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Definitions

• the present invention is directed to methods for increasing the efficiencies with which recombinant adeno-associated virus (rAAV) are packaged, so as to increase their production titers. More specifically, the invention relates to a method for increasing the production titer of rAAV by transfected cells by increasing the ionic strength of the cell culture media through the administration of additional ions.
• rAAV recombinant adeno-associated virus
• AAV Adeno-Associated Virus
• Adeno-Associated Virus is a small, naturally-occurring, non- pathogenic virus belonging to the Dependovirus genus of the Parvoviridae (Balakrishnan, B. et al. (2014)“ Basic Biology of Adeno-Associated Virus (AAV) Vectors Used in Gene Therapy ,” Curr. Gene Ther. 14(2):86-100; Zinn, E. et al. (2014) ‘ del ⁇ lo-A ssociated Virus: Fit To Serve f Curr. Opin. Virol. 0:90-97). Despite not causing disease, AAV is known to be able to infect humans and other primates and is prevalent in human populations (Johnson, F.B. et al.
• AAV infect a broad range of different cell types (e.g. , cells of the central nervous system, heart, kidney, liver, lung, pancreas, retinal pigment epithelium or photoreceptor cells, or skeletal muscle cells). Twelve serotypes of the virus (e.g, AAV2, AAV5, AAV6, etc.), exhibiting different tissue infection capabilities (“tropisms”), have been identified (Colella, P. et al.
• AAV is a single-stranded DNA virus that is composed of approximately 4,700 nucleotides.
• the viral genome may be described as having a 5’ half and a 3’ half which together comprise the genes that encode the virus’ proteins ( 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, pl9 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.
• 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).
• 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.
• 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. etal. (2016) “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 of AAV Vectors ,” Mol. Ther. Meth. Clin. Devel.
• 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 Therapyf Curr. Gene Ther. 14(2):86-100; Colella, P. et al. (2016)“Emerging Issues in AAV-Mediated In Vivo Gene Therapyf Molec. Ther. Meth. Clin. Develop. 8:87-104).
• ITR AAV-specific palindromic inverted terminal repeated sequences
• AAV is an inherently defective virus, lacking the capacity to perform at least two critical functions: the ability to initiate the synthesis of viral-specific products and the ability to assemble such products to form the icosahedral protein shell (capsid) of the mature infectious viral particle. It thus requires a co-infecting“helper” virus, such as adenovirus (Ad), herpes simplex virus (HSV), cytomegalovirus (CMV), vaccinia virus or human papillomavirus to provide the viral-associated ( A) 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.
• the AAV genome does not include genes that encode the viral proteins El a, Elb, E2a, and E4 of Ad; thus, these proteins must also be provided by a co-infecting“helper” virus.
• the El a 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.
• 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.
• 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 b-globin intron, a cloning site into which a therapeutic gene of choice (transgene) has been inserted, and a poly-adenylation (“poly A”) site.
• the inverted terminal repeated sequences (ITR) of the rAAV are, however, retained, so that the transgene expression cassette of the rAAV plasmid vector is flanked by AAV ITR sequences (Colella, P. et al. (2016)“Emerging Issues in AAV-Mediated In Vivo Gene Therapy ,” Molec. Ther.
• the rAAV comprises a 5’ ITR, the transgene expression cassette of the rAAV, and a 3’ ITR.
• rAAV have been used to deliver a transgene to patients suffering from any of a multitude of genetic diseases (e.g. , hereditary lipoprotein lipase deficiency (LPLD), Leber’s congenital amaurosis (LCA), aromatic L-amino acid decarboxylase deficiency (AADC), choroideremia and hemophilia), and have utility in new clinical modalities, such as in interfering RNA (RNAi) therapy and gene-modifying strategies such as Crispr/Cas9 (US Patents No. 8,697,359, 10,000,772, 10,113,167, 10,227,611; Lino, C.A. etal.
• LPLD hereditary lipoprotein lipase deficiency
• LCA congenital amaurosis
• AADC aromatic L-amino acid decarboxylase deficiency
• choroideremia and hemophilia choroideremia and hemophilia
• RNAi interfer
• AAV serotypes are described in US Patents No.
• rAAV containing a desired transgene expression cassette are typically produced by human cells (such as HEK293) grown in either adhesion or suspension. Since, as described above, rAAV are defective viruses, additional functions must be provided in order to replicate and package rAAV.
• rAAV are produced by transiently transfecting cells with an rAAV plasmid vector and a second plasmid vector that comprises an AAV helper function providing polynucleotide that provides the Rep52 and Rep78 genes that are required for vector transcription control and replication, and for the packaging of viral genomes into the viral capsule (Rep40 and Rep68 are not required for rAAV production) and the cap genes that were excised from the AAV in order to produce the rAAV.
• 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. (2016)“ Pharmacology of Recombinant Adeno-associated Virus Production,” Molec. Ther. Meth. Clin. Develop. 8: 166-180).
• 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,
• Else 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.
