Suspension system for the production of adeno-associated viruses

Abstract

To provide a large-scale method for producing an AAV vector and a high titer.SOLUTION: The present technology relates to a production system that produces AAV vectors at high titers in a serum-free suspension platform. This technology uses reagents including media, cells, transfection reagents, AAV enhancers, and lysis buffer, each of which is designed to provide maximal AAV production from a suspension culture of mammalian cells, e.g., HEK293 cells. This new system can deliver up to about 2*10 11 viral genomes per milliliter of non-concentrated AAV vector (vg / mL).SELECTED DRAWING: None

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 809,407, filed February 22, 2019. The entire contents of the foregoing application are incorporated herein by reference.

[0003] Adeno-associated viruses (AAV) are small DNA viruses that infect human and some non-human primate cells. AAV is not known to cause disease and has low immunogenicity in humans. AAV vectors can be engineered to contain a desired DNA sequence with few or no viral genes. These advantages have led to the use of AAV vectors in gene therapy and other clinical and research applications. Summary of the Invention [Problem to be solved by the invention]

[0004] Large-scale methods for producing AAV vectors and producing high titers are needed. [Means for solving the problem]

[0005] This technology generally relates to a new AAV system for producing vectors at high titers in a serum-free suspension platform. The technology employs a newly developed, suitable set of Good Manufacturing Process (GMP) reagents, including media, cells, transfection reagents, AAV enhancers, and lysis buffers, each designed to provide maximum AAV production from suspension cultures of mammalian cells. Using this new system, it is possible to produce up to approximately 2 x 10 vectors per milliliter of unconcentrated AAV vector (i.e., vector that has not been further concentrated after harvest using the harvesting methods described herein). 11 of viral genome (vg / mL) can be delivered.

[0006] In one aspect, provided herein is a method for producing an AAV vector, comprising: (i) culturing mammalian cells; (ii) transfecting the mammalian cells with an AAV transfer vector using a transfection reagent; (iii) contacting the transfected cells with an AAV enhancer; and (iv) culturing the transfected cells in suspension culture for a time sufficient to package the AAV vector, thereby producing a transfected AAV cell culture. In embodiments, the mammalian cells are cultured in suspension culture. In embodiments, the method comprises harvesting AAV from the transfected AAV cell culture. In embodiments, the AAV is harvested using a lysis buffer. In embodiments, the transfecting comprises contacting the cells with a transfection booster.

[0007] In embodiments, the method includes titrating the harvested AAV. In embodiments, the AAV is titrated using quantitative PCR. In embodiments, the harvested AAV is titrated at least about 2 x 10 per milliliter. 10 In embodiments, the harvested AAV has a titer of about 2 x 10 viral genomes (vg / mL). 10 vg / mL to approximately 2 × 10 11 It has a titer of between 1000 and 1000 mg / mL.

[0008] In embodiments, the cells are cultured in a volume of about 10 milliliters (mL) to about 800 liters (L). In embodiments, the cells are cultured in a volume of about 1 L to about 10 L. In embodiments, the cells are transfected in a volume of about 15 milliliters (mL) to about 200 liters (L). In embodiments, the cells are transfected in a volume of about 1 L to about 2 L.

[0009] In an embodiment, the cells are cultured in a bioreactor.

[0010] In embodiments, the cells are cultured in a medium that supports the growth and proliferation of HEK293 cells. In embodiments, the cells are contacted with an AAV production enhancer during culture (e.g., after transfection).

[0011] In one aspect, provided herein is an AAV production system comprising HEK293 cells, an AAV transfer vector, a packaging plasmid, an AAV production enhancer, and a cell culture medium that supports the growth and proliferation of HEK293 cells. In embodiments, the AAV production system comprises a transfection reagent. In embodiments, the AAV production system comprises a transfection booster. In embodiments, the AAV production system comprises a lysis buffer. In embodiments, the HEK293 cells are at least about 0.3 x 10 6 In embodiments, the HEK293 cells are present at a density of at least about 2 x 10 cells / mL. 6 In embodiments, the HEK293 cells are present at a density of about 0.3 x 10 cells / mL. 6 cells / mL to approximately 1 x 10 7 In one embodiment, the AAV vector is present at a density of between about 2 x 10 cells / mL after harvest. 10 The virus is present at a titer of 10 ...

[0012] In embodiments, the lysis buffer contains a detergent. In embodiments, the detergent is Triton-100, Triton-alter, NP-40, poloxamer 188, or NDSB-201. In embodiments, the lysis buffer does not contain a detergent. In embodiments, the lysis buffer contains at least one of Tris-HCl, Tricine HCl, sodium citrate, sodium chloride, citric acid, EDTA, tripotassium EDTA, sodium hydroxide, and sodium dihydrogen phosphate. In embodiments, the lysis buffer contains at least one detergent. In embodiments, the detergent is CHAP, CHAPS, CHAPSO, Big CHAP, octylthioglucoside, and / or sodium deoxycholate.

[0013] In embodiments, the AAV production enhancer comprises one or more of a histone deacetylase (HDAC) inhibitor, sodium propionate, egg lecithin, lithium acetate, trichostatin hydroxyurea, nocodazole-DMSO, NaCl, and caffeine. In embodiments, the HDAC inhibitor is apicidin, belinstat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A, and / or valproic acid. In embodiments, the HDAC inhibitor is sodium butyrate, sodium phenylbutyrate, trichostatin A, and / or valproic acid. In embodiments, the AAV enhancer is added between about 0 hours and about 6 hours after transfection.

[0014] In embodiments, the transfection reagent comprises a cationic lipid. In embodiments, the transfection reagent further comprises a peptide. In embodiments, the transfection booster comprises a cationic lipid. In embodiments, the transfection booster comprises a peptide. In embodiments, the peptide is a membrane-permeable peptide. Non-limiting examples of membrane-permeable peptides are provided in U.S. Pat. No. 9,856,496, which is incorporated herein by reference in its entirety. In embodiments, the transfection booster is used at a ratio of between 5:1 and about 1:5 (volume / weight) transfection booster:DNA.

[0015] In some embodiments, the mammalian cells are HEK293 cells or derivatives of HEK293 cells. In some embodiments, the HEK293 cells are adapted for high AAV expression in an AAV vector production system. In some embodiments, the HEK293 cells are at least 0.3 x 10 per milliliter in suspension culture. 6 In embodiments, the HEK293 cells can be grown at a density of up to 1.2 x 10 cells per milliliter in suspension culture. 7 In embodiments, the cells can be grown at a density of about 2.5 x 10 cells / mL. 6 and approximately 4 × 10 6 Transfect at a cell density between 100 and 200 cells / mL.

[0016] In embodiments, no helper virus is used. In embodiments, the method includes transfecting the cells with a packaging plasmid. In embodiments, the AAV production system includes a packaging plasmid. In embodiments, the packaging plasmid includes pRC and pHelper.

[0017] In embodiments, the cells are not centrifuged prior to harvesting the AAV.

[0018] In embodiments, the cells do not contain large T antigen.

[0019] In one aspect, provided herein is a kit for producing adeno-associated virus (AAV). In embodiments, the kit includes HEK293 cells, a transfection reagent comprising an enhancer and a cationic lipid, and a cell culture medium that supports the growth and proliferation of HEK293 cells. In embodiments, the transfection reagent comprises a cationic lipid and a peptide.

[0020] In embodiments, the kit also includes a transfection booster. In embodiments, the transfection booster includes a peptide.

[0021] In embodiments, the kit also includes a lysis buffer. In embodiments, the lysis buffer includes at least one detergent. In embodiments, the detergent is Triton-100, Triton-alter, NP-40, poloxamer 188, and / or NDSB-201. In embodiments, the lysis buffer includes Tris-HCl, sodium citrate, tricine HCl, sodium chloride, citric acid, EDTA, tripotassium EDTA, sodium hydroxide, and / or sodium dihydrogen phosphate. In embodiments, the lysis buffer includes at least one detergent. In embodiments, the detergent is CHAP, CHAPS, CHAPSO, big CHAP, deoxyl big CHAP, Triton X-114, octylthioglucoside, and / or sodium deoxycholate. [Brief explanation of the drawings]

[0022] [Figure 1]The effect of different conditions on AAV2 production was shown. AAV2 virus was produced by adherent HEK293 cells (6-well plates) transfected with polyethyleneimine (PEI) under three different conditions: Prot-1 (enhancer 1 and supplement 1), Prot-2 (supplement 1, no enhancer), and Prot-3 (enhancer 1, no supplement) compared to System 1. [Figure 2] 1 shows a comparison of AAV2 production in HEK293F cells adapted to four different types of culture media. [Figure 3] Figure 1 shows a comparison of three different systems for AAV production. HEK293 cells adapted to medium 4 were tested in System 1, System 2, and System 3, as detailed in the Examples. [Figure 4A] Growth characteristics of clonal HEK293 cells adapted to Medium 4 (Clone 45) are shown. The culture medium supported Clone 45 cells (~11 x 10 cells / mL) growing to high density with high cell viability (Figure 4A). As shown in Figure 4B, there is very limited aggregation of cells at high density. [Figure 4B] Growth characteristics of clonal HEK293 cells adapted to Medium 4 (Clone 45) are shown. The culture medium supported Clone 45 cells (~11 x 10 cells / mL) growing to high density with high cell viability (Figure 4A). As shown in Figure 4B, there is very limited aggregation of cells at high density. [Figure 5A] Figure 1 shows the production of different AAV serotypes in clonal HEK293 cells. Control: parental HEK293 cells in medium 4. Expi45: clone 45 in Expi293 medium. Cl45, Cl12, Cl22 and Cl51: HEK293 clones in medium 4. [Figure 5B] Figure 1 shows the production of different AAV serotypes in clonal HEK293 cells. Control: parental HEK293 cells in medium 4. Expi45: clone 45 in Expi293 medium. Cl45, Cl12, Cl22 and Cl51: HEK293 clones in medium 4. [Figure 5C]Figure 1 shows the production of different AAV serotypes in clonal HEK293 cells. Control: parental HEK293 cells in medium 4. Expi45: clone 45 in Expi293 medium. Cl45, Cl12, Cl22 and Cl51: HEK293 clones in medium 4. [Figure 5D] Figure 1 shows the production of different AAV serotypes in clonal HEK293 cells. Control: parental HEK293 cells in medium 4. Expi45: clone 45 in Expi293 medium. Cl45, Cl12, Cl22 and Cl51: HEK293 clones in medium 4. [Figure 5E] Figure 1 shows the production of different AAV serotypes in clonal HEK293 cells. Control: parental HEK293 cells in medium 4. Expi45: clone 45 in Expi293 medium. Cl45, Cl12, Cl22 and Cl51: HEK293 clones in medium 4. [Figure 6] Comparison of transfection reagents: Transfection reagent 1 (TR1) was compared with transfection reagent 2 (TR2) for the production of AAV2. [Figure 7] 1 shows the effect of the timing of enhancer addition on viral titer in an AAV production system. [Figure 8] 1 shows the effect of various detergents in AAV lysis buffer on virus titer. [Figure 9] AAV titers in extracts from cell pellets versus whole transfected cell cultures are shown for different AAV serotypes. [Figure 10A] A comparison of AAV production (FIG. 10A) and cell viability (FIG. 10B) after transfection between clone 45, subclonal lines C13 and C20, and LV293 cells (VPC) is shown. [Figure 10B] A comparison of AAV production (FIG. 10A) and cell viability (FIG. 10B) after transfection between clone 45, subclonal lines C13 and C20, and LV293 cells (VPC) is shown. [Figure 11A]Figure 11 shows a comparison of AAV production between clone 45 and LV293 cells (VPC). Figure 11A compares the viral titers (vg / mL) obtained for different AAV serotypes. Figure 11B compares the infectivity (as % GFP in Ht1080) of AAV2 and AAV6 harvested from each clonal cell line. [Figure 11B] Figure 11 shows a comparison of AAV production between clone 45 and LV293 cells (VPC). Figure 11A compares the viral titers (vg / mL) obtained for different AAV serotypes. Figure 11B compares the infectivity (as % GFP in Ht1080) of AAV2 and AAV6 harvested from each clonal cell line. [Figure 12A] A comparison of the clone 45 AAV system and the LV293-PEI system in post-harvest AAV6 production (vg / mL, FIG. 12A) and AAV6 infection (as % GFP in Ht1080, FIG. 12B) is shown. [Figure 12B] A comparison of the clone 45 AAV system and the LV293-PEI system in post-harvest AAV6 production (vg / mL, FIG. 12A) and AAV6 infection (as % GFP in Ht1080, FIG. 12B) is shown. [Figure 13A] Figure 13 shows a comparison of AAV production between the Clone 45 AAV system and the HEK293T-PEI system. Figure 13A compares the viral titers (vg / mL) obtained for different AAV serotypes. Figure 13B compares the infectivity (as % GFP in Ht1080) of AAV2, AAV6, and AAV-dj harvested from each system. [Figure 13B] Figure 13 shows a comparison of AAV production between the Clone 45 AAV system and the HEK293T-PEI system. Figure 13A compares the viral titers (vg / mL) obtained for different AAV serotypes. Figure 13B compares the infectivity (as % GFP in Ht1080) of AAV2, AAV6, and AAV-dj harvested from each system. [Figure 14] AAV titers in crude lysates before and after diatomaceous earth filtration are shown. DETAILED DESCRIPTION OF THE INVENTION

[0023] After reading this description, it will become apparent to one skilled in the art how to implement the present invention in various alternative embodiments and alternative applications. However, not all various embodiments of the present invention are described herein. It will be understood that the embodiments presented herein are presented by way of example only, and not by way of limitation. Therefore, this detailed description of various alternative embodiments should not be construed as limiting the scope or breadth of the present invention, as described below.

