RU2802520C2 - AMPLIFIER AGENTS TO INCREASE CELL TRANSFECTION AND/OR rAAV VECTOR PRODUCTION - Google Patents

Abstract

FIELD: molecular biology and biotechnology.

SUBSTANCE: invention relates to the compositions and methods of producing transduced (transfected) cells, where the said cells optionally produce an adeno-associated viral vector (rAAV). Cells transduced/transfected at high efficiency with plasmids containing (i) nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding helper proteins; and (ii) a transgene that encodes a protein or is transcribed into a transcript of interest can produce a large amount of the recombinant rAAV vector.

EFFECT: obtaining amplifier agents to increase cell transfection and/or rAAV vector production.

60 cl, 8 dwg, 8 ex

Description

Information on related applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/516,432, filed June 7, 2017, and U.S. Provisional Patent Application No. 62/531,626, filed July 12, 2017. The contents of the foregoing applications are incorporated herein in their entirety by references, including all text, tables, sequence lists, and figures.

Field of technology to which the invention relates

[0002] The present invention relates to the field of transduction (transfection) of cells with nucleic acid, for example, plasmids. More specifically, the invention relates to compositions and methods for producing transduced (transfected) cells, wherein said cells optionally produce an adeno-associated viral vector (rAAV).

Introduction

[0003] Several publications and patent documents are cited throughout the application to describe the prior art to which the present invention relates. Each of these documents cited is incorporated herein by reference as if set forth in its entirety.

The essence of the invention

[0004] The invention provides compositions and methods for transfecting cells with at least one nucleic acid sequence. In one embodiment, the transfection composition or method comprises: (a) contacting cells with at least one nucleic acid formulated with a solution containing polyethylenimine (PEI); (b) incubating or culturing cells with nucleic acid and polyethylenimine (PEI) solution; (c) adding an enhancing agent during or immediately after step (a) or before but less than 3 hours after step (a) to obtain a mixture; and (d) incubating said mixture from step (c), thereby transfecting the cells with the nucleic acid sequence.

[0005] The invention also provides compositions and methods for producing cells that produce recombinant viral vectors, such as the rAAV vector. In one embodiment, the composition or method includes: (a) providing a PEI/plasmid mixture of components (i), (ii) and (iii), wherein (i) is one or more plasmids containing nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding helper proteins; (ii) is a plasmid containing a transgene that encodes a protein or is transcribed into a transcript of interest; and (iii) providing a polyethylenimine (PEI) solution, (b) contacting the cells with the plasmid/PEI mixture from step (a) to obtain a cell culture with the plasmid/PEI mixture; (c) adding an enhancing agent to the cell culture with the plasmid/PEI mixture to obtain a second mixture; and (d) incubating said second mixture from step (c) to obtain transfected cells that produce the recombinant rAAV vector.

[0006] In various further embodiments of the invention, the compositions and methods include one or more additional optional steps.

[0007] In a specific aspect, the additional step comprises (e) collecting said transfected cells obtained in step (d) and/or culture medium from the transfected cells obtained in step (d) to obtain a cell collection and/or culture medium.

[0008] In a particular aspect, a further optional step includes (f) culturing, expanding, isolating or selecting cells that have been transfected with the nucleic acid or plasmids.

[0009] In a specific aspect, the additional optional step includes (e) isolating and/or purifying the recombinant AAV vector from the transfected cells obtained in step (d) and/or the culture medium and/or from the transfected cells obtained in step (d) .

[0010] In a particular aspect, an additional optional step includes (f) isolating and/or purifying the recombinant AAV vector from the transfected cells and/or collecting the culture medium obtained in step (e).

[0011] In yet another embodiment, the nucleic acid sequence(s) comprises a vector and/or plasmid.

[0012] In yet another embodiment, the nucleic acid sequence(s) comprise a viral vector and/or a viral plasmid. In a particular aspect, the viral vector or viral plasmid includes a lentiviral vector or plasmid or an adeno-associated viral vector (AAV) or plasmid.

[0013] In yet another embodiment, the vector contains a transgene that encodes a protein or is transcribed into a transcript of interest. In a particular aspect, the transgene encodes a wild-type or functional variant coagulation factor, apoE2, TPP1, argininosuccinate synthase, copper-transporting ATPase 2, acid alpha-glucosidase, β-glucocerebrosidase, α-galactosidase, or a C1 serine protease inhibitor. In a particular aspect, the wild-type or functional variant clotting factor is factor VII, factor VIII, or factor IX.

[0014] In yet another embodiment, the enhancing agent is added before step (a), during step (a), or immediately after step (a).

[0015] In yet another embodiment, the enhancing agent is added before step (a), during step (a), or no more than 16 hours after step (a).

[0016] In yet another embodiment, the enhancing agent is added before step (a), during step (a), or before, but not less than 3 hours after step (a).

[0017] In yet another embodiment, the enhancing agent is added before step (a), during step (a), the enhancing agent is added two or more times before or after steps (a) or (b).

[0018] In yet another embodiment, the enhancing agent is first added during or immediately after step (a) or no more than 16 hours after step (a) and added again 16-72 hours after steps (a) or (b ), or added again 16-48 hours after steps (a) or (b), or added again 16-24 hours after steps (a) or (b).

[0019] In yet another embodiment, the enhancing agent is first added during or immediately after step (a) or before, but not less than 3 hours after step (a), and added again 12-72 hours after steps (a ) or (b), or added again 12-48 hours after steps (a) or (b), or added again 12-24 hours after steps (a) or (b).

[0020] In yet another embodiment, the enhancing agent is first added 12-72 hours before step (a) and added again during or immediately after step (a) or no more than 16 hours after step (a).

[0021] In yet another embodiment, the enhancing agent is first added 12-72 hours before step (a) and added again during or immediately after step (a) or before, but not less than 3 hours after step (a) .

[0022] In yet another embodiment, plasmids (i) and (ii) are in a molar ratio range of about 1:0.01 to about 1:100, or in a molar ratio range of about 100:1 to about 1:0, 01, and wherein the mixture of components (i), (ii) and (iii) are optionally incubated for a period of time from about 10 seconds to about 4 hours before step (b).

[0023] In yet another embodiment, the nucleic acid or plasmids are in a PEI:nucleic acid or PEI:plasmid weight ratio ranging from about 0.1:1 to about 5:1, or a PEI:nucleic acid or PEI:plasmid weight ratio of ranging from about 5:1 to about 0.1:1.

[0024] In yet another embodiment, the nucleic acid or plasmids are in a PEI:nucleic acid or PEI:plasmid weight ratio ranging from about 1:1 to about 5:1 or in a PEI:nucleic acid or PEI:plasmid weight ratio in the range from about 5:1 to about 1:1.

[0025] In yet another embodiment, the nucleic acid or plasmids are in a PEI:nucleic acid or PEI:plasmid weight ratio ranging from about 1:1 to about 3:1.

[0026] In yet another embodiment, the nucleic acid or plasmids are in a PEI:nucleic acid or PEI:plasmid weight ratio in the range of about 1:1, about 1.5:1, about 2:1, about 2.5:1, or approximately 3:1.

[0027] In yet another embodiment, the compositions or methods further comprise adding free PEI to the cells.

[0028] In yet another embodiment, free PEI is added to the cells before, during or after steps (a) or (b), or before or during or after step (c).

[0029] In yet another embodiment, free PEI is added to the cells during or after steps (a) or (b) or during or after step (c).

[0030] In yet another embodiment, free PEI is added such that the PEI:nucleic acid or PEI:plasmid weight ratio is in the range of about 0.1:1 to about 5:1 or in the range of about 5:1 to about 0 ,1:1.

[0031] In yet another embodiment, free PEI is added such that the weight ratio of PEI:nucleic acid or PEI:plasmid is in the range of about 1:1 to about 5:1 or in the range of about 5:1 to about 1:1 .

[0032] In yet another embodiment, the PEI in the PEI:nucleic acid and/or PEI:plasmid mixture and/or free PEI contains linear polyethylenimine.

[0033] In yet another embodiment, the PEI in the PEI:nucleic acid and/or PEI:plasmid mixture and/or free PEI contains hydrolyzed linear polyethylenimine.

[0034] In yet another embodiment, the PEI in the PEI:nucleic acid and/or PEI:plasmid mixture and/or free PEI contains hydrolyzed linear polyethylenimine with a molecular weight in the range of about 4,000 to about 160,000 and/or with a molecular weight in the range of about from 2500 to about 250,000 in free base form.

[0035] In yet another embodiment, the PEI in the PEI:nucleic acid and/or PEI:plasmid mixture and/or free PEI contains hydrolyzed linear polyethylenimine with a molecular weight of about 40,000 and/or a molecular weight of about 25,000 in free base form.

[0036] In yet another embodiment, the molar ratio of nitrogen (N) in total PEI to phosphate (P) in the nucleic acid:PEI and/or plasmid:PEI mixture ranges from about 1:1 to about 50:1 (N: P).

[0037] In yet another embodiment, the molar ratio of nitrogen (N) in total PEI to phosphate (P) in the nucleic acid:PEI and/or plasmid:PEI mixture is from about 5:1 to about 10:1.

[0038] In yet another embodiment, the molar ratio of nitrogen (N) in total PEI to phosphate (P) in the nucleic acid:PEI and/or plasmid:PEI mixture is any of about 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1 (N:P).

[0039] In yet another embodiment, the amount of free PEI is from about 10% to about 90% of the total PEI.

[0040] In another embodiment, the amount of free PEI is from about 25% to about 75% of the total PEI.

[0041] In another embodiment, the amount of free PEI is approximately 50% of the total PEI.

[0042] In another embodiment, the concentration of free PEI is from about 0.1 μg/ml to about 10 μg/ml.

[0043] In another embodiment, the concentration of free PEI is from about 1.0 μg/ml to about 5 μg/ml.

[0044] In yet another embodiment, the PEI and/or free PEI solution contains a solution having a pH of from about 7.0 to about 8.0.

[0045] In yet another embodiment, the nucleic acid sequence and PEI are incubated together for about 10 seconds to about 4 hours before step (a).

[0046] In another embodiment, the nucleic acid sequence and PEI are incubated together for about 30 seconds to about 4 hours before step (a).

[0047] In another embodiment, the nucleic acid sequence and PEI are incubated together for about 1 minute to about 30 minutes before step (a).

[0048] In yet another embodiment, the mixture of components (i), (ii) and (iii) are incubated together for about 10 seconds to about 4 hours before step (b).

[0049] In yet another embodiment, the mixture of components (i), (ii) and (iii) are incubated together for about 30 seconds to about 4 hours before step (b).

[0050] In yet another embodiment, the mixture of components (i), (ii) and (iii) are incubated together for about 1 minute to about 4 hours before step (b).

[0051] In another embodiment, the incubation in step (d) is at least about 4 hours.

[0052] In another embodiment, the incubation in step (d) is from about 4 hours to about 140 hours.

[0053] In another embodiment, the incubation in step (d) is from about 4 hours to about 96 hours.

[0054] In yet another embodiment, the cells include mammalian cells. In particular aspects, the cells are human embryonic kidney (HEK) cells or Chinese hamster ovary (CHO) cells. In particular aspects, the cells include human embryonic kidney (HEK) 293 cells. In particular aspects, the cells are HEK 293E, HEK 293F, or HEK 293T cells.

[0055] In yet another embodiment, the cells are stably or transiently transfected.

[0056] In another embodiment, the cells are in suspension culture.

[0057] In another embodiment, the cells are attached to the substrate.

[0058] In yet another embodiment, the cells are cultured or maintained in a serum-free culture medium.

[0059] In yet another embodiment, the cells have a density ranging from about 1x10 ⁵ cells/ml to about 1x10 ⁸ cells/ml when contacted with said nucleic acid sequence or said plasmid/PEI mixture and/or when contacted with specified free PEI.

[0060] In yet another embodiment, the cells have a density ranging from about 5 x 10 ⁵ cells/mL to about 1 x 10 ⁷ cells/mL when contacted with said nucleic acid sequence or said plasmid/PEI mixture and/or when contacted with specified free PEI.

[0061] In yet another embodiment, the cells have a density ranging from about 1 x 10 ⁶ cells/mL to about 5 x 10 ⁶ cells/mL when contacted with said nucleic acid sequence or said plasmid/PEI mixture and/or when contacted with specified free PEI.

[0062] In yet another embodiment, the cell viability when contacted with said nucleic acid sequence or plasmid/PEI mixture or with said free PEI is about 60% or greater than 60%, or wherein said cells are in logarithmic growth phase when contacted with said sequence nucleic acid or plasmid/PEI mixture.

[0063] In yet another embodiment, the cell viability when contacted with said nucleic acid sequence or plasmid/PEI mixture or with said free PEI is about 90% or greater than 90%, or wherein said cells are in logarithmic growth phase when contacted with said sequence nucleic acid or a mixture of plasmid/PEI or with the specified free PEI.

[0064] In yet another embodiment, the total amount of nucleic acid sequence or plasmid is in the range of from about 0.1 μg to about 15 μg per ml of cells.

[0065] In yet another embodiment, the molar ratio of the plasmid containing the transgene to one or more plasmids containing nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding helper proteins is from about 1:5 to about 1: 1, or from about 1:1 to about 5:1.

[0066] In yet another embodiment, the one or more plasmids comprise a first plasmid containing nucleic acids encoding AAV packaging proteins and a second plasmid containing nucleic acids encoding helper proteins.

[0067] In yet another embodiment, the molar ratio of the plasmid containing the transgene to the first plasmid containing nucleic acids encoding AAV packaging proteins to the second plasmid containing nucleic acids encoding helper proteins is in the range of about 1-5:1: 1, or 1:1-5:1, or 1:1:1-5.

[0068] In yet another embodiment, the encoded AAV packaging proteins include AAV rep and/or AAV cap.

[0069] In yet another embodiment, the encoded AAV packaging proteins include the AAV rep and/or AAV cap proteins of any AAV serotype.

[0070] In yet another embodiment, the encoded helper proteins include adenoviral E2 and/or E4, VARNA proteins and/or non-AAV helper proteins.

[0071] In yet another embodiment, the recombinant AAV vector comprises any of AAV serotypes 1-12, AAV capsid protein VP1, VP2 and/or VP3, or a modified or variant AAV capsid protein VP1, VP2 and/or VP3, or AAV capsid protein VP1 , VP2 and/or wild-type VP3.

[0072] In yet another embodiment, the adeno-associated viral vector (AAV) comprises a VP1, VP2 and/or VP3 capsid protein sequence or an inverted terminal repeat sequence having 70% or more sequence identity with a capsid protein sequence or an inverted terminal repeat (ITR) sequence. any capsid protein or ITR sequence selected from serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10.

[0073] In yet another embodiment, the adeno-associated viral vector (AAV) comprises a VP1, VP2 and/or VP3 capsid protein sequence having 70% or more sequence identity with a capsid protein sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2.

[0074] In yet another embodiment, the AAV vector comprises an AAV serotype or an AAV pseudotype, wherein the AAV pseudotype comprises an AAV capsid serotype other than an ITR serotype.

[0075] In yet another embodiment, the AAV vector comprises a VP1, VP2 and/or VP3 capsid protein or inverted terminal repeat of any serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, SEQ ID NO: 1 and SEQ ID NO: 2.

[0076] In yet another embodiment, the AAV vector further comprises an intron, an expression control element, one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs), and/or a filler polynucleotide sequence.

[0077] In yet another embodiment, the intron is located within or flanks a nucleic acid that encodes a protein, or is transcribed into a transcript of interest.

[0078] In yet another embodiment, the expression control element is operably linked to a nucleic acid that encodes a protein or is transcribed into a transcript of interest.

[0079] In yet another embodiment, the AAV ITR(s) flank the 5' or 3' end of a nucleic acid that encodes a protein or is transcribed into a transcript of interest.

[0080] In yet another embodiment, a filler polynucleotide sequence flanks the 5' or 3' end of a nucleic acid that encodes a protein or is transcribed into a transcript of interest.

[0081] In yet another embodiment, the expression control element includes a constitutive or regulated control element or a tissue-specific expression control element or promoter.

[0082] In yet another embodiment, the expression control element includes an element that provides expression in the liver.

[0083] In another embodiment, the ITR includes one or more ITRs of any of the AAV2 or AAV6 serotypes, or a combination thereof.

[0084] In another embodiment, the cells are subcultured to restored cell density before contacting the specified nucleic acid sequence or plasmid/PEI mixture.

[0085] In yet another embodiment, the cells are subcultured to a cell density ranging from about 0.1 x 10 ⁵ cells/ml to about 5.0 x 10 ⁶ cells/ml before contacting the specified nucleic acid sequence or plasmid/PEI mixture.

[0086] In another embodiment, the cells are contacted with the specified nucleic acid sequence or plasmid/PEI mixture for a period of 2 days to 5 days after subculture.

[0087] In yet another embodiment, the cells are contacted with the specified nucleic acid sequence or plasmid/PEI mixture for a period of 3 days to 4 days after subculture.

[0088] In yet another embodiment, the amount of nucleic acid sequence or plasmids inserted into said transfected cells is at least 50% greater during the step of adding free PEI to the cell culture compared to an option without adding free PEI to the cell culture.

