitis B; hepatitis C; Huntington's disease; idiopathic Parkinson's disease; late-infantile neuronal ceroid lipofuscinosis (LINCL, an infantile form of Batten disease); Leber congenital amaurosis (LCA); Leber's hereditary optic neuropathy (LHON); limb girdle muscular dystrophy 1B (LGMD1B); limb girdle muscular dystrophy 1C (LGMD1C); limb girdle muscular dystrophy 2A (LGMD2A); limb girdle muscular dystrophy 2B (LGMD2B); limb girdle muscular dystrophy 2I (LGMD2I); limb girdle muscular dystrophy 2L (LGMD2L); lipoprotein lipase (LPL) deficiency; metachromatic leukodystrophy; neurological disability; neuromotor deficit; neuroskeletal impairment; Parkinson's disease; rheumatoid arthritis; Sanfilippo A syndrome; spinal muscular atrophy (SMA); X-linked retinoschisis (XLRS); .alpha.-sarcoglycan deficiency (LGMD2D); .beta.-sarcoglycan deficiency (LGMD2E); .gamma.-sarcoglycan deficiency (LGMD2C) and .delta.-sarcoglycan deficiency (LGMD2F).

IV. Embodiments of the Invention

[0101] The invention concerns a method for increasing the production titer of recombinantly-modified adeno-associated virus (rAAV), the recombinantly-modified adeno-associated virus (AAV) helper vector produced from such method, and uses and compositions thereof. It is particularly directed to the following embodiments E1-E19: [0102] E1. A method for increasing the production titer of recombinantly-modified adeno-associated virus (rAAV), wherein the method comprises the steps: [0103] (A) culturing cells that have been transfected with the rAAV in an initial culture medium for an initial period under conditions sufficient to permit the production of rAAV, wherein the cells additionally contain an AAV helper function-providing polynucleotide and a non-AAV helper function-providing polynucleotide; [0104] (B) changing the ionic strength of the culture medium after the initial period by adding one or more ions other than Na.sup.+ to the culture medium; and [0105] (C) continuing the culturing of the cells to thereby produce a production titer of with the rAAV that is greater than a titer obtained in the absence of step (B). [0106] E2. The method of E1, wherein each of the added ion(s) is provided in an amount sufficient to increase the concentration of such ion in the initial culture medium by from about 10 mM to about 80 mM. [0107] E3. The method of any one of E1 or E2, wherein the production titer is at least 50% greater than the titer obtained from a similarly conducted cell culturing in the absence of the step (B). [0108] E4. The method of any one of E1-E3, wherein the rAAV comprises a transgene cassette that encodes a protein, or comprises a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition. [0109] E5. The method of any one of E1-E4, wherein the rAAV belongs to the rAAV1, rAAV2, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9 or rAAV10 serotype, or to a hybrid of the serotypes. [0110] E6. The method of E5, wherein the rAAV belongs to the rAAV2, rAAV5, or rAAV9 serotype, or to a hybrid of the serotypes. [0111] E7. The method of any one of E1-E6, wherein the added ions comprise one or more of K.sup.+, Ca.sup.++, or Mg.sup.++. [0112] E8. The method of any one of E1-E7, wherein the added ions comprise one or more of CO.sub.3.sup..dbd., HCO.sub.3.sup.-, HPO.sub.4.sup.-, PO.sub.4.sup..dbd., SCN.sup.-, SO.sub.4.sup..dbd., HSO.sub.4.sup.-, and Cl.sup.-. [0113] E9. The method of any one of E1-E7, wherein the added ions comprise one or more of acetate, aspartate, biphthalate, bitartrate, butoxyethoxy acetate, caprylate, citrate, dehydroacetate, diacetate, dihydroxy glycinate, d-saccharate, gluconate, glutamate, glycinate, glycosulfate, hydroxymethane sulfonate, lactate, methionate, oxalate, phenate, phenosulfonate, propionate, propionate, saccharin, salicylate, sarcosinate, sorbate, thioglycolate, and toluene sulfonate. [0114] E10. The method of any one of E1-E8, wherein the added ions comprise K.sup.+ and CO.sub.3.sup..dbd.. [0115] E11. The method of any one of E1-E10, wherein the cells are human embryonic kidney cells. [0116] E12. The method of E11, wherein the cells are HEK293 cells. [0117] E13. The method of any one of E1-E10, wherein the cells are baby hamster kidney cells. [0118] E14. The method of E13, wherein the cells are BHK21 cells. [0119] E15. The method of any one of E1-E10, wherein the cells are sf9 insect cells. [0120] E16. The method of any one of E1-E15, wherein the initial culture medium is Dulbecco's Modified Eagle's Medium. [0121] E17. The method of E16, wherein the initial culture medium is supplemented with serum. [0122] E18. A pharmaceutical composition that comprises: [0123] (A) a preparation of recombinantly-modified adeno-associated virus (rAAV) produced by the method of any one of E1-E17, wherein the rAAV comprises a transgene cassette that encodes a protein, or a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition, and wherein the pharmaceutical composition contains an effective amount of the rAAV preparation; and [0124] (B) a pharmaceutically acceptable carrier. [0125] E19. The preparation of recombinantly-modified adeno-associated virus (rAAV) produced by the method of any one of E1-E17, wherein the rAAV comprises a transgene cassette that encodes a protein, or a transcribed nucleic acid, or the pharmaceutical composition of E18, for use in the treatment of a genetic or heritable disease or condition.

EXAMPLES

[0126] Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention unless specified.

Example 1

Effect of Cation and Cation Concentration on rAAV Production

[0127] The effect of cation and cation concentration on AAV production was demonstrated using cultured HEK293 cells. The culture medium was changed, and then, one hour later, the cells were transfected with: [0128] (1) the plasmid vector pAAV-RC2, which is capable of expressing the AAV rep and cap gene functions that are required for the replication and packaging of an rAAV; [0129] (2) the plasmid vector pHelper, which is capable of providing the viral transcription and translation factors (E1a, E1b, E2a, VA and E4) required for AAV proliferation; and [0130] (3) the rAAV plasmid vector pAV-CMV-EGFP, which comprises the transgene cassette encoding the enhanced green fluorescent protein (EGFP) and the AAV ITRs.