[0024] Before the present invention is disclosed and described, it is to be understood that the embodiments described below are not limited to particular compositions, and that the methods of preparing such compositions or their uses as such may, of course, vary. It is understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0025] The detailed description of the present invention has been divided into various sections for the convenience of the reader only, and disclosure found in any section may be combined with disclosure from another section. Titles or subtitles may be used herein for the convenience of the reader and are not intended to affect the scope of the invention. definition

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In this specification and the claims that follow, reference will be made to a number of terms that are defined herein.

[0027] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0028] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes instances in which the event or circumstance occurs and instances in which it does not occur.

[0029] The term "about," when used before numerical designations such as temperature, time, amount, concentration, and such others, including ranges, indicates approximations that may vary by (+) or (-) 10%, 5%, 1%, or any subrange or subvalue therebetween. Preferably, when used in reference to the amount of a dose, the term "about" means that the dose may vary by + / - 10%.

[0030] "Comprising" or "comprises" is intended to mean that compositions and methods include the recited elements, but do not exclude others. When used to define compositions and methods, "consisting essentially" shall mean excluding other elements that are essential to the combination for the stated purpose. Thus, a composition consisting essentially of the elements defined herein will not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention. "Consisting of" shall mean excluding more than trace elements and substantial method steps of other components. Embodiments defined by each of these transition terms are within the scope of the present invention. Adeno-associated virus (AAV) production system

[0031] As used herein, the term "cell" refers to and includes all types of eukaryotic and prokaryotic cells. In some embodiments, the term refers to eukaryotic cells, particularly mammalian cells. In certain exemplary, but non-limiting, embodiments, the term "cell" is meant to refer to human embryonic kidney (HEK) cells or human 293 cells, or variants thereof, such as, for example, 293 (HEK293) variants that can grow in suspension. In some embodiments, variants of 293 cells that can be grown, propagated, and transfected in suspension culture, particularly at high densities (e.g., at least about 2×10 6 cells / mL, at least approximately 3 x 10 6 cells / mL, or optionally at least about 4 x 10 6 cells / mL or approximately 1.2 x 10 6 This mutant can be cultured at 1000 kJ / mL.

[0032] In some embodiments, the term "high density" when used in the context of culturing cells and performing transfection workflows generally refers to culturing cells at a density of at least about 2 x 10 cells while still retaining the ability to be transfected with high efficiency. 6 cells / mL, at least approximately 3 x 10 6 cells / mL, or optionally at least about 4 x 10 6 They can be grown or cultured in appropriate cell culture medium to a density of 100 cells / mL and produce high titers (e.g., 5 x 10 cells per mL). 10 This refers to a known cell line, or a variant of a known cell line, that is capable of expressing a target AAV vector at a viable genome (vg / mL) or higher.

[0033] In some embodiments, these cells are adapted for high-density cell culture. This refers to a cell line or a (non-clonal) cell population derived from the same parent cell line that has been adapted to grow at high density in high-density culture medium while maintaining a cell viability of about 80% or more. Such cells can be isolated or selected from a parent population of cells by maintaining the cells at high density for about 40, 50, 60, 70, or 80 or more serial passages and gradually replacing a proportion of the growth medium with the desired high-density culture medium. Optionally, during the process, different pools of cells may be individually propagated and subjected to a selection procedure, while simultaneously evaluating transfection efficiency and / or AAV vector production efficiency, thereby selecting a clonal population of cells that can persist and grow at high density, be highly transfected, and express high titers of AAV. Clonal populations of cells can be produced using known methods and techniques, such as flow cytometry sorting and / or single-cell cloning. In embodiments, flow cytometry sorting is used to isolate cell clones adapted for high-density cell culture and used in the AAV production system. In some embodiments, cell clones adapted for use in high-density cell culture and AAV production systems are obtained via single-cell cloning and confirmed as single-cell clones using known techniques, such as imaging. While it will be readily apparent to those skilled in the art that various cell types and lineages can undergo this selection procedure, it has been determined that cell lines derived from 293 human embryonic kidney cells are particularly suitable for the selection process for adaptation to high-density growth conditions. In some scenarios, cells adapted to high-density growth culture and suitable for use herein can also be highly efficiently transfected and / or transfect at least about 5 x 10 of unconcentrated AAV vectors. 9 vg / mL to approximately 5 × 10 12 vg / mL, approximately 1 × 10 10 vg / mL to approximately 2 × 10 11 vg / mL, approximately 1 × 10 10 vg / mL to approximately 1 × 10 11 vg / mL, approximately 8 × 10 10 vg / mL to approximately 3 × 1011 vg / mL, or approximately 5 × 10 10 vg / mL to approximately 2 × 10 11 In some scenarios, high density culture-adapted cells are used at yields of approximately 1 x 10 6 to approximately 2 × 10 7 cells / mL, approximately 1×10 6 to about 3 × 10 6 cells / mL, approximately 2×10 6 From about 4 × 10 6 cells / mL, or approximately 2.5 x 10 6 From about 4 × 10 6 Cells can be maintained and transfected at densities ranging from approximately 1×10 cells / mL. In some embodiments, cells can be adapted to high density cultures, reaching approximately 1×10 6 to approximately 2 × 10 7 cells / mL, approximately 1×10 6 From about 4 × 10 6 cells / mL, approximately 1×10 6 to about 3 × 10 6 cells / mL, approximately 1×10 6 to approximately 2 × 10 6 Densities ranging from 100 cells / mL can be transfected.

[0034] In some embodiments, the cells grow in a suspension culture. This refers to a cell culture in which most or all of the cells in the culture vessel are in suspension, with a small number of cells in the culture vessel adhering to the surface of the vessel or to another surface within the vessel, or neither. In some embodiments, a suspension culture is one in which at least about 75% of the cells in the culture vessel are in suspension and do not adhere to any surface on or within the culture vessel. In some embodiments, a suspension culture is one in which at least about 85% of the cells in the culture vessel are in suspension and do not adhere to any surface on or within the culture vessel. In some embodiments, a suspension culture is one in which at least about 95% of the cells in the culture vessel are in suspension and do not adhere to any surface on or within the culture vessel.

[0035] The AAV production system can produce approximately 0.3 x 10 293 cells or cells derived therefrom with less than 20% cell death after 5 days.6 cells / mL to approximately 20 x 10 6 In embodiments, the cells can be grown at a density of about 0.3 x 10 cells / mL with less than 20% cell death after 5 days. 6 cells / mL to approximately 12 x 10 6 In some embodiments, the 293 cells provided herein can grow to a density of about 0.3 x 10 cells / mL with less than 20% cell death after 6, 7, or 8 days. 6 cells / mL to approximately 20 x 10 6 cells / mL, or approximately 0.3 x 10 6 cells / mL to approximately 12 x 10 6 In embodiments, the 293 cells can be grown to a high density, such as about 0.3 x 10 cells / mL, with less than 10% cell death after 5, 6, 7, or 8 days. 6 cells / mL to approximately 20 x 10 6 cells / mL, or approximately 0.3 x 10 6 cells / mL to approximately 12 x 10 6 They can be grown at high densities, such as cells / mL.

[0036] Alternatively stated, the AAV production system provided herein allows the 293 cells, or cells derived therefrom, to produce approximately 0.3×10 cells with a cell viability of greater than 80% after 5 days in culture. 6 cells / mL to approximately 20 x 10 6 In embodiments, the cells can be grown at a density of about 0.3 x 10 cells / mL with greater than 80% cell viability after 5 days in culture. 6 cells / mL to approximately 12 x 10 6 In some embodiments, the 293 cells provided herein can grow to a density of about 0.3 x 10 cells / mL with greater than 80% cell viability after 6, 7, or 8 days in culture. 6 cells / mL to approximately 20 x 10 6 cells / mL, or approximately 0.3 x 10 6 cells / mL to approximately 12 x 10 6In embodiments, the 293 cells can grow to a high density, such as about 0.3 x 10 cells / mL, with greater than 90% cell viability after 5, 6, 7, or 8 days in culture. 6 cells / mL to approximately 20 x 10 6 cells / mL, or approximately 0.3 x 10 6 cells / mL to approximately 12 x 10 6 They can be grown at high densities, such as cells / mL.

[0037] In some embodiments, suspension cultures of high-density adapted 293 cells provided herein, such as Clone 45, subclones of Clone 45, and other clones described herein, have at least 10% higher viability than HEK293F cells, Expi293F™ cells, or LV293 (LV-MAX virus-producing cells) (all available from Thermo Fisher Scientific) after 3 days of high-density culture. In some embodiments, suspension cultures of high-density adapted 293 cells provided herein have at least 10% higher viability than HEK 293F, Expi293F, or LV293 cells after 4, 5, 6, 7, or 8 days of high-density culture. In some embodiments, suspension cultures of high-density adapted 293 cells provided herein have at least 15% higher viability than HEK 293F, Expi293F, or LV293 cells after 3, 4, 5, 6, 7, or 8 days of high-density culture. In some embodiments, suspension cultures of high-density adapted 293 cells provided herein have at least 20% higher viability than HEK 293F, Expi293F, or LV293 cells after 3, 4, 5, 6, 7, or 8 days of high-density culture. In some embodiments, suspension cultures of high-density adapted 293 cells provided herein have at least 25% higher viability than HEK 293F, Expi293F, or LV293 cells after 3, 4, 5, 6, 7, or 8 days of high-density culture. In some embodiments, suspension cultures of high-density adapted 293 cells provided herein have at least 30% higher viability than HEK 293F, Expi293F, or LV293 cells after 3, 4, 5, 6, 7, or 8 days of high-density culture. In some embodiments, the high density adapted suspension cultures of 293 cells provided herein have at least 40% higher viability than HEK 293F, Expi293F, or LV293 cells after 3, 4, 5, 6, 7, or 8 days of high density culture.In embodiments, suspension cultures of high density adapted 293 cells provided herein have about 10% to about 30% higher viability than HEK 293F, Expi293F, or LV293 cells after 3, 4, 5, 6, 7, or 8 days of high density culture. In some embodiments, suspension cultures of high density adapted 293 cells provided herein have about 20% to about 40% higher viability than HEK 293F, Expi293F, or LV293 cells after 3, 4, 5, 6, 7, or 8 days of high density culture. In some embodiments, the high density adapted suspension cultures of 293 cells provided herein have about 25% to about 50% higher viability than HEK 293F, Expi293F, or LV293 cells after 3, 4, 5, 6, 7, or 8 days of high density culture.

[0038] In embodiments, suspension cultures of high-density adapted 293 cells provided herein, such as Clone 45, subclones of Clone 45, and other clones described herein, result in significantly higher AAV vector yields and titers (vg / mL) than equivalent amounts of HEK293F cells, Expi293F™ cells, or LV293 (LV-MAX virus producer cells) (all available from Thermo Fisher Scientific). In some embodiments, suspension cultures of high-density adapted 293 cells provided herein result in harvested AAV titers (vg / mL) that are at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 18-fold, or at least 20-fold higher than the titers harvested from equivalent amounts of HEK 293F, Expi293F, or LV293 cells. In some embodiments, the high density adapted suspension cultures of 293 cells provided herein result in a harvested AAV titer (vg / mL) that is about 2 to about 20-fold, about 2 to about 5-fold, about 2 to about 10-fold, about 5 to about 15-fold, about 5 to about 10-fold, about 7 to about 20-fold, or about 10 to about 15-fold higher than the titer harvested from the same amount of HEK 293F, Expi293F, or LV293 cells.

[0039] In embodiments, the cells may be, for example, about 8 x 10 6 In embodiments, the cells are at a very high density, greater than about 9 x 10 cells / mL, showing limited aggregation. 6 In embodiments, the cells exhibit limited aggregation of more than about 10 x 10 cells / mL. 6 In embodiments, the cells exhibit limited aggregation at very high densities grown under the conditions described herein in culture medium.

[0040] In embodiments, the cells have a diameter of between about 15 μm and about 20 μm, e.g., between about 16 μm and about 19 μm, or between about 16.5 μm and about 19 μm. In embodiments, the cells have a diameter of about 0.3×10 6 cells / mL to approximately 20 x 10 6 In embodiments, the cells have a diameter of between about 15 μm and about 20 μm and are grown at a density of about 1×10 cells / mL. 6 cells / mL to approximately 10 x 10 6 Grown at a density of 16 cells / mL, they have a diameter between about 16 μm and about 19 μm.

[0041] In embodiments, 293 cells are adapted to high density growth culture and can also be transfected with high efficiency, and / or have a density of approximately 5×10 9 vg / mL to approximately 5 × 10 12 293 cells, which can also express AAV vectors at yields up to 10 ...

