Suspension system for adeno-associated virus production

Abstract

The present technology relates to