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. 20 26 23 39 90 16 S ep 2 02 6 2 0 2 6 2 3 3 9 9 0 1 6 2 0 2 6 S e p

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 July 15, 2019, and having a size of 84,101 bytes), which file is herein incorporated by reference in its entirety. BACKGROUND OF THE INVENTION: I. Adeno-Associated Virus (AAV)

[0003] 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 f Curr. Gene Ther. 14(2):86-100; Zinn, E. et al. (2014) “Adeno-Associated Virus: Fit To Servef Curr. Opin. Virol. 0:90-97). Despite not 2026233990   16 Sep 2026 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, etcf exhibiting different tissue infection capabilities (“tropisms”), have been identified (Colella, P. et al. (2018) “ Emerging Issues in AAV-MediatedIn 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).

[0004] 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 (Figure 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 p!9 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-RegulatedDNA 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 p!9 Promoter by Rep Protein,” J. Virol. 2026233990   16 Sep 2026 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 (Biining, 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) “EmergingIssues in AAV-MediatedIn Vivo Gene Therapy,” Molec. Ther. Meth. Clin. Develop. 8:87-104; Biining, 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, el04596:l-10).

[0005] 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) “EmergingIssues in AAV-MediatedIn Vivo Gene Therapy,” Molec. Ther. Meth. Clin. Develop. 8:87-104).

[0006] 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 2026233990   16 Sep 2026 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 Ela, Elb, E2a, and E4; thus, these proteins must also be provided by a co-infecting “helper” virus. The Ela protein greatly stimulate viral gene transcription during the productive infection. The Elb 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. 507:229254; Dyson, N. et al. (1992) “Adenovirus El A Targets Key Regulators Of Cell Proliferation,” Cane. Surv. 12:161-195; Jones N.C. (1990) “Transformation By The Human Adenoviruses,” Semin. Cancer Biol. l(6):425-435; Ben-Israel, H. et al. (2002) “Adenovirus and Cell Cycle Control,” Front. Biosci. 7:dl369-dl395; Hoeben, R.C. et al. (2013)“AdenovirusDNA Replication,” Cold Spring Harb. Perspect. Biol. 5:a013003 (pages 1-11); Berk, A.J. (2013) “Adenoviridae: The Viruses And Their Replication, In: Fields Virology, 6th 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. 10:11061117).

[0007] 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. 2026233990   16 Sep 2026 II. rAAV and Their Use in Gene Therapy

[0008] 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) “Ade no-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 AndLentivirus 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; Biining, 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 OfMPTP-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; US Patents No.: 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).

[0009] rAAV are typically produced using circular plasmids (“rAAV plasmid vector”). The AAV rep and cap genes are typically deleted from such constructs and replaced with a promoter, a P-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 2026233990   16 Sep 2026 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; Biining, H. et al. (2019) “ Capsid Modifications for Targeting and Improving the Efficacy ofAA V Vectorsf 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.

[0010] 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 (US Patents No. 8,697,359, 10,000,772, 10,113,167, 10,227,611; Lino, C.A. etal. (2018) “Delivering CRISPR: A Review Of The Challenges And Approaches f Drug Deliv. 25(1): 1234-1237; Ferreira, V. etal. (2014) “Immune Responses To AAV-Vectors, The Glybera Example From Bench To Bedside" Front. Immunol. 5(82):1-15), Biining, 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. 28:361372); Berns, K. I. etal. (2017) “AAV: An Overview of Unanswered Questions" Human Gene Ther. 28(4):308-313). More than 150 clinical trials involving rAAV have been instituted (Biining, H. etal. (2019) “Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors,” Mol. Ther. Meth. Clin. Devel. 12:P248-P265; Clement, N. et al. (2016) “Manufacturing Of Recombinant Adeno-Associated Viral Vectors For Clinical Trials,” Meth. Clin. Develop. 3:16002:1-7). The most commonly used AAV serotype for such recombinantly-modified AAV is AAV2, which is capable of infecting cells of the central nervous system, kidney, retinal pigment epithelium and photoreceptor cells. 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 US Patents No. 10,301,650; 10,266,846; 10,265,417; 10,214,785; 10,214,566; 10,202,657; 10,046,016; 2026233990   16 Sep 2026 9,884,071;  9,856,539;  9,737,618;  9,677,089;  9,458,517;  9,457,103;  9,441,244; 9,193,956;  8,846,389;  8,507,267;  7,906,111;  7,479,554;  7,186,552;  7,105,345; 6,984,517; 6,962,815; and 6,733,757. III. Methods of rAAV Production

[0011] 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.

[0012] 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 (Ela, Elb, 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).

[0013] 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 (Figure 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 2026233990   16 Sep 2026 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).

[0014] 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. etal. (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).

[0015] rAAV may alternatively be produced in insect cells (e.g., sf9 cells) using baculoviral vectors (see, e.g., US Patents No.: 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. 507:229254, and in Penaud-Budloo, M. et al. (2018) “Pharmacology of Recombinant Adeno-associated Virus Production,” Molec. Ther. Meth. Clin. Develop. 8:166-180. 2026233990   16 Sep 2026 IV. Methods of rAAV Purification and Recovery

[0016] After production, rAAV are typically collected and purified by one or more overnight CsCl gradient centrifugations (Zolotukhin, S. et al. (1999) “Recombinant Adeno-Associated Virus Purification Using Novel Methods Improves Infectious Titer And Yield” Gene Ther. 6:973-985), followed by desalting to form a purified rAAV production stock. Titers of 1012-1013 infectious rAAV capsids / mL are obtainable.

[0017] 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 cu / j-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 US Patents No. 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.

[0018] 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) 2026233990   16 Sep 2026 “Engineering Adeno-Associated Viruses For Clinical Gene Therapy,” Nat. Rev. Genet. 15(7):445-451; Kwon, I. el 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).

[0019] 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:

[0020] 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.

[0021] 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.

[0022] 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. 2026233990   16 Sep 2026

[0023] The invention also particularly includes the embodiment of such recombinantly-modified adeno-associated virus (AAV) helper vector wherein the nonnative AAV serotype P5 or P40 promoter sequence replaces a native AAV serotype promoter sequence.

[0024] 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.

[0025] 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: (1)   the rAAV; (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.

[0026] 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: (1)   the rAAV; (2)    any of the above-described recombinantly-modified adeno-associated virus (AAV) helper vectors; and (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 2026233990   16 Sep 2026 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.

[0027] 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.

[0028] The invention also particularly includes the embodiment of such methods, wherein: (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; (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; (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; (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; (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 2026233990   16 Sep 2026 AAV1, AAV3, AAV4, AAV6, AAV7 or AAV8, or a hybrid of one or more of the serotypes; (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; (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; (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.

[0029] 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.

[0030] 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:

[0031] Figure 1 provides a schematic genetic map of the wild-type (Wt) AAV genome.

[0032] Figure 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 2026233990   16 Sep 2026 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 Ela, Elb, E2a, VA and E4.

[0033] Figure 3 shows a map of the AAV helper plasmid vector pAAV-RCl (SEQ ID NO:1)

[0034] Figure 4 shows a map of the AAV helper plasmid vector pAAV-RC2 (SEQ ID NO:2)

[0035] Figure 5 shows a map of the AAV helper plasmid vector pAAV-RC5 (SEQ ID NO:3)

[0036] Figure 6 shows a map of the AAV helper plasmid vector pAAV-RC6 (SEQ ID NO:4)

[0037] Figure 7 shows a map of the AAV helper plasmid vector pAAV-RC7 (SEQ ID NO:5)

[0038] Figure 8 shows a map of the non-AAV helper plasmid vector pHelper-Kan (SEQ ID NO:6)

[0039] Figure 9 shows a map of the rAAV plasmid vector pAV-CMV-EGFP (SEQ ID NO:7)

[0040] Figure 10 shows a map of the rAAV plasmid vector pAV-TBG-EGFP (SEQ ID NO:8)

[0041] Figure 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 2026233990   16 Sep 2026 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.

[0042] 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.

[0043] Figures 12A-12B show the production titers of rAAV obtained by modifying a parental RC2 vector to comprise a non-native P5 promoter sequence (Figure 11; Figure 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 2026233990   16 Sep 2026 both native AAV2 serotype promoter sequences (solid black rectangles). Figure 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.

[0044] Figures 13A-13B show the production titers of rAAV obtained by modifying a parental RC2 vector to comprise a non-native P40 promoter sequence (Figure 11; Figure 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). Figure 13B shows the production titers of rAAV obtained using such AAV helper plasmid vectors. The following constructs were employed: Parent-RC2, P40(l)-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.

[0045] Figures 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 nonnative P40 promoter sequence (Figure 11; Figure 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(l)-RC2, P5(2) / P40(l)-RC2, P5(3) / P40(l)-RC2, and P5(5) / P40(l)-RC2. The sequences of the promoter regions are shown in Table 1. Figure 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. 2026233990   16 Sep 2026

[0046] Figures 15A-15C show the production titers of rAAV obtained by modifying a parental RC6 vector to comprise a non-native P5 promoter sequence (Figure 11; Figure 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 (Figures 15B-15C) using a triple plasmid transfection system with an rAAV, and an Ad helper plasmid that provided the required adenoviral functions.

[0047] Figures 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 (Figure 11; Figure 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-RCl, Parent-RC5, 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 (Figure 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: I. The Methods of the Present Invention

[0048] 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 2026233990   16 Sep 2026 serotype. The use of such substitute or additional promoter sequences causes increased production of recombinantly-modified adeno-associated virus.

[0049] 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 US Patents No. 10,214,730; 7,122,348; 6,821,511; 6,753,419; 9,441,206; and 7,115,391.

[0050] 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 A 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; US Patents No. 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.

[0051] 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.

[0052] 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 2026233990   16 Sep 2026 “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 functionproviding polynucleotide, and the rAAV plasmid vector (Figure 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. A. Illustrative AAV Helper Function-Providing Polynucleotides

[0053] 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-RCl, pAAV-RC2, pAAV-RC5, pAAV-RC6, and pAAV-RC7 1. Plasmid pAAV-RCl