• helper plasmids rather than helper viruses, permits rAAV to be produced without additionally producing particles of the helper virus (Franqois, A. et al. (2016)“ 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).
• 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) (Franqois, A. el al. (2016)“ Accurate Titration of Infectious AAV Particles Requires Measurement of Biologically Active Vector Genomes and Suitable Controls ,” Molec. Ther. Meth. Clin. Develop. 10:223-236).
• rAAV are typically collected and purified by one or more overnight CsCl gradient centrifugations (Zolotukhin, S. et al. (1999)“ Recombinant Adeno-Associated Virus Purification Using Novel Methods Improves Infectious Titer And Yield,” Gene Ther. 6:973-985), followed by desalting to form a purified rAAV production stock. Titers of 10 12 -10 13 infectious rAAV capsids/mL are obtainable.
• TCID50 median tissue culture infective dose
• a HeLa-derived AAV2 rep- and c «/;-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.
• infectious titer of an rAAV preparation can be measured using the infectious center assay (ICA).
• ICA infectious center assay
• 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.
• Lock, M. et al. (2010) disclose a PEI transfection-based- and supernatant harvest-based-technique for facilitating the recovery of rAAV particles (Lock, M. et al. (2010)“Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale f Hum. Gene Ther. 21 : 1259-1271).
• the method is based on the observation that rAAV belonging to AAV serotypes other than AAV2 were released primarily into the culture medium of calcium phosphate-transfected cells and were not retained in the cell lysate.
• the production culture medium represents a relatively pure source of rAAV plasmid vector that possesses a lower level of cellular contaminants and that these factors improve the loading capacity and resolution of purification gradients.
• rAAV including rAAV belonging to AAV2 serotype, were transfected into HEK293 cells using calcium phosphate. Seventy-two hours (or 120 hours) post-transfection, serum-free media was added and the incubation was continued for an additional 28 hours.
• Benzonase® a genetically engineered endonuclease that degrades all forms for DNA and RNA, was then added to the culture supernatant.
• the Limited Tissue-Specific Tropism of rAAV One such problem reflects the limited tissue-specific tropisms of AAV and rAAV.
• the use of multiple helper plasmids, encoding capsid proteins of differing serotypes (i.e., “mosaic” capsids) has been exploited as a way to increase the range of tissue types that can be infected by rAAV (Hauck, B. et al. (2003) “ Generation And Characterization Of Chimeric Recombinant AAV Vectors ,” Mol. Ther. 7:419- 425; Rabinowitz, J.E. et al.
• a second such problem reflects the fact that 30-80% of humans have been naturally exposed to AAV infection (mainly AAV2) and 20-67% of humans harbor titers of neutralizing anti-AAV capsid antibodies in their blood and other bodily fluids (Liu, Q. et al. (2014)“ Neutralizing Antibodies against AAV2, AAV 5 And AAV8 In Healthy And HIV- 1 -Infected Subjects In China: Implications For Gene Therapy Using AAV Vectors ,” Gene Ther. 21 :732-738; Vandamme, C. et al. (2017) “ Unraveling the Complex Story of Immune Responses to AAV Vectors Trial After Trial f Hum. Gene. Ther.
• AAV antigen that can lead to unwanted immune responses and toxicity.
• improved methods for increasing packaging efficiency and obtaining high production titers are of great importance.
• the present invention is directed to improved methods for increasing the efficiency of rAAV packaging by altering the concentration of ions in a culturing medium during the production of rAAV.
• the present invention is directed to methods for increasing the efficiency with which recombinant adeno-associated virus (rAAV) are packaged, so as to increase their production titers. More specifically, the invention relates to a method for increasing the production titer of rAAV by transfected cells by increasing the ionic strength of the cell culture media through the administration of additional ions.
• rAAV recombinant adeno-associated virus
• the invention provides a method for increasing the production titer of recombinantly-modified adeno-associated virus (rAAV), wherein the method comprises the steps:
• step (C) continuing the culturing of the cells to thereby produce a production titer of with the rAAV that is greater than a titer obtained in the absence of step (B).
• the invention additionally provides the embodiment of such method wherein each of the added ion(s) is provided in an amount sufficient to increase the concentration of such ion in the initial culture medium by from about 10 mM to about 80 mM.
• the invention additionally provides the embodiment of such methods wherein the production titer is at least 50% greater than the titer obtained from a similarly conducted cell culturing in the absence of the step (B).
• the invention additionally provides the embodiment of such methods wherein the rAAV comprises a transgene cassette that encodes a protein, or comprises a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition.
• the invention additionally provides the embodiment of such methods wherein the rAAV belongs to the rAAVl, rAAV2, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9 or rAAVIO serotype, or to a hybrid of such serotypes.
• the invention additionally provides the embodiment of such methods wherein the rAAV belongs to the rAAV2, rAAV5, or rAAV9 serotype, or to a hybrid of the serotypes.
• the invention additionally provides the embodiment of such methods wherein the added ions comprise one or more of K + , Ca ++ , or Mg ++ .