[0089] In yet another embodiment, the amount of nucleic acid sequence or plasmids inserted into said transfected cells is at least 50% greater with the step of adding an enhancing agent compared to without the addition of an enhancing agent.

[0090] In yet another embodiment, the amount of recombinant AAV vector produced is at least 50% or more with the step of adding free PEI to the plasmid/PEI mixture cell culture compared to without adding free PEI to the plasmid mixture cell culture /PEI.

[0091] In yet another embodiment, the amount of recombinant AAV vector produced is 1-5, 5-8, 8-10, or 10-20 times greater with the step of adding free PEI to the cell culture with the plasmid/PEI mixture compared to without the addition free PEI to cell culture with a plasmid/PEI mixture.

[0092] In yet another embodiment, the amount of recombinant AAV vector produced is 1-5, 5-8, 8-10, or 10-15 times greater with the enhancer step compared to without the enhancer added in the cell culture with the plasmid mixture /PEI.

[0093] In yet another embodiment, the cells are in a culture volume of about 10-500 mL, 500 mL-2 L, 2-20 L, 20-50 L, 50-100 L, 100-500 L, 500-1000 L, or 1000-2000 l.

[0094] In yet another embodiment, the transgene has a size of about 4.0 kb. to about 6.0 kb.

[095] In yet another embodiment, the transgene has a size of about 4.5 kb. to about 6.0 kb.

[0096] In yet another embodiment, the transgene has a size of about 4.5 kb. to about 5.5 kb.

[0097] In yet another embodiment, the transgene has a size of about 4.5 kb. to about 5.0 kb.

[0098] In yet another embodiment, the enhancing agent includes valproic acid, a salt or derivative thereof. In a particular aspect, the valproic acid salt includes a sodium or potassium salt. In a particular aspect, the valproic acid derivative includes an amino acid linked or conjugated thereto.

[0099] In yet another embodiment, the enhancing agent includes isobutyric acid, a salt or derivative thereof. In a particular aspect, the isobutyric acid salt includes a sodium or potassium salt. In a particular aspect, the isobutyric acid derivative includes an amino acid linked or conjugated thereto.

[0100] In yet another embodiment, the enhancing agent includes isovaleric acid, a salt or derivative thereof. In a particular aspect, the isovaleric acid salt includes a sodium or potassium salt. In a particular aspect, the isovaleric acid derivative includes an amino acid linked or conjugated thereto.

[0101] In another embodiment, after addition, the enhancing agent(s) are present at a concentration of from about 0.1 mM to about 25 mM.

[0102] In another embodiment, after addition, the enhancing agent(s) are present at a concentration of from about 0.5 mM to about 10 mM.

[0103] In another embodiment, after addition, the enhancing agent(s) are present at a concentration of from about 0.5 mM to about 5 mM.

[0104] In yet other embodiments, after addition, the enhancing agent(s) are at a concentration of about 1 mM to about 10 mM, about 1 mM to about 9 mM, about 1 mM to about 8 mM, about 1 mM to about 7 mM, about 1 mM to about 6 mM, about 1 mM to about 5 mM, about 1 mM to about 4 mM, about 1 mM to about 3 mM, or about 1 mM to about 2 mM.

[0105] In still other embodiments, any of steps (a)-(f) or conditions set forth in any of claims 1-94 are performed as set forth in any of Examples 1-7.

Brief description of the figures

[0106] In FIG. Figure 1 shows a comparison of rAAV-FVIII vector production. HEK 293F cells in spinner flasks were transfected with three plasmids: the pAd helper plasmid containing helper genes from the adenovirus for the production of rAAV; pAAV rep/cap expresses the AAV Rep and Cap genes; pAAVhFVIII, containing a human factor VIII expression cassette flanked by the AAV ITR. The total amount of DNA used was 1.86 μg/ml with a molar ratio of 1:1:1. A PEI/DNA ratio of 1:1 (by weight) was used to prepare the PEI/DNA complex and transfect cells. Additional free PEI (the same amount used to prepare the PEI/DNA complex) was also added to the cell culture separately during transfection. Enhancers 1 and 2 from the ExpiFectamine™ 293 Transfection Kit were added to the cells either simultaneously with transfection or 16-18 hours after transfection (according to the manufacturer's instructions). Amplifiers 1 and 2 were used in a ratio of 1:200 and 1:20 to the culture volume, respectively. The cell culture was harvested 72 h after transfection, and the rAAV-FVIII vector was detected by qPCR assay. The rAAV-FVIII vector titer was 2- to 3-fold higher when enhancers were added during transfection compared to when enhancers were added 16 h posttransfection or when enhancers were not added.

[0107] In FIG. Figure 2 shows optimization of rAAV-FVIII vector production using different amounts of DNA and PEI/DNA ratios. HEK 293F cells in spinner flasks were transfected with total amounts of 1.2, 1.86, or 2.8 μg/ml DNA, respectively, using three plasmids. The ratio of plasmid DNA was the same as described above. However, the PEI/DNA ratios used in this study were 1:1, 1.5:1, 2:1, 2.5:1, and 3:1. The concentration of free PEI used was 1.5 μg/mL. Enhancers 1 and 2 from the ExpiFectamine™ 293 transfection kit were used as described above. The cell density during transfection was 2-3×10 ⁶ cells/ml. Samples were collected 48 h (#) or 72 h (*) after transfection, and the rAAV vector titer was determined by qPCR assay. The highest yields of the rAAV vector were observed under conditions with 1.2 μg/ml DNA at a PEI/DNA ratio of 2-2.5:1 and with amplifiers.

[0108] In FIG. 3 confirms the presence of rAAV-FVIII vector production in the bioreactor. HEK 293F cells were cultured in 400 ml bioreactors and transfected with three plasmids (total amount of plasmid DNA was 1.2 or 2.8 μg/ml). Plasmid DNA molar ratio was 1:1:1, PEI/DNA ratio (by weight) was 1.5:1, 2:1, 2.5:1 and 3:1. Free PEI at a concentration of 1.5 μg/ml and enhancers 1 and 2 were also used as described above. The cell density during transfection was 2-3×10 ⁶ cells/ml. The highest vector production (qPCR data) was observed under conditions with 1.2 μg/ml total DNA with PEI/DNA ratios of 2.5-3:1.

[0109] In FIG. Figure 4 shows that high rAAV vector production is maintained when the DASGIP bioreactor is fully scaled up. Optimized transfection conditions were further evaluated for rAAV production in a 1.2 L volume, full scale-up of the DASGIP bioreactor. HEK 293F cells were cultured in 1.2 L DASGIP bioreactors with 2 or 3 inclined impeller mixers at stirring speeds of 130 rpm, 150 rpm and 170 rpm. Cells were transfected with three plasmids with a total plasmid DNA of 1.2 μg/ml, and a PEI/DNA ratio of 2.5:1 was used to obtain the PEI/DNA complex; DNA molar ratio and amount of free PEI and transfection enhancers were as described above. qPCR data showed that vector production at 1.2 L volume remained comparable to that observed at smaller volumes, indicating that optimized transfection can be scaled up. Without wishing to be bound by theory, the qPCR data suggest that high mixing rates may further enhance vector production.

[0110] Figure 5 shows that one amplifier 1 increased the production of rAAV to the same level as was obtained using both amplifiers 1 and 2. To further optimize the rAAV production method, studies were conducted using only one amplifier: only amplifier 1 or only enhancer 2 during transfection, to compare vector production with that using both enhancers. qPCR data showed that rAAV production using booster 1 was comparable to that using both boosters 1 and 2. However, booster 2 resulted in decreased rAAV production, suggesting that booster 2 does not have a beneficial effect on rAAV production. In addition, reducing the amount of enhancers 1 and 2 during transfection resulted in a decrease in rAAV titer.

[0111] In FIG. Figure 6 shows that repeated use of enhancers 1 and 2 as during transfection (TF), 24 hours or 48 hours after transfection, or use of enhancers 1 and 2 three times again, during transfection and 24 hours and 48 hours after transfection, slightly increased RAAV titers. Enhancers 1 and 2 were used at a ratio of 1:200 and 1:20 to culture volume, respectively, initially during transfection. Additional amounts of enhancers 1 and 2 were added after 24 hours or 48 hours, or 24 hours and 48 hours after transfection into the cell culture. The transfection conditions used in this study are shown in Fig. 5. qPCR data showed that rAAV vector production increased slightly (less than 1-fold) with repeated use of enhancers.

[0112] Figure 7 shows data indicating increased rAAV production by valproic acid. HEK 293F cells in a spinner flask were transfected with the three plasmids using the PEI transfection method as described above. Enhancers from the ExpiFectamine™ 293 transfection kit were not used in this study. Various concentrations of valproic acid were added to the cells during transfection. Cells were transfected with 1.2 μg/ml total DNA of the three plasmids, the molar ratio of the DNA of the three plasmids was 1:1:1, the PEI/DNA ratio (by weight) was 2:1, and 1.5 μg/ml free PEI was used. The effectiveness of valproic acid was assessed in amounts from 0.25 to 2 mM. qPCR data showed that when 2 mM valproic acid was used during transfection, there was a 10-fold increase in rAAV vector production compared to the amount of vector produced in the absence of valproic acid.

[0113] In FIG. Figure 8 shows the production of the rAAV-FVIII vector using valproic acid at various concentrations, as determined by qPCR. Valproic acid in amounts ranging from 2 to 8 mM (2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM and 8 mM) was used to transfect HEK 293F cells when cultured in a 1 L bioreactor. Valproic acid at a concentration of ≥4 mM resulted in a 1.2- to 1.8-fold increase in AAV vector production compared with 2 mM valproic acid. In this study, 1.2 μg/mL DNA, DNA molar ratio 1:1:1, PEI/DNA mass ratio 2.5, and 1.5 μg/mL free PEI were used.

Detailed Description of the Invention

[0114] Disclosed herein are compositions and methods for transducing cells with a molecule, such as a nucleic acid (eg, a plasmid), with high efficiency. Such highly efficient transduced cells can, when transduced with a nucleic acid (plasmid) that encodes a protein or contains a sequence that is transcribed into a transcript of interest, can produce the protein and/or transcript with high efficiency. In addition, such cells, when transduced with sequences such as plasmids that encode viral packaging proteins and/or helper proteins and a transgene that encodes a protein or is transcribed into a transcript of interest, can produce recombinant vectors that include a transgene that encodes protein or contains a sequence that is transcribed into a transcript of interest, which in turn produces recombinant viral vectors in high yield.

[0115] The invention provides a platform for cell transfection/transduction and/or production of a viral vector (eg, AAV) that includes features that differentiate it from existing "industry standard" methods for producing a vector virus (eg, AAV). The compositions and methods of the invention are characterized by mixing PEI with nucleic acids under certain conditions. Mixing PEI with nucleic acids results in efficient PEI-induced compression of nucleic acids to form stable complexes called polyplexes. Compositions and methods for transfecting cells with nucleic acids involve contacting cells with nucleic acid mixed with PEI under certain conditions.

[0116] The compositions and methods of the invention further include adding an enhancing agent to the cells. In certain embodiments, the enhancing agent is added before, about, or simultaneously when the cells are contacted with the nucleic acid/PEI mixture. In some embodiments, the enhancing agent is added after the cells are contacted with the nucleic acid/PEI mixture. In certain aspects, the enhancing agent is added 5-30 or 30-60 seconds after the cells are contacted with the nucleic acid/PEI mixture. In certain aspects, the enhancing agent is added 1-2, 2-5, 5-10, 10-20, 20-30, 30-60 minutes after the cells are contacted with the nucleic acid/PEI mixture. In certain aspects, the enhancing agent is added 1-2, 2-4, 4-6, 6-12, 12-24, 24-36, 36-48, or 48-72 hours after the cells are contacted with the nucleic acid/PEI mixture.

[0117] The enhancing agent can be maintained in contact with the cells for a certain period of time. In some embodiments, the enhancing agent is in contact with the cells for 5 minutes to 72 hours after the cells have been contacted with the nucleic acid/PEI mixture. In some embodiments, the enhancing agent is in contact with the cells for 1-2, 2-5, 5-10, 10-20, 20-30, 30-60 minutes after the cells have been contacted with the nucleic acid/PEI mixture. In certain aspects, the enhancing agent is in contact with the cells for 1-72 hours, 6-48 hours, 12-36 hours, 24-48 hours, or 36-72 hours after the cells have been contacted with the nucleic acid/PEI mixture. In certain aspects, the enhancing agent is in contact with the cells for 1-2, 2-4, 4-6, 6-12, 12-24, 24-36, 36-48, or 48-72 hours after the cells are contacted with the nucleic acid mixture acid/PEI.

[0118] In some embodiments, cells are contacted with free PEI, or the methods include contacting cells with free PEI in a specific sequence relative to the step of contacting cells with the PEI/nucleic acid mixture. In some embodiments, the cells are contacted with free PEI at approximately the same time that the cells are contacted with the nucleic acid/PEI mixture. In specific embodiments, the cells are contacted with free PEI after the cells have been contacted with the nucleic acid/PEI mixture.

[0119] In some embodiments, cells are contacted with free PEI, or the methods include contacting cells with free PEI in a specific sequence relative to the step of adding an enhancing agent to the cells contacted with the nucleic acid/PEI mixture. In some embodiments, the cells are contacted with free PEI at approximately the same time that the cells are contacted with the enhancing agent. In certain embodiments, the cells are contacted with free PEI after the cells have been contacted with the enhancing agent.

[0120] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein and refer to all forms of nucleic acid, oligonucleotides including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids and polynucleotides include genomic DNA, cDNA and antisense DNA, as well as spliced or unspliced mRNA, rRNA tRNA and inhibitory DNA or RNA (RNA, e.g. small or short hairpin (sh)RNA, microRNA (miRNA), small or short interfering (si)RNA, trans-spliced RNA, or antisense RNA). Nucleic acids and polynucleotides include natural, synthetic, and intentionally modified or altered sequences (eg, variant nucleic acid).

[0121] Nucleic acid or plasmid can also refer to a sequence that encodes a protein. Such proteins may be wild type or a variant, modified or chimeric protein. "Variant protein" can mean a modified protein such that the modified protein has an amino acid change compared to the wild type protein.

[0122] Proteins encoded by a nucleic acid or plasmid include therapeutic proteins. Non-limiting examples include coagulation factor (e.g., factor XIII, factor IX, factor X, factor VIII, factor VIIa, or protein C), apoE2, TPP1, arginine succinate synthase, copper transport ATPase 2, acid alpha-glucosidase, β-glucocerebrosidase, α -galactosidase, C1 serine protease inhibitor, CFTR (cystic fibrosis transmembrane protein regulator), antibody, pigment epithelium-specific protein with a molecular weight of 65 kDa (RPE65), erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin , spectrin, α-antitrypsin, adenosine deaminase (ADA), metal transporter (ATP7A or ATP7), sulfamidase, enzyme involved in lysosomal storage disease (ARSA), hypoxanthine-guanine phosphoribosyltransferase, β-25-glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormone, growth factor (eg, insulin-like growth factors 1 and 2, platelet-derived growth factor, epidermal growth factor, nerve growth factor, neurotrophic factor-3 and factor-4, brain-derived neurotrophic factor, glial growth factor, transforming growth factor α and β, etc.), cytokine (for example, interferon-α, interferon-β, interferon-γ, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, etc. .), a suicide gene product (e.g., herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, etc.), drug resistance protein (e.g., which mediates drug resistance, used in cancer therapy), tumor suppressor protein (e.g. p53, Rb, Wt-1, NF1, von Hippel-Lindau protein (VHL), adenomatous polyposis coli (APC)-associated protein), peptide with immunomodulatory properties, tolerogenic or immunogenic peptide or protein Tregitopes or hCDR1, insulin, glucokinase, guanylate cyclase 2D (LCA-GUCY2D), Rab excort protein 1 (choroideremia), LCA 5 (LCA-Lebercillin), ornithine ketoacid aminotransferase (hydrate atrophy), retinoschisis 1 ( X-linked congenital retinoschisis), USH1C (Usher syndrome 1C), GTPase in X-linked retinitis pigmentosa (XLRP), MERTK (AR forms of RP: retinitis pigmentosa), DFNB1 (connexin 26-related hearing loss), ACHM 2, 3 and 4 (achromatopsia), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CLN2, proteins whose gene deficiency causes lysosomal storage disease (eg, sulfatases, N-acetylglucosamine-1-phosphate transferase, cathepsin A, GM2- AP, NPC1, VPC2, sphingolipid activator proteins, etc.), one or more zinc finger nucleases for genome editing, or donor sequences used as repair templates for genome editing.

[0123] Nucleic acid or plasmid can also refer to a sequence that produces a transcript when transcribed. Such transcripts may be RNA, such as inhibitory RNA (RNAi, e.g., small or short hairpin RNA (sh), microRNA (miRNA), small or short interfering (si)RNA, trans-spliced RNA, or antisense RNA) .