[0054] Plasmid pAAV-RCl (SEQ ID NO:1; Figure 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-RCl are AAV2 serotype promoters (SEQ ID NO: 10 and SEQ ID NO: 18, respectively). 2026233990   16 Sep 2026 Coding Strand of Plasmid pAAV-RCl (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 2026233990   16 Sep 2026 ttttcaccac gcccaagaga cgacgaatga caagtcttct gcaccagggc aatacggcta tccttttact caactttacc acgcgcacag tacctgtatt caaggacttg ccaaaaactg acaaaaacag atataacctc cctcacacaa atttttggaa catgattaca aaagatttgg gcgaccggag agatagagac cagatggaca aacccgcctc tccggcggag ccacaggaca agcaagcgct tgccaacgtt gccccattgg gttgcttgtt ctctgcgtat aacggccgcc ccatgctact cacgtgagtg tttaagcccg aacgcgcagc gctcgactag ttagtgaggg ctgtgtgaaa agcataaagt aattgcgttg agctgcatta gggcgctctt gctgcggcga cagaatcagg aaggccagga ccgcccccct gaaacccgac ctcgtgcgct ctttctccct atctcagttc ccccccgttc gtgactggca ctcaacttca tggcgtcaca cggactcgga tgcctccctc cctgacgctc gcctggaata ttcagctaca ccagagcctg acctgaacag ctgtttagcc gctacctgga acaacaacaa aatgggcgtg agacgacgaa aagagagcgc gacgaagagg gaccgtggca atgtgcatgc gtgtacctgc ctttcacccg ctcagatcct ttttcagcta agtgagtgtg ggaatcccga gattttactg cacccgttac aatcaataaa ttctttctta agtgtgctgg tatctacgta ttttgcgaca agtgagcacg cgccaagccg agcggccgcc ttaattgcgc ttgttatccg gtaaagcctg cgctcactgc atgaatcggc ccgcttcctc gcggtatcag ggataacgca accgtaaaaa gacgagcatc aggactataa ctcctgttcc tcgggaagcg ggtgtaggtc agcccgaccg gcgactcatc aactcttcaa accatcgcta gtaccagctt cgttcccggc aacaatggca tttcccttct cctttgagga gaccggctga aactcaaaat gtgggtctcc ccctgttatc cagcaatttt aatccatcat gacaagttct cggagcttca aaattaaagc gtcaatttcc tatgggagca agggtcccat tctcctctta catcaaaaac caaagtttgc gaaattgaat agtgcagtac tggacaacaa cttacccgtc ccgtttaatt tctagtttcc aattcggctt gccatgctct ttttgcgaca cagggtctcc aattctgcag accgcggtgg gcttggcgta ctcacaattc gggtgcctaa ccgctttcca caacgcgcgg gctcactgac ctcactcaaa ggaaagaaca ggccgcgttg acaaaaatcg agataccagg gaccctgccg tggcgctttc gttcgctcca ctgcgcctta aacaacaatt catccaagtc ataaccttac ccgtacgtcc ggacgtgttc gccaagccgt cagatgctga agtgcctttc tgaatcctct cagtccggaa agctggcatg ggcagcagcg acctggactg caaccctggc ttcccatgag aacactgcat cactaaccct agagcagcag ttacctggca ttgggccaaa tgggcggctt acgcctgttc ttcattcatc gggagctgca acatccaatt tggactttat ccctgtaagg cgtttcagtt atgctctagg tgtagttaat agaggtcctg ccatgtggtc attttgaagc atatccaaac agctccagct atcatggtca cacacaacat tgagtgagct gtcgggaaac ggagaggcgg tcgctgcgct ggcggtaata tgtgagcaaa ctggcgtttt acgctcaagt cgtttccccc cttaccggat tcatagctca agctgggctg tccggtaact ggggattccg aaggaggtca cagcacggtt tcggctctgc atgattccgc gggacgttca gaacgggcaa cacagcagct catcgaccaa gtgcccaaaa tctgttcagc cgtttctaaa gtgcttcaaa actgctatgg cggtgtcatg tggacaatgt gtggccaccg cacagaccct tggtgtggca attcctcaca tggactcaag ctgcgaatcc acccaatact gaaagaaaac atgcaaaatc actgagcctc cgcgccaccg gaactttggt atccactagt gattaacccg tattagaggt acgctgggta gggaggtttg actggcggcc tttgttccct tagctgtttc acgagccgga aactcacatt ctgtcgtgcc tttgcgtatt cggtcgttcg cggttatcca aggccagcaa tccataggct cagaggtggc tggaagctcc acctgtccgc cgctgtaggt tgtgcacgaa atcgtcttga 2026233990   16 Sep 2026 gtccaacccg acaggattag tggtggccta tctgctgaag gcaaacaaac attacgcgca ggggtctgac tgagattatc agttttaaat ccaatgctta catccatagt ggcttaccat accggctcca gcagaagtgg tgccgggaag tgttgccatt cttcattcag atgttgtgca aagtaagttg attctcttac tactcaacca ttgcccggcg aagtgctcat ttaccgctgt atcttcagca gaaggcaaaa atactcatac ttgtctcatg taggggttcc ttaatatttt tttaaccaat gaccgagata taaagaacgt gatggcccac gtgccgtaaa cttgacgggg aaaggagcgg aaccaccaca cgccattcag tcgctattac ttgggtaacg gtgagcgcgc cccctcgagg gcgggaggtt attaaggtcc ctttgtgaac acatggatct ctgcagcgcg ggctcttttc acgtgctcgt gtaagacacg cagagcgagg actacggcta ccagttacct caccgctggt gaaaaaaagg gctcagtgga aaaaaggatc caatctaaag atcagtgagg tgcctgactc ctggccccag gatttatcag tcctgcaact ctagagtaag gctacaggca ctccggttcc aaaaagcggt gccgcagtgt tgtcatgcca agtcattctg tcaatacggg cattggaaaa tgagatccag tcttttactt tgccgcaaaa tcttcctttt agcggataca gcgcacattt gttaaaattc aggccgaaat gggttgagtg ggactccaac tacgtgaacc gcactaaatc aaagccggcg gcgctagggc cccgccgcgc gctgcgcaac gccagctggc ccagggtttt gtaatacgac tcgacggtat tgaacgcgca ccagcgacct tgggtggccg gaatctgatt actttctgac tttgtgcaat ggaaaccacc acttatcgcc tatgtaggcg cactagaaga tcggaaaaag agcggtggtt atctcaagaa acgaaaactc ttcacctaga tatatatgag cacctatctc cccgtcgtgt tgctgcaatg caataaacca ttatccgcct tagttcgcca tcgtggtgtc caacgatcaa tagctccttc tatcactcat tccgtaagat agaatagtgt ataataccgc cgttcttcgg ttcgatgtaa tcaccagcgt aagggaataa tcaatattat tatttgaatg ccccgaaaag gcgttaaatt cggcaaaatc ttgttccagt gtcaaagggc atcaccctaa ggaaccctaa aacgtggcga gctggcaagt ttaatgcgcc tgttgggaag gaaaggggga cccagtcacg tcactatagg cgggggagct gccgccatgc tgacgagcat agaaggaatg gagcaggcac ggaatggcgc ttgagaaggg ggggtgaaat actggcagca gtgctacaga acagtatttg agttggtagc tttttgtttg gatcctttga acgttaaggg tccttttaaa taaacttggt agcgatctgt agataactac ataccgcgag gccagccgga ccatccagtc gttaatagtt acgctcgtcg ggcgagttac ggtcctccga ggttatggca gcttttctgt atgcggcgac gccacatagc ggcgaaaact cccactcgtg ttctgggtga gggcgacacg tgaagcattt tatttagaaa tgccacctaa tttgttaaat ccttataaat ttggaacaag gaaaaaccgt tcaagttttt agggagcccc gaaaggaagg gtagcggtca gctacagggc ggcgatcggt tgtgctgcaa acgttgtaaa gcgaattggg cgcagggtct cggggtttta ctgcccggca ggagttgccg ccctgaccgt cgtgtgagta agagagctac c gccactggta gttcttgaag gtatctgcgc tcttgatccg caagcagcag tcttttctac attttggtca ttaaaaatga ctgacagtta ctatttcgtt gatacgggag acccacgctc agggccgagc tattaattgt tgcgcaacgt tttggtatgg atgatccccc tcgttgtcag gcactgcata gactggtgag cgagttgctc agaactttaa ctcaaggatc cacccaactg gcaaaaacag gaaatgttga atcagggtta aataaacaaa attgtaagcg cagctcattt caaaagaata agtccactat ctatcagggc tggggtcgag cgatttagag gaagaaagcg cgctgcgcgt gcgtcccatt gcgggcctct ggcgattaag acgacggcca taccgggccc ccattttgaa cgagattgtg tttctgacag ccagattctg ggccgagaag aggccccgga ttccacatgc 2026233990   16 Sep 2026

[0055] In SEQ ID NO:1, residues 1-1561 of pAAV-RCl 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 avaM13 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 (Figure 3). 2. Plasmid pAAV-RC2

[0056] Plasmid pAAV-RC2 (SEQ ID NO:2; Figure 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). 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 2026233990   16 Sep 2026 ggccaaagcc agtcctcggc aacatgtgcg gccgttgcaa atgactttgg gcaaaggatc tggagccaag aacgggtgcg tcgatcaact catgaatctg attcaaatat cccgtgtcag actgtgctac cctgcgatct atgatttaaa aggacactct ccaccaccac tgtgcttcct gagagccggt tacgaccggc cgccgacgcg gcaacctcgg ctgggcctgg ggtagagcac cgggccagca gcagactcag ctctggtctg cagacaataa cattgcgatt aacctgggcc gccaatcagg tgggggtatt ctggcaaaga acttcaagct acgacgacga ctcggagtac tcccgccgtt accctgaaca ggagtacttt gctacacttt agtctggacc gagcagaaca tttctcaggc cctggaccct caacaacagt gcagagactc gatgaagaaa aggctcagag aagaggaaat gtatctacca attctcggag ccagatagac ccgtgattga gaccggatgt gaaggtcacc acgtggttga aaaagacccg cgagtcagtt acgcagacag atgctgtttc ctgcttcact aatctcaacc attcatcata ggtcaatgtg tcaggtatgg ctctgaagga caaagcccgc gggtacaagt caacgaggca agctcgacag gagtttcagg acgagcagtc ttgaggaacc tctcctgtgg gcctgcaaga tacctgaccc ggaactaata cgagggcgcc ccacatggat ctgcccacct agcctcgaac ttgacttcaa ctcatcaaca ctttaacatt ttgccaataa cagctcccgt cccagcagac acgggagtca ccttctcaga tgaggacgtt gtctcatgaa aacactccaa cggagcgagt gttaccgcca gaatactcgt tctggtgaat agttttttcc aaaacaaatg caggacaacc acctccagag gaagcaaggt ccgactcccg cgggaactca tcaaatttga aagcaggaag ggtggagcat cccccagtga gcgcagccat gtaccaaaac cctgcagaca cacggacaga cgtttctgtc tcatgggaaa gatttggatg ctgccgatgg ataagacagt agagcggcat acctcggacc gacgccgcgg cggagacaac agcgccttaa ttccaggcga tgttaagacg agccagactc aaaagattga ccagcctctc cgatggctac gacggagtgg gggcgacaga acaacaacca gacaatcact cagattccac acaactgggg caagtcaaag ccttaccagc acgtcctcgg gtcttcatgg ggcagtagga tgctgcgtac cctttccaca tcctctcatc gtggaaccac gacattcggg gcagcgagta ggactggagc ccgggcccgg tcagagcggg tggacattga aatcccgtgg aggcaacaga gcgcgtggac tgatcgtcac acgaccttcg actcacccgc tcaaagactt gaattctacg cgcagatata cgacgtcaga aaatgttctc atgcgagaga aagactgttt gtcaaaaagg ggtgccagac actgcatctt ttatcttcca ggtggaagct aaggacgaca cttcaacgga ccctcgagca ccgtacctca agaagatacg aaaagagggt gctccgggaa ctcctcggga attttggtca ggacagccac aggcagtggc gtaattcctc gtcatcacca cctctacaaa actttggcta tgccactttt attccgaccc aggtcacgca acggttcagg ctcggcgcat tgccacagta cgctcttcat cggaaacaac gcagctacgc gaccagtacc cacgcagtca accagtctag tcaaagacat taccaagtac ccatggcaag gttctcatct aaaggtcatg ctacggagca caagcagcta cagaaatgca ctccaacacc aacaccagca cgtctggatc tttccggtgg tcaaaaaggg agtgagccca cgcggaagct gtcacgtggg atgaatcaga agagtgcttt cgtatcagaa gcttgcactg tgaacaataa gattggctcg caaacctggc gcaggggtct ctcgacaagg cgacaaagcc agtacaacca tcttttgggg tcttgaacct aaaagaggcc accggaaagg gactggagac cagcagcccc gcaccaatgg gggaaattgg ccagcacccg caaatttcca cagcacccct caccacgtga aagagactca gaatgacggt tgtttactga caaggatgcc tggatacctc tttactgcct tttaccttca tcacagccag tgtattactt aggcttcagt gaactggctt ctgcggataa cacctcaatg ccacaaggac ttgggaagca attacagacg gtatggttct ccgcagatgt 2026233990   16 Sep 2026 caacacacaa accttcaggg cacccctctc gattctcatc gtgcggcaaa agcgtggaga tcccgaaatt ttactgtgga agatacctga attcgtttca tccatgctct ctttgtagtt tctagaggtc acaccatgtg tccattttga cagatatcca tggagctcca gtaatcatgg ttccacacaa taatgagtga ccagtcggga cggggagagg gactcgctgc aaaggcggta acatgtgagc ttgctggcgt tcgacgctca aggcgtttcc ccgcttaccg ttctcatagc ccaagctggg ttatccggta gccactggca gcggtgctac agaacagtat aagagttggt gtttttttgt gaagatcctt ctcacgttaa agatcctttt gagtaaactt ctcagcgatc tgtagataac atgataccgc ccagccagcc cctccatcca ccagttaata gtcacgctcg caaggcgagt ttcggtcctc ggcgttcttc gcccatctgg ccctcatggg aagaacaccc gtttgcttcc tcgagtggga cagtacactt cactaatggc ctcgtaatct gttgaacttt aggatccact aatgattaac ctgtattaga gtcacgctgg agcgggaggt aacactggcg gcttttgttc tcatagctgt catacgagcc gctaactcac aacctgtcgt cggtttgcgt gctcggtcgt atacggttat aaaaggccag ttttccatag agtcagaggt ccctggaagc gatacctgtc tcacgctgta ctgtgtgcac actatcgtct gcagccactg agagttcttg ttggtatctg agctcttgat ttgcaagcag tgatcttttc gggattttgg aaattaaaaa ggtctgacag tgtctatttc tacgatacgg gagacccacg ggaagggccg gtctattaat gtttgcgcaa tcgtttggta tacatgatcc cgatcgttgt caggcatggt gcaaagattc tggattcgga cggtacctgc ttcatcacac gctgcagaag ccaactacaa gtgtattcag gtaattgctt ggtctctgcg agtaacggcc ccgccatgct ggtcacgtga gtatttaagc ttgaacgcgc gccgctcgac cctttagtga ttcctgtgtg ggaagcataa attaattgcg gccagctgca attgggcgct tcggctgcgg ccacagaatc caaaaggcca gctccgcccc ggcgaaaccc tccctcgtgc cgcctttctc ggtatctcag gaaccccccg tgagtccaac gtaacaggat aagtggtggc cgctctgctg ccggcaaaca cagattacgc tacggggtct tcatgagatt tgaagtttta ttaccaatgc gttcatccat gagggcttac ctcaccggct agcgcagaag tgttgccggg cgttgttgcc tggcttcatt cccatgttgt cagaagtaag ctggcaggac cacacacgga cttaaacacc gaatccttcg agtactccac gaaaacagca caagtctgtt agcctcgccc gttaatcaat tatttctttc gccagtgtgc acttatctac gtgttttgcg ccgagtgagc agccgccaag tagagcggcc gggttaattg aaattgttat agtgtaaagc ttgcgctcac ttaatgaatc cttccgcttc cgagcggtat aggggataac ggaaccgtaa cctgacgagc gacaggacta gctctcctgt ccttcgggaa ttcggtgtag ttcagcccga ccggtaagac tagcagagcg ctaactacgg aagccagtta aaccaccgct gcagaaaaaa gacgctcagt atcaaaaagg aatcaatcta ttaatcagtg agttgcctga catctggccc ccagatttat tggtcctgca aagctagagt attgctacag cagctccggt gcaaaaaagc ttggccgcag agagatgtgt cggacatttt ctcctccaca accaccttca gggacaggtc aacgctggaa aatgtggact cattggcacc aaaccgttta ttatctagtt tggaattcgg gtagccatgc acattttgcg acgcagggtc ccgaattctg gccaccgcgg cgcgcttggc ccgctcacaa ctggggtgcc tgcccgcttt ggccaacgcg ctcgctcact cagctcactc gcaggaaaga aaaggccgcg atcacaaaaa taaagatacc tccgaccctg gcgtggcgct gtcgttcgct ccgctgcgcc acgacttatc aggtatgtag ctacactaga ccttcggaaa ggtagcggtg aggatctcaa ggaacgaaaa atcttcacct aagtatatat aggcacctat ctccccgtcg cagtgctgca cagcaataaa actttatccg aagtagttcg gcatcgtggt tcccaacgat ggttagctcc tgttatcact 2026233990   16 Sep 2026 catggttatg gatgcttttc tgtatgcggc cgcgccacat cggggcgaaa taacccactc cgtttctggg taagggcgac tattgaagca atgtatttag aagtgccacc atttttgtta atcccttata agtttggaac ggcgaaaaac taatcaagtt taaagggagc cgagaaagga agtgtagcgg gccgctacag aagggcgatc ggatgtgctg acgacgttgt agggcgaatt gcagcactgc tgtgactggt gaccgagttg agcagaactt actctcaagg gtgcacccaa tgagcaaaaa acggaaatgt tttatcaggg aaaaataaac taaattgtaa aatcagctca aatcaaaaga aagagtccac cgtctatcag ttttggggtc ccccgattta agggaagaaa tcacgctgcg ggcgcgtccc ggtgcgggcc caaggcgatt aaaacgacgg gggta ataattctct gagtactcaa ctcttgcccg taaaagtgct atcttaccgc ctgatcttca caggaaggca tgaatactca ttattgtctc aaataggggt gcgttaatat ttttttaacc atagaccgag tattaaagaa ggcgatggcc gaggtgccgt gagcttgacg gcgaaaggag cgtaaccacc attcgccatt tcttcgctat aagttgggta ccagtgagcg tactgtcatg ccaagtcatt gcgtcaatac catcattgga tgttgagatc gcatctttta aaatgccgca tactcttcct atgagcggat tccgcgcaca tttgttaaaa aataggccga atagggttga cgtggactcc cactacgtga aaagcactaa gggaaagccg cgggcgctag acacccgccg caggctgcgc tacgccagct acgccagggt cgcgtaatac ccatccgtaa ctgagaatag gggataatac aaacgttctt cagttcgatg ctttcaccag aaaaagggaa ttttcaatat acatatttga tttccccgaa ttcgcgttaa aatcggcaaa gtgttgttcc aacgtcaaag accatcaccc atcggaaccc gcgaacgtgg ggcgctggca cgcttaatgc aactgttggg ggcgaaaggg tttcccagtc gactcactat

