Recombinantly-modified Adeno-associated Virus Helper Vectors And Their Use To Improve The Packaging Efficiency Of Recombinantly-modified Adeno-associated Virus

Abstract

The present invention is directed to recombinantly-modified adeno-associated virus (AAV) helper vectors that are capable of increasing the packaging efficiency of recombinantly-modified adeno-associated virus (rAAV) and their use to improve the packaging efficiency of such rAAV. The present invention is particularly directed to recombinantly-modified adeno-associated virus (AAV) helper vectors that have been further modified to replace (or augment) the P5 and/or P40 promoter sequences that are natively associated with the Rep proteins encoded by such rAAV with AAV P5 and/or P40 promoters that are associated with the Rep proteins of an rAAV of different serotype. The use of such substitute or additional promoter sequences causes increased production of recombinantly-modified adeno-associated virus.

Description

FIELD OF THE INVENTION

[0001] The present invention is directed to recombinantly-modified adeno-associated virus (AAV) helper vectors that are capable of increasing the packaging efficiency of recombinantly-modified adeno-associated virus (rAAV) and their use to improve the packaging efficiency of such rAAV. The present invention is particularly directed to recombinantly-modified adeno-associated virus (AAV) helper vectors that have been further modified to replace (or augment) the P5 and/or P40 promoter sequences that are natively associated with the Rep proteins encoded by such rAAV with AAV P5 and/or P40 promoters that are associated with the Rep proteins of an rAAV of different serotype. The use of such substitute or additional promoter sequences causes increased production of recombinantly-modified adeno-associated virus.

REFERENCE TO SEQUENCE LISTING

[0002] 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: 2650-0004US_ST25.txt, created on Jul. 15, 2019, and having a size of 84,101 bytes), which file is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

[0003] I. Adeno-Associated Virus (AAV)

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

[0005] AAV is a single-stranded DNA virus that is composed of approximately 4,800 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). In the presence of viral proteins (such as Ad proteins), the P5 promoter becomes activated and mediates the transcription of the Rep68 and Rep78 proteins, which are involved in transcriptional control, in latency, in rescue, and in viral DNA replication and thus function as master controllers of the AAV life cycle (Murphy, M. et al. (2007) "Adeno-Associated Virus Type 2 p5 Promoter: a Rep-Regulated DNA Switch Element Functioning in Transcription, Replication, and Site-Specific Integration," J. Virol. 81(8):3721-3730). Expression of the Rep68 and Rep78 proteins activates the P19 promoter, which is responsible for the transcription of the Rep40 and Rep52 proteins (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; Ogasawara, Y. et al. (1998) "The Use of Heterologous Promoters for Adeno Associated Virus (AAV) Protein Expression in AAV Vector Production," Microbiol. Immunol. 42(3):177-185). The 3' half the AAV genome comprises the AAV capsid gene (cap), which encodes three capsid proteins (VP): VP1, VP2 and VP3. The three capsid proteins are translated from a single mRNA transcript that is controlled by a single promoter (P40 in case of AAV2). The 3' half of the AAV genome also comprises the AAP gene, which encodes the AAV assembly-activating protein (AAP). Sixty VP monomers (comprising approximately 5 copies of VP1, 5 copies of VP2, and 50 copies of VP3) self-assemble around the AAV genome to form the icosahedral protein shell (capsid) of the mature viral particle (Buning, H. et al. (2019) "Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors," Mol. Ther. Meth. Clin. Devel. 12:P248-P265; Van Vliet K. M. et al. (2008) The Role of the Adeno-Associated Virus Capsid in Gene Transfer. In: DRUG DELIVERY SYSTEMS, Jain, K. K. (eds.), Meth. Molec. Biol. 437:51-91). The AAV AAP protein is believed to be required for stabilizing and transporting newly produced VP proteins from the cytoplasm into the cell nucleus. The 3' half of the AAV genome also comprises the AAV X gene, which is believed to encode a protein that supports genome replication (Colella, P. et al. (2018) "Emerging Issues in AAV-Mediated In Vivo Gene Therapy," Molec. Ther. Meth. Clin. Develop. 8:87-104; Buning, H. et al. (2019) "Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors," Mol. Ther. Meth. Clin. Devel. 12:P248-P265; Cao, M. et al. (2014) "The X Gene Of Adeno-Associated Virus 2 (AAV2) Is Involved In Viral DNA Replication," PLoS ONE 9, e104596:1-10).

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

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

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

[0009] II. rAAV and Their Use in Gene Therapy

[0010] In light of AAV's properties, recombinantly-modified versions of AAV (rAAV) have found substantial utility as vectors for gene therapy (see, Naso, M. F. et al. (2017) "Adeno Associated Virus (AAV) as a Vector for Gene Therapy," BioDrugs 31:317-334; Berns, K. I. et al. (2017) "AAV. An Overview of Unanswered Questions," Human Gene Ther. 28(4):308-313; Berry, G. E. et al. (2016) "Cellular Transduction Mechanisms Of Adeno-Associated Viral Vectors," Curr. Opin. Virol. 21:54-60; Blessing, D. et al. (2016) "Adeno-Associated Virus And Lentivirus Vectors: A Refined Toolkit For The Central Nervous System," 21:61-66; Santiago-Ortiz, J. L. (2016) "Adeno-Associated Virus (AAV) Vectors in Cancer Gene Therapy," J. Control Release 240:287-301; Salganik, M. et al. (2015) "Adeno-Associated Virus As A Mammalian DNA Vector," Microbiol. Spectr. 3(4):1-32; Hocquemiller, M. et al. (2016) "Adeno-Associated Virus-Based Gene Therapy for CNS Diseases," Hum. Gene Ther. 27(7):478-496; Lykken, E. A. et al. (2018) "Recent Progress And Considerations For AAV Gene Therapies Targeting The Central Nervous System," J. Neurodevelop. Dis. 10:16:1-10; Buning, H. et al. (2019) "Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors," Mol. Ther. Meth. Clin. Devel. 12:P248-P265; During, M. J. et al. (1998) "In Vivo Expression Of Therapeutic Human Genes For Dopamine Production In The Caudates Of MPTP-Treated Monkeys Using An AAV Vector," Gene The. 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; U.S. Pat. Nos. 10,266,845; 10,081,659; 9,890,396; 9,840,719; 9,839,696; 9,834,789; 9,803,218; 9,783,825; 9,777,291; 9,540,659; 9,527,904; 8,236,557; 7,972,593 and 7,943,374).

[0011] 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, .alpha. .beta.-globin intron, a cloning site into which a therapeutic gene of choice (transgene) has been inserted, and a poly-adenylation ("polyA") site. The inverted terminal repeated sequences (ITR) of the rAAV are, however, retained, so that the transgene expression cassette of the rAAV plasmid vector is flanked by AAV ITR sequences (Colella, P. et al. (2018) "Emerging Issues in AAV-Mediated In Vivo Gene Therapy," Molec. Ther. Meth. Clin. Develop. 8:87-104; Buning, H. et al. (2019) "Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors," Mol. Ther. Meth. Clin. Devel. 12:P248-P265). Thus, in the 5' to 3' direction, the rAAV comprises a 5' ITR, the transgene expression cassette of the rAAV, and a 3' ITR.

[0012] rAAV have been used to deliver a transgene to patients suffering from any of a multitude of genetic diseases (e.g., hereditary lipoprotein lipase deficiency (LPLD), Leber's congenital amaurosis (LCA), aromatic L-amino acid decarboxylase deficiency (AADC), choroideremia and hemophilia), and have utility in new clinical modalities, such as in interfering RNA (RNAi) therapy and gene-modifying strategies such as Crispr/Cas9 (U.S. Pat. Nos. 8,697,359, 10,000,772, 10,113,167, 10,227,611; Lino, C. A. et al. (2018) "Delivering CRISPR: A Review Of The Challenges And Approaches," Drug Deliv. 25(1):1234-1237; Ferreira, V. et al. (2014) "Immune Responses To AAV-Vectors, The Glybera Example From Bench To Bedside" Front. Immunol. 5(82):1-15), Buning, H. et al. (2019) "Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors," Mol. Ther. Meth. Clin. Devel. 12:P248-P265; Rastall, D. P. W. (2017) "Current and Future Treatments for Lysosomal Storage Disorders," Curr. Treat Options Neurol. 19(12):45; Kay, M. et al. (2017) "Future Of rAAV Gene Therapy: Platform For 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 (Buning, H. et al. (2019) "CapsidModifications 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. 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.

[0013] III. Methods of rAAV Production

[0014] rAAV containing a desired transgene expression cassette are typically produced by human cells (such as HEK293) grown in suspension. Since, as described above, rAAV are defective viruses, additional functions must be provided in order to replicate and package rAAV.

[0015] rAAV can be 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).

[0016] 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 (e.g., 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).

[0017] The transient transfection of plasmid DNAs comprising the rAAV plasmid vector, the AAV rep and cap genes, and the trans-acting AAD helper genes 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).

[0018] 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.

[0019] IV. Methods of rAAV Purification and Recovery

[0020] After production, rAAV are typically collected and purified by one or more overnight CsCl gradient centrifugations (Zolotukhin, S. et al. (1999) "Recombinant Adeno Associated Virus Purification Using Novel Methods Improves Infectious Titer And Yield," Gene Ther. 6:973-985), followed by desalting to form a purified rAAV production stock. Titers of 10.sup.12-10.sup.13 infectious rAAV capsids/mL are obtainable.

[0021] 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.

[0022] Despite all such prior advances, 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).

[0023] The present invention is directed to improved methods for increasing the efficiency of AAV and rAAV packaging through regulation of the expression of the AAV rep and cap genes.

SUMMARY OF THE INVENTION

[0024] The present invention is directed to recombinantly-modified adeno-associated virus (AAV) helper vectors that are capable of increasing the packaging efficiency of recombinantly-modified adeno-associated virus (rAAV) and their use to improve the packaging efficiency of such rAAV. The present invention is particularly directed to recombinantly-modified adeno-associated virus (AAV) helper vectors that have been further modified to replace (or augment) the P5 and/or P40 promoter sequences that are natively associated with the Rep proteins encoded by such rAAV with AAV P5 and/or P40 promoters that are associated with the Rep proteins of an rAAV of different serotype. The use of such substitute or additional promoter sequences causes increased production of recombinantly-modified adeno-associated virus.

[0025] In detail, the invention provides a recombinantly-modified adeno-associated virus (AAV) helper vector that comprises an AAV helper function-providing polynucleotide, and especially an AAV helper function-providing polynucleotide that is a plasmid vector, wherein the polynucleotide comprises a non-native AAV serotype P5 or P40 promoter sequence.

[0026] The invention particularly includes the embodiment of such recombinantly-modified adeno-associated virus (AAV) helper vector wherein the AAV helper function-providing polynucleotide vector comprises a non-native AAV serotype P5 promoter sequence and/or a non-native AAV serotype P40 promoter sequence.

[0027] The invention also particularly includes the embodiment of such recombinantly-modified adeno-associated virus (AAV) helper vector wherein the non-native AAV serotype P5 or P40 promoter sequence replaces a native AAV serotype promoter sequence.

[0028] The invention also particularly includes the embodiment of such recombinantly-modified adeno-associated virus (AAV) helper vector wherein the vector additionally comprises a non-AAV helper function-providing polynucleotide.

[0029] The invention additionally provides a method for increasing the production titer of a recombinantly-modified adeno-associated virus (rAAV) that comprises a transgene cassette, wherein the method comprises culturing cells that have been transfected with: [0030] (1) the rAAV; [0031] (2) the above-described recombinantly-modified adeno-associated virus (AAV) helper vector that additionally comprises a non-AAV helper function-providing polynucleotide; wherein the culturing is conducted in a culture medium under conditions sufficient to permit the production of the rAAV, and wherein the presence of the non-native AAV serotype P5 or P40 promoter sequence causes the cells to produce the rAAV at an increased production titer relative to that which would be attained if the AAV helper function-providing polynucleotide contained native serotype P5 and P40 promoters.

[0032] The invention additionally provides a method for increasing the production titer of recombinantly-modified adeno-associated virus (rAAV) that comprises a transgene cassette, wherein the method comprises culturing cells that have been transfected with: [0033] (1) the rAAV; [0034] (2) any of the above-described recombinantly-modified adeno-associated virus (AAV) helper vectors; and [0035] (3) an additional vector, especially a plasmid vector, that comprises a non-AAV helper function-providing polynucleotide; wherein the culturing is conducted in a culture medium under conditions sufficient to permit the production of the rAAV, and wherein the presence of the non-native AAV serotype P5 or P40 promoter sequence causes the cells to produce the rAAV at an increased production titer relative to that which would be attained if the AAV helper function-providing polynucleotide contained native serotype P5 and P40 promoters.

[0036] The invention particularly includes the embodiment of such methods, wherein the transgene cassette encodes a protein, or comprises a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition.

[0037] The invention also particularly includes the embodiment of such methods, wherein: [0038] (A) the AAV helper function-providing polynucleotide of the vector encodes an AAV1 Cap protein, and the non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of the serotypes; [0039] (B) the AAV helper function-providing polynucleotide of the vector encodes an AAV2 Cap protein, and the non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of the serotypes; [0040] (C) the AAV helper function-providing polynucleotide of the vector encodes an AAV3 Cap protein, and the non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV4, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of the serotypes; [0041] (D) the AAV helper function-providing polynucleotide of the vector encodes an AAV4 Cap protein, and the non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of the serotypes; [0042] (E) the AAV helper function-providing polynucleotide of the vector encodes an AAV5 Cap protein, and the non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV6, AAV7 or AAV8, or a hybrid of one or more of the serotypes; [0043] (F) the AAV helper function-providing polynucleotide of the vector encodes an AAV6 Cap protein, and the non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV7 or AAV8, or a hybrid of one or more of the serotypes; [0044] (G) the AAV helper function-providing polynucleotide of the vector encodes an AAV7 Cap protein, and the non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV6 or AAV8, or a hybrid of one or more of the serotypes; [0045] (H) the AAV helper function-providing polynucleotide of the vector encodes an AAV8 Cap protein, and the non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV6 or AAV7, or a hybrid of one or more of the serotypes.

[0046] The invention also particularly includes the embodiment of such methods, wherein the cells are human embryonic kidney cells, baby hamster kidney cells or sf9 insect cells.

[0047] The invention additionally provides a pharmaceutical composition that comprises the recombinantly-modified adeno-associated virus (rAAV) produced by any of the above-listed methods, and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG. 1 provides a schematic genetic map of the wild-type (Wt) AAV genome.

[0049] 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, Eta, VA and E4.

[0050] FIG. 3 shows a map of the AAV helper plasmid vector pAAV-RC1 (SEQ ID NO:1).

[0051] FIG. 4 shows a map of the AAV helper plasmid vector pAAV-RC2 (SEQ ID NO:2).

[0052] FIG. 5 shows a map of the AAV helper plasmid vector pAAV-RC5 (SEQ ID NO:3).

[0053] FIG. 6 shows a map of the AAV helper plasmid vector pAAV-RC6 (SEQ ID NO:4).

[0054] FIG. 7 shows a map of the AAV helper plasmid vector pAAV-RC7 (SEQ ID NO:5).

[0055] FIG. 8 shows a map of the non-AAV helper plasmid vector pHelper-Kan (SEQ ID NO:6).

[0056] FIG. 9 shows a map of the rAAV plasmid vector pAV-CMV-EGFP (SEQ ID NO:7).

[0057] FIG. 10 shows a map of the rAAV plasmid vector pAV-TBG-EGFP (SEQ ID NO:8).

[0058] FIG. 11 shows the overall structure and approach followed for the development of the exemplary AAV helper constructs described herein. The parent construct (pAAV-RC2; Parent-RC) comprises AAV2 serotype promoter sequences for the P5 and P19 promoters (solid black boxes) that direct expression of the native AAV2 rep gene (white boxed gene), which encodes the Rep proteins, as well as the AAV2 serotype promoter sequence of the P40 promoter (solid black box) that directs expression of the native AAV2 cap gene (gray boxed gene), which encodes the Cap proteins.

[0059] P5-RC constructs are derivatives of parental plasmid AAV RC that have been modified to direct expression of the AAV rep gene using a non-native P5 promoter (i.e., an AAV P5 promoter that is not natively present within the AAV rep gene of the vector (downward striped box)) in lieu of the native AAV serotype P5 promoter (solid black box); P5-RC constructs direct expression of the AAV rep and cap genes using the native AAV serotype P19 and P40 promoter sequences (solid black boxes) of the parent vector. P40-RC constructs are derivatives of parental plasmid AAV RC that have been modified to direct expression of the AAV cap gene using a non-native P40 promoter (i.e., an AAV P40 promoter that is not natively present within the AAV rep gene (upward striped box)) of the vector in lieu of the native AAV serotype P40 promoter (solid black box); P40-RC constructs direct expression of the AAV rep gene using the native AAV serotype P5 and P19 promoter sequences (solid black boxes) of the parent vector. P5/P40-RC constructs are derivatives of parental plasmid AAV RC that have been modified to direct expression of the AAV rep gene using a non-native P5 promoter (i.e., an AAV P5 promoter that is not natively present within the AAV rep gene of the vector (downward striped box)) in lieu of the native AAV serotype P5 promoter (solid black box). P5/P40-RC constructs have additionally been modified to direct expression to direct expression of the AAV cap gene using a non-native P40 promoter (i.e., an AAV P40 promoter that is not natively present within the AAV rep gene (upward striped box)) of the vector in lieu of the native AAV serotype P40 promoter (solid black box). P40-RC constructs direct expression of the AAV rep gene using the native AAV serotype P19 promoter sequences (solid black boxes) of the parent vector. The sequences of the promoter regions are shown in Table 1.

[0060] FIGS. 12A-12B show the production titers of rAAV obtained by modifying a parental RC2 vector to comprise a non-native P5 promoter sequence (FIG. 11; FIG. 12A; downward striped rectangle) in lieu of the AAV2 P5 promoter that is natively associated with the rep gene of such vector. The P19 and P40 promoters are both native AAV2 serotype promoter sequences (solid black rectangles). FIG. 12B shows the production titers of rAAV obtained using such AAV helper plasmid vectors. The following constructs were employed: Parent-RC2, P5(2)-RC2, P5(1)-RC2, P5(3)-RC2, P5(4)-RC2, P5(5)-RC2, P5(7)-RC2, and P5(8)-RC2. The sequences of the promoter regions are shown in Table 1. The production titers of rAAV were obtained using a triple plasmid transfection system with an rAAV, and an Ad helper plasmid that provided the required adenoviral functions.

[0061] FIGS. 13A-13B show the production titers of rAAV obtained by modifying a parental RC2 vector to comprise a non-native P40 promoter sequence (FIG. 11; FIG. 13A; upward striped rectangle) in lieu of the AAV2 serotype P40 promoter of the parental vector. The P5 and P19 promoters are both native AAV2 serotype promoter sequences (solid black rectangle). FIG. 13B shows the production titers of rAAV obtained using such AAV helper plasmid vectors. The following constructs were employed: Parent-RC2, P40(1)-RC2, P40(3)-RC2, P40(4)-RC2, P40(5)-RC2, P40(6)-RC2, P40(7)-RC2, and P40(8)-RC2. The sequences of the promoter regions are shown in Table 1. The production titers of rAAV were obtained using a triple plasmid transfection system with an rAAV, and an Ad helper plasmid that provided the required adenoviral functions.

[0062] FIGS. 14A-14B show the production titers of rAAV obtained by modifying a parental RC2 vector to comprise a non-native P5 promoter sequence and/or a non-native P40 promoter sequence (FIG. 11; FIG. 14A; P5, downward striped rectangle; P40, upward striped rectangle) in lieu of the AAV2 serotype P5 and P40 promoters of the parental vector. The P19 promoter is a native AAV2 serotype promoter sequences (solid black rectangle). The following constructs were employed: Parent-RC2, P5(2)-RC2, P5(3)-RC2, P5(5)-RC2, P40(1)-RC2, P5(2)/P40(1)-RC2, P5(3)/P40(1)-RC2, and P5(5)/P40(1)-RC2. The sequences of the promoter regions are shown in Table 1. FIG. 14B shows the production titers of rAAV obtained using such AAV helper plasmid vectors. The production titers of rAAV were obtained using a triple plasmid transfection system with an rAAV, and an Ad helper plasmid that provided the required adenoviral functions.

[0063] FIGS. 15A-15C show the production titers of rAAV obtained by modifying a parental RC6 vector to comprise a non-native P5 promoter sequence (FIG. 11; FIG. 15A; downward striped rectangle) in lieu of the AAV2 serotype P5 promoter that is natively associated with the rep gene of such vector. The P19 and P40 promoters are both native AAV2 serotype promoter sequences (solid black rectangles). The following constructs were employed: Parent-RC6, P5(1)-RC6, P5(2)-RC6, P5(3)-RC6, P5(7)-RC6 and P5(8)-RC6. The sequences of the promoter regions are shown in Table 1. The production titers of rAAV were obtained (FIGS. 15B-15C) using a triple plasmid transfection system with an rAAV, and an Ad helper plasmid that provided the required adenoviral functions.

[0064] FIGS. 16A-16B show the production titers of rAAV obtained by modifying a parental RC1, RC5, or RC7 vector to comprise a non-native P5 promoter sequence (FIG. 11; FIG. 16A; downward striped rectangle) in lieu of the AAV2 serotype P5 promoter that is natively associated with the rep gene of such vectors. The P19 and P40 promoters are both native AAV2 serotype promoter sequences (solid black rectangles). The following constructs were employed: Parent-RC1, Parent-RCS, Parent-RC7, P5(2)-RC1, P5(7)-RC1, P5(8)-RC1, P5(7)-RC5, P5(2)-RC7, P5(7)-RC7 and P5(8)-RC7. The sequences of the promoter regions are shown in Table 1. The production titers of rAAV (FIG. 16B) were obtained using a triple plasmid transfection system with an rAAV, and an Ad helper plasmid that provided the required adenoviral functions.

DETAILED DESCRIPTION OF THE INVENTION:

[0065] I. The Methods of the Present Invention

[0066] The present invention is directed to recombinantly-modified adeno-associated virus (AAV) helper vectors that are capable of increasing the packaging efficiency of recombinantly-modified adeno-associated virus (rAAV) and their use to improve the packaging efficiency of such rAAV. The present invention is particularly directed to recombinantly-modified adeno-associated virus (AAV) helper vectors that have been further modified to replace (or augment) the P5 and/or P40 promoter sequences that are natively associated with the Rep proteins encoded by such rAAV with AAV P5 and/or P40 promoters that are associated with the Rep proteins of an rAAV of different serotype. The use of such substitute or additional promoter sequences causes increased production of recombinantly-modified adeno-associated virus.

[0067] The present invention is based in part on the recognition that high levels of Rep and Cap proteins increase the amount of rAAV genomes particles produced and, consequently, the efficiency of rAAV packaging, and thus result in high production titers of rAAV stocks. It has been unexpectedly found that by replacing the AAV P5 and/or P40 promoters that direct the expression of the Cap proteins with different AAV P5 and/or P40 promoters, or by adding such different AAV P5 and/or P40 promoters in addition to those initially present, causes the desired high levels of rAAV to be attained. AAV Rep proteins are described in U.S. Pat. Nos. 10,214,730; 7,122,348; 6,821,511; 6,753,419; 9,441,206; and 7,115,391.

[0068] As discussed above, AAV and rAAV are characterized based on their serotype, which is determined by their capsid proteins (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; 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). By forming AAV and rAAV in the presence of AAV helper function-providing polynucleotides that encode two or more capsid proteins of different serotype, one can produce AAV and rAAV having "hybrid" serotypes. Such AAV and rAAV exhibit the combined trophism of AAV and rAAV having each of such capsid proteins.