[0124] Non-limiting examples include inhibitory nucleic acids that inhibit the expression of: the huntingtin gene (HTT), a gene associated with dentatorubropallidoluis autorophy (eg, utrophin 1, ATN1); androgen receptor on the X chromosome in spinobulbar muscular atrophy, Cav2.1 P/Q voltage-gated calcium channel encoded (CACNA1A), TATA-binding protein, opposite chain of ataxin 8, also known as ATXN8OS, beta isoform of the serine B regulatory subunit /threonine protein phosphatase 2A 55 kDa in spinocerebellar ataxia (type 1, 2, 3, 6, 7, 8, 12 17), FMR1 (mental retardation 1 associated with fragile X) in fragile X syndrome, FMR1 (mental retardation 1 associated with fragile X syndrome), FMR1 (mental retardation 1 associated with fragile X syndrome), fragile X-associated mental retardation 1) in fragile X-associated tremor/ataxia syndrome, FMR1 (fragile X-associated mental retardation 2) or AF4/FMR2 family member 2 in fragile X-associated mental retardation XE chromosome, myotonin protein kinase (MT-PK) in myotonic dystrophy; frataxin for Friedreich's ataxia; mutant superoxide dismutase 1 (SOD1) gene in amyotrophic lateral sclerosis; a gene involved in the pathogenesis of Parkinson's disease and/or Alzheimer's disease; apolipoprotein B (APOB) and proprotein convertase subtilisin/kexin type 9 (PCSK9), hypercholesterolemia; HIV Tat, the human immunodeficiency virus transcription transactivator gene in HIV infection; HIV TAR, HIV TAR, human immunodeficiency virus transactivator response element gene for HIV infection; C-C chemokine receptor (CCR5) in HIV infection; nucleocapsid protein of Rous sarcoma virus (RSV) for RSV infection, liver-specific microRNA (miR-122) for infection caused by hepatitis C virus; p53 for acute kidney injury or delayed renal graft function; protein kinase N3 (PKN3) in advanced recurrent or metastatic solid malignancies; LMP2, LMP2, also known as proteasome subunit beta type 9 (PSMB 9) in metastatic melanoma; LMP7, also known as proteasome subunit beta type 8 (PSMB 8) in metastatic melanoma; MECL1 also known as proteasome subunit beta type 10 (PSMB 10) in metastatic melanoma; vascular endothelial growth factor (VEGF) for solid tumors; kinesin, spindle protein in solid tumors, suppressor of apoptosis of B-cell CLL/lymphoma (BCL-2) in chronic myeloid leukemia; ribonucleotide reductase M2 (RRM2) in solid tumors; furin for solid tumors; polo-like kinase 1 (PLK1) for liver tumors, diacylglycerol acyltransferase 1 (DGAT1) for hepatitis C virus infection, beta-catenin for familial adenomatous polyposis; adrenergic receptor beta2 for glaucoma; RTP801/Redd1, also known as DAN transcript damage-inducible protein 4, in diabetic macular edema (DME) or age-related macular degeneration; vascular endothelial growth factor receptor I (VEGFR1) in age-related macular degeneration or choroidal neovascularization, caspase 2 in non-arterial ischemic optic neuropathy; mutant protein N17K keratin 6A in congenital pachyonychia; genomic/gene sequences of influenza A virus during infection caused by influenza virus; genomic/gene sequences of the coronavirus that causes severe acute respiratory syndrome (SARS) during SARS infection; genomic/gene sequences of respiratory syncytial virus in respiratory syncytial virus infection; Ebola virus genomic/gene sequences during Ebola virus infection; genomic/gene sequences of hepatitis B and C virus in hepatitis B and C; genomic/gene sequences of herpes simplex virus (HSV) in HSV infection, genomic/gene sequences of Coxsackie B3 virus in infection caused by Coxsackie B3 virus; silencing of a pathogenic allele of a gene (allele-specific silencing), such as the torsin A gene (TOR1A), in primary dystonia, silencing of the class I pan gene and a graft-specific HLA allele; mutant rhodopsin gene (RHO) in autosomal dominant congenital retinitis pigmentosa (adRP); or an inhibitory nucleic acid binds to a transcript of any of the above genes or sequences.

[0125] Nucleic acids (plasmids) can be single-stranded, double-stranded or triple-stranded, linear or cyclic, and can be of any length. When discussing nucleic acids (plasmids), the sequence or structure of a particular polynucleotide may be described herein according to the convention of presenting the sequence in the 5' to 3' direction.

[0126] A “plasmid” is a form of nucleic acid or polynucleotide that typically has additional elements for expression (eg, transcription, replication, etc.) or propagation (replication) of the plasmid. The term "plasmid" can be used to refer to a sequence of nucleic acids and polynucleotides. Therefore, in all aspects, the compositions and methods of the invention are applicable to plasmids, nucleic acids and polynucleotides, for example, for introducing a plasmid, nucleic acid or polynucleotide into cells, for transducing cells with a plasmid, nucleic acid or polynucleotide, for producing transduced (transfected) ) cells that contain a plasmid, nucleic acid or polynucleotide, to produce cells that produce viral vectors (eg, AAV), to produce viral (eg, AAV) vectors, to produce a cell culture medium that contains viral vectors (eg, AAV) etc.

[0127] The compositions and methods of the present invention include polyethylenimine (PEI). PEI is a cationic polymer and is capable of forming a stable complex with a nucleic acid called a polyplex. However, without wishing to be bound by any theory, it is believed that the polyplex is introduced into cells via endocytosis.

[0128] The PEI may be a linear PEI or a branched PEI. PEI may be in salt or free base form. In certain embodiments, the PEI is linear PEI, such as optionally hydrolyzed linear PEI. Hydrolyzed PEI may be fully or partially hydrolyzed. Hydrolyzed linear PEI has a higher relative amount of free (protonated) nitrogen atoms compared to non-hydrolyzed linear PEI, which typically has at least 1-5% more free (protonated) nitrogen atoms compared to non-hydrolyzed linear PEI, more typically at 5-10% more free (protonated) nitrogen atoms compared to non-hydrolyzed linear PEI or generally has 10-15% more free (protonated) nitrogen atoms compared to non-hydrolyzed linear PEI.

[0129] In particular embodiments, PEI may have a molecular weight ranging from about 4,000 to about 160,000 and/or a molecular weight ranging from about 2,500 to about 250,000 in free base form. In still other specific embodiments, PEI may have a molecular weight of about 40,000 and/or a molecular weight of about 25,000 in free base form. In particular, linear PEI has a molecular weight of about 40,000 and/or a molecular weight of about 25,000 in free base form. In addition, chemically modified linear PEI or branched PEI can be used. PEI is commercially available (eg, from Polysciences, Inc., Warrington, PA, USA).

[0130] In the compositions and methods of the invention, a nucleic acid, such as a plasmid, is mixed with PEI to form a PEI mixture or solution. Such a mixture or solution may be referred to as a "plasmid/PEI mixture" or a "nucleic acid/PEI mixture". Therefore, the terms “plasmid/PEI mixture” and “nucleic acid/PEI mixture” mean that PEI is mixed with a nucleic acid/plasmid. Therefore, PEI, as indicated herein, can be mixed with the nucleic acid (plasmid) before or substantially simultaneously with contacting the cells for transduction/transfection.

[0131] As used herein, the term “free PEI” means PEI that is substantially or completely free of nucleic acid (plasmid). Therefore, PEI, as indicated herein, can also be in the form of free PEI. Therefore, “plasmid/PEI mixture” or “nucleic acid/PEI mixture” is different from free PEI. If free PEI is substantially free, then the amount of nucleic acid sequences (plasmid) present will be no more than about 5%, as determined by molecular weight or weight. Of course, the amount may be below 5%, such as about 4.5% or below, about 4% or below, about 3.5% or below, about 3% or below, about 2.5% or below, about 2% or lower, about 1.5% or lower, about 1% or lower, or about 0.5% or lower.

[0132] As used herein, the term “total PEI” means the total amount of PEI present in the PEI/plasmid mixture and as free PEI. Therefore, general PEI includes PEI that is mixed with a plasmid and PEI that is substantially or completely free of nucleic acid sequences, such as a plasmid.

[0133] The disclosure of PEI amounts, ratios, compositions, solutions, solvents and buffers, pH, salts, and time and duration of cell contact and incubation applies to any of, any two of, or all three of: 1) PEI in plasmid/PEI mixtures or nucleic acid/PEI mixtures; 2) PEI as free PEI (i.e., PEI that is substantially or completely free of nucleic acid or polynucleotide sequences such as a plasmid, and 3) total PEI (PEI in a plasmid/PEI mixture or in a nucleic acid/PEI+ mixture free PEI).

[0134] In certain embodiments, the PEI is a solution, such as an aqueous (eg, water) solution. In further specific embodiments, the PEI is acidified or neutralized PEI. The term "acidified PEI" means a solution of PEI that is prepared by dissolving PEI in an acidic solvent. The acidity of the acidified PEI solution is typically a pH of about 0 to about 3.0, more typically a pH of about 0.5 to about 2.0. The term "neutralized PEI" means a solution of PEI that is prepared by dissolving PEI in a neutral solvent or buffer. Neutralized PEI solutions may have a pH in the range of about 6.0 to about 8.0, typically a pH in the range of about 6.5 to about 7.5, more typically a pH in the range of about 6.8 to about 7.2, and most typically a pH in the range of about 7.0 to about 7.2, such as about 7.1.

[0135] Any solvent or buffer can be used to adjust or maintain the pH of the PEI solution within the above range without interfering with the transfection activity of the PEI. Examples of acidic solvents include mineral acids such as hydrochloric acid (HCl) and organic acids with a pH in the acidic range such as glycine-hydrochloric acid solution. Non-limiting examples of neutral solvents/buffers include Tris(trisma base) and HEPES. Buffers can range in concentration from about 1 mM to about 100 mM, more typically from about 2 mM to about 50 mM, and most typically from about 5 mM to about 20 mM.

[0136] PEI solutions may optionally include salts. Non-limiting examples of salts include sodium (Na), potassium (K) and magnesium (Mg) salts. In particular aspects, salt concentrations in the PEI solution range from about 50 mM to about 500 mM, more typically from about 100 mM to about 250 mM, and most typically from about 125 mM to about 175 mM.

[0137] A mixture of nucleic acids (plasmids) and PEI is prepared by mixing nucleic acids (plasmids) and PEI in a solution. Mixing can take place in any solution compatible with PEI-based cell transduction. Non-limiting examples include those listed here. Once mixed, the nucleic acid (plasmid)/PEI mixture can be incubated for a period of about 1 minute to about 8 hours; about 10 seconds to about 4 hours; from about 1 minute to about 60 minutes; from about 1 minute to about 30 minutes; about 10 minutes to about 45 minutes; about 10 minutes to about 30 minutes; and/or about 20 minutes to about 30 minutes. Typically, times include about 1 minute, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, and about 30 minutes.

[0138] PEI and nucleic acids (plasmid) are mixed in a ratio that is not particularly limited. Typical ratios include a mixture of plasmids in a molar (or mass) ratio range from about 1:0.01 to about 1:100, or in a molar (or mass) ratio range from about 100:1 to about 1:0.01 to obtain a mixture plasmid/PEI. More typical molar (or mass) ratios include a mixture of plasmids in a molar (or mass) ratio range of about 1:1 to about 1:5, or in a molar (or mass) ratio range of about 1:2 to about 1:4, so that obtain plasmid/PEI mixture. In additional embodiments, the PEI:plasmid weight ratio is in the range of about 0.1:1 to about 5:1, or in the range of about 5:1 to about 0.1:1. In still other embodiments, free PEI/plasmid/PEI in cell culture has a PEI:plasmid weight ratio ranging from about 0.1:1 to about 5:1 or has a PEI:plasmid weight ratio ranging from about 5:1 to about 0.1:1. In specific embodiments, the plasmid/PEI mixture has a PEI:plasmid weight ratio ranging from about 1:1 to about 5:1, or in the range from about 5:1 to about 1:1. In still further specific embodiments, free PEI/plasmid/PEI in cell culture has a PEI:plasmid weight ratio in the range of about 1:1 to about 5:1, or in the range of about 5:1 to about 1:1.

[0139] The amount of nucleic acids (plasmids) used to produce cell transduction compositions and methods varies. In specific embodiments, the molar ratio of nitrogen (N) in total PEI to phosphate (P) in the plasmid ranges from about 1:1 to about 50:1 (N:P), in free PEI/plasmid/PEI in cell culture, or the molar ratio of nitrogen (N) in total PEI to phosphate (P) in the plasmid is approximately 1:1 to 10:1 (N:P) in free PEI/plasmid/PEI in cell culture, or the molar ratio of nitrogen (N) in general the PEI to phosphate (P) in the plasmid is approximately 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1 (N:P) in free PEI/plasmid/PEI in cell culture . In additional specific embodiments, the total amount of a plasmid containing a nucleic acid that encodes a protein or is transcribed into a transcript of interest, and one or more plasmids containing nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding helper proteins, is in range from about 0.1 μg to about 15 μg per ml of cells.

[0140] Application of the nucleic acid (plasmid)/PEI mixture to cells is accomplished by adding the nucleic acid (plasmid)/PEI mixture to the cells such that the nucleic acid (plasmid)/PEI mixture is in contact with the cells. The cells to which the nucleic acid (plasmid)/PEI mixture is added (which are contacted) may be adherent cells or cells in suspension. Such cells may include co-cultures with other cells.

[0141] Cells are contacted with the nucleic acid (plasmid)/PEI mixture for a period of time, which is not particularly limited, to achieve cell transduction. Contacting cells with free PEI usually occurs simultaneously with (or immediately after) or after the cells have been contacted with the nucleic acid (plasmid)/PEI mixture. If there is to be a time interval between contacting the cells with the nucleic acid (plasmid)/PEI mixture and contacting the cells with free PEI, the time interval may be from about 1 second to about 140 hours, typically from about 1 second to about 96 hours, more typically from about 1 second to about 48 hours or about 72 hours, most typically from about 1 second to about 24 hours or less, such as about 16 hours, about 12 hours, about 8 hours, or about 6 hours or less.

[0142] Upon prolonged contact, cells may be affected by the cytotoxicity of PEI, leading to an increase in the number of dead (non-viable) cells, which reduces transfection efficiency. Incubation time after cells are exposed to common PEI can vary from seconds to days. In particular, cells can be contacted with nucleic acids (plasmids)/PEI or total PEI, for example, for a period of time from about 1 minute to about 48 hours; from about 1 minute to about 24 hours; from about 1 minute to about 16 hours; about 1 minute to about 8 hours; about 1 minute to about 4 hours; from about 1 minute to about 120 minutes; from about 5 minutes to about 60 minutes; about 10 minutes to about 45 minutes; or about 10 minutes to about 30 minutes.

[0143] To reduce the cytotoxicity of PEI, the culture medium can be replaced with fresh culture medium after contacting the cells with the nucleic acid (plasmid)/PEI mixture. Replacing the culture medium after transfection can minimize the cytotoxicity of PEI without significant loss of cell transfection efficiency.

[0144] The cells to be transfected, before or during contact with the plasmid/PEI mixture, and/or contact with the enhancing agent, and/or contact with free PEI, have a density ranging from about 1×10 ⁵ cells/ml to about 1×10 ⁸ cells/ml. Typically, cells have a density ranging from about 2x10 ⁵ cells/ml to about 5x10 ⁶ cells/ml. More typically, the cells have a density ranging from about 3x10 ⁵ cells/ml to about 4x10 ⁶ cells/ml, such as about 4x10 ⁵ cells/ml to about 3x10 ⁶ cells/ml, or about from 5x10 ⁵ cells/ml to about 2x10 ⁶ cells/ml. In yet other embodiments, the cells have a density ranging from about 5x10 ⁵ cells/ml to about 5x10 ⁶ cells/ml, such as about 6x10 ⁵ cells/ml to about 4x10 ⁶ cells/ml, or from about 7x10 ⁵ cells/ml to about 3x10 ⁶ cells/ml. In further embodiments, the cells have a density ranging from about 1x10 ⁶ cells/mL to about 5x10 ⁶ cells/mL, such as about 1x10 ⁶ cells/mL to about 4x10 ⁶ cells/mL or about from 2x10 ⁶ cells/ml to about 3x10 ⁶ cells/ml.

[0145] The cells to be transfected, before or during contact with the plasmid/PEI mixture, and/or contact with the enhancing agent, and/or contact with free PEI, may optionally be in a logarithmic (exponential) growth phase. Cells to be transfected, before or during contact with the plasmid/PEI mixture, and/or contact with an enhancing agent, and/or contact with free PEI, may have a viability of 60% or greater than 60%, for example, a viability of 70%, 80% or 90% or above 90%.

[0146] Cells that can be contacted as specified herein include mammalian cells, such as human cells. Such cells may represent primary cells or cell lines that are capable of growing or maintaining viability in vitro, or have been adapted for tissue culture in vitro. Examples of cell lines include HEK (human embryonic kidney) cells, which include HEK293 cells such as HEK293F (293F) and HEK293T (293T) cells.