[0057] 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 Ml3 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 (Figure 4). 3. Plasmid pAAV-RC5

[0058] Plasmid pAAV-RC5 (SEQ ID NO:3; Figure 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 2026233990   16 Sep 2026 pAAV-RC5 are AAV2 serotype promoters (SEQ ID NO: 10 and SEQ ID NO: 18, respectively). 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 2026233990   16 Sep 2026 tacaacaacc caacgccaac ttaaccgctt aacaactact cattcaagtc acaacctcac ccctacgtcg gcaggtcttt acacagaaaa cccagcaaga tgaggaggtg agctggccaa aataacactg caacacctac ggaacctggg accaatagga gaacggcatg acactatgat tacctcgagg gaaccgcgtg gctccaccac cccggcagcg ggccaagatc gcggattcgg cctgtgcccg catcacccag tcaagaagga aactacaacg atacagaacc aaggcgcgcc agttgaactt taggatccac taatgattaa cctgtattag ggtcacgctg aagcgggagg aaacactggc agcttttgtt gtcatagctg acatacgagc agctaactca aaacctgtcg gcggtttgcg cgctcggtcg aatacggtta caaaaggcca tttttccata aagtcagagg cccctggaag ggatacctgt accagtaccg gcctactttg ccacagccac ggggcttcag aaagaggtca ctccaccgtc tcggcaacgg acgctgccgc tcccaccgag tgctgagaac cccttccact cccgctggtg gcggagtcca aaaaactggt ctccggggtc tggagctcga accaacaacc cttcaacagc gcaacatgct gcgtacaacg tgcccccgcg tgtggatgga ccagagacgg actcaaacac gaaatatcac tacagcaccg aaactccaag acccccagtt accagaccta accggttgct tggtctctgc tagtaacggc cccgccatgc aggtcacgtg ggtatttaag tttgaacgcg ggccgctcga ccctttagtg tttcctgtgt cggaagcata cattaattgc tgccagctgc tattgggcgc ttcggctgcg tccacagaat gcaaaaggcc ggctccgccc tggcgaaacc ctccctcgtg ccgcctttct agagatcaaa gatacagcac tggagccccc accccggtcc cggtgcagga caagtgttta gaccgaggga agtacggtta aggagcagct gggcaacaac ccagcttcgc gaccagtact gttcaacaag tcccggggcc aaccgcgcca gggcgcgagt tccagggcag cagccggcga catcaccagc tcggcgggca accggcacgt gagggacgtg gggcgcactt ccaccgccca cagcttctcg ggcaggtcac aggtggaacc tgtggacttt tcggaacccg tgttaatcaa gtatttcttt cgccagtgtg tacttatcta agtgttttgc cccgagtgag cagccgccaa ctagagcggc agggttaatt gaaattgtta aagtgtaaag gttgcgctca attaatgaat tcttccgctt gcgagcggta caggggataa aggaaccgta ccctgacgag cgacaggact cgctctcctg cccttcggga agcggctccg cccctggggg gagactggca ctcagagtca ctccaccacc cggacgacga tgcctgccgg cgcgacgctg tcttctgcct tttgagttta tcccagtcag tgtaccgctt aacctggccg catgggccga gtgtcagcgc taccaggtgc caacacctat acccgggcac gagagegaga gatggccacc acaacctcca tacctccaag tcacccctct tgatgctcat gacgtgcccg cgtggagatg cagagatcca gccccggaca ataccttacc taaaccgttt ettatetagt etggaatteg cgtagccatg gacattttgc cacgcagggt geegaattet cgccaccgcg gegegettgg tccgctcaca cctggggtgc ctgcccgctt cggccaacgc cctcgctcac tcagctcact egeaggaaag aaaaggeege catcacaaaa ataaagatac ttccgaccct agegtggege tegaeggaag tactttgact aagactcatc aaatcttcaa accatcgcca ctaccagctg ccttccctcc aaccgcgaca agagtaettt cctacaactt aacctgttca cgtgagcaca ggagataege acccagggct cttcgccacg ccccgcagcc gccctggaga caccgccacg cgcagccggt aacaaccaga ggaaatcgtg gacccatctg ccggccatgg caagaacacg tcagcagctt gagtgggagc gtacacaaac gcaccgggga cgaccccttt aattcgtttc ttccatgctc gctttgtagt ctctagaggt gacaccatgt ctccattttg gcagatatcc gtggagctcc cgtaatcatg attccacaca ctaatgagtg tccagtcggg gcggggagag tgactcgctg caaaggcggt aacatgtgag gttgctggcg atcgacgctc caggcgtttc gccgcttacc tttctcatag 2026233990   16 Sep 2026 ctcacgctgt gctgtgtgca aactatcgtc agcagccact cagagttctt tttggtatct tagctcttga tttgcaagca ttgatctttt agggattttg taaattaaaa tggtctgaca ctgtctattt ctacgatacg cgagacccac cggaagggcc agtctattaa agtttgcgca gtcgtttggt ttacatgatc ccgatcgttg ggcagcactg ctgtgactgg cgaccgagtt tagcagaact aactctcaag cgtgcaccca gtgagcaaaa cacggaaatg atttatcagg gaaaaataaa ctaaattgta aaatcagctc aaatcaaaag caagagtcca ccgtctatca tttttggggt cccccgattt aagggaagaa gtcacgctgc gggcgcgtcc cggtgcgggc gcaaggcgat taaaacgacg tgggtaccgg gtctccattt tttacgagat ggcatttctg gccgccagat ccgtggccga aggtatctca cgaacccccc ttgagtccaa ggtaacagga gaagtggtgg gcgctctgct tccggcaaac gcagattacg ctacggggtc gtcatgagat atgaagtttt gttaccaatg cgttcatcca ggagggctta gctcaccggc gagcgcagaa ttgttgccgg acgttgttgc atggcttcat ccccatgttg tcagaagtaa cataattctc tgagtactca gctcttgccc ttaaaagtgc gatcttaccg actgatcttc acaggaaggc ttgaatactc gttattgtct caaatagggg agcgttaata attttttaac aatagaccga ctattaaaga gggcgatggc cgaggtgccg agagcttgac agcgaaagga gcgtaaccac cattcgccat ctcttcgcta taagttgggt gccagtgagc gccccccctc tgaagcggga tgtgattaag acagctttgt tctgacatgg gaagctgcag gttcggtgta gttcagcccg cccggtaaga ttagcagagc cctaactacg gaagccagtt aaaccaccgc cgcagaaaaa tgacgctcag tatcaaaaag aaatcaatct cttaatcagt tagttgcctg ccatctggcc tccagattta gtggtcctgc gaagctagag cattgctaca tcagctccgg tgcaaaaaag gttggccgca ttactgtcat accaagtcat ggcgtcaata tcatcattgg ctgttgagat agcatctttt aaaatgccgc atactcttcc catgagcgga ttccgcgcac ttttgttaaa caataggccg gatagggttg acgtggactc ccactacgtg taaagcacta ggggaaagcc gcgggcgcta cacacccgcc tcaggctgcg ttacgccagc aacgccaggg gcgcgtaata gaggtcgacg ggtttgaacg gtccccagcg gaactgggtg atctgaatct cgcgactttc ggtcgttcgc accgctgcgc cacgacttat gaggtatgta gctacactag accttcggaa tggtagcggt aaggatctca tggaacgaaa gatcttcacc aaagtatata gaggcaccta actccccgtc ccagtgctgc tcagcaataa aactttatcc taagtagttc ggcatcgtgg ttcccaacga cggttagctc gtgttatcac gccatccgta tctgagaata cgggataata aaaacgttct ccagttcgat actttcacca aaaaaaggga tttttcaata tacatatttg atttccccga attcgcgtta aaatcggcaa agtgttgttc caacgtcaaa aaccatcacc aatcggaacc ggcgaacgtg gggcgctggc gcgcttaatg caactgttgg tggcgaaagg ttttcccagt cgactcacta gtatcggggg cgcagccgcc accttgacga gccgagaagg gattgagcag tgacggaatg tccaagctgg cttatccggt cgccactggc ggcggtgcta aagaacagta aaagagttgg ggtttttttg agaagatcct actcacgtta tagatccttt tgagtaaact tctcagcgat gtgtagataa aatgataccg accagccagc gcctccatcc gccagttaat tgtcacgctc tcaaggcgag cttcggtcct tcatggttat agatgctttt gtgtatgcgg ccgcgccaca tcggggcgaa gtaacccact gcgtttctgg ataagggcga ttattgaagc aatgtattta aaagtgccac aatttttgtt aatcccttat cagtttggaa gggcgaaaaa ctaatcaagt ctaaagggag gcgagaaagg aagtgtagcg cgccgctaca gaagggcgat gggatgtgct cacgacgttg tagggcgaat agctcgcagg atgccggggt gcatctgccc aatgggagtt gcacccctga gcgccgtgtg 2026233990   16 Sep 2026 agtaaggccc cggaggctct tttctttgtg caatttgaga agggagagag ctacttccac atgcacgtgc tcgtggaaac caccggggtg aaatc

[0059] 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 Ml3 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 (Figure 5). 4. Plasmid pAAV-RC6