[0131] Five hours after such transfection, salt (either NaCl, KCl, CaCl.sub.2 or MgCl.sub.2) was provided to a final concentration of 0, 20, 40, 60 80 or 100 mM. FIG. 7A shows the extent of expression of EGFP in the transfected cells and the titering of the rAAV stocks using the infectious center assay. FIG. 7B is a graph of the fold-change in infectious centers as a function of such cation and cation concentrations. FIG. 7C is a graph of the fold-change in Total Genomes (TG) of AAV as a function of such cation and cation concentrations. The results show that the provision of cations affected the total genomes (TG) produced and that the provision of NaCl, KCl and MgCl.sub.2 increased AAV genome replication and AAV production. Provision of NaCl and KCl was found to cause the highest titers of total genomes and the greatest increase in AAV production, with the greatest increase seen at NaCl and KCl concentrations that are sufficient to increase the concentrations of such ions in the culture medium by between about 40 mM to about 80 mM. The provision of higher concentrations of cations was found to inhibit EGFP expression (NaCl .gtoreq.180 mM; KCl .gtoreq.100 mM; MgCl.sub.2 .gtoreq.60 mM).

Example 2

Effect of Anion and Anion Concentration on rAAV Production

[0132] The effect of anion and anion concentration on AAV production was also demonstrated using cultured HEK293 cells. As in Example 1, the culture medium was changed, and one hour later, the cells were transfected with the Ad helper plasmid, the AAV helper plasmid, and the pAAV-ITR plasmid vector that provides the AAV ITRs and transgene cassette encoding the enhanced green fluorescent protein. Five hours after such transfection, salt (either K.sub.2CO.sub.3, KHCO.sub.3, KH.sub.2PO.sub.4, KCH.sub.3COO (potassium acetate), KCNS, K.sub.2SO.sub.4, KNO.sub.3, K.sub.3C.sub.6H.sub.5O.sub.7 (potassium citrate) or KCL) was provided in an amount sufficient to increase the concentrations of such ions in the culture medium by 40, 50, 60, or 70 mM. The fold-change in rAAV infectious centers was determined after 72 hours. Provision of KHCO.sub.3 was found to cause the greatest increase in rAAV production, with the greatest increase seen at concentrations sufficient to increase the concentrations of such ions by between about 40 mM to about 50 mM (FIG. 8A). FIG. 8B is a graph of the fold-change in the titer of rAAV vector as a function of such anion and anion concentrations. The results show that the provision of anions affected the total genomes (TG) produced. The provision of high concentrations of ions (>60 mM) was found to attenuate rAAV production. The results demonstrate that the provision of KHCO.sub.3 in an amount sufficient to increase the concentrations of such ions in the culture medium by between about 30 mM and about 50 mM provided unexpectedly better results than those obtained with other salts (FIGS. 9A-9B). An increase in concentration by about 30 mM was considered optimum.

Example 3

Effect of Time of Provision of KHCO.sub.3 on rAAV Production

[0133] The effect caused by providing KHCO.sub.3 at differing times post-transfection was also investigated. HEK293 cells were cultured and co-transfected with: (1) the above-described Ad helper plasmid, (2) the pAAV-ITR plasmid vector that provides the AAV ITRs and transgene cassette encoding the enhanced green fluorescent protein and (3) an AAV2 helper plasmid or an AAV8 helper plasmid in order to provide the AAV rep and cap gene functions. Culture medium had been changed one hour before the co-transfections. At 2, 4, 6, 8, and 10 hours post-transfection, KHCO.sub.3 was added in an amount sufficient to increase the concentrations of such ions in the culture medium by a concentration of 30 mM and the fold-change of rAAV that had been released into the medium was assessed at 72 hours. The fold-change in the total amount of rAAV produced was also assessed (FIG. 10). The results indicate that the greatest enhancement was seen when salts were added 4-8 hours post-transfection.

Example 4

Effect of Serotype on rAAV Production

[0134] As discussed above, prior methods for enhancing the production of rAAV were not successful for rAAV having the AAV2 serotype (Lock, M. et al. (2010) "Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale," Hum. Gene Ther. 21:1259-1271). In order to assess the ability of KHCO.sub.3 addition to enhance the production of rAAV of different serotypes, AAV2 helper plasmid encoding Cap proteins of serotypes 1, 2, 5, 6, 7, 8, 9 or 10 were transfected into HEK293 cells along with the above-described Ad helper plasmid and a pAAV-ITR plasmid vector (pAV-TBG-EGFP) that provides the AAV ITRs and a transgene cassette encoding the enhanced green fluorescent protein. Four hours post-transfection, KHCO.sub.3 was added to a final concentration of 30 mM and the fold-change of rAAV released into the medium was assessed at 72 hours. The results of this study are shown in FIGS. 11A-11B, and indicate that the addition of ions, and specifically the addition of KHCO.sub.3, significantly increased the production titer of rAAV of all serotypes tested, including the rAAV2 serotype.

Example 5

Effect of Ion Provision on Large-Scale rAAV Production

[0135] In order to demonstrate that the provision of ions enhanced production of rAAV in large-scale preparations, rAAV of serotypes 1, 5, 6 and 9 with transgene cassettes encoding the green fluorescent protein or other exogenous molecules were produced in large-scale in the presence or absence of a total concentration of 30 mM KHCO.sub.3 in five 15 cm dishes. AAV titers were obtained after purification. The results of this demonstration are shown in Table 2 (pDNA_001 donor construct, PiBFXNco3 and PiBFXNco11 are control vectors).

TABLE-US-00006 TABLE 2 Effect of KHCO.sub.3 Provision on Large-Scale AAV Production Yield Fold- AAV Transgene (per mL) Change KHCO.sub.3 Addition AAV1 A5514-1 pAV-CMV-EGFP 1.17 .times. 10.sup.13 None A5514-2 pAV-CMV-EGFP 3.8 .times. 10.sup.13 3.2 30 mM, 4 hours post-transfection AAV5 A5658 pAV-CMV-EGFP 1.14 .times. 10.sup.13 None A5659 pAV-CMV-GFP 3.03 .times. 10.sup.13 2.7 30 mM, 4 hours post-transfection AAV6 A5516-1 pAV-CMV-EGFP 1.25 .times. 10.sup.13 None A5516-2 pAV-CMV-EGFP 2.69 .times. 10.sup.13 2.2 30 mM, 4 hours post-transfection A5555 pDNA_001 donor 8.99 .times. 10.sup.12 None construct A5556 pDNA_001 donor 2.64 .times. 10.sup.13 2.9 30 mM, 4 hours construct post-transfection AAV9 A5474-1 PiBFXNco3 1.34 .times. 10.sup.13 None A5474-2 PiBFXNco3 1.61 .times. 10.sup.13 1.2 30 mM, overnight post-transfection A5475-1 PiBFXNco11 3.14 .times. 10.sup.12 None A5475-2 PiBFXNco11 1.46 .times. 10.sup.13 4.6 30 mM, overnight post-transfection

[0136] As indicated in Table 2, the provision of ions, and particularly the provision of KHCO.sub.3, resulted in an increase in rAAV production of 1.2 to 4.6 fold, with an average fold-increase of about 3-fold.