[0147] More generally, such cells that contact as defined herein may be referred to as “host cells.” “Host cell” means, for example, microorganisms, yeast cells, insect cells and mammalian cells that can or have been used as recipients of nucleic acid (plasmid) encoding packaging proteins, such as AAV packaging proteins, nucleic acid (plasmid) encoding helper proteins, a nucleic acid (plasmid) that encodes a protein or is transcribed into a transcript of interest, or other nucleic acid transfer (plasmid). The term includes the progeny of the original cell that has been transduced or transfected. Thus, as used herein, “host cell” generally refers to a cell that has been transduced or transfected with an exogenous nucleic acid sequence. It is understood that the progeny of one parent cell may not necessarily be completely identical in morphology or in genomic or complete nucleic acid of the original parent due to natural, accidental or intentional mutation.

[0148] Numerous cell culture media suitable for maintaining cell viability or promoting cell growth and/or proliferation are commercially available or can be easily obtained. Examples of such media include serum-free eukaryotic culture media, such as media for maintaining the viability or growth of mammalian cells (eg, human cells). Non-limiting examples include Ham's F12 or F12K medium (Sigma-Aldrich), FreeStyle™ ^M (FS) F17 medium (Thermo-Fisher Scientific), MEM, DMEM, RPMI-1640 (Thermo-Fisher Scientific), and mixtures thereof. Such a medium may be supplemented with vitamins and/or microelements and/or salts and/or amino acids, such as essential amino acids for mammalian cells (eg human cells).

[0149] “Enhancing agents,” otherwise referred to herein as “transfection enhancers” or in the same context simply “enhancers,” are compounds that enhance the transduction/transfection of cells by a nucleic acid (plasmid). In specific embodiments, the enhancing agent includes or consists of valproic acid, a salt or derivative thereof. In some embodiments, the valproic acid salt includes or consists of a sodium or potassium salt. In some embodiments, the valproic acid derivative includes or consists of an amino acid linked or conjugated thereto. Additional examples of enhancing agents include, for example, and without limitation, those described in US Patent Publications No. 2013/0316400 and 2017/0016043, incorporated herein by reference in their entirety, as well as other transfection enhancers known in the art.

[0150] Enhancing agents including valproic acid can be used at a concentration, for example, and without limitation, in the range of from about 0.1 to about 25 mM or in any subranges or concentrations covered therein. In certain embodiments, the concentration of the enhancing agent is from about 0.5 mM to about 10 mM. In certain embodiments, the concentration of the enhancing agent is from about 0.5 mM to about 5 mM. In certain embodiments, the concentration of the enhancing agent is from about 1 mM to about 4 mM, from about 1 mM to about 3 mM, or from about 1 mM to about 2 mM.

[0151] The terms “transduce” and “transfect” refer to the introduction of a molecule, such as a nucleic acid (plasmid), into a host cell. A cell has been "transduced" or "transfected" when an exogenous nucleic acid has been introduced into the cell membrane. Therefore, a “transduced cell” is a cell into which a “nucleic acid” or “polynucleotide” has been introduced, or its progeny into which an exogenous nucleic acid has been introduced. In specific embodiments, a “transduced” cell (eg, in a mammal, such as a cell, tissue, or organ cell) is a genetic change in the cell following the incorporation of an exogenous molecule, such as a nucleic acid (eg, a transgene). "Transduced" or "transfected" cells can proliferate and the introduced nucleic acid is transcribed and/or a protein is expressed.

[0152] In a "transduced" or "transfected" cell, the nucleic acid (plasmid) may or may not be integrated into the genomic nucleic acid of the recipient cell. If the introduced nucleic acid becomes integrated into the nucleic acid (genomic DNA) of a recipient cell or organism, it can be stably maintained in that cell or organism and further transmitted or inherited by descendants of the cells or organism, or by the recipient cell or organism. Finally, the introduced nucleic acid may exist in the recipient cell or host organism extrachromosomally or only transiently. A number of methods are known (see, for example, Graham et al. (1973) Virology, 52: 456, Sambrook et al. (1989), Molecular Cloning, laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) ) Basic Methods in Molecular Biology, Elsevier, and Chu et al (1981) Gene 13: 197). Such methods can be used to introduce one or more exogenous DNA fragments into suitable host cells.

[0153] The term “vector” refers to a small nucleic acid carrier molecule, plasmid, virus (eg, AAV vector) or other carrier that can be manipulated by insertion or inclusion of a nucleic acid. Such vectors can be used for genetic manipulation (ie, "cloning vectors"), for the introduction/transfer of polynucleotides into cells, and for transcription or translation of the inserted polynucleotide in cells. An "expression vector" is a specialized vector that contains a gene or nucleic acid sequence with the necessary regulatory regions required for expression in a host cell. The vector nucleic acid sequence typically contains at least an origin of replication for propagation in the cell and optionally additional elements such as a heterologous polynucleotide sequence, an expression control element (eg, promoter, enhancer), intron, ITR(s), selectable marker (eg , conferring resistance to antibiotics), a polyadenylation signal. For purposes of the invention, “vector” as defined herein is included within the scope of the term “plasmid” as that term is used herein.

[0154] A viral vector is derived from or based on one or more nucleic acid elements that make up the viral genome. Specific viral vectors include lentiviral, pseudotyped lentiviral and parvoviral vectors, such as adeno-associated virus (AAV) vectors.

[0155] The term "recombinant" as a designator of a vector, such as a recombinant viral, e.g., lentiviral or parvoviral (e.g., AAV) vector, and as a designator of sequences, such as recombinant polynucleotides and polypeptides, means that the compositions have been manipulated (i.e., e. designed), which are usually absent in nature. A specific example of a recombinant vector, such as an AAV vector, is one where a polynucleotide that is not normally present in the wild-type viral genome (eg, AAV) is inserted into the viral genome, i.e., is heterologous. Although the term “recombinant” is not always used herein in relation to vectors, such as viral and AAV vectors, as well as sequences, such as polynucleotides, recombinant forms, including polynucleotides, are covered by the present invention notwithstanding any such omission.

[0156] A recombinant viral "vector" or "AAV vector" is prepared from the genome of a wild-type virus, such as AAV, using molecular techniques to remove the wild-type genome from the virus (e.g., AAV) and replace it with a non-natural nucleic acid, such as a nucleic acid, which is transcribed into a transcript or encodes a protein. Typically, for AAV, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained in the AAV vector. A “recombinant” viral vector (eg, AAV) differs from a viral (eg, AAV) genome because all or part of the viral genome is replaced by a non-natural (ie, heterologous) sequence relative to the viral (eg, AAV) genomic nucleic acid. Therefore, the inclusion of a non-natural sequence allows the viral vector (eg, AAV) to be defined as a “recombinant” vector, which in the case of AAV may be referred to as a “rAAV vector.”

[0157] The sequence of a recombinant vector (eg, lentiviral, parvovirus, AAV) can be packaged, referred to herein as a “particle,” for subsequent infection (transduction) of a cell ex vivo, in vitro, or in vivo. When the recombinant vector sequence is encapsidated or packaged into an AAV particle, the particle may also be referred to as "rAAV". Such particles include proteins that encapsidate or package the vector genome. Specific examples include viral envelope proteins, and in the case of AAV, capsid proteins such as AAV VP1, VP2 and VP3.

[0158] A vector “genome” refers to that portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form a viral (eg, AAV) particle. In cases where recombinant plasmids are used to construct or produce recombinant vectors, the vector genome does not include a portion of the “plasmid” that does not match the vector genome sequence of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid refers to the “plasmid backbone,” which is important for cloning and amplification of the plasmid, a process that is necessary for propagation and production of the recombinant virus, but is not itself packaged or encapsidated into viral (e.g., AAV) particles . Thus, a vector "genome" refers to a nucleic acid that is packaged or encapsidated by a virus (eg, AAV).

[0159] The terms “empty capsid” and “empty particle” refer to an AAV virion that includes the AAV protein coat but lacks all or part of the nucleic acid that encodes the protein or is transcribed into the transcript of interest flanked by the AAV ITR. Therefore, the empty capsid does not function to carry nucleic acid that encodes a protein or is transcribed into a transcript of interest in the host cell. However, empty capsid formulations are used in other applications such as ELISA.

[0160] The term “packaging proteins” refers to non-AAV-derived viral and/or cellular functions on which AAV replication depends. Thus, the term covers proteins and RNAs that are required for AAV replication, including such fragments that are involved in the transcriptional activation of the AAV gene, the specific splicing step of AAV mRNA, the replication of AAV DNA, the synthesis of Cap expression products, and the assembly of the AAV capsid. Viral helper functions can be derived from any of the known helper viruses, such as adenovirus, herpes virus (other than herpes simplex virus type 1), and vaccinia virus.

[0161] As used herein, the term “AAV packaging proteins” refers to derivatives of AAV sequences that function in trans configuration for productive AAV replication. Thus, AAV packaging proteins are encoded by the AAV major open reading frames (ORFs), rep and cap. rep proteins have been shown to have many functions including, but not limited to: recognition, binding and nicking of the AAV origin of DNA replication; DNA helicase activity; and modulation of transcription from AAV (or other heterologous) promoters. Cap proteins provide essential packaging functions. AAV packaging proteins are used here to complement AAV functions in trans configuration that are missing in AAV vectors.

[0162] The term “nucleic acids encoding AAV packaging proteins” generally refers to a nucleic acid molecule that includes nucleotide sequences providing AAV functions removed from an AAV vector to be used to produce a transducing recombinant AAV vector. Nucleic acids encoding AAV packaging proteins are commonly used to enable transient expression of AAV rep and/or cap genes to complement missing AAV functions that are required for AAV replication; however, the nucleic acid constructs do not have the AAV ITR and cannot replicate or package themselves. Nucleic acids encoding AAV packaging proteins may be in the form of a plasmid, phage, transposon, cosmid, virus, or virion. A number of nucleic acid constructs have been described, such as the commonly used plasmids pAAV/Ad and pIM29+45, which encode both Rep and Cap expression products. See, for example, Samulski et al. (1989) J. Virol. 63: 3822-3828; and McCarty et al. (1991) J. Virol. 65: 2936-2945. A number of vectors have been described that encode Rep and/or Cap expression products (eg, US Pat. Nos. 5,139,941 and 6,376,237).

[0163] The term “nucleic acids encoding helper proteins” generally refers to nucleic acid molecule(s) that include nucleotide sequences encoding proteins that provide helper function(s). A vector with nucleic acid(s) encoding the helper protein(s) can be transfected into a suitable host cell, where the vector is then capable of supporting AAV virion production in the host cell. The term expressly excludes infectious viral particles as they exist naturally, such as adenovirus, herpesvirus, or vaccinia virus particles.

[0164] Thus, helper protein vectors may be in the form of a plasmid, phage, transposon, or cosmid. In particular, it was shown that the full set of adenovirus genes is not required for helper functions. For example, adenoviral mutants incapable of DNA replication and late gene synthesis have been shown to be competent for AAV replication. Ito et al., (1970) J. Gen. Virol. 9: 243; Ishibashi et al. (1971) Virology 45: 317.

[0165] Mutants in the E2B and E3 regions have been shown to support AAV replication, indicating that the E2B and E3 regions are likely not involved in mediating helper function. Carter et al., (1983), Virology 126: 505. However, adenoviruses defective in the El region or with the E4 region deleted cannot support AAV replication. Thus, for adenoviral helper proteins, the EIA and E4 regions are likely required for AAV replication, either directly or indirectly. Laughlin et al. (1982) J. Virol., 41: 868; Janik et al. (1981) Proc. Natl. Academician Sci. USA, 78: 1925; Carter et al. (1983) Virology, 126: 505. Other characterized Ad mutants include: EIB (Laughlin et al. (1982), supra; Janik et al. (1981), supra; Ostrove et al. (1980) Virology, 104: 502) ; E2A (Handa et al. (1975) J. Gen. Virol., 29: 239; Strauss et al. (1976) J. Virol., 17: 140; Myers et al. (1980) J. Virol., 35: 665; Jay et al. (1981) Proc. Natl. Acad. Sci. USA, 78: 2927; Myers et al. (1981) J. Biol. Chem., 256: 567); E2B (Carter, Adeno-Associated Virus Helper Functions, in I CRC Handbook of Parvoviruses (P. Tijssen ed., 1990)); E3 (Carter et al. (1983), supra); and E4 (Carter et al. (1983), supra; Carter (1995)).

[0166] Studies of helper proteins mediated by adenoviruses having mutations in E1B have shown that E1 B55k is required for AAV virion production, while E1B 19k is not. In addition, in the publication of international application WO 97/17458 and in the publication of Matshushita et al. (1998) Gene Therapy, 5: 938-945 describe vectors with helper functions encoding various Ad genes. An example helper vector contains an adenovirus RNA VA coding region, an adenovirus E4 ORF6 coding region, an adenovirus 72 kDa E2A coding region, an adenovirus E1A coding region, and an adenovirus E1B region lacking an intact EI BS5k coding region (see, for example, International Application Publication WO 01 /83797).

[0167] The term “transgene” is used herein to refer to a nucleic acid that is intended or has been introduced into a cell or organism. Transgenes include any nucleic acid, such as a gene, that is transcribed into a transcript or that encodes a polypeptide or protein.

[0168] “Expression control element” refers to nucleic acid sequence(s) that influence the expression of an operably linked nucleic acid. Control elements, including expression control elements as defined herein, such as promoters and enhancers, vector sequences, including AAV vectors, may include one or more “expression control elements”. Typically, such elements are included to facilitate proper transcription of the heterologous polynucleotide and, if necessary, translation (e.g., promoter, enhancer, intronic splicing signal, maintaining the correct reading frame of the gene, to ensure in-frame translation of mRNA, stop codons, etc.). d.). Such elements typically function in a cis configuration, and are referred to as a “cis-acting” element, but can also act in a trans configuration.

[0169] Expression control can occur at the level of transcription, translation, splicing, transcript stability, etc. Typically, the expression control element that modulates transcription is located near the 5' end (i.e., upstream) of the transcribed nucleic acid . Expression control elements can also be located at the 3' end (i.e., downstream) of the transcribed sequence or within the transcript (eg, in an intron). Expression control elements may be located adjacent to or at a distance from the transcribed sequence (eg, 1-10, 10-25, 25-50, 50-100, 100-500 or more nucleotides from the polynucleotide), even at greater distances. However, due to the length limitations of some vectors, such as AAV vectors, expression control elements are typically located within 1-1000 nucleotides of the transcribed nucleic acid.

[0170] Functionally, the expression of an operably linked nucleic acid is at least partially regulated by an element (eg, a promoter), such that the element modulates transcription of the nucleic acid and, if necessary, translation of the transcript. A specific example of an expression control element is a promoter, which is typically located at the 5' end of the transcribed sequence. A promoter generally increases the amount expressed from an operably linked nucleic acid compared to the amount expressed when the promoter is absent.

[0171] As used here, the term “enhancer” can refer to a sequence that is adjacent to a heterologous polynucleotide. Enhancer elements are usually located upstream of a promoter element, but also function and can be located downstream or within the nucleic acid sequence. Therefore, the enhancer element may be located 100 base pairs, 200 base pairs, or 300 or more base pairs upstream or downstream from the nucleic acid. Enhancer elements generally increase the expression of an operably linked nucleic acid relative to the expression provided by a promoter element.

[0172] An expression construct may include regulatory elements that serve to control expression in a particular cell or tissue type. Expression control elements (eg, promoters) include those that are active in a specific tissue or cell type, referred to herein as “tissue-specific expression control elements/promoters.” Tissue-specific expression control elements are usually active in a specific cell or tissue (eg, liver). Expression control elements are typically active in specific cells, tissues, or organs because they are recognized by transcriptional activator proteins or other transcriptional regulators that are unique to a particular cell, tissue, or organ type. Such regulatory elements are known to those skilled in the art (see, for example, Sambrook et al. (1989) and Ausubel et al. (1992)).

[0173] The inclusion of tissue-specific regulatory elements in the plasmids of the invention provides at least partial tissue tropism for nucleic acid expression. Examples of promoters that are active in the liver include the TTR promoter (eg, mutant TTR promoter), human alpha-1 antitrypsin (hAAT) promoter; albumin promoter, Miyatake et al., J. Virol., 71: 5124-32 (1997); hepatitis B virus core antigen promoter, Sandig et al., Gene Ther., 3: 1002-9 (1996); alpha-fetoprotein (AFP) promoter, Arbuthnot et al., Hum. Gen. Ther., 7: 1503-14 (1996)], among others. An example of an enhancer active in the liver is the apolipoprotein E (apoE) enhancer of HCR-1 and HCR-2 (Allan et al., J. Biol. Chem., 272: 29113-19 (1997)).

[0174] Expression control elements also include ubiquitous or non-selective promoters/enhancers that are capable of directing expression of a polynucleotide in many different cell types. Such elements include, but are not limited to, cytomegalovirus (CMV) early promoter/enhancer sequences, Rous sarcoma virus (RSV) promoter/enhancer sequences, and other viral promoters/enhancers active in various mammalian cell types, or synthetic elements that are not naturally occurring. (see, for example, Boshart et al., Cell, 41: 521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic β-actin promoter and the phosphoglycerol kinase (PGK) promoter.