[0069] The Rep proteins of the different AAV serotypes differ, however, since such proteins are not structural proteins, the differences do not contribute to the observed serotype of an AAV.

[0070] 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 discussed above, rAAV typically lack certain AAV genes and thus are produced using a double plasmid transfection system, or more preferably a triple plasmid transfection system that comprises a plasmid vector that comprises an AAV helper function-providing polynucleotide, a plasmid vector that comprises a non-AAV helper function-providing polynucleotide, and the rAAV plasmid vector (FIG. 2). In one embodiment, the AAV helper function-providing polynucleotide of such double or triple transfection systems may comprise more than one rep and/or cap gene, so as to be capable of forming rAAV having hybrid serotypes. In another embodiment, a second or additional AAV helper function-providing polynucleotide (for example on a second or additional plasmid vector) may be provided to permit the formation of rAAV having hybrid serotypes.

[0071] A. Illustrative AAV Helper Function-Providing Polynucleotides

[0072] 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 include pAAV-RC (Agilent; Addgene; Cell Biolabs), pAAV-RC1, pAAV-RC2, pAAV-RC5, pAAV-RC6, and pAAV-RC7.

[0073] 1. Plasmid pAAV-RC1

[0074] Plasmid pAAV-RC1 (SEQ ID NO:1; FIG. 3) is an AAV helper plasmid that expresses AAV1 serotype capsid proteins that may be used in accordance with the present invention to provide AAV helper functions. The P5 and P40 promoters of pAAV-RC1 are AAV2 serotype promoters (SEQ ID NO:10 and SEQ ID NO:18, respectively).

TABLE-US-00001 Coding Strand of Plasmid pAAV-RC1 (SEQ ID NO: 1): catggttttg ggacgtttcc tgagtcagat tcgcgaaaaa ctgattcaga gaatttaccg cgggatcgag ccgactttgc caaactggtt cgcggtcaca aagaccagaa atggcgccgg aggcgggaac aaggtggtgg atgagtgcta catccccaat tacttgctcc ccaaaaccca gcctgagctc cagtgggcgt ggactaatat ggaacagtat ttaagcgcct gtttgaatct cacggagcgt aaacggttgg tggcgcagca tctgacgcac gtgtcgcaga cgcaggagca gaacaaagag aatcagaatc ccaattctga tgcgccggtg atcagatcaa aaacttcagc caggtacatg gagctggtcg ggtggctcgt ggacaagggg attacctcgg agaagcagtg gatccaggag gaccaggcct catacatctc cttcaatgcg gcctccaact cgcggtccca aatcaaggct gccttggaca atgcgggaaa gattatgagc ctgactaaaa ccgcccccga ctacctggtg ggccagcagc ccgtggagga catttccagc aatcggattt ataaaatttt ggaactaaac gggtacgatc cccaatatgc ggcttccgtc tttctgggat gggccacgaa aaagttcggc aagaggaaca ccatctggct gtttgggcct gcaactaccg ggaagaccaa catcgcggag gccatagccc acactgtgcc cttctacggg tgcgtaaact ggaccaatga gaactttccc ttcaacgact gtgtcgacaa gatggtgatc tggtgggagg aggggaagat gaccgccaag gtcgtggagt cggccaaagc cattctcgga ggaagcaagg tgcgcgtgga ccagaaatgc aagtcctcgg cccagataga cccgactccc gtgatcgtca cctccaacac caacatgtgc gccgtgattg acgggaactc aacgaccttc gaacaccagc agccgttgca agaccggatg ttcaaatttg aactcacccg ccgtctggat catgactttg ggaaggtcac caagcaggaa gtcaaagact ttttccggtg ggcaaaggat cacgtggttg aggtggagca tgaattctac gtcaaaaagg gtggagccaa gaaaagaccc gcccccagtg acgcagatat aagtgagccc aaacgggtgc gcgagtcagt tgcgcagcca tcgacgtcag acgcggaagc ttcgatcaac tacgcagaca ggtaccaaaa caaatgttct cgtcacgtgg gcatgaatct gatgctgttt ccctgcagac aatgcgagag aatgaatcag aattcaaata tctgcttcac tcacggacag aaagactgtt tagagtgctt tcccgtgtca gaatctcaac ccgtttctgt cgtcaaaaag gcgtatcaga aactgtgcta cattcatcat atcatgggaa aggtgccaga cgcttgcact gcctgcgatc tggtcaatgt ggatttggat gactgcatct ttgaacaata aatgatttaa atcaggtatg gctgccgatg gttatcttcc agattggctc gaggacaacc tctctgaggg cattcgcgag tggtgggact tgaaacctgg agccccgaag cccaaagcca accagcaaaa gcaggacgac ggccggggtc tggtgcttcc tggctacaag tacctcggac ccttcaacgg actcgacaag ggggagcccg tcaacgcggc ggacgcagcg gccctcgagc acgacaaggc ctacgaccag cagctcaaag cgggtgacaa tccgtacctg cggtataacc acgccgacgc cgagtttcag gagcgtctgc aagaagatac gtcttttggg ggcaacctcg ggcgagcagt cttccaggcc aagaagcggg ttctcgaacc tctcggtctg gttgaggaag gcgctaagac ggctcctgga aagaaacgtc cggtagagca gtcgccacaa gagccagact cctcctcggg catcggcaag acaggccagc agcccgctaa aaagagactc aattttggtc agactggcga ctcagagtca gtccccgatc cacaacctct cggagaacct ccagcaaccc ccgctgctgt gggacctact acaatggctt caggcggtgg cgcaccaatg gcagacaata acgaaggcgc cgacggagtg ggtaatgcct caggaaattg gcattgcgat tccacatggc tgggcgacag agtcatcacc accagcaccc gcacctgggc cttgcccacc tacaataacc acctctacaa gcaaatctcc agtgcttcaa cgggggccag caacgacaac cactacttcg gctacagcac cccctggggg tattttgatt tcaacagatt ccactgccac ttttcaccac gtgactggca gcgactcatc aacaacaatt ggggattccg gcccaagaga ctcaacttca aactcttcaa catccaagtc aaggaggtca cgacgaatga tggcgtcaca accatcgcta ataaccttac cagcacggtt caagtcttct cggactcgga gtaccagctt ccgtacgtcc tcggctctgc gcaccagggc tgcctccctc cgttcccggc ggacgtgttc atgattccgc aatacggcta cctgacgctc aacaatggca gccaagccgt gggacgttca tccttttact gcctggaata tttcccttct cagatgctga gaacgggcaa caactttacc ttcagctaca cctttgagga agtgcctttc cacagcagct acgcgcacag ccagagcctg gaccggctga tgaatcctct catcgaccaa tacctgtatt acctgaacag aactcaaaat cagtccggaa gtgcccaaaa caaggacttg ctgtttagcc gtgggtctcc agctggcatg tctgttcagc ccaaaaactg gctacctgga ccctgttatc ggcagcagcg cgtttctaaa acaaaaacag acaacaacaa cagcaatttt acctggactg gtgcttcaaa atataacctc aatgggcgtg aatccatcat caaccctggc actgctatgg cctcacacaa agacgacgaa gacaagttct ttcccatgag cggtgtcatg atttttggaa aagagagcgc cggagcttca aacactgcat tggacaatgt catgattaca gacgaagagg aaattaaagc cactaaccct gtggccaccg aaagatttgg gaccgtggca gtcaatttcc agagcagcag cacagaccct gcgaccggag atgtgcatgc tatgggagca ttacctggca tggtgtggca agatagagac gtgtacctgc agggtcccat ttgggccaaa attcctcaca cagatggaca ctttcacccg tctcctctta tgggcggctt tggactcaag aacccgcctc ctcagatcct catcaaaaac acgcctgttc ctgcgaatcc tccggcggag ttttcagcta caaagtttgc ttcattcatc acccaatact ccacaggaca agtgagtgtg gaaattgaat gggagctgca gaaagaaaac agcaagcgct ggaatcccga agtgcagtac acatccaatt atgcaaaatc tgccaacgtt gattttactg tggacaacaa tggactttat actgagcctc gccccattgg cacccgttac cttacccgtc ccctgtaagg cgcgccaccg gttgcttgtt aatcaataaa ccgtttaatt cgtttcagtt gaactttggt ctctgcgtat ttctttctta tctagtttcc atgctctagg atccactagt aacggccgcc agtgtgctgg aattcggctt tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct agaggtcctg tattagaggt cacgtgagtg ttttgcgaca ttttgcgaca ccatgtggtc acgctgggta tttaagcccg agtgagcacg cagggtctcc attttgaagc gggaggtttg aacgcgcagc cgccaagccg aattctgcag atatccaaac actggcggcc gctcgactag agcggccgcc accgcggtgg agctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga

atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattggg taccgggccc cccctcgagg tcgacggtat cgggggagct cgcagggtct ccattttgaa gcgggaggtt tgaacgcgca gccgccatgc cggggtttta cgagattgtg attaaggtcc ccagcgacct tgacgagcat ctgcccggca tttctgacag ctttgtgaac tgggtggccg agaaggaatg ggagttgccg ccagattctg acatggatct gaatctgatt gagcaggcac ccctgaccgt ggccgagaag ctgcagcgcg actttctgac ggaatggcgc cgtgtgagta aggccccgga ggctcttttc tttgtgcaat ttgagaaggg agagagctac ttccacatgc acgtgctcgt ggaaaccacc ggggtgaaat c

[0075] In SEQ ID NO:1, residues 1-1561 of pAAV-RC1 encode the Rep protein, Rep78 (with residues 95-221 corresponding to the AAV2 P19 promoter and residues 1075-1254 corresponding to the AAV2 P40 promoter (SEQ ID NO:18)); residues 1578-3788 encode the AAV1 VP1 capsid protein; residues 7127-7431 encode a portion of the Rep68 protein; residues 3984-4114 correspond to AAV2 P5 promoter sequences (SEQ ID NO:10); residues 4237-4253 are M13 Rev sequences; residues 4261-4277 are Lac operator sequences; 4285-4315 are Lac promoter sequences; residues 4578-5302 correspond to pMB ori sequences, residues 5398-6258 encode an ampicillin resistance determinant; and residues 6259-6357 are bla promoter sequences (FIG. 3).

[0076] 2. Plasmid pAAV-RC2

[0077] Plasmid pAAV-RC2 (SEQ ID NO:2; FIG. 4) is an AAV helper plasmid that expresses AAV2 serotype capsid proteins that may be used in accordance with the present invention to provide AAV helper functions. The P5 and P40 promoters of pAAV-RC2 are AAV2 serotype promoters (SEQ ID NO:10 and SEQ ID NO:18, respectively).

TABLE-US-00002 Coding Strand of Plasmid pAAV-RC2 (SEQ ID NO: 2): 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

[0078] In SEQ ID NO:2, residues 85-1950 of pAAV-RC2 encode the Rep protein, Rep78 (with residues 484-663 corresponding to the AAV2 P19 promoter, residues 1464-1643 corresponding to the AAV2 P40 promoter (SEQ ID NO:18) and residues 1668-1676 being a donor site); residues 1967-4174 encode the AAV2 VP1 capsid protein; residues 1992-2016 encode a portion of the Rep68 protein; residues 4175-4256 encode a polyA sequence; residues 4357-4487 correspond to the AAV2 P5 promoter sequences of SEQ ID NO:10); 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. 4).

[0079] 3. Plasmid pAAV-RC5

[0080] Plasmid pAAV-RC5 (SEQ ID NO:3; FIG. 5) is an AAV helper plasmid that expresses AAV5 serotype capsid proteins that may be used in accordance with the present invention to provide AAV helper functions. The P5 and P40 promoters of pAAV-RC5 are AAV2 serotype promoters (SEQ ID NO:10 and SEQ ID NO:18, respectively).

TABLE-US-00003 Coding Strand of Plasmid pAAV-RC5 (SEQ ID NO: 3): catggttttg ggacgtttcc tgagtcagat tcgcgaaaaa ctgattcaga gaatttaccg cgggatcgag ccgactttgc caaactggtt cgcggtcaca aagaccagaa atggcgccgg aggcgggaac aaggtggtgg atgagtgcta catccccaat tacttgctcc ccaaaaccca gcctgagctc cagtgggcgt ggactaatat ggaacagtat ttaagcgcct gtttgaatct cacggagcgt aaacggttgg tggcgcagca tctgacgcac gtgtcgcaga cgcaggagca gaacaaagag aatcagaatc ccaattctga tgcgccggtg atcagatcaa aaacttcagc caggtacatg gagctggtcg ggtggctcgt ggacaagggg attacctcgg agaagcagtg gatccaggag gaccaggcct catacatctc cttcaatgcg gcctccaact cgcggtccca aatcaaggct gccttggaca atgcgggaaa gattatgagc ctgactaaaa ccgcccccga ctacctggtg ggccagcagc ccgtggagga catttccagc aatcggattt ataaaatttt ggaactaaac gggtacgatc cccaatatgc ggcttccgtc tttctgggat gggccacgaa aaagttcggc aagaggaaca ccatctggct gtttgggcct gcaactaccg ggaagaccaa catcgcggag gccatagccc acactgtgcc cttctacggg tgcgtaaact ggaccaatga gaactttccc ttcaacgact gtgtcgacaa gatggtgatc tggtgggagg aggggaagat gaccgccaag gtcgtggagt cggccaaagc cattctcgga ggaagcaagg tgcgcgtgga ccagaaatgc aagtcctcgg cccagataga cccgactccc gtgatcgtca cctccaacac caacatgtgc gccgtgattg acgggaactc aacgaccttc gaacaccagc agccgttgca agaccggatg ttcaaatttg aactcacccg ccgtctggat catgactttg ggaaggtcac caagcaggaa gtcaaagact ttttccggtg ggcaaaggat cacgtggttg aggtggagca tgaattctac gtcaaaaagg gtggagccaa gaaaagaccc gcccccagtg acgcagatat aagtgagccc aaacgggtgc gcgagtcagt tgcgcagcca tcgacgtcag acgcggaagc ttcgatcaac tacgcagaca ggtaccaaaa caaatgttct cgtcacgtgg gcatgaatct gatgctgttt ccctgcagac aatgcgagag aatgaatcag aattcaaata tctgcttcac tcacggacag aaagactgtt tagagtgctt tcccgtgtca gaatctcaac ccgtttctgt cgtcaaaaag gcgtatcaga aactgtgcta cattcatcat atcatgggaa aggtgccaga cgcttgcact gcctgcgatc tggtcaatgt ggatttggat gactgcatct ttgaacaata aatgatttaa atcaggtatg tcttttgttg atcaccctcc agattggttg gaagaagttg gtgaaggtct tcgcgagttt ttgggccttg aagcgggccc accgaaacca aaacccaatc agcagcatca agatcaagcc cgtggtcttg tgctgcctgg ttataactat ctcggacccg gaaacggtct cgatcgagga gagcctgtca acagggcaga cgaggtcgcg cgagagcacg acatctcgta caacgagcag cttgaggcgg gagacaaccc ctacctcaag tacaaccacg cggacgccga gtttcaggag aagctcgccg acgacacatc cttcggggga aacctcggaa aggcagtctt tcaggccaag aaaagggttc tcgaaccttt tggcctggtt gaagagggtg ctaagacggc ccctaccgga aagcggatag acgaccactt tccaaaaaga aagaaggctc ggaccgaaga ggactccaag ccttccacct cgtcagacgc cgaagctgga cccagcggat cccagcagct gcaaatccca gcccaaccag cctcaagttt gggagctgat acaatgtctg cgggaggtgg cggcccattg ggcgacaata accaaggtgc cgatggagtg ggcaatgcct cgggagattg gcattgcgat tccacgtgga tgggggacag agtcgtcacc aagtccaccc gaacctgggt gctgcccagc tacaacaacc accagtaccg agagatcaaa agcggctccg tcgacggaag caacgccaac gcctactttg gatacagcac cccctggggg tactttgact ttaaccgctt ccacagccac tggagccccc gagactggca aagactcatc aacaactact ggggcttcag accccggtcc ctcagagtca aaatcttcaa cattcaagtc aaagaggtca cggtgcagga ctccaccacc accatcgcca acaacctcac ctccaccgtc caagtgttta cggacgacga ctaccagctg ccctacgtcg tcggcaacgg gaccgaggga tgcctgccgg ccttccctcc gcaggtcttt acgctgccgc agtacggtta cgcgacgctg aaccgcgaca acacagaaaa tcccaccgag aggagcagct tcttctgcct agagtacttt cccagcaaga tgctgagaac gggcaacaac tttgagttta cctacaactt tgaggaggtg cccttccact ccagcttcgc tcccagtcag aacctgttca agctggccaa cccgctggtg gaccagtact tgtaccgctt cgtgagcaca aataacactg gcggagtcca gttcaacaag aacctggccg ggagatacgc caacacctac aaaaactggt tcccggggcc catgggccga acccagggct ggaacctggg ctccggggtc aaccgcgcca gtgtcagcgc cttcgccacg accaatagga tggagctcga gggcgcgagt taccaggtgc ccccgcagcc gaacggcatg accaacaacc tccagggcag caacacctat gccctggaga acactatgat cttcaacagc cagccggcga acccgggcac caccgccacg tacctcgagg gcaacatgct catcaccagc gagagcgaga cgcagccggt gaaccgcgtg gcgtacaacg tcggcgggca gatggccacc aacaaccaga gctccaccac tgcccccgcg accggcacgt acaacctcca ggaaatcgtg cccggcagcg tgtggatgga gagggacgtg tacctccaag gacccatctg ggccaagatc ccagagacgg gggcgcactt tcacccctct ccggccatgg gcggattcgg actcaaacac ccaccgccca tgatgctcat caagaacacg cctgtgcccg gaaatatcac cagcttctcg gacgtgcccg tcagcagctt catcacccag tacagcaccg ggcaggtcac cgtggagatg gagtgggagc tcaagaagga aaactccaag aggtggaacc cagagatcca gtacacaaac aactacaacg acccccagtt tgtggacttt gccccggaca gcaccgggga atacagaacc accagaccta tcggaacccg ataccttacc cgaccccttt aaggcgcgcc accggttgct tgttaatcaa taaaccgttt aattcgtttc agttgaactt tggtctctgc gtatttcttt cttatctagt ttccatgctc taggatccac tagtaacggc cgccagtgtg ctggaattcg gctttgtagt taatgattaa cccgccatgc tacttatcta cgtagccatg ctctagaggt cctgtattag aggtcacgtg agtgttttgc gacattttgc gacaccatgt ggtcacgctg ggtatttaag cccgagtgag cacgcagggt ctccattttg aagcgggagg tttgaacgcg cagccgccaa gccgaattct gcagatatcc aaacactggc ggccgctcga ctagagcggc cgccaccgcg gtggagctcc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 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 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg gccccccctc gaggtcgacg gtatcggggg agctcgcagg gtctccattt tgaagcggga ggtttgaacg cgcagccgcc atgccggggt tttacgagat tgtgattaag gtccccagcg accttgacga gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga gaagctgcag cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggctct tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac caccggggtg aaatc

[0081] In SEQ ID NO:3, residues 1-1561 of pAAV-RC5 encode the Rep protein, Rep78 (with residues 91-221 corresponding to the AAV2 P19 promoter, and residues 1075-1254 corresponding to the P40 promoter (SEQ ID NO:18)); residues 1578-3749 encode the AAV5 VP1 capsid protein; residues 7091-7395 encode a portion of the Rep68 protein; residues 3948-4078 correspond to the AAV2 P5 promoter sequences of SEQ ID NO:10); residues 4201-4217 are M13 Rev sequences; residues 4225-4241 are Lac operator sequences; 4249-4279 are Lac promoter sequences; residues 4542-5266 correspond to pMB ori sequences, residues 5362-6222 encode an ampicillin resistance determinant; and residues 6223-6321 are bla promoter sequences (FIG. 5).

[0082] 4. Plasmid pAAV-RC6

[0083] Plasmid pAAV-RC6 (SEQ ID NO:4; FIG. 6) is an AAV helper plasmid that expresses AAV6 serotype capsid proteins that may be used in accordance with the present invention to provide AAV helper functions. The P5 and P40 promoters of pAAV-RC6 are AAV2 serotype promoters (SEQ ID NO:10 and SEQ ID NO:18, respectively).