Example 6

Effect of the Provision of Ions on the Production of rAAV by Cells Grown in Suspension

[0137] In order to demonstrate that the provision of ions enhanced production of rAAV by cells that were grown in suspension, HEK293 cells were co-transfected with: (1) the above-described Ad helper plasmid, (2) the pAAV-ITR plasmid vector that provides the AAV ITRs and transgene cassette encoding the enhanced green fluorescent protein and (3) an AAV5 helper plasmid or an AAV6 helper plasmid in order to provide the AAV rep and cap gene functions. KHCO.sub.3 was added in an amount sufficient to increase the concentrations of such ions in the culture medium by 10, 20, 30, 40, 50 or 60 mM at 5 hours or 20 hours post-transfection. Total Genomes of produced rAAV was determined at 72 hours post-transfection. Suspension cells were cultured at 37.degree. C., 5% CO.sub.2 with an agitation speed of 120 rpm. The ability of cells cultured in suspension to produce enhanced levels of rAAV in response to the provision of ions is shown in FIG. 8. As shown in FIG. 12, provision of ions at a final concentration of greater than about 20 mM enhanced production of rAAV5 and rAAV6.

[0138] All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

Sequence CWU 1

1

417415DNAArtificial SequenceCoding Strand of Plasmid pAAV-RC2 1ccgggccccc cctcgaggtc gacggtatcg ggggagctcg cagggtctcc attttgaagc 60gggaggtttg aacgcgcagc cgccatgccg gggttttacg agattgtgat taaggtcccc 120agcgaccttg acgagcatct gcccggcatt tctgacagct ttgtgaactg ggtggccgag 180aaggaatggg agttgccgcc agattctgac atggatctga atctgattga gcaggcaccc 240ctgaccgtgg ccgagaagct gcagcgcgac tttctgacgg aatggcgccg tgtgagtaag 300gccccggagg ctcttttctt tgtgcaattt gagaagggag agagctactt ccacatgcac 360gtgctcgtgg aaaccaccgg ggtgaaatcc atggttttgg gacgtttcct gagtcagatt 420cgcgaaaaac tgattcagag aatttaccgc gggatcgagc cgactttgcc aaactggttc 480gcggtcacaa agaccagaaa tggcgccgga ggcgggaaca aggtggtgga tgagtgctac 540atccccaatt acttgctccc caaaacccag cctgagctcc agtgggcgtg gactaatatg 600gaacagtatt taagcgcctg tttgaatctc acggagcgta aacggttggt ggcgcagcat 660ctgacgcacg tgtcgcagac gcaggagcag aacaaagaga atcagaatcc caattctgat 720gcgccggtga tcagatcaaa aacttcagcc aggtacatgg agctggtcgg gtggctcgtg 780gacaagggga ttacctcgga gaagcagtgg atccaggagg accaggcctc atacatctcc 840ttcaatgcgg cctccaactc gcggtcccaa atcaaggctg ccttggacaa tgcgggaaag 900attatgagcc tgactaaaac cgcccccgac tacctggtgg gccagcagcc cgtggaggac 960atttccagca atcggattta taaaattttg gaactaaacg ggtacgatcc ccaatatgcg 1020gcttccgtct ttctgggatg ggccacgaaa aagttcggca agaggaacac catctggctg 1080tttgggcctg caactaccgg gaagaccaac atcgcggagg ccatagccca cactgtgccc 1140ttctacgggt gcgtaaactg gaccaatgag aactttccct tcaacgactg tgtcgacaag 1200atggtgatct ggtgggagga ggggaagatg accgccaagg tcgtggagtc ggccaaagcc 1260attctcggag gaagcaaggt gcgcgtggac cagaaatgca agtcctcggc ccagatagac 1320ccgactcccg tgatcgtcac ctccaacacc aacatgtgcg ccgtgattga cgggaactca 1380acgaccttcg aacaccagca gccgttgcaa gaccggatgt tcaaatttga actcacccgc 1440cgtctggatc atgactttgg gaaggtcacc aagcaggaag tcaaagactt tttccggtgg 1500gcaaaggatc acgtggttga ggtggagcat gaattctacg tcaaaaaggg tggagccaag 1560aaaagacccg cccccagtga cgcagatata agtgagccca aacgggtgcg cgagtcagtt 1620gcgcagccat cgacgtcaga cgcggaagct tcgatcaact acgcagacag gtaccaaaac 1680aaatgttctc gtcacgtggg catgaatctg atgctgtttc cctgcagaca atgcgagaga 1740atgaatcaga attcaaatat ctgcttcact cacggacaga aagactgttt agagtgcttt 1800cccgtgtcag aatctcaacc cgtttctgtc gtcaaaaagg cgtatcagaa actgtgctac 1860attcatcata tcatgggaaa ggtgccagac gcttgcactg cctgcgatct ggtcaatgtg 1920gatttggatg actgcatctt tgaacaataa atgatttaaa tcaggtatgg ctgccgatgg 1980ttatcttcca gattggctcg aggacactct ctctgaagga ataagacagt ggtggaagct 2040caaacctggc ccaccaccac caaagcccgc agagcggcat aaggacgaca gcaggggtct 2100tgtgcttcct gggtacaagt acctcggacc cttcaacgga ctcgacaagg gagagccggt 2160caacgaggca gacgccgcgg ccctcgagca cgacaaagcc tacgaccggc agctcgacag 2220cggagacaac ccgtacctca agtacaacca cgccgacgcg gagtttcagg agcgccttaa 2280agaagatacg tcttttgggg gcaacctcgg acgagcagtc ttccaggcga aaaagagggt 2340tcttgaacct ctgggcctgg ttgaggaacc tgttaagacg gctccgggaa aaaagaggcc 2400ggtagagcac tctcctgtgg agccagactc ctcctcggga accggaaagg cgggccagca 2460gcctgcaaga aaaagattga attttggtca gactggagac gcagactcag tacctgaccc 2520ccagcctctc ggacagccac cagcagcccc ctctggtctg ggaactaata cgatggctac 2580aggcagtggc gcaccaatgg cagacaataa cgagggcgcc gacggagtgg gtaattcctc 2640gggaaattgg cattgcgatt ccacatggat gggcgacaga gtcatcacca ccagcacccg 2700aacctgggcc ctgcccacct acaacaacca cctctacaaa caaatttcca gccaatcagg 2760agcctcgaac gacaatcact actttggcta