[0175] Expression control elements can also provide expression in a regulated manner, ie, a signal or stimuli increases or decreases the expression of an operably linked heterologous polynucleotide. A regulated element that increases the expression of an operably linked polynucleotide in response to a signal or stimuli is also called an “inducible element” (ie, inducible by a signal). Specific examples include, but are not limited to, a hormone-inducible (eg, steroid) promoter. Generally, the degree of increase or decrease provided by such elements is proportional to the amount of signal or stimuli present; the higher the amount of signal or stimuli, the greater the increase or decrease in expression. Specific non-limiting examples include the zinc-inducible ovine metallothionein (MT) promoter; steroid hormone-inducible mouse mammary tumor virus (MMTV) promoter; T7 polymerase promoter system (WO 98/10088); tetracycline-repressible system (Gossen et al., Proc. Natl. Acad. Sci. USA, 89: 5547-5551 (1992)); tetracycline-inducible system (Gossen et al., Science, 268: 1766-1769 (1995) ); see also Harvey et al., Curr. Opin. Chem. Biol. 2: 512-518 (1998)); RU486-inducible system (Wang et al., Nat. Biotech., 15: 239-243 (1997) and Wang et al., Gene Ther., 4: 432-441 (1997)]; and rapamycin-inducible system (Magari et al., J. Clin. Invest., 100: 2865-2872 (1997); Rivera et al., Nat. Medicine, 2: 1028-1032 (1996). Other adjustable controls that may be suitable in this regard context, represent those that are regulated by a certain physiological state, for example, temperature, acute phase, development.

[0176] Expression control elements also include element(s) native to the nucleic acid. A native control element (eg, a promoter) can be used when it is desired that expression of a heterologous polynucleotide mimic native expression. A native element can be used when the expression of a heterologous polynucleotide is subject to temporal or developmental stage-dependent regulation, or tissue-specific regulation, or in response to specific transcriptional stimuli. Other native expression control elements such as introns, polyadenylation sites or Kozak consensus sequences may also be used.

[0177] The term "operably linked" means that the regulatory sequences required for the expression of the coding sequence are located at appropriate positions relative to the coding sequence, allowing them to influence the expression of the coding sequence. The same definition is sometimes applied to the location of coding sequences and transcriptional control elements (eg, promoters, enhancers, and termination elements) in an expression vector. The same definition is also sometimes applied to the arrangement of the nucleic acid sequences of the first and second nucleic acid molecules when a hybrid nucleic acid molecule is generated.

[0178] In the example of an expression control element in operably linked to a nucleic acid, the relationship is that the control element modulates the expression of the nucleic acid. More specifically, for example, two DNAs are operably linked means if two DNA sequences are arranged (cis or trans) such that at least one of the DNA sequences is capable of exerting a physiological effect on the other sequence.

[0179] Therefore, additional elements for vectors include, but are not limited to, an expression control element (e.g., a promoter/enhancer), a transcription termination signal or stop codon, 5' or 3' untranslated regions (e.g., polyadenylation (polyA) sequences ) that flank a sequence, such as one or more copies of an AAV ITR sequence or an intron.

[0180] Other elements include, for example, polynucleotide filler sequences or spacer sequences, for example, to improve packaging and reduce the presence of contaminating nucleic acid. AAV vectors typically accept DNA inserts having a varying range of sizes, which typically range from about 4 kb. to about 5.2 kb. or a little more. Thus, for shorter sequences, a spacer sequence or filler is included to bring the length of the insert to a size close to or equal to the normal size of the viral genomic sequence suitable for packaging the AAV vector into a viral particle. In various embodiments, the filler/spacer nucleic acid sequence is a non-translated (non-protein coding) segment of the nucleic acid. For a nucleic acid sequence less than 4.7 kb, the filler polynucleotide sequence or spacer sequence has a length that, when combined (e.g., inserted into a vector) with the sequence, has a total length of approximately 3.0-5.5 kb. p.n. or approximately 4.0-5.0 kb. or approximately 4.3-4.8 kb.

[0181] The intron may also function as a polynucleotide filler sequence or spacer sequence to achieve a length of the AAV vector suitable for packaging into a viral particle. Introns and fragments of introns that function as a polynucleotide filler sequence or spacer sequence can also enhance expression.

[0182] “Polypeptides,” “proteins,” and “peptides” encoded by “nucleic acid” or “plasmids” include the full-length native sequences of naturally occurring wild-type proteins, as well as functional subsequences, modified forms, or sequence variants, provided that the subsequence, modified form, or variant retains some degree of functional properties of the native full-length protein. For example, a protein may have a deletion, substitution, or addition and retain at least partial function or activity.

[0183] The terms “modified form” or “variant form” and their grammatical variations mean that the nucleic acid or polypeptide differs from the reference sequence. Therefore, modified and variant sequences may have substantially the same, greater or lesser expression, activity, or function than the reference sequence, but at least retain partial activity or function of the reference sequence.

[0184] Non-limiting examples of modifications include one or more nucleotide or amino acid substitutions (e.g., 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40 -50, 50-100, 100-150, 150-200, 200-250, 250-500, 500-750, 750-850 or more nucleotides or residues).

[0185] An example of an amino acid modification is a conservative amino acid substitution or deletion (eg, subsequences or fragments) in a reference sequence. In certain embodiments, the modified or variant sequence retains at least a portion of the function or activity of the unmodified sequence.

[0186] Included are all forms of mammalian and non-mammalian nucleic acids that are transcribed and nucleic acids that encode proteins. Thus, the invention includes non-mammalian genes and proteins, non-human mammals, and humans whose genes and proteins function in a substantially similar manner to human genes and proteins.

[0187] Following transfection of cells and/or production of recombinant viral vectors (e.g., AAV) as described herein, if desired, viral (e.g., rAAV) virions can be obtained/harvested from the cells/cell culture and optionally purified and/or isolated from the transfected cells using various conventional methods. Such methods include column chromatography, CsCl gradients and the like. For example, multiple column purification steps may be used, such as anion exchange column, affinity column, and/or cation exchange column purification. (See, for example, International Application Publication WO 02/12455 and US Application Publication No. 20030207439). Alternatively or in addition, CsCl gradient purification steps can be used. (See, for example, US Application Publication No. 20120135515; and 20130072548). Also, if an infectious virus is used to express the packaging and/or helper proteins, the residual virus can be inactivated in various ways. For example, the adenovirus can be inactivated by heating to a temperature of about 60°C for, for example, 20 minutes or more. This treatment effectively inactivates the helper virus because AAV is heat stable and the helper adenovirus is heat labile.

[0188] The term “isolated,” when used as a qualifier for a composition, means that the compositions are artificially produced or separated, in whole or at least in part, from their natural in vivo environment. Typically, isolated compositions are substantially free of one or more substances with which they are typically associated in nature, for example, one or more contaminants such as protein, nucleic acid, lipid, carbohydrate, cell membrane.

[0189] With respect to RNA molecules, the term “isolated” generally refers to the RNA molecule encoded by the isolated DNA molecule, as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from the RNA molecules with which it is associated in its natural state (i.e., in cells or tissues) such that it is in "substantially pure" form (the term "substantially pure" is defined below).

[0190] In relation to protein, the term "isolated protein" or "isolated and purified protein" is sometimes used herein. The term refers generally to a protein produced by the expression of an isolated nucleic acid molecule. Alternatively, the term may refer to a protein that has been sufficiently separated from other proteins with which it is associated in nature to be in an "essentially pure" form.

[0191] The term “isolated” does not exclude artificially produced combinations, such as a recombinant vector sequence (eg, rAAV) or a viral particle that packages or encapsidates the vector genome and a pharmaceutical composition. The term “isolated” also does not exclude alternative physical forms of the composition, such as hybrids/chimeras, multimers/oligomers, forms resulting from modification (eg, such as phosphorylation, glycosylation, lipidation) or derivatized forms or forms expressed in host cells , obtained artificially.

[0192] The term “substantially pure” refers to a preparation containing at least 50-60% by weight of the compound of interest (eg, nucleic acid, oligonucleotide, protein, etc.). The formulation may contain at least 75% by weight or about 90-99% by weight of the compound of interest. Purity is measured by methods appropriate to the compound of interest (eg, chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).

[0193] Nucleic acid molecules, expression vectors (eg, vector genomes), plasmids can be produced using recombinant DNA technology methods. The availability of information on nucleotide sequences makes it possible to obtain isolated nucleic acid molecules in various ways. For example, nucleic acids (eg, plasmids) can be produced using a variety of standard cloning techniques, recombinant DNA technology, through cellular expression or in vitro translation, and chemical synthesis techniques. Purity can be determined by sequencing, gel electrophoresis and the like. For example, nucleic acids can be isolated using hybridization or computer database screening methods. Such methods include, but are not limited to: (1) hybridization of genomic DNA or cDNA libraries with probes to detect homologous nucleotide sequences; (2) screening antibodies to detect polypeptides having common structural features, for example, using an expression library; (3) polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to the nucleic acid sequence of interest; (4) computer search for related sequences in databases; and (5) differential subtraction nucleic acid library screening.

[0194] Nucleic acids can be maintained as DNA in any convenient cloning vector. In one embodiment, the nucleic acids are contained in a plasmid. Alternatively, the nucleic acids may be maintained in a vector suitable for expression in mammalian cells.

[0195] Nucleic acids, plasmids, vectors, expression vectors (eg, rAAV) and recombinant virus particles, methods and applications allow for the treatment of genetic diseases. In deficiency diseases, gene transfer can be used to introduce a normal gene into affected tissues for replacement therapy, as well as to create animal models of the disease using antisense mutations. For unbalanced disease states, gene transfer can be used to reproduce the disease state in a model system, which can then be used to counteract the disease state. Site-specific integration of nucleic acid sequences can also be used to correct defects.

[0196] Viral vectors, such as lentiviral and parvoviral vectors, including AAV serotypes and variants thereof, provide a means for delivering nucleic acid to cells ex vivo, in vitro, and in vivo that encode proteins such that the cells express the encoded proteins. AAVs are viruses useful as vectors for gene therapy because they can enter cells and introduce nucleic acid/genetic material, ensuring stable maintenance of the nucleic acid/genetic material in the cells. In addition, these viruses can, for example, introduce nucleic acid/genetic material at specific sites. Because AAVs are not associated with pathogenic disease in humans, AAV vectors are capable of delivering heterologous polynucleotide sequences (eg, therapeutic proteins and agents) to patients without causing significant AAV-mediated pathogenesis or disease.

[0197] Viral vectors that may be used include, but are not limited to, adeno-associated virus (AAV) vectors of numerous serotypes (e.g., AAV-1 to AAV-12 and others) and AAV hybrid/chimeric vectors, lentiviral vectors, and pseudotyped lentiviral vectors (eg, Ebola virus, vesicular stomatitis virus (VSV) and feline immunodeficiency virus (FIV)), herpes simplex virus vectors, adenoviral vectors (with or without tissue-specific promoters/enhancers), vaccinia virus vectors, retroviral vectors, lentiviral vectors, non-viral vectors and others.

[0198] AAV and lentiviral particles can be used as carriers for efficient gene delivery. Such virions have a number of desirable properties for such applications, including tropism for dividing and non-dividing cells. Early clinical experience with these vectors also demonstrated a lack of sustained toxicity, and immune responses were minimal or undetectable. AAV is known to infect a wide range of cell types in vivo and in vitro via receptor-mediated endocytosis or transcytosis. These vector systems have been tested in humans, targeting retinal epithelium, liver, skeletal muscle, airway, brain, joints, and hematopoietic stem cells. Non-viral vectors, such as those based on plasmid DNA or minicycles, are also suitable gene carriers.

[0199] Therefore, in various embodiments of the invention, the vector includes a lentiviral or parvoviral vector, such as an adenoviral vector. In specific embodiments, the recombinant vector is a parvovirus vector. Parvoviruses are small viruses whose genome contains single-stranded DNA. “Adeno-associated viruses” (AAV) belong to the parvovirus family.

[0200] AAV vectors and lentiviral vectors generally do not include viral genes associated with pathogenesis. Such vectors typically have one or more wild-type AAV genes completely or partially deleted, such as the rep and/or cap genes, but retain at least one functional ITR flanking sequence necessary for rescue, replication, and packaging of the recombinant vector into a vector AAV particle. For example, only the essential parts of the vector are included, such as the ITR and LTR elements, respectively. Therefore, the AAV vector genome must include sequences required in cis configuration for replication and packaging (eg, functional ITR sequences).

[0201] The recombinant AAV vector, as well as methods and uses thereof, include any viral strain or serotype. As a non-limiting example, the recombinant AAV vector may be based on the genome of any AAV, such as AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10 , -11, -12 or AAV-2i8. Such vectors may be based on the same strain or serotype (or subgroup or variant) or may differ from each other. As a non-limiting example, a recombinant AAV vector based on a single serotype genome may be identical to one or more capsid proteins that package the vector. In addition, the genome of the recombinant AAV vector may be based on the genome of an AAV serotype (eg, AAV2) other than one or more AAV capsid proteins that package the vector. For example, the AAV vector genome may be AAV2-based, while at least one of the three capsid proteins may be AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or AAV- 2i8 or its variant, for example. AAV variants include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 AAV-2i8 variants and chimeras, capsids SEQ ID NO1 and SEQ ID NO: 2.

[0202] In specific embodiments, adeno-associated virus (AAV) vectors include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, SEQ ID NO1 and SEQ ID NO: 2, as well as variants thereof (e.g. capsid variants such as amino acid insertions, additions, substitutions and deletions), for example as described in WO 2013/158879 (international application PCT/US2013/037170), WO 2015/013313 ( international application PCT/US2014/047670) and US patent application 2013/0059732 (US patent application 13/594773, discloses LK01, LK02, LK03, etc.).

[0203] AAV and AAV variant serotypes (e.g., capsid variants) (e.g., VP1, VP2, and/or VP3 sequences) may or may not be different from other AAV serotypes, including, for example, AAV1-AAV12 (e.g., different from VP1 sequences , VP2 and/or VP3 of any of the serotypes AAV1-AAV12).

[0204] As used herein, the term “serotype” is a distinction used to designate a capsid-containing AAV that is serologically distinct from other AAV serotypes. Serological difference is determined based on the lack of cross-reactivity between antibodies to one AAV compared with another AAV. Such differences in cross-reactivity are usually due to differences in capsid protein/antigenic determinant sequences (eg, due to differences in the VP1, VP2 and/or VP3 sequences of AAV serotypes). Although it is possible that AAV variants, including capsid variants, may not differ serologically from the reference AAV or another AAV serotype, they differ by at least one nucleotide or amino acid residue compared to the reference AAV or AAV of another serotype.

[0205] According to the traditional definition, a serotype means that the virus of interest has been tested on a serum specific to all existing and characterized serotypes for neutralizing activity, and no antibodies have been detected that neutralize the virus of interest. Since a large number of natural virus isolates have been discovered and/or capsid mutants have been generated, serological differences with any of the currently existing serotypes may or may not exist. Thus, in cases where a new virus (eg, AAV) is not serologically distinct, the new virus (eg, AAV) will represent a subgroup or variant of the corresponding serotype. In many cases, serological testing for neutralizing activity has not yet been performed on mutant viruses with capsid sequence modifications to determine whether they are of a different serotype according to the traditional definition of serotype. Therefore, for convenience and to avoid repetition, the term "serotype" broadly refers to both serologically distinct viruses (e.g., AAV) and viruses (e.g., AAV) that are not serologically distinct, and which may be found in a subgroup or variant of this serotype.

[0206] In various example embodiments, an AAV vector of a reference serotype has a polynucleotide, polypeptide, or subsequence thereof that includes or consists of at least 80% or more of the sequence (e.g., 85%, 90%, 95 %, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, etc.) identical to one or more AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, SEQ ID NO1 or SEQ ID NO: 2 (for example, such as ITR or VP1, VP2 and/or VP3 sequences ).

[0207] The compositions, methods and uses of the invention include AAV sequences (polypeptides and nucleotides) and subsequences thereof that exhibit less than 100% sequence identity to a reference AAV serotype, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 , AAV8, AAV9, AAV10, AAV11, AAV12, or AAV-2i8, but are different from and not identical to known AAV genes or proteins such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 , AAV12 or AAV-2i8, genes or proteins, etc. In one embodiment, the AAV polypeptide or subsequence thereof includes or consists of a sequence that is at least 75% or more identical, e.g., 80%, 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2 %, 99.3%, 99.4%, 99.5%, etc., up to 100% identical to any AAV reference sequence or subsequence thereof, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, SEQ ID NO1 or SEQ ID NO: 2 (for example, capsid VP1, VP2 and/or VP3 or ITR). In particular aspects, the AAV variant has 1, 2, 3, 4, 5, 5-10, 10-15, 15-20, or more amino acid substitutions.

[0208] Recombinant AAV vectors, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV-2i8, SEQ ID NO1 or SEQ ID NO: 2 and a variant associated with Accordingly, hybrid and chimeric sequences can be constructed using recombinant techniques known to those skilled in the art to include one or more nucleic acid sequences (transgenes) flanked by one or more functional AAV ITR sequences.

[0209] Nucleic acids (plasmids), vectors, recombinant vectors (eg, rAAV), and recombinant virus particles can be included in pharmaceutical compositions. Such pharmaceutical compositions are suitable, inter alia, for administration and delivery to a subject in vivo or ex vivo. In certain embodiments, the pharmaceutical compositions contain a pharmaceutically acceptable carrier or excipient. Such excipients include any pharmaceutical agent that does not itself induce an immune response harmful to the subject receiving the composition and that can be administered without causing excessive toxicity.