[0060] Plasmid pAAV-RC6 (SEQ ID NO:4; Figure 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). Coding Strand of Plasmid pAAV-RC6 (SEQ ID NO:4): catggttttg gaatttaccg aagaccagaa catccccaat ggactaatat aaacggttgg gaacaaagag aaacttcagc attacctcgg cttcaatgcg atgcgggaaa ggccagcagc ggaactaaac gggccacgaa gcaactaccg cttctacggg gtgtcgacaa gtcgtggagt ccagaaatgc cctccaacac gaacaccagc ccgtctggat ggacgtttcc cgggatcgag atggcgccgg tacttgctcc ggaacagtat tggcgcagca aatcagaatc caggtacatg agaagcagtg gcctccaact gattatgagc ccgtggagga gggtacgatc aaagttcggc ggaagaccaa tgcgtaaact gatggtgatc cggccaaagc aagtcctcgg caacatgtgc agccgttgca catgactttg tgagtcagat ccgactttgc aggcgggaac ccaaaaccca ttaagcgcct tctgacgcac ccaattctga gagctggtcg gatccaggag cgcggtccca ctgactaaaa catttccagc cccaatatgc aagaggaaca catcgcggag ggaccaatga tggtgggagg cattctcgga cccagataga gccgtgattg agaccggatg ggaaggtcac tcgcgaaaaa caaactggtt aaggtggtgg gcctgagctc gtttgaatct gtgtcgcaga tgcgccggtg ggtggctcgt gaccaggcct aatcaaggct ccgcccccga aatcggattt ggcttccgtc ccatctggct gccatagccc gaactttccc aggggaagat ggaagcaagg cccgactccc acgggaactc ttcaaatttg caagcaggaa ctgattcaga cgcggtcaca atgagtgcta cagtgggcgt cacggagcgt cgcaggagca atcagatcaa ggacaagggg catacatctc gccttggaca ctacctggtg ataaaatttt tttctgggat gtttgggcct acactgtgcc ttcaacgact gaccgccaag tgcgcgtgga gtgatcgtca aacgaccttc aactcacccg gtcaaagact 2026233990   16 Sep 2026 ttttccggtg gtcaaaaagg aagtgagccc acgcggaagc cgtcacgtgg aatgaatcag tagagtgctt gcgtatcaga cgcttgcact ttgaacaata agattggctc tgaaacctgg ggccggggtc actcgacaag acgacaaggc cggtataacc gtcttttggg ttctcgaacc aagaaacgtc cattggcaag agactggcga ccagcaaccc cgcaccaatg caggaaattg accagcaccc gcaaatctcc gctacagcac ttctcaccac gcccaagaga cgacgaatga caagtcttct gcaccagggc agtacggcta tccttttact taactttacc acgcgcacag tacctgtatt caaggacttg ccaaaaactg acaaaaacag atataacctt cctcacacaa atttttggaa catgatcaca aaagatttgg gcgaccggag agacagagac cggatggaca cacccgcctc tccggcagag ggcaaaggat gtggagccaa aaacgggtgc ttcgatcaac gcatgaatct aattcaaata tcccgtgtca aactgtgcta gcctgcgatc aatgatttaa gaggacaacc agccccgaaa tggtgcttcc ggggagcccg ctacgaccag acgccgacgc ggcaacctcg ttttggtctg cggtagagca acaggccagc ctcagagtca ccgctgctgt gcagacaata gcattgcgat gaacatgggc agtgcttcaa cccctggggg gtgactggca ctcaacttca tggcgtcacg cggactcgga tgcctccctc cctaacgctc gcctggaata ttcagctaca ccagagcctg acctgaacag ctgtttagcc gctacctgga acaacaacaa aatgggcgtg agacgacaaa aggagagcgc gacgaagagg gactgtggca atgtgcatgt gtatacctgc ctttcacccg ctcagatcct ttttcggcta cacgtggttg gaaaagaccc gcgagtcagt tacgcagaca gatgctgttt tctgcttcac gaatctcaac cattcatcat tggtcaatgt atcaggtatg tctctgaggg cccaaagcca tggctacaag tcaacgcggc cagctcaaag cgagtttcag ggcgagcagt gttgaggaag gtcgccacaa agcccgctaa gtccccgacc gggacctact acgaaggcgc tccacatggc cttgcccacc cgggggccag tattttgatt gcgactcatc agctcttcaa accatcgcta gtaccagttg cgttcccggc aacaatggca tttcccatcg ccttcgagga gaccggctga aactcagaat gggggtctcc ccctgttacc cagcaacttt aatctataat gacaagttct cggagcttca aaatcaaagc gtcaatctcc tatgggagcc agggtcctat tctcctctca catcaaaaac caaagtttgc aggtggagca gcccccagtg tgcgcagcca ggtaccaaaa ccctgcagac tcacggacag ccgtttctgt atcatgggaa ggatttggat gctgccgatg cattcgcgag accagcaaaa tacctcggac ggatgcagcg cgggtgacaa gagcgtctgc cttccaggcc gtgctaagac gagccagact aaagagactc cacaacctct acaatggctt cgacggagtg tgggcgacag tataacaacc caacgacaac tcaacagatt aacaacaatt catccaagtc ataaccttac ccgtacgtcc ggacgtgttc gccaggcagt cagatgctga cgtgcctttc tgaatcctct cagtccggaa agctggcatg ggcagcagcg acctggactg caaccctggc ttcccatgag aacactgcat cactaacccc agagcagcag ttacctggaa ttgggccaaa tgggcggctt acgcctgttc ttcattcatc tgaattctac acgcagatat tcgacgtcag caaatgttct aatgcgagag aaagactgtt cgtcaaaaag aggtgccaga gactgcatct gttatcttcc tggtgggact gcaggacgac ccttcaacgg gccctcgagc tccgtacctg aagaagatac aagaagaggg ggctcctgga cctcctcggg aattttggtc cggagaacct caggcggtgg ggtaatgcct agtcatcacc acctctacaa cactacttcg ccactgccat ggggattccg aaggaggtca cagcacggtt tcggctctgc atgattccgc gggacggtca gaacgggcaa cacagcagct catcgaccag gtgcccaaaa tctgttcagc cgtttctaaa gtgcttcaaa actgctatgg cggtgtcatg tggacaatgt gtggccaccg cacagaccct tggtgtggca attcctcaca tggacttaag ctgcgaatcc acccagtatt 2026233990   16 Sep 2026 ccacaggaca agcaaacgct tgccaacgtt gccccattgg gttgcttgtt ctctgcgtat aacggccgcc ccatgctact cacgtgagtg tttaagcccg aacgcgcagc gctcgactag ttagtgaggg ctgtgtgaaa agcataaagt aattgcgttg agctgcatta gggcgctctt gctgcggcga cagaatcagg aaggccagga ccgcccccct gaaacccgac ctcgtgcgct ctttctccct atctcagttc ccccccgttc gtccaacccg acaggattag tggtggccta tctgctgaag gcaaacaaac attacgcgca ggggtctgac tgagattatc agttttaaat ccaatgctta catccatagt ggcttaccat accggctcca gcagaagtgg tgccgggaag tgttgccatt cttcattcag atgttgtgca aagtaagttg attctcttac tactcaacca ttgcccggcg aagtgctcat agtgagcgtg ggaatcccga gatttcactg cacccgttac aatcaataaa ttctttctta agtgtgctgg tatctacgta ttttgcgaca agtgagcacg cgccaagccg agcggccgcc ttaattgcgc ttgttatccg gtaaagcctg cgctcactgc atgaatcggc ccgcttcctc gcggtatcag ggataacgca accgtaaaaa gacgagcatc aggactataa ctcctgttcc tcgggaagcg ggtgtaggtc agcccgaccg gtaagacacg cagagcgagg actacggcta ccagttacct caccgctggt gaaaaaaagg gctcagtgga aaaaaggatc caatctaaag atcagtgagg tgcctgactc ctggccccag gatttatcag tcctgcaact ctagagtaag gctacaggca ctccggttcc aaaaagcggt gccgcagtgt tgtcatgcca agtcattctg tcaatacggg cattggaaaa gagattgaat agtgcagtat tggacaacaa ctcacccgtc ccgtttaatt tctagtttcc aattcggctt gccatgctct ttttgcgaca cagggtctcc aattctgcag accgcggtgg gcttggcgta ctcacaattc gggtgcctaa ccgctttcca caacgcgcgg gctcactgac ctcactcaaa ggaaagaaca ggccgcgttg acaaaaatcg agataccagg gaccctgccg tggcgctttc gttcgctcca ctgcgcctta acttatcgcc tatgtaggcg cactagaaga tcggaaaaag agcggtggtt atctcaagaa acgaaaactc ttcacctaga tatatatgag cacctatctc cccgtcgtgt tgctgcaatg caataaacca ttatccgcct tagttcgcca tcgtggtgtc caacgatcaa tagctccttc tatcactcat tccgtaagat agaatagtgt ataataccgc cgttcttcgg gggagctgca acatctaact tggactttat ccctgtaagg cgtttcagtt atgctctagg tgtagttaat agaggtcctg ccatgtggtc attttgaagc atatccaaac agctccagct atcatggtca cacacaacat tgagtgagct gtcgggaaac ggagaggcgg tcgctgcgct ggcggtaata tgtgagcaaa ctggcgtttt acgctcaagt cgtttccccc cttaccggat tcatagctca agctgggctg tccggtaact actggcagca gtgctacaga acagtatttg agttggtagc tttttgtttg gatcctttga acgttaaggg tccttttaaa taaacttggt agcgatctgt agataactac ataccgcgag gccagccgga ccatccagtc gttaatagtt acgctcgtcg ggcgagttac ggtcctccga ggttatggca gcttttctgt atgcggcgac gccacatagc ggcgaaaact gaaagaaaac atgcaaaatc actgagcctc cgcgccaccg gaactttggt atccactagt gattaacccg tattagaggt acgctgggta gggaggtttg actggcggcc tttgttccct tagctgtttc acgagccgga aactcacatt ctgtcgtgcc tttgcgtatt cggtcgttcg cggttatcca aggccagcaa tccataggct cagaggtggc tggaagctcc acctgtccgc cgctgtaggt tgtgcacgaa atcgtcttga gccactggta gttcttgaag gtatctgcgc tcttgatccg caagcagcag tcttttctac attttggtca ttaaaaatga ctgacagtta ctatttcgtt gatacgggag acccacgctc agggccgagc tattaattgt tgcgcaacgt tttggtatgg atgatccccc tcgttgtcag gcactgcata gactggtgag cgagttgctc agaactttaa ctcaaggatc 2026233990   16 Sep 2026 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

[0061] 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 Ml3 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 (Figure 6). 5. Plasmid pAAV-RC7

[0062] Plasmid pAAV-RC7 (SEQ ID NO:5; Figure 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 2026233990   16 Sep 2026 pAAV-RC7 are AAV2 serotype promoters (SEQ ID NO: 10 and SEQ ID NO: 18, respectively). 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 2026233990   16 Sep 2026 accaccagca caagcaaatc tcggctacag cacttctcac ccggcccaag tcacgacgaa attcaggtat tgcgcaccag ctcagtacgg tcctccttct caacaacttt gctacgcaca cagtacttgt tggcaatcgg aacaagccaa tccaaaacgc caccaaatat ccatggcaac gtcctgattt tgtgttaatg cggaagaata gcccagacac gcagaaccgg acacggatgg aaacatccgc tcctccggag acagcaccgg aacagcaagc gcagactggt ctcgccctat ccggttgctt ggtctctgcg agtaacggcc ccgccatgct ggtcacgtga gtatttaagc ttgaacgcgc gccgctcgac cctttagtga ttcctgtgtg ggaagcataa attaattgcg gccagctgca attgggcgct tcggctgcgg ccacagaatc caaaaggcca gctccgcccc ggcgaaaccc tccctcgtgc cccgaacctg tccagtgaaa caccccctgg cacgtgactg aagctgcggt tgacggcgtt tctcggactc ggctgcctgc ctacctgact actgcctgga gagttcagct cagccagagc actacctggc gaactgcagt gaattggtta tggatcaaaa cacctgaacg tcacaaggac ttggaaaaac acaaatgaag cgggatagtc aagttgtcaa gacgtgtacc caactttcac ctcctcagat gtgtttactc acaagtcagc gctggaaccc gtggactttg tggcactcgt gttaatcaat tatttctttc gccagtgtgc acttatctac gtgttttgcg ccgagtgagc agccgccaag tagagcggcc gggttaattg aaattgttat agtgtaaagc ttgcgctcac ttaatgaatc cttccgcttc cgagcggtat aggggataac ggaaccgtaa cctgacgagc gacaggacta gctctcctgt ggccctgccc ctgcaggtag gggtattttg gcagcgactc tcaagctctt acgaccatcg ggaataccag ctccgttccc ctcaacaatg gtacttcccc acagcttcga ctggaccggc cagaacacag tttaccaggg cctggacctt caacaacagc gcagaaactc gacgaggacc tggagcaact aagaaattcg agcagcaact caaccaggga tgcagggtcc ccgtctcctt cctgatcaag ctgccaagtt gtggaaatcg ggagattcag ccgttgacag tacctcaccc aaaccgttta ttatctagtt tggaattcgg gtagccatgc acattttgcg acgcagggtc ccgaattctg gccaccgcgg cgcgcttggc ccgctcacaa ctggggtgcc tgcccgcttt ggccaacgcg ctcgctcact cagctcactc gcaggaaaga aaaggccgcg atcacaaaaa taaagatacc tccgaccctg acctacaaca taccaacgac actttaacag atcaacaaca caacatccag ctaataacct ctgccgtacg ggcggacgtc gcagtcagtc tctcagatgc ggacgtgcct tgatgaatcc agtaacccag cgggccttca gcttccggca aactttgctt gttggttaat gctttttccc aacaaaacta tcctactaat tacaagcggc gccttacctg catctgggcc tgatgggcgg aacactcccg tgcttcgttc agtgggagct tacacctcca ccagggtgtt gtaatctgta attcgtttca tccatgctct ctttgtagtt tctagaggtc acaccatgtg tccattttga cagatatcca tggagctcca gtaatcatgg ttccacacaa taatgagtga ccagtcggga cggggagagg gactcgctgc aaaggcggta acatgtgagc ttgctggcgt tcgacgctca aggcgtttcc ccgcttaccg accacctcta aacacctact attccactgc actggggatt gtcaaggagg taccagcacg tcctcggctc ttcatgattc tgtgggacgt tgagaacggg ttccacagca cctcatcgac gaggcacagc actatggccg acaaagagtc ggactggtgc cccggcgtcg atccagcgga cattggaaaa cctgtagcca taatactgca gcatggtctg aagattcctc ctttggactt ttcccgctaa atcacacagt gcagaaggaa actttgaaaa tactctgagc aggcgcgcca gttgaacttt aggatccact aatgattaac ctgtattaga gtcacgctgg agcgggaggt aacactggcg gcttttgttc tcatagctgt catacgagcc gctaactcac aacctgtcgt cggtttgcgt gctcggtcgt atacggttat aaaaggccag ttttccatag agtcagaggt ccctggaagc gatacctgtc 2026233990   16 Sep 2026 cgcctttctc ggtatctcag gaaccccccg tgagtccaac gtaacaggat aagtggtggc cgctctgctg ccggcaaaca cagattacgc tacggggtct tcatgagatt tgaagtttta ttaccaatgc gttcatccat gagggcttac ctcaccggct agcgcagaag tgttgccggg cgttgttgcc tggcttcatt cccatgttgt cagaagtaag ataattctct gagtactcaa ctcttgcccg taaaagtgct atcttaccgc ctgatcttca caggaaggca tgaatactca ttattgtctc aaataggggt gcgttaatat ttttttaacc atagaccgag tattaaagaa ggcgatggcc gaggtgccgt gagcttgacg gcgaaaggag cgtaaccacc attcgccatt tcttcgctat aagttgggta ccagtgagcg ccccccctcg gaagcgggag gtgattaagg cagctttgtg ctgacatgga ccttcgggaa ttcggtgtag ttcagcccga ccggtaagac tagcagagcg ctaactacgg aagccagtta aaccaccgct gcagaaaaaa gacgctcagt atcaaaaagg aatcaatcta ttaatcagtg agttgcctga catctggccc ccagatttat tggtcctgca aagctagagt attgctacag cagctccggt gcaaaaaagc ttggccgcag tactgtcatg ccaagtcatt gcgtcaatac catcattgga tgttgagatc gcatctttta aaatgccgca tactcttcct atgagcggat tccgcgcaca tttgttaaaa aataggccga atagggttga cgtggactcc cactacgtga aaagcactaa gggaaagccg cgggcgctag acacccgccg caggctgcgc tacgccagct acgccagggt cgcgtaatac aggtcgacgg gtttgaacgc tccccagcga aactgggtgg tctgaatctg gcgtggcgct gtcgttcgct ccgctgcgcc acgacttatc aggtatgtag ctacactaga ccttcggaaa ggtagcggtg aggatctcaa ggaacgaaaa atcttcacct aagtatatat aggcacctat ctccccgtcg cagtgctgca cagcaataaa actttatccg aagtagttcg gcatcgtggt tcccaacgat ggttagctcc tgttatcact ccatccgtaa ctgagaatag gggataatac aaacgttctt cagttcgatg ctttcaccag aaaaagggaa ttttcaatat acatatttga tttccccgaa ttcgcgttaa aatcggcaaa gtgttgttcc aacgtcaaag accatcaccc atcggaaccc gcgaacgtgg ggcgctggca cgcttaatgc aactgttggg ggcgaaaggg tttcccagtc gactcactat tatcggggga gcagccgcca ccttgacgag ccgagaagga attgagcagg ttctcatagc ccaagctggg ttatccggta gccactggca gcggtgctac agaacagtat aagagttggt gtttttttgt gaagatcctt ctcacgttaa agatcctttt gagtaaactt ctcagcgatc tgtagataac atgataccgc ccagccagcc cctccatcca ccagttaata gtcacgctcg caaggcgagt ttcggtcctc catggttatg gatgcttttc tgtatgcggc cgcgccacat cggggcgaaa taacccactc cgtttctggg taagggcgac tattgaagca atgtatttag aagtgccacc atttttgtta atcccttata agtttggaac ggcgaaaaac taatcaagtt taaagggagc cgagaaagga agtgtagcgg gccgctacag aagggcgatc ggatgtgctg acgacgttgt agggcgaatt gctcgcaggg tgccggggtt catctgcccg atgggagttg cacccctgac tcacgctgta ctgtgtgcac actatcgtct gcagccactg agagttcttg ttggtatctg agctcttgat ttgcaagcag tgatcttttc gggattttgg aaattaaaaa ggtctgacag tgtctatttc tacgatacgg gagacccacg ggaagggccg gtctattaat gtttgcgcaa tcgtttggta tacatgatcc cgatcgttgt gcagcactgc tgtgactggt gaccgagttg agcagaactt actctcaagg gtgcacccaa tgagcaaaaa acggaaatgt tttatcaggg aaaaataaac taaattgtaa aatcagctca aatcaaaaga aagagtccac cgtctatcag ttttggggtc ccccgattta agggaagaaa tcacgctgcg ggcgcgtccc ggtgcgggcc caaggcgatt aaaacgacgg gggtaccggg tctccatttt ttacgagatt gcatttctga ccgccagatt cgtggccgag 2026233990   16 Sep 2026 aagctgcagc gcgactttct gacggaatgg cgccgtgtga gtaaggcccc ggaggctctt ttctttgtgc aatttgagaa gggagagagc tacttccaca tgcacgtgct cgtggaaacc accggggtga aatc