cagcacccct tgggggtatt ttgacttcaa 2820cagattccac tgccactttt caccacgtga ctggcaaaga ctcatcaaca acaactgggg 2880attccgaccc aagagactca acttcaagct ctttaacatt caagtcaaag aggtcacgca 2940gaatgacggt acgacgacga ttgccaataa ccttaccagc acggttcagg tgtttactga 3000ctcggagtac cagctcccgt acgtcctcgg ctcggcgcat caaggatgcc tcccgccgtt 3060cccagcagac gtcttcatgg tgccacagta tggatacctc accctgaaca acgggagtca 3120ggcagtagga cgctcttcat tttactgcct ggagtacttt ccttctcaga tgctgcgtac 3180cggaaacaac tttaccttca gctacacttt tgaggacgtt cctttccaca gcagctacgc 3240tcacagccag agtctggacc gtctcatgaa tcctctcatc gaccagtacc tgtattactt 3300gagcagaaca aacactccaa gtggaaccac cacgcagtca aggcttcagt tttctcaggc 3360cggagcgagt gacattcggg accagtctag gaactggctt cctggaccct gttaccgcca 3420gcagcgagta tcaaagacat ctgcggataa caacaacagt gaatactcgt ggactggagc 3480taccaagtac cacctcaatg gcagagactc tctggtgaat ccgggcccgg ccatggcaag 3540ccacaaggac gatgaagaaa agttttttcc tcagagcggg gttctcatct ttgggaagca 3600aggctcagag aaaacaaatg tggacattga aaaggtcatg attacagacg aagaggaaat 3660caggacaacc aatcccgtgg ctacggagca gtatggttct gtatctacca acctccagag 3720aggcaacaga caagcagcta ccgcagatgt caacacacaa ggcgttcttc caggcatggt 3780ctggcaggac agagatgtgt accttcaggg gcccatctgg gcaaagattc cacacacgga 3840cggacatttt cacccctctc ccctcatggg tggattcgga cttaaacacc ctcctccaca 3900gattctcatc aagaacaccc cggtacctgc gaatccttcg accaccttca gtgcggcaaa 3960gtttgcttcc ttcatcacac agtactccac gggacaggtc agcgtggaga tcgagtggga 4020gctgcagaag gaaaacagca aacgctggaa tcccgaaatt cagtacactt ccaactacaa 4080caagtctgtt aatgtggact ttactgtgga cactaatggc gtgtattcag agcctcgccc 4140cattggcacc agatacctga ctcgtaatct gtaattgctt gttaatcaat aaaccgttta 4200attcgtttca gttgaacttt ggtctctgcg tatttctttc ttatctagtt tccatgctct 4260aggatccact agtaacggcc gccagtgtgc tggaattcgg ctttgtagtt aatgattaac 4320ccgccatgct acttatctac gtagccatgc tctagaggtc ctgtattaga ggtcacgtga 4380gtgttttgcg acattttgcg acaccatgtg gtcacgctgg gtatttaagc ccgagtgagc 4440acgcagggtc tccattttga agcgggaggt ttgaacgcgc agccgccaag ccgaattctg 4500cagatatcca aacactggcg gccgctcgac tagagcggcc gccaccgcgg tggagctcca 4560gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt 4620ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 4680agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 4740tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 4800cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 4860gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 4920ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 4980ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 5040atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5100aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5160gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 5220ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5280ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5340acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5400gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 5460ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 5520ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 5580gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 5640ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 5700agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 5760ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 5820gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 5880catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 5940cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 6000cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 6060gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 6120tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 6180gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 6240tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 6300gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 6360gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 6420taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 6480tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 6540ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 6600taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 6660tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 6720aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat 6780tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga 6840aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc 6900agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac 6960cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc 7020gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg 7080gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag 7140ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc 7200gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc 7260ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt 7320aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg 7380cgcgtaatac gactcactat agggcgaatt gggta 7415211569DNAArtificial SequenceCoding Strand of Plasmid pHelper-Kan 2ggtacccaac tccatgctta acagtcccca ggtacagccc accctgcgtc gcaaccagga 60acagctctac agcttcctgg agcgccactc gccctacttc cgcagccaca gtgcgcagat 120taggagcgcc acttcttttt gtcacttgaa aaacatgtaa aaataatgta ctaggagaca 180ctttcaataa aggcaaatgt ttttatttgt acactctcgg gtgattattt accccccacc 240cttgccgtct gcgccgttta aaaatcaaag gggttctgcc gcgcatcgct atgcgccact 300ggcagggaca cgttgcgata ctggtgttta gtgctccact taaactcagg cacaaccatc 360cgcggcagct cggtgaagtt ttcactccac aggctgcgca ccatcaccaa cgcgtttagc 420aggtcgggcg ccgatatctt gaagtcgcag ttggggcctc cgccctgcgc gcgcgagttg 480cgatacacag ggttgcagca ctggaacact atcagcgccg ggtggtgcac gctggccagc 540acgctcttgt cggagatcag atccgcgtcc aggtcctccg cgttgctcag ggcgaacgga 600gtcaactttg gtagctgcct tcccaaaaag ggtgcatgcc caggctttga gttgcactcg 660caccgtagtg gcatcagaag gtgaccgtgc ccggtctggg cgttaggata cagcgcctgc 720atgaaagcct tgatctgctt aaaagccacc tgagcctttg cgccttcaga gaagaacatg 780ccgcaagact tgccggaaaa ctgattggcc ggacaggccg cgtcatgcac gcagcacctt 840gcgtcggtgt tggagatctg caccacattt cggccccacc ggttcttcac gatcttggcc 900ttgctagact gctccttcag cgcgcgctgc ccgttttcgc tcgtcacatc catttcaatc 960acgtgctcct tatttatcat aatgctcccg tgtagacact taagctcgcc ttcgatctca 1020gcgcagcggt gcagccacaa cgcgcagccc gtgggctcgt ggtgcttgta ggttacctct 1080gcaaacgact gcaggtacgc ctgcaggaat cgccccatca tcgtcacaaa ggtcttgttg 1140ctggtgaagg tcagctgcaa cccgcggtgc tcctcgttta gccaggtctt gcatacggcc 1200gccagagctt ccacttggtc aggcagtagc ttgaagtttg cctttagatc gttatccacg 1260tggtacttgt ccatcaacgc gcgcgcagcc tccatgccct tctcccacgc agacacgatc 1320ggcaggctca gcgggtttat caccgtgctt tcactttccg cttcactgga ctcttccttt 1380tcctcttgcg tccgcatacc ccgcgccact gggtcgtctt cattcagccg ccgcaccgtg 1440cgcttacctc ccttgccgtg cttgattagc accggtgggt tgctgaaacc caccatttgt 1500agcgccacat cttctctttc ttcctcgctg tccacgatca cctctgggga tggcgggcgc 1560tcgggcttgg gagaggggcg cttctttttc tttttggacg caatggccaa atccgccgtc 1620gaggtcgatg gccgcgggct gggtgtgcgc ggcaccagcg catcttgtga cgagtcttct 1680tcgtcctcgg actcgagacg ccgcctcagc cgcttttttg ggggcgcgcg gggaggcggc 1740ggcgacggcg acggggacga cacgtcctcc atggttggtg gacgtcgcgc cgcaccgcgt 1800ccgcgctcgg gggtggtttc gcgctgctcc tcttcccgac tggccatttc cttctcctat 1860aggcagaaaa agatcatgga gtcagtcgag aaggaggaca gcctaaccgc cccctttgag 1920ttcgccacca ccgcctccac cgatgccgcc aacgcgccta ccaccttccc cgtcgaggca 1980cccccgcttg aggaggagga agtgattatc gagcaggacc caggttttgt aagcgaagac 2040gacgaggatc gctcagtacc aacagaggat aaaaagcaag accaggacga cgcagaggca 2100aacgaggaac aagtcgggcg gggggaccaa aggcatggcg actacctaga tgtgggagac 2160gacgtgctgt tgaagcatct gcagcgccag tgcgccatta tctgcgacgc gttgcaagag 2220cgcagcgatg tgcccctcgc catagcggat gtcagccttg cctacgaacg ccacctgttc 2280tcaccgcgcg taccccccaa acgccaagaa aacggcacat gcgagcccaa cccgcgcctc 2340aacttctacc ccgtatttgc cgtgccagag gtgcttgcca cctatcacat ctttttccaa 2400aactgcaaga tacccctatc ctgccgtgcc aaccgcagcc gagcggacaa gcagctggcc 2460ttgcggcagg gcgctgtcat acctgatatc gcctcgctcg acgaagtgcc aaaaatcttt 2520gagggtcttg gacgcgacga gaaacgcgcg gcaaacgctc tgcaacaaga aaacagcgaa 2580aatgaaagtc actgtggagt gctggtggaa cttgagggtg acaacgcgcg cctagccgtg 2640ctgaaacgca gcatcgaggt cacccacttt gcctacccgg cacttaacct accccccaag 2700gttatgagca cagtcatgag cgagctgatc gtgcgccgtg cacgacccct ggagagggat 2760gcaaacttgc aagaacaaac cgaggagggc ctacccgcag ttggcgatga gcagctggcg 2820cgctggcttg agacgcgcga gcctgccgac ttggaggagc gacgcaagct aatgatggcc 2880gcagtgcttg ttaccgtgga gcttgagtgc atgcagcggt tctttgctga cccggagatg 2940cagcgcaagc tagaggaaac gttgcactac acctttcgcc agggctacgt gcgccaggcc 3000tgcaaaattt ccaacgtgga gctctgcaac ctggtctcct accttggaat tttgcacgaa 3060aaccgcctcg ggcaaaacgt gcttcattcc acgctcaagg gcgaggcgcg ccgcgactac 3120gtccgcgact gcgtttactt atttctgtgc tacacctggc aaacggccat gggcgtgtgg 3180cagcaatgcc tggaggagcg caacctaaag gagctgcaga agctgctaaa gcaaaacttg 3240aaggacctat ggacggcctt caacgagcgc tccgtggccg cgcacctggc ggacattatc 3300ttccccgaac gcctgcttaa aaccctgcaa cagggtctgc cagacttcac cagtcaaagc 3360atgttgcaaa actttaggaa ctttatccta gagcgttcag gaattctgcc cgccacctgc 3420tgtgcgcttc ctagcgactt tgtgcccatt aagtaccgtg aatgccctcc gccgctttgg 3480ggtcactgct accttctgca gctagccaac taccttgcct accactccga catcatggaa 3540gacgtgagcg gtgacggcct actggagtgt cactgtcgct gcaacctatg