[0042] A variety of cell culture media can be used to culture the cells of the AAV production system. Serum-free media are often desired by researchers. Any medium can be used, including serum-free media that supports the growth of the cells described herein. The medium can also be protein-free.

[0043] "Serum-free medium" (sometimes referred to as "SFM medium") is a medium that does not contain serum (e.g., fetal bovine serum (FBS), calf serum, horse serum, goat serum, human serum, etc.) and is generally designated by the letters SFM. The phrase "protein-free" culture medium refers to a culture medium that does not contain protein (e.g., without serum proteins such as serum albumin or adhesion factors, nutrient proteins such as growth factors, or metal ion carrier proteins such as transferrin and ceruloplasmin). In some embodiments, if peptides are present, they are smaller peptides, e.g., dipeptides or tripeptides. In some implementations, peptides of decapeptide length or greater represent less than about 1%, less than about 0.1%, and less than about 0.01% of the amino acids present in the protein-free medium.

[0044] In some embodiments, high density culture media may be used, including any culture medium capable of supporting the growth of mammalian cells. In some embodiments, cells may be grown in a density of about 2×10 cells while maintaining greater than about 80%, e.g., greater than about 90%, viability of the cells and maintaining the ability of the suspension cells to be efficiently transfected and express large amounts of AAV vector. 7 cells / ml, e.g., about 12 x 10 6 The cells are grown in suspension at a cell density of 1000 cells / ml. The high-density culture medium used may vary for different applications and uses and may depend on the nature of the cell line used, the nature of the transfection modality selected for the transfer of the expression vector into the cells, and the amount and nature of any expression enhancers added to the system described herein. In embodiments, the high-density culture medium used in the present systems and methods is serum-free and protein-free. In embodiments, the cell culture medium can accommodate up to about 2 x 10 7 cells / mL, e.g., up to approximately 1.2 x 10 7 cells / mL, or approximately 2 x 10 6 cells / mL to approximately 1 x 10 7In embodiments, the culture medium used allows for the cultivation and growth of suspension cells at densities up to 1 x 10 cells / mL. In embodiments, the culture medium used allows for the viral titer produced in a transient expression system to be at least 1 x 10 10 vg / mL up to approximately 1 × 10 12 vg / mL, or up to approximately 2 × 10 11 In some embodiments, the high density culture medium used can be greater than about 1 x 10 6 From about 20 x 10 6 cells / ml, approximately 1×10 6 From about 4 × 10 6 cells / ml, or approximately 2.5 x 10 6 to about 3 × 10 6 Facilitates transfection of cells at densities ranging from 1000 cells / ml.

[0045] Examples of such high density culture media suitable for use herein include, but are not limited to, HuMEC Basal Serum-Free Medium, KNOCKOUT™ CTS™ XenoFREE ESC / iPSC Medium, STEMPRO™-34 SFM Medium, STEMPRO™ NSC Medium, ESSENTIAL™-8 Medium, Medium 254, Medium 106, Medium 131, Medium 154, Medium 171, Medium 171, Medium 200, Medium 231, HeptoZYME-SFM, Human Endothelial-SFM, GIBCO® FREESTYLE™ 293 Expression Medium, Medium 154CF / PRF, Medium 154C, Medium 154 CF, Medium 106, Medium 200PRF, Medium 131, Essential™-6 Medium, STEMPRO™-34 Medium, Gibco® Astrocyte Medium, AIM V® Medium CTS™, AMINOMAX™ C-100 Basal Medium, AMINOMAX™-II Complete Medium, CD FORTICHO™ Medium, CD CHO AGT Medium, CHO-S-SFM Medium, GIBCO® FREESTYLE™ CHO Expression Medium, CD OPTICHO™ Medium, CD CHO Medium, CD DG44 Medium, SF-900™ Medium, EXPI293™ Expression Medium, LHC Basal Medium, LHC-8 Medium, 293 SFM Medium, CD 293 Medium, AEM Growth Medium, PER.C6® Cell Medium, AIM V® Medium, EXPILIFE® Medium, Keratinocyte SFM Medium, LHC Medium, LHC-8 Medium, LHC-9 Medium, and any derivatives or variations thereof.In certain non-limiting embodiments, the high-density culture medium can be CD FORTICHO™ Medium, CD CHO AGT Medium, CHO-S-SFM Medium, GIBCO® FREESTYLE™ CHO Expression Medium, CD OPTICHO™ Medium, CD CHO Medium, CD DG44 Medium, GIBCO® FREESTYLE™ 293 Expression Medium, EXPI293™ Expression Medium, LV-MAX™ Production Medium, FREESTYLE™ F17 Expression Medium, DYNAMIS™ Medium, BALANCD® HEK293 Medium, or a similar medium, or a variant thereof. The culture medium can be any medium suitable (e.g., formulated) for the high-density growth, proliferation, transfection, and maintenance of 293 cells, 293 cell variants, or any other cells adapted for use in a high-density culture system.

[0046] The AAV production system also includes a transfection reagent or composition that facilitates entry of macromolecules into cells. In embodiments, the transfection reagent includes a cationic lipid. In embodiments, the transfection reagent is a cationic lipid described in U.S. Patent No. 9,856,496, the entire contents of which are incorporated herein by reference.

[0047] In some embodiments, the reagent for the introduction of macromolecules into cells may include one or more lipids, which may be cationic lipids and / or neutral lipids. Preferred lipids include N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), dioleoylphosphatidylcholine (DOPE), 1,2-bis(oleoyloxy)-3-(4'-trimethylammonio)propane (DOTAP), dihydroxyl-dimyristylspermine tetrahydrochloride (DHDMS), hydroxyl-dimyristylspermine tetrahydrochloride (HDMS), 1,2-dioleoyl-3-(4'-trimethylammonio)butanoyl-sn-glycerol (DOTB), 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC), cholesteryl (4'-trimethylammonio), 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC ... (2-trimethylammonio)butanoate (ChoTB), cetyltrimethylammonium bromide (CTAB), 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORI), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DOME), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), O,O´-didodecyl-N-[p(2-trimethylammonioethyloxy)benzoyl]-N,N,N-trimethylammonium chloride, spermine conjugated to one or more lipids (e.g., 5-carboxyspermylglycine dioctadecylamide (DOGS), N,N I ,N II ,N III -Tetramethyl-N,N I ,N II ,N III-tet-lapalmitylspermine (TM-TPS), and dipalmitoylphosphatidylethanolamine 5-carboxyspermylamine (DPPES)), lipopolylysine (polylysine conjugated to DOPE), TRIS (tris(hydroxymethyl)aminomethane, tromethamine)-conjugated fatty acids (TFAs), and / or peptides, such as trilysyl-alanyl-TRIS mono-, di-, and tripalmitate (3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DCChol), N-(α-trimethylammonioacetyl)-didodecyl-D-glutamate chloride (TMAG), dimethyldioctadecylammonium bromide (DMBA), Examples of suitable transfection reagents include, but are not limited to, dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DDAB), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), and combinations thereof. In some cases, the transfection reagent may further comprise at least one additional helper lipid. Helper lipids are known in the art and preferably include, but are not limited to, neutral lipids selected from the group consisting of DOPE, DOPC, and cholesterol. In embodiments, the transfection reagent comprises at least one cationic lipid and at least one neutral lipid.

[0048] Those skilled in the art will appreciate that certain combinations of the aforementioned lipids have been shown to be particularly suitable for introducing nucleic acids into cells. For example, a 3:1 (w / w) combination of DOSPA and DOPE is available from Life Technologies Corporation (Carlsbad, Calif.) under the trade name LIPOFECTAMINE™; a 1:1 (w / w) combination of DOTMA and DOPE is available from Thermo Fisher Scientific under the trade name LIPOFECTIN®; a 1:1 (M / M) combination of DIVIRIE and cholesterol is available from Life Technologies Corporation (Carlsbad, Calif.) under the trade name DIVIRIE-C reagent; and a 1:1.5 (M / M) combination of TM-TPS and DOPE is available from Life Technologies. In some embodiments, the transfection reagent is a cationic lipid transfection reagent. In some embodiments, the transfection reagent is a polymer-based transfection reagent. Other commercially available cationic lipid transfection reagents include, but are not limited to, TRANSFAST™ (available from Promega Corporation), LYOVEC™ (available from InvivoGen), DOTAP liposome transfection reagent (available from Roche), TRANSIT® transfection reagent (available from Mirus), and insect GENEJUICE® transfection reagent (EMD Millipore).Additional transfection reagents that may be used herein include, but are not limited to, LIPOFECTAMINE® 2000, LIPOFECTAMINE® 3000, VIAFECT™ Transfection Reagent, FUGENE® 6 Transfection Reagent, and FUGENE® HD Transfection Reagent available from Thermo Fisher Scientific (each of which is available from Promega Corporation), and TRANSFECTIN™ Lipid Reagent available from BioRad Laboratories, Inc.

[0049] In embodiments, the transfection reagent is combined with a transfection booster. In embodiments, the transfection booster comprises a peptide. In embodiments, the transfection booster comprises at least one peptide. In embodiments, the at least one peptide of the transfection booster is a naturally occurring or non-naturally occurring membrane-permeable peptide. In embodiments, the at least one peptide of the transfection booster comprises a naturally occurring or non-naturally occurring membrane-permeable peptide sequence. Non-limiting examples of suitable membrane-permeable peptides and peptide sequences are provided in U.S. Pat. No. 9,856,496, which is incorporated herein by reference in its entirety. In embodiments, the at least one peptide of the transfection booster is a fusogenic peptide, a cell-permeable peptide, a nuclear localization peptide, a cell surface adhesion peptide, or a plant virus movement peptide. In embodiments, the at least one peptide of the transfection booster comprises a fusogenic peptide sequence, a cell-permeable peptide sequence, a nuclear localization peptide sequence, a cell surface adhesion peptide sequence, or a plant virus movement peptide sequence. Non-limiting examples of suitable fusogenic, cell-penetrating, nuclear localization, cell surface adhesion, and plant virus movement peptides and peptide sequences are provided in U.S. Patent Application Publication No. 2017 / 0253888 A1, which is incorporated herein by reference in its entirety.

[0050] In embodiments, the transfection reagent is combined with a transfection booster and the AAV transfer vector to form a transfection complex. In embodiments, the transfection reagent is combined with a transfection booster, a rep / cap plasmid (pRC), a pHelper plasmid (encoding helper virus components), and the AAV transfer vector to form a transfection complex.

[0051] In embodiments, the AAV production system includes a lysis buffer. The lysis buffer is a buffer used to disrupt open cells and release their contents, e.g., AAV vectors. In embodiments, the lysis buffer is provided at a 5x concentration (5x the final concentration contacted with the cells). In embodiments, the lysis buffer is provided at a 10x concentration (10x the final concentration contacted with the cells).

[0052] In embodiments, the lysis buffer comprises a detergent. In embodiments, the lysis buffer comprises at least one detergent selected from CHAP, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO), N,N-bis-(3-D-gluconamidopropyl)deoxycholamide (big CHAP), octylthioglucoside (OTG), and sodium deoxycholate. In embodiments, the lysis buffer comprises one detergent selected from CHAP, CHAPS, CHAPSO, big CHAP, OTG, and sodium deoxycholate. In embodiments, the lysis buffer comprises two detergents selected from CHAP, CHAPS, CHAPSO, big CHAP, OTG, and sodium deoxycholate. In embodiments, the lysis buffer comprises three detergents selected from CHAP, CHAPS, CHAPSO, big CHAP, OTG, and sodium deoxycholate. In embodiments, the lysis buffer comprises four or more detergents selected from CHAP, CHAPS, CHAPSO, big CHAP, OTG, and sodium deoxycholate.

[0053] In embodiments, the lysis buffer comprises CHAP at a concentration (final concentration in contact with cells) of between about 0.005% and about 1% (w / v). In embodiments, CHAP is present in the lysis buffer (final concentration in contact with cells) at a concentration of between about 0.01% and about 1% (w / v). In embodiments, CHAP is present in the lysis buffer (final concentration in contact with cells) at a concentration of between about 0.01% and about 0.8% (w / v). The concentrations can be any value or subrange within the recited ranges, including the endpoints.

[0054] In embodiments, the lysis buffer comprises CHAPS at a concentration (final concentration in contact with cells) of between about 0.005% and about 1% (w / v). In embodiments, CHAPS is present in the lysis buffer (final concentration in contact with cells) at a concentration of between about 0.01% and about 1% (w / v). In embodiments, CHAPS is present in the lysis buffer (final concentration in contact with cells) at a concentration of between about 0.01% and about 0.8% (w / v). The concentrations can be any value or subrange within the recited ranges, including endpoints.

[0055] In embodiments, the lysis buffer comprises CHAPSO at a concentration (final concentration in contact with cells) of between about 0.005% and about 1% (w / v). In embodiments, CHAPSO is present in the lysis buffer (final concentration in contact with cells) at a concentration of between about 0.01% and about 1% (w / v). In embodiments, CHAPSO is present in the lysis buffer (final concentration in contact with cells) at a concentration of between about 0.01% and about 0.8% (w / v). The concentrations can be any value or subrange within the recited ranges, including the endpoints.