• the invention additionally provides the embodiment of such methods wherein the added ions comprise one or more of acetate, aspartate, biphthalate, bitartrate, butoxyethoxy acetate, caprylate, citrate, dehydroacetate, diacetate, dihydroxy glycinate, d-saccharate, gluconate, glutamate, glycinate, glycosulfate, hydroxymethane sulfonate, lactate, methionate, oxalate, phenate, phenosulfonate, propionate, propionate, saccharin, salicylate, sarcosinate, sorbate, thioglycolate, and toluene sulfonate.
• the added ions comprise one or more of acetate, aspartate, biphthalate, bitartrate, butoxyethoxy acetate, caprylate, citrate, dehydroacetate, diacetate, dihydroxy glycinate, d-saccharate
• the invention additionally provides the embodiment of such methods wherein the cells are human embryonic kidney cells, baby hamster kidney cells or sf9 insect cells.
• the invention additionally provides the embodiment of such methods wherein the cells are HEK293 human embryonic kidney cells.
• the invention additionally provides the embodiment of such methods wherein the cells are BHK21 baby hamster kidney cells.
• the invention additionally provides the embodiment of such methods wherein the initial culture medium is Dulbecco’s Modified Eagle’s Medium or Dulbecco’s Modified Eagle’s Medium supplemented with serum.
• the invention additionally provides a pharmaceutical composition that comprises:
• rAAV recombinantly-modified adeno-associated virus
• the invention additionally provides a preparation of recombinantly-modified adeno-associated virus (rAAV) produced by any of the above-described methods, wherein the rAAV comprises a transgene cassette that encodes a protein, or a transcribed nucleic acid, or the above-described pharmaceutical composition for use in the treatment of a genetic or heritable disease or condition.
• rAAV recombinantly-modified adeno-associated virus
• Figure 1 provides a schematic genetic map of the wild-type (Wt) AAV genome.
• 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”
• the wild-type (Wt) AAV2 (1 ) is composed of AAV-specific palindromic inverted terminal repeated sequences (ITR), a 5’ half containing genes that encode the Rep proteins and a 3’ half containing genes that encode the Cap proteins.
• the rAAV (2) is formed by replacing the Rep- and Cap-encoding genes of the wild-type (Wt) AAV2 (1 ) with a transgene cassette that comprises a promoter (Pro), the exogenous transgene of interest, and a polyadenylation site (pA).
• a complementing“AAV helper” plasmid vector (3) and an adenovirus helper plasmid vector (Ad helper plasmid)
• 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.
• Figure 3 shows a map of the AAV helper plasmid vector pAAV-RC2.
• Figure 4 shows a map of the non-AAV helper plasmid vector pHelper-Kan.
• Figure 5 shows a map of the rAAV plasmid vector pAV-CMV-EGFP.
• Figure 6 shows a map of the rAAV plasmid vector pAV-TBG-EGFP.
• Figures 7A-7C show the effect of cation and cation concentration on the production of rAAV by transfected cells.
• Figure 7A shows the extent of expression of the enhanced green fluorescent protein (EGFP) in the transfected cells and the titering of the rAAV stocks using the infectious center assay.
• Stocks were produced by growing transfected HEK293 cells in Dulbecco’s Modified Eagle’s Medium in the presence of additionally added NaCl, KC1, CaCb or MgCh. The additional concentration of such provided salt is 0, 20, 40, 60, 80 or 100 mM.
• Figure 7A shows the infectious center assay.
• Figure 7B is a graph of the fold-change in the titers of AAV vectors and salt concentration.
• Figure 7C is a graph of the fold-change in Total Genomes (TG) of AAV as a function of cation and cation concentration.
• the concentration shown in the Figure is the concentration increase in the culturing medium provided by the addition of such salts.
• Figures 8A-8B show the effect of cation and cation concentration on the production of rAAV stocks.
• Figure 8A shows the extent of expression of the enhanced green fluorescent protein (EGFP) in the transfected cells and the titering of the rAAV stocks using the infectious center assay.
• Stocks were produced by growing transfected HEK293 cells in Dulbecco’s Modified Eagle’s Medium in the presence of additionally added 12 salts.
• EGFP enhanced green fluorescent protein
• Figure 8A shows the infectious center assay.
• Figure 8B is a graph of the fold- change in the titers of AAV vectors and salt concentration.
• the Figure shows the fold- change in rAAV titer for rAAV that were produced in the presence of different anions and differing additionally provided concentrations of such anions.
• the concentration shown in the Figure is the concentration increase in the culturing medium provided by the addition of such anions.
• Figures 9A-9B demonstrate that the provision of KHCO3 caused unexpectedly higher titers of rAAV, relative to other ions ( Figure 9A: fold-change in AAV titer in culture medium; Figure 9B: fold-change in Total Genomes).
• the concentration shown in the Figure (40, 50, 60 or 70 mM) is the concentration increase in the culturing medium provided by the addition of such KHCO3.
