Use of ion concentrations to increase the packaging efficiency of recombinant adeno-associated virus

Description

FIELD OF THE INVENTION

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

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)

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).

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 (Büning, 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; Büning, 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).

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).

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^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).

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

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; Büning, 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).

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 β-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; Büning, 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.

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), Büning, 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 RNAi, 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 (Büning, 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

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.

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).

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).

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).

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

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 10¹²-10¹³ infectious rAAV capsids/mL are obtainable.

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.

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.

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.

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⁺ or K⁺. 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).

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®, 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 (2.2×10¹⁴ genome copies), yields of other rAAV serotypes were significantly lower (e.g., 6.7×10¹³ 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.

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 CaCl₂, 1.0 mm MgCl₂, 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).

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:

• (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).
• (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).
  (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 a. (2017) “Adeno-associated Virus (AAV) Dual Vector Strategies for Gene Therapy Encoding Large Transgenes,” Yale J. Biol. Med. 90:611-623).
• (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).
  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.

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

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.

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:

• • (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;
  • (B) changing the ionic strength of the culture medium after the initial period by adding one or more ions other than Na⁺ to the culture medium; and
  • (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).

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.

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).

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.

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.

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.

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

The invention additionally provides the embodiment of such methods wherein the added ions comprise one or more of CO₃^═, HCO₃⁻, HPO₄⁻, PO₄^═, SCN⁻, SO₄^═, HSO₄⁻, and Cl⁻.

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.

The invention additionally provides the embodiment of such methods wherein the added ions comprise K⁺ and CO₃^═.

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.

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

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

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.

The invention additionally provides a pharmaceutical composition that comprises:

• • (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
  • (B) a pharmaceutically acceptable carrier.

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

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

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.

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

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

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

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

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, CaCl₂ or MgCl₂. 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.

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.

FIGS. 9A-9B demonstrate that the provision of KHCO₃ 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 KHCO₃.

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, KHCO₃ 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.

FIGS. 11A-11B show the effect of providing KHCO₃ 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; KHCO₃-30 denotes that KHCO₃ was added to produce an additional concentration of 30 mM in the culturing medium; KHCO₃-55 denotes that KHCO₃ was added to produce an additional concentration of 55 mM in the culturing medium.

FIG. 12 shows the ability of cells cultured in suspension to produce enhanced levels of rAAV in response to the provision of KHCO₃. Provision of KHCO₃ sufficient to increase the concentration of KHCO₃ 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

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.

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.

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.

	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

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).

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.

	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

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).

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×His-tag sites for facilitating recovery or localization of expressed proteins, an SV40 poly(A) site and a 3′ ITR.

	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

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×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).

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×His-tag sites for facilitating recovery or localization of expressed proteins, an SV40 poly(A) site and a 3′ ITR.

	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

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×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).

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).

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.

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.

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.

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.

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).

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.

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® 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.

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 1
		Concentration

	Inorganic Salt	Formula	mg/L	Molarity

	Calcium Chloride	CaCl2	200	1.80 mM
	Ferric Nitrate	Fe(NO3)3—9H2O	0.1	0.25 μM
	Magnesium Sulfate (Anhyd.)	MgSO4	97.67	0.81 mM
	Potassium Chloride	KCl	400	5.37 mM
	Sodium Bicarbonate	NaHCO3	3700	44.04 mM
	Sodium Chloride	NaCl	6400	109.5 mM
	Sodium Phosphate Monobasic	NaH2PO4—H2O	125	0.78 mM
	Sodium Phosphate Dibasic	Na2HPO4—H2O

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, 1×10⁶ HEK293 or BHK cells per well on a multi-well plate, or 1.5×10⁷ 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 6×10⁸ 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.

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⁺, K⁺, Ca⁺⁺, and Mg⁺⁺. 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: CO₃^═, HCO₃⁻, HPO₄⁻, PO₄^═, SCN⁻ (thiocyanate), SO₄^═, HSO₄⁻, and Cl⁻, and organic ions, such as: acetate (CH₃COO⁻), aspartate, biphthalate, bitartrate, butoxyethoxy acetate, caprylate, citrate (C₆HSO₇^≡), 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.

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.

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.

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.

Thus, for example, since Dulbecco's Modified Eagle's Medium has an initial K⁺ concentration of about 5.37 mM, a provision of additional K⁺ sufficient to increase the concentration of such cation by about 30 mM would cause the culture medium to have a resultant Na⁺ concentration of about 35.4 mM. Likewise, since Dulbecco's Modified Eagle's Medium has an initial HCO₃⁻ concentration of about 44.04 mM, a provision of additional HCO₃⁻ sufficient to increase the concentration of such cation by about 30 mM would cause the culture medium to have a resultant HCO₃⁻ concentration of about 74.04 mM.

In particular, the present invention thus provides a method for increasing the production titer of recombinantly-modified AAV (rAAV) that comprises the steps:

• (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;
• (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^t, 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;
• (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).