[0210] As used herein, the terms "pharmaceutically acceptable" and "physiologically acceptable" mean a biologically acceptable composition, gaseous, liquid or solid, or a mixture thereof, that is suitable for one or more routes of administration, delivery or contact in vivo. A "pharmaceutically acceptable" or "physiologically acceptable" composition is a substance that is not biologically or otherwise undesirable, for example, the substance can be administered to a subject without significant adverse biological effects. Thus, such a pharmaceutical composition can be used, for example, to administer a nucleic acid, vector, viral particle or protein to a subject.

[0211] Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerin, sugars, and ethanol. Pharmaceutically acceptable salts may also be included, for example, salts of mineral acids such as hydrochlorides, hydrobromides, phosphates, sulfates and the like; and salts of organic acids such as acetates, propionates, malonates, benzoates and the like. In addition, such carriers may contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like.

[0212] The pharmaceutical composition may be provided as a salt, and the salt may be formed with many acids, including, but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic acid, etc. Salts tend to be more soluble in water or other protic solvents than the corresponding free base forms. In other cases, the preparation may be a lyophilized powder that may contain any or all of the following: 1-50 mM histidine, 0.1%-2% sucrose and 2%-7% mannitol at a pH range of 4.5 to 5.5 , and which is concatenated with the buffer before use.

[0213] Pharmaceutical compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (eg, oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersions and suspensions, carriers, coatings , isotonic and stimulant and absorption-decreasing agents compatible with pharmaceutical administration or contact or in vivo delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickeners. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules and crystals. Additional active compounds (eg, preservatives, antibacterial, antiviral and antifungal agents) may also be included in the compositions.

[0214] Pharmaceutical compositions may be formulated to be compatible with a particular route of administration or delivery. Thus, pharmaceutical compositions include carriers, diluents or excipients suitable for administration by various routes.

[0215] The compositions and methods may be sterile. The compositions can be prepared and the methods can be carried out in containers suitable for such processes. Such containers include cuvettes, flasks, roller bottles, bags, bioreactors, vessels, test tubes, vials, etc. Containers can be made from materials that include, but are not limited to, glass, plastic and polymers such as polystyrene, polybutylene, polypropylene, etc.

[0216] The compositions and method steps may be performed in a predetermined order or in a rearranged order. The process steps may be performed in stages or at specified intervals. In other words, a method step may be performed and then there may be a time interval between the next step, where such intervals vary, for example, from about 1 second to about 60 seconds; from about 1 minute to about 60 minutes; from about 1 hour to about 24 hours; from about 1 day to about 7 days; or from about 1 week to about 48 weeks.

[0217] Protocols for the production of adenoviral vectors are described in US Pat. No. 5,998,205; 6228646; 6093699 and 6100242; and international patent applications WO 94/17810 and WO 94/23744, which are incorporated herein by reference in their entirety.

[0218] The invention is suitable for obtaining cells and vectors for subsequent use in medicine and veterinary medicine. Therefore, suitable subjects include mammals such as humans, as well as mammals other than humans. The term "subject" refers to an animal, usually a mammal such as a human, a non-human primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (poultry such as chickens) and ducks, horses, cows, goats, sheep, pigs) and experimental animals (mice, rats, rabbits, guinea pigs). Human subjects include fetuses, newborns, infants, adolescents and adults. Subjects include animal models of diseases, such as mice and other animal models of coagulation disorders such as HemA and others known to those skilled in the art.

[0219] As used herein, the term “unit dosage form” refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit contains a predetermined amount, optionally in combination with a pharmaceutical carrier (excipient, diluent, vehicle or excipient), which, when administered in one or more doses, is calculated to produce the desired effect (eg, prophylactic or therapeutic effect). Unit dosage forms may be, for example, in ampoules and vials, which may include a liquid composition or a composition in a freeze-dried or lyophilized state; for example, a sterile liquid carrier may be added prior to administration or in vivo delivery. Individual unit dosage forms may be included in multi-dose kits or containers. Recombinant vector sequences (eg, rAAV), recombinant virus particles, and pharmaceutical compositions thereof may be packaged into one or multiple unit dosage forms for ease of administration and uniformity of dosage.

[0220] The invention provides kits with packaging material and one or more components therein. The kit typically includes a label or package insert that includes a description of the components or instructions for use of the components contained within. The kit may contain a number of such components, for example, nucleic acid (plasmid), PEI, enhancing agent, cells.

[0221] The term “kit” refers to the physical structure that houses one or more components of the kit. The packaging material may contain the components in a sterile state and may be made from material commonly used for such purposes (eg, paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0222] Labels or inserts may include identifying information for one or more components. Labels or inserts may contain information identifying the manufacturer, lot numbers, place and date of manufacture, and expiration date. Labels or inserts may include identifying information about the manufacturer, lot numbers, manufacturer's location, and date. Labels or package inserts may include instructions for use of one or more components of the kit in a method, use, or manufacturing protocol. The instructions may include instructions for preparing the compositions or using any of the methods described herein.

[0223] Labels or inserts include “printed materials,” such as paper or cardboard, either separately or attached to a component, kit, or packaging material (e.g., a box), or affixed to an ampoule, tube, or vial containing a kit component. The labels or inserts may further include a machine-readable medium such as a printed bar code label, a disk, an optical disk such as a CD or DVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage medium such as RAM and ROM , or hybrids thereof, such as magnetic/optical storage media, flash media or memory cards.

[0224] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention relates. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0225] All patents, patent applications, publications and other references, GenBank data and ATCC data cited herein are incorporated by reference in their entirety. In the event of a conflict, the description, including definitions, will control.

[0226] Various terms relating to the biological molecules of the invention are used above and in the description and claims.

[0227] All features disclosed herein may be combined in any combination. Each feature disclosed in the specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, the disclosed features (e.g., PEI, plasmid, vector (e.g., rAAV or recombinant viral particle) are an example of a type of equivalent or similar features.

[0228] As used herein, the singular forms "a", "and" and "the" include multiple references unless the context clearly indicates otherwise. Thus, for example, a reference to a “plasmid” or “nucleic acid” includes a plurality of such plasmids or nucleic acids, a reference to a “vector” includes a plurality of such vectors, a reference to a “virus” or “particle” includes a plurality of such viruses/particles , and reference to “enhancing agent” includes a variety of such agents.

[0229] As used herein, all numeric values or numeric ranges include integers within such ranges and fractions of values or integers within the ranges unless the context clearly indicates otherwise. Thus, for illustration purposes, reference to 80% identical or greater includes 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, etc., as well as 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, etc., 82.1%, 82, 2%, 82.3%, 82.4%, 82.5%, etc., etc.

[0230] A reference to an integer greater than or less than includes any number greater than or less than the reference number, respectively. Thus, for example, a reference to less than 100 includes 99, 98, 97, etc., up to the number one (1); and less than 10, including 9, 8, 7, etc. up to the number one (1).

[0231] As used herein, all numerical values or ranges include fractions of values and integers in such ranges and fractions of integers in such ranges, unless the context clearly indicates otherwise. Thus, for illustration purposes, reference to a numeric range such as 1-10 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1. 3, 1.4, 1.5, etc. etc. Range reference 1-50 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, etc. up to and including 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc. etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc., etc.

[0232] A range series reference includes ranges that combine the boundary values of different ranges within the series. Thus, for illustration purposes, reference is made to a number of ranges, for example, 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150 150-200 , 200-250, 250-300, 300-400, 400-500, 500-750, 750-850, includes ranges 1-20, 1-30, 1-40, 1-50, 1-60, 10-30 , 10-40, 10-50, 10-60, 10-70, 10-80, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 50-75, 50 -100, 50-150, 50-200, 50-250, 100-200, 100-250, 100-300, 100-350, 100-400, 100-500, 150-250, 150-300, 150-350 , 150-400, 150-450, 150-500, etc.

[0233] The invention is generally disclosed herein using affirmative expressions to describe numerous embodiments and aspects. The invention also particularly includes embodiments in which specific subject matter of the invention is excluded, in whole or in part, such as substances or materials, method steps and conditions, protocols or procedures. For example, in certain embodiments or aspects of the invention, materials and/or method steps are eliminated. Thus, even though the invention as a whole is not expressed herein in terms that the invention does not include aspects that are not expressly excluded by the invention, it is nevertheless disclosed herein.

[0234] A number of embodiments of the invention have been described. However, one skilled in the art, without departing from the spirit and scope of the invention, can make various changes and modifications to the invention to adapt it to different applications and conditions. Accordingly, the following examples are intended to illustrate, but not to limit, the scope of the invention claimed in any way.

Example 1

Representative materials and methods

[0235] Cell culture. FreeStyle™ 293F (HEK 293F) cells obtained from Thermo Fisher Scientific (R79007) were cultured in FreeStyle™ F17 (F17) expression medium (Thermo Fisher Scientific, A1383501), supplemented with 1× GlutaMAX™ (Thermo Fisher Scientific, 35050-061 ) and 1× antibiotic-antimycotic (Thermo Fisher Scientific, 15240). Cells were cultured in spinner flasks (Corning, 3152 or 3153), shake flasks (Corning 431143 or 431145), or bioreactors. For the spin flask/shaken flask, cells were cultured in an incubator at 37°C with stirring at 170 rpm and in a humidified atmosphere of 8% CO ₂ ; for bioreactors (Eppendorf, DASGIP Parallel Bioreactor system, glass vessels and disposable vessels), cell culture was controlled according to programmed parameters (DO 40%, pH 7.2, stirring at 130 rpm, 150 rpm or 170 rpm). Typically, cells were seeded at a titer of 0.25-0.5×10 ⁶ /ml, subcultured every 2-3 days by adding fresh cell culture medium when the cell density reached approximately 2-3×10 ⁶ /ml. Cell density and viability were determined using a Vi-cell™ XR Cell Viability Analyzer (Beckman Coulter).

[0236] Plasmids: Three plasmids were used to produce recombinant adeno-associated viral vectors (rAAV): 1) transgenic plasmid containing hFVIII flanked by ITR, 2) packaging plasmid containing rep and cap genes, 3) adenoviral helper plasmid containing adenoviral E2 genes , E4 and VARNA. All plasmids were purchased and manufactured by Aldevron.

[0237] Preparation of PEI solutions. The transfection reagent used was linear polyethylenimine (PEI) “Max” 40 kDa (Polysciences, 24765-2, linear PEI hydrochloride with a molecular weight of 25 kDa). For transfection optimization studies, PEI "Max" was dissolved in 5 mM Tris buffer to produce a 0.5 mg/ml solution, and the pH of the solution was adjusted to various pH values, including pH 7.0, 7.1, 7.2, 7 .3, 7.4, 7.6, 7.8 or 8.0. After several experiments, PEI pH 7.1 solution was chosen for all studies unless otherwise noted due to better transfection and rAAV production.

[0238] Use of enhancing agents in PEI-mediated transfection: Potential transfection enhancing agents assessed for their effect on transfection and rAAV production were enhancers 1 and 2 from the ExpiFectamine™ 293 transfection kit (Thermo Fisher Scientific, A14525), P3000 reagent from Lipofectamine® 3000 Transfection Kit (Thermo Fisher Scientific, L3000015), enhancer from Effectene™ Transfection Kit (Qiagen, 301427). Various compounds including valproic acid, etoposide, teniposide, siomycin A, and vorinostat were also studied to test their efficacy in PEI cell transfection and rAAV production.

[0239] HEK 293F cells were grown in F17 medium plus 1x GlutaMAX™ supplement and 1x antibiotic-antimycotic in spinner flasks or shake flasks. One day before transfection, cells were seeded at a rate of 0.5-2×10 ⁶ cells/ml by adding fresh medium. After 24 hours, cells were transfected at a cell density of 1-4×10 ⁶ cells/ml. Three transfection plasmids, hFVIII, Rep/cap, Ad2 helper, were used in a molar ratio of 1:1:1, 0.5:1:1 and 1:2:2 and a mass ratio of 0.75:0.75:0 .75, 1:1:1 and 1.5:1.5:1.5. The total amount of DNA used for transfection ranged from 0.5 to 4.2 μg per ml cell culture volume. The PEI/DNA complex was prepared with different mass ratios of PEI and DNA at 1:1, 1.5:1, 2:1, 2.5:1 and 3:1, incubated at room temperature for 1, 5, 10, 15 , 20, 25 and 30 min, then the DNA/PEI complexes were added to the cell culture. Free PEI (without DNA) at a concentration of 0.5 to 5.6 μg/ml was added to the cell culture immediately after the DNA/PEI complexes. Enhancing agents were added directly to cells at the time of transfection, either 24 h before transfection or 16–18 h after transfection. Various amounts of enhancing agents were evaluated. Samples, including cells and cell culture media, were collected at 48 and 72 h posttransfection for cell counting and cell viability assessment, and cell cultures were harvested at 72 h posttransfection.

[0240] Production of rAAV vectors in bioreactors: To scale up the vector production method, a DASGIP 2L parallel bioreactor system (Eppendorf) equipped with two or three inclined impeller mixers was used. The final working volume was adjusted to 400 ml or 1.2 l. Stirring was set at 130 rpm, 150 rpm or 170 rpm, and the temperature was maintained at 37°C. The pH value was tested at 6.3, 6.8, 7.2, 7.4, 7.6, or 8. pH 7.2 was selected due to better cell growth and rAAV production. Dissolved oxygen was maintained at 40% by adding a gas mixture of oxygen, carbon dioxide and air. All these parameters were monitored and controlled by the DASGIP control system with DASGIP Control 4.0 software. HEK 293F cells cultured in F17 medium were inoculated at a cell density of 0.4×10 ⁶ cells/ml with a viability of more than 95%. Cells were subcultured on day 3 after seeding by adding fresh medium. Cell density was adjusted to approximately 0.5-1.7 x 10 ⁶ cells/ml after subculture. After 24 h of subculture, cells were transfected with the PEI/DNA complex, free PEI, and transfection enhancers as described above and in the figure legends. During transfection, the cell density was approximately 1-3×10 ⁶ cells/ml. 1.2-4.2 µg/ml DNA, PEI/DNA mixture in a mass ratio of 1:1, 1.5:1, 2:1, 2.5:1 or 3:1 with 0.5-4.2 μg/ml free PEI and enhancing agents were analyzed for effects on plasmid transfection and RAAV production. The cell culture was harvested 72 h after transfection.

[0241] Quantification of rAAV vectors: rAAV vectors were isolated from a harvest of transfected HEK 293F cells by microfluidization (microfluidizer TM, Microfluidics) or triple sonication. Cellular debris was pelleted by centrifugation, and supernatants were collected and analyzed using real-time PCR.

[0242] The rAAV vector genome copy number was determined by real-time polymerase chain reaction (qPCR) (Thermo Fisher Scientific, QuanStudio 7) using TaqMan Master Mix (Thermo Fisher Scientific, 4304437). 10 μl of cell lysate was first treated with 2 μl of universal RNA (Biochain, R423565), and then with 7.6 U of DNase I (Qiagen, 79254) to digest contaminating unfolded DNA. The solution was then treated with 0.2% SDS/5 mM EDTA/0.2 M NaCl and heated at 95°C for 10 min to inactivate DNase I and release vector DNA. Primers and probe for detecting the hFVIII transgene sequence: forward primer: 5'-TGAGGAGGCTGAAGACTAT-3' (SEQ ID NO:3), reverse primer 5'-CCACAGACCTGATCTGAATGAA -3' (SEQ ID NO:4) and probe/5'-6FAM /TGGATGTGG /ZEN/TGAGGTTTGATGATGACA/3IABkFQ/-3' (SEQ ID NO:5). The standard was prepared by linearization of the pAAV-hFVIII plasmid. All samples were analyzed in triplicate.

[0243] Western blot analysis: rAAV vectors were isolated from transfected HEK 293F cells using microfluidization (microfluidizer TM, Microfluidics) or three times sonication. Cellular debris was pelleted by centrifugation, and supernatants were collected for Western blotting. Cell lysate was mixed with 4× NuPAGE LDS sample buffer (Thermo Fisher Scientific, NP0007) and then heated at 95°C for 5 min. Samples were separated by SDS-PAGE and transferred to a PVDF membrane (Thermo Fisher Scientific, LC2002). After blocking with Odyssey blocking buffer (Li-COR Biosciences, 927-50000) for 1 h, the membranes were incubated at room temperature for 2 h with a 1:500 dilution of mouse monoclonal anti-AAV VP1,2,3 antibody (American Research Products, Inc., 03-65158). After three washes, membranes were incubated at room temperature for 1 h with Alexa Fluo-680-conjugated goat anti-mouse IgG secondary antibody at a dilution of 1:5000 (Invitrogen, A21057). Membranes were scanned using an Odyssey® CLx imager (Li-COR Biosciences).

Example 2

[0244] Transfection enhancers increased production of the rAAV-FVIII vector.