caccccgcac 3600cgctccctgg tctgcaattc gcaactgctt agcgaaagtc aaattatcgg tacctttgag 3660ctgcagggtc cctcgcctga cgaaaagtcc gcggctccgg ggttgaaact cactccgggg 3720ctgtggacgt cggcttacct tcgcaaattt gtacctgagg actaccacgc ccacgagatt 3780aggttctacg aagaccaatc ccgcccgcca aatgcggagc ttaccgcctg cgtcattacc 3840cagggccaca tccttggcca attgcaagcc atcaacaaag cccgccaaga gtttctgcta 3900cgaaagggac ggggggttta cctggacccc cagtccggcg aggagctcaa cccaatcccc 3960ccgccgccgc agccctatca gcagccgcgg gcccttgctt cccaggatgg cacccaaaaa 4020gaagctgcag ctgccgccgc cgccacccac ggacgaggag gaatactggg acagtcaggc 4080agaggaggtt ttggacgagg aggaggagat gatggaagac tgggacagcc tagacgaagc 4140ttccgaggcc gaagaggtgt cagacgaaac accgtcaccc tcggtcgcat tcccctcgcc 4200ggcgccccag aaattggcaa ccgttcccag catcgctaca acctccgctc ctcaggcgcc 4260gccggcactg cctgttcgcc gacccaaccg tagatgggac accactggaa ccagggccgg 4320taagtctaag cagccgccgc cgttagccca agagcaacaa cagcgccaag gctaccgctc 4380gtggcgcggg cacaagaacg ccatagttgc ttgcttgcaa gactgtgggg gcaacatctc 4440cttcgcccgc cgctttcttc tctaccatca cggcgtggcc ttcccccgta acatcctgca 4500ttactaccgt catctctaca gcccctactg caccggcggc agcggcagcg gcagcaacag 4560cagcggtcac acagaagcaa aggcgaccgg atagcaagac tctgacaaag cccaagaaat 4620ccacagcggc ggcagcagca ggaggaggag cgctgcgtct ggcgcccaac gaacccgtat 4680cgacccgcga gcttagaaat aggatttttc ccactctgta tgctatattt caacaaagca 4740ggggccaaga acaagagctg aaaataaaaa acaggtctct gcgctccctc acccgcagct 4800gcctgtatca caaaagcgaa gatcagcttc ggcgcacgct ggaagacgcg gaggctctct 4860tcagcaaata ctgcgcgctg actcttaagg actagtttcg cgccctttct caaatttaag 4920cgcgaaaact acgtcatctc cagcggccac acccggcgcc agcacctgtc gtcagcgcca 4980ttatgagcaa ggaaattccc acgccctaca tgtggagtta ccagccacaa atgggacttg 5040cggctggagc tgcccaagac tactcaaccc gaataaacta catgagcgcg ggaccccaca 5100tgatatcccg ggtcaacgga atccgcgccc accgaaaccg aattctcctc gaacaggcgg 5160ctattaccac cacacctcgt aataacctta atccccgtag ttggcccgct gccctggtgt 5220accaggaaag tcccgctccc accactgtgg tacttcccag agacgcccag gccgaagttc 5280agatgactaa ctcaggggcg cagcttgcgg gcggctttcg tcacagggtg cggtcgcccg 5340ggcgttttag ggcggagtaa cttgcatgta ttgggaattg tagttttttt aaaatgggaa 5400gtgacgtatc gtgggaaaac ggaagtgaag atttgaggaa gttgtgggtt ttttggcttt 5460cgtttctggg cgtaggttcg cgtgcggttt tctgggtgtt ttttgtggac tttaaccgtt 5520acgtcatttt ttagtcctat atatactcgc tctgtacttg gcccttttta cactgtgact 5580gattgagctg gtgccgtgtc gagtggtgtt ttttaatagg tttttttact ggtaaggctg 5640actgttatgg ctgccgctgt ggaagcgctg tatgttgttc tggagcggga gggtgctatt 5700ttgcctaggc aggagggttt ttcaggtgtt tatgtgtttt tctctcctat taattttgtt 5760atacctccta tgggggctgt aatgttgtct ctacgcctgc gggtatgtat tcccccgggc 5820tatttcggtc gctttttagc actgaccgat gttaaccaac ctgatgtgtt taccgagtct 5880tacattatga ctccggacat gaccgaggaa ctgtcggtgg tgctttttaa tcacggtgac 5940cagttttttt acggtcacgc cggcatggcc gtagtccgtc ttatgcttat aagggttgtt 6000tttcctgttg taagacaggc ttctaatgtt taaatgtttt tttttttgtt attttatttt 6060gtgtttaatg caggaacccg cagacatgtt tgagagaaaa atggtgtctt tttctgtggt 6120ggttccggaa cttacctgcc tttatctgca tgagcatgac tacgatgtgc ttgctttttt 6180gcgcgaggct ttgcctgatt ttttgagcag caccttgcat tttatatcgc cgcccatgca 6240acaagcttac ataggggcta cgctggttag catagctccg agtatgcgtg tcataatcag 6300tgtgggttct tttgtcatgg ttcctggcgg ggaagtggcc gcgctggtcc gtgcagacct 6360gcacgattat gttcagctgg ccctgcgaag ggacctacgg gatcgcggta tttttgttaa 6420tgttccgctt ttgaatctta tacaggtctg tgaggaacct gaatttttgc aatcatgatt 6480cgctgcttga ggctgaaggt ggagggcgct ctggagcaga tttttacaat ggccggactt 6540aatattcggg atttgcttag agacatattg ataaggtggc gagatgaaaa ttatttgggc 6600atggttgaag gtgctggaat gtttatagag gagattcacc ctgaagggtt tagcctttac 6660gtccacttgg acgtgagggc agtttgcctt ttggaagcca ttgtgcaaca tcttacaaat 6720gccattatct gttctttggc tgtagagttt gaccacgcca ccggagggga gcgcgttcac 6780ttaatagatc ttcattttga ggttttggat aatcttttgg aataaaaaaa aaaaaacatg 6840gttcttccag ctcttcccgc tcctcccgtg tgtgactcgc agaacgaatg tgtaggttgg 6900ctgggtgtgg cttattctgc ggtggtggat gttatcaggg cagcggcgca tgaaggagtt 6960tacatagaac ccgaagccag ggggcgcctg gatgctttga gagagtggat atactacaac 7020tactacacag agcgagctaa gcgacgagac cggagacgca gatctgtttg tcacgcccgc 7080acctggtttt gcttcaggaa atatgactac gtccggcgtt ccatttggca tgacactacg 7140accaacacga tctcggttgt ctcggcgcac tccgtacagt agggatcgcc tacctccttt 7200tgagacagag acccgcgcta ccatactgga ggatcatccg ctgctgcccg aatgtaacac 7260tttgacaatg cacaacgtga gttacgtgcg aggtcttccc tgcagtgtgg gatttacgct 7320gattcaggaa tgggttgttc cctgggatat ggttctgacg cgggaggagc ttgtaatcct 7380gaggaagtgt atgcacgtgt gcctgtgttg tgccaacatt gatatcatga cgagcatgat 7440gatccatggt tacgagtcct gggctctcca ctgtcattgt tccagtcccg gttccctgca