TABLE-US-00004 Coding Strand of Plasmid pAAV-RC6 (SEQ ID NO: 4): catggttttg ggacgtttcc tgagtcagat tcgcgaaaaa ctgattcaga gaatttaccg cgggatcgag ccgactttgc caaactggtt cgcggtcaca aagaccagaa atggcgccgg aggcgggaac aaggtggtgg atgagtgcta catccccaat tacttgctcc ccaaaaccca gcctgagctc cagtgggcgt ggactaatat ggaacagtat ttaagcgcct gtttgaatct cacggagcgt aaacggttgg tggcgcagca tctgacgcac gtgtcgcaga cgcaggagca gaacaaagag aatcagaatc ccaattctga tgcgccggtg atcagatcaa aaacttcagc caggtacatg gagctggtcg ggtggctcgt ggacaagggg attacctcgg agaagcagtg gatccaggag gaccaggcct catacatctc cttcaatgcg gcctccaact cgcggtccca aatcaaggct gccttggaca atgcgggaaa gattatgagc ctgactaaaa ccgcccccga ctacctggtg ggccagcagc ccgtggagga catttccagc aatcggattt ataaaatttt ggaactaaac gggtacgatc cccaatatgc ggcttccgtc tttctgggat gggccacgaa aaagttcggc aagaggaaca ccatctggct gtttgggcct gcaactaccg ggaagaccaa catcgcggag gccatagccc acactgtgcc cttctacggg tgcgtaaact ggaccaatga gaactttccc ttcaacgact gtgtcgacaa gatggtgatc tggtgggagg aggggaagat gaccgccaag gtcgtggagt cggccaaagc cattctcgga ggaagcaagg tgcgcgtgga ccagaaatgc aagtcctcgg cccagataga cccgactccc gtgatcgtca cctccaacac caacatgtgc gccgtgattg acgggaactc aacgaccttc gaacaccagc agccgttgca agaccggatg ttcaaatttg aactcacccg ccgtctggat catgactttg ggaaggtcac caagcaggaa gtcaaagact ttttccggtg ggcaaaggat cacgtggttg aggtggagca tgaattctac gtcaaaaagg gtggagccaa gaaaagaccc gcccccagtg acgcagatat aagtgagccc aaacgggtgc gcgagtcagt tgcgcagcca tcgacgtcag acgcggaagc ttcgatcaac tacgcagaca ggtaccaaaa caaatgttct cgtcacgtgg gcatgaatct gatgctgttt ccctgcagac aatgcgagag aatgaatcag aattcaaata tctgcttcac tcacggacag aaagactgtt tagagtgctt tcccgtgtca gaatctcaac ccgtttctgt cgtcaaaaag gcgtatcaga aactgtgcta cattcatcat atcatgggaa aggtgccaga cgcttgcact gcctgcgatc tggtcaatgt ggatttggat gactgcatct ttgaacaata aatgatttaa atcaggtatg gctgccgatg gttatcttcc agattggctc gaggacaacc tctctgaggg cattcgcgag tggtgggact tgaaacctgg agccccgaaa cccaaagcca accagcaaaa gcaggacgac ggccggggtc tggtgcttcc tggctacaag tacctcggac ccttcaacgg actcgacaag ggggagcccg tcaacgcggc ggatgcagcg gccctcgagc acgacaaggc ctacgaccag cagctcaaag cgggtgacaa tccgtacctg cggtataacc acgccgacgc cgagtttcag gagcgtctgc aagaagatac gtcttttggg ggcaacctcg ggcgagcagt cttccaggcc aagaagaggg ttctcgaacc ttttggtctg gttgaggaag gtgctaagac ggctcctgga aagaaacgtc cggtagagca gtcgccacaa gagccagact cctcctcggg cattggcaag acaggccagc agcccgctaa aaagagactc aattttggtc agactggcga ctcagagtca gtccccgacc cacaacctct cggagaacct ccagcaaccc ccgctgctgt gggacctact acaatggctt caggcggtgg cgcaccaatg gcagacaata acgaaggcgc cgacggagtg ggtaatgcct caggaaattg gcattgcgat tccacatggc tgggcgacag agtcatcacc accagcaccc gaacatgggc cttgcccacc tataacaacc acctctacaa gcaaatctcc agtgcttcaa cgggggccag caacgacaac cactacttcg gctacagcac cccctggggg tattttgatt tcaacagatt ccactgccat ttctcaccac gtgactggca gcgactcatc aacaacaatt ggggattccg gcccaagaga ctcaacttca agctcttcaa catccaagtc aaggaggtca cgacgaatga tggcgtcacg accatcgcta ataaccttac cagcacggtt caagtcttct cggactcgga gtaccagttg ccgtacgtcc tcggctctgc gcaccagggc tgcctccctc cgttcccggc ggacgtgttc atgattccgc agtacggcta cctaacgctc aacaatggca gccaggcagt gggacggtca tccttttact gcctggaata tttcccatcg cagatgctga gaacgggcaa taactttacc ttcagctaca ccttcgagga cgtgcctttc cacagcagct acgcgcacag ccagagcctg gaccggctga tgaatcctct catcgaccag tacctgtatt acctgaacag aactcagaat cagtccggaa gtgcccaaaa caaggacttg ctgtttagcc gggggtctcc agctggcatg tctgttcagc ccaaaaactg gctacctgga ccctgttacc ggcagcagcg cgtttctaaa acaaaaacag acaacaacaa cagcaacttt acctggactg gtgcttcaaa atataacctt aatgggcgtg aatctataat caaccctggc actgctatgg cctcacacaa agacgacaaa gacaagttct ttcccatgag cggtgtcatg atttttggaa aggagagcgc cggagcttca aacactgcat tggacaatgt catgatcaca gacgaagagg aaatcaaagc cactaacccc gtggccaccg aaagatttgg gactgtggca gtcaatctcc agagcagcag cacagaccct gcgaccggag atgtgcatgt tatgggagcc ttacctggaa tggtgtggca agacagagac gtatacctgc agggtcctat ttgggccaaa attcctcaca cggatggaca ctttcacccg tctcctctca tgggcggctt tggacttaag cacccgcctc ctcagatcct catcaaaaac acgcctgttc ctgcgaatcc tccggcagag ttttcggcta caaagtttgc ttcattcatc acccagtatt ccacaggaca agtgagcgtg gagattgaat gggagctgca gaaagaaaac agcaaacgct ggaatcccga agtgcagtat acatctaact atgcaaaatc tgccaacgtt gatttcactg tggacaacaa tggactttat actgagcctc gccccattgg cacccgttac ctcacccgtc ccctgtaagg cgcgccaccg gttgcttgtt aatcaataaa ccgtttaatt cgtttcagtt gaactttggt ctctgcgtat ttctttctta tctagtttcc atgctctagg atccactagt aacggccgcc agtgtgctgg aattcggctt tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct agaggtcctg tattagaggt cacgtgagtg ttttgcgaca ttttgcgaca ccatgtggtc acgctgggta tttaagcccg agtgagcacg cagggtctcc attttgaagc gggaggtttg aacgcgcagc cgccaagccg aattctgcag atatccaaac actggcggcc gctcgactag agcggccgcc accgcggtgg agctccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg

gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac tcactatagg gcgaattggg taccgggccc cccctcgagg tcgacggtat cgggggagct cgcagggtct ccattttgaa gcgggaggtt tgaacgcgca gccgccatgc cggggtttta cgagattgtg attaaggtcc ccagcgacct tgacgagcat ctgcccggca tttctgacag ctttgtgaac tgggtggccg agaaggaatg ggagttgccg ccagattctg acatggatct gaatctgatt gagcaggcac ccctgaccgt ggccgagaag ctgcagcgcg actttctgac ggaatggcgc cgtgtgagta aggccccgga ggctcttttc tttgtgcaat ttgagaaggg agagagctac ttccacatgc acgtgctcgt ggaaaccacc ggggtgaaat c

[0084] In SEQ ID NO:4, residues 1-1561 of pAAV-RC6 encode the Rep protein, Rep78 (with residues 91-221 corresponding to the AAV2 P19 promoter, and residues 1075-1254 corresponding to the P40 promoter (SEQ ID NO:18)); residues 1578-3788 encode the AAV6 VP1 capsid protein; residues 736-1281 encode a portion of the Rep68 protein; residues 3984-4114 correspond to the AAV2 P5 promoter sequences of SEQ ID NO:10); residues 4237-4253 are M13 Rev sequences; residues 4261-4277 are Lac operator sequences; 4285-4315 are Lac promoter sequences; residues 4578-5302 correspond to pMB ori sequences, residues 5398-6258 encode an ampicillin resistance determinant; and residues 6259-6357 are bla promoter sequences (FIG. 6).

[0085] 5. Plasmid pAAV-RC7

[0086] Plasmid pAAV-RC7 (SEQ ID NO:5; FIG. 7) is an AAV helper plasmid that expresses AAV6 serotype capsid proteins that may be used in accordance with the present invention to provide AAV helper functions. The P5 and P40 promoters of pAAV-RC7 are AAV2 serotype promoters (SEQ ID NO:10 and SEQ ID NO:18, respectively).

TABLE-US-00005 Coding Strand of Plasmid pAAV-RC7 (SEQ ID NO: 5): catggttttg ggacgtttcc tgagtcagat tcgcgaaaaa ctgattcaga gaatttaccg cgggatcgag ccgactttgc caaactggtt cgcggtcaca aagaccagaa atggcgccgg aggcgggaac aaggtggtgg atgagtgcta catccccaat tacttgctcc ccaaaaccca gcctgagctc cagtgggcgt ggactaatat ggaacagtat ttaagcgcct gtttgaatct cacggagcgt aaacggttgg tggcgcagca tctgacgcac gtgtcgcaga cgcaggagca gaacaaagag aatcagaatc ccaattctga tgcgccggtg atcagatcaa aaacttcagc caggtacatg gagctggtcg ggtggctcgt ggacaagggg attacctcgg agaagcagtg gatccaggag gaccaggcct catacatctc cttcaatgcg gcctccaact cgcggtccca aatcaaggct gccttggaca atgcgggaaa gattatgagc ctgactaaaa ccgcccccga ctacctggtg ggccagcagc ccgtggagga catttccagc aatcggattt ataaaatttt ggaactaaac gggtacgatc cccaatatgc ggcttccgtc tttctgggat gggccacgaa aaagttcggc aagaggaaca ccatctggct gtttgggcct gcaactaccg ggaagaccaa catcgcggag gccatagccc acactgtgcc cttctacggg tgcgtaaact ggaccaatga gaactttccc ttcaacgact gtgtcgacaa gatggtgatc tggtgggagg aggggaagat gaccgccaag gtcgtggagt cggccaaagc cattctcgga ggaagcaagg tgcgcgtgga ccagaaatgc aagtcctcgg cccagataga cccgactccc gtgatcgtca cctccaacac caacatgtgc gccgtgattg acgggaactc aacgaccttc gaacaccagc agccgttgca agaccggatg ttcaaatttg aactcacccg ccgtctggat catgactttg ggaaggtcac caagcaggaa gtcaaagact ttttccggtg ggcaaaggat cacgtggttg aggtggagca tgaattctac gtcaaaaagg gtggagccaa gaaaagaccc gcccccagtg acgcagatat aagtgagccc aaacgggtgc gcgagtcagt tgcgcagcca tcgacgtcag acgcggaagc ttcgatcaac tacgcagaca ggtaccaaaa caaatgttct cgtcacgtgg gcatgaatct gatgctgttt ccctgcagac aatgcgagag aatgaatcag aattcaaata tctgcttcac tcacggacag aaagactgtt tagagtgctt tcccgtgtca gaatctcaac ccgtttctgt cgtcaaaaag gcgtatcaga aactgtgcta cattcatcat atcatgggaa aggtgccaga cgcttgcact gcctgcgatc tggtcaatgt ggatttggat gactgcatct ttgaacaata aatgatttaa atcaggtatg gctgccgatg gttatcttcc agattggctc gaggacaacc tctctgaggg cattcgcgag tggtgggacc tgaaacctgg agccccgaaa cccaaagcca accagcaaaa gcaggacaac ggccggggtc tggtgcttcc tggctacaag tacctcggac ccttcaacgg actcgacaag ggggagcccg tcaacgcggc ggacgcagcg gccctcgagc acgacaaggc ctacgaccag cagctcaaag cgggtgacaa tccgtacctg cggtataacc acgccgacgc cgagtttcag gagcgtctgc aagaagatac gtcatttggg ggcaacctcg ggcgagcagt cttccaggcc aagaagcggg ttctcgaacc tctcggtctg gttgaggaag gcgctaagac ggctcctgca aagaagagac cggtagagcc gtcacctcag cgttcccccg actcctccac gggcatcggc aagaaaggcc agcagcccgc cagaaagaga ctcaatttcg gtcagactgg cgactcagag tcagtccccg accctcaacc tctcggagaa cctccagcag cgccctctag tgtgggatct ggtacagtgg ctgcaggcgg tggcgcacca atggcagaca ataacgaagg tgccgacgga gtgggtaatg cctcaggaaa ttggcattgc gattccacat ggctgggcga cagagtcatt accaccagca cccgaacctg ggccctgccc acctacaaca accacctcta caagcaaatc tccagtgaaa ctgcaggtag taccaacgac aacacctact tcggctacag caccccctgg gggtattttg actttaacag attccactgc cacttctcac cacgtgactg gcagcgactc atcaacaaca actggggatt ccggcccaag aagctgcggt tcaagctctt caacatccag gtcaaggagg tcacgacgaa tgacggcgtt acgaccatcg ctaataacct taccagcacg attcaggtat tctcggactc ggaataccag ctgccgtacg tcctcggctc tgcgcaccag ggctgcctgc ctccgttccc ggcggacgtc ttcatgattc ctcagtacgg ctacctgact ctcaacaatg gcagtcagtc tgtgggacgt tcctccttct actgcctgga gtacttcccc tctcagatgc tgagaacggg caacaacttt gagttcagct acagcttcga ggacgtgcct ttccacagca gctacgcaca cagccagagc ctggaccggc tgatgaatcc cctcatcgac cagtacttgt actacctggc cagaacacag agtaacccag gaggcacagc tggcaatcgg gaactgcagt tttaccaggg cgggccttca actatggccg aacaagccaa gaattggtta cctggacctt gcttccggca acaaagagtc tccaaaacgc tggatcaaaa caacaacagc aactttgctt ggactggtgc caccaaatat cacctgaacg gcagaaactc gttggttaat cccggcgtcg ccatggcaac tcacaaggac gacgaggacc gctttttccc atccagcgga gtcctgattt ttggaaaaac tggagcaact aacaaaacta cattggaaaa tgtgttaatg acaaatgaag aagaaattcg tcctactaat cctgtagcca cggaagaata cgggatagtc agcagcaact tacaagcggc taatactgca gcccagacac aagttgtcaa caaccaggga gccttacctg gcatggtctg gcagaaccgg gacgtgtacc tgcagggtcc catctgggcc aagattcctc acacggatgg caactttcac ccgtctcctt tgatgggcgg ctttggactt aaacatccgc ctcctcagat cctgatcaag aacactcccg ttcccgctaa tcctccggag gtgtttactc ctgccaagtt tgcttcgttc atcacacagt acagcaccgg acaagtcagc gtggaaatcg agtgggagct gcagaaggaa aacagcaagc gctggaaccc ggagattcag tacacctcca actttgaaaa gcagactggt gtggactttg ccgttgacag ccagggtgtt tactctgagc ctcgccctat tggcactcgt tacctcaccc gtaatctgta aggcgcgcca ccggttgctt 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 gggtaccggg ccccccctcg aggtcgacgg tatcggggga gctcgcaggg tctccatttt gaagcgggag gtttgaacgc gcagccgcca tgccggggtt ttacgagatt gtgattaagg tccccagcga ccttgacgag catctgcccg gcatttctga cagctttgtg aactgggtgg ccgagaagga atgggagttg ccgccagatt ctgacatgga tctgaatctg attgagcagg cacccctgac cgtggccgag aagctgcagc gcgactttct gacggaatgg cgccgtgtga gtaaggcccc ggaggctctt ttctttgtgc aatttgagaa gggagagagc tacttccaca tgcacgtgct cgtggaaacc accggggtga aatc

[0087] In SEQ ID NO:5, residues 1-1561 of pAAV-RC7 encode the Rep protein, Rep78 (with residues 91-221 corresponding to the AAV2 P19 promoter, and residues 1075-1254 corresponding to the P40 promoter (SEQ ID NO:18)); residues 1578-3791 encode the AAV7 VP1 capsid protein; residues 736-1281 encode a portion of the Rep68 protein; residues 3987-4117 correspond to the AAV2 P5 promoter sequences of SEQ ID NO:10); residues 4240-4256 are M13 Rev sequences; residues 4264-4280 are Lac operator sequences; 4288-4318 are Lac promoter sequences; residues 4581-5305 correspond to pMB ori sequences, residues 5401-6261 encode an ampicillin resistance determinant; and residues 6262-6360 are bla promoter sequences (FIG. 7).

[0088] B. Illustrative Non-AAV Helper Function-Providing Polynucleotides

[0089] 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 (such as those commercially available from Cell Biolabs, Inc., Invitrogen, Stratagene and other sources), 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).

[0090] Plasmid pHelper-Kan (SEQ ID NO:6; FIG. 8) is a non-AAV helper function-providing polynucleotide that may be used in accordance with the present invention to provide non-AAV helper functions.

TABLE-US-00006 Coding Strand of Plasmid pHelper-Kan (SEQ ID NO: 6): 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

[0091] In SEQ ID NO:6, 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. 8).

[0092] C. Illustrative rAAV Plasmid Vectors

[0093] 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:7; FIG. 9) which comprises a 5' ITR, a U6 promoter, CMV enhancer and promoter sequences, a polynucleotide encoding the enhanced green fluorescent protein (EGFP) (Gambotto, A. et al. (2000) "Immunogenicity Of Enhanced Green Fluorescent Protein (EGFP) In BALB/C Mice: Identification Of An H2-Kd-Restricted CTL Epitope," Gene Ther. 7(23):2036-2040; Tsien, R. Y. (1998) "The Green Fluorescent Protein," Annu. Rev. Biochem. 67:509-544; Cinelli, R. A. et al. (2000) "The Enhanced Green Fluorescent Protein As A Tool For The Analysis Of Protein Dynamics And Localization: Local Fluorescence Study At The Single-Molecule Level," Photochem. Photobiol. 71(6):771-776; Chopra A. (2008) "Recombinant Adenovirus With Enhanced Green Fluorescent Protein," In: MOLECULAR IMAGING AND CONTRAST AGENT DATABASE (MICAD), National Center for Biotechnology Information, Bethesda Md.), FLAG-tag and 6.times.His-tag sites for facilitating recovery or localization of expressed proteins, an SV40 poly(A) site and a 3' ITR.

TABLE-US-00007 Coding Strand of Plasmid pAV-CMV-EGFP (SEQ ID NO: 7): 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

[0094] In SEQ ID NO:7, residues 1-128 of pAV-CMV-EGFP correspond to the 5' ITR; residues 201-441 are U6 promoter sequences; residues 562-865 are human cytomegalovirus (CMV) immediate early enhancer sequences; residues 866-1068 comprise the CMV immediate early promoter; residues 1192-1911 comprise a mammalian codon-optimized polynucleotide that encodes the EGFP; residues 1918-1941 encode the FLAG-tag; residues 1951-1968 encode the 6.times.His-tag; residues 2139-2260 encode the SV40 poly(A) sequence; residues 2293-2433 correspond to the 3' ITR; residues 2508-22963 correspond to F1 ori sequences; residues 3350-4210 encode an ampicillin resistance determinant and its signal sequence (residues 3350-3418) expressed by a bla promoter sequence (residues 3245-3349); residues 4381-4969 correspond to an ori sequence (FIG. 9).

[0095] A second illustrative rAAV plasmid vector that may be used in accordance with the present invention is pAV-TBG-EGFP (SEQ ID NO:8; FIG. 10) which comprises a 5' ITR, a thyroid hormone-binding globulin (TBG) promoter, a polynucleotide encoding the enhanced green fluorescent protein (EGFP), FLAG-tag and 6.times.His-tag sites for facilitating recovery or localization of expressed proteins, an SV40 poly(A) site and a 3' ITR.

TABLE-US-00008 Coding Strand of Plasmid pAV-TBG-EGFP (SEQ ID NO: 8): 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

[0096] In SEQ ID NO:8, 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 6xHis-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. 10).

[0097] As used herein, the term "native AAV serotype promoter sequence" is intended to denote a promoter sequence that natively controls the transcription of an AAV rep gene or is natively present within such rep gene. For example: [0098] AAV1 P5 promoter sequences natively control the transcription of the rep gene of AAV1 and AAV1 P40 promoter sequences are natively found within the rep gene of AAV1. Thus, the AAV1 P5 promoter sequences and the AAV1 P40 promoter sequences are native AAV serotype promoter sequences of the rep gene of AAV1; [0099] AAV2 P5 promoter sequences natively control the transcription of the rep gene of AAV2 and the AAV2 P40 promoter sequences are natively found within the rep gene of AAV2. Thus, the AAV2 P5 promoter sequences and the AAV2 P40 promoter sequences are native AAV serotype promoter sequences of the rep gene of AAV2; [0100] AAV5 P5 promoter sequences natively control the transcription of the rep gene of AAV5 and the AAVS P40 promoter sequences are natively found within the rep gene of AAV5. Thus, the AAVS P5 promoter sequences and the AAV5 P40 promoter sequences are native AAV serotype promoter sequences of the rep gene of AAV5; [0101] AAV6 P5 promoter sequences natively control the transcription of the rep gene of AAV6 and the AAV6 P40 promoter sequences are natively found within the rep gene of AAV6. Thus, the AAV6 P5 promoter sequences and the AAV6 P40 promoter sequences are native AAV serotype promoter sequences of the rep gene of AAV6; and [0102] AAV7 P5 promoter sequences natively control the transcription of the rep gene of AAV7 and the AAV7 P40 promoter sequences are natively found within the rep gene of AAV7. Thus, the AAV7 P5 promoter sequences and the AAV7 P40 promoter sequences are native AAV serotype promoter sequences of the rep gene of AAV7;

[0103] Native AAV P5 and P40 promoter sequences for AAV serotypes 1-8 are shown in Table 1. Such sequences, or subsequences thereof that are capable of mediating transcription, may be used in accordance with the methods of the present invention.

TABLE-US-00009 TABLE 1 AAV Native SEQ ID Pro- Sero- NO moter type Sequence SEQ ID P5 AAV1 ggtcctgtat tagctgtcac gtgagtgctt NO: 9 ttgcgacatt ttgcgacacc acgtggccat ttagggtata tatggccgag tgagcgagca ggatctccat tttgaccgcg aaatttgaac gagcagcagc c SEQ ID P5 AAV2 ggtcctgtat tagaggtcac gtgagtgttt NO: 10 tgcgacattt tgcgacacca tgtggtcacg ctgggtattt aagcccgagt gagcacgcag ggtctccatt ttgaagcggg aggtttgaac gcgcagccgc c SEQ ID P5 AAV3 ccagctgcgt cagcagtcag gtgacccttt NO: 11 tgcgacagtt tgcgacacca cgtggccgct gagggtatat attctcgagt gagcgaacca ggagctccat tttgaccgcg aaatttgaac gagcagcagc c SEQ ID P5 AAV4 ggtccctgta ttagcagtca cgtgagtgtc NO: 12 gtatttcgcg gagcgtagcg gagcgcatac caagctgcca cgtcacagcc acgtggtccg tttgcgacag tttgcgacac catgtggtca ggagggtata taaccgcgag tgagccagcg aggagctcca ttttgcccgc gaattttgaa cgagcagcag cc SEQ ID P5 AAV5 atgtgatgtg ttttatccaa taggaagaaa NO: 13 gcgcgcgtat gagttctcgc gagacttccg gggtataaaa gaccgagtga acgagcccgc cgccattctt tgctctggac tgctagagga ccctcgctgc c SEQ ID P5 AAV6 ggtcctgtat tagaggtcac gtgagtgttt NO: 14 tgcgacattt tgcgacacca tgtggtcacg ctgggtattt aagcccgagt gagcacgcag ggtctccatt ttgaagcggg aggtttgaac gcgcagcgcc SEQ ID P5 AAV7 ggtcctgtat tagctgtcac gtgagtgctt NO: 15 ttgcgacatt ttgcgacacc acgtggccat ttgaggtata tatggccgag tgagcgagca ggatctccat tttgaccgcg aaatttgaac gagcagcagc c SEQ ID P5 AAV8 ggtcctgtat tagctgtcac gtgagtgctt NO: 16 ttgcggcatt ttgcgacacc acgtggccat ttgaggtata tatggccgag tgagcgagca ggatctccat tttgaccgcg aaatttgaac gagcagcagc c SEQ ID P40 AAV1 ggtgacaaag caggaagtca aagagttctt NO: 17 ccgctgggcg caggatcacg tgaccgaggt ggcgcatgag ttctacgtca gaaagggtgg agccaacaaa agacccgccc ccgatgacgc ggataaaagc gagcccaagc gggcctgccc ctcagtcgcg gatccatcga cgtcagacgc SEQ ID P40 AAV2 ggtcaccaag caggaagtca aagacttttt NO: 18 ccggtgggca aaggatcacg tggttgaggt ggagcatgaa ttctacgtca aaaagggtgg agccaagaaa agacccgccc ccagtgacgc agatataagt gagcccaaac gggtgcgcga gtcagttgcg cagccatcga cgtcagacgc SEQ ID P40 AAV3 ggtcaccaaa caggaagtaa aggacttttt NO: 19 ccggtgggct tccgatcacg tgactgacgt ggctcatgag ttctacgtca gaaagggtgg agctaagaaa cgccccgcct ccaatgacgc ggatgtaagc gagccaaaac gggagtgcac gtcacttgcg cagccgacaa cgtcagacgc SEQ ID P40 AAV4 ggtcaccaag caggaagtca aagacttttt NO: 20 ccggtgggcg tcagatcacg tgaccgaggt gactcacgag ttttacgtca gaaagggtgg agctagaaag aggcccgccc ccaatgacgc agatataagt gagcccaagc gggcctgtcc gtcagttgcg cagccatcga cgtcagacgc SEQ ID P40 AAV5 gattactaag caggaagtca aggacttttt NO: 21 tgcttgggca aaggtcaatc aggtgccggt gactcacgag tttaaagttc ccagggaatt ggcgggaact aaaggggcgg agaaatctct aaaacgccca ctgggtgacg tcaccaatac tagctataaa agtctggaga agcgggcctg gagcatgagg ctctcatttg ttcccgagac gcctcgcagt tcagacg SEQ ID P40 AAV6 ggtgacaaag caggaagtca aagagttctt NO: 22 ccgctgggcg caggatcacg tgaccgaggt ggcgcatgag ttctacgtca gaaagggtgg agccaacaag agacccgccc ccgatgacgc ggataaaagc gagcccaagc gggcctgccc ctcagtcgcg gatccatcga cgtcagacgc SEQ ID P40 AAV7 ggtgacgaag caggaagtca aagagttctt NO: 23 ccgctgggcc agtgatcacg tgaccgaggt ggcgcatgag ttctacgtca gaaagggcgg agccagcaaa agacccgccc ccgatgacgc ggatataagc gagcccaagc gggcctgccc ctcagtcgcg gatccatcga cgtcagacgc SEQ ID P40 AAV8 ggtgacaaag caggaagtca aagagttctt NO: 24 ccgctgggcc agtgatcacg tgaccgaggt ggcgcatgag ttttacgtca gaaagggcgg agccagcaaa agacccgccc ccgatgacgc ggataaaagc gagcccaagc gggcctgccc ctcagtcgcg gatccatcga cgtcagacgc

[0104] In contrast, the term "non-native AAV serotype promoter sequence" is intended to denote a promoter sequence that does not natively control a rep gene of an AAV and is not natively found within such rep gene. Illustrative, non-limiting examples of non-native AAV serotype promoter sequences include: the AAV1 P5 promoter when used to direct the expression of an AAV2, AAV5, AAV6, or AAV7 rep gene; the AAV2 P5 promoter when used to direct the expression of an AAV1, AAV5, AAV6, or AAV7 rep gene; the AAV5 P5 promoter when used to direct the expression of an AAV1, AAV2, AAV6, or AAV7 rep gene; the AAV6 P5 promoter when used to direct the expression of an AAV1, AAV2, AAV5, or AAV7 rep gene; the AAV7 P5 promoter when used to direct the expression of an AAV1, AAV2, AAV5, or AAV6 rep gene; the AAV1 P40 promoter, when present within an AAV2, AAV5, AAV6, or AAV7 rep gene; the AAV2 P40 promoter, when present within an AAV1, AAV5, AAV6, or AAV7 rep gene; the AAV5 P40 promoter, when present within an AAV1, AAV2, AAV6, or AAV7 rep gene; the AAV6 P40 promoter, when present within an AAV1, AAV2, AAV5, or AAV7 rep gene; the AAV7 P40 promoter, when present within an AAV1, AAV2, AAV5, or AAV6 rep gene, etc.