[0244] A highly efficient PEI-based transfection method using PEI "Max" as the transfection reagent was developed to transfect three plasmids into HEK 293F cells in F17 medium to produce rAAV vectors. The best cell culture window for plasmid transfection to obtain maximum rAAV production has been described. However, when used to produce rAAV-FVIII vectors, vector production was relatively low (vector titer at 2-3E+10 vg/ml). The reason for the low production may be that the FVIII transgene is quite large compared to other transgenes such as eGFP, FIX.

[0245] A search has been made for agents that can improve transfection efficiency. Various transfection enhancers from several transfection kits were evaluated, including the ExpiFectamine™ 293 Transfection Kit, the Lipofectamine® 3000 Transfection Kit, and the Effectene™ Transfection Reagent Kit. Data show that transfection enhancers from the ExpiFectamine™ 293 Transfection Kit can significantly increase rAAV vector production. Enhancers from other transfection kits did not significantly improve rAAV production. Enhancers 1 and 2 from the ExpiFectamine™ 293 kit were used at a ratio of 1:200 and 1:20 to culture volume, respectively. ExpiFectamine™ 293 Transfection Kit Enhancers 1 and 2 increased rAAV titer 2- to 3-fold during transfection compared to their addition 16 to 18 h posttransfection or without enhancers (Figure 1). Addition of enhancers 1 and 2 before transfection did not increase rAAV production. Enhancer 1 and Enhancer 2 are components of the ExpiFectamine™ 293 Transfection Kit, which is a set of cationic lipid-based transfection reagents for protein expression from Expi293F™ cells cultured in Expi293™ expression medium. Manufacturers state that the enhancers are chemically defined, protein-free, serum-free reagents of animal origin. These amps have been used in a new application that differs from the original design of this kit. These enhancers were used to generate rAAV vectors instead of protein expression. The kit suggests using enhancers 1 and 2 16-18 hours after transfection to enhance transfection and protein expression. However, the results of the studies disclosed herein show that enhancer 1 and enhancer 2 added simultaneously during transfection result in the highest rAAV production, 2-3 times higher than use after transfection, suggesting that enhancers play a role another role in rAAV production through protein expression. The addition of these enhancers to the PEI-mediated transfection method described here and in PCT/US16/64414 enhanced transfection of the large transgene plasmid into cells, resulting in a significant increase in rAAV titer.

[0246] Transfection parameters including transfection cell density, amount of DNA, PEI/DNA ratio, amount of free PEI in the presence or absence of these enhancers were further optimized. The findings indicate that increasing transfection cell density and PEI/DNA ratio using less DNA and free PEI resulted in the greatest rAAV production in the presence of enhancers.

[0247] Figure 2 shows the production of the rAAV-FVIII vector using different amounts of DNA and PEI/DNA ratio in a spinner flask, which was determined by qPCR. HEK 293F cells in spinner flasks were transfected with 1.2, 1.86, 2.8 μg/ml DNA, the PEI/DNA (N/P) ratio used was 1:1, 1.5:1, 2:1, 2. 5:1, 3:1 and 1.5 µg/ml free PEI. Enhancers 1 and 2 from the ExpiFectamine™ 293 Transfection Kit were added to cells during transfection. Amplifier 1 and amplifier 2 were used in amounts of 1:200 and 1:20 of the culture volume, respectively. The cell density during transfection was 2.5-3×10 ⁶ cells/ml. The best condition for vector production was the use of 1.2 μg/ml DNA with a PEI/DNA ratio of 2-2.5:1 and 1.5 μg/ml free PEI and enhancer 1 (1:200 dilution) and enhancer 2 (1 dilution :20) upon transfection.

[0248] Conditions in 2L DASGIP bioreactors were evaluated and further optimized on a larger scale. Figure 3 shows the production of the rAAV-FVIII vector using different amounts of DNA and PEI/DNA ratio in bioreactors, as determined by qPCR. HEK 293F cells were cultured in 400 ml F17 at 37°C, pH 7.2, DO 40%, with stirring at 150 rpm in bioreactors. Cells were transfected with DNA molar ratios of 1:1:1 and 1.2 or 2.8 μg/ml DNA, PEI/DNA (N/P) ratios of 1.5:1, 2:1, 2.5:1, or 3 :1 and 1.5 µg/ml free PEI. Enhancers 1 and 2 from the ExpiFectamine™ 293 Transfection Kit were added to cells during transfection. Enhancer 1 and Enhancer 2 were used at a ratio of 1:200 and 1:20 to culture volume, respectively. The cell density was 2-3×10 ⁶ cells/ml during transfection. The condition that resulted in the highest rAAV vector production was the use of a DNA molar ratio of 1:1:1 and 1.2 μg/ml DNA with a PEI/DNA ratio of 2.5-3:1 and 1.5 μg/ml free PEI and amplifiers.

[0249] Cell culture volume was increased from 400 ml to 1.2 L in bioreactors to produce rAAV. HEK 293F cells were cultured at 37°C, pH 7.2, DO 40%, agitation 130 rpm, 150 rpm or 170 rpm with 2 or 3 impeller mixers. Cells were transfected with a DNA molar ratio of 1:1:1, 1.2 μg/ml DNA, a PEI/DNA (N/P) ratio of 2.5:1, and 1.5 μg/ml free PEI. Enhancers 1 and 2 from the ExpiFectamine™ 293 transfection kit were used as described above. In fig. Figure 4 shows the titers of rAAV obtained in these experiments. The data showed that optimized conditions from a small volume of 50 mL in a spinner flask could be scaled up to a volume of 400 mL, and then to a volume of 1.2 L. The use of enhancing agents under optimized PEI-mediated transfection conditions can increase rAAV vector production by 8- to 10-fold in serum-free suspension culture. This process is very efficient, safe and scalable.

[0250] To determine whether both enhancers 1 and 2 are required upon transfection to enhance rAAV production and to further quantify these enhancers, they were assessed separately. The data showed that booster 1 alone could increase rAAV production to the same level as both boosters 1 and 2 combined. Enhancer 2 alone did not increase rAAV production (Fig. 5). The number of amplifiers 1 and 2 was also reduced to further determine the optimal conditions of the method. Reducing the amount of enhancers 1 and 2 resulted in a decrease in rAAV titer (Fig. 5). Increased amounts of enhancer 1 (1:100 or 1:150 dilution) and enhancer 2 (1:10 or 1:15 dilution) during transfection did not markedly improve rAAV production. In addition to the use of enhancers 1 and 2 during transfection, they were evaluated when added at 24 h or 48 h or at 24 h and 48 h posttransfection to determine whether they could then further increase the titer of rAAV. In fig. Figure 6 shows that repeated use of enhancers slightly increased rAAV production.

Example 3

Valproic acid increases production of the rAAV-FVIII vector.

[0251] Various compounds, including valproic acid, etoposide, teniposide, siomycin A, and vorinostat, were evaluated to determine their effects on rAAV transfection and production. Among these compounds, valproic acid significantly increased rAAV production. Valproic acid is a histone deacetylase inhibitor and an FDA-approved drug for the treatment of epileptic conditions. Various concentrations of valproic acid, 0.25 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 5 mM, 7.5 mM and 10 mM were used for transfection in spinner/shaken flask culture, and when cultivated in a DASGIP bioreactor. The use of valproic acid in the optimized PEI-mediated transfection increased the titer of the rAAV-FVIII vector by approximately 10-fold compared to the no-enhancer variant and achieved the same level of rAAV production or higher as with enhancers 1 and 2 in a serum-free suspension culture system (Fig. 7). 1.2 μg/ml DNA, DNA molar ratio 1:1:1, PEI/DNA mass ratio 2:1 or 2.5:1, and 1.5 μg/ml free PEI were used in these valproic acid experiments. After adding the PEI/DNA complex and free PEI, valproic acid was added to the cells during transfection at various concentrations. No enhancers from the ExpiFectamine™ 293 transfection kit were used in this study. Figure 8 shows increased AAV vector production at elevated concentrations of valproic acid in a 1 L bioreactor, for example, in the presence of valproic acid at a concentration of 4 mM or higher, such as 5 -8 mm.

Example 4

[0252] One ExpiFectamineTM 293 transfection kit enhancer 1 and valproic acid, used separately in an optimized PEI-based transfection method, can significantly increase rAAV vector production, approximately 10-fold, than the currently reported production protocol. using similar technologies. The use of only enhancer 1 or valproic acid in the rAAV production system provides a new scalable rAAV production platform that can be used to efficiently produce any serotype of rAAV vectors in serum-free suspension cell culture. This process is particularly useful for producing rAAV vectors with large transgenes and for producing rAAV vectors that are difficult to generate. The method is fully scalable, cGMP compliant, and a versatile rAAV production system that is suitable for large-scale rAAV production.

Example 5

[0253] The data in Figure 4 demonstrates that optimized conditions from a small volume of 50 mL in a spinner flask can be scaled up to a volume of 400 mL, and then to a volume of 1.2 L. Further scalability to larger volumes can be achieved. For example, the methods are expected to be scalable to volumes of 2 L, 2-20 L, 20-50 L, or 50-100 L. Even larger volumes such as 100-500 L, 500-1000 L or 1000 L or more are considered. Such scaling up may involve cloning cells and selecting clones that produce large quantities of the rAAV vector. The establishment of master cell banks of such clones that express the rAAV vector in large quantities provides a reliable and reproducible source of cells applicable to the compositions and methods of the present invention.

[0254] Further improvements in cell culture conditions, transfection conditions, cell lysis, and/or culture supernatant collection for rAAV vector isolation, contaminant removal, and downstream purification conditions may also contribute to significant increases in scalability.

Example 6

1.0 Exemplary, non-limiting, method development acceptance criteria, general outline, process description, and parameters modified to scale up the production of rAAV vector (e.g., LK03-FVIII) using suspension cell cultures.

1.1 Thawing and cell expansion

Process criteria Units Instrument used for analysis Viable Cell Density (VCD) Sufficient number of viable cells/ml to proceed to the next stage of expansion Vi-CELL™ XR

1.2 Production of rAAV vector in a volume of 1.2 l in a bioreactor

Process criteria Units Test used for analysis Products when collected ≥ 5 × 10 ¹⁰ vg/ml Viral genome titer according to qPCR data

2.0 General view of the process

2.1 Process of cell thawing and cell expansion

2.1.1 Process flow diagram

Below is a typical flow diagram for the cell thawing and upstream cell expansion section to obtain the rAAV vector:

2.1.2 Description of the technological process

Below is a typical process description for the thawing and upstream cell expansion portion of the rAAV vector (e.g., LK03-FVIII):

Stage or operation Description Thawing cells Cells are thawed at 37°C for ≥ 2.5 min
Cells are placed in 29 ml of pre-warmed Freestyle™ F17 medium with 1X GlutaMAX™
30 ml cells in a 125 ml shake flask
Cell cultivation at 37°C for 4 days at 8% CO ₂ , 125 rpm Passage 1 Subculture cells to 0.3-0.5 x ¹⁰⁶ live cells/ml using Freestyle™ F17 pre-warmed media with 1X GlutaMAX™
150 ml cells in a 500 ml shake flask
Cell cultivation at 37°C for 3-4 days at 8% CO ₂ , 125 rpm Passage 2 Subculture cells to 0.3-0.5 x ¹⁰⁶ live cells/ml using Freestyle™ F17 pre-warmed media with 1X GlutaMAX™
400 cells in each of 2 x 1000 ml shake flasks (800 ml total)
Cell cultivation at 37°C for 3-4 days at 8% CO ₂ , 125 rpm Passage 3 Subculture cells to 0.3-0.5 x ¹⁰⁶ live cells/ml using Freestyle™ F17 pre-warmed media with 1X GlutaMAX™
400 cells in each of 3 x 1000 ml shake flasks (1200 ml total)
Cell cultivation at 37°C for 3-4 days at 8% CO ₂ , 125 rpm Next stage Shake flask cells are used to seed DAS GIP bioreactors to produce rAAV vector, e.g. LK03-FVIII

2.1.3 Parameters changed during cell thawing and cell expansion studies

The following parameters were evaluated during the development of the cell thawing and cell expansion process of 293-F cells:

Stage or operation Parameter under study Ranges studied (if applicable) Thawing cells Minimum defrost time Time required to see one ice crystal remaining in the vial Thawing cells Maximum defrost time 2X and 3X from a certain minimum time Thawing cells Time spent on dry ice for vials with cells before thawing 0-2 h Cell expansion Seeding density 0.1-1.1 × 10 ⁶ live cells/ml Cell expansion Upper limit of cell density for seeding in culture 4-6 × 10 ⁶ live cells/ml Cell expansion Shaker speed limits (2.5 cm stroke) 125±15 rpm

Example 7

2.3. Exemplary, non-limiting rAAV vector (e.g., LK03-FVIII) production at 1.2 L bioreactor scale, process flow, and parameters modified to scale up rAAV vector protein production

2.3.1. Process flow diagram

Below is a typical flow diagram for rAAV vector production (e.g. LK03-FVIII) in a 1.2 L bioreactor:

2.3.2 Description of the technological process

Below is a description of the process flow for rAAV vector production (e.g. LK03-FVIII) in a 1.2 L bioreactor:

Stage or operation Description Sowing in DAS GIP bioreactors
(day 0) Inoculate each of the 8 x 2 L DAS GIP bioreactors at a seeding density of 0.4 x ¹⁰⁶ live cells/ml using Freestyle™ F17 pre-warmed media with 1X GlutaMAX™
Addition of Pluronic™ F-68 to the medium, final content 0.1%
Working volume is 400 ml
Cell cultivation at 37°C, pH 7.2, DO 40%, 150 rpm for 3 days Breeding and bringing bioreactors to full operating volume
(day 3) Diluting the bioreactor contents to a final working volume of 1200 ml using Freestyle™ F17 pre-warm media with 1X GlutaMAX™
Cell density is 1-1.5 × 10 ⁶ living cells/ml
Cultivation of cells at 37°C, pH 7.2, DO 40%, 150 rpm for 1 day Transfection
(4 days) Transfection in each bioreactor by adding 3 plasmids/PEI, free PEI, booster(s)
Cell density is 2-3 × 10 ⁶ living cells/ml
Cell cultivation at 37°C, pH 7.2, DO 40%, 150 rpm for 3 days End of production
(day 7) Sampling from each bioreactor 72 h post-transfection for production using titer of viral genomes using qPCR assay Next stage Collection of material from each bioreactor and subsequent processing

2.3.3 Parameters changed during rAAV vector production development (e.g. LK03-FVIII)

The following parameters were evaluated during the development of a method for producing rAAV vector (e.g. LK03-FVIII) in a 1.2 L bioreactor:

Stage or operation Parameter under study Ranges studied (if applicable) Cell cultivation in DAS GIP bioreactors Seeding density 0.25-0.5 × 10 ⁶ live cells/ml Mixing speed 130-170 rpm pH 6.3-8.0 Final displacement 400 ml, 1200 ml Transfection pH of PEI solution 7.0-8.0 Molar ratios of plasmids:FVIII:Rep/cap:Ad2 helper 1:1:1, 0.5:1:1, 1:2:2 Mass ratios of plasmids:FVIII:Rep/cap:Ad2 helper 0.75:0.75:0.75, 1:1:1, 1.5:1.5:1.5 Mass ratio of PEI complex/DNA 1:1, 1.5:1, 2:1, 2.5:1, 3:1 Incubation time of the PEI/DNA complex 1-30 min Total amount of DNA 0.5-4.2 µg/ml Amount of free PEI 0.5-5.6 µg/ml Amplifiers Ranges Add time Type Used together ExpiFectamine™ 293 Kit
amplifier 1 1:100-1:800 24 h before transfection - 18 h after transfection ExpiFectamine™ 293 Kit
amplifier 2 1:10-1:80 24 h before transfection - 18 h after transfection ExpiFectamine™ 293 Kit
amplifier 1 1:200-1:800 24 h before transfection - 18 h after transfection Valproic acid 0.25-10 mM 24 h before transfection - 18 h after transfection End of production Collection time 48, 72 h after transfection

Example 8

[255] Representative AAA capsid proteins (VP1)

AAV-SPK VP1 capsid (SEQ ID NO:1)

1 MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLD

61 KGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ

121 AKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDS

181 ESVPDPQPIGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRV

241 ITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQ

301 RLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSA

361 HQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFED

421 VPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNW

481 LPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSS

541 GVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNS

601 QGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADP

661 PTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTE

721 GTYSEPRPIGTRYLTRNL

AAV-LK03 VP1 capsid (SEQ ID NO:2)

MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQ PLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNN GSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYG TVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRPL

--->

List of sequences

<110> SPARK THERAPEUTICS, INC.

Qu, Guang

Lu, Lin

Josue-Almqvist, Jesusa

Wright, John F.