• Figure 10 shows the fold-change in the total amount of rAAV produced, and in the amount of rAAV released into the medium by cells that had been co-transfected with an Ad helper plasmid, a plasmid that provides the AAV ITRs, an enhanced green fluorescent protein-encoding transgene cassette and either an AAV2 helper plasmid or an AAV8 helper plasmid in order to provide the AAV rep and cap gene functions.
• Ad helper plasmid a plasmid that provides the AAV ITRs
• an enhanced green fluorescent protein-encoding transgene cassette and either an AAV2 helper plasmid or an AAV8 helper plasmid in order to provide the AAV rep and cap gene functions.
• KHCO3 was added to produce an additional concentration of 30 mM in the culturing medium and the fold-change of rAAV that had been released into the medium (AAV2-medium and AAV8-medium) and the total genomes in the cell lysis (AAV2-total and AAV8-total) were assessed at 72 hours post transfection.
• Figures 11A-11B show the effect of providing KHCO3 on the enhancement of the production of rAAV of different serotypes.
• Figure 11 A shows the fold-change of rAAV released into the medium after 24 hours;
• Figure 11B shows the fold-change of total genomes of rAAV;
• KHCO3-30 denotes that KHCO3 was added to produce an additional concentration of 30 mM in the culturing medium;
• KHCCb-55 denotes that KHCCb was added to produce an additional concentration of 55 mM in the culturing medium.
• Figure 12 shows the ability of cells cultured in suspension to produce enhanced levels of rAAV in response to the provision of KHCCb. Provision of KHCCb sufficient to increase the concentration of KHCCb in the culturing medium by greater than about 20 mM enhanced production of rAAV5 and rAAV6 after 20 hours.
• the present invention is directed to methods for increasing the efficiencies with which recombinant adeno-associated virus (rAAV) are packaged, so as to increase their production titers. More specifically, the invention relates to a method for increasing the production titer of rAAV by transfected cells by increasing the ionic strength of the cell culture media through the administration of additional ions.
• rAAV recombinant adeno-associated virus
• 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.
• the term “rAAV” is intended to denote a recombinantly-modified version of AAV that comprises a polynucleotide sequence not of AAV origin (z.e., a polynucleotide heterologous to AAV).
• the rAAV may be single-stranded or double-stranded, and may be composed of deoxyribonucleotides or ribonucleotides.
• 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.
• AAV helper functions are provided by an“AAV helper function-providing polynucleotide,” which as such term is used herein is a virus, plasmid vector, a non-plasmid vector, or a polynucleotide that has been integrated into a cellular chromosome, that provides AAV helper functions.
• AAV helper plasmids that may be used in accordance with the present invention to provide AAV helper functions, such as pAAV-RC (Agilent; Addgene; Cell Biolabs), pAAV-RC2 (Cell Biolabs), etc., are commercially available.
• Plasmid pAAV-RC2 (SEQ ID NO:l; Figure 3) is an AAV helper plasmid that may be used in accordance with the present invention to provide AAV helper functions.
• residues 85-1950 of pAAV-RC2 encode the Rep protein, Rep78 (with residues 484-663 corresponding to the P19 promoter, residues 1464-1643 corresponding to the P40 promoter and residues 1668-1676 being a donor site); residues 1967-4174 encode the capsid protein, VP1; residues 1992-2016 encodes a portion of the Rep68 protein; residues 4175-4256 encode a polyA sequence; residues 4610-4626 are M13 Rev sequences; residues 4634-4650 are Lac operator sequences; 4658-4688 are Lac promoter sequences; residues 4951-5675 correspond to pMB ori sequences, residues 5771-6631 encode an ampicillin resistance determinant; and residues 6632- 6730 are bla promoter sequences ( Figure 3).
• non-AAV helper functions denotes proteins of Ad, CMV, HSV or other non-AAD viruses (e.g, El a, 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.
• 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 are suitable non-AAV helper function providing polynucleotide (see, e.g., Matsushita, T. el al.
• Plasmid pHelper-Kan (SEQ ID NO:2; Figure 4) is a non-AAV helper function-providing polynucleotide that may be used in accordance with the present invention to provide non-AAV helper functions.
• residues 1-5343 of pHelper-Kan are derived from adenovirus, and include a polynucleotide encoding the E2A protein (residues 258- 1847); residues 5344-8535 are derived from adenovirus, and include a polynucleotide encoding the E4orf6 protein; residues 9423-10011 correspond to ori sequences; residues 10182-10976 encode a kanamycin resistance determinant expressed by a bla promoter sequence (residues 10977-11081); residues 11107-11561 correspond to fl ori sequences ( Figure 4).
• AAV helper function-providing polynucleotides and non-AAV helper function-providing polynucleotides are typically employed in conceit with an rAAV plasmid vector to comprise a triple plasmid transfection system.
• rAAV plasmid vectors e.g ., pAV-CMV-EGFP, pGOI, etc. (Cell Biolabs, Inc., Invitrogen and Stratagene) may be used in accordance with the present invention.