7500gtgcatagcc ggcgggcagg ttttggccag ctggtttagg atggtggtgg atggcgccat 7560gtttaatcag aggtttatat ggtaccggga ggtggtgaat tacaacatgc caaaagaggt 7620aatgtttatg tccagcgtgt ttatgagggg tcgccactta atctacctgc gcttgtggta 7680tgatggccac gtgggttctg tggtccccgc catgagcttt ggatacagcg ccttgcactg 7740tgggattttg aacaatattg tggtgctgtg ctgcagttac tgtgctgatt taagtgagat 7800cagggtgcgc tgctgtgccc ggaggacaag gcgtctcatg ctgcgggcgg tgcgaatcat 7860cgctgaggag accactgcca tgttgtattc ctgcaggacg gagcggcggc ggcagcagtt 7920tattcgcgcg ctgctgcagc accaccgccc tatcctgatg cacgattatg actctacccc 7980catgtaggcg tggacttccc cttcgccgcc cgttgagcaa ccgcaagttg gacagcagcc 8040tgtggctcag cagctggaca gcgacatgaa cttaagcgag ctgcccgggg agtttattaa 8100tatcactgat gagcgtttgg ctcgacagga aaccgtgtgg aatataacac ctaagaatat 8160gtctgttacc catgatatga tgctttttaa ggccagccgg ggagaaagga ctgtgtactc 8220tgtgtgttgg gagggaggtg gcaggttgaa tactagggtt ctgtgagttt gattaaggta 8280cggtgatcaa tataagctat gtggtggtgg ggctatacta ctgaatgaaa aatgacttga 8340aattttctgc aattgaaaaa taaacacgtt gaaacataac atgcaacagg ttcacgattc 8400tttattcctg ggcaatgtag gagaaggtgt aagagttggt agcaaaagtt tcagtggtgt 8460attttccact ttcccaggac catgtaaaag acatagagta agtgcttacc tcgctagttt 8520ctgtggattc actagaatcg atgtaggatg ttgcccctcc tgacgcggta ggagaagggg 8580agggtgccct gcatgtctgc cgctgctctt gctcttgccg ctgctgagga ggggggcgca 8640tctgccgcag caccggatgc atctgggaaa agcaaaaaag gggctcgtcc ctgtttccgg 8700aggaatttgc aagcggggtc ttgcatgacg gggaggcaaa cccccgttcg ccgcagtccg 8760gccggcccga gactcgaacc gggggtcctg cgactcaacc cttggaaaat aaccctccgg 8820ctacagggag cgagccactt aatgctttcg ctttccagcc taaccgctta cgccgcgcgc 8880ggccagtggc caaaaaagct agcgcagcag ccgccgcgcc tggaaggaag ccaaaaggag 8940cgctcccccg ttgtctgacg tcgcacacct gggttcgaca cgcgggcggt aaccgcatgg 9000atcacggcgg acggccggat ccggggttcg aaccccggtc gtccgccatg atacccttgc 9060gaatttatcc accagaccac ggaagagtgc ccgcttacag gctctccttt tgcacggtct 9120agagcgtcaa cgactgcgca cgcctcaccg gccagagcgt cccgaccatg gagcactttt 9180tgccgctgcg caacatctgg aaccgcgtcc gcgactttcc gcgcgcctcc accaccgccg 9240ccggcatcac ctggatgtcc aggtacatct acggattacg tcgacgttta aaccatatga 9300tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 9360aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 9420tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 9480tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 9540cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 9600agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 9660tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 9720aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 9780ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 9840cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 9900accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 9960ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 10020ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 10080gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 10140aaatcaatct aaagtatata tgagtaaact tggtctgaca gtcagaagaa ctcgtcaaga 10200aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag 10260cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc 10320tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt 10380tccaccatga tattcggcaa gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg 10440ggcatgctcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg 10500tccagatcat cctgatcgac aagaccggct tccatccgag tacgtgctcg ctcgatgcga 10560tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt 10620gcatcagcca tgatggatac tttctcggca ggagcaaggt gagatgacag gagatcctgc 10680cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagtaca 10740gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcttgcagt 10800tcattcaggg caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac 10860agccggaaca cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat 10920agcctctcca cccaagcggc cggagaacct gcgtgcaatc catcttgttc aatcatactc 10980ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 11040tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 11100ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca 11160gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga 11220ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg 11280actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat 11340caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag 11400ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga 11460agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa 11520ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gatggatcc 1156935030DNAArtificial SequenceCoding Strand of Plasmid pAV-CMV-EGFP 3cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60ggtcgcccgg ccctccagtg agcgagcgcg cagagaggga gtggccaact ccatcactag 120gggttcctgc ggccgcacgc gtctagttat taatagtaat cgaattcgtg ttactcataa 180ctagtaaggt cgggcaggaa gagggcctat ttcccatgat tccttcatat ttgcatatac 240gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag 300tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat 360gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttgggtt 420tatatatctt gtggaaagga cgcgggatcc actggaccag gcagcagcgt cagaagactt 480ttttggaaaa gcttgactag taatactgta atagtaatca attacggggt cattagttca 540tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc 600gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat 660agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt 720acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc 780cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta 840cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg 900atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt 960gttttgcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg 1020caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac 1080cgtcagatcc gctagagatc cggtaccgag gagatctgcc gccgcgatcg ccggcgcgcc 1140agatctcacg cttaactagc tagcggaccg acgcgtacgc ggccgctcga gatggtgagc 1200aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta 1260aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg 1320accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc 1380accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac 1440ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac 1500gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc 1560atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag 1620tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag 1680gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac 1740cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc 1800acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag 1860ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta agtcgaggat 1920tataaggatg acgacgataa attcgtcgag caccaccacc accaccacta ataaggttta 1980tccgatccac cggatctaga taagatatcc gatccaccgg atctagataa ctgatcataa 2040tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc 2100tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata 2160atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 2220attctagttg tggtttgtcc aaactcatca atgtatctta acgcggtaac cacgtgcgga 2280ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct 2340cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct 2400cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta 2460cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag 2520cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacctgccag 2580cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt 2640tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca 2700cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata 2760gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca 2820aactggaaca acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc 2880gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa 2940caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc 3000atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 3060gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 3120gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt 3180ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa 3240tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 3300gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca 3360acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca 3420cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta 3480catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt 3540tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc 3600cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc 3660accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc 3720cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa 3780ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga 3840accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat 3900ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca 3960attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc 4020ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat 4080tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag 4140tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa 4200gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca 4260tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc 4320ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 4380ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 4440agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 4500cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 4560caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 4620tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 4680ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 4740ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 4800gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 4860gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 4920tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 4980cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 503044742DNAArtificial SequenceCoding Strand of Plasmid pAV-TBG-EGFP 4cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120aggggttcct gcggccggtc gcgtctagta ctagtaggtt aatttttaaa aagcagtcaa 180aagtccaagt ggcccttggc agcatttact ctctctgttt gctctggtta ataatctcag 240gagcacaaac attccagatc caggttaatt tttaaaaagc agtcaaaagt ccaagtggcc 300cttggcagca tttactctct ctgtttgctc tggttaataa tctcaggagc acaaacattc 360cagatccggc gcgccagggc tggaagctac ctttgacatc atttcctctg cgaatgcatg 420tataatttct acagaaccta ttagaaagga tcacccagcc tctgcttttg tacaactttc 480ccttaaaaaa ctgccaattc cactgctgtt tggcccaata gtgagaactt tttcctgctg 540cctcttggtg cttttgccta tggcccctat tctgcctgct gaagacactc ttgccagcat 600ggacttaaac ccctccagct ctgacaatcc tctttctctt ttgttttaca tgaagggtct 660ggcagccaaa gcaatcactc aaagttcaaa ccttatcatt ttttgctttg ttcctcttgg 720ccttggtttt gtacatcagc tttgaaaata ccatcccagg gttaatgctg gggttaattt 780ataactaaga gtgctctagt tttgcaatac aggacatgct ataaaaatgg aaagatgttg 840ctttctgaga gacaggtacc gaggagatct gccgccgcga tcgccaccat ggtgagcaag 900ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 960ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacttacgg caagctgacc 1020ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc 1080ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc 1140ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac 1200ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc 1260gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac 1320aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg 1380aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag 1440cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc 1500cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 1560gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtagac gcgtacgcgg 1620ccgctcgagg attataagga tgacgacgat aaattcgtcg agcaccacca ccaccaccac 1680taataaggtt tatccgatcc accggatcta gataagatat ccgatccacc ggatctagat 1740aactgatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc 1800cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta 1860ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 1920ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct taacgcggta 1980accacgtgcg gacccaacgg ccgcaggaac ccctagtgat ggagttggcc actccctctc 2040tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg 2100cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt 2160attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta 2220cgcgccctgt agcggcacat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc 2280tacacctgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac 2340gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag 2400tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc 2460atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 2520actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata 2580agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa 2640cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg 2700ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg 2760acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg 2820catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat 2880acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac 2940ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 3000gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag 3060tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc 3120tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc 3180acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc 3240cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc 3300ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt 3360ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt 3420atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat 3480cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct 3540tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat 3600gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc 3660ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg 3720ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc 3780tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta 3840cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc 3900ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga 3960tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat 4020gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat 4080caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa 4140accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa 4200ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt 4260aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt 4320accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata 4380gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt 4440ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac 4500gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga 4560gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 4620ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 4680aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 4740gt 4742