[0063] 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 ate. Ml3 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 (Figure 7). B.    Illustrative Non-AAV Helper Function-Providing Polynucleotides

[0064] As used herein, the term “non-AAV helper functions” denotes proteins of Ad, CMV, HSV or other non-AAD viruses (e.g., Ela, Elb, 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 Virusf 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 f Brain Res. Bull. 81(2-3):273-278).

[0065] Plasmid pHelper-Kan (SEQ ID NO:6; Figure 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. Coding Strand of Plasmid pHelper-Kan (SEQ ID NO:6): ggtacccaac tccatgctta acagtcccca ggtacagccc accctgcgtc gcaaccagga acagctctac agcttcctgg agcgccactc gccctacttc 2026233990   16 Sep 2026 cgcagccaca aaacatgtaa ttttatttgt gcgccgttta ggcagggaca cacaaccatc ccatcaccaa ttggggcctc ctggaacact cggagatcag gtcaactttg gttgcactcg cgttaggata tgagcctttg ctgattggcc tggagatctg ttgctagact catttcaatc taagctcgcc gtgggctcgt ctgcaggaat tcagctgcaa gccagagctt gttatccacg tctcccacgc tcactttccg ccgcgccact ccttgccgtg agcgccacat tggcgggcgc caatggccaa ggcaccagcg ccgcctcagc acggggacga ccgcgctcgg cttctcctat gcctaaccgc aacgcgccta agtgattatc gctcagtacc aacgaggaac tgtgggagac tctgcgacgc gtcagccttg acgccaagaa ccgtatttgc aactgcaaga gcagctggcc acgaagtgcc gcaaacgctc gtgcgcagat aaataatgta acactctcgg aaaatcaaag cgttgcgata cgcggcagct cgcgtttagc cgccctgcgc atcagcgccg atccgcgtcc gtagctgcct caccgtagtg cagcgcctgc cgccttcaga ggacaggccg caccacattt gctccttcag acgtgctcct ttcgatctca ggtgcttgta cgccccatca cccgcggtgc ccacttggtc tggtacttgt agacacgatc cttcactgga gggtcgtctt cttgattagc cttctctttc tcgggcttgg atccgccgtc catcttgtga cgcttttttg cacgtcctcc gggtggtttc aggcagaaaa cccctttgag ccaccttccc gagcaggacc aacagaggat aagtcgggcg gacgtgctgt gttgcaagag cctacgaacg aacggcacat cgtgccagag tacccctatc ttgcggcagg aaaaatcttt tgcaacaaga taggagcgcc ctaggagaca gtgattattt gggttctgcc ctggtgttta cggtgaagtt aggtcgggcg gcgcgagttg ggtggtgcac aggtcctccg tcccaaaaag gcatcagaag atgaaagcct gaagaacatg cgtcatgcac cggccccacc cgcgcgctgc tatttatcat gcgcagcggt ggttacctct tcgtcacaaa tcctcgttta aggcagtagc ccatcaacgc ggcaggctca ctcttccttt cattcagccg accggtgggt ttcctcgctg gagaggggcg gaggtcgatg cgagtcttct ggggcgcgcg atggttggtg gcgctgctcc agatcatgga ttcgccacca cgtcgaggca caggttttgt aaaaagcaag gggggaccaa tgaagcatct cgcagcgatg ccacctgttc gcgagcccaa gtgcttgcca ctgccgtgcc gcgctgtcat gagggtcttg aaacagcgaa acttcttttt ctttcaataa accccccacc gcgcatcgct gtgctccact ttcactccac ccgatatctt cgatacacag gctggccagc cgttgctcag ggtgcatgcc gtgaccgtgc tgatctgctt ccgcaagact gcagcacctt ggttcttcac ccgttttcgc aatgctcccg gcagccacaa gcaaacgact ggtcttgttg gccaggtctt ttgaagtttg gcgcgcagcc gcgggtttat tcctcttgcg ccgcaccgtg tgctgaaacc tccacgatca cttctttttc gccgcgggct tcgtcctcgg gggaggcggc gacgtcgcgc tcttcccgac gtcagtcgag ccgcctccac cccccgcttg aagcgaagac accaggacga aggcatggcg gcagcgccag tgcccctcgc tcaccgcgcg cccgcgcctc cctatcacat aaccgcagcc acctgatatc gacgcgacga aatgaaagtc gtcacttgaa aggcaaatgt cttgccgtct atgcgccact taaactcagg aggctgcgca gaagtcgcag ggttgcagca acgctcttgt ggcgaacgga caggctttga ccggtctggg aaaagccacc tgccggaaaa gcgtcggtgt gatcttggcc tcgtcacatc tgtagacact cgcgcagccc gcaggtacgc ctggtgaagg gcatacggcc cctttagatc tccatgccct caccgtgctt tccgcatacc cgcttacctc caccatttgt cctctgggga tttttggacg gggtgtgcgc actcgagacg ggcgacggcg cgcaccgcgt tggccatttc aaggaggaca cgatgccgcc aggaggagga gacgaggatc cgcagaggca actacctaga tgcgccatta catagcggat taccccccaa aacttctacc ctttttccaa gagcggacaa gcctcgctcg gaaacgcgcg actgtggagt 2026233990   16 Sep 2026 gctggtggaa gcatcgaggt gttatgagca ggagagggat ttggcgatga ttggaggagc gcttgagtgc tagaggaaac tgcaaaattt tttgcacgaa gcgaggcgcg tacacctggc caacctaaag ggacggcctt ttccccgaac cagtcaaagc gaattctgcc aagtaccgtg gctagccaac gtgacggcct cgctccctgg tacctttgag ggttgaaact gtacctgagg ccgcccgcca tccttggcca cgaaagggac cccaatcccc cccaggatgg ggacgaggag aggaggagat gaagaggtgt ggcgccccag ctcaggcgcc accactggaa agagcaacaa ccatagttgc cgctttcttc ttactaccgt gcagcaacag tctgacaaag cgctgcgtct aggatttttc acaagagctg gcctgtatca gaggctctct cgccctttct acccggcgcc acgccctaca tgcccaagac cttgagggtg cacccacttt cagtcatgag gcaaacttgc gcagctggcg gacgcaagct atgcagcggt gttgcactac ccaacgtgga aaccgcctcg ccgcgactac aaacggccat gagctgcaga caacgagcgc gcctgcttaa atgttgcaaa cgccacctgc aatgccctcc taccttgcct actggagtgt tctgcaattc ctgcagggtc cactccgggg actaccacgc aatgcggagc attgcaagcc ggggggttta ccgccgccgc cacccaaaaa gaatactggg gatggaagac cagacgaaac aaattggcaa gccggcactg ccagggccgg cagcgccaag ttgcttgcaa tctaccatca catctctaca cagcggtcac cccaagaaat ggcgcccaac ccactctgta aaaataaaaa caaaagcgaa tcagcaaata caaatttaag agcacctgtc tgtggagtta tactcaaccc acaacgcgcg gcctacccgg cgagctgatc aagaacaaac cgctggcttg aatgatggcc tctttgctga acctttcgcc gctctgcaac ggcaaaacgt gtccgcgact gggcgtgtgg agctgctaaa tccgtggccg aaccctgcaa actttaggaa tgtgcgcttc gccgctttgg accactccga cactgtcgct gcaactgctt cctcgcctga ctgtggacgt ccacgagatt ttaccgcctg atcaacaaag cctggacccc agccctatca gaagctgcag acagtcaggc tgggacagcc accgtcaccc ccgttcccag cctgttcgcc taagtctaag gctaccgctc gactgtgggg cggcgtggcc gcccctactg acagaagcaa ccacagcggc gaacccgtat tgctatattt acaggtctct gatcagcttc ctgcgcgctg cgcgaaaact gtcagcgcca ccagccacaa gaataaacta cctagccgtg cacttaacct gtgcgccgtg cgaggagggc agacgcgcga gcagtgcttg cccggagatg agggctacgt ctggtctcct gcttcattcc gcgtttactt cagcaatgcc gcaaaacttg cgcacctggc cagggtctgc ctttatccta ctagcgactt ggtcactgct catcatggaa gcaacctatg agcgaaagtc cgaaaagtcc cggcttacct aggttctacg cgtcattacc cccgccaaga cagtccggcg gcagccgcgg ctgccgccgc agaggaggtt tagacgaagc tcggtcgcat catcgctaca gacccaaccg cagccgccgc gtggcgcggg gcaacatctc ttcccccgta caccggcggc aggcgaccgg ggcagcagca cgacccgcga caacaaagca gcgctccctc ggcgcacgct actcttaagg acgtcatctc ttatgagcaa atgggacttg catgagcgcg ctgaaacgca accccccaag cacgacccct ctacccgcag gcctgccgac ttaccgtgga cagcgcaagc gcgccaggcc accttggaat acgctcaagg atttctgtgc tggaggagcg aaggacctat ggacattatc cagacttcac gagcgttcag tgtgcccatt accttctgca gacgtgagcg caccccgcac aaattatcgg gcggctccgg tcgcaaattt aagaccaatc cagggccaca gtttctgcta aggagctcaa gcccttgctt cgccacccac ttggacgagg ttccgaggcc tcccctcgcc acctccgctc tagatgggac cgttagccca cacaagaacg cttcgcccgc acatcctgca agcggcagcg atagcaagac ggaggaggag gcttagaaat ggggccaaga acccgcagct ggaagacgcg actagtttcg cagcggccac ggaaattccc cggctggagc ggaccccaca 2026233990   16 Sep 2026 tgatatcccg gaacaggcgg ttggcccgct tacttcccag cagcttgcgg ggcggagtaa gtgacgtatc ttttggcttt ttttgtggac tctgtacttg gagtggtgtt ctgccgctgt ttgcctaggc taattttgtt gggtatgtat gttaaccaac gaccgaggaa acggtcacgc tttcctgttg attttatttt atggtgtctt tgagcatgac ttttgagcag ataggggcta tgtgggttct gtgcagacct gatcgcggta tgaggaacct ggagggcgct atttgcttag atggttgaag tagcctttac ttgtgcaaca gaccacgcca ggttttggat ctcttcccgc ctgggtgtgg tgaaggagtt