• An illustrative rAAV plasmid vector that may be used in accordance with the present invention is pAV-CMV-EGFP (SEQ ID NO:3; Figure 5) which comprises a 5’ ITR, a U6 promoter, CMV enhancer and promoter sequences, a polynucleotide encoding the enhanced green fluorescent protein (EGFP) (Gambotto, A. et al. (2000)“ Immunogenicity Of Enhanced Green Fluorescent Protein (EGFP) In BALB/C Mice: Identification Of An H2-Kd-Restricted CTL Epitope ,” Gene Ther. 7(23):2036-2040; Tsien, R.Y. (1998)“The Green Fluorescent Protein ,” Annu. Rev.
• EGFP enhanced green fluorescent protein
• residues 1-128 of pAV-CMV-EGFP correspond to the 5’ ITR; residues 201-441 are U6 promoter sequences; residues 562-865 are human cytomegalovirus (CMV) immediate early enhancer sequences; residues 866-1068 comprise the CMV immediate early promoter; residues 1192-1911 comprise a mammalian codon-optimized polynucleotide that encodes the EGFP; residues 1918- 1941 encode the FLAG-tag; residues 1951-1968 encode the 6xHis-tag; residues 2139- 2260 encode the SV40 poly(A) sequence; residues 2293-2433 correspond to the 3’ ITR; residues 2508-22963 correspond to FI 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 the 5’ ITR; residues 20
• a second illustrative rAAV plasmid vector that may be used in accordance with the present invention is pAV-TBG-EGFP (SEQ ID NO:4; Figure 6) which comprises a 5’ ITR, a thyroid hormone-binding globulin (TBG) promoter, a polynucleotide encoding the enhanced green fluorescent protein (EGFP), FLAG-tag and 6xHis-tag sites for facilitating recovery or localization of expressed proteins, an SV40 poly(A) site and a 3’ ITR.
• Plasmid pAV-TBG-EGFP (SEQ ID NO:4):
• the term“production titer” is intended to denote the amount of concentration of infectious rAAV in a preparation. Such amounts or concentrations are preferably determined by titering the AAV or rAAV in such preparation.
• the production titers of the rAAV preparations of the present invention are preferably titered after subjecting producing cells (e.g ., HEK293 transformed with an rAAV plasmid vector, an AAV helper vector providing Rep and Cap proteins, and an Ad helper vector providing required adenovirus transcription and translation factors) to three rounds of freeze / thawing, followed by sonication to release the rAAV particles. The preparation is then centrifuged.
• the employed AAV vector is localized to the supernatant.
• An aliquot of the preparation is treated with proteinase K, and the number of AAV genomes is determined.
• An aliquot of the preparation is infected into HeLa- 32C2 cells (which express AAV2 Rep and Cap proteins), and infectious titer is measured using the infectious center assay (ICA) (Franqois, A. et al. (2016)“ 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. el al. (2004) “ Infectious Titer Assay For Adeno-Associated Virus Vectors With Sensitivity Sufficient To Detect Single Infectious Events ,” Hum. Gene Ther. 15:709- 715).
• ICA infectious center as
• 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
• the rAAV whose production titer may be increased using the methods of the present invention may comprise any transgene cassette that permits the rAAV to be packaged into an rAAV plasmid vector that may be encapsidated within an AAV capsid particle.
• 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.
• 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.
• a protein e.g ., an enzyme, hormone, antibody, receptor, ligand, etc
• a transcribed nucleic acid that is relevant to a genetic or heritable disease or condition, such that it may be used in gene therapy to treat such disease or condition.
• the methods of the present invention may be used to increase the production titer of rAAV and rAAV plasmid vectors in cells that have been transfected with a desired rAAV or rAAV plasmid vector, and with such one or more viruses and/or helper plasmids that can provide proteins or RNA molecules that are not provided by such rAAV or rAAV plasmid vectors, but are required for their production.
• proteins or RNA molecules include the genes encoding the Rep52 and Rep78 proteins that are required for vector transcription control and replication, and for the packaging of viral genomes into the viral capsule, and, in the case of rAAV, cap genes that encode VP capsid proteins required to form infectious particles.
• Such proteins or RNA molecules also include the viral transcription and translation factors (El a, Elb, E2a, VA and E4) required for AAV proliferation.
• all of these genes and RNA molecules are provided on the same helper virus (or more preferably, helper vector) so as to comprise, in concert with an rAAV, a double plasmid transfection system.
• the required rep and cap genes are provided by one plasmid, and the genes that encode the viral transcription and translation factors are provided on a second plasmid, so that such plasmids, in concert with the rAAV, comprise a triple plasmid transfection system.
• the methods of the present invention may be employed to increase the production titer of rAAV belonging to any serotype, including the AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10 serotypes and the rAAVl, rAAV2, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9, and rAAVIO serotypes, and including hybrid serotypes ( e.g ., AAV2/5 and rAAV2/5, which is a hybrid of serotypes 2 and 5 and thus has the trophism of both such serotypes).