[0105] In one embodiment, one or more of such AAV serotype promoter sequences can be genetically engineered into recombinant AAV helper plasmids that are designed to provide the Rep and Cap proteins to replace or augment the existing P5 or P40 promoters of such plasmids. Such modification is preferably accomplished using well-known methods of recombinant DNA technology.

[0106] The identity of the serotype of promoter sequences is indicated herein by denoting the involved promoter (e.g., P5, P40, etc.), the serotype of the rep gene with which it is natively associated, and the name of the vector. Thus, for example, a pAAV-RC2 plasmid that comprises a P5 promoter sequence that is natively associated with AAV2 is denoted as P5(2)-RC2; a pAAV-RC2 plasmid that comprises a P5 promoter sequence that is natively associated with AAV3 is denoted as P5(3)-RC2; a pAAV-RC5 plasmid that comprises a P40 promoter sequence that is natively associated with AAV7 is denoted as P40(7)-RCS; a pAAV-RC2 plasmid that comprises a P5 promoter sequence that is natively associated with AAV3 and a P40 promoter sequence that is natively associated with AAV8 is denoted as P5(3)/P40(8)-RC2; etc.

[0107] In one embodiment, the introduced AAV serotype promoter sequence will replace an initially present AAV serotype promoter sequence. In other embodiments, the introduced AAV serotype promoter sequence will be present in addition to such initially present AAV serotype promoter sequence, and will be positioned 5' to, or 3' to, such initially present AAV serotype promoter sequence. The introduced nucleotide sequence may be positioned adjacent to, or apart from, such initially present AAV serotype promoter sequence.

[0108] The substitution or addition of one or more of such AAV serotype promoter sequences invention increases rAAV production titers. 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 helper 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).

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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 additionally transfected with: [0113] (1) an AAD helper vector possessing a non-native AAV serotype promoter sequence and capable of expressing proteins or RNA molecules that are not natively 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 cap genes that encode VP capsid proteins required to form infectious particles; and [0114] (2) an Ad helper vector that can provide the non-AAV helper proteins (e.g., E1a, E1b, E2a, VA and E4) or RNA molecules that are not provided by such rAAV or rAAV plasmid vectors, but are required for their production.

[0115] In one embodiment for producing the rAAV of the present invention, all of such 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 AAV helper function-providing polynucleotide that provides the required rep and cap genes and such non-native AAV serotype promoter sequences are provided on a vector that is separate from the vector that comprises the non-AAV helper function-providing polynucleotide, so that such vectors or plasmids, in concert with the rAAV, comprise a triple plasmid transfection system.

[0116] The invention thus derives in part from the recognition that the production of rAAV may be increased by causing the expression of Rep and Cap proteins to be directed by promoter sequences that are not native promoter sequences. Thus, by modifying a particular rAAV to replace its native P5 and/or P40 AAV serotype promoter sequence(s) with a non-native P5 and/or P40 AAV serotype promoter sequence (or by incorporating a non-native P5 and/or P40 AAV serotype promoter sequence into such rAAV), 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 including hybrid serotypes (e.g., AAV2/5 and rAAV2/5, which is a hybrid of AAV serotypes 2 and 5 and thus has the trophism of both such serotypes).

[0117] The methods of the present invention may be employed to increase 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.

[0118] The methods of the present invention may be employed to increase the production titers of rAAV produced by cells in adherent monolayer culture or in suspension culture, and may be used with any method capable of producing rAAV. Preferably, however, rAAV is produced by transfecting baby hamster kidney (BHK) cells, or more preferably, human embryonic kidney (HEK) cells grown in tissue culture with the plasmid vectors described above. The BHK cell line BHK-21 (ATCC CCL-10), which lacks endogenous retroviruses is a preferred BHK cell line. The HEK cell line HEK293 (ATCC CRL-1573) and its derivatives, such as HEK293T (ATCC CRL-3216, which is a highly transfectable derivative of the HEK293 cell line into which the temperature-sensitive gene for SV40 T-antigen was inserted) or HEK293T/17 (ATCC.RTM. CRL-11268, which was selected for its ease of transfection) are particularly preferred. The HEK293T/17 SF cell line (ATCC ACS-4500) is a derivative of the 293T/17 cell line (ATCC CRL-11268), adapted to serum-free medium and suspension, and may be employed if desired.

[0119] 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. 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).

[0120] Cells to be used for such transfection are preferably passaged twice weekly to maintain them in exponential growth phase. For small-scale transfections, an aliquot of, for example, 1.times.10.sup.6 HEK293 or BHK cells per well on a multi-well plate, or 1.5.times.10.sup.7 HEK293 cells per 15-cm dish, may be employed. For large-scale production HEK293 or BHK cells may be collected from multiple confluent 15-cm plates, and split into two 10-layer cell stacks (Corning, Corning, N.Y.) containing 1 liter of complete culturing medium. In one embodiment, such cells are grown for 4 days in such medium before transfection. The day before transfection, the two cell stacks may be trypsinized and the cells (e.g., approximately 6.times.10.sup.8 cells) may be resuspended in 200 ml of medium. Preferably, the cells are allowed to attach for 24 hours before transfection. Confluency of the cell stacks may be monitored using a Diaphot inverted microscope (Nikon, Melville, N.Y.) from which the phase-contrast hardware had been removed in order to accommodate the cell stack on the microscope stage.

[0121] In particular, the present invention thus provides a method for increasing the production titer of a recombinantly-modified adeno-associated virus (rAAV) that comprises a transgene cassette, wherein the method comprises culturing cells that have been transfected with: [0122] (1) the rAAV; [0123] (2) a recombinantly-modified adeno-associated virus (AAV) helper vector that comprises an AAV helper function-providing polynucleotide, wherein such polynucleotide comprises a non-native AAV serotype P5 or P40 promoter sequence in replacement of, or in addition to, a native AAV serotype promoter sequence; and [0124] (3) a vector that comprises a non-AAV helper function-providing polynucleotide; wherein the culturing is conducted in a culture medium under conditions sufficient to permit the production of the rAAV, and wherein the presence of the non-native AAV serotype P5 or P40 promoter sequence causes the cells to produce the rAAV at an increased production titer relative to that which would be attained if the AAV helper function-providing polynucleotide contained native serotype P5 and P40 promoters.

[0125] The present invention further provides a method for increasing the production titer of a recombinantly-modified adeno-associated virus (rAAV) that comprises a transgene cassette, wherein the method comprises culturing cells that have been transfected with: [0126] (1) the rAAV; and [0127] (2) a recombinantly-modified adeno-associated virus (AAV) helper vector that comprises: [0128] (a) an AAV helper function-providing polynucleotide, wherein such polynucleotide comprises a non-native AAV serotype P5 or P40 promoter sequence in replacement of, or in addition to, a native AAV serotype promoter sequence; and [0129] (b) a non-AAV helper function-providing polynucleotide; wherein the culturing is conducted in a culture medium under conditions sufficient to permit the production of the rAAV, and wherein the presence of the non-native AAV serotype P5 or P40 promoter sequence causes the cells to produce the rAAV at an increased production titer relative to that which would be attained if the AAV helper function-providing polynucleotide contained native serotype P5 and P40 promoters.

[0130] In preferred embodiments, the transgene cassette of such rAAV encodes a protein, or comprises a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition.

[0131] II. Pharmaceutical Compositions of the Present Invention

[0132] 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")

[0133] The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. 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.

[0134] 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.

[0135] 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.

[0136] The rAAV of such pharmaceutical compositions is preferably packaged in a hermetically sealed container, such as a vial, an ampoule or sachette indicating the quantity of the molecule, and optionally including instructions for use. In one embodiment, the rAAV of such kit is supplied as a dry sterilized lyophilized powder or water-free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water, saline, or other diluent to the appropriate concentration for administration to a subject. The lyophilized material should be stored at between 2.degree. C. and 8.degree. C. in their original container and the material should be administered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted. In another embodiment, the rAAV of such kit is supplied as an aqueous solution in a hermetically sealed container and can be diluted, e.g., with water, saline, or other diluent, to the appropriate concentration for administration to a subject. The kit can further comprise one or more other prophylactic and/or therapeutic agents useful for the treatment of the disease or condition, in one or more containers; and/or the kit can further comprise one or more cytotoxic antibodies that bind one or more cancer antigens associated with cancer. In certain embodiments, the other prophylactic or therapeutic agent is a chemotherapeutic. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic.

[0137] III. Uses of the Invention

[0138] The methods of the present invention may be used to facilitate the production of rAAV, and may particularly be used to facilitate the production of rAAV that comprise transgene cassettes that encode a protein (e.g., an enzyme, hormone, antibody, receptor, ligand, etc.), or of rAAV that comprise a transcribed nucleic acid, that is relevant to a genetic or heritable disease or condition, such that it may be used in gene therapy to treat such disease or condition. Examples of such diseases and conditions include: achromatopsia (ACHM); alpha-1 antitrypsin (AAT) deficiency; Alzheimer's Disease; aromatic L-amino acid decarboxylase (AADC) deficiency; choroideremia (CHM); cancer; Duchenne muscular dystrophy; dysferlin deficiency; follistatin gene deficiency (BMDSIBM); hemophilia A; hemophilia B; hepatitis A; hepatitis B; hepatitis C; Huntington's disease; idiopathic Parkinson's disease; late-infantile neuronal ceroid lipofuscinosis (LINCL, an infantile form of Batten disease); Leber congenital amaurosis (LCA); Leber' s hereditary optic neuropathy (LHON); limb girdle muscular dystrophy 1B (LGMD1B); limb girdle muscular dystrophy 1C (LGMD1C); limb girdle muscular dystrophy 2A (LGMD2A); limb girdle muscular dystrophy 2B (LGMD2B); limb girdle muscular dystrophy 2I (LGMD2I); limb girdle muscular dystrophy 2L (LGMD2L); lipoprotein lipase (LPL) deficiency; metachromatic leukodystrophy; neurological disability; neuromotor deficit; neuroskeletal impairment; Parkinson's disease; rheumatoid arthritis; Sanfilippo A syndrome; spinal muscular atrophy (SMA); X-linked retinoschisis (XLRS); .alpha.-sarcoglycan deficiency (LGMD2D); .beta.-sarcoglycan deficiency (LGMD2E); .gamma.-sarcoglycan deficiency (LGMD2C) and .delta.-sarcoglycan deficiency (LGMD2F).

IV. Embodiments of the Invention

[0139] The invention concerns a recombinantly-modified adeno-associated virus (AAV) helper vector that comprises an AAV helper function-providing polynucleotide, and uses and compositions thereof. It is particularly directed to the following embodiments E1-E16: [0140] E1. A recombinantly-modified adeno-associated virus (AAV) helper vector that comprises an AAV helper function-providing polynucleotide, wherein the polynucleotide comprises a non-native AAV serotype P5 or P40 promoter sequence. [0141] E2. The recombinantly-modified adeno-associated virus (AAV) helper vector of E1, wherein the AAV helper function-providing polynucleotide vector comprises a non-native AAV serotype P5 promoter sequence. [0142] E3. The recombinantly-modified adeno-associated virus (AAV) helper vector of any one of E1 or E2, wherein the AAV helper function-providing polynucleotide vector comprises a non-native AAV serotype P40 promoter sequence. [0143] E4. The recombinantly-modified adeno-associated virus (AAV) helper vector of any one of E1-E3, wherein the vector is a plasmid vector. [0144] E5. The recombinantly-modified adeno-associated virus (AAV) helper vector of E1, wherein the non-native AAV serotype P5 or P40 promoter sequence replaces a native AAV serotype promoter sequence. [0145] E6. The recombinantly-modified adeno-associated virus (AAV) helper vector of any one of E1-E5, wherein the vector additionally comprises a non-AAV helper function-providing polynucleotide. [0146] E7. A method for increasing the production titer of a recombinantly-modified adeno-associated virus (rAAV) that comprises a transgene cassette, wherein the method comprises culturing cells that have been transfected with: [0147] (1) the rAAV; [0148] (2) the recombinantly-modified adeno-associated virus (AAV) helper vector of E6; [0149] wherein the culturing is conducted in a culture medium under conditions sufficient to permit the production of the rAAV and wherein the presence of the non-native AAV serotype P5 or P40 promoter sequence causes the cells to produce the rAAV at an increased production titer relative to that which would be attained if the AAV helper function-providing polynucleotide contained native serotype P5 and P40 promoters. [0150] E8. A method for increasing the production titer of a recombinantly-modified adeno-associated virus (rAAV) that comprises a transgene cassette, wherein the method comprises culturing cells that have been transfected with: [0151] (1) the rAAV; [0152] (2) the recombinantly-modified adeno-associated virus (AAV) helper vector of any one of E1-E6; and [0153] (3) an additional vector, especially a plasmid vector, that comprises a non-AAV helper function-providing polynucleotide; wherein the culturing is conducted in a culture medium under conditions sufficient to permit the production of the rAAV, and wherein the presence of the non-native AAV serotype P5 or P40 promoter sequence causes the cells to produce the rAAV at an increased production titer relative to that which would be attained if the AAV helper function-providing polynucleotide contained native serotype P5 and P40 promoters. [0154] E9. The method of any one of E7-E8, wherein: [0155] (A) the AAV helper function-providing polynucleotide of the vector encodes an AAV1 Cap protein, and the non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of the serotypes; [0156] (B) the AAV helper function-providing polynucleotide of the vector encodes an AAV2 Cap protein, and the non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of the serotypes; 7 [0157] (C) the AAV helper function-providing polynucleotide of the vector encodes an AAV3 Cap protein, and the non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV4, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of the serotypes; [0158] (D) the AAV helper function-providing polynucleotide of the vector encodes an AAV4 Cap protein, and the non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of the serotypes; [0159] (E) the AAV helper function-providing polynucleotide of the vector encodes an AAV5 Cap protein, and the non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV6, AAV7 or AAV8, or a hybrid of one or more of the serotypes; [0160] (F) the AAV helper function-providing polynucleotide of the vector encodes an AAV6 Cap protein, and the non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV7 or AAV8, or a hybrid of one or more of the serotypes; [0161] (G) the AAV helper function-providing polynucleotide of the vector encodes an AAV7 Cap protein, and the non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV6 or AAV8, or a hybrid of one or more of the serotypes; or [0162] (H) the AAV helper function-providing polynucleotide of the vector encodes an AAV8 Cap protein, and the non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV6 or AAV7, or a hybrid of one or more of the serotypes. [0163] E10. The method of any one of E7-E9, wherein the cells are human embryonic kidney cells, baby hamster kidney cells or sf9 insect cells. [0164] E11. The method of E10, wherein the cells are HEK293 human embryonic kidney cells. [0165] E12. The method of E10, wherein the cells are BHK21 baby hamster kidney cells. [0166] E13. The method of any one of E7-E12, wherein the transgene cassette encodes a protein, or comprises a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition. [0167] E14. A preparation of the recombinantly-modified adeno-associated virus (rAAV) produced by the method of E13. [0168] E15. A pharmaceutical composition that comprises the recombinantly-modified adeno-associated virus (rAAV) produced by the method of E13, and a pharmaceutically acceptable carrier. [0169] E16. The preparation of recombinantly-modified adeno-associated virus (rAAV) of E14, or the pharmaceutical composition of E15, for use in the treatment of the genetic or heritable disease or condition.

EXAMPLES

[0170] 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

Comparison Of rAAV Production Titers By Cells Transfected With AAV RC2 Helper Plasmid Vectors Having A Non-Native AAV Serotype P5 Promoter Sequence

[0171] In order to demonstrate the ability of non-native AAV serotype promoter sequences to affect the production titer of rAAV, derivatives of AAV helper plasmid AAV RC2 (having an AAV2 rep gene and a cap gene that encodes Cap protein of the AAV2 serotype) were constructed that comprised a non-native AAV serotype promoter sequence (FIG. 11) in lieu of the native AAV2 serotype P5 promoter of such plasmid (FIG. 12A; downward striped rectangle). The P19 and P40 promoters of the constructs were not changed, and thus were both native AAV2 serotype promoter sequences (FIG. 12A; solid black rectangles).

[0172] The following constructs were employed; the sequences of the promoter regions are shown in Table 1: [0173] (1) Parent-RC2--pAAV-RC2 (SEQ ID NO:2), which contains the AAV2 rep gene and a partial portion of the full AAV2 serotype P5 promoter sequence (SEQ ID NO:10), and the AAV2 cap gene, whose expression is controlled by a native AAV2 P40 promoter sequence (SEQ ID NO:18); [0174] (2) P5(1)-RC2--a derivative of plasmid vector pAAV-RC2 in which native AAV serotype P5 promoter sequences had been replaced with the P5 promoter sequences of AAV serotype 1 (SEQ ID NO:9); [0175] (3) P5(2)-RC2--a derivative of plasmid vector pAAV-RC2 in which the partial AV2 serotype P5 promoter sequences of Parent-RC2 had been replaced with the full-length P5 promoter sequences of AAV serotype 2 (SEQ ID NO:10); [0176] (4) P5(3)-RC2--a derivative of plasmid vector pAAV-RC2 in which native AAV2 serotype P5 promoter sequences had been replaced with the P5 promoter sequences of AAV serotype 3 (SEQ ID NO:11); [0177] (5) P5(4)-RC2--a derivative of plasmid vector pAAV-RC2 in which native AAV2 serotype P5 promoter sequences had been replaced with the P5 promoter sequences of AAV serotype 4 (SEQ ID NO:12); [0178] (6) P5(5)-RC2--a derivative of plasmid vector pAAV-RC2 in which native AAV2 serotype P5 promoter sequences had been replaced with the P5 promoter sequences of AAV serotype 5 (SEQ ID NO:13); [0179] (7) P5(6)-RC2--a derivative of plasmid vector pAAV-RC2 in which native AAV2 serotype P5 promoter sequences had been replaced with the P5 promoter sequences of AAV serotype 6 (SEQ ID NO:14); [0180] (8) P5(7)-RC2--a derivative of plasmid vector pAAV-RC2 in which native AAV2 serotype P5 promoter sequences had been replaced with the P5 promoter sequences of AAV serotype 7 (SEQ ID NO:15); and [0181] (9) P5(8)-RC2--a derivative of plasmid vector pAAV-RC2 in which native AAV2 serotype P5 promoter sequences had been replaced with the P5 promoter sequences of AAV serotype 8 (SEQ ID NO:16).

[0182] FIG. 12B shows the production titers of rAAV obtained using such AAV helper plasmid vectors. The production titers of rAAV were obtained using a triple plasmid transfection system with an rAAV (pGOI; BBa K404119), and an Ad helper plasmid (pHelper) that provided the required adenoviral functions. Plasmid pGOI is an rAAV plasmid vector that comprises, in the 5' to 3' direction, a 5' ITR, a CMV promoter, a .beta.-globin intron, a polynucleotide encoding the yellow fluorescent protein mVenus (Nagai, T. et al. (2002) "A Variant Of Yellow Fluorescent Protein With Fast And Efficient Maturation For Cell-Biological Applications," Nat. Biotechnol. 20(1):87-90), the polyA domain of human growth hormone and a 3' ITR. FIG. 12B reveals that the serotype of the P5 promoter affects rAAV production titers, and indicates that replacing the native AAV2 P5 promoter of the plasmid vector pAAV-RC2 with an AAVS serotype P5 promoter greatly decreased rAAV production titer, whereas replacing the native AAV2 P5 promoter of the plasmid vector pAAV-RC2 with a P5 promoter of AAV serotype 1, 3, 5, 7 or 8 greatly increased rAAV production titer.

Example 2

Comparison Of rAAV Production Titers By Cells Transfected With AAV RC2 Helper Plasmid Vectors Having A Non-Native AAV Serotype P40 Promoter Sequence

[0183] In order to further demonstrate the ability of non-native AAV serotype promoter sequences to affect the production titer of rAAV, derivatives of AAV helper uplasmid AAV RC2 (having an AAV2 rep gene and a cap gene that encodes Cap protein of the AAV2 serotype) were constructed that comprised a non-native AAV serotype promoter sequence (FIG. 11) in lieu of the native serotype P40 promoter of such plasmid (FIG. 13A; upward striped rectangle). The P5 and P19 promoters of the constructs were not changed, and thus were both native AAV2 serotype promoter sequences (FIG. 13A; solid black rectangles).