gagagtggat cggagacgca atatgactac tctcggttgt tgagacagag aatgtaacac tgcagtgtgg ggttctgacg gcctgtgttg tacgagtcct gtgcatagcc atggcgccat ggtcaacgga ctattaccac gccctggtgt agacgcccag gcggctttcg cttgcatgta gtgggaaaac cgtttctggg tttaaccgtt gcccttttta ttttaatagg ggaagcgctg aggagggttt atacctccta tcccccgggc ctgatgtgtt ctgtcggtgg cggcatggcc taagacaggc gtgtttaatg tttctgtggt tacgatgtgc caccttgcat cgctggttag tttgtcatgg gcacgattat tttttgttaa gaatttttgc ctggagcaga agacatattg gtgctggaat gtccacttgg tcttacaaat ccggagggga aatcttttgg tcctcccgtg cttattctgc tacatagaac atactacaac gatctgtttg gtccggcgtt ctcggcgcac acccgcgcta tttgacaatg gatttacgct cgggaggagc tgccaacatt gggctctcca ggcgggcagg gtttaatcag atccgcgccc cacacctcgt accaggaaag gccgaagttc tcacagggtg ttgggaattg ggaagtgaag cgtaggttcg acgtcatttt cactgtgact tttttttact tatgttgttc ttcaggtgtt tgggggctgt tatttcggtc taccgagtct tgctttttaa gtagtccgtc ttctaatgtt caggaacccg ggttccggaa ttgctttttt tttatatcgc catagctccg ttcctggcgg gttcagctgg tgttccgctt aatcatgatt tttttacaat ataaggtggc gtttatagag acgtgagggc gccattatct gcgcgttcac aataaaaaaa tgtgactcgc ggtggtggat ccgaagccag tactacacag tcacgcccgc ccatttggca tccgtacagt ccatactgga cacaacgtga gattcaggaa ttgtaatcct gatatcatga ctgtcattgt ttttggccag aggtttatat accgaaaccg aataacctta tcccgctccc agatgactaa cggtcgcccg tagttttttt atttgaggaa cgtgcggttt ttagtcctat gattgagctg ggtaaggctg tggagcggga tatgtgtttt aatgttgtct gctttttagc tacattatga tcacggtgac ttatgcttat taaatgtttt cagacatgtt cttacctgcc gcgcgaggct cgcccatgca agtatgcgtg ggaagtggcc ccctgcgaag ttgaatctta cgctgcttga ggccggactt gagatgaaaa gagattcacc agtttgcctt gttctttggc ttaatagatc aaaaaacatg agaacgaatg gttatcaggg ggggcgcctg agcgagctaa acctggtttt tgacactacg agggatcgcc ggatcatccg gttacgtgcg tgggttgttc gaggaagtgt cgagcatgat tccagtcccg ctggtttagg ggtaccggga aattctcctc atccccgtag accactgtgg ctcaggggcg ggcgttttag aaaatgggaa gttgtgggtt tctgggtgtt atatactcgc gtgccgtgtc actgttatgg gggtgctatt tctctcctat ctacgcctgc actgaccgat ctccggacat cagttttttt aagggttgtt tttttttgtt tgagagaaaa tttatctgca ttgcctgatt acaagcttac tcataatcag gcgctggtcc ggacctacgg tacaggtctg ggctgaaggt aatattcggg ttatttgggc ctgaagggtt ttggaagcca tgtagagttt ttcattttga gttcttccag tgtaggttgg cagcggcgca gatgctttga gcgacgagac gcttcaggaa accaacacga tacctccttt ctgctgcccg aggtcttccc cctgggatat atgcacgtgt gatccatggt gttccctgca atggtggtgg ggtggtgaat 2026233990   16 Sep 2026 tacaacatgc tcgccactta tggtccccgc aacaatattg cagggtgcgc tgcgaatcat gagcggcggc tatcctgatg cttcgccgcc cagctggaca tatcactgat ctaagaatat ggagaaagga tactagggtt gtggtggtgg aattgaaaaa tttattcctg tcagtggtgt agtgcttacc ttgcccctcc cgctgctctt caccggatgc aggaatttgc ccgcagtccg cttggaaaat ctttccagcc agcgcagcag ttgtctgacg atcacggcgg atacccttgc gctctccttt gccagagcgt aaccgcgtcc ctggatgtcc tcagctcact cgcaggaaag aaaaggccgc catcacaaaa ataaagatac ttccgaccct agcgtggcgc ggtcgttcgc accgctgcgc cacgacttat gaggtatgta gctacactag accttcggaa tggtagcggt aaggatctca tggaacgaaa caaaagaggt atctacctgc catgagcttt tggtgctgtg tgctgtgccc cgctgaggag ggcagcagtt cacgattatg cgttgagcaa gcgacatgaa gagcgtttgg gtctgttacc ctgtgtactc ctgtgagttt ggctatacta taaacacgtt ggcaatgtag attttccact tcgctagttt tgacgcggta gctcttgccg atctgggaaa aagcggggtc gccggcccga aaccctccgg taaccgctta ccgccgcgcc tcgcacacct acggccggat gaatttatcc tgcacggtct cccgaccatg gcgactttcc aggtacatct caaaggcggt aacatgtgag gttgctggcg atcgacgctc caggcgtttc gccgcttacc tttctcatag tccaagctgg cttatccggt cgccactggc ggcggtgcta aagaacagta aaagagttgg ggtttttttg agaagatcct actcacgtta aatgtttatg gcttgtggta ggatacagcg ctgcagttac ggaggacaag accactgcca tattcgcgcg actctacccc ccgcaagttg cttaagcgag ctcgacagga catgatatga tgtgtgttgg gattaaggta ctgaatgaaa gaaacataac gagaaggtgt ttcccaggac ctgtggattc ggagaagggg ctgctgagga agcaaaaaag ttgcatgacg gactcgaacc ctacagggag cgccgcgcgc tggaaggaag gggttcgaca ccggggttcg accagaccac agagcgtcaa gagcactttt gcgcgcctcc acggattacg aatacggtta caaaaggcca tttttccata aagtcagagg cccctggaag ggatacctgt ctcacgctgt gctgtgtgca aactatcgtc agcagccact cagagttctt tttggtatct tagctcttga tttgcaagca ttgatctttt agggattttg tccagcgtgt tgatggccac ccttgcactg tgtgctgatt gcgtctcatg tgttgtattc ctgctgcagc catgtaggcg gacagcagcc ctgcccgggg aaccgtgtgg tgctttttaa gagggaggtg cggtgatcaa aatgacttga atgcaacagg aagagttggt catgtaaaag actagaatcg agggtgccct ggggggcgca gggctcgtcc gggaggcaaa gggggtcctg cgagccactt ggccagtggc ccaaaaggag cgcgggcggt aaccccggtc ggaagagtgc cgactgcgca tgccgctgcg accaccgccg tcgacgttta tccacagaat gcaaaaggcc ggctccgccc tggcgaaacc ctccctcgtg ccgcctttct aggtatctca cgaacccccc ttgagtccaa ggtaacagga gaagtggtgg gcgctctgct tccggcaaac gcagattacg ctacggggtc gtcatgagat ttatgagggg gtgggttctg tgggattttg taagtgagat ctgcgggcgg ctgcaggacg accaccgccc tggacttccc tgtggctcag agtttattaa aatataacac ggccagccgg gcaggttgaa tataagctat aattttctgc ttcacgattc agcaaaagtt acatagagta atgtaggatg gcatgtctgc tctgccgcag ctgtttccgg cccccgttcg cgactcaacc aatgctttcg caaaaaagct cgctcccccg aaccgcatgg gtccgccatg ccgcttacag cgcctcaccg caacatctgg ccggcatcac aaccatatga caggggataa aggaaccgta ccctgacgag cgacaggact cgctctcctg cccttcggga gttcggtgta gttcagcccg cccggtaaga ttagcagagc cctaactacg gaagccagtt aaaccaccgc cgcagaaaaa tgacgctcag tatcaaaaag 2026233990   16 Sep 2026 gatcttcacc aaagtatata aggcgataga cacgaggaag gggtagccaa cagtcgatga gcaggcatcg ccttgagcct tccagatcat ctcgatgcga gcgtatgcag ggagcaaggt cagccagtcc gaacgcccgt tcattcaggg ctgcgctgac gtgcccagtc gcgtgcaatc aagcatttat tttagaaaaa ccacctaaat tgttaaatca ttataaatca ggaacaagag aaaaccgtct aagttttttg ggagcccccg aaggaaggga agcggtcacg tacagggcgc tagatccttt tgagtaaact aggcgatgcg cggtcagccc cgctatgtcc atccagaaaa ccatgggtca ggcgaacagt cctgatcgac tgtttcgctt ccgccgcatt gagatgacag cttcccgctt cgtggccagc caccggacag agccggaaca atagccgaat catcttgttc cagggttatt taaacaaata tgtaagcgtt gctcattttt aaagaataga tccactatta atcagggcga gggtcgaggt atttagagct agaaagcgaa ctgcgcgtaa gatggatcc taaattaaaa tggtctgaca ctgcgaatcg attcgccgcc tgatagcggt gcggccattt cgacgagatc tcggctggcg aagaccggct ggtggtcgaa gcatcagcca gagatcctgc cagtgacaac cacgatagcc gtcggtcttg cggcggcatc agcctctcca aatcatactc gtctcatgag ggggttccgc aatattttgt taaccaatag ccgagatagg aagaacgtgg tggcccacta gccgtaaagc tgacggggaa aggagcgggc ccaccacacc atgaagtttt gtcagaagaa ggagcggcga aagctcttca ccgccacacc tccaccatga ctcgccgtcg cgagcccctg tccatccgag tgggcaggta tgatggatac cccggcactt gtcgagtaca gcgctgcctc acaaaaagaa agagcagccg cccaagcggc ttcctttttc cggatacata gcacatttcc taaaattcgc gccgaaatcg gttgagtgtt actccaacgt cgtgaaccat actaaatcgg agccggcgaa gctagggcgc cgccgcgctt aaatcaatct ctcgtcaaga taccgtaaag gcaatatcac cagccggcca tattcggcaa ggcatgctcg atgctcttcg tacgtgctcg gccggatcaa tttctcggca cgcccaatag gctgcgcaag gtcttgcagt ccgggcgccc attgtctgtt cggagaacct aatattattg tttgaatgta ccgaaaagtg gttaaatttt gcaaaatccc gttccagttt caaagggcga caccctaatc aaccctaaag cgtggcgaga tggcaagtgt aatgcgccgc