• hybrid serotypes e.g ., AAV2/5 and rAAV2/5, which is a hybrid of serotypes 2 and 5 and thus has the trophism of both such serotypes.
• the methods of the present invention may be employed to enhance the production titers of rAAV that are to be produced using“helper” RNA or proteins provided by an adenovirus, a herpes simplex virus, a cytomegalovirus, a vaccinia virus or a papillomavirus.
• the methods of the present invention may be employed to enhance the production titers of rAAV produced by cells in adherent monolayer culture or in suspension culture, and may be used with any method capable of producing rAAV.
• 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.
• BHK cell line BHK-21 ATCC CCL- 10
• which lacks endogenous retroviruses is a preferred BHK cell line.
• the HEK cell line HEK293 (ATCC CRL-1573) and its derivatives, such as HEK293T (ATCC CRL- 3216, which is a highly transfectable derivative of the HEK293 cell line into which the temperature-sensitive gene for SV40 T-antigen was inserted) or HEK293T/17 (ATCC® CRL-11268, which was selected for its ease of transfection) are particularly preferred.
• the HEK293T/17 SF cell line (ATCC ACS-4500) is a derivative of the 293T/17 cell line (ATCC CRL-11268), adapted to serum-free medium and suspension, and may be employed if desired.
• the preferred base medium of the present invention for culturing such cells is Eagle’s Minimum Essential Medium (ATCC Catalog No. 30-2003) or Dulbecco’s Modified Eagle’s Medium (DMEM; Mediatech, Manassas, VA). Fetal bovine serum (e.g ., FBS; HyClone Laboratories, South Logan, 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.
• Dulbecco’ s Modified Eagle’s Medium (commercially available from, e.g., ThermoFisher Scientific) additionally contains approximately the inorganic salts shown in Table 1.
• the media differ in that Dulbecco's modified Eagle's medium contains approximately four times as much of the vitamins and amino acids present in the original formula of Eagle’s Minimum Essential Medium, and two to four times as much glucose. Additionally, it contains iron in the form of ferric sulfate and phenol red for pH indication (Yao, T et al. (2017)“ Animal-Cell Culture Media: History, Characteristics, And Current Issues,” Reproduc. Med. Biol. 16(2): 99-117).
• Cells to be used for such transfection are preferably passaged twice weekly to maintain them in exponential growth phase.
• an aliquot of, for example, 1 x 10 6 HEK293 or BHK cells per well on a multi-well plate, or 1.5 x 10 7 HEK293 cells per 15-cm dish may be employed.
• 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 two cell stacks may be trypsinized and the cells (e.g ., approximately 6 x 10 8 cells) may be resuspended in 200 ml of medium. Preferably, the cells are allowed to attach for 24 hours before transfection. Confluency of the cell stacks may be monitored using a Diaphot inverted microscope (Nikon, Melville, NY) from which the phase-contrast hardware had been removed in order to accommodate the cell stack on the microscope stage.
• the phrase “ionic strength” is intended to denote the concentration of ions in a solution.
• the present invention enhances rAAV production titers by increasing the ionic strength of the culture medium by providing additional ions to the medium used to culture rAAV transfected cells.
• the provided ions are cations. Suitable cations include Na + , K + , Ca ++ , and Mg ++ . Such cations may be provided as an inorganic salt or as a salt of organic molecule. In another embodiment, the provided ions are anions.
• CbCOO- acetate
• aspartate biphthalate
• Such cations or anions may be provided at any concentration sufficient to enhance rAAV production titers over the titers produced in the same culture medium without any such additionally provided cations.
• Suitable concentrations of such cations or anions include concentrations sufficient to increase the initial concentration of such ion in a culturing medium by from about 30 mM to about 80 mM, by from about 40 mM to about 80 mM, by from about 50 mM to about 80 mM, by from about 60 mM to about 80 mM, by from about 70 mM to about 80 mM, or by about 80 mM, with such concentrations being in addition to any concentration of such ion present in such culture medium prior to such addition.
• such culture medium did not initially contain the ions to be administered, then such added ions are preferably provided in an amount sufficient to provide concentrations of the provided ions in such culture medium of from about 30 mM to about 80 mM, by from about 40 mM to about 80 mM, by from about 50 mM to about 80 mM, by from about 60 mM to about 80 mM, by from about 70 mM to about 80 mM or to about 80 mM.
• the ions or salts that are to be added to the initial culture medium may be added at any time prior to the harvesting of produced rAAV.
• such ions or salts will have been added at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 12 hours, at least about 15 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, or at least about 24 hours after the initiation of the culturing.
• the term“about” when used with reference to a concentration, amount, or time is intended to denote such concentration and also a range of concentrations that is within ⁇ 40% of such concentration, and more preferably within ⁇ 30% of such concentration, and still more preferably within ⁇ 20% of such concentration, and still more preferably within ⁇ 10% of such concentration, and still more preferably within ⁇ 5% of such concentration.