[0184] The following constructs were employed; the sequences of the promoter regions are shown in Table 1: [0185] (1) Parent-RC2--pAAV-RC2 (SEQ ID NO:2), which contains the AAV2 rep gene and a partial portion of the full AAV2 serotype P5 promoter sequence (SEQ ID NO:10), and the AAV2 cap gene, whose expression is controlled by a native AAV2 P40 promoter sequence (SEQ ID NO:18); [0186] (2) P40(1)-RC2--a derivative of plasmid vector pAAV-RC2 in which native AAV2 serotype P40 promoter sequences had been replaced with the P40 promoter sequences of AAV serotype 1 (SEQ ID NO:17); [0187] (3) P40(2)-RC2--a derivative of plasmid vector pAAV-RC2 in which the AAV2 serotype P40 promoter sequences of Parent-RC2 had been replaced with the P40 promoter sequences of AAV serotype 2 (SEQ ID NO:18); [0188] (4) P40(3)-RC2--a derivative of plasmid vector pAAV-RC2 in which native AAV2 serotype P40 promoter sequences had been replaced with the P40 promoter sequences of AAV serotype 3 (SEQ ID NO:19); [0189] (5) P40(4)-RC2--a derivative of plasmid vector pAAV-RC2 in which native AAV2 serotype P40 promoter sequences had been replaced with the P40 promoter sequences of AAV serotype 4 (SEQ ID NO:20); [0190] (6) P40(5)-RC2--a derivative of plasmid vector pAAV-RC2 in which native AAV2 serotype P40 promoter sequences had been replaced with the P40 promoter sequences of AAV serotype 5 (SEQ ID NO:21); [0191] (7) P40(6)-RC2--a derivative of plasmid vector pAAV-RC2 in which native AAV2 serotype P40 promoter sequences had been replaced with the P40 promoter sequences of AAV serotype 6 (SEQ ID NO:22); [0192] (8) P40(7)-RC2--a derivative of plasmid vector pAAV-RC2 in which native AAV2 serotype P40 promoter sequences had been replaced with the P40 promoter sequences of AAV serotype 7 (SEQ ID NO:23); and [0193] (9) P40(8)-RC2--a derivative of plasmid vector pAAV-RC2 in which native AAV2 serotype P40 promoter sequences had been replaced with the P40 promoter sequences of AAV serotype 8 (SEQ ID NO:24).

[0194] FIG. 13B shows the production titers of rAAV obtained using such AAV helper plasmid vectors. Production titers of rAAV were obtained essentially as described in Example 1. The results of the investigation reveal that the serotype of the P40 promoter also affects rAAV production titers, and indicate that replacing the native AAV2 P40 promoter of the plasmid vector pAAV-RC2 with an AAV5 serotype P40 promoter greatly decreased rAAV production titer, whereas replacing the native AAV2 P40 promoter of the plasmid vector pAAV-RC2 with an AAV1 serotype P40 promoter or with an AAV8 serotype P40 promoter greatly increased rAAV production titer.

Example 3

Comparison Of rAAV Production Titers By Cells Transfected With AAV RC2 Helper Plasmid Vectors Having Non-Native AAV Serotype P5 and P40 Promoter Sequences

[0195] In order to further demonstrate the ability of non-native AAV serotype promoter sequences to affect the production titer of rAAV, derivatives of AAV helper plasmid AAV RC2 (having an AAV2 rep gene and a cap gene that encodes Cap protein of the AAV2 serotype) were constructed that comprised non-native AAV serotype promoter sequences (FIG. 11) in lieu of the native AAV2 serotype P5 (FIG. 14A; downward striped rectangle) and P40 (FIG. 14A; upward striped rectangle) promoters of such plasmid. The AAV2 P19 promoter of the constructs were not changed, and thus was the native AAV2 serotype promoter sequence (FIG. 14A; solid black rectangle).

[0196] The following constructs were employed; the sequences of the promoter regions are shown in Table 1: [0197] (1) Parent-RC2--pAAV-RC2 (SEQ ID NO:2), which contains the AAV2 rep gene and a partial portion of the full AAV2 serotype P5 promoter sequence (SEQ ID NO:10), and the AAV2 cap gene, whose expression is controlled by a native AAV2 P40 promoter sequence (SEQ ID NO:18); [0198] (2) P5(2)-RC2--a derivative of plasmid vector pAAV-RC2 in which the partial

[0199] AAV2 serotype P5 promoter sequences of Parent-RC2 had been replaced with the full-length P5 promoter sequences of AAV serotype 2 (SEQ ID NO:10); [0200] (3) P5(3)-RC2--a derivative of plasmid vector pAAV-RC2 in which native AAV2 serotype P5 promoter sequences had been replaced with the P5 promoter sequence of AAV3 (SEQ ID NO:11); [0201] (4) P5(5)-RC2--a derivative of plasmid vector pAAV-RC2 in which native AAV2 serotype P5 promoter sequences had been replaced with the P5 promoter sequence of AAV5 (SEQ ID NO:13); [0202] (5) P40(1)-RC2--a derivative of plasmid vector pAAV-RC2 in which native AAV2 serotype P40 promoter sequences had been replaced with the P40 promoter sequence of AAV1 (SEQ ID NO:17); [0203] (6) P5(2)/P40(1)-RC2--a derivative of plasmid vector pAAV-RC2 in which native AAV2 serotype P5 promoter sequences had been replaced with the P5 promoter sequence of AAV2 (SEQ ID NO:10) and in which native P40 promoter sequences had been replaced with the P40 promoter sequence of AAV1 (SEQ ID NO:17); [0204] (7) P5(3)/P40(1)-RC2--a derivative of plasmid vector pAAV-RC2 in which native AAV2 serotype P5 promoter sequences had been replaced with the P5 promoter sequence of AAV3 (SEQ ID NO:11) and in which native P40 promoter sequences had been replaced with the P40 promoter sequence of AAV1 (SEQ ID NO:17); and [0205] (8) P5(5)/P40(1)-RC2--a derivative of plasmid vector pAAV-RC2 in which native AAV2 serotype P5 promoter sequences had been replaced with the P5 promoter sequence of AAV5 (SEQ ID NO:13) and in which native P40 promoter sequences had been replaced with the P40 promoter sequence of AAV1 (SEQ ID NO:17).

[0206] Production titers of rAAV were obtained essentially as described in Example 1. FIG. 14B shows the production titers of rAAV obtained using such AAV helper plasmid vectors. As shown in FIG. 14B, the replacement of the native P5 and P40 promoters of pAAV-RC2 with the P5 promoter sequence of AAV3 or AAV5 and the P40 promoter sequence of AAV1 synergistically increased rAAV production titers.

Example 4

Comparison Of rAAV Production Titers By Cells Transfected With AAV RC6 Helper Plasmid Vectors Having Non-Native AAV Serotype P5 and P40 Promoter Sequences

[0207] In order to further demonstrate the ability of non-native AAV serotype promoter sequences to affect the production titer of rAAV, derivatives of AAV helper plasmid AAV RC6 (having an AAV2 rep gene and a cap gene that encodes Cap protein of the AAV6 serotype) were constructed that comprised non-native AAV serotype promoter sequences (FIG. 11) in lieu of the native AAV2 serotype P5 (FIG. 15A; downward striped rectangle) and P40 (FIG. 15A; downward striped rectangle) promoters of such plasmid. The AAV2 P19 promoter of the constructs were not changed, and thus was the native AAV2 serotype promoter sequence (FIG. 15A; solid black rectangle).

[0208] The following constructions were employed; the sequences of the promoter regions are shown in Table 1: [0209] (1) Parent-RC6--pAAV-RC6 (SEQ ID NO:4), which contains the AAV2 rep gene and its native AAV2 serotype P5 promoter sequence (SEQ ID NO:10), and the AAV6 cap gene, whose expression is controlled by a native AAV2 P40 promoter sequence (SEQ ID NO:18); [0210] (2) P5(1)-RC6--a derivative of plasmid vector pAAV-RC6 in which native AAV2 serotype P5 promoter sequences had been replaced with the P5 promoter sequence of AAV1 (SEQ ID NO:9); [0211] (3) P5(2)-RC6--a derivative of plasmid vector pAAV-RC6 in which native AAV2 serotype P5 promoter sequences had been replaced with the P5 promoter sequence of AAV2 (SEQ ID NO:10); [0212] (4) P5(3)-RC6--a derivative of plasmid vector pAAV-RC6 in which native AAV2 serotype P5 promoter sequences had been replaced with the P5 promoter sequence of AAV3 (SEQ ID NO:11); [0213] (5) P5(7)-RC6--a derivative of plasmid vector pAAV-RC6 in which native AAV2 serotype P5 promoter sequences had been replaced with the P5 promoter sequence of AAV7 (SEQ ID NO:15); and [0214] (6) P5(8)-RC6--a derivative of plasmid vector pAAV-RC6 in which native AAV2 serotype P5 promoter sequences had been replaced with the P5 promoter sequence of AAV8 (SEQ ID NO:16).

[0215] FIG. 15B shows the production titers of rAAV obtained using such AAV helper plasmid vectors. Production titers of rAAV were obtained essentially as described in Example 1.

[0216] The results of the investigation are shown in FIGS. 15B and 15C, and reveal that the production titers of rAAV obtained using such AAV helper plasmid vectors. As shown in such Figures, the replacement of the native P5 and P40 promoters of pAAV-RC6 with the P5 promoter sequence of AAV serotype 1, 2, 3, 7 or 8 increased rAAV production titers.

Example 5

Comparison Of rAAV Production Titers By Cells Transfected With AAV RC1, AAV RC5 or AAV RC7 Helper Plasmid Vectors Having Non-Native AAV Serotype P5 and P40 Promoter Sequences

[0217] In order to further demonstrate the ability of non-native AAV serotype promoter sequences to affect the production titer of rAAV, derivatives of AAV helper plasmid AAV RC1 (having an AAV2 rep gene and a cap gene that encodes Cap protein of the AAV1 serotype), derivatives of AAV helper plasmid AAV RCS (having an AAV2 rep gene and a cap gene that encodes Cap protein of the AAVS serotype) and derivatives of AAV helper plasmid AAV RC7 (having an AAV2 rep gene and a cap gene that encodes Cap protein of the AAV7 serotype) were constructed that comprised non-native AAV serotype promoter sequences (FIG. 11) in lieu of the native AAV2 serotype P5 (FIG. 16A; downward striped rectangle) and/or P40 (FIG. 16A; upward striped rectangle) promoter sequences of such plasmids.

[0218] The following constructions were employed; the sequences of the promoter regions are shown in Table 1: [0219] (1) Parent-RC1--pAAV-RC1 (SEQ ID NO:1), which contains the AAV2 rep gene and its native AAV2 serotype P5 promoter sequence (SEQ ID NO:10), and the AAV1 cap gene, whose expression is controlled by a native AAV2 P40 promoter sequence (SEQ ID NO:18); [0220] (2) Parent-RC5--pAAV-RC5 (SEQ ID NO:3), which contains the AAV2 rep gene and its native AAV2 serotype P5 promoter sequence (SEQ ID NO:10), and the AAV5 cap gene, whose expression is controlled by a native AAV2 P40 promoter sequence (SEQ ID NO:18); [0221] (3) Parent-RC7--pAAV-RC7 (SEQ ID NO:5), which contains the AAV2 rep gene and its native AAV2 serotype P5 promoter sequence (SEQ ID NO:10), and the AAV7 cap gene, whose expression is controlled by a native AAV2 P40 promoter sequence (SEQ ID NO:18); [0222] (4) P5(2)-RC1-- a derivative of plasmid vector pAAV-RC1 in which native AAV1 serotype P5 promoter sequences had been replaced with the P5 promoter sequence of AAV2 (SEQ ID NO:10); [0223] (5) P5(7)-RC1-- a derivative of plasmid vector pAAV-RC1 in which native AAV1 serotype P5 promoter sequences had been replaced with the P5 promoter sequence of AAV7 (SEQ ID NO:15); [0224] (6) P5(8)-RC1-- a derivative of plasmid vector pAAV-RC1 in which native AAV1 serotype P5 promoter sequences had been replaced with the P5 promoter sequence of AAV8 (SEQ ID NO:16); [0225] (7) P5(7)-RC5--a derivative of plasmid vector pAAV-RC5 in which native AAV5 serotype P5 promoter sequences had been replaced with the P5 promoter sequence of AAV7 (SEQ ID NO:15); [0226] (8) P5(2)-RC7--a derivative of plasmid vector pAAV-RC7 in which native AAV7 serotype P5 promoter sequences had been replaced with the P5 promoter sequence of AAV2 (SEQ ID NO:10). [0227] (9) P5(7)-RC7--a derivative of plasmid vector pAAV-RC7 in which native AAV7 serotype P5 promoter sequences had been replaced with the P5 promoter sequence of AAV7 (SEQ ID NO:15); and [0228] (10) P5(8)-RC7--a derivative of plasmid vector pAAV-RC7 in which native AAV7 serotype P5 promoter sequences had been replaced with the P5 promoter sequence of AAV8 (SEQ ID NO:16).

[0229] Production titers of rAAV were obtained essentially as described in Example 1. The results of the investigation are shown in FIG. 16B, and reveals that the replacement of the native P5 promoter sequences of pAAV-RC1, pAAV-RCS, and pAAV-RC7 with P5 promoter sequence of AAV serotype 2, 7 or 8 increased rAAV production titers.

[0230] All publications and patents mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth.

Sequence CWU 1

1

2417431DNAArtificial SequenceCoding Strand of Plasmid pAAV-RC1 1catggttttg ggacgtttcc tgagtcagat tcgcgaaaaa ctgattcaga gaatttaccg 60cgggatcgag ccgactttgc caaactggtt cgcggtcaca aagaccagaa atggcgccgg 120aggcgggaac aaggtggtgg atgagtgcta catccccaat tacttgctcc ccaaaaccca 180gcctgagctc cagtgggcgt ggactaatat ggaacagtat ttaagcgcct gtttgaatct 240cacggagcgt aaacggttgg tggcgcagca tctgacgcac gtgtcgcaga cgcaggagca 300gaacaaagag aatcagaatc ccaattctga tgcgccggtg atcagatcaa aaacttcagc 360caggtacatg gagctggtcg ggtggctcgt ggacaagggg attacctcgg agaagcagtg 420gatccaggag gaccaggcct catacatctc cttcaatgcg gcctccaact cgcggtccca 480aatcaaggct gccttggaca atgcgggaaa gattatgagc ctgactaaaa ccgcccccga 540ctacctggtg ggccagcagc ccgtggagga catttccagc aatcggattt ataaaatttt 600ggaactaaac gggtacgatc cccaatatgc ggcttccgtc tttctgggat gggccacgaa 660aaagttcggc aagaggaaca ccatctggct gtttgggcct gcaactaccg ggaagaccaa 720catcgcggag gccatagccc acactgtgcc cttctacggg tgcgtaaact ggaccaatga 780gaactttccc ttcaacgact gtgtcgacaa gatggtgatc tggtgggagg aggggaagat 840gaccgccaag gtcgtggagt cggccaaagc cattctcgga ggaagcaagg tgcgcgtgga 900ccagaaatgc aagtcctcgg cccagataga cccgactccc gtgatcgtca cctccaacac 960caacatgtgc gccgtgattg acgggaactc aacgaccttc gaacaccagc agccgttgca 1020agaccggatg ttcaaatttg aactcacccg ccgtctggat catgactttg ggaaggtcac 1080caagcaggaa gtcaaagact ttttccggtg ggcaaaggat cacgtggttg aggtggagca 1140tgaattctac gtcaaaaagg gtggagccaa gaaaagaccc gcccccagtg acgcagatat 1200aagtgagccc aaacgggtgc gcgagtcagt tgcgcagcca tcgacgtcag acgcggaagc 1260ttcgatcaac tacgcagaca ggtaccaaaa caaatgttct cgtcacgtgg gcatgaatct 1320gatgctgttt ccctgcagac aatgcgagag aatgaatcag aattcaaata tctgcttcac 1380tcacggacag aaagactgtt tagagtgctt tcccgtgtca gaatctcaac ccgtttctgt 1440cgtcaaaaag gcgtatcaga aactgtgcta cattcatcat atcatgggaa aggtgccaga 1500cgcttgcact gcctgcgatc tggtcaatgt ggatttggat gactgcatct ttgaacaata 1560aatgatttaa atcaggtatg gctgccgatg gttatcttcc agattggctc gaggacaacc 1620tctctgaggg cattcgcgag tggtgggact tgaaacctgg agccccgaag cccaaagcca 1680accagcaaaa gcaggacgac ggccggggtc tggtgcttcc tggctacaag tacctcggac 1740ccttcaacgg actcgacaag ggggagcccg tcaacgcggc ggacgcagcg gccctcgagc 1800acgacaaggc ctacgaccag cagctcaaag cgggtgacaa tccgtacctg cggtataacc 1860acgccgacgc cgagtttcag gagcgtctgc aagaagatac gtcttttggg ggcaacctcg 1920ggcgagcagt cttccaggcc aagaagcggg ttctcgaacc tctcggtctg gttgaggaag 1980gcgctaagac ggctcctgga aagaaacgtc cggtagagca gtcgccacaa gagccagact 2040cctcctcggg catcggcaag acaggccagc agcccgctaa aaagagactc aattttggtc 2100agactggcga ctcagagtca gtccccgatc cacaacctct cggagaacct ccagcaaccc 2160ccgctgctgt gggacctact acaatggctt caggcggtgg cgcaccaatg gcagacaata 2220acgaaggcgc cgacggagtg ggtaatgcct caggaaattg gcattgcgat tccacatggc 2280tgggcgacag agtcatcacc accagcaccc gcacctgggc cttgcccacc tacaataacc 2340acctctacaa gcaaatctcc agtgcttcaa cgggggccag caacgacaac cactacttcg 2400gctacagcac cccctggggg tattttgatt tcaacagatt ccactgccac ttttcaccac 2460gtgactggca gcgactcatc aacaacaatt ggggattccg gcccaagaga ctcaacttca 2520aactcttcaa catccaagtc aaggaggtca cgacgaatga tggcgtcaca accatcgcta 2580ataaccttac cagcacggtt caagtcttct cggactcgga gtaccagctt ccgtacgtcc 2640tcggctctgc gcaccagggc tgcctccctc cgttcccggc ggacgtgttc atgattccgc 2700aatacggcta cctgacgctc aacaatggca gccaagccgt gggacgttca tccttttact 2760gcctggaata tttcccttct cagatgctga gaacgggcaa caactttacc ttcagctaca 2820cctttgagga agtgcctttc cacagcagct acgcgcacag ccagagcctg gaccggctga 2880tgaatcctct catcgaccaa tacctgtatt acctgaacag aactcaaaat cagtccggaa 2940gtgcccaaaa caaggacttg ctgtttagcc gtgggtctcc agctggcatg tctgttcagc 3000ccaaaaactg gctacctgga ccctgttatc ggcagcagcg cgtttctaaa acaaaaacag 3060acaacaacaa cagcaatttt acctggactg gtgcttcaaa atataacctc aatgggcgtg 3120aatccatcat caaccctggc actgctatgg cctcacacaa agacgacgaa gacaagttct 3180ttcccatgag cggtgtcatg atttttggaa aagagagcgc cggagcttca aacactgcat 3240tggacaatgt catgattaca gacgaagagg aaattaaagc cactaaccct gtggccaccg 3300aaagatttgg gaccgtggca gtcaatttcc agagcagcag cacagaccct gcgaccggag 3360atgtgcatgc tatgggagca ttacctggca tggtgtggca agatagagac gtgtacctgc 3420agggtcccat ttgggccaaa attcctcaca cagatggaca ctttcacccg tctcctctta 3480tgggcggctt tggactcaag aacccgcctc ctcagatcct catcaaaaac acgcctgttc 3540ctgcgaatcc tccggcggag ttttcagcta caaagtttgc ttcattcatc acccaatact 3600ccacaggaca agtgagtgtg gaaattgaat gggagctgca gaaagaaaac agcaagcgct 3660ggaatcccga agtgcagtac acatccaatt atgcaaaatc tgccaacgtt gattttactg 3720tggacaacaa tggactttat actgagcctc gccccattgg cacccgttac cttacccgtc 3780ccctgtaagg cgcgccaccg gttgcttgtt aatcaataaa ccgtttaatt cgtttcagtt 3840gaactttggt ctctgcgtat ttctttctta tctagtttcc atgctctagg atccactagt 3900aacggccgcc agtgtgctgg aattcggctt tgtagttaat gattaacccg ccatgctact 3960tatctacgta gccatgctct agaggtcctg tattagaggt cacgtgagtg ttttgcgaca 4020ttttgcgaca ccatgtggtc acgctgggta tttaagcccg agtgagcacg cagggtctcc 4080attttgaagc gggaggtttg aacgcgcagc cgccaagccg aattctgcag atatccaaac 4140actggcggcc gctcgactag agcggccgcc accgcggtgg agctccagct tttgttccct 4200ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa 4260ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg 4320gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca 4380gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 4440tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4500gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4560ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4620ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4680acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4740tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4800ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 4860ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4920ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4980actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 5040gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc 5100tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 5160caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 5220atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 5280acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 5340ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 5400ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 5460tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 5520tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 5580gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 5640tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 5700tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 5760ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 5820tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 5880ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 5940gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 6000ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 6060cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 6120ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 6180ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 6240gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 6300ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 6360gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc 6420gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc 6480ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag 6540agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc 6600gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa 6660gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg 6720aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt 6780gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc 6840gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct 6900tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg 6960ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac 7020tcactatagg gcgaattggg taccgggccc cccctcgagg tcgacggtat cgggggagct 7080cgcagggtct ccattttgaa gcgggaggtt tgaacgcgca gccgccatgc cggggtttta 7140cgagattgtg attaaggtcc ccagcgacct tgacgagcat ctgcccggca tttctgacag 7200ctttgtgaac tgggtggccg agaaggaatg ggagttgccg ccagattctg acatggatct 7260gaatctgatt gagcaggcac ccctgaccgt ggccgagaag ctgcagcgcg actttctgac 7320ggaatggcgc cgtgtgagta aggccccgga ggctcttttc tttgtgcaat ttgagaaggg 7380agagagctac ttccacatgc acgtgctcgt ggaaaccacc ggggtgaaat c 743127415DNAArtificial SequenceCoding Strand of Plasmid pAAV-RC2 2ccgggccccc cctcgaggtc gacggtatcg ggggagctcg cagggtctcc attttgaagc 60gggaggtttg aacgcgcagc cgccatgccg gggttttacg agattgtgat taaggtcccc 120agcgaccttg acgagcatct gcccggcatt tctgacagct ttgtgaactg ggtggccgag 180aaggaatggg agttgccgcc agattctgac atggatctga atctgattga gcaggcaccc 240ctgaccgtgg ccgagaagct gcagcgcgac tttctgacgg aatggcgccg tgtgagtaag 300gccccggagg ctcttttctt tgtgcaattt gagaagggag agagctactt ccacatgcac 360gtgctcgtgg aaaccaccgg ggtgaaatcc atggttttgg gacgtttcct gagtcagatt 420cgcgaaaaac tgattcagag aatttaccgc gggatcgagc cgactttgcc aaactggttc 480gcggtcacaa agaccagaaa tggcgccgga ggcgggaaca aggtggtgga tgagtgctac 540atccccaatt acttgctccc caaaacccag cctgagctcc agtgggcgtg gactaatatg 600gaacagtatt taagcgcctg tttgaatctc acggagcgta aacggttggt ggcgcagcat 660ctgacgcacg tgtcgcagac gcaggagcag aacaaagaga atcagaatcc caattctgat 720gcgccggtga tcagatcaaa aacttcagcc aggtacatgg agctggtcgg gtggctcgtg 780gacaagggga ttacctcgga gaagcagtgg atccaggagg accaggcctc atacatctcc 840ttcaatgcgg cctccaactc gcggtcccaa atcaaggctg ccttggacaa tgcgggaaag 900attatgagcc tgactaaaac cgcccccgac tacctggtgg gccagcagcc cgtggaggac 960atttccagca atcggattta taaaattttg gaactaaacg ggtacgatcc ccaatatgcg 1020gcttccgtct ttctgggatg ggccacgaaa aagttcggca agaggaacac catctggctg 1080tttgggcctg caactaccgg gaagaccaac atcgcggagg ccatagccca cactgtgccc 1140ttctacgggt gcgtaaactg gaccaatgag aactttccct tcaacgactg tgtcgacaag 1200atggtgatct ggtgggagga ggggaagatg accgccaagg tcgtggagtc ggccaaagcc 1260attctcggag gaagcaaggt gcgcgtggac cagaaatgca agtcctcggc ccagatagac 1320ccgactcccg tgatcgtcac ctccaacacc aacatgtgcg ccgtgattga cgggaactca 1380acgaccttcg aacaccagca gccgttgcaa gaccggatgt tcaaatttga actcacccgc 1440cgtctggatc atgactttgg gaaggtcacc aagcaggaag tcaaagactt tttccggtgg 1500gcaaaggatc acgtggttga ggtggagcat gaattctacg tcaaaaaggg tggagccaag 1560aaaagacccg cccccagtga cgcagatata agtgagccca aacgggtgcg cgagtcagtt 1620gcgcagccat cgacgtcaga cgcggaagct tcgatcaact acgcagacag gtaccaaaac 1680aaatgttctc gtcacgtggg catgaatctg atgctgtttc cctgcagaca atgcgagaga 1740atgaatcaga attcaaatat ctgcttcact cacggacaga aagactgttt agagtgcttt 1800cccgtgtcag aatctcaacc cgtttctgtc gtcaaaaagg cgtatcagaa actgtgctac 1860attcatcata tcatgggaaa ggtgccagac gcttgcactg cctgcgatct ggtcaatgtg 1920gatttggatg actgcatctt tgaacaataa atgatttaaa tcaggtatgg ctgccgatgg 1980ttatcttcca gattggctcg aggacactct ctctgaagga ataagacagt ggtggaagct 2040caaacctggc ccaccaccac caaagcccgc agagcggcat aaggacgaca gcaggggtct 2100tgtgcttcct gggtacaagt acctcggacc cttcaacgga ctcgacaagg gagagccggt 2160caacgaggca gacgccgcgg ccctcgagca cgacaaagcc tacgaccggc agctcgacag 2220cggagacaac ccgtacctca agtacaacca cgccgacgcg gagtttcagg agcgccttaa 2280agaagatacg tcttttgggg gcaacctcgg acgagcagtc ttccaggcga aaaagagggt 2340tcttgaacct ctgggcctgg ttgaggaacc tgttaagacg gctccgggaa aaaagaggcc 2400ggtagagcac tctcctgtgg agccagactc ctcctcggga accggaaagg cgggccagca 2460gcctgcaaga aaaagattga attttggtca gactggagac gcagactcag tacctgaccc 2520ccagcctctc ggacagccac cagcagcccc ctctggtctg ggaactaata cgatggctac 2580aggcagtggc gcaccaatgg cagacaataa cgagggcgcc gacggagtgg gtaattcctc 2640gggaaattgg cattgcgatt ccacatggat gggcgacaga gtcatcacca ccagcacccg 2700aacctgggcc ctgcccacct acaacaacca cctctacaaa caaatttcca gccaatcagg 2760agcctcgaac gacaatcact actttggcta cagcacccct tgggggtatt ttgacttcaa 2820cagattccac tgccactttt caccacgtga ctggcaaaga ctcatcaaca acaactgggg 2880attccgaccc aagagactca acttcaagct ctttaacatt caagtcaaag aggtcacgca 2940gaatgacggt acgacgacga ttgccaataa ccttaccagc acggttcagg tgtttactga 3000ctcggagtac cagctcccgt acgtcctcgg ctcggcgcat caaggatgcc tcccgccgtt 3060cccagcagac gtcttcatgg tgccacagta tggatacctc accctgaaca acgggagtca 3120ggcagtagga cgctcttcat tttactgcct ggagtacttt ccttctcaga tgctgcgtac 3180cggaaacaac tttaccttca gctacacttt tgaggacgtt cctttccaca gcagctacgc 3240tcacagccag agtctggacc gtctcatgaa tcctctcatc gaccagtacc tgtattactt 3300gagcagaaca aacactccaa gtggaaccac cacgcagtca aggcttcagt tttctcaggc 3360cggagcgagt gacattcggg accagtctag gaactggctt cctggaccct gttaccgcca 3420gcagcgagta tcaaagacat ctgcggataa caacaacagt gaatactcgt ggactggagc 3480taccaagtac cacctcaatg gcagagactc tctggtgaat ccgggcccgg ccatggcaag 3540ccacaaggac gatgaagaaa agttttttcc tcagagcggg gttctcatct ttgggaagca 3600aggctcagag aaaacaaatg tggacattga aaaggtcatg attacagacg aagaggaaat 3660caggacaacc aatcccgtgg ctacggagca gtatggttct gtatctacca acctccagag 3720aggcaacaga caagcagcta ccgcagatgt caacacacaa ggcgttcttc caggcatggt 3780ctggcaggac agagatgtgt accttcaggg gcccatctgg gcaaagattc cacacacgga 3840cggacatttt cacccctctc ccctcatggg tggattcgga cttaaacacc ctcctccaca 3900gattctcatc aagaacaccc cggtacctgc gaatccttcg accaccttca gtgcggcaaa 3960gtttgcttcc ttcatcacac agtactccac gggacaggtc agcgtggaga tcgagtggga 4020gctgcagaag gaaaacagca aacgctggaa tcccgaaatt cagtacactt ccaactacaa 4080caagtctgtt aatgtggact ttactgtgga cactaatggc gtgtattcag agcctcgccc 4140cattggcacc agatacctga ctcgtaatct gtaattgctt gttaatcaat aaaccgttta 4200attcgtttca gttgaacttt ggtctctgcg tatttctttc ttatctagtt tccatgctct 4260aggatccact agtaacggcc gccagtgtgc tggaattcgg ctttgtagtt aatgattaac 4320ccgccatgct acttatctac gtagccatgc tctagaggtc ctgtattaga ggtcacgtga 4380gtgttttgcg acattttgcg acaccatgtg gtcacgctgg gtatttaagc ccgagtgagc 4440acgcagggtc tccattttga agcgggaggt ttgaacgcgc agccgccaag ccgaattctg 4500cagatatcca aacactggcg gccgctcgac tagagcggcc gccaccgcgg tggagctcca 4560gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt 4620ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 4680agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 4740tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 4800cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 4860gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 4920ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 4980ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 5040atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 5100aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 5160gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 5220ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 5280ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 5340acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 5400gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 5460ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 5520ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 5580gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 5640ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 5700agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 5760ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 5820gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 5880catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 5940cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 6000cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 6060gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 6120tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 6180gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 6240tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 6300gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 6360gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 6420taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 6480tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 6540ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 6600taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 6660tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac 6720aaataggggt tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat 6780tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga 6840aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc 6900agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac 6960cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc 7020gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg 7080gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag 7140ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc 7200gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc 7260ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt 7320aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg 7380cgcgtaatac gactcactat agggcgaatt gggta 741537395DNAArtificial SequenceCoding Strand of Plasmid pAAV-RC5