[0066] In SEQ ID NO:6, residues 1-5343 of pHelper-Kan are derived from adenovirus, and include a polynucleotide encoding the E2A protein (residues 2581847); 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 fl ori sequences (Figure 8). C. Illustrative rAAV Plasmid Vectors

[0067] 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, 2026233990   16 Sep 2026 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; Figure 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 O / 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 6xHis-tag sites for facilitating recovery or localization of expressed proteins, an SV40 poly(A) site and a 3 ’ ITR. Coding Strand cctgcaggca ggcgaccttt gtggccaact taatagtaat gagggcctat tgttagagag tacaaaatac ttaaaattat gtatttcgat actggaccag taatactgta atggagttcc gcccaacgac taacgccaat taaactgccc ccctattgac acatgacctt atcgctatta atagcggttt atgggagttt acaactccgc gtctatataa cggtaccgag cttaactagc of Plasmid pA gctgcgcgct ggtcgcccgg ccatcactag cgaattcgtg ttcccatgat ataattagaa gtgacgtaga gttttaaaat ttcttgggtt gcagcagcgt atagtaatca gcgttacata ccccgcccat agggactttc acttggcagt gtcaatgacg atgggacttt ccatggtgat gactcacggg gttttgcacc cccattgacg gcagagctgg gagatctgcc tagcggaccg -CMV-EGFP cgctcgctca ccctccagtg gggttcctgc ttactcataa tccttcatat ttaatttgac aagtaataat ggactatcat tatatatctt cagaagactt attacggggt acttacggta tgacgtcaat cattgacgtc acatcaagtg gtaaatggcc cctacttggc gcggttttgg gatttccaag aaaatcaacg caaatgggcg tttagtgaac gccgcgatcg acgcgtacgc SEQ ID NO:7) ctgaggccgc agcgagcgcg ggccgcacgc ctagtaaggt ttgcatatac tgtaaacaca ttcttgggta atgcttaccg gtggaaagga ttttggaaaa cattagttca aatggcccgc aatgacgtat aatgggtgga tatcatatgc cgcctggcat agtacatcta cagtacatca tctccacccc ggactttcca gtaggcgtgt cgtcagatcc ccggcgcgcc ggccgctcga ccgggcgtcg cagagaggga gtctagttat cgggcaggaa gatacaaggc aagatattag gtttgcagtt taacttgaaa cgcgggatcc gcttgactag tagcccatat ctggctgacc gttcccatag gtatttacgg caagtacgcc tatgcccagt cgtattagtc atgggcgtgg attgacgtca aaatgtcgta acggtgggag gctagagatc agatctcacg gatggtgagc 2026233990   16 Sep 2026 aagggcgagg cggcgacgta atgccaccta ctgcccgtgc gtgcttcagc ccgccatgcc gacggcaact ggtgaaccgc tcctggggca atggccgaca caacatcgag cccccatcgg acccagtccg cctgctggag tgtacaagta caccaccacc taagatatcc cacatttgta tgaacctgaa gcagcttata taaagcattt atgtatctta ctagtgatgg ggccgggcga cagtgagcga tttctcctta ccatagtacg tacgcgcagc tcgctttctt gctctaaatc cctcgacccc cgccctgata aatagtggac ctattctttt aaaatgagct acgtttacaa atagttaagc gggcttgtct gggagctgca acgaaagggc taatggtttc acccctattt gagacaataa tgagtattca tgccttcctg tgaagatcag gcggtaagat agcactttta cgggcaagag ttgagtactc agctgttcac aacggccaca cggcaagctg cctggcccac cgctaccccg cgaaggctac acaagacccg atcgagctga caagctggag agcagaagaa gacggcagcg cgacggcccc ccctgagcaa ttcgtgaccg agtcgaggat accaccacta gatccaccgg gaggttttac acataaaatg atggttacaa ttttcactgc acgcggtaac agttggccac ccaaaggtcg gcgagcgcgc cgcatctgtg cgccctgtag gtgaccgcta cccttccttt gggggctccc aaaaaacttg gacggttttt tcttgttcca gatttataag gatttaacaa ttttatggtg cagccccgac gctcccggca tgtgtcagag ctcgtgatac ttagacgtca gtttattttt ccctgataaa acatttccgt tttttgctca ttgggtgcac ccttgagagt aagttctgct caactcggtc accagtcaca cggggtggtg agttcagcgt accctgaagt cctcgtgacc accacatgaa gtccaggagc cgccgaggtg agggcatcga tacaactaca cggcatcaag tgcagctcgc gtgctgctgc agaccccaac ccgccgggat tataaggatg ataaggttta atctagataa ttgctttaaa aatgcaattg ataaagcaat attctagttg cacgtgcgga tccctctctg cccgacgccc agctgcctgc cggtatttca cggcgcatta cacctgccag ctcgccacgt tttagggttc atttgggtga cgccctttga aactggaaca ggattttgcc aaatttaacg cactctcagt acccgccaac tccgcttaca gttttcaccg gcctattttt ggtggcactt ctaaatacat tgcttcaata gtcgccctta cccagaaacg gagtgggtta tttcgccccg atgtggcgcg gccgcataca gaaaagcatc cccatcctgg gtccggcgag tcatctgcac accctgacct gcagcacgac gcaccatctt aagttcgagg cttcaaggag acagccacaa gtgaacttca cgaccactac ccgacaacca gagaagcgcg cactctcggc acgacgataa tccgatccac ctgatcataa aaacctccca ttgttgttaa agcatcacaa tggtttgtcc ccgagcggcc cgcgctcgct gggctttgcc aggggcgcct caccgcatac agcgcggcgg cgccttagcg tcgccggctt cgatttagtg tggttcacgt cgttggagtc acactcaacc gatttcggcc cgaattttaa acaatctgct acccgctgac gacaagctgt tcatcaccga ataggttaat ttcggggaaa tcaaatatgt atattgaaaa ttcccttttt ctggtgaaag catcgaactg aagaacgttt gtattatccc ctattctcag ttacggatgg tcgagctgga ggcgagggcg caccggcaag acggcgtgca ttcttcaagt cttcaaggac gcgacaccct gacggcaaca cgtctatatc agatccgcca cagcagaaca ctacctgagc atcacatggt atggacgagc attcgtcgag cggatctaga tcagccatac cacctccccc cttgtttatt atttcacaaa aaactcatca gcaggaaccc cgctcactga cgggcggcct gatgcggtat gtcaaagcaa gtgtggtggt cccgctcctt tccccgtcaa ctttacggca agtgggccat cacgttcttt ctatctcggg tattggttaa caaaatatta ctgatgccgc gcgccctgac gaccgtctcc aacgcgcgag gtcatgataa tgtgcgcgga atccgctcat aggaagagta tgcggcattt taaaagatgc gatctcaaca tccaatgatg gtattgacgc aatgacttgg catgacagta 2026233990   16 Sep 2026 agagaattat cttacttctg acaacatggg aatgaagcca ggcaacaacg cccggcaaca cttctgcgct agccggtgag gtaagccctc atggatgaac gcattggtaa taaaacttca aatctcatga agaccccgta gcgtaatctg tgtttgccgg cagcagagcg gccaccactt atcctgttac gttggactca cggggggttc ctgagatacc gagaaaggcg gcacgaggga gggtttcgcc ggggcggagc tggccttttg gcagtgctgc acaacgatcg ggatcatgta taccaaacga ttgcgcaaac attaatagac cggcccttcc cgtgggtctc ccgtatcgta gaaatagaca ctgtcagacc tttttaattt ccaaaatccc gaaaagatca ctgcttgcaa atcaagagct cagataccaa caagaactct cagtggctgc agacgatagt gtgcacacag tacagcgtga gacaggtatc gcttccaggg acctctgact ctatggaaaa ctggcctttt cataaccatg gaggaccgaa actcgccttg cgagcgtgac tattaactgg tggatggagg ggctggctgg gcggtatcat gttatctaca gatcgctgag aagtttactc aaaaggatct ttaacgtgag aaggatcttc acaaaaaaac accaactctt atactgtcct gtagcaccgc tgccagtggc taccggataa cccagcttgg gctatgagaa cggtaagcgg ggaaacgcct tgagcgtcga acgccagcaa gctcacatgt agtgataaca ggagctaacc atcgttggga accacgatgc cgaactactt cggataaagt tttattgctg tgcagcactg cgacggggag ataggtgcct atatatactt aggtgaagat ttttcgttcc ttgagatcct caccgctacc tttccgaagg tctagtgtag ctacatacct gataagtcgt ggcgcagcgg agcgaacgac agcgccacgc cagggtcgga ggtatcttta tttttgtgat cgcggccttt ctgcggccaa gcttttttgc accggagctg ctgtagcaat actctagctt tgcaggacca ataaatctgg gggccagatg tcaggcaact cactgattaa tagattgatt cctttttgat actgagcgtc ttttttctgc agcggtggtt taactggctt ccgtagttag cgctctgcta gtcttaccgg tcgggctgaa ctacaccgaa ttcccgaagg acaggagagc tagtcctgtc gctcgtcagg ttacggttcc

[0068] 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 19181941 encode the FLAG-tag; residues 1951-1968 encode the 6xHis-tag; residues 21392260 encode the SV40 poly(A) sequence; residues 2293-2433 correspond to the 3’ ITR; residues 2508-22963 correspond to Fl 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 (Figure 9).

[0069] A second illustrative rAAV plasmid vector that may be used in accordance with the present invention is pAV-TBG-EGFP (SEQ ID NO:8; Figure 10) which comprises a 5’ ITR, a thyroid hormone-binding globulin (TBG) promoter, a 2026233990   16 Sep 2026 polynucleotide encoding the enhanced green fluorescent protein (EGFP), FLAG-tag and 6xHis-tag sites for facilitating recovery or localization of expressed proteins, an SV40 poly(A) site and a 3’ ITR. Coding Strand of Plasmid pAV-TBG-EGFP (SEQ ID NO: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 2026233990   16 Sep 2026 acgtcaaagc gggtgtggtg cgcccgctcc tttccccgtc tgctttacgg gtagtgggcc tccacgttct ctctatctcg tctattggtt aacaaaatat ctctgatgcc acgcgccctg gtgaccgtct gaaacgcgcg atgtcatgat aatgtgcgcg gtatccgctc aaaggaagag tttgcggcat agtaaaagat tggatctcaa tttccaatga ccgtattgac agaatgactt ggcatgacag cactgcggcc ccgctttttt gaaccggagc gcctgtagca ttactctagc gttgcaggac tgataaatct tggggccaga agtcaggcaa ctcactgatt tttagattga atcctttttg ccactgagcg ctttttttct ccagcggtgg ggtaactggc agccgtagtt ctcgctctgc gtgtcttacc ggtcgggctg acctacaccg gcttcccgaa gaacaggaga tatagtcctg aaccatagta gttacgcgca tttcgctttc aagctctaaa cacctcgacc atcgccctga ttaatagtgg ggctattctt aaaaaatgag taacgtttac gcatagttaa acgggcttgt ccgggagctg agacgaaagg aataatggtt gaacccctat atgagacaat tatgagtatt tttgccttcc gctgaagatc cagcggtaag tgagcacttt gccgggcaag ggttgagtac taagagaatt aacttacttc gcacaacatg tgaatgaagc atggcaacaa ttcccggcaa cacttctgcg ggagccggtg tggtaagccc ctatggatga aagcattggt tttaaaactt ataatctcat tcagaccccg gcgcgtaatc tttgtttgcc ttcagcagag aggccaccac taatcctgtt gggttggact aacggggggt aactgagata gggagaaagg gcgcacgagg tcgggtttcg cgcgccctgt gcgtgaccgc ttcccttcct tcgggggctc ccaaaaaact tagacggttt actcttgttc ttgatttata ctgatttaac aattttatgg gccagccccg ctgctcccgg catgtgtcag gcctcgtgat tcttagacgt ttgtttattt aaccctgata caacatttcc tgtttttgct agttgggtgc atccttgaga taaagttctg agcaactcgg tcaccagtca atgcagtgct tgacaacgat ggggatcatg cataccaaac cgttgcgcaa caattaatag ctcggccctt agcgtgggtc tcccgtatcg acgaaataga aactgtcaga catttttaat gaccaaaatc tagaaaagat tgctgcttgc ggatcaagag cgcagatacc ttcaagaact accagtggct caagacgata tcgtgcacac cctacagcgt cggacaggta gagcttccag ccacctctga agcggcacat tacacctgcc ttctcgccac cctttagggt tgatttgggt ttcgcccttt caaactggaa agggattttg aaaaatttaa tgcactctca acacccgcca catccgctta aggttttcac acgcctattt caggtggcac ttctaaatac aatgcttcaa gtgtcgccct cacccagaaa acgagtgggt gttttcgccc ctatgtggcg tcgccgcata cagaaaagca gccataacca cggaggaccg taactcgcct gacgagcgtg actattaact actggatgga ccggctggct tcgcggtatc tagttatcta cagatcgctg ccaagtttac ttaaaaggat ccttaacgtg caaaggatct aaacaaaaaa ctaccaactc aaatactgtt ctgtagcacc gctgccagtg gttaccggat agcccagctt gagctatgag tccggtaagc ggggaaacgc cttgagcgtc taagcgcggc agcgccttag gttcgccggc tccgatttag gatggttcac gacgttggag caacactcaa ccgatttcgg cgcgaatttt gtacaatctg acacccgctg cagacaagct cgtcatcacc ttataggtta ttttcgggga attcaaatat taatattgaa tattcccttt cgctggtgaa tacatcgaac cgaagaacgt cggtattatc cactattctc tcttacggat tgagtgataa aaggagctaa tgatcgttgg acaccacgat ggcgaactac ggcggataaa ggtttattgc attgcagcac cacgacgggg agataggtgc tcatatatac ctaggtgaag agttttcgtt tcttgagatc accaccgcta tttttccgaa cttctagtgt gcctacatac gcgataagtc aaggcgcagc ggagcgaacg aaagcgccac ggcagggtcg ctggtatctt gatttttgtg 2026233990   16 Sep 2026 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gt

[0070] 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 22202675 correspond to Fl 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 (Figure 10).

[0071] 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: • AAV 1 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 AAV 1. Thus, the AAV 1 P5 promoter sequences and the AAV 1 P40 promoter sequences are native AAV serotype promoter sequences of the rep gene of AAV 1; • 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; • AAV5 P5 promoter sequences natively control the transcription of the rep gene of AAV5 and the AAV5 P40 promoter sequences are natively found within the rep gene of AAV5. Thus, the AAV5 P5 promoter sequences and the AAV5 P40 promoter sequences are native AAV serotype promoter sequences of the rep gene of AAV5; • 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 2026233990   16 Sep 2026 P40 promoter sequences are native AAV serotype promoter sequences of the rep gene of AAV6; and • 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;

[0072] 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 1 SEQID NO AAV Promoter Native Serotype Sequence SEQID NO:9 P5 AAV1 ggtcctgtat tagctgtcac gtgagtgctt ttgcgacatt ttgcgacacc acgtggccat ttagggtata tatggccgag tgagcgagca ggatctccat tttgaccgcg aaatttgaac gagcagcagc c SEQID NQ:10 P5 AAV2 ggtcctgtat tagaggtcac gtgagtgttt tgcgacattt tgcgacacca tgtggtcacg ctgggtattt aagcccgagt gagcacgcag ggtctccatt ttgaagcggg aggtttgaac gcgcagccgc c SEQID NO:11 P5 AAV3 ccagctgcgt cagcagtcag gtgacccttt tgcgacagtt tgcgacacca cgtggccgct gagggtatat attctcgagt gagcgaacca ggagctccat tttgaccgcg aaatttgaac gagcagcagc c SEQID NO:12 P5 AAV4 ggtccctgta ttagcagtca cgtgagtgtc gtatttcgcg gagcgtagcg gagcgcatac caagctgcca cgtcacagcc acgtggtccg tttgcgacag tttgcgacac catgtggtca ggagggtata taaccgcgag tgagccagcg aggagctcca ttttgcccgc gaattttgaa cgagcagcag cc SEQID NO:13 P5 AAV5 atgtgatgtg ttttatccaa taggaagaaa gcgcgcgtat gagttctcgc gagacttccg gggtataaaa gaccgagtga acgagcccgc cgccattctt tgctctggac tgctagagga ccctcgctgc c 2026233990   16 Sep 2026 Table 1 SEQID NO AAV Promoter Native Serotype Sequence SEQID NO:14 P5 AAV6 ggtcctgtat tagaggtcac gtgagtgttt tgcgacattt tgcgacacca tgtggtcacg ctgggtattt aagcccgagt gagcacgcag ggtctccatt ttgaagcggg aggtttgaac gcgcagcgcc SEQID NO:15 P5 AAV7 ggtcctgtat tagctgtcac gtgagtgctt ttgcgacatt ttgcgacacc acgtggccat ttgaggtata tatggccgag tgagcgagca ggatctccat tttgaccgcg aaatttgaac gagcagcagc c SEQID NO:16 P5 AAV8 ggtcctgtat tagctgtcac gtgagtgctt ttgcggcatt ttgcgacacc acgtggccat ttgaggtata tatggccgag tgagcgagca ggatctccat tttgaccgcg aaatttgaac gagcagcagc c SEQID NO:17 P40 AAV1 ggtgacaaag caggaagtca aagagttctt ccgctgggcg caggatcacg tgaccgaggt ggcgcatgag ttctacgtca gaaagggtgg agccaacaaa agacccgccc ccgatgacgc ggataaaagc gagcccaagc gggcctgccc ctcagtcgcg gatccatcga cgtcagacgc SEQID NO:18 P40 AAV2 ggtcaccaag caggaagtca aagacttttt ccggtgggca aaggatcacg tggttgaggt ggagcatgaa ttctacgtca aaaagggtgg agccaagaaa agacccgccc ccagtgacgc agatataagt gagcccaaac gggtgcgcga gtcagttgcg cagccatcga cgtcagacgc SEQID NO:19 P40 AAV3 ggtcaccaaa caggaagtaa aggacttttt ccggtgggct tccgatcacg tgactgacgt ggctcatgag ttctacgtca gaaagggtgg agctaagaaa cgccccgcct ccaatgacgc ggatgtaagc gagccaaaac gggagtgcac gtcacttgcg cagccgacaa cgtcagacgc SEQID NO:20 P40 AAV4 ggtcaccaag caggaagtca aagacttttt ccggtgggcg tcagatcacg tgaccgaggt gactcacgag ttttacgtca gaaagggtgg agctagaaag aggcccgccc ccaatgacgc agatataagt gagcccaagc gggcctgtcc gtcagttgcg cagccatcga cgtcagacgc 2026233990   16 Sep 2026 Table 1 SEQID NO AAV Promoter Native Serotype Sequence SEQID NO:21 P40 AAV5 gattactaag caggaagtca aggacttttt tgcttgggca aaggtcaatc aggtgccggt gactcacgag tttaaagttc ccagggaatt ggcgggaact aaaggggcgg agaaatctct aaaacgccca ctgggtgacg tcaccaatac tagctataaa agtctggaga agcgggcctg gagcatgagg ctctcatttg ttcccgagac gcctcgcagt tcagacg SEQID NO:22 P40 AAV6 ggtgacaaag caggaagtca aagagttctt ccgctgggcg caggatcacg tgaccgaggt ggcgcatgag ttctacgtca gaaagggtgg agccaacaag agacccgccc ccgatgacgc ggataaaagc gagcccaagc gggcctgccc ctcagtcgcg gatccatcga cgtcagacgc SEQID NO:23 P40 AAV7 ggtgacgaag caggaagtca aagagttctt ccgctgggcc agtgatcacg tgaccgaggt ggcgcatgag ttctacgtca gaaagggcgg agccagcaaa agacccgccc ccgatgacgc ggatataagc gagcccaagc gggcctgccc ctcagtcgcg gatccatcga cgtcagacgc SEQID NO:24 P40 AAV8 ggtgacaaag caggaagtca aagagttctt ccgctgggcc agtgatcacg tgaccgaggt ggcgcatgag ttttacgtca gaaagggcgg agccagcaaa agacccgccc ccgatgacgc ggataaaagc gagcccaagc gggcctgccc ctcagtcgcg gatccatcga cgtcagacgc

[0073] 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 ofan AAV1, AAV2, AAV5, or AAV6rep 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, 2026233990   16 Sep 2026 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.