The invention particularly contemplates the use of KHCO₃ to enhance rAAV production titer. Such KHCO₃ is preferably provided in an amount sufficient to increase the concentrations of K⁺ and HCO₃⁻ 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⁺ 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 HCO₃⁻ 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⁺ and HCO₃⁻ ions, then such KHCO₃ is preferably provided in an amount sufficient to provide concentrations of K⁺ and HCO₃⁻ in such culture medium of about 20 mM, of about 30 mM, or of about 40 mM.

II. Pharmaceutical Compositions of the Present Invention

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”)

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.

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.

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.

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° C. and 8° 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

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 21 (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); α-sarcoglycan deficiency (LGMD2D); β-sarcoglycan deficiency (LGMD2E); γ-sarcoglycan deficiency (LGMD2C) and δ-sarcoglycan deficiency (LGMD2F).

IV. Embodiments of the Invention

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:

• E1. A method for increasing the production titer of recombinantly-modified adeno-associated virus (rAAV), wherein the method comprises the steps:
  • (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;
  • (B) changing the ionic strength of the culture medium after the initial period by adding one or more ions other than Na⁺ to the culture medium; and
  • (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).
• 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.
• 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).
• 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.
• 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.
• E6. The method of E5, wherein the rAAV belongs to the rAAV2, rAAV5, or rAAV9 serotype, or to a hybrid of the serotypes.
• E7. The method of any one of E1-E6, wherein the added ions comprise one or more of K⁺, Ca⁺⁺, or Mg⁺⁺.
• E8. The method of any one of E1-E7, wherein the added ions comprise one or more of CO₃^═, HCO₃⁻, HPO₄⁻, PO₄^═, SCN⁻, SO₄^═, HSO₄⁻, and Cl⁻.
• 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.
• E10. The method of any one of E1-E8, wherein the added ions comprise K⁺ and CO₃^═.
• E10. The method of any one of E1-E10, wherein the cells are human embryonic kidney cells.
• E12. The method of E11, wherein the cells are HEK293 cells.
• E13. The method of any one of E1-E10, wherein the cells are baby hamster kidney cells.
• E14. The method of E13, wherein the cells are BHK21 cells.
• E15. The method of any one of E1-E10, wherein the cells are sf9 insect cells. E16. The method of any one of E1-E15, wherein the initial culture medium is Dulbecco's Modified Eagle's Medium.
• E17. The method of E16, wherein the initial culture medium is supplemented with serum.
• E18. A pharmaceutical composition that comprises:
  • (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
  • (B) a pharmaceutically acceptable carrier.
• 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

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

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:

• (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;
• (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
• (3) the rAAV plasmid vector pAV-CMV-EGFP, which comprises the transgene cassette encoding the enhanced green fluorescent protein (EGFP) and the AAV ITRs.

Five hours after such transfection, salt (either NaCl, KCl, CaCl₂ or MgCl₂) 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 MgCl₂ 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≥180 mM; KCl≥100 mM; MgCl₂≥60 mM).

Example 2

Effect of Anion and Anion Concentration on rAAV Production

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 K₂CO₃, KHCO₃, KH₂PO₄, KCH₃COO (potassium acetate), KCNS, K₂SO₄, KNO₃, K₃C₆HSO₇ (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 KHCO₃ 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 KHCO₃ 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 KHCO₃ on rAAV Production

The effect caused by providing KHCO₃ 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, KHCO₃ 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

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 KHCO₃ 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, KHCO₃ 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 KHCO₃, 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

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 KHCO₃ 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 2
Effect of KHCO3 Provision on Large-Scale AAV Production

		Yield	Fold-
AAV	Transgene	(per mL)	Change	KHCO3 Addition

AAV1

A5514-1	pAV-CMV-EGFP	1.17 × 1013	—	None
A5514-2	pAV-CMV-EGFP	3.8 × 1013	3.2	30 mM, 4 hours
				post-transfection

AAV5

A5658	pAV-CMV-EGFP	1.14 × 1013	—	None
A5659	pAV-CMV-GFP	3.03 × 1013	2.7	30 mM, 4 hours
				post-transfection

AAV6

A5516-1	pAV-CMV-EGFP	1.25 × 1013	—	None
A5516-2	pAV-CMV-EGFP	2.69 × 1013	2.2	30 mM, 4 hours
				post-transfection
A5555	pDNA_001 donor	8.99 × 1012		None
	construct
A5556	pDNA_001 donor	2.64 × 1013	2.9	30 mM, 4 hours
				post-transfection
	construct

AAV9

A5474-1	PiBFXNco3	1.34 × 1013	—	None
A5474-2	PiBFXNco3	1.61 × 1013	1.2	30 mM, overnight
				post-transfection
A5475-1	PiBFXNco11	3.14 × 1012	—	None
A5475-2	PiBFXNco11	1.46 × 1013	4.6	30 mM, overnight
				post-transfection

As indicated in Table 2, the provision of ions, and particularly the provision of KHCO₃, 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

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. KHCO₃ 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° C., 5% CO₂ 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.

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.