<120> ENHANCED AGENTS FOR INCREASED CELL TRANSFECTION AND/OR

rAAV VECTOR PRODUCTS

<130> 023637-0459431

<140> PCT/US2018/036344

<141> 2018-06-06

<150> 62/516,432

<151> 2017-06-07

<150> 62/531,626

<151> 2017-07-12

<160> 5

<170> Patent In version 3.5

<210> 1

<211> 738

<212> Protein

<213> Adeno-associated virus

<400> 1

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Asp Leu Lys Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asn Gly Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Ser Pro Val Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Glu Pro Ser Pro Gln Arg Ser Pro Asp Ser Ser Thr Gly Ile

145 150 155 160

Gly Lys Lys Gly Gln Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln

165 170 175

Thr Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro

180 185 190

Pro Ala Ala Pro Ser Gly Val Gly Pro Asn Thr Met Ala Ala Gly Gly

195 200 205

Gly Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser

210 215 220

Ser Ser Gly Asn Trp His Cys Asp Ser Thr Trp Leu Gly Asp Arg Val

225 230 235 240

Ile Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His

245 250 255

Leu Tyr Lys Gln Ile Ser Asn Gly Thr Ser Gly Gly Ser Thr Asn Asp

260 265 270

Asn Thr Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn

275 280 285

Arg Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn

290 295 300

Asn Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn

305 310 315 320

Ile Gln Val Lys Glu Val Thr Gln Asn Glu Gly Thr Lys Thr Ile Ala

325 330 335

Asn Asn Leu Thr Ser Thr Ile Gln Val Phe Thr Asp Ser Glu Tyr Gln

340 345 350

Leu Pro Tyr Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe

355 360 365

Pro Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn

370 375 380

Asn Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr

385 390 395 400

Phe Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Glu Phe Ser Tyr

405 410 415

Asn Phe Glu Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser

420 425 430

Leu Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu

435 440 445

Ser Arg Thr Gln Ser Thr Gly Gly Thr Ala Gly Thr Gln Gln Leu Leu

450 455 460

Phe Ser Gln Ala Gly Pro Asn Asn Met Ser Ala Gln Ala Lys Asn Trp

465 470 475 480

Leu Pro Gly Pro Cys Tyr Arg Gln Gln Arg Val Ser Thr Thr Leu Ser

485 490 495

Gln Asn Asn Asn Ser Asn Phe Ala Trp Thr Gly Ala Thr Lys Tyr His

500 505 510

Leu Asn Gly Arg Asp Ser Leu Val Asn Pro Gly Val Ala Met Ala Thr

515 520 525

His Lys Asp Asp Glu Glu Arg Phe Phe Pro Ser Ser Gly Val Leu Met

530 535 540

Phe Gly Lys Gln Gly Ala Gly Lys Asp Asn Val Asp Tyr Ser Ser Val

545 550 555 560

Met Leu Thr Ser Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr

565 570 575

Glu Gln Tyr Gly Val Val Ala Asp Asn Leu Gln Gln Gln Asn Ala Ala

580 585 590

Pro Ile Val Gly Ala Val Asn Ser Gln Gly Ala Leu Pro Gly Met Val

595 600 605

Trp Gln Asn Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile

610 615 620

Pro His Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe

625 630 635 640

Gly Leu Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val

645 650 655

Pro Ala Asp Pro Pro Thr Thr Phe Asn Gln Ala Lys Leu Ala Ser Phe

660 665 670

Ile Thr Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu

675 680 685

Leu Gln Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr

690 695 700

Ser Asn Tyr Tyr Lys Ser Thr Asn Val Asp Phe Ala Val Asn Thr Glu

705 710 715 720

Gly Thr Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg

725 730 735

Asn Leu

<210> 2

<211> 736

<212> Protein

<213> Adeno-associated virus

<400> 2

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser

1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Gln Pro Gly Ala Pro Lys Pro

20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro

35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro

50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp

65 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala

85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg

130 135 140

Pro Val Asp Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Val Gly

145 150 155 160

Lys Ser Gly Lys Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr

165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro

180 185 190

Ala Ala Pro Thr Ser Leu Gly Ser Asn Thr Met Ala Ser Gly Gly Gly

195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser

210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile

225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu

245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr

260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His

275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp

290 295 300

Gly Phe Arg Pro Lys Lys Leu Ser Phe Lys Leu Phe Asn Ile Gln Val

305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu

325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr

340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp

355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser

370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser

385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Thr Phe Glu

405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg

420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg Thr

435 440 445

Gln Gly Thr Thr Ser Gly Thr Thr Asn Gln Ser Arg Leu Leu Phe Ser

450 455 460

Gln Ala Gly Pro Gln Ser Met Ser Leu Gln Ala Arg Asn Trp Leu Pro

465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Leu Ser Lys Thr Ala Asn Asp Asn

485 490 495

Asn Asn Ser Asn Phe Pro Trp Thr Ala Ala Ser Lys Tyr His Leu Asn

500 505 510

Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys

515 520 525

Asp Asp Glu Glu Lys Phe Phe Pro Met His Gly Asn Leu Ile Phe Gly

530 535 540

Lys Glu Gly Thr Thr Ala Ser Asn Ala Glu Leu Asp Asn Val Met Ile

545 550 555 560

Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln

565 570 575

Tyr Gly Thr Val Ala Asn Asn Leu Gln Ser Ser Asn Thr Ala Pro Thr

580 585 590

Thr Arg Thr Val Asn Asp Gln Gly Ala Leu Pro Gly Met Val Trp Gln

595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His

610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu

625 630 635 640

Lys His Pro Pro Pro Gln Ile Met Ile Lys Asn Thr Pro Val Pro Ala

645 650 655

Asn Pro Pro Thr Thr Phe Ser Pro Ala Lys Phe Ala Ser Phe Ile Thr

660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln

675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn

690 695 700

Tyr Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val

705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu

725 730 735

<210> 3

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<223> forward primer for hFVIII transgene

<400> 3

tgaggaggct gaagactat

19

<210> 4

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223> reverse primer for hFVIII transgene

<400> 4

ccacagacct gatctgaatg aa

22

<210> 5

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> probe for hFVIII transgene

<400> 5

tggatgtggt gaggtttgat gatgaca

27

<---

Claims (67)

1. A method for producing cells that produce a recombinant adeno-associated virus (rAAV) vector, comprising the steps of: (a) providing a PEI/plasmid mixture of components (i), (ii) and (iii): (i) one or more plasmids containing nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding helper functions; (ii) a plasmid containing a transgene that encodes a protein or is transcribed into a transcript of interest; And (iii) polyethylenimine (PEI) solution, (b) contacting the cells with the plasmid/PEI mixture from step (a) to obtain a cell culture with the plasmid/PEI mixture; (c) adding valproic acid, a salt or derivative thereof, to the cell culture with the plasmid/PEI mixture to form a second mixture; And (d) incubating said second mixture from step (c) to obtain transfected cells that produce the rAAV vector. 2. The method according to claim 1, further comprising step (e) collecting said transfected cells obtained in step (d) and/or culture medium from the transfected cells obtained in step (d), obtaining a cell collection and/or culture environment. 3. The method according to claim 1 or 2, further comprising step (e) of culturing, expanding, isolating or selecting cells that have been transfected with plasmids. 4. The method according to claim 1 or 2, further comprising step (e) isolating and/or purifying the recombinant AAV vector from the transfected cells obtained in step (d) and/or the culture medium from the transfected cells obtained in step (d) . 5. The method of claim 4, further comprising the step of (f) isolating and/or purifying the recombinant AAV vector from the transfected cells and/or collecting the culture medium obtained in step (e). 6. The method according to any one of claims 1 to 5, wherein the transgene encodes a wild-type or functional variant coagulation factor, apoE2, TPP1, argininosuccinate synthase, copper-transporting ATPase 2, acid alpha-glucosidase, β-glucocerebrosidase, α-galactosidase, or C1 serine protease inhibitor. 7. The method of claim 6, wherein the wild-type or functional variant coagulation factor is factor VII, factor VIII or factor IX. 8. Method according to any one of claims 1 to 7, where valproic acid is a salt or derivative thereof. 9. Method according to any one of claims 1 to 8, wherein valproic acid, its salt or derivative is added before step (a), during step (a) or immediately after step (a) or before, but not less than 3 h after stage (a). 10. Method according to any one of claims 1 to 9, wherein valproic acid, a salt or derivative thereof is added two or more times after steps (a) or (b). 11. The method according to claim 10, wherein valproic acid, its salt or derivative is first added during or immediately after step (a) or before, but not less than 3 hours after step (a) and is added again after 12-72 h after steps (a) or (b), or added again 12-48 h after steps (a) or (b), or added again 12-24 h after steps (a) or (b). 12. The method according to any one of claims 1 to 11, wherein said plasmids (i) and (ii) are in the range of molar ratios from about 1:0.01 to about 1:100 and where the mixture of components (i), (ii) and (iii) optionally incubated for a period of time from about 10 seconds to about 4 hours before step (b). 13. The method according to any one of claims 1 to 12, wherein said plasmids are present in a PEI:plasmid weight ratio ranging from about 0.1:1 to about 5:1 or a PEI:plasmid weight ratio in the range from about 5:1 to approximately 0.1:1. 14. The method according to any one of claims 1 to 13, further comprising adding free PEI to the cells before, during or after steps (a) or (b), before or during or after step (c). 15. The method according to any one of claims 1 to 13, further comprising adding free PEI to the cells during or after steps (a) or (b) or during or after step (c). 16. The method of any one of claims 1 to 15, wherein said free PEI is added such that the PEI:plasmid weight ratio is in the range of about 0.1:1 to about 5:1 or in the range of about 5:1 to about 0.1:1. 17. Method according to any one of claims 1 to 16, where the PEI in the PEI:plasmid mixture and/or free PEI contains linear polyethylenimine. 18. Method according to any one of claims 1 to 16, where the PEI in the PEI:plasmid and/or free PEI mixture contains hydrolyzed linear polyethylenimine. 19. The method according to any one of claims 1 to 18, wherein the PEI in the PEI:plasmid mixture and/or free PEI contains hydrolyzed linear polyethylenimine with a molecular weight in the range of about 4000 to about 160,000 and/or with a molecular weight in the range of about 2500 up to approximately 250,000 in free base form. 20. The method of any one of claims 1 to 19, wherein the molar ratio of nitrogen (N) in total PEI to phosphate (P) in the plasmid:PEI mixture ranges from about 1:1 to about 50:1 (N:P) . 21. The method according to any one of claims 14 to 20, wherein the amount of free PEI is from about 10% to about 90% of the total PEI. 22. The method according to any one of claims 14 to 21, wherein the concentration of free PEI is from about 0.1 μg/ml to about 10 μg/ml. 23. The method according to any one of claims 1 to 22, wherein the PEI and/or free PEI solution contains a solution having a pH of from about 7.0 to about 8.00. 24. The method of any one of claims 1 to 23, wherein said plasmids and PEI are incubated together for about 10 seconds to about 4 hours before step (a). 25. The method according to any one of claims 1 to 24, wherein said plasmids and PEI are incubated together for about 1 minute to about 30 minutes before step (a). 26. The method according to any one of claims 1 to 25, wherein the mixture of components (i), (ii) and (iii) are incubated together for about 10 seconds to about 4 hours before step (b). 27. The method according to any one of claims 1 to 26, wherein the incubation in step (d) is at least about 4 hours. 28. The method according to any one of claims 1 to 27, wherein the cells include mammalian cells. 29. The method according to any one of claims 1 to 28, wherein the cells are human embryonic kidney (HEK) cells or Chinese hamster ovary (CHO) cells. 30. The method according to any one of claims 1 to 29, where the cells include human embryonic kidney (HEK) 293 cells. 31. The method according to any one of claims 1 to 30, wherein said cells are HEK 293E, HEK 293F or HEK 293T cells. 32. Method according to any one of claims 1 to 31, wherein the cells are stably or transiently transfected. 33. Method according to any one of claims 1-32, where the cells are in suspension culture. 34. Method according to any one of claims 1 to 32, wherein the cells are attached to the substrate. 35. The method according to any one of claims 1 to 34, wherein said cells are cultured or maintained in a serum-free culture medium. 36. The method according to any one of claims 1 to 35, wherein said cells have a density ranging from about 1x10 ⁵ cells/ml to about 1x10 ⁸ cells/ml when contacted with said plasmid/PEI mixture and/or when contacted with specified free PEI. 37. The method according to any one of claims 1 to 36, wherein the cell viability when contacted with said plasmid/PEI mixture or with said free PEI is about 60% or greater than 60%, or wherein said cells are in logarithmic growth phase when contacted with said plasmid/PEI mixture. 38. The method of any one of claims 1 to 37, wherein the total amount of plasmid is in the range of from about 0.1 μg to about 15 μg per ml of cells. 39. The method according to any one of claims 1 to 37, where the molar ratio of the plasmid containing the transgene to one or more plasmids containing nucleic acids encoding AAV packaging proteins and/or nucleic acids encoding helper functions is from about 1: 5 to about 1:1 or is from about 1:1 to about 5:1. 40. The method according to any one of claims 1 to 39, wherein said one or more plasmids comprise a first plasmid containing nucleic acids encoding AAV packaging proteins and a second plasmid containing nucleic acids encoding helper functions. 41. The method of claim 40, wherein the molar ratio of the plasmid containing the transgene to the first plasmid containing nucleic acids encoding AAV packaging proteins to the second plasmid containing nucleic acids encoding helper functions is in the range of about 1-5:1 :1, or 1:1-5:1, or 1:1:1-5. 42. The method according to any one of claims 1 to 41, wherein the encoded AAV packaging proteins include AAV rep and/or AAV cap. 43. The method according to any one of claims 1 to 42, wherein the encoded helper functions include adenoviral E2 and/or E4 proteins, VA RNA and/or non-AAV helper functions. 44. The method according to any one of claims 1-43, where the recombinant AAV vector contains any of AAV serotypes 1-12, AAV capsid protein VP1, VP2 and/or VP3, or a modified or variant AAV capsid protein VP1, VP2 and/or VP3, or wild-type AAV capsid protein VP1, VP2 and/or VP3. 45. The method according to any one of claims 1 to 44, wherein the adeno-associated viral vector (AAV) contains a VP1, VP2 and/or VP3 capsid protein sequence or an inverted terminal repeat sequence having 70% or more sequence identity with a capsid protein sequence or an inverted terminal repeat sequence. terminal repeat (ITR) of any capsid protein sequence or ITR selected from serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9 and AAV10. 46. The method according to any one of claims 1 to 45, wherein the adeno-associated viral vector (AAV) contains a capsid protein sequence VP1, VP2 and/or VP3 having 70% or more sequence identity with a capsid protein sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2. 47. The method of claim 1-46, wherein the AAV vector comprises an AAV serotype or an AAV pseudotype, wherein said AAV pseudotype comprises an AAV capsid serotype other than an ITR serotype. 48. The method according to any one of claims 1 to 47, where the AAV vector contains a capsid protein VP1, VP2 and/or VP3 or an inverted terminal repeat of any serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, SEQ ID NO: 1 and SEQ ID NO: 2. 49. The method according to any one of claims 1 to 48, wherein the AAV vector further comprises an intron, an expression control element, one or more adeno-associated virus (AAV) inverted terminal repeats (ITRs), and/or a filler polynucleotide sequence. 50. The method of claim 49, wherein the intron is within or flanks a nucleic acid that encodes a protein or is transcribed into a transcript of interest, or wherein the expression control element is operably linked to a nucleic acid that encodes a protein or is transcribed into a transcript of interest, or wherein the AAV ITR(s) flank the 5' or 3' end of a nucleic acid that encodes a protein or is transcribed into a transcript of interest, or where the filler polynucleotide sequence flanks the 5' or 3' end of a nucleic acid that encodes a protein or transcribed into the transcript of interest. 51. The method according to any one of claims 1 to 50, wherein the cells are subcultured to restored cell density before contacting with said plasmid/PEI mixture. 52. The method according to any one of claims 1 to 51, where the cells are contacted with the specified plasmid/PEI mixture for a period of 2 days to 5 days after subculture. 53. The method according to any one of claims 1 to 52, wherein the number of plasmids inserted into said transfected cells is at least 50% greater at the stage of adding free PEI to the cell culture compared to the option without adding free PEI to the cell culture. 54. The method according to any one of claims 1 to 52, wherein the number of plasmids inserted into said transfected cells is at least 50% greater with the step of adding valproic acid, a salt or derivative thereof, compared to a variant without adding valproic acid, its salt or derivative. 55. The method according to any one of claims 1 to 54, wherein the amount of recombinant AAV vector produced is at least 50% or more with the step of adding free PEI to the cell culture with the plasmid/PEI mixture compared to the option without adding free PEI to cell culture with a plasmid/PEI mixture. 56. The method according to any one of claims 1-54, where the amount of the produced recombinant AAV vector is 1-5, 5-8, 8-10 or 10-20 times greater with the step of adding free PEI to the cell culture with a plasmid/PEI mixture according to compared to the option without adding free PEI to a cell culture with a plasmid/PEI mixture. 57. The method according to any one of claims 1-55, where the amount of the produced recombinant AAV vector is 1-5, 5-8, 8-10 or 10-15 times greater with the step of adding valproic acid, its salt or derivative compared to the variant without adding valproic acid, its salt or derivative to the cell culture with the plasmid/PEI mixture. 58. The method according to any one of claims 1 to 57, where valproic acid is presented in the form of its derivative. 59. The method according to any one of claims 1 to 58, where after step (c) valproic acid, a salt or derivative thereof is present in a concentration of from about 0.1 to about 25 mM. 60. The method according to any one of claims 1 to 58, where any of stages (a)-(f) or conditions described in any of claims. 1-58 is performed as described in any of Examples 1-7.

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