3catggttttg ggacgtttcc tgagtcagat tcgcgaaaaa ctgattcaga gaatttaccg 60cgggatcgag ccgactttgc caaactggtt cgcggtcaca aagaccagaa atggcgccgg 120aggcgggaac aaggtggtgg atgagtgcta catccccaat tacttgctcc ccaaaaccca 180gcctgagctc cagtgggcgt ggactaatat ggaacagtat ttaagcgcct gtttgaatct 240cacggagcgt aaacggttgg tggcgcagca tctgacgcac gtgtcgcaga cgcaggagca 300gaacaaagag aatcagaatc ccaattctga tgcgccggtg atcagatcaa aaacttcagc 360caggtacatg gagctggtcg ggtggctcgt ggacaagggg attacctcgg agaagcagtg 420gatccaggag gaccaggcct catacatctc cttcaatgcg gcctccaact cgcggtccca 480aatcaaggct gccttggaca atgcgggaaa gattatgagc ctgactaaaa ccgcccccga 540ctacctggtg ggccagcagc ccgtggagga catttccagc aatcggattt ataaaatttt 600ggaactaaac gggtacgatc cccaatatgc ggcttccgtc tttctgggat gggccacgaa 660aaagttcggc aagaggaaca ccatctggct gtttgggcct gcaactaccg ggaagaccaa 720catcgcggag gccatagccc acactgtgcc cttctacggg tgcgtaaact ggaccaatga 780gaactttccc ttcaacgact gtgtcgacaa gatggtgatc tggtgggagg aggggaagat 840gaccgccaag gtcgtggagt cggccaaagc cattctcgga ggaagcaagg tgcgcgtgga 900ccagaaatgc aagtcctcgg cccagataga cccgactccc gtgatcgtca cctccaacac 960caacatgtgc gccgtgattg acgggaactc aacgaccttc gaacaccagc agccgttgca 1020agaccggatg ttcaaatttg aactcacccg ccgtctggat catgactttg ggaaggtcac 1080caagcaggaa gtcaaagact ttttccggtg ggcaaaggat cacgtggttg aggtggagca 1140tgaattctac gtcaaaaagg gtggagccaa gaaaagaccc gcccccagtg acgcagatat 1200aagtgagccc aaacgggtgc gcgagtcagt tgcgcagcca tcgacgtcag acgcggaagc 1260ttcgatcaac tacgcagaca ggtaccaaaa caaatgttct cgtcacgtgg gcatgaatct 1320gatgctgttt ccctgcagac aatgcgagag aatgaatcag aattcaaata tctgcttcac 1380tcacggacag aaagactgtt tagagtgctt tcccgtgtca gaatctcaac ccgtttctgt 1440cgtcaaaaag gcgtatcaga aactgtgcta cattcatcat atcatgggaa aggtgccaga 1500cgcttgcact gcctgcgatc tggtcaatgt ggatttggat gactgcatct ttgaacaata 1560aatgatttaa atcaggtatg tcttttgttg atcaccctcc agattggttg gaagaagttg 1620gtgaaggtct tcgcgagttt ttgggccttg aagcgggccc accgaaacca aaacccaatc 1680agcagcatca agatcaagcc cgtggtcttg tgctgcctgg ttataactat ctcggacccg 1740gaaacggtct cgatcgagga gagcctgtca acagggcaga cgaggtcgcg cgagagcacg 1800acatctcgta caacgagcag cttgaggcgg gagacaaccc ctacctcaag tacaaccacg 1860cggacgccga gtttcaggag aagctcgccg acgacacatc cttcggggga aacctcggaa 1920aggcagtctt tcaggccaag aaaagggttc tcgaaccttt tggcctggtt gaagagggtg 1980ctaagacggc ccctaccgga aagcggatag acgaccactt tccaaaaaga aagaaggctc 2040ggaccgaaga ggactccaag ccttccacct cgtcagacgc cgaagctgga cccagcggat 2100cccagcagct gcaaatccca gcccaaccag cctcaagttt gggagctgat acaatgtctg 2160cgggaggtgg cggcccattg ggcgacaata accaaggtgc cgatggagtg ggcaatgcct 2220cgggagattg gcattgcgat tccacgtgga tgggggacag agtcgtcacc aagtccaccc 2280gaacctgggt gctgcccagc tacaacaacc accagtaccg agagatcaaa agcggctccg 2340tcgacggaag caacgccaac gcctactttg gatacagcac cccctggggg tactttgact 2400ttaaccgctt ccacagccac tggagccccc gagactggca aagactcatc aacaactact 2460ggggcttcag accccggtcc ctcagagtca aaatcttcaa cattcaagtc aaagaggtca 2520cggtgcagga ctccaccacc accatcgcca acaacctcac ctccaccgtc caagtgttta 2580cggacgacga ctaccagctg ccctacgtcg tcggcaacgg gaccgaggga tgcctgccgg 2640ccttccctcc gcaggtcttt acgctgccgc agtacggtta cgcgacgctg aaccgcgaca 2700acacagaaaa tcccaccgag aggagcagct tcttctgcct agagtacttt cccagcaaga 2760tgctgagaac gggcaacaac tttgagttta cctacaactt tgaggaggtg cccttccact 2820ccagcttcgc tcccagtcag aacctgttca agctggccaa cccgctggtg gaccagtact 2880tgtaccgctt cgtgagcaca aataacactg gcggagtcca gttcaacaag aacctggccg 2940ggagatacgc caacacctac aaaaactggt tcccggggcc catgggccga acccagggct 3000ggaacctggg ctccggggtc aaccgcgcca gtgtcagcgc cttcgccacg accaatagga 3060tggagctcga gggcgcgagt taccaggtgc ccccgcagcc gaacggcatg accaacaacc 3120tccagggcag caacacctat gccctggaga acactatgat cttcaacagc cagccggcga 3180acccgggcac caccgccacg tacctcgagg gcaacatgct catcaccagc gagagcgaga 3240cgcagccggt gaaccgcgtg gcgtacaacg tcggcgggca gatggccacc aacaaccaga 3300gctccaccac tgcccccgcg accggcacgt acaacctcca ggaaatcgtg cccggcagcg 3360tgtggatgga gagggacgtg tacctccaag gacccatctg ggccaagatc ccagagacgg 3420gggcgcactt tcacccctct ccggccatgg gcggattcgg actcaaacac ccaccgccca 3480tgatgctcat caagaacacg cctgtgcccg gaaatatcac cagcttctcg gacgtgcccg 3540tcagcagctt catcacccag tacagcaccg ggcaggtcac cgtggagatg gagtgggagc 3600tcaagaagga aaactccaag aggtggaacc cagagatcca gtacacaaac aactacaacg 3660acccccagtt tgtggacttt gccccggaca gcaccgggga atacagaacc accagaccta 3720tcggaacccg ataccttacc cgaccccttt aaggcgcgcc accggttgct tgttaatcaa 3780taaaccgttt aattcgtttc agttgaactt tggtctctgc gtatttcttt cttatctagt 3840ttccatgctc taggatccac tagtaacggc cgccagtgtg ctggaattcg gctttgtagt 3900taatgattaa cccgccatgc tacttatcta cgtagccatg ctctagaggt cctgtattag 3960aggtcacgtg agtgttttgc gacattttgc gacaccatgt ggtcacgctg ggtatttaag 4020cccgagtgag cacgcagggt ctccattttg aagcgggagg tttgaacgcg cagccgccaa 4080gccgaattct gcagatatcc aaacactggc ggccgctcga ctagagcggc cgccaccgcg 4140gtggagctcc agcttttgtt ccctttagtg agggttaatt gcgcgcttgg cgtaatcatg 4200gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc 4260cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc 4320gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat 4380cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac 4440tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt 4500aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca 4560gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc 4620ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact 4680ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct 4740gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag 4800ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca 4860cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 4920cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc 4980gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag 5040aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg 5100tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca 5160gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc 5220tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag 5280gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata 5340tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat 5400ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg 5460ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc 5520tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc 5580aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc 5640gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc 5700gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc 5760ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa 5820gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat 5880gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata 5940gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca 6000tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag 6060gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc 6120agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc 6180aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata 6240ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 6300gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctaaattgta 6360agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac 6420caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga gatagggttg 6480agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc caacgtcaaa 6540gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc ctaatcaagt 6600tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag cccccgattt 6660agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga 6720gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc 6780gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg caactgttgg 6840gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct 6900gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg 6960gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg gccccccctc 7020gaggtcgacg gtatcggggg agctcgcagg gtctccattt tgaagcggga ggtttgaacg 7080cgcagccgcc atgccggggt tttacgagat tgtgattaag gtccccagcg accttgacga 7140gcatctgccc ggcatttctg acagctttgt gaactgggtg gccgagaagg aatgggagtt 7200gccgccagat tctgacatgg atctgaatct gattgagcag gcacccctga ccgtggccga 7260gaagctgcag cgcgactttc tgacggaatg gcgccgtgtg agtaaggccc cggaggctct 7320tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac 7380caccggggtg aaatc 739547431DNAArtificial SequenceCoding Strand of Plasmid pAAV-RC6 4catggttttg ggacgtttcc tgagtcagat tcgcgaaaaa ctgattcaga gaatttaccg 60cgggatcgag ccgactttgc caaactggtt cgcggtcaca aagaccagaa atggcgccgg 120aggcgggaac aaggtggtgg atgagtgcta catccccaat tacttgctcc ccaaaaccca 180gcctgagctc cagtgggcgt ggactaatat ggaacagtat ttaagcgcct gtttgaatct 240cacggagcgt aaacggttgg tggcgcagca tctgacgcac gtgtcgcaga cgcaggagca 300gaacaaagag aatcagaatc ccaattctga tgcgccggtg atcagatcaa aaacttcagc 360caggtacatg gagctggtcg ggtggctcgt ggacaagggg attacctcgg agaagcagtg 420gatccaggag gaccaggcct catacatctc cttcaatgcg gcctccaact cgcggtccca 480aatcaaggct gccttggaca atgcgggaaa gattatgagc ctgactaaaa ccgcccccga 540ctacctggtg ggccagcagc ccgtggagga catttccagc aatcggattt ataaaatttt 600ggaactaaac gggtacgatc cccaatatgc ggcttccgtc tttctgggat gggccacgaa 660aaagttcggc aagaggaaca ccatctggct gtttgggcct gcaactaccg ggaagaccaa 720catcgcggag gccatagccc acactgtgcc cttctacggg tgcgtaaact ggaccaatga 780gaactttccc ttcaacgact gtgtcgacaa gatggtgatc tggtgggagg aggggaagat 840gaccgccaag gtcgtggagt cggccaaagc cattctcgga ggaagcaagg tgcgcgtgga 900ccagaaatgc aagtcctcgg cccagataga cccgactccc gtgatcgtca cctccaacac 960caacatgtgc gccgtgattg acgggaactc aacgaccttc gaacaccagc agccgttgca 1020agaccggatg ttcaaatttg aactcacccg ccgtctggat catgactttg ggaaggtcac 1080caagcaggaa gtcaaagact ttttccggtg ggcaaaggat cacgtggttg aggtggagca 1140tgaattctac gtcaaaaagg gtggagccaa gaaaagaccc gcccccagtg acgcagatat 1200aagtgagccc aaacgggtgc gcgagtcagt tgcgcagcca tcgacgtcag acgcggaagc 1260ttcgatcaac tacgcagaca ggtaccaaaa caaatgttct cgtcacgtgg gcatgaatct 1320gatgctgttt ccctgcagac aatgcgagag aatgaatcag aattcaaata tctgcttcac 1380tcacggacag aaagactgtt tagagtgctt tcccgtgtca gaatctcaac ccgtttctgt 1440cgtcaaaaag gcgtatcaga aactgtgcta cattcatcat atcatgggaa aggtgccaga 1500cgcttgcact gcctgcgatc tggtcaatgt ggatttggat gactgcatct ttgaacaata 1560aatgatttaa atcaggtatg gctgccgatg gttatcttcc agattggctc gaggacaacc 1620tctctgaggg cattcgcgag tggtgggact tgaaacctgg agccccgaaa cccaaagcca 1680accagcaaaa gcaggacgac ggccggggtc tggtgcttcc tggctacaag tacctcggac 1740ccttcaacgg actcgacaag ggggagcccg tcaacgcggc ggatgcagcg gccctcgagc 1800acgacaaggc ctacgaccag cagctcaaag cgggtgacaa tccgtacctg cggtataacc 1860acgccgacgc cgagtttcag gagcgtctgc aagaagatac gtcttttggg ggcaacctcg 1920ggcgagcagt cttccaggcc aagaagaggg ttctcgaacc ttttggtctg gttgaggaag 1980gtgctaagac ggctcctgga aagaaacgtc cggtagagca gtcgccacaa gagccagact 2040cctcctcggg cattggcaag acaggccagc agcccgctaa aaagagactc aattttggtc 2100agactggcga ctcagagtca gtccccgacc cacaacctct cggagaacct ccagcaaccc 2160ccgctgctgt gggacctact acaatggctt caggcggtgg cgcaccaatg gcagacaata 2220acgaaggcgc cgacggagtg ggtaatgcct caggaaattg gcattgcgat tccacatggc 2280tgggcgacag agtcatcacc accagcaccc gaacatgggc cttgcccacc tataacaacc 2340acctctacaa gcaaatctcc agtgcttcaa cgggggccag caacgacaac cactacttcg 2400gctacagcac cccctggggg tattttgatt tcaacagatt ccactgccat ttctcaccac 2460gtgactggca gcgactcatc aacaacaatt ggggattccg gcccaagaga ctcaacttca 2520agctcttcaa catccaagtc aaggaggtca cgacgaatga tggcgtcacg accatcgcta 2580ataaccttac cagcacggtt caagtcttct cggactcgga gtaccagttg ccgtacgtcc 2640tcggctctgc gcaccagggc tgcctccctc cgttcccggc ggacgtgttc atgattccgc 2700agtacggcta cctaacgctc aacaatggca gccaggcagt gggacggtca tccttttact 2760gcctggaata tttcccatcg cagatgctga gaacgggcaa taactttacc ttcagctaca 2820ccttcgagga cgtgcctttc cacagcagct acgcgcacag ccagagcctg gaccggctga 2880tgaatcctct catcgaccag tacctgtatt acctgaacag aactcagaat cagtccggaa 2940gtgcccaaaa caaggacttg ctgtttagcc gggggtctcc agctggcatg tctgttcagc 3000ccaaaaactg gctacctgga ccctgttacc ggcagcagcg cgtttctaaa acaaaaacag 3060acaacaacaa cagcaacttt acctggactg gtgcttcaaa atataacctt aatgggcgtg 3120aatctataat caaccctggc actgctatgg cctcacacaa agacgacaaa gacaagttct 3180ttcccatgag cggtgtcatg atttttggaa aggagagcgc cggagcttca aacactgcat 3240tggacaatgt catgatcaca gacgaagagg aaatcaaagc cactaacccc gtggccaccg 3300aaagatttgg gactgtggca gtcaatctcc agagcagcag cacagaccct gcgaccggag 3360atgtgcatgt tatgggagcc ttacctggaa tggtgtggca agacagagac gtatacctgc 3420agggtcctat ttgggccaaa attcctcaca cggatggaca ctttcacccg tctcctctca 3480tgggcggctt tggacttaag cacccgcctc ctcagatcct catcaaaaac acgcctgttc 3540ctgcgaatcc tccggcagag ttttcggcta caaagtttgc ttcattcatc acccagtatt 3600ccacaggaca agtgagcgtg gagattgaat gggagctgca gaaagaaaac agcaaacgct 3660ggaatcccga agtgcagtat acatctaact atgcaaaatc tgccaacgtt gatttcactg 3720tggacaacaa tggactttat actgagcctc gccccattgg cacccgttac ctcacccgtc 3780ccctgtaagg cgcgccaccg gttgcttgtt aatcaataaa ccgtttaatt cgtttcagtt 3840gaactttggt ctctgcgtat ttctttctta tctagtttcc atgctctagg atccactagt 3900aacggccgcc agtgtgctgg aattcggctt tgtagttaat gattaacccg ccatgctact 3960tatctacgta gccatgctct agaggtcctg tattagaggt cacgtgagtg ttttgcgaca 4020ttttgcgaca ccatgtggtc acgctgggta tttaagcccg agtgagcacg cagggtctcc 4080attttgaagc gggaggtttg aacgcgcagc cgccaagccg aattctgcag atatccaaac 4140actggcggcc gctcgactag agcggccgcc accgcggtgg agctccagct tttgttccct 4200ttagtgaggg ttaattgcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa 4260ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg 4320gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca 4380gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 4440tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 4500gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 4560ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 4620ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 4680acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 4740tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 4800ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 4860ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 4920ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 4980actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 5040gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc 5100tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 5160caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 5220atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 5280acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 5340ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 5400ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 5460tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 5520tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 5580gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 5640tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 5700tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 5760ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 5820tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 5880ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 5940gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 6000ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 6060cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 6120ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 6180ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 6240gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 6300ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 6360gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc 6420gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc 6480ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag 6540agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc 6600gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa 6660gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg 6720aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt 6780gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc 6840gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct 6900tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg 6960ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac 7020tcactatagg gcgaattggg taccgggccc cccctcgagg tcgacggtat cgggggagct 7080cgcagggtct ccattttgaa gcgggaggtt tgaacgcgca gccgccatgc cggggtttta 7140cgagattgtg attaaggtcc ccagcgacct tgacgagcat ctgcccggca tttctgacag 7200ctttgtgaac tgggtggccg agaaggaatg ggagttgccg ccagattctg acatggatct 7260gaatctgatt gagcaggcac ccctgaccgt ggccgagaag ctgcagcgcg actttctgac 7320ggaatggcgc cgtgtgagta aggccccgga ggctcttttc tttgtgcaat ttgagaaggg 7380agagagctac ttccacatgc acgtgctcgt ggaaaccacc ggggtgaaat c 743157434DNAArtificial SequenceCoding Strand of Plasmid pAAV-RC7 5catggttttg ggacgtttcc tgagtcagat tcgcgaaaaa ctgattcaga gaatttaccg