[0056] In embodiments, the lysis buffer comprises Big CHAP at a concentration (final concentration in contact with cells) of between about 0.005% and about 1% (w / v). In embodiments, Big CHAP is present in the lysis buffer (final concentration in contact with cells) at a concentration of between about 0.01% and about 1% (w / v). In embodiments, Big CHAP is present in the lysis buffer (final concentration in contact with cells) at a concentration of between about 0.01% and about 0.8% (w / v). The concentrations can be any value or subrange within the recited ranges, including the endpoints.

[0057] In embodiments, the lysis buffer comprises OTG at a concentration (final concentration in contact with cells) of between about 0.005% and about 1% (w / v). In embodiments, OTG is present in the lysis buffer (final concentration in contact with cells) at a concentration of between about 0.01% and about 1% (w / v). In embodiments, OTG is present in the lysis buffer (final concentration in contact with cells) at a concentration of between about 0.01% and about 0.8% (w / v). The concentrations can be any value or subrange within the recited ranges, including the endpoints.

[0058] In embodiments, the lysis buffer comprises sodium deoxycholate at a concentration (final concentration in contact with cells) of between about 0.005% and about 1% (w / v). In embodiments, sodium deoxycholate is present in the lysis buffer (final concentration in contact with cells) at a concentration of between about 0.01% and about 1% (w / v). In embodiments, sodium deoxycholate is present in the lysis buffer (final concentration in contact with cells) at a concentration of between about 0.01% and about 0.8% (w / v). The concentrations can be any value or subrange within the recited ranges, including the endpoints.

[0059] In embodiments, the lysis buffer comprises at least one surfactant selected from Triton-100, Triton-alter, NP40, and poloxamer 188 (a copolymer of polyoxyethylene and polyoxypropylene, Pluronic® F-68). In embodiments, the lysis buffer comprises one surfactant selected from Triton-100, Triton-alter, NP40, and poloxamer 188. In embodiments, the lysis buffer comprises two surfactants selected from Triton-100, Triton-alter, NP40, and poloxamer 188. In embodiments, the lysis buffer comprises three surfactants selected from Triton-100, Triton-alter, NP40, and poloxamer 188. In embodiments, the lysis buffer comprises four surfactants selected from Triton-100, Triton-alter, NP40, and poloxamer 188. The formula (I) of Poloxamer 188 is: [ka] (I)

[0060] In embodiments, the lysis buffer comprises Triton-100 or Triton-alter at a concentration (final concentration in contact with cells) of between about 0.01% and about 0.1% (w / v). In embodiments, the lysis buffer comprises Triton-100 or Triton-alter at a concentration (final concentration in contact with cells) of between about 0.05% and about 0.1% (w / v). In embodiments, the lysis buffer comprises Triton-100 or Triton-alter at a concentration (final concentration in contact with cells) of between about 0.01% and about 0.05% (w / v). The concentrations can be any value or subrange within the recited ranges, including the endpoints.

[0061] In embodiments, the lysis buffer comprises NP40 at a concentration (final concentration in contact with cells) between about 0.05% and about 0.5% (w / v). In embodiments, the lysis buffer comprises NP40 at a concentration (final concentration in contact with cells) between about 0.1% and about 0.5% (w / v). In embodiments, the lysis buffer comprises NP40 at a concentration (final concentration in contact with cells) between about 0.2% and about 0.5% (w / v). In embodiments, the lysis buffer comprises NP40 at a concentration (final concentration in contact with cells) between about 0.3% and about 0.5% (w / v). In embodiments, the lysis buffer comprises NP40 at a concentration (final concentration in contact with cells) between about 0.4% and about 0.5% (w / v). In embodiments, the lysis buffer comprises NP40 at a concentration (final concentration in contact with cells) between about 0.05% and about 0.4% (w / v). In embodiments, the lysis buffer comprises NP40 at a concentration (final concentration in contact with cells) of between about 0.05% and about 0.3% (w / v). In embodiments, the lysis buffer comprises NP40 at a concentration (final concentration in contact with cells) of between about 0.05% and about 0.2% (w / v). In embodiments, the lysis buffer comprises NP40 at a concentration (final concentration in contact with cells) of between about 0.05% and about 0.1% (w / v). The concentrations can be any value or subrange within the recited ranges, including the endpoints.

[0062] In embodiments, the lysis buffer comprises poloxamer 188 at a concentration (final concentration in contact with cells) between about 0.08% and about 0.2% (w / v). In embodiments, the lysis buffer comprises poloxamer 188 at a concentration (final concentration in contact with cells) between about 0.09% and about 0.2% (w / v). In embodiments, the lysis buffer comprises poloxamer 188 at a concentration (final concentration in contact with cells) between about 0.1% and about 0.2% (w / v). In embodiments, the lysis buffer comprises poloxamer 188 at a concentration (final concentration in contact with cells) between about 0.08% and about 0.15% (w / v). In embodiments, the lysis buffer comprises poloxamer 188 at a concentration (final concentration in contact with cells) between about 0.08% and about 0.1% (w / v). The concentrations can be any value or subrange within the recited ranges, including the endpoints.

[0063] In embodiments, the lysis buffer comprises at least one salt, which in embodiments is sodium citrate, sodium chloride, potassium chloride, ammonium sulfate, ammonium phosphate, and / or sodium phosphate (e.g., sodium dihydrogen phosphate, sodium disodium phosphate, sodium triphosphate).

[0064] In embodiments, the lysis buffer comprises sodium citrate at a concentration of between about 1 mM and about 1000 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium citrate at a concentration of between about 1 mM and about 500 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium citrate at a concentration of between about 1 mM and about 400 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium citrate at a concentration of between about 1 mM and about 300 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium citrate at a concentration of between about 1 mM and about 200 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium citrate at a concentration of between about 1 mM and about 100 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium citrate at a concentration of between about 10 mM and about 1000 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium citrate at a concentration of between about 10 mM and about 500 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium citrate at a concentration of between about 10 mM and about 400 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium citrate at a concentration of between about 10 mM and about 300 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium citrate at a concentration of between about 10 mM and about 200 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium citrate at a concentration of between about 10 mM and about 100 mM (final concentration in contact with cells). The concentrations can be any value or subrange within the recited ranges, including the endpoints.

[0065] In embodiments, the lysis buffer comprises sodium chloride at a concentration of between about 1 mM and about 1000 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium chloride at a concentration of between about 1 mM and about 500 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium chloride at a concentration of between about 1 mM and about 400 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium chloride at a concentration of between about 1 mM and about 300 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium chloride at a concentration of between about 1 mM and about 200 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium chloride at a concentration of between about 1 mM and about 100 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium chloride at a concentration of between about 10 mM and about 1000 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium chloride at a concentration of between about 10 mM and about 500 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium chloride at a concentration of between about 10 mM and about 400 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium chloride at a concentration of between about 10 mM and about 300 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium chloride at a concentration of between about 10 mM and about 200 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium chloride at a concentration of between about 10 mM and about 100 mM (final concentration in contact with cells). The concentrations can be any value or subrange within the recited ranges, including the endpoints.

[0066] In embodiments, the lysis buffer comprises ammonium phosphate at a concentration of between about 0.5 mM and about 500 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises ammonium phosphate at a concentration of between about 0.5 mM and about 250 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises ammonium phosphate at a concentration of between about 0.5 mM and about 100 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises ammonium phosphate at a concentration of between about 0.5 mM and about 50 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises ammonium phosphate at a concentration of between about 0.5 mM and about 10 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises ammonium phosphate at a concentration of between about 1 mM and about 500 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises ammonium phosphate at a concentration of between about 1 mM and about 250 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises ammonium phosphate at a concentration (final concentration in contact with cells) between about 1 mM and about 100 mM. In embodiments, the lysis buffer comprises ammonium phosphate at a concentration (final concentration in contact with cells) between about 1 mM and about 50 mM. In embodiments, the lysis buffer comprises ammonium phosphate at a concentration (final concentration in contact with cells) between about 1 mM and about 10 mM. The concentrations can be any value or subrange within the recited ranges, including the endpoints.

[0067] In embodiments, the lysis buffer comprises sodium phosphate (e.g., sodium dihydrogen phosphate, disodium phosphate, trisodium phosphate) at a concentration of between about 0.5 mM and about 500 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium phosphate (e.g., sodium dihydrogen phosphate, disodium phosphate, trisodium phosphate) at a concentration of between about 0.5 mM and about 250 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium phosphate (e.g., sodium dihydrogen phosphate, disodium phosphate, trisodium phosphate) at a concentration of between about 0.5 mM and about 100 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium phosphate (e.g., sodium dihydrogen phosphate, disodium phosphate, trisodium phosphate) at a concentration of between about 0.5 mM and about 50 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium phosphate (e.g., sodium dihydrogen phosphate, disodium phosphate, trisodium phosphate) at a concentration of between about 0.5 mM and about 10 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium phosphate (e.g., sodium dihydrogen phosphate, disodium phosphate, trisodium phosphate) at a concentration of between about 1 mM and about 500 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium phosphate (e.g., sodium dihydrogen phosphate, disodium phosphate, trisodium phosphate) at a concentration of between about 1 mM and about 250 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium phosphate (e.g., sodium dihydrogen phosphate, disodium phosphate, trisodium phosphate) at a concentration of between about 1 mM and about 100 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises sodium phosphate (e.g., sodium dihydrogen phosphate, disodium phosphate, trisodium phosphate) at a concentration (final concentration in contact with cells) of between about 1 mM and about 50 mM, hi embodiments, the lysis buffer comprises sodium phosphate (e.g., sodium dihydrogen phosphate, disodium phosphate, trisodium phosphate) at a concentration (final concentration in contact with cells) of between about 1 mM and about 10 mM.The concentration can be any value or subrange within the stated range, including the endpoints.

[0068] In embodiments, the lysis buffer comprises a chelating agent, which in embodiments is ethylenediaminetetraacetic acid (EDTA), tripotassium EDTA, and / or ethylene glycol tetraacetic acid (EGTA).

[0069] In embodiments, the lysis buffer comprises EDTA at a concentration of between about 0.1 mM and about 50 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EDTA at a concentration of between about 0.1 mM and about 40 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EDTA at a concentration of between about 0.1 mM and about 30 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EDTA at a concentration of between about 0.1 mM and about 20 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EDTA at a concentration of between about 0.1 mM and about 10 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EDTA at a concentration of between about 0.1 mM and about 5 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EDTA at a concentration of between about 1 mM and about 50 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EDTA at a concentration of between about 1 mM and about 40 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EDTA at a concentration of between about 1 mM and about 30 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EDTA at a concentration of between about 1 mM and about 20 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EDTA at a concentration of between about 1 mM and about 10 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EDTA at a concentration of between about 1 mM and about 5 mM (final concentration in contact with cells). The concentrations can be any value or subrange within the recited ranges, including the endpoints.

[0070] In embodiments, the lysis buffer comprises tripotassium EDTA at a concentration of between about 0.1 mM and about 50 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises tripotassium EDTA at a concentration of between about 0.1 mM and about 40 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises tripotassium EDTA at a concentration of between about 0.1 mM and about 30 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises tripotassium EDTA at a concentration of between about 0.1 mM and about 20 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises tripotassium EDTA at a concentration of between about 0.1 mM and about 10 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises tripotassium EDTA at a concentration of between about 0.1 mM and about 5 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises tripotassium EDTA at a concentration of between about 1 mM and about 50 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises tripotassium EDTA at a concentration of between about 1 mM and about 40 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises tripotassium EDTA at a concentration of between about 1 mM and about 30 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises tripotassium EDTA at a concentration of between about 1 mM and about 20 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises tripotassium EDTA at a concentration of between about 1 mM and about 10 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises tripotassium EDTA at a concentration of between about 1 mM and about 5 mM (final concentration in contact with cells). The concentrations can be any value or subrange within the recited ranges, including the endpoints.

[0071] In embodiments, the lysis buffer comprises EGTA at a concentration of between about 0.1 mM and about 50 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EGTA at a concentration of between about 0.1 mM and about 40 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EGTA at a concentration of between about 0.1 mM and about 30 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EGTA at a concentration of between about 0.1 mM and about 20 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EGTA at a concentration of between about 0.1 mM and about 10 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EGTA at a concentration of between about 0.1 mM and about 5 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EGTA at a concentration of between about 1 mM and about 50 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EGTA at a concentration of between about 1 mM and about 40 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EGTA at a concentration of between about 1 mM and about 30 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EGTA at a concentration of between about 1 mM and about 20 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EGTA at a concentration of between about 1 mM and about 10 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises EGTA at a concentration of between about 1 mM and about 5 mM (final concentration in contact with cells). The concentrations can be any value or subrange within the recited ranges, including the endpoints.