[0074] 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.

[0075] 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)-RC5; 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.

[0076] 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.

[0077] 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 2026233990   16 Sep 2026 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).

[0078] 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.

[0079] 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 2026233990   16 Sep 2026 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.

[0080] 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.

[0081] 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: (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 (2) an Ad helper vector that can provide the non-AAV helper proteins (e.g., Ela, Elb, E2a, VA and E4) or RNA molecules that are not provided by such rAAV or rAAV plasmid vectors, but are required for their production.

[0082] 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 functionproviding polynucleotide, so that such vectors or plasmids, in concert with the rAAV, comprise a triple plasmid transfection system. 2026233990   16 Sep 2026

[0083] 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).

[0084] 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.

[0085] 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® 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.

[0086] 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 2026233990   16 Sep 2026 Modified Eagle’s Medium (DMEM; Mediatech, Manassas, VA). Fetal bovine serum (e.g., FBS; HyClone Laboratories, South Logan, UT) 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 etal. (2017) “Animal-Cell Culture Media: History, Characteristics, And Current Issues,” Reproduc. Med. Biol. 16(2): 99-117).

[0087] 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 x 106 HEK293 or BHK cells per well on a multi-well plate, or 1.5 x 107 HEK293 cells per 15-cm dish, may be employed. For large-scale production HEK293 or BHK cells may be collected from multiple confluent 15-cm plates, and split into two 10-layer cell stacks (Corning, Corning, NY) 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 x 108 cells) may be resuspended in 200 ml of medium. Preferably, the cells are allowed to attach for 24 hours before transfection. Confluency of the cell stacks may be monitored using a Diaphot inverted microscope (Nikon, Melville, NY) from which the phase-contrast hardware had been removed in order to accommodate the cell stack on the microscope stage.

[0088] 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: (1)   the rAAV; (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 2026233990   16 Sep 2026 or P40 promoter sequence in replacement of, or in addition to, a native AAV serotype promoter sequence; and (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.

[0089] 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: (1) the rAAV; and (2) a recombinantly-modified adeno-associated virus (AAV) helper vector that comprises: (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 (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.

[0090] 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. 2026233990   16 Sep 2026 II. Pharmaceutical Compositions of the Present Invention

[0091] 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”)

[0092] 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 US Patents No. 8,852,607; 8,192,975; 6,764,845; 6,759,050; and 7,598,070.

[0093] 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. 2026233990   16 Sep 2026 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.

[0094] 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.

[0095] The rAAV of such pharmaceutical compositions is preferably packaged in a hermetically sealed container, such as a vial, an ampoule or sachette indicating the quantity of the molecule, and optionally including instructions for use. In one embodiment, the rAAV of such kit is supplied as a dry sterilized lyophilized powder or water-free concentrate in a hermetically sealed container and can be reconstituted, e.g., with water, saline, or other diluent to the appropriate concentration for administration to a subject. The lyophilized material should be stored at between 2°C and 8°C in their original container and the material should be administered within 12 hours, preferably within 6 hours, within 5 hours, within 3 hours, or within 1 hour after being reconstituted. In another embodiment, the rAAV of such kit is supplied as an aqueous solution in a hermetically sealed container and can be diluted, e.g., with water, saline, or other diluent, to the appropriate concentration for administration to a subject. The kit can further comprise one or more other prophylactic and / or therapeutic agents useful for the treatment of the disease or condition, in one or more containers; and / or the kit can further comprise one or more cytotoxic antibodies that bind one or more cancer antigens associated with cancer. In certain embodiments, the other prophylactic or therapeutic agent is a chemotherapeutic. In other embodiments, the prophylactic or therapeutic agent is a biological or hormonal therapeutic. III. Uses of the Invention

[0096] 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 2026233990   16 Sep 2026 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 IB (LGMD1B); limb girdle muscular dystrophy IC (LGMD1C); limb girdle muscular dystrophy 2A (LGMD2A); limb girdle muscular dystrophy 2B (LGMD2B); limb girdle muscular dystrophy 21 (LGMD2I); limb girdle muscular dystrophy 2L (LGMD2L); lipoprotein lipase (LPL) deficiency; metachromatic leukodystrophy; neurological disability; neuromotor deficit; neuroskeletal impairment; Parkinson’s disease; rheumatoid arthritis; Sanfilippo A syndrome; spinal muscular atrophy (SMA); X-linked retinoschisis (XLRS); a-sarcoglycan deficiency (LGMD2D); P-sarcoglycan deficiency (LGMD2E); y-sarcoglycan deficiency (LGMD2C) and 6-sarcoglycan deficiency (LGMD2F). IV. Embodiments of the Invention

[0097] 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: El. 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. E2. The recombinantly-modified adeno-associated virus (AAV) helper vector of El, wherein the AAV helper function-providing polynucleotide vector comprises a non-native AAV serotype P5 promoter sequence. 2026233990   16 Sep 2026 E3. The recombinantly-modified adeno-associated virus (AAV) helper vector of any one of El or E2, wherein the AAV helper function-providing polynucleotide vector comprises a non-native AAV serotype P40 promoter sequence. E4. The recombinantly-modified adeno-associated virus (AAV) helper vector of any one of E1-E3, wherein the vector is a plasmid vector. E5. The recombinantly-modified adeno-associated virus (AAV) helper vector of El, wherein the non-native AAV serotype P5 or P40 promoter sequence replaces a native AAV serotype promoter sequence. 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. 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: (1)   the rAAV; (2)    the recombinantly-modified adeno-associated virus (AAV) helper vector of E6; 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. 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: (1)   the rAAV; (2)    the recombinantly-modified adeno-associated virus (AAV) helper vector of any one of E1-E6; and (3) an additional vector, especially a plasmid vector, that comprises a non-AAV helper function-providing polynucleotide; 2026233990   16 Sep 2026 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. E9. The method of any one of E7-E8, wherein: (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; (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; (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; (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; (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; 2026233990   16 Sep 2026 (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; (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 (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. 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. Ell. The method of E10, wherein the cells are HEK293 human embryonic kidney cells. Ell. The method of E10, wherein the cells are BHK21 baby hamster kidney cells. 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. E14. A preparation of the recombinantly-modified adeno-associated virus (rAAV) produced by the method of E13. E15. A pharmaceutical composition that comprises the recombinantly-modified adeno-associated virus (rAAV) produced by the method of E13, and a pharmaceutically acceptable carrier. 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. 2026233990   16 Sep 2026 EXAMPLES

[0098] 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

[0099] 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 (Figure 11) in lieu of the native AAV2 serotype P5 promoter of such plasmid (Figure 12A; downward striped rectangle). The P19 and P40 promoters of the constructs were not changed, and thus were both native AAV2 serotype promoter sequences (Figure 12A; solid black rectangles).

[00100] The following constructs were employed; the sequences of the promoter regions are shown in Table 1: (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); (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); (3)    P5(2)-RC2 - a derivative of plasmid vector pAAV-RC2 in which the partial 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); (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); 2026233990   16 Sep 2026 (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); (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); (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); (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 (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).

[00101] Figure 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 P-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 f Nat. Biotechnol. 20(1):87-90), the polyA domain of human growth hormone and a 3’ ITR. Figure 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 AAV5 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. 2026233990   16 Sep 2026 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

[00102] 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 a non-native AAV serotype promoter sequence (Figure 11) in lieu of the native serotype P40 promoter of such plasmid (Figure 13A; upward striped rectangle). The P5 and P19 promoters of the constructs were not changed, and thus were both native AAV2 serotype promoter sequences (Figure 13A; solid black rectangles).

[00103] The following constructs were employed; the sequences of the promoter regions are shown in Table 1: (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); (2)    P40(l)-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); (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); (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); (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); (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); 2026233990   16 Sep 2026 (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); (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 (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).

[00104] Figure 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

[00105] 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 (Figure 11) in lieu of the native AAV2 serotype P5 (Figure 14A; downward striped rectangle) and P40 (Figure 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 (Figure 14A; solid black rectangle). 2026233990   16 Sep 2026

[00106] The following constructs were employed; the sequences of the promoter regions are shown in Table 1: (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); (2)    P5(2)-RC2 - a derivative of plasmid vector pAAV-RC2 in which the partial 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); (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); (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); (5)    P40(l)-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); (6)    P5(2) / P40(l)-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); (7)    P5(3) / P40(l)-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 (8)    P5(5) / P40(l)-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) 2026233990   16 Sep 2026

[00107] Production titers of rAAV were obtained essentially as described in Example 1. Figure 14B shows the production titers of rAAV obtained using such AAV helper plasmid vectors. As shown in Figure 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

[00108] 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 (Figure 11) in lieu of the native AAV2 serotype P5 (Figure 15A; downward striped rectangle) and P40 (Figure 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 (Figure 15A; solid black rectangle).

[00109] The following constructions were employed; the sequences of the promoter regions are shown in Table 1: (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); (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); (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); 2026233990   16 Sep 2026 (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); (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 (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).

[00110] Figure 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.

[00111] The results of the investigation are shown in Figures 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

[00112] 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 RC5 (having an AAV2 rep gene and a cap gene that encodes Cap protein of the AAV5 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 (Figure 11) in lieu of the native AAV2 serotype P5 (Figure 16A; downward striped rectangle) and / or P40 (Figure 16A; upward striped rectangle) promoter sequences of such plasmids. 2026233990   16 Sep 2026

[00113] The following constructions were employed; the sequences of the promoter regions are shown in Table 1: (1) Parent-RCl - pAAV-RCl (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); (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); (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); (4)    P5(2)-RC1 - a derivative of plasmid vector pAAV-RCl in which native AAV 1 serotype P5 promoter sequences had been replaced with the P5 promoter sequence of AAV2 (SEQ ID NO: 10); (5)    P5(7)-RC1 - a derivative of plasmid vector pAAV-RCl in which native AAV 1 serotype P5 promoter sequences had been replaced with the P5 promoter sequence of AAV7 (SEQ ID NO: 15); (6)    P5(8)-RC1 - a derivative of plasmid vector pAAV-RCl in which native AAV 1 serotype P5 promoter sequences had been replaced with the P5 promoter sequence of AAV8 (SEQ ID NO: 16); (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); (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). (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 2026233990   16 Sep 2026 (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).

[00114] Production titers of rAAV were obtained essentially as described in Example 1. The results of the investigation are shown in Figure 16B, and reveals that the replacement of the native P5 promoter sequences of pAAV-RCl, pAAV-RC5, and pAAV-RC7 with P5 promoter sequence of AAV serotype 2, 7 or 8 increased rAAV production titers.

[00115] 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.

Claims

Claim 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. Claim 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. Claim 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. Claim 4. The recombinantly-modified adeno-associated virus (AAV) helper vector of claim 1, wherein said vector is a plasmid vector. Claim 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. Claim 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. Claim 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;2026233990   16 Sep 2026wherein 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.Claim 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.Claim 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;2026233990   16 Sep 2026(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.Claim 10. The method of claim 7, wherein said cells are human embryonic kidney cells, baby hamster kidney cells or sf9 insect cells.Claim 11. The method of claim 10, wherein said cells are HEK293 human embryonic kidney cells.Claim 12. The method of claim 10, wherein said cells are BHK21 baby hamster kidney cells.2026233990   16 Sep 2026Claim 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 claim 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.Claim 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.Claim 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 AAV2026233990   16 Sep 2026Claim 16.Claim 17.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.The method of claim 13, wherein said cells are human embryonic kidney cells, baby hamster kidney cells or sf9 insect cells.The method of claim 16, wherein said cells are HEK293 human embryonic kidney cells.2026233990   16 Sep 2026Claim 18. The method of claim 16, wherein said cells are BHK21 baby hamster kidney cells. Claim 19. A pharmaceutical composition that comprises recombinantly-modified adeno-associated virus (rAAV) produced by the method of claim 6, and a pharmaceutically acceptable carrier. Claim 20. A pharmaceutical composition that comprises recombinantly-modified adeno-associated virus (rAAV) produced by the method of claim 13, and a pharmaceutically acceptable carrier.

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