• a recited concentration of 10.0 mM denotes a concentration of 10.0 mM, as well as a concentration between 6-14 mM, and more preferably a concentration between 7-13 mM and still more preferably a concentration between 8-12 mM, and still more preferably a concentration between 9-11 mM, and still more preferably a concentration between 9.5-10.5 mM.
• the present invention thus provides a method for increasing the production titer of recombinantly-modified AAV (rAAV) that comprises the steps:
• step (C) continuing the culturing of the cells to thereby produce a production titer of rAAV that is greater than a titer obtained in the absence of step (B).
• the invention particularly contemplates the use of KHCCb to enhance rAAV production titer.
• KHCCb is preferably provided in an amount sufficient to increase the concentrations of K + and HCCb- in the culture medium by about 20 mM, by about 30 mM, by about 40 mM, or by about 50 mM.
• Such addition would cause the K + concentration in Dulbecco’s Modified Eagle’s Medium to be about 25 mM, about 35 mM, about 45 mM, or about 55 mM, and would cause the HCCb- concentration in such medium to be about 64 mM, about 74 mM, about 84 mM or about 94 mM.
• such culture medium did not contain K + and HCCb- ions, then such KHCCb is preferably provided in an amount sufficient to provide concentrations of K + and HCCb- in such culture medium of about 20 mM, of about 30 mM, or of about 40 mM.
• the invention additionally includes pharmaceutical compositions that comprise a pharmaceutically acceptable preparation of rAAV produced in accordance with the methods of the present invention, and a pharmaceutically acceptable carrier.
• the rAAV of such pharmaceutical compositions comprises a transgene cassette that encodes a protein, or comprises a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition, and is present in such pharmaceutical composition in an amount effective to (“effective amount”)
• the term“pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the US Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
• the term“carrier” refers to a diluent, adjuvant (e.g ., Freund’s adjuvant complete and incomplete), excipient, or vehicle with which the therapeutic is administered.
• Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
• Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
• Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
• the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
• Suitable pharmaceutical excipients are described in US Patents No. 8,852,607; 8,192,975; 6,764,845; 6,759,050; and 7,598,070.
• 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.
• a hermetically sealed container such as a vial, an ampoule or sachette indicating the quantity of active agent.
• the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
• an ampoule of sterile water for injection or saline, or other diluent can be provided so that the ingredients may be mixed prior to administration.
• the invention also provides a pharmaceutical pack or kit comprising one or more containers such pharmaceutical composition.
• container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
• the rAAV of such pharmaceutical compositions is preferably packaged in a hermetically sealed container, such as a vial, an ampoule or sachette indicating the quantity of the molecule, and optionally including instructions for use.
• 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.
• the rAAV of such kit is supplied as an aqueous solution in a hermetically sealed container and can be diluted, e.g.
• 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.
• the other prophylactic or therapeutic agent is a chemotherapeutic.
• the prophylactic or therapeutic agent is a biological or hormonal therapeutic.
• the methods of the present invention may be used to facilitate the production of rAAV, and may particularly be used to facilitate the production of rAAV that comprise transgene cassettes that encode a protein (e.g, an enzyme, hormone, antibody, receptor, ligand, etc.), or of rAAV that comprise a transcribed nucleic acid, that is relevant to a genetic or heritable disease or condition, such that it may be used in gene therapy to treat such disease or condition.
• a protein e.g, an enzyme, hormone, antibody, receptor, ligand, etc.
• achromatopsia ACLM
• alpha-1 antitrypsin AAT
• AADC aromatic L-amino acid decarboxylase
• CHM choroideremia
• BMDSIBM 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 1C (LGMD1C); limb girdle muscular dys
• the invention concerns a method for increasing the production titer of recombinantly-modified adeno-associated virus (rAAV), the recombinantly-modified adeno-associated virus (AAV) helper vector produced from such method, and uses and compositions thereof. It is particularly directed to the following embodiments E1-E19: El.
• step (C) continuing the culturing of the cells to thereby produce a production titer of with the rAAV that is greater than a titer obtained in the absence of step (B).
• each of the added ion(s) is provided in an amount sufficient to increase the concentration of such ion in the initial culture medium by from about 10 mM to about 80 mM.
• E3. The method of any one of El or E2, wherein the production titer is at least 50% greater than the titer obtained from a similarly conducted cell culturing in the absence of the step (B).
• E4 The method of any one of E1-E3, wherein the rAAV comprises a transgene cassette that encodes a protein, or comprises a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition.
• E5 The method of any one of E1-E4, wherein the rAAV belongs to the rAAVl, rAAV2, rAAV5, rAAV6, rAAV7, rAAV8, rAAV9 or rAAVIO serotype, or to a hybrid of the serotypes.
• E6 The method of E5, wherein the rAAV belongs to the rAAV2, rAAV5, or rAAV9 serotype, or to a hybrid of the serotypes.