60cgggatcgag ccgactttgc caaactggtt cgcggtcaca aagaccagaa atggcgccgg 120aggcgggaac aaggtggtgg atgagtgcta catccccaat tacttgctcc ccaaaaccca 180gcctgagctc cagtgggcgt ggactaatat ggaacagtat ttaagcgcct gtttgaatct 240cacggagcgt aaacggttgg tggcgcagca tctgacgcac gtgtcgcaga cgcaggagca 300gaacaaagag aatcagaatc ccaattctga tgcgccggtg atcagatcaa aaacttcagc 360caggtacatg gagctggtcg ggtggctcgt ggacaagggg attacctcgg agaagcagtg 420gatccaggag gaccaggcct catacatctc cttcaatgcg gcctccaact cgcggtccca 480aatcaaggct gccttggaca atgcgggaaa gattatgagc ctgactaaaa ccgcccccga 540ctacctggtg ggccagcagc ccgtggagga catttccagc aatcggattt ataaaatttt 600ggaactaaac gggtacgatc cccaatatgc ggcttccgtc tttctgggat gggccacgaa 660aaagttcggc aagaggaaca ccatctggct gtttgggcct gcaactaccg ggaagaccaa 720catcgcggag gccatagccc acactgtgcc cttctacggg tgcgtaaact ggaccaatga 780gaactttccc ttcaacgact gtgtcgacaa gatggtgatc tggtgggagg aggggaagat 840gaccgccaag gtcgtggagt cggccaaagc cattctcgga ggaagcaagg tgcgcgtgga 900ccagaaatgc aagtcctcgg cccagataga cccgactccc gtgatcgtca cctccaacac 960caacatgtgc gccgtgattg acgggaactc aacgaccttc gaacaccagc agccgttgca 1020agaccggatg ttcaaatttg aactcacccg ccgtctggat catgactttg ggaaggtcac 1080caagcaggaa gtcaaagact ttttccggtg ggcaaaggat cacgtggttg aggtggagca 1140tgaattctac gtcaaaaagg gtggagccaa gaaaagaccc gcccccagtg acgcagatat 1200aagtgagccc aaacgggtgc gcgagtcagt tgcgcagcca tcgacgtcag acgcggaagc 1260ttcgatcaac tacgcagaca ggtaccaaaa caaatgttct cgtcacgtgg gcatgaatct 1320gatgctgttt ccctgcagac aatgcgagag aatgaatcag aattcaaata tctgcttcac 1380tcacggacag aaagactgtt tagagtgctt tcccgtgtca gaatctcaac ccgtttctgt 1440cgtcaaaaag gcgtatcaga aactgtgcta cattcatcat atcatgggaa aggtgccaga 1500cgcttgcact gcctgcgatc tggtcaatgt ggatttggat gactgcatct ttgaacaata 1560aatgatttaa atcaggtatg gctgccgatg gttatcttcc agattggctc gaggacaacc 1620tctctgaggg cattcgcgag tggtgggacc tgaaacctgg agccccgaaa cccaaagcca 1680accagcaaaa gcaggacaac ggccggggtc tggtgcttcc tggctacaag tacctcggac 1740ccttcaacgg actcgacaag ggggagcccg tcaacgcggc ggacgcagcg gccctcgagc 1800acgacaaggc ctacgaccag cagctcaaag cgggtgacaa tccgtacctg cggtataacc 1860acgccgacgc cgagtttcag gagcgtctgc aagaagatac gtcatttggg ggcaacctcg 1920ggcgagcagt cttccaggcc aagaagcggg ttctcgaacc tctcggtctg gttgaggaag 1980gcgctaagac ggctcctgca aagaagagac cggtagagcc gtcacctcag cgttcccccg 2040actcctccac gggcatcggc aagaaaggcc agcagcccgc cagaaagaga ctcaatttcg 2100gtcagactgg cgactcagag tcagtccccg accctcaacc tctcggagaa cctccagcag 2160cgccctctag tgtgggatct ggtacagtgg ctgcaggcgg tggcgcacca atggcagaca 2220ataacgaagg tgccgacgga gtgggtaatg cctcaggaaa ttggcattgc gattccacat 2280ggctgggcga cagagtcatt accaccagca cccgaacctg ggccctgccc acctacaaca 2340accacctcta caagcaaatc tccagtgaaa ctgcaggtag taccaacgac aacacctact 2400tcggctacag caccccctgg gggtattttg actttaacag attccactgc cacttctcac 2460cacgtgactg gcagcgactc atcaacaaca actggggatt ccggcccaag aagctgcggt 2520tcaagctctt caacatccag gtcaaggagg tcacgacgaa tgacggcgtt acgaccatcg 2580ctaataacct taccagcacg attcaggtat tctcggactc ggaataccag ctgccgtacg 2640tcctcggctc tgcgcaccag ggctgcctgc ctccgttccc ggcggacgtc ttcatgattc 2700ctcagtacgg ctacctgact ctcaacaatg gcagtcagtc tgtgggacgt tcctccttct 2760actgcctgga gtacttcccc tctcagatgc tgagaacggg caacaacttt gagttcagct 2820acagcttcga ggacgtgcct ttccacagca gctacgcaca cagccagagc ctggaccggc 2880tgatgaatcc cctcatcgac cagtacttgt actacctggc cagaacacag agtaacccag 2940gaggcacagc tggcaatcgg gaactgcagt tttaccaggg cgggccttca actatggccg 3000aacaagccaa gaattggtta cctggacctt gcttccggca acaaagagtc tccaaaacgc 3060tggatcaaaa caacaacagc aactttgctt ggactggtgc caccaaatat cacctgaacg 3120gcagaaactc gttggttaat cccggcgtcg ccatggcaac tcacaaggac gacgaggacc 3180gctttttccc atccagcgga gtcctgattt ttggaaaaac tggagcaact aacaaaacta 3240cattggaaaa tgtgttaatg acaaatgaag aagaaattcg tcctactaat cctgtagcca 3300cggaagaata cgggatagtc agcagcaact tacaagcggc taatactgca gcccagacac 3360aagttgtcaa caaccaggga gccttacctg gcatggtctg gcagaaccgg gacgtgtacc 3420tgcagggtcc catctgggcc aagattcctc acacggatgg caactttcac ccgtctcctt 3480tgatgggcgg ctttggactt aaacatccgc ctcctcagat cctgatcaag aacactcccg 3540ttcccgctaa tcctccggag gtgtttactc ctgccaagtt tgcttcgttc atcacacagt 3600acagcaccgg acaagtcagc gtggaaatcg agtgggagct gcagaaggaa aacagcaagc 3660gctggaaccc ggagattcag tacacctcca actttgaaaa gcagactggt gtggactttg 3720ccgttgacag ccagggtgtt tactctgagc ctcgccctat tggcactcgt tacctcaccc 3780gtaatctgta aggcgcgcca ccggttgctt gttaatcaat aaaccgttta attcgtttca 3840gttgaacttt ggtctctgcg tatttctttc ttatctagtt tccatgctct aggatccact 3900agtaacggcc gccagtgtgc tggaattcgg ctttgtagtt aatgattaac ccgccatgct 3960acttatctac gtagccatgc tctagaggtc ctgtattaga ggtcacgtga gtgttttgcg 4020acattttgcg acaccatgtg gtcacgctgg gtatttaagc ccgagtgagc acgcagggtc 4080tccattttga agcgggaggt ttgaacgcgc agccgccaag ccgaattctg cagatatcca 4140aacactggcg gccgctcgac tagagcggcc gccaccgcgg tggagctcca gcttttgttc 4200cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg 4260aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc 4320ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt 4380ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg 4440cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt 4500tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc 4560aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa 4620aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa 4680tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 4740ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc 4800cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag 4860ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga 4920ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc 4980gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac 5040agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg 5100cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca 5160aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa 5220aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa 5280ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt 5340aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag 5400ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat 5460agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc 5520cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa 5580ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca 5640gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa 5700cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt 5760cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc 5820ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact 5880catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc 5940tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg 6000ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct 6060catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc 6120cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag 6180cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac 6240acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg 6300ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt 6360tccgcgcaca tttccccgaa aagtgccacc taaattgtaa gcgttaatat tttgttaaaa 6420ttcgcgttaa atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa 6480atcccttata aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac 6540aagagtccac tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag 6600ggcgatggcc cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt 6660aaagcactaa atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg 6720gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca 6780agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag 6840ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc 6900tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta 6960acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgagcg cgcgtaatac 7020gactcactat agggcgaatt gggtaccggg ccccccctcg aggtcgacgg tatcggggga 7080gctcgcaggg tctccatttt gaagcgggag gtttgaacgc gcagccgcca tgccggggtt 7140ttacgagatt gtgattaagg tccccagcga ccttgacgag catctgcccg gcatttctga 7200cagctttgtg aactgggtgg ccgagaagga atgggagttg ccgccagatt ctgacatgga 7260tctgaatctg attgagcagg cacccctgac cgtggccgag aagctgcagc gcgactttct 7320gacggaatgg cgccgtgtga gtaaggcccc ggaggctctt ttctttgtgc aatttgagaa 7380gggagagagc tacttccaca tgcacgtgct cgtggaaacc accggggtga aatc 7434611569DNAArtificial SequenceCoding Strand of Plasmid pHelper-Kan 6ggtacccaac tccatgctta acagtcccca ggtacagccc accctgcgtc gcaaccagga 60acagctctac agcttcctgg agcgccactc gccctacttc cgcagccaca gtgcgcagat 120taggagcgcc acttcttttt gtcacttgaa aaacatgtaa aaataatgta ctaggagaca 180ctttcaataa aggcaaatgt ttttatttgt acactctcgg gtgattattt accccccacc 240cttgccgtct gcgccgttta aaaatcaaag gggttctgcc gcgcatcgct atgcgccact 300ggcagggaca cgttgcgata ctggtgttta gtgctccact taaactcagg cacaaccatc 360cgcggcagct cggtgaagtt ttcactccac aggctgcgca ccatcaccaa cgcgtttagc 420aggtcgggcg ccgatatctt gaagtcgcag ttggggcctc cgccctgcgc gcgcgagttg 480cgatacacag ggttgcagca ctggaacact atcagcgccg ggtggtgcac gctggccagc 540acgctcttgt cggagatcag atccgcgtcc aggtcctccg cgttgctcag ggcgaacgga 600gtcaactttg gtagctgcct tcccaaaaag ggtgcatgcc caggctttga gttgcactcg 660caccgtagtg gcatcagaag gtgaccgtgc ccggtctggg cgttaggata cagcgcctgc 720atgaaagcct tgatctgctt aaaagccacc tgagcctttg cgccttcaga gaagaacatg 780ccgcaagact tgccggaaaa ctgattggcc ggacaggccg cgtcatgcac gcagcacctt 840gcgtcggtgt tggagatctg caccacattt cggccccacc ggttcttcac gatcttggcc 900ttgctagact gctccttcag cgcgcgctgc ccgttttcgc tcgtcacatc catttcaatc 960acgtgctcct tatttatcat aatgctcccg tgtagacact taagctcgcc ttcgatctca 1020gcgcagcggt gcagccacaa cgcgcagccc gtgggctcgt ggtgcttgta ggttacctct 1080gcaaacgact gcaggtacgc ctgcaggaat cgccccatca tcgtcacaaa ggtcttgttg 1140ctggtgaagg tcagctgcaa cccgcggtgc tcctcgttta gccaggtctt gcatacggcc 1200gccagagctt ccacttggtc aggcagtagc ttgaagtttg cctttagatc gttatccacg 1260tggtacttgt ccatcaacgc gcgcgcagcc tccatgccct tctcccacgc agacacgatc 1320ggcaggctca gcgggtttat caccgtgctt tcactttccg cttcactgga ctcttccttt 1380tcctcttgcg tccgcatacc ccgcgccact gggtcgtctt cattcagccg ccgcaccgtg 1440cgcttacctc ccttgccgtg cttgattagc accggtgggt tgctgaaacc caccatttgt 1500agcgccacat cttctctttc ttcctcgctg tccacgatca cctctgggga tggcgggcgc 1560tcgggcttgg gagaggggcg cttctttttc tttttggacg caatggccaa atccgccgtc 1620gaggtcgatg gccgcgggct gggtgtgcgc ggcaccagcg catcttgtga cgagtcttct 1680tcgtcctcgg actcgagacg ccgcctcagc cgcttttttg ggggcgcgcg gggaggcggc 1740ggcgacggcg acggggacga cacgtcctcc atggttggtg gacgtcgcgc cgcaccgcgt 1800ccgcgctcgg gggtggtttc gcgctgctcc tcttcccgac tggccatttc cttctcctat 1860aggcagaaaa agatcatgga gtcagtcgag aaggaggaca gcctaaccgc cccctttgag 1920ttcgccacca ccgcctccac cgatgccgcc aacgcgccta ccaccttccc cgtcgaggca 1980cccccgcttg aggaggagga agtgattatc gagcaggacc caggttttgt aagcgaagac 2040gacgaggatc gctcagtacc aacagaggat aaaaagcaag accaggacga cgcagaggca 2100aacgaggaac aagtcgggcg gggggaccaa aggcatggcg actacctaga tgtgggagac 2160gacgtgctgt tgaagcatct gcagcgccag tgcgccatta tctgcgacgc gttgcaagag 2220cgcagcgatg tgcccctcgc catagcggat gtcagccttg cctacgaacg ccacctgttc 2280tcaccgcgcg taccccccaa acgccaagaa aacggcacat gcgagcccaa cccgcgcctc 2340aacttctacc ccgtatttgc cgtgccagag gtgcttgcca cctatcacat ctttttccaa 2400aactgcaaga tacccctatc ctgccgtgcc aaccgcagcc gagcggacaa gcagctggcc 2460ttgcggcagg gcgctgtcat acctgatatc gcctcgctcg acgaagtgcc aaaaatcttt 2520gagggtcttg gacgcgacga gaaacgcgcg gcaaacgctc tgcaacaaga aaacagcgaa 2580aatgaaagtc actgtggagt gctggtggaa cttgagggtg acaacgcgcg cctagccgtg 2640ctgaaacgca gcatcgaggt cacccacttt gcctacccgg cacttaacct accccccaag 2700gttatgagca cagtcatgag cgagctgatc gtgcgccgtg cacgacccct ggagagggat 2760gcaaacttgc aagaacaaac cgaggagggc ctacccgcag ttggcgatga gcagctggcg 2820cgctggcttg agacgcgcga gcctgccgac ttggaggagc gacgcaagct aatgatggcc 2880gcagtgcttg ttaccgtgga gcttgagtgc atgcagcggt tctttgctga cccggagatg 2940cagcgcaagc tagaggaaac gttgcactac acctttcgcc agggctacgt gcgccaggcc 3000tgcaaaattt ccaacgtgga gctctgcaac ctggtctcct accttggaat tttgcacgaa 3060aaccgcctcg ggcaaaacgt gcttcattcc acgctcaagg gcgaggcgcg ccgcgactac 3120gtccgcgact gcgtttactt atttctgtgc tacacctggc aaacggccat gggcgtgtgg 3180cagcaatgcc tggaggagcg caacctaaag gagctgcaga agctgctaaa gcaaaacttg 3240aaggacctat ggacggcctt caacgagcgc tccgtggccg cgcacctggc ggacattatc 3300ttccccgaac gcctgcttaa aaccctgcaa cagggtctgc cagacttcac cagtcaaagc 3360atgttgcaaa actttaggaa ctttatccta gagcgttcag gaattctgcc cgccacctgc 3420tgtgcgcttc ctagcgactt tgtgcccatt aagtaccgtg aatgccctcc gccgctttgg 3480ggtcactgct accttctgca gctagccaac taccttgcct accactccga catcatggaa 3540gacgtgagcg gtgacggcct actggagtgt cactgtcgct gcaacctatg caccccgcac 3600cgctccctgg tctgcaattc gcaactgctt agcgaaagtc aaattatcgg tacctttgag 3660ctgcagggtc cctcgcctga cgaaaagtcc gcggctccgg ggttgaaact cactccgggg 3720ctgtggacgt cggcttacct tcgcaaattt gtacctgagg actaccacgc ccacgagatt 3780aggttctacg aagaccaatc ccgcccgcca aatgcggagc ttaccgcctg cgtcattacc 3840cagggccaca tccttggcca attgcaagcc atcaacaaag cccgccaaga gtttctgcta 3900cgaaagggac ggggggttta cctggacccc cagtccggcg aggagctcaa cccaatcccc 3960ccgccgccgc agccctatca gcagccgcgg gcccttgctt cccaggatgg cacccaaaaa 4020gaagctgcag ctgccgccgc cgccacccac ggacgaggag gaatactggg acagtcaggc 4080agaggaggtt ttggacgagg aggaggagat gatggaagac tgggacagcc tagacgaagc 4140ttccgaggcc gaagaggtgt cagacgaaac accgtcaccc tcggtcgcat tcccctcgcc 4200ggcgccccag aaattggcaa ccgttcccag catcgctaca acctccgctc ctcaggcgcc 4260gccggcactg cctgttcgcc gacccaaccg tagatgggac accactggaa ccagggccgg 4320taagtctaag cagccgccgc cgttagccca agagcaacaa cagcgccaag gctaccgctc 4380gtggcgcggg cacaagaacg ccatagttgc ttgcttgcaa gactgtgggg gcaacatctc 4440cttcgcccgc cgctttcttc tctaccatca cggcgtggcc ttcccccgta acatcctgca 4500ttactaccgt catctctaca gcccctactg caccggcggc agcggcagcg gcagcaacag 4560cagcggtcac acagaagcaa aggcgaccgg atagcaagac tctgacaaag cccaagaaat 4620ccacagcggc ggcagcagca ggaggaggag cgctgcgtct ggcgcccaac gaacccgtat 4680cgacccgcga gcttagaaat aggatttttc ccactctgta tgctatattt caacaaagca 4740ggggccaaga acaagagctg aaaataaaaa acaggtctct gcgctccctc acccgcagct 4800gcctgtatca caaaagcgaa gatcagcttc ggcgcacgct ggaagacgcg gaggctctct 4860tcagcaaata ctgcgcgctg actcttaagg actagtttcg cgccctttct caaatttaag 4920cgcgaaaact acgtcatctc cagcggccac acccggcgcc agcacctgtc gtcagcgcca 4980ttatgagcaa ggaaattccc acgccctaca tgtggagtta ccagccacaa atgggacttg 5040cggctggagc tgcccaagac tactcaaccc gaataaacta catgagcgcg ggaccccaca 5100tgatatcccg ggtcaacgga atccgcgccc accgaaaccg aattctcctc gaacaggcgg 5160ctattaccac cacacctcgt aataacctta atccccgtag ttggcccgct gccctggtgt 5220accaggaaag tcccgctccc accactgtgg tacttcccag agacgcccag gccgaagttc 5280agatgactaa ctcaggggcg cagcttgcgg gcggctttcg tcacagggtg cggtcgcccg 5340ggcgttttag ggcggagtaa cttgcatgta ttgggaattg tagttttttt aaaatgggaa 5400gtgacgtatc gtgggaaaac ggaagtgaag atttgaggaa gttgtgggtt ttttggcttt 5460cgtttctggg cgtaggttcg cgtgcggttt tctgggtgtt ttttgtggac tttaaccgtt 5520acgtcatttt ttagtcctat atatactcgc tctgtacttg gcccttttta cactgtgact 5580gattgagctg gtgccgtgtc gagtggtgtt ttttaatagg tttttttact ggtaaggctg 5640actgttatgg ctgccgctgt ggaagcgctg tatgttgttc tggagcggga gggtgctatt 5700ttgcctaggc aggagggttt ttcaggtgtt tatgtgtttt tctctcctat taattttgtt 5760atacctccta tgggggctgt aatgttgtct ctacgcctgc gggtatgtat tcccccgggc 5820tatttcggtc gctttttagc actgaccgat gttaaccaac ctgatgtgtt taccgagtct 5880tacattatga ctccggacat gaccgaggaa ctgtcggtgg tgctttttaa tcacggtgac 5940cagttttttt acggtcacgc cggcatggcc gtagtccgtc ttatgcttat aagggttgtt 6000tttcctgttg taagacaggc ttctaatgtt taaatgtttt tttttttgtt attttatttt 6060gtgtttaatg caggaacccg cagacatgtt tgagagaaaa atggtgtctt tttctgtggt 6120ggttccggaa cttacctgcc tttatctgca tgagcatgac tacgatgtgc ttgctttttt 6180gcgcgaggct ttgcctgatt ttttgagcag caccttgcat tttatatcgc cgcccatgca 6240acaagcttac ataggggcta cgctggttag catagctccg agtatgcgtg tcataatcag 6300tgtgggttct tttgtcatgg ttcctggcgg ggaagtggcc gcgctggtcc gtgcagacct 6360gcacgattat gttcagctgg ccctgcgaag ggacctacgg gatcgcggta tttttgttaa 6420tgttccgctt ttgaatctta tacaggtctg tgaggaacct gaatttttgc aatcatgatt 6480cgctgcttga ggctgaaggt ggagggcgct ctggagcaga tttttacaat ggccggactt 6540aatattcggg atttgcttag agacatattg ataaggtggc gagatgaaaa ttatttgggc 6600atggttgaag gtgctggaat gtttatagag gagattcacc ctgaagggtt tagcctttac 6660gtccacttgg acgtgagggc agtttgcctt ttggaagcca ttgtgcaaca tcttacaaat 6720gccattatct gttctttggc tgtagagttt gaccacgcca ccggagggga gcgcgttcac 6780ttaatagatc ttcattttga ggttttggat aatcttttgg aataaaaaaa aaaaaacatg 6840gttcttccag ctcttcccgc tcctcccgtg tgtgactcgc agaacgaatg tgtaggttgg 6900ctgggtgtgg cttattctgc ggtggtggat gttatcaggg cagcggcgca tgaaggagtt 6960tacatagaac ccgaagccag ggggcgcctg gatgctttga gagagtggat atactacaac 7020tactacacag agcgagctaa gcgacgagac cggagacgca gatctgtttg tcacgcccgc 7080acctggtttt gcttcaggaa atatgactac gtccggcgtt ccatttggca tgacactacg 7140accaacacga tctcggttgt ctcggcgcac tccgtacagt agggatcgcc tacctccttt 7200tgagacagag acccgcgcta ccatactgga ggatcatccg ctgctgcccg aatgtaacac 7260tttgacaatg cacaacgtga gttacgtgcg aggtcttccc tgcagtgtgg gatttacgct 7320gattcaggaa tgggttgttc cctgggatat ggttctgacg cgggaggagc ttgtaatcct 7380gaggaagtgt atgcacgtgt gcctgtgttg tgccaacatt gatatcatga cgagcatgat 7440gatccatggt tacgagtcct gggctctcca ctgtcattgt tccagtcccg gttccctgca 7500gtgcatagcc ggcgggcagg ttttggccag ctggtttagg atggtggtgg atggcgccat 7560gtttaatcag aggtttatat ggtaccggga ggtggtgaat tacaacatgc caaaagaggt