[0072] In embodiments, the lysis buffer comprises at least one additional compound. For example, the lysis buffer may comprise 3-(1-pyridinio)propanesulfonate (NDSB 201, non-detergent sulfobetaine 201). In embodiments, the lysis buffer comprises Tris-HCl. In embodiments, the lysis buffer comprises citric acid. In embodiments, the lysis buffer comprises sodium hydroxide (NaOH).

[0073] In embodiments, the lysis buffer comprises NDSB-201 at a concentration of between about 0.5M and about 1M (final concentration in contact with cells). In embodiments, the lysis buffer comprises NDSB-201 at a concentration of between about 0.6M and about 1M (final concentration in contact with cells). In embodiments, the lysis buffer comprises NDSB-201 at a concentration of between about 0.7M and about 1M (final concentration in contact with cells). In embodiments, the lysis buffer comprises NDSB-201 at a concentration of between about 0.8M and about 1M (final concentration in contact with cells). In embodiments, the lysis buffer comprises NDSB-201 at a concentration of between about 0.9M and about 1M (final concentration in contact with cells). In embodiments, the lysis buffer comprises NDSB-201 at a concentration of between about 0.5M and about 0.9M (final concentration in contact with cells). In embodiments, the lysis buffer comprises NDSB-201 at a concentration (final concentration in contact with cells) of between about 0.5 M and about 0.8 M. In embodiments, the lysis buffer comprises NDSB-201 at a concentration (final concentration in contact with cells) of between about 0.5 M and about 0.7 M. In embodiments, the lysis buffer comprises NDSB-201 at a concentration (final concentration in contact with cells) of between about 0.5 M and about 0.6 M. The concentrations can be any value or subrange within the recited ranges, including the endpoints.

[0074] In embodiments, the lysis buffer comprises Tris-HCl at a concentration of between about 5 mM and about 20 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises Tris-HCl at a concentration of between about 6 mM and about 20 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises Tris-HCl at a concentration of between about 8 mM and about 20 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises Tris-HCl at a concentration of between about 10 mM and about 20 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises Tris-HCl at a concentration of between about 12 mM and about 20 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises Tris-HCl at a concentration of between about 14 mM and about 20 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises Tris-HCl at a concentration of between about 15 mM and about 20 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises Tris-HCl at a concentration of between about 10 mM and about 20 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises Tris-HCl at a concentration of between about 12 mM and about 20 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises Tris-HCl at a concentration of between about 15 mM and about 20 mM (final concentration in contact with cells). The concentrations can be any value or subrange within the recited ranges, including the endpoints.

[0075] In embodiments, the lysis buffer comprises citric acid at a concentration of between about 20 mM and about 100 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises citric acid at a concentration of between about 40 mM and about 100 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises citric acid at a concentration of between about 50 mM and about 100 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises citric acid at a concentration of between about 60 mM and about 100 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises citric acid at a concentration of between about 80 mM and about 100 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises citric acid at a concentration of between about 20 mM and about 80 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises citric acid at a concentration (final concentration in contact with cells) of between about 20 mM and about 60 mM. In embodiments, the lysis buffer comprises citric acid at a concentration (final concentration in contact with cells) of between about 20 mM and about 40 mM. The concentration can be any value or subrange within the recited range, including the endpoints.

[0076] In embodiments, the lysis buffer comprises NaOH at a concentration of between about 1 mM and about 50 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises NaOH at a concentration of between about 10 mM and about 50 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises NaOH at a concentration of between about 20 mM and about 50 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises NaOH at a concentration of between about 30 mM and about 50 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises NaOH at a concentration of between about 40 mM and about 50 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises NaOH at a concentration of between about 1 mM and about 40 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises NaOH at a concentration of between about 1 mM and about 30 mM (final concentration in contact with cells). In embodiments, the lysis buffer comprises NaOH at a concentration (final concentration in contact with cells) of between about 1 mM and about 20 mM. In embodiments, the lysis buffer comprises NaOH at a concentration (final concentration in contact with cells) of between about 1 mM and about 10 mM. The concentration can be any value or subrange within the stated range, including the endpoints.

[0077] The AAV production system also includes an AAV production enhancer (AAV enhancer), which includes one or more of a histone deacetylase (HDAC) inhibitor, sodium propionate, sodium butyrate, theobromine, and caffeine.

[0078] In embodiments, the HDAC inhibitor is selected from apicidin, belinstat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A, and valproic acid. In embodiments, the HDAC inhibitor is sodium butyrate, sodium phenylbutyrate, trichostatin A, and / or valproic acid.

[0079] In embodiments, the sodium propionate and / or HDAC inhibitor is provided in water. In embodiments, the caffeine is provided in a cell culture expression medium, such as an Expi293™ expression medium.

[0080] In embodiments, sodium propionate is present at a concentration of about 1 mM to 50 mM (final concentration in contact with the cells). In embodiments, sodium propionate is present at about 1 mM to 40 mM. In embodiments, sodium propionate is present at about 1 mM to 30 mM. In embodiments, sodium propionate is present at about 1 mM to 20 mM. In embodiments, sodium propionate is present at about 1 mM to 10 mM. In embodiments, sodium propionate is present at about 1 mM to 5 mM. In embodiments, sodium propionate is present at about 2 mM to 30 mM. In embodiments, sodium propionate is present at about 2 mM to 20 mM. In embodiments, sodium propionate is present at about 2 mM to 10 mM. In embodiments, sodium propionate is present at about 2 mM to 5 mM. The concentration can be any value or subrange within the stated range, including the endpoints.

[0081] In embodiments, the HDAC inhibitor is present at a concentration of about 0.1 mM to about 100 mM (final concentration contacting the cells). In embodiments, the HDAC inhibitor is present at a concentration of about 0.1 mM to about 75 mM. In embodiments, the HDAC inhibitor is present at a concentration of about 0.1 mM to about 50 mM. In embodiments, the HDAC inhibitor is present at a concentration of about 0.1 mM to about 25 mM. In embodiments, the HDAC inhibitor is present at a concentration of about 0.1 mM to about 10 mM. In embodiments, the HDAC inhibitor is present at a concentration of about 0.1 mM to about 9 mM. In embodiments, the HDAC inhibitor is present at a concentration of about 0.1 mM to about 8 mM. In embodiments, the HDAC inhibitor is present at a concentration of about 0.1 mM to about 7 mM. In embodiments, the HDAC inhibitor is present at a concentration of about 0.1 mM to about 6 mM. In embodiments, the HDAC inhibitor is present at a concentration of about 0.1 mM to about 5 mM. In embodiments, the HDAC inhibitor is present at a concentration of about 1 mM to about 100 mM. In embodiments, the HDAC inhibitor is present at a concentration of about 1 mM to about 50 mM. In embodiments, the HDAC inhibitor is present at a concentration of about 1 mM to about 25 mM. In embodiments, the HDAC inhibitor is present at a concentration of about 1 mM to about 10 mM. In embodiments, the HDAC inhibitor is present at a concentration of about 1 mM to about 9 mM. In embodiments, the HDAC inhibitor is present at a concentration of about 1 mM to about 8 mM. In embodiments, the HDAC inhibitor is present at a concentration of about 1 mM to about 7 mM. In embodiments, the HDAC inhibitor is present at a concentration of about 1 mM to about 6 mM. In embodiments, the HDAC inhibitor is present at a concentration of about 1 mM to about 5 mM. The concentration can be any value or subrange within the stated range, including the endpoints.

[0082] In embodiments, caffeine is present at a concentration of about 0.1 mM to about 50 mM (final concentration in contact with the cells). In embodiments, caffeine is present at a concentration of about 0.1 mM to about 25 mM. In embodiments, caffeine is present at a concentration of about 0.1 mM to about 15 mM. In embodiments, caffeine is present at a concentration of about 0.1 mM to about 10 mM. In embodiments, caffeine is present at a concentration of about 0.1 mM to about 9 mM. In embodiments, caffeine is present at a concentration of about 0.1 mM to about 8 mM. In embodiments, caffeine is present at a concentration of about 0.1 mM to about 7 mM. In embodiments, caffeine is present at a concentration of about 0.1 mM to about 6 mM. In embodiments, caffeine is present at a concentration of about 0.1 mM to about 5 mM. In embodiments, caffeine is included at a concentration of about 0.1 mM to about 4 mM. In embodiments, caffeine is included at a concentration of about 0.1 mM to about 3 mM. In embodiments, caffeine is included at a concentration of about 0.1 mM to about 2 mM. In embodiments, caffeine is included at a concentration of about 0.5 mM to about 50 mM. In embodiments, caffeine is included at a concentration of about 0.5 mM to about 10 mM. In embodiments, caffeine is included at a concentration of about 0.5 mM to about 5 mM. In embodiments, caffeine is included at a concentration of about 0.5 mM to about 4 mM. In embodiments, caffeine is included at a concentration of about 0.5 mM to about 3 mM. In embodiments, caffeine is included at a concentration of about 0.5 mM to about 2 mM. The concentration can be any value or subrange within the stated ranges, including the endpoints.

[0083] In some embodiments, the AAV enhancer is added at one or more time points, such as from the time of transfection (about 0 hours) to about 48 hours post-transfection. The AAV enhancer can be added about 1 hour to about 16 hours post-transfection to promote cellular packaging of the AAV vector. In some embodiments, the AAV enhancer can be added at the time of transfection. In some embodiments, the AAV enhancer can be added at the time of transfection and about 1 hour to about 16 hours post-transfection. In some embodiments, the AAV enhancer can be added about 4 to 5 hours post-transfection. In some embodiments, the AAV enhancer can be added about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 24, 36, or 48 hours post-transfection. The AAV enhancer can be added at any time (or subrange) within the stated ranges, including the endpoints.

[0084] The design and production of AAV vectors are known in the art. See, for example, U.S. Patent Nos. 5,354,678, 6,759,237, 5,753,500, and 5,474,935. Packaging plasmids can be used to properly package AAV. These plasmids encode the genes necessary for packaging AAV vectors. Such genes include genes expressing capsid proteins (cap) and replication (rep) genes. Alternatively, the genes can be stably expressed by cells. The AAV genes can be from any serotype of AAV, including, but not limited to, serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, DJ, or DJ / 8. Packaging plasmids encoding the AAV rep and cap genes are often referred to as pAAV-RC, pRep / Cap, or pRC plasmids. AAV transfer vectors, packaging plasmids, packaging cell lines, and other products for AAV production are commercially available from, for example, Cell Biolabs, Inc., Vector Biolabs, Addgene, Clontech, and Thermo Fisher Scientific.

[0085] In embodiments, helper virus components (e.g., from adenovirus or herpesvirus) are required for proper function of the AAV production system. The helper virus components may be present on a plasmid (further often referred to as pAAV-Helper or pHelper plasmid) or may be otherwise present in the cell. Helper virus components include, but are not limited to, E1A, E1B, E2A, E4, and / or VA.

[0086] Provided herein is a kit for producing adeno-associated virus (AAV). In embodiments, the kit includes 293 cells adapted to high-density suspension culture, an AAV production enhancer, a transfection reagent containing a cationic lipid, and a cell culture medium that supports the growth and proliferation of 293 cells. In embodiments, the transfection reagent includes a cationic lipid and a peptide. In embodiments, the transfection reagent includes at least one cationic lipid and at least one neutral lipid. In embodiments, the AAV production enhancer includes one or more of an HDAC inhibitor, sodium propionate, sodium butyrate, theobromine, and caffeine. In embodiments, the 293 cells do not contain large T antigen.

[0087] In embodiments, the kit also includes a transfection booster. In embodiments, the transfection booster includes a peptide. In embodiments, the transfection booster includes a membrane-permeable peptide.

[0088] In embodiments, the kit also includes a lysis buffer. In embodiments, the lysis buffer includes at least one detergent. In embodiments, the detergent is Triton-100, Triton-alter, NP-40, poloxamer 188, and / or NDSB-201. In embodiments, the lysis buffer includes Tris-HCl, sodium citrate, tricine HCl, sodium chloride, citric acid, EDTA, tripotassium EDTA, sodium hydroxide, and / or sodium dihydrogen phosphate. In embodiments, the lysis buffer includes at least one detergent. In embodiments, the detergent is CHAP, CHAPS, CHAPSO, big CHAP, deoxyl big CHAP, Triton X-114, octylthioglucoside, and / or sodium deoxycholate. How to Use the Adeno-Associated Virus (AAV) Production System

[0089] The AAV production systems described herein can be used to produce AAV vectors, which in embodiments are produced at high titers.

[0090] In one aspect, a method for producing an AAV vector is provided, comprising: (i) culturing mammalian cells; (ii) transfecting the mammalian cells with an AAV transfer vector using a transfection reagent; and (iii) culturing the transfected cells in suspension culture for a time sufficient for expression of the AAV vector. In embodiments, the mammalian cells are cultured in suspension culture. In embodiments, the method comprises harvesting AAV from the transfected AAV cell culture. In embodiments, the transfecting comprises contacting the cells with a transfection booster. In embodiments, the cells are contacted with an enhancer after transfection.

[0091] In embodiments, the transfection reagent is combined with the AAV transfer vector to form a DNA / transfection reagent complex prior to addition to the cells. In embodiments, the transfection reagent is combined with the AAV transfer vector, the pRep / Cap plasmid, and the pHelper plasmid to form a DNA / transfection reagent complex prior to addition to the cells.