• E7 The method of any one of E1-E6, wherein the added ions comprise one or more of K + , Ca ++ , or Mg ++ .
• E9 The method of any one of E1-E7, wherein the added ions comprise one or more of acetate, aspartate, biphthalate, bitartrate, butoxyethoxy acetate, caprylate, citrate, dehydroacetate, diacetate, dihydroxy glycinate, d-saccharate, gluconate, glutamate, glycinate, glycosulfate, hydroxymethane sulfonate, lactate, methionate, oxalate, phenate, phenosulfonate, propionate, propionate, saccharin, salicylate, sarcosinate, sorbate, thioglycolate, and toluene sulfonate.
• the added ions comprise one or more of acetate, aspartate, biphthalate, bitartrate, butoxyethoxy acetate, caprylate, citrate, dehydroacetate, diacetate, dihydroxy glycinate, d
• E13 The method of any one of E1-E10, wherein the cells are baby hamster kidney cells.
• E14 The method of E13, wherein the cells are BHK21 cells.
• E15 The method of any one of E1-E10, wherein the cells are sf9 insect cells.
• E17 The method of E16, wherein the initial culture medium is supplemented with serum.
• a pharmaceutical composition that comprises:
• rAAV recombinantly-modified adeno-associated virus
• rAAV produced by the method of any one of El -El 7, wherein the rAAV comprises a transgene cassette that encodes a protein, or a transcribed nucleic acid, or the pharmaceutical composition of E18, for use in the treatment of a genetic or heritable disease or condition.
• the plasmid vector pAAV-RC2 which is capable of expressing the AAV rep and cap gene functions that are required for the replication and packaging of an rAAV
• the plasmid vector pHelper which is capable of providing the viral transcription and translation factors (Ela, Elb, E2a, VA and E4) required for AAV proliferation
• rAAV plasmid vector pAV-CMV-EGFP which comprises the transgene cassette encoding the enhanced green fluorescent protein (EGFP) and the AAV ITRs.
• Figure 7A shows the extent of expression of EGFP in the transfected cells and the titering of the rAAV stocks using the infectious center assay.
• Figure 7B is a graph of the fold-change in infectious centers as a function of such cation and cation concentrations.
• Figure 7C is a graph of the fold-change in Total Genomes (TG) of AAV as a function of such cation and cation concentrations.
• TG Total Genomes
• salt either K2CO3, KHCCb, KFbPCri, KCFbCOO (potassium acetate), KCNS, K2SO4, KNCb, K3C6H5O7 (potassium citrate) or KCL
• KHCCb was found to cause the greatest increase in rAAV production, with the greatest increase seen at concentrations sufficient to increase the concentrations of such ions by between about 40 mM to about 50 mM ( Figure 8A).
• Figure 8B is a graph of the fold-change in the titer of rAAV vector as a function of such anion and anion concentrations.
• the results show that the provision of anions affected the total genomes (TG) produced.
• the provision of high concentrations of ions > 60 mM was found to attenuate rAAV production.
• the results demonstrate that the provision of KHCO3 in an amount sufficient to increase the concentrations of such ions in the culture medium by between about 30 mM and about 50 mM provided unexpectedly better results than those obtained with other salts ( Figures 9A-9B). An increase in concentration by about 30 mM was considered optimum.
• HEK293 cells were cultured and co-transfected with: (1) the above-described Ad helper plasmid, (2) the pAAV-ITR plasmid vector that provides the AAV ITRs and transgene cassette encoding the enhanced green fluorescent protein and (3) an AAV2 helper plasmid or an AAV8 helper plasmid in order to provide the AAV rep and cap gene functions. Culture medium had been changed one hour before the co-transfections.
• KHCCb was added in an amount sufficient to increase the concentrations of such ions in the culture medium by a concentration of 30 mM and the fold-change of rAAV that had been released into the medium was assessed at 72 hours. The fold-change in the total amount of rAAV produced was also assessed ( Figure 10). The results indicate that the greatest enhancement was seen when salts were added 4-8 hours post-transfection.
• AAV2 helper plasmid encoding Cap proteins of serotypes 1, 2, 5, 6, 7, 8, 9 or 10 were transfected into HEK293 cells along with the above-described Ad helper plasmid and a pAAV-ITR plasmid vector (pAV-TBG-EGFP) that provides the AAV ITRs and a transgene cassette encoding the enhanced green fluorescent protein.
• pAV-TBG-EGFP pAAV-ITR plasmid vector
• HEK293 cells were co-transfected with: (1) the above-described Ad helper plasmid, (2) the pAAV-ITR plasmid vector that provides the AAV ITRs and transgene cassette encoding the enhanced green fluorescent protein and (3) an AAV5 helper plasmid or an AAV6 helper plasmid in order to provide the AAV rep and cap gene functions.
• KHCCb was added in an amount sufficient to increase the concentrations of such ions in the culture medium by 10, 20, 30, 40, 50 or 60 mM at 5 hours or 20 hours post-transfection.

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