7620aatgtttatg tccagcgtgt ttatgagggg tcgccactta atctacctgc gcttgtggta 7680tgatggccac gtgggttctg tggtccccgc catgagcttt ggatacagcg ccttgcactg 7740tgggattttg aacaatattg tggtgctgtg ctgcagttac tgtgctgatt taagtgagat 7800cagggtgcgc tgctgtgccc ggaggacaag gcgtctcatg ctgcgggcgg tgcgaatcat 7860cgctgaggag accactgcca tgttgtattc ctgcaggacg gagcggcggc ggcagcagtt 7920tattcgcgcg ctgctgcagc accaccgccc tatcctgatg cacgattatg actctacccc 7980catgtaggcg tggacttccc cttcgccgcc cgttgagcaa ccgcaagttg gacagcagcc 8040tgtggctcag cagctggaca gcgacatgaa cttaagcgag ctgcccgggg agtttattaa 8100tatcactgat gagcgtttgg ctcgacagga aaccgtgtgg aatataacac ctaagaatat 8160gtctgttacc catgatatga tgctttttaa ggccagccgg ggagaaagga ctgtgtactc 8220tgtgtgttgg gagggaggtg gcaggttgaa tactagggtt ctgtgagttt gattaaggta 8280cggtgatcaa tataagctat gtggtggtgg ggctatacta ctgaatgaaa aatgacttga 8340aattttctgc aattgaaaaa taaacacgtt gaaacataac atgcaacagg ttcacgattc 8400tttattcctg ggcaatgtag gagaaggtgt aagagttggt agcaaaagtt tcagtggtgt 8460attttccact ttcccaggac catgtaaaag acatagagta agtgcttacc tcgctagttt 8520ctgtggattc actagaatcg atgtaggatg ttgcccctcc tgacgcggta ggagaagggg 8580agggtgccct gcatgtctgc cgctgctctt gctcttgccg ctgctgagga ggggggcgca 8640tctgccgcag caccggatgc atctgggaaa agcaaaaaag gggctcgtcc ctgtttccgg 8700aggaatttgc aagcggggtc ttgcatgacg gggaggcaaa cccccgttcg ccgcagtccg 8760gccggcccga gactcgaacc gggggtcctg cgactcaacc cttggaaaat aaccctccgg 8820ctacagggag cgagccactt aatgctttcg ctttccagcc taaccgctta cgccgcgcgc 8880ggccagtggc caaaaaagct agcgcagcag ccgccgcgcc tggaaggaag ccaaaaggag 8940cgctcccccg ttgtctgacg tcgcacacct gggttcgaca cgcgggcggt aaccgcatgg 9000atcacggcgg acggccggat ccggggttcg aaccccggtc gtccgccatg atacccttgc 9060gaatttatcc accagaccac ggaagagtgc ccgcttacag gctctccttt tgcacggtct 9120agagcgtcaa cgactgcgca cgcctcaccg gccagagcgt cccgaccatg gagcactttt 9180tgccgctgcg caacatctgg aaccgcgtcc gcgactttcc gcgcgcctcc accaccgccg 9240ccggcatcac ctggatgtcc aggtacatct acggattacg tcgacgttta aaccatatga 9300tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 9360aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 9420tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 9480tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 9540cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 9600agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 9660tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 9720aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 9780ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 9840cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt 9900accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 9960ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 10020ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 10080gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 10140aaatcaatct aaagtatata tgagtaaact tggtctgaca gtcagaagaa ctcgtcaaga 10200aggcgataga aggcgatgcg ctgcgaatcg ggagcggcga taccgtaaag cacgaggaag 10260cggtcagccc attcgccgcc aagctcttca gcaatatcac gggtagccaa cgctatgtcc 10320tgatagcggt ccgccacacc cagccggcca cagtcgatga atccagaaaa gcggccattt 10380tccaccatga tattcggcaa gcaggcatcg ccatgggtca cgacgagatc ctcgccgtcg 10440ggcatgctcg ccttgagcct ggcgaacagt tcggctggcg cgagcccctg atgctcttcg 10500tccagatcat cctgatcgac aagaccggct tccatccgag tacgtgctcg ctcgatgcga 10560tgtttcgctt ggtggtcgaa tgggcaggta gccggatcaa gcgtatgcag ccgccgcatt 10620gcatcagcca tgatggatac tttctcggca ggagcaaggt gagatgacag gagatcctgc 10680cccggcactt cgcccaatag cagccagtcc cttcccgctt cagtgacaac gtcgagtaca 10740gctgcgcaag gaacgcccgt cgtggccagc cacgatagcc gcgctgcctc gtcttgcagt 10800tcattcaggg caccggacag gtcggtcttg acaaaaagaa ccgggcgccc ctgcgctgac 10860agccggaaca cggcggcatc agagcagccg attgtctgtt gtgcccagtc atagccgaat 10920agcctctcca cccaagcggc cggagaacct gcgtgcaatc catcttgttc aatcatactc 10980ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 11040tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 11100ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca 11160gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga 11220ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg 11280actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat 11340caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag 11400ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga 11460agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa 11520ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gatggatcc 1156975030DNAArtificial SequenceCoding Strand of Plasmid pAV-CMV-EGFP 7cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60ggtcgcccgg ccctccagtg agcgagcgcg cagagaggga gtggccaact ccatcactag 120gggttcctgc ggccgcacgc gtctagttat taatagtaat cgaattcgtg ttactcataa 180ctagtaaggt cgggcaggaa gagggcctat ttcccatgat tccttcatat ttgcatatac 240gatacaaggc tgttagagag ataattagaa ttaatttgac tgtaaacaca aagatattag 300tacaaaatac gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat 360gttttaaaat ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttgggtt 420tatatatctt gtggaaagga cgcgggatcc actggaccag gcagcagcgt cagaagactt 480ttttggaaaa gcttgactag taatactgta atagtaatca attacggggt cattagttca 540tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc ctggctgacc 600gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag taacgccaat 660agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt 720acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc 780cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc agtacatcta 840cgtattagtc atcgctatta ccatggtgat gcggttttgg cagtacatca atgggcgtgg 900atagcggttt gactcacggg gatttccaag tctccacccc attgacgtca atgggagttt 960gttttgcacc aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg 1020caaatgggcg gtaggcgtgt acggtgggag gtctatataa gcagagctgg tttagtgaac 1080cgtcagatcc gctagagatc cggtaccgag gagatctgcc gccgcgatcg ccggcgcgcc 1140agatctcacg cttaactagc tagcggaccg acgcgtacgc ggccgctcga gatggtgagc 1200aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta 1260aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg 1320accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc 1380accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac 1440ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac 1500gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc 1560atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag 1620tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag 1680gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac 1740cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc 1800acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag 1860ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta agtcgaggat 1920tataaggatg acgacgataa attcgtcgag caccaccacc accaccacta ataaggttta 1980tccgatccac cggatctaga taagatatcc gatccaccgg atctagataa ctgatcataa 2040tcagccatac cacatttgta gaggttttac ttgctttaaa aaacctccca cacctccccc 2100tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata 2160atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc 2220attctagttg tggtttgtcc aaactcatca atgtatctta acgcggtaac cacgtgcgga 2280ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct 2340cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct 2400cagtgagcga gcgagcgcgc agctgcctgc aggggcgcct gatgcggtat tttctcctta 2460cgcatctgtg cggtatttca caccgcatac gtcaaagcaa ccatagtacg cgccctgtag 2520cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacctgccag 2580cgccttagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt 2640tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca 2700cctcgacccc aaaaaacttg atttgggtga tggttcacgt agtgggccat cgccctgata 2760gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca 2820aactggaaca acactcaacc ctatctcggg ctattctttt gatttataag ggattttgcc 2880gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa 2940caaaatatta acgtttacaa ttttatggtg cactctcagt acaatctgct ctgatgccgc 3000atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct 3060gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag 3120gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac gcctattttt 3180ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa 3240tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat 3300gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca 3360acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca 3420cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta 3480catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt 3540tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc 3600cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc 3660accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc 3720cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa 3780ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga 3840accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat 3900ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca 3960attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc 4020ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat 4080tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag 4140tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa 4200gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca 4260tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc 4320ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc 4380ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc 4440agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt 4500cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt 4560caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc 4620tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa 4680ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac 4740ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg 4800gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga 4860gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact 4920tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa 4980cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 503084742DNAArtificial SequenceCoding Strand of Plasmid pAV-TBG-EGFP 8cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120aggggttcct gcggccggtc gcgtctagta ctagtaggtt aatttttaaa aagcagtcaa 180aagtccaagt ggcccttggc agcatttact ctctctgttt gctctggtta ataatctcag 240gagcacaaac attccagatc caggttaatt tttaaaaagc agtcaaaagt ccaagtggcc 300cttggcagca tttactctct ctgtttgctc tggttaataa tctcaggagc acaaacattc 360cagatccggc gcgccagggc tggaagctac ctttgacatc atttcctctg cgaatgcatg 420tataatttct acagaaccta ttagaaagga tcacccagcc tctgcttttg tacaactttc 480ccttaaaaaa ctgccaattc cactgctgtt tggcccaata gtgagaactt tttcctgctg 540cctcttggtg cttttgccta tggcccctat tctgcctgct gaagacactc ttgccagcat 600ggacttaaac ccctccagct ctgacaatcc tctttctctt ttgttttaca tgaagggtct 660ggcagccaaa gcaatcactc aaagttcaaa ccttatcatt ttttgctttg ttcctcttgg 720ccttggtttt gtacatcagc tttgaaaata ccatcccagg gttaatgctg gggttaattt 780ataactaaga gtgctctagt tttgcaatac aggacatgct ataaaaatgg aaagatgttg 840ctttctgaga gacaggtacc gaggagatct gccgccgcga tcgccaccat ggtgagcaag 900ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac 960ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacttacgg caagctgacc 1020ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc 1080ctgacctacg gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc 1140ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac 1200ggcaactaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc 1260gagctgaagg gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac 1320aactacaaca gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg 1380aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag 1440cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc 1500cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc 1560gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtagac gcgtacgcgg 1620ccgctcgagg attataagga tgacgacgat aaattcgtcg agcaccacca ccaccaccac 1680taataaggtt tatccgatcc accggatcta gataagatat ccgatccacc ggatctagat 1740aactgatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc 1800cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta 1860ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 1920ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct taacgcggta 1980accacgtgcg gacccaacgg ccgcaggaac ccctagtgat ggagttggcc actccctctc 2040tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg 2100cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc ctgatgcggt 2160attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc aaccatagta 2220cgcgccctgt agcggcacat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc 2280tacacctgcc agcgccttag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac 2340gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag 2400tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac gtagtgggcc 2460atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 2520actcttgttc caaactggaa caacactcaa ctctatctcg ggctattctt ttgatttata 2580agggattttg ccgatttcgg tctattggtt aaaaaatgag ctgatttaac aaaaatttaa 2640cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca gtacaatctg 2700ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg 2760acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg 2820catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat 2880acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac 2940ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat 3000gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag 3060tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc 3120tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc 3180acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc 3240cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc 3300ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt 3360ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt 3420atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat 3480cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct 3540tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat 3600gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc 3660ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg 3720ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc 3780tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta 3840cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc 3900ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga 3960tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat 4020gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat 4080caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa 4140accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa 4200ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt 4260aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt 4320accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata 4380gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt 4440ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac 4500gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga 4560gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg 4620ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa 4680aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat 4740gt 47429131DNAadeno-associated virus 1misc_feature(1)..(131)P5 Promoter of AAV1 9ggtcctgtat tagctgtcac gtgagtgctt ttgcgacatt ttgcgacacc acgtggccat 60ttagggtata tatggccgag tgagcgagca ggatctccat tttgaccgcg aaatttgaac 120gagcagcagc c 13110131DNAadeno-associated virus 2misc_feature(1)..(131)P5 Promoter of AAV2 10ggtcctgtat tagaggtcac gtgagtgttt tgcgacattt tgcgacacca tgtggtcacg 60ctgggtattt aagcccgagt gagcacgcag ggtctccatt ttgaagcggg aggtttgaac 120gcgcagccgc c 13111131DNAadeno-associated virus 3'misc_feature(1)..(131)P5 Promoter of AAV3 11ccagctgcgt cagcagtcag gtgacccttt tgcgacagtt tgcgacacca cgtggccgct 60gagggtatat attctcgagt gagcgaacca ggagctccat tttgaccgcg aaatttgaac 120gagcagcagc c 13112192DNAadeno-associated virus 4misc_feature(1)..(192)P5 Promoter of AAV4 12ggtccctgta ttagcagtca cgtgagtgtc gtatttcgcg gagcgtagcg gagcgcatac 60caagctgcca cgtcacagcc acgtggtccg tttgcgacag tttgcgacac catgtggtca 120ggagggtata taaccgcgag tgagccagcg aggagctcca ttttgcccgc gaattttgaa 180cgagcagcag cc 19213131DNAadeno-associated virus 5misc_feature(1)..(131)P5 Promoter of AAV5 13atgtgatgtg ttttatccaa taggaagaaa gcgcgcgtat gagttctcgc gagacttccg

60gggtataaaa gaccgagtga acgagcccgc cgccattctt tgctctggac tgctagagga 120ccctcgctgc c 13114130DNAadeno-associated virus 6misc_feature(1)..(130)P5 Promoter of AAV6 14ggtcctgtat tagaggtcac gtgagtgttt tgcgacattt tgcgacacca tgtggtcacg 60ctgggtattt aagcccgagt gagcacgcag ggtctccatt ttgaagcggg aggtttgaac 120gcgcagcgcc 13015131DNAadeno-associated virus 7misc_feature(1)..(131)P5 Promoter of AAV7 15ggtcctgtat tagctgtcac gtgagtgctt ttgcgacatt ttgcgacacc acgtggccat 60ttgaggtata tatggccgag tgagcgagca ggatctccat tttgaccgcg aaatttgaac 120gagcagcagc c 13116131DNAadeno-associated virus 8misc_feature(1)..(131)P5 Promoter of AAV8 16ggtcctgtat tagctgtcac gtgagtgctt ttgcggcatt ttgcgacacc acgtggccat 60ttgaggtata tatggccgag tgagcgagca ggatctccat tttgaccgcg aaatttgaac 120gagcagcagc c 13117180DNAadeno-associated virus 1misc_feature(1)..(180)P40 Promoter of AAV1 17ggtgacaaag caggaagtca aagagttctt ccgctgggcg caggatcacg tgaccgaggt 60ggcgcatgag ttctacgtca gaaagggtgg agccaacaaa agacccgccc ccgatgacgc 120ggataaaagc gagcccaagc gggcctgccc ctcagtcgcg gatccatcga cgtcagacgc 18018180DNAadeno-associated virus 2misc_feature(1)..(180)P40 Promoter of AAV2 18ggtcaccaag caggaagtca aagacttttt ccggtgggca aaggatcacg tggttgaggt 60ggagcatgaa ttctacgtca aaaagggtgg agccaagaaa agacccgccc ccagtgacgc 120agatataagt gagcccaaac gggtgcgcga gtcagttgcg cagccatcga cgtcagacgc 18019180DNAadeno-associated virus 3misc_feature(1)..(180)P40 Promoter of AAV3 19ggtcaccaaa caggaagtaa aggacttttt ccggtgggct tccgatcacg tgactgacgt 60ggctcatgag ttctacgtca gaaagggtgg agctaagaaa cgccccgcct ccaatgacgc 120ggatgtaagc gagccaaaac gggagtgcac gtcacttgcg cagccgacaa cgtcagacgc 18020180DNAadeno-associated virus 4misc_feature(1)..(180)P40 Promoter of AAV4 20ggtcaccaag caggaagtca aagacttttt ccggtgggcg tcagatcacg tgaccgaggt 60gactcacgag ttttacgtca gaaagggtgg agctagaaag aggcccgccc ccaatgacgc 120agatataagt gagcccaagc gggcctgtcc gtcagttgcg cagccatcga cgtcagacgc 18021227DNAadeno-associated virus 5misc_feature(1)..(227)P40 Promoter of AAV5 21gattactaag caggaagtca aggacttttt tgcttgggca aaggtcaatc aggtgccggt 60gactcacgag tttaaagttc ccagggaatt ggcgggaact aaaggggcgg agaaatctct 120aaaacgccca ctgggtgacg tcaccaatac tagctataaa agtctggaga agcgggcctg 180gagcatgagg ctctcatttg ttcccgagac gcctcgcagt tcagacg 22722180DNAadeno-associated virus 6misc_feature(1)..(180)P40 Promoter of AAV6 22ggtgacaaag caggaagtca aagagttctt ccgctgggcg caggatcacg tgaccgaggt 60ggcgcatgag ttctacgtca gaaagggtgg agccaacaag agacccgccc ccgatgacgc 120ggataaaagc gagcccaagc gggcctgccc ctcagtcgcg gatccatcga cgtcagacgc 18023180DNAadeno-associated virus 7misc_feature(1)..(180)P40 Promoter of AAV7 23ggtgacgaag caggaagtca aagagttctt ccgctgggcc agtgatcacg tgaccgaggt 60ggcgcatgag ttctacgtca gaaagggcgg agccagcaaa agacccgccc ccgatgacgc 120ggatataagc gagcccaagc gggcctgccc ctcagtcgcg gatccatcga cgtcagacgc 18024180DNAadeno-associated virus 8misc_feature(1)..(180)P40 Promoter of AAV8 24ggtgacaaag caggaagtca aagagttctt ccgctgggcc agtgatcacg tgaccgaggt 60ggcgcatgag ttttacgtca gaaagggcgg agccagcaaa agacccgccc ccgatgacgc 120ggataaaagc gagcccaagc gggcctgccc ctcagtcgcg gatccatcga cgtcagacgc 180

Claims

1. A recombinantly-modified adeno-associated virus (AAV) helper vector that comprises an AAV helper function-providing polynucleotide, wherein said polynucleotide comprises a non-native AAV serotype P5 or P40 promoter sequence.

2. The recombinantly-modified adeno-associated virus (AAV) helper vector of claim 1, wherein said AAV helper function-providing polynucleotide vector comprises a non-native AAV serotype P5 promoter sequence.

3. The recombinantly-modified adeno-associated virus (AAV) helper vector of claim 1, wherein said AAV helper function-providing polynucleotide vector comprises a non-native AAV serotype P40 promoter sequence.

4. The recombinantly-modified adeno-associated virus (AAV) helper vector of claim 1, wherein said vector is a plasmid vector.

5. The recombinantly-modified adeno-associated virus (AAV) helper vector of claim 1, wherein said non-native AAV serotype P5 or P40 promoter sequence replaces a native AAV serotype promoter sequence.

6. The recombinantly-modified adeno-associated virus (AAV) helper vector of claim 1, wherein said vector additionally comprises a non-AAV helper function-providing polynucleotide.

7. A method for increasing the production titer of a recombinantly-modified adeno-associated virus (rAAV) that comprises a transgene cassette, wherein said method comprises culturing cells that have been transfected with: (1) said rAAV; and (2) the recombinantly-modified adeno-associated virus (AAV) helper vector of claim 6; wherein said culturing is conducted in a culture medium under conditions sufficient to permit the production of said rAAV and wherein the presence of said non-native AAV serotype P5 or P40 promoter sequence causes said cells to produce said rAAV at an increased production titer relative to that which would be attained if said AAV helper function-providing polynucleotide contained native serotype P5 and P40 promoters.

8. The method of claim 7, wherein said transgene cassette encodes a protein, or comprises a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition.

9. The method of claim 7, wherein: (A) said AAV helper function-providing polynucleotide of said vector encodes an AAV1 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of said serotypes; (B) said AAV helper function-providing polynucleotide of said vector encodes an AAV2 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of said serotypes; (C) said AAV helper function-providing polynucleotide of said vector encodes an AAV3 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV4, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of said serotypes; (D) said AAV helper function-providing polynucleotide of said vector encodes an AAV4 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of said serotypes; (E) said AAV helper function-providing polynucleotide of said vector encodes an AAV5 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV6, AAV7 or AAV8, or a hybrid of one or more of said serotypes; (F) said AAV helper function-providing polynucleotide of said vector encodes an AAV6 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV7 or AAV8, or a hybrid of one or more of said serotypes; (G) said AAV helper function-providing polynucleotide of said vector encodes an AAV7 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV6 or AAV8, or a hybrid of one or more of said serotypes; or (H) said AAV helper function-providing polynucleotide of said vector encodes an AAV8 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV6 or AAV7, or a hybrid of one or more of said serotypes.

10. The method of claim 7, wherein said cells are human embryonic kidney cells, baby hamster kidney cells or sf9 insect cells.

11. The method of claim 10, wherein said cells are HEK293 human embryonic kidney cells.

12. The method of claim 10, wherein said cells are BHK21 baby hamster kidney cells.

13. A method for increasing the production titer of a recombinantly-modified adeno-associated virus (rAAV) that comprises a transgene cassette, wherein said method comprises culturing cells that have been transfected with: (1) said rAAV; (2) the recombinantly-modified adeno-associated virus (AAV) helper vector of claims 1; and (3) an additional vector that comprises a non-AAV helper function-providing polynucleotide; wherein said culturing is conducted in a culture medium under conditions sufficient to permit the production of said rAAV and wherein the presence of said non-native AAV serotype P5 or P40 promoter sequence causes said cells to produce said rAAV at an increased production titer relative to that which would be attained if said AAV helper function-providing polynucleotide contained native serotype P5 and P40 promoters.

14. The method of claim 13, wherein said transgene cassette encodes a protein, or comprises a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition.

15. The method of claim 13, wherein: (A) said AAV helper function-providing polynucleotide of said vector encodes an AAV1 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of said serotypes; (B) said AAV helper function-providing polynucleotide of said vector encodes an AAV2 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of said serotypes; (C) said AAV helper function-providing polynucleotide of said vector encodes an AAV3 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV4, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of said serotypes; (D) said AAV helper function-providing polynucleotide of said vector encodes an AAV4 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of said serotypes; (E) said AAV helper function-providing polynucleotide of said vector encodes an AAV5 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV6, AAV7 or AAV8, or a hybrid of one or more of said serotypes; (F) said AAV helper function-providing polynucleotide of said vector encodes an AAV6 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV7 or AAV8, or a hybrid of one or more of said serotypes; (G) said AAV helper function-providing polynucleotide of said vector encodes an AAV7 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV6 or AAV8, or a hybrid of one or more of said serotypes; or (H) said AAV helper function-providing polynucleotide of said vector encodes an AAV8 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV6 or AAV7, or a hybrid of one or more of said serotypes.

16. The method of claim 13, wherein said cells are human embryonic kidney cells, baby hamster kidney cells or sf9 insect cells.

17. The method of claim 16, wherein said cells are HEK293 human embryonic kidney cells.

18. The method of claim 16, wherein said cells are BHK21 baby hamster kidney cells.

19. A pharmaceutical composition that comprises recombinantly-modified adeno-associated virus (rAAV) produced by the method of claim 6, and a pharmaceutically acceptable carrier.

20. A pharmaceutical composition that comprises recombinantly-modified adeno-associated virus (rAAV) produced by the method of claim 13, and a pharmaceutically acceptable carrier.