[0092] In other embodiments, the transfection booster is combined with the AAV transfer vector to form a DNA / transfection booster mixture, and then the transfection reagent is combined with the DNA / transfection booster mixture before adding to the cells. In embodiments, the transfection booster is combined with the AAV transfer vector, the pRep / Cap plasmid, and the pHelper plasmid to form a DNA / transfection booster mixture, and then the transfection reagent is combined with the DNA / transfection booster mixture before adding to the cells. In some embodiments, the DNA and transfection booster are combined in a tube, the transfection reagent is diluted in medium in a second tube, and then the diluted transfection reagent is added to the DNA / transfection booster mixture to form a DNA / transfection booster / transfection reagent complex. In other embodiments, the DNA and transfection booster are combined in a tube and then the transfection reagent is added to the same tube to form a DNA / transfection booster / transfection reagent complex. In other embodiments, the DNA and transfection reagent are combined in a tube and then the transfection booster is added to the same tube to form a DNA / transfection booster / transfection reagent complex.

[0093] In embodiments, the transfection booster is used at a transfection booster:DNA ratio of between 5:1 and about 1:5 (volume / weight). In embodiments, the transfection reagent is combined with the transfection booster, rep / cap plasmid (pRC), pHelper plasmid (encoding helper virus components), and the AAV transfer vector to form a transfection complex.

[0094] In some embodiments, the AAV is harvested using a lysis buffer. In some embodiments, the cells are not centrifuged before harvesting the AAV. In some embodiments, the lysis buffer is added directly to the transfected cell culture (e.g., cells and culture medium).

[0095] In some embodiments, crude culture lysate containing AAV is filtered before downstream processing, such as nuclease treatment and purification. In some embodiments, the crude lysate is mixed with diatomaceous earth, and then the mixture is passed through a filter, for example, a 2-micron filter, to recover the collected AAV. Alternatively, a cellulose filtration step can be used with the crude lysate to produce an AAV preparation ready for downstream processing. By subjecting the crude AAV lysate to such a filtration step, for example, using diatomaceous earth, cellulose, or the like, the number of filters required can be reduced, and the filtration and processing time of the AAV lysate before the purification process can be reduced.

[0096] In embodiments, the cells are cultured in a bioreactor. In embodiments, the cells are cultured in a flask.

[0097] In embodiments, the method includes titrating the harvested AAV. AAV can be titrated using any method. In embodiments, AAV is titrated using polymerase chain reaction (PCR). In embodiments, AAV is titrated using quantitative PCR (qPCR). In embodiments, AAV is titrated using digital droplet PCR. In embodiments, AAV is titrated using ELISA. In embodiments, AAV is titrated using a virus titer kit, e.g., QUICKTITER™ AAV Quantitation Kit (Cell BioLabs, Inc.). See also U.S. Patent No. 6,841,357, which is incorporated herein by reference in its entirety. In embodiments, AAV is titrated by determining the concentration of viral particles capable of transducing cells (infectious titer), e.g., by a cell transduction assay. In embodiments, AAV is titrated using DNA dot blotting.

[0098] In embodiments, the harvested AAV is at least about 1 x 10 per milliliter. 10 In embodiments, the harvested AAV has a titer of at least about 2 x 10 viral genomes (vg / mL). 10 In embodiments, the harvested AAV has a titer of at least about 3 x 10 vg / mL. 10 In embodiments, the harvested AAV has a titer of at least about 4 x 10 vg / mL. 10 In embodiments, the harvested AAV has a titer of at least about 5 x 10 vg / mL. 10 In embodiments, the harvested AAV has a titer of at least about 6 x 10 vg / mL. 10 In embodiments, the harvested AAV has a titer of at least about 7 x 10 vg / mL. 10 In embodiments, the harvested AAV has a titer of at least about 8 x 10 vg / mL. 10 In embodiments, the harvested AAV has a titer of at least about 9 x 10 vg / mL. 10In embodiments, the harvested AAV has a titer of at least about 1 x 10 vg / mL. 11 In embodiments, the harvested AAV has a titer of at least about 2 x 10 vg / mL. 11 In embodiments, the harvested AAV has a titer of at least about 3 x 10 vg / mL. 11 In embodiments, the harvested AAV has a titer of at least about 4 x 10 vg / mL. 11 In embodiments, the harvested AAV has a titer of at least about 5 x 10 vg / mL. 11 In embodiments, the harvested AAV has a titer of at least about 6 x 10 vg / mL. 11 In embodiments, the harvested AAV has a titer of at least about 7 x 10 vg / mL. 11 In embodiments, the harvested AAV has a titer of at least about 8 x 10 vg / mL. 11 In embodiments, the harvested AAV has a titer of at least about 9 x 10 vg / mL. 11 It has a titer of 0.05 mg / mL.

[0099] In embodiments, the harvested AAV is about 1 x 10 10 vg / mL to approximately 1 × 10 12 In embodiments, the harvested AAV has a titer of between about 2 x 10 vg / mL. 10 vg / mL to approximately 1 × 10 12 In embodiments, the harvested AAV has a titer of between about 3 x 10 vg / mL. 10 vg / mL to approximately 1 × 10 12 In embodiments, the harvested AAV has a titer of between about 4 x 10 vg / mL. 10 vg / mL to approximately 1 × 10 12 In embodiments, the harvested AAV has a titer of between about 5 x 10 vg / mL. 10 vg / mL to approximately 1 × 10 12 In embodiments, the harvested AAV has a titer of between about 6 x 10 vg / mL. 10 vg / mL to approximately 1 × 10 12 In embodiments, the harvested AAV has a titer of between about 7 x 10 vg / mL.10 vg / mL to approximately 1 × 10 12 In embodiments, the harvested AAV has a titer of between about 8 x 10 vg / mL. 10 vg / mL to approximately 1 × 10 12 In embodiments, the harvested AAV has a titer of between about 9 x 10 vg / mL. 10 vg / mL to approximately 1 × 10 12 In embodiments, the harvested AAV has a titer of between about 1 x 10 vg / mL. 11 vg / mL to approximately 1 × 10 12 In embodiments, the harvested AAV has a titer of between about 2 x 10 vg / mL. 11 vg / mL to approximately 1 × 10 12 In embodiments, the harvested AAV has a titer of between about 3 x 10 vg / mL. 11 vg / mL to approximately 1 × 10 12 In embodiments, the harvested AAV has a titer of between about 4 x 10 vg / mL. 11 vg / mL to approximately 1 × 10 12 In embodiments, the harvested AAV has a titer of between about 5 x 10 vg / mL. 11 vg / mL to approximately 1 × 10 12 In embodiments, the harvested AAV has a titer of between about 2 x 10 vg / mL. 10 vg / mL to approximately 9 × 10 11 In embodiments, the harvested AAV has a titer of between about 2 x 10 vg / mL. 10 vg / mL to approximately 8 × 10 11 In embodiments, the harvested AAV has a titer of between about 2 x 10 vg / mL. 10 vg / mL to approximately 7 × 10 11 In embodiments, the harvested AAV has a titer of between about 2 x 10 vg / mL. 10 vg / mL to approximately 6 × 10 11 In embodiments, the harvested AAV has a titer of between about 2 x 10 vg / mL. 10 vg / mL to approximately 5 × 10 11 In embodiments, the harvested AAV has a titer of between about 2 x 10 vg / mL. 10 vg / mL to approximately 4 × 1011 In embodiments, the harvested AAV has a titer of between about 2 x 10 vg / mL. 10 vg / mL to approximately 3 × 10 11 In embodiments, the harvested AAV has a titer of between about 2 x 10 vg / mL. 10 vg / mL to approximately 2 × 10 11 In embodiments, the harvested AAV has a titer of between about 2 x 10 vg / mL. 10 vg / mL to approximately 1 × 10 11 The titer may be any value or subrange within the stated range, including the endpoints.

[0100] The cells may be cultured in any amount of cell culture medium that supports cell growth and AAV production. In embodiments, the cells are cultured in a volume of about 15 milliliters (mL) to about 200 liters (L). In embodiments, the cells are cultured in a volume of about 30 mL to about 200 L. In embodiments, the cells are cultured in a volume of about 50 mL to about 200 L. In embodiments, the cells are cultured in a volume of about 100 mL to about 200 L. In embodiments, the cells are cultured in a volume of about 500 mL to about 200 L. In embodiments, the cells are cultured in a volume of about 1 L to about 200 L. In embodiments, the cells are cultured in a volume of about 10 L to about 200 L. In embodiments, the cells are cultured in a volume of about 15 mL to about 100 L. In embodiments, the cells are cultured in a volume of about 15 mL to about 50 L. In embodiments, the cells are cultured in a volume of about 15 mL to about 20 L. In embodiments, the cells are cultured in a volume of about 15 mL to about 20 L. In embodiments, the cells are cultured in a volume of about 15 mL to about 5 L. In embodiments, the cells are cultured in a volume of about 15 mL to about 1 L. In embodiments, the cells are cultured in a volume of about 15 mL to about 500 L. In embodiments, the cells are cultured in a volume of about 500 mL to about 10 L. In embodiments, the cells are cultured in a volume of about 1 L to about 10 L. The culture volume can be any value or subrange within the recited range, including the endpoints.

[0101] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes will be suggested to those skilled in the art in light thereof, and are to be included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. example

[0102] Those skilled in the art will understand that the descriptions of making and using the particles described herein are for illustrative purposes only, and that the present disclosure is not limited by these examples. Example 1: Effect of culture conditions on virus titer

[0103] Adherent HEK293T and HEK293F cells were cultured in an incubator at 37°C, 8% CO2, and 80% humidity. Cells were cultured at approximately 4 × 10 in 6-well plates using PEI (HEK293T) or System 1 (HEK293F). 6Cells were transfected at a concentration of 1000 cells / mL with the AAV transfer vectors, pAAV-RC, and pAAV-Helper. The conditions were Prot-1 (Enhancer 1 and Supplement 1), Prot-2 (Supplement 1, no Enhancer), or Prot-3 (Enhancer 1, no Supplement). System 1 included a medium supporting HEK293 cell growth and proliferation (LV-MAX™ Production Medium, GIBCO™, Thermo Fisher Scientific, catalog number A3583401), LV-MAX™ Transfection Reagent, LV-MAX™ Supplement, and LV-MAX™ Enhancer (GIBCO™, Thermo Fisher Scientific, catalog number A35348). AAV was extracted by adding 200 μL of 5X AAV lysis buffer to 800 μL of transfected cell culture, mixing thoroughly, and incubating at room temperature for 30 minutes. After incubation, the tube was inverted to completely lyse the cells, then spun at maximum speed in a tabletop centrifuge for 10 minutes. The supernatant containing crude AAV was collected.

[0104] AAV was titrated by qPCR using primers and probes against ITR2. Briefly, extracts were treated with DNase and proteinase K. Extracts were diluted 1:50 in water, and qPCR was performed and compared to a standard curve (digested AAV plasmid).

[0105] The results are provided in Figure 1. For HEK293F cells, System 1 without LV-MAX™ supplement produced the highest AAV titers. Example 2 Effect of culture medium on virus titer

[0106] HEK293F cells were established in four different types of commercially available media that support HEK293F cells in suspension culture. Cells were frozen in their adapted media and thawed before use. The thawed cells were transfected with an AAV transfer vector (pAAV-GFP). The AAV transfer vector, pAAV-RC2, and pAAV-Helper were complexed with AAV transfection reagent at a 1:4 w / w ratio and transfection booster at a 2:1 (v / w) ratio in Opti-MEM complex solution buffer and incubated at room temperature for 10 minutes. After incubation, the DNA / transfection reagent complex mixture was added directly to the prepared cell culture. AAV2 production occurred at cell passage 6 in all four cell systems / media types.

[0107] FIG. 2 shows that HEK293F cells adapted to medium 4 produced more AAV2 than the other media tested. Example 3 Characteristics of Medium 4-adapted Cells

[0108] To determine the optimal conditions for transfection of HEK293F cells established in Medium 4, the effects of different transfection reagents were evaluated. Cells were transfected with AAV2, AAV6, or AAV-Dj using System 1 (HEK293F cells with LV-MAX transfection reagent, enhancer, and supplements (see Example 1)), System 2 (HEK293F cells with a different transfection reagent (Transfection Reagent 2) with LV-MAX enhancer and supplements), and System 3 (Medium 4-adapted HEK293 cells with Transfection Reagent 2 and LV-MAX enhancer, but without supplements). Viral titers in Medium 4-adapted HEK293 cells were highest with System 3 for all AAV serotypes (Figure 3). Transfection Reagent 2 is a cationic lipid transfection reagent containing a peptide-containing transfection booster (described herein). Example 4 Clone 45 cells

[0109] The growth characteristics of a clonal population of Medium 4-adapted HEK293 cells (clone 45) were evaluated. Clone 45 cells were cultured at 0.3 x 10 in 250 mL shake flasks in Medium 4. 6 Cells were split at passage 5 to a cell density of 11 x 10 viable cells / mL. Cell density and cell viability were collected daily for 9 days. The growth curve is shown in Figure 4A (inset: cell viability). Medium 4 produced a maximum of approximately 11 x 10 6 Clone 45 cells supported high cell densities of 1000 cells / mL and exhibited high cell viability in high-density cultures. As shown in Figure 4B, Clone 45 cells showed little clumping, even at high densities.