Claims

1. A method for increasing the production titer of recombinantly-modified adeno-associated virus (rAAV), wherein said method comprises the steps: (A) culturing cells that have been transfected with said rAAV in an initial culture medium for an initial period under conditions sufficient to permit the production of rAAV, wherein said cells additionally contain an AAV helper function-providing polynucleotide and a non-AAV helper function-providing polynucleotide; (B) changing the ionic strength of said culture medium after said initial period by adding one or more ions other than Na.sup.+ to said culture medium; and (C) continuing said culturing of said cells to thereby produce a production titer of with said rAAV that is greater than a titer obtained in the absence of step (B).

2. The method of claim 1, wherein each of said added ion(s) is provided in an amount sufficient to increase the concentration of such ion in said initial culture medium by from about 10 mM to about 80 mM.

3. The method of any one of claim 1 or claim 2, wherein the production titer is at least 50% greater than the titer obtained from a similarly conducted cell culturing in the absence of said step (B).

4. The method of any one of claims 1-3, wherein said rAAV comprises a transgene cassette that encodes a protein, or comprises a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition.

5. The method of any one of claims 1-4, wherein said rAAV belongs to the rAAV1, rAAV2, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9 or rAAV10 serotype, or to a hybrid of said serotypes.

6. The method of claim 5, wherein said rAAV belongs to the rAAV2, rAAV5, or rAAV9 serotype, or to a hybrid of said serotypes.

7. The method of any one of claims 1-6, wherein said added ions comprise one or more of K.sup.+, Ca.sup.++, or Mg.sup.++.

8. The method of any one of claims 1-7, wherein said added ions comprise one or more of CO.sub.3.sup..dbd., HCO.sub.3.sup.-, HPO.sub.4.sup.-, PO.sub.4.sup..dbd., SCN.sup.-, SO.sub.4.sup..dbd., HSO.sub.4.sup.-, and Cl.sup.-.

9. The method of any one of claims 1-7, wherein said added ions comprise one or more of acetate, aspartate, biphthalate, bitartrate, butoxyethoxy acetate, caprylate, citrate, dehydroacetate, diacetate, dihydroxy glycinate, d-saccharate, gluconate, glutamate, glycinate, glycosulfate, hydroxymethane sulfonate, lactate, methionate, oxalate, phenate, phenosulfonate, propionate, propionate, saccharin, salicylate, sarcosinate, sorbate, thioglycolate, and toluene sulfonate.

10. The method of any one of claims 1-8, wherein said added ions comprise K.sup.+ and CO.sub.3.sup..dbd..

11. The method of any one of claims 1-10, wherein said cells are human embryonic kidney cells.

12. The method of claim 11, wherein said cells are HEK293 cells.

13. The method of any one of claims 1-10, wherein said cells are baby hamster kidney cells.

14. The method of claim 13, wherein said cells are BHK21 cells.

15. The method of any one of claims 1-10, wherein said cells are sf9 insect cells.

16. The method of any one of claims 1-15, wherein said initial culture medium is Dulbecco's Modified Eagle's Medium.

17. The method of claim 16, wherein said initial culture medium is supplemented with serum.

18. A pharmaceutical composition that comprises: (A) a preparation of recombinantly-modified adeno-associated virus (rAAV) produced by the method of any one of claims 1-17, wherein said rAAV comprises a transgene cassette that encodes a protein, or a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition, and wherein said pharmaceutical composition contains an effective amount of said rAAV preparation; and (B) a pharmaceutically acceptable carrier.

19. The preparation of recombinantly-modified adeno-associated virus (rAAV) produced by the method of any one of claims 1-17, wherein the rAAV comprises a transgene cassette that encodes a protein, or a transcribed nucleic acid, or the pharmaceutical composition of claim 18, for use in the treatment of a genetic or heritable disease or condition.