[0110] The ability of Clone 45 cells and three other Medium 4-adapted clonal lines to produce acceptable titers of different AAV serotypes in Medium 4 was evaluated. Clone 45 cells were grown in EXPI293™ Expression Medium (Expi45, GIBCO™ Thermo Fisher Scientific, catalog number A1435101) or Medium 4 (Cl45) and transfected with AAV2, AAV6, AAV-dj, AAV8, or AAV9 vectors corresponding to the transgene encoding GFP. Similarly, the Medium 4-adapted clonal lines Clone 12 (Cl12), Clone 22 (Cl22), and Clone 51 (Cl51) were grown in Medium 4 and transfected with vectors for five AAV serotypes. Transfections were performed using Transfection Reagent 2 as described above. Production in Clone 45 cells was compared to the parental cell line (parental HEK293, control) or the other three Medium 4-adapted clonal lines. The resulting AAV titers were determined using qPCR with AAV-GFP primers and probes, as described elsewhere herein. Clone 45 cells grown in Medium 4 yielded approximately 1 x 10 AAV titers for each serotype (Figure 5A-E). 11 vg / mL to approximately 2 × 10 11 This resulted in high viral titers between 100 and 2000 vg / mL.

[0111] The effect of different transfection reagents on virus production by Clone 45 cells was also evaluated. Cells were transfected with LV-MAX™ Transfection Reagent (TR1) or Transfection Reagent 2 (TR2). Plasmid delivery via Transfection Reagent 2 produced more AAV virus than LV-MAX™ Transfection Reagent under these conditions (Figure 6). Example 5: Improvement of virus production by adding an enhancer

[0112] Clone 45 cells in medium 4 were transfected with AAV plasmids using transfection reagent 2 with or without enhancers added at 0, 4.5, 8, 15.5, 24, or 28 hours post-transfection. The addition of enhancers increases AAV production, particularly between 0 and 15.5 hours post-transfection (Figure 7). Enhancers include HDAC inhibitors, sodium propionate, sodium butyrate, and caffeine. Example 6 Screening of Lysis Buffer Reagents

[0113] Using the JMP® Design of Experiments (DOE) platform, a screening experiment was designed using four core chemicals (Triton-alter, CHAPSO, Big CHAP, and NDSB-201) as potential detergents in the lysis buffer.

[0114] The DOE platform allowed researchers to simultaneously vary multiple parameters, instead of varying each parameter individually and then examining each optimized parameter across the entire optimized formulation. When second-order effects between parameters can affect results, the DOE platform allows for more efficient and accurate results by simultaneously varying all candidate parameters. Experiments using the DOE platform also require fewer runs than traditional experimental methods, making them more economical. For theoretical considerations regarding the DOE platform, see Kauffman et al., Optimization of Lipid Nanoparticle Formulations for mRNA Delivery in Vivo with Fractional Factorial and Definitive Screening Designs, NanoLetters 15:7300-7306 (2015) and supplementary materials.

[0115] Triton-alter, CHAPSO, Big CHAP, and NDSB-201 were evaluated at different concentrations, as shown in Table 1. Percentages are provided as weight per volume (w / v). JPEG2026035576000002.jpg165169

[0116] The results were analyzed using Jmp software to determine the effect of detergents during the cell lysis process, and the results are provided in Figure 8 for three different AAV serotypes.

[0117] Lysis buffer was also tested on AAV extracts from transfected cell cultures and cell pellets. Clone 45 cells were transfected with AAV plasmids using transfection reagent 2 and enhancer. AAV was extracted 70 to 72 hours after transfection by adding 1x lysis buffer to the cell pellet or 5x lysis buffer to the whole cell culture (transfected cells and medium). The resulting AAV titers are provided in Figure 9. Example 7 Initial AAV Production Protocol

[0118] Follow these guidelines for suspension culture of AAV producer cells: Cells are grown according to standard AAV suspension cell culture protocols. Cells are typically cultured at approximately 3 x 10 cells every 3 to 4 days. 6 From 6.5 x 10 6 Cells are subcultured when they reach a density of 0.3 x 10 viable cells / mL after approximately 3 or 4 days of culture. 6 to 0.6 × 10 6 Cells are split into 100ml of medium. Cell growth is monitored by counting cells daily at approximately the same time each day. During cell culture, an elliptical orbital shaker (19 mm elliptical diameter) is used at approximately 125 rpm in 125 mL to 1 L shake flasks. The incubator is set to approximately 37 °C, approximately 8% CO2, and approximately 75-80% humidity.

[0119] Reagents and materials: 125mL, 250mL, and 1L Polycarbonate, Disposable, Sterile, Vented, Unbaffled Erlenmeyer Flasks 50mL sterile conical tube Opti-MEM I medium Clone 45 cells AAV293 culture medium AAV transfection reagents and transfection boosters AAV enhancer 5X AAV lysis buffer

[0120] 0.55 × 10 cells 6 For example, if cells are split on Friday morning to a cell count of approximately 4.0 x 10 cells / mL, they can be cultured for 3 days in a 1 L flask with approximately 300 mL of culture medium. On Monday morning, for example, cells are prepared by counting the cells and counting approximately 4.0 x 10 cells / mL. 6 A cell density of approximately 3.0 x 10 cells / mL can be expected. 6 The cells / mL can be diluted and cultured for a further 24 hours (approximately).

[0121] Transfection can be performed, for example, on Tuesday. Cells are cultured at approximately 2.5 x 10 in 30 mL of cell culture medium in a 125 mL flask. 6 cells / mL to approximately 4 x 10 6 The cells / mL can be counted and diluted. Table 2 shows the amount of each plasmid at various ratios. Table 3 provides additional transfection guidelines. JPEG2026035576000003.jpg119167 JPEG2026035576000004.jpg141169

[0122] The DNA / transfection reagent is prepared as follows: Two tubes are labeled Tube-1 and Tube-2. In Tube-1, 4.5 mL of OPTI-MEM™ I medium is mixed with 135 μg of DNA (ratio shown in Table 2) and 270 μL of transfection booster is added. In Tube-2, 4.5 mL of OPTI-MEM™ I medium is mixed with 540 μL of transfection reagent and incubated at room temperature for 1 minute. Tube-1 and Tube-2 are combined by adding the Tube-2 solution to Tube-1 with mixing and incubating at room temperature for 10 minutes. Approximately 3.2 mL of DNA / AAV transfection reagent complex is added to the cells in each flask. The AAV enhancer is added at 1% v / v per flask at the time of transfection.

[0123] AAV is harvested 70-72 hours after transfection, e.g., on a Friday morning. Alternatively, the transfected cell culture can be stored at -80°C (an 800µL sample should be stored separately at -80°C for titer determination). Add 5x AAV lysis buffer at a 1:5 dilution (200µL of lysis buffer per 800µL transfected cell culture sample) and mix by pipetting up and down and vortexing. Incubate the sample for approximately 30 minutes, then invert by hand 25-30 times. The cells are completely lysed when the culture medium becomes clear and clumps of cell debris are observed.

[0124] Spin the lysed cells for 10 min at 4 °C (maximum speed in a benchtop centrifuge). Transfer the supernatant containing the crude AAV to a new tube and store at 4 °C. The sample is then titered, for example by qPCR. Example 8 Measurement of AAV titers from the production of Example 7

[0125] The supernatant from Example 7 was mixed thoroughly and 100 μL of crude AAV sample was aliquoted into each of two wells of a 96-well round-bottom plate with a lid. The sample was digested with DNase I by adding 2 μL of crude AAV sample to 2 μL of 10x DNase I buffer and 137 units of DNase I in a total volume of 20 μL and incubating at 37°C for 60 minutes, 95°C for 20 minutes, and then 4°C.

[0126] The DNase-treated samples are digested with proteinase K by adding 19 μL of 2x PK buffer and 20 μg of proteinase K to each sample and incubating at 60 °C for 60 min, 95 °C for 10 min, and then at 4 °C.

[0127] After proteinase K digestion, the sample is diluted 1:50 in water. Quantitative PCR (qPCR) is performed using primers specific for the AAV-GFP gene and a labeled probe.

[0128] A standard curve is generated using linearized AAV transfer plasmids. In the case of pAAV-GFP, the plasmid is linearized by digestion with HindIII or BamHI, and then the DNA concentration is determined. Linearization can be determined by loading the cleaved and uncut plasmids onto a 0.8% agarose gel and visualizing the resulting bands after electrophoresis. Uncut plasmids should appear as a smear, while cut plasmids should appear as a single large band of approximately 5 kb.

[0129] qPCR is performed in a 384-well qPCR sample plate using premixed ROX (Thermo Fisher Scientific) 2X EXPRESS qPCR Supermix according to the manufacturer's instructions. Briefly, 3 μL of diluted sample (or standard curve) is combined with 7.5 μL of 2X Supermix, 0.11 μL of AAV-GFP probe (FAM / TAMRA), 1.13 μL of GFP-specific primers (forward and reverse primers mixed at 10 μM), and 3.26 μL of water. Samples are run in a qPCR machine using a cycling program of 50°C for 2 min (UDG incubation), 95°C for 2 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Example 9 AAV-GFP Virus Infectivity Test Protocol

[0130] AAV can be tested for its ability to infect target cells (e.g., Ht1080 or HEK293). Approximately 4 hours prior to infection, cells are seeded into 96-well plates at a density of 7000 cells / well in 100 μL of culture medium.

[0131] Infect cells by adding 1 μL of crude AAV preparation to each well. Incubate cells for approximately 3 days. For AAV containing an expressible green fluorescent protein (GFP) gene, perform flow cytometry to measure the percentage of cells expressing GFP. Example 10 Comparison of AAV production systems

[0132] AAV vector production from clone 45 and two subclonal lines derived from clone 45 was compared with production from the HEK293F derivative LV293 (LV-MAX virus producer cells (VPC), Thermo Fisher Scientific). Cells were grown according to the culture protocol described above and transfected with pAAV-GFP, pAAV-Helper, and pAAV-RC for AAV8. Transfections were performed using transfection reagent 2 with transfection boosters as described above. Approximately 72 hours after transfection, AAV titers (via qPCR for GFP (Example 8)) and cell viability of the cultures were determined. Clone 45 and subclonal lines C13 and C20 produced approximately 1.5 × 10 AAV vectors compared to the same system with LV293 (VPC, Figure 10A). 11 to approximately 2.5 x 10 11 These results yielded significantly higher AAV8 titers (between 100 and 150 vg / mL). Furthermore, AAV production cultures using clones 45, C13, and C20 had significantly higher cell viability than cultures using LV293 (VPC, Figure 10B).

[0133] Vector production across various AAV serotypes in Clone 45 cells was compared to that in LV293 (VPC) cells. Cells were grown according to the suspension cell culture protocol for AAV as described above, and the cells were cultured in 125 mL flasks in 30 mL of cell culture medium-4 (3 x 10 6 cells / mL clone 45 and 2.5 x 10 6The cells were diluted in a 1000-µL LV293 solution (1000µL / mL LV293 cells). Cells were transfected with pAAV-GFP, pAAV-Helper, and pAAV-RC for AAV2, AAV6, AAV8, and AAV9. Transfections were performed using transfection reagent 2 along with transfection boosters as described above. AAV was harvested from the culture approximately 72 hours post-transfection. AAV titers were determined via qPCR and infectivity testing protocols for the GFP method (Example 9). Exemplary results are shown in Figure 11. Compared to an identical system with LV293 (VPC, Figure 11A), the AAV production system with clone 45 as producer cells produced approximately 1.3 x 10 cells across multiple serotypes. 11 to approximately 2.15 x 10 11 As shown in Figure 11B, the AAV production system with clone 45 also produced AAV2 and AAV6 with higher infectivity than LV293 VPC cells.

[0134] AAV vector production using the AAV vector production system provided herein was compared with two polyethyleneimine (PEI)-based production systems. The PEI-based transfection system was performed according to the manufacturer's instructions and known methods. In one analysis, cells were transfected with pAAV-GFP, pAAV-Helper, and pAAV-RC for AAV6. (1) LV293 cells were transfected using PEIpro™ (Polyplus Transfection) without an enhancer, and (2) Clone 45 cells were transfected with Transfection Reagent 2 along with the transfection booster and AAV production enhancer described above. After the culture period, AAV was harvested and titers were determined via qPCR and infectivity testing protocols for the GFP method. Exemplary results are shown in Figure 12. The AAV production system provided herein produced significantly higher AAV6 titers (Figure 12A) and infectivities (Figure 12B) than LV293 with the PEIpro system.

[0135] In another analysis, we compared vector production across various AAV serotypes. Here, we used (1) HEK293T adherent cells using PEI-MAX transfection reagent (Polysciences, Inc.) and (2) Clone 45 cells using transfection reagent 2 and transfection booster as described above. Cells were transfected with pAAV-GFP, pAAV-Helper, and pAAV-RC for AAV2, AAV6, AAV8, AAV9, and AAV-dj. After the culture period, AAV was harvested and titers were determined via qPCR and infectivity testing protocols using the GFP method. Exemplary results are shown in Figures 13A and 13B. The AAV production system provided herein produced significantly higher AAV titers across four of the five serotypes tested than HEK293T with the PEI-MAX system (Figure 13A). As shown in Figure 13B, the AAV production system provided herein also resulted in AAV2 and AAV6 with higher infectivity than with the PEI-MAX system. Example 11 Post-harvest processing

[0136] Following transfection, virus production, and cell lysis, crude AAV lysates were filtered using diatomaceous earth before downstream processing. Two samples from the same AAV culture were lysed either by the freeze-thaw method (without lysis buffer) or by adding 10X AAV lysis buffer and incubating for 30 minutes (as described above) to form crude AAV lysates. Immediately after lysis, diatomaceous earth (DE) was mixed with the crude AAV lysate, and the mixture was passed through a 2-micron filter. Various amounts of DE per mL of lysate were tested, including 0.5 g of DE vs. 30 mL of lysate and 1 g of DE vs. 100 mL of lysate. To measure AAV recovery after the filtration step, samples were taken from each lysate before and after DE filtration. AAV titers from the samples were determined using GFP qPCR, and exemplary results are shown in Figure 14. Forming crude lysates with the AAV lysis buffer method resulted in high recovery of AAV titers during DE filtration, whereas the freeze-thaw cell lysis method resulted in significantly lower recovery. For example, lysis buffer resulted in approximately 100% titer recovery compared to approximately 10% recovery with freeze-thaw, as shown in Figure 14. The single DE filtration step reduced the number of filters required and significantly reduced filtration and processing time of the AAV lysate prior to downstream processing, such as nuclease treatment and purification processes.

Claims

1. 1. A method for adeno-associated virus (AAV) vector production, comprising: (a) culturing mammalian cells in suspension culture; (b) transfecting said mammalian cells with an AAV transfer vector using a transfection reagent; (c) contacting the transfected cell with an enhancer; (d) culturing the transfected cells in suspension culture for a time sufficient for expression of the AAV vector, thereby producing a transfected AAV cell culture; (e) harvesting AAV from the transfected AAV cell culture; and A method comprising:

2. 2. The method of claim 1, wherein harvesting the AAV comprises contacting the transfected AAV cell culture with a lysis buffer.

3. 3. The method of claim 2, wherein the lysis buffer comprises at least one surfactant selected from Triton-100, Triton-alter, NP-40, poloxamer 188, and NDSB-201.

4. 4. The method of claim 2 or 3, wherein the lysis buffer comprises at least one of Tris-HCl, sodium citrate, sodium chloride, citric acid, EDTA, tripotassium EDTA, sodium hydroxide, and sodium dihydrogen phosphate.

5. 5. The method of claim 2, wherein the lysis buffer comprises at least one detergent selected from CHAP, CHAPS, CHAPSO, big CHAP, octylthioglucoside, and sodium deoxycholate.

6. 4. The method of claim 0, wherein the enhancer comprises one or more of a histone deacetylase (HDAC) inhibitor, sodium propionate, and caffeine.

7. 7. The method of claim 6, wherein the HDAC inhibitor is selected from apicidin, belinstat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A, and valproic acid.

8. 7. The method of claim 6, wherein the HDAC inhibitor is sodium butyrate, sodium phenylbutyrate, trichostatin A, and / or valproic acid.

9. 9. The method of claim 1, wherein the enhancer is added between about 0 and about 12 hours after transfection.

10. 10. The method of claim 1, wherein the transfection reagent comprises a cationic lipid.

11. The method of claim 10 , wherein the transfection reagent further comprises a peptide.

12. 12. The method of any one of claims 1 to 11, wherein step 0 further comprises contacting the cells with a transfection booster.

13. 13. The method of claim 12, wherein the transfection booster comprises a peptide.

14. 14. The method of claim 12 or 13, wherein the transfection booster is used at a transfection booster:DNA ratio of 5:1 to about 1:5 (volume / weight).

15. 15. The method of any one of claims 1 to 14, wherein the mammalian cell is a HEK293 cell or a derivative of a HEK293 cell.

16. 16. The method of claim 15, wherein the HEK293 cells are adapted for high AAV expression in the AAV vector production system.

17. The HEK293 cells were grown in suspension at a density of at least 5 x 10 cells per milliliter. 6 17. The method of claim 15 or 16, wherein the cells can be grown at a density of 10 ...

18. The HEK293 cells were grown in suspension culture at a density of up to 1.1 x 10 cells per milliliter. 7 17. The method of claim 15 or 16, wherein the cells can be grown at a density of 10 ...

19. The cells were approximately 1.5 x 10 6 and approximately 5 × 10 6 19. The method of any one of claims 1 to 18, wherein the cells are transfected at a cell density of between 100 and 150 cells / mL.

20. 20. The method of any one of claims 1 to 19, wherein no helper virus is used.

21. 21. The method of any one of claims 1 to 20, wherein the cells are not centrifuged before harvesting the AAV.

22. 22. The method of any one of claims 1 to 21, wherein the cells do not contain large T antigen.

23. 23. The method of any one of claims 1 to 22, further comprising titrating the harvested AAV.

24. 24. The method of claim 23, wherein the AAV is titered using quantitative PCR.

25. The harvested AAV is at least about 2 x 10 per milliliter 10 25. The method of claim 23 or 24, wherein the viral genome (vg / mL) is at a titer of 0.01% or less.

26. The collected AAV was approximately 2 x 10 10 vg / mL to about 1 x 10 12 26. The method of claim 25, wherein the antibody has a titer of between 1000 and 10 ... vg / mL.

27. 27. The method of any one of claims 1 to 26, wherein step 0 further comprises transfecting the cells with a packaging plasmid.

28. 28. The method of claim 27, wherein the packaging plasmid comprises pRC and pHelper.

29. 29. The method of any one of claims 1 to 28, wherein the cells are cultured in a volume of about 15 milliliters (mL) to about 200 liters (L).

30. 30. The method of any one of claims 1 to 29, wherein the cells are cultured in a volume of about 1 L to about 10 L.

31. 31. The method of any one of claims 1 to 30, wherein the cells are transfected in a volume of about 15 milliliters (mL) to about 200 liters (L).

32. 32. The method of any one of claims 1 to 31, wherein the cells are transfected in a volume of about 1 L to about 10 L.

33. 33. The method of any one of claims 1 to 32, wherein the cells are cultured in a bioreactor.

34. 34. The method of any one of claims 1 to 33, wherein the cells are cultured in a medium that supports the growth and proliferation of HEK293 cells.

35. 35. The method of any one of claims 1 to 34, wherein during step 0 and / or 1, the cells are contacted with a culture supplement.

36. 1. An adeno-associated virus (AAV) production system comprising: (a) at least about 2×10 6 HEK293 cells at a density of 1000 cells / mL; (b) an AAV transfer vector; and (c) a packaging plasmid; and (d) an enhancer comprising one or more of a histone deacetylase (HDAC) inhibitor, sodium propionate, sodium butyrate, and caffeine; (e) a cell culture medium that supports the growth and proliferation of the HEK293 cells; An AAV production system comprising:

37. The HEK293 cells were approximately 2 x 10 6 cells / mL to approximately 2 x 10 7 37. The AAV production system of claim 36, wherein the density is between 100 and 150 cells / mL.

38. 38. An AAV production system according to claim 36 or 37, further comprising a transfection reagent.

39. 39. The AAV production system of claim 38, wherein the transfection reagent comprises a cationic lipid.

40. 40. The AAV production system of claim 38 or 39, wherein the transfection reagent further comprises a peptide.

41. 41. An AAV production system according to any one of claims 36 to 40, further comprising a transfection booster.

42. 42. The AAV production system of claim 41, wherein the transfection booster comprises a cationic lipid.

43. 43. The AAV production system of claim 41 or 42, wherein the transfection booster comprises a peptide.

44. 44. An AAV production system according to any one of claims 36 to 43, further comprising a lysis buffer.

45. The AAV production system of claim 4443, wherein the lysis buffer comprises at least one surfactant selected from Triton-100, Triton-alter, NP-40, poloxamer 188, and NDSB-201.

46. The AAV production system of claim 44 or 45, wherein the lysis buffer comprises at least one of Tris-HCl, sodium citrate, sodium chloride, citric acid, EDTA, tripotassium EDTA, sodium hydroxide, and sodium dihydrogen phosphate.

47. 47. The AAV production system of any one of claims 44 to 46, wherein the lysis buffer comprises at least one detergent selected from CHAP, CHAPS, CHAPSO, big CHAP, octylthioglucoside, and sodium deoxycholate.

48. The AAV production system according to any one of claims 36 to 47, wherein the HDAC inhibitor is selected from apicidin, belinostat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A, and valproic acid.

49. The AAV production system of claim 48, wherein the HDAC inhibitor is sodium butyrate, sodium phenylbutyrate, trichostatin A, and / or valproic acid.

50. The AAV vectors have a concentration of at least about 2 x 10 per milliliter after harvest. 10 50. The AAV production system of any one of claims 36 to 49, wherein the titer of the AAV production system is 0.01% or more of the viral genome (vg / mL).

51. 1. An adeno-associated virus (AAV) production system comprising: (a) HEK293 cells; (b) at least about 2 x 10 per milliliter after collection 10 an AAV vector having a titer of viral genome (vg / mL); (c) a packaging plasmid; and (d) an enhancer comprising one or more of a histone deacetylase (HDAC) inhibitor, sodium propionate, sodium butyrate, and caffeine; (e) a cell culture medium that supports the growth and proliferation of the HEK293 cells; An AAV production system comprising:

52. The HEK293 cells were approximately 2 x 10 6 cells / mL to approximately 2 x 10 7 52. The AAV production system of claim 51, wherein the density is between 100 and 150 cells / mL.

53. 53. An AAV production system according to claim 51 or 52, further comprising a transfection reagent.

54. 54. The AAV production system of claim 53, wherein the transfection reagent comprises a cationic lipid.

55. 55. The AAV production system of claim 54, wherein the transfection reagent further comprises a peptide.

56. 56. An AAV production system according to any one of claims 51 to 55, further comprising a transfection booster.

57. 57. The AAV production system of claim 56, wherein the transfection booster comprises a cationic lipid.

58. 58. The AAV production system of claim 56 or 57, wherein the transfection booster comprises a peptide.

59. 59. An AAV production system according to any one of claims 51 to 58, further comprising a lysis buffer.

60. The AAV production system according to claim 59, wherein the lysis buffer comprises at least one surfactant selected from Triton-100, Triton-alter, NP-40, poloxamer 188, and NDSB-201.

61. The AAV production system of claim 59 or 60, wherein the lysis buffer comprises at least one of Tris-HCl, sodium citrate, sodium chloride, citric acid, EDTA, tripotassium EDTA, sodium hydroxide, and sodium dihydrogen phosphate.

62. 62. The AAV production system of any one of claims 59 to 61, wherein the lysis buffer comprises at least one detergent selected from CHAP, CHAPS, CHAPSO, big CHAP, octylthioglucoside, and sodium deoxycholate.

63. The AAV production system according to any one of claims 51 to 62, wherein the HDAC inhibitor is selected from apicidin, belinstat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A, and valproic acid.

64. 64. The AAV production system of claim 63, wherein the HDAC inhibitor is sodium butyrate, sodium phenylbutyrate, trichostatin A, and / or valproic acid.

65. A kit for producing adeno-associated virus (AAV), comprising: (a) HEK293 cells; (b) an enhancer comprising one or more of a histone deacetylase (HDAC) inhibitor, sodium propionate, and caffeine; (c) a transfection reagent comprising a cationic lipid; (d) a cell culture medium that supports the growth and proliferation of HEK293 cells; A kit comprising:

66. 66. The kit of claim 65, further comprising a transfection booster.

67. 67. The kit of claim 66, wherein the transfection booster comprises a cationic lipid and / or a peptide.

68. 68. The kit of any one of claims 65 to 67, further comprising a lysis buffer.

69. The AAV production system of claim 68, wherein the lysis buffer comprises at least one surfactant selected from Triton-100, Triton-alter, NP-40, poloxamer 188, and NDSB-201.

70. The AAV production system of claim 68 or 69, wherein the lysis buffer comprises at least one of Tris-HCl, sodium citrate, sodium chloride, citric acid, EDTA, tripotassium EDTA, sodium hydroxide, and sodium dihydrogen phosphate.

71. 71. The AAV production system of any one of claims 68 to 70, wherein the lysis buffer comprises at least one detergent selected from CHAP, CHAPS, CHAPSO, big CHAP, octylthioglucoside, and sodium deoxycholate.

72. The AAV production system according to any one of claims 65 to 71, wherein the HDAC inhibitor is selected from apicidin, belinostat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A, and valproic acid.

73. 73. The AAV production system of claim 72, wherein the HDAC inhibitor is sodium butyrate, sodium phenylbutyrate, trichostatin A, and / or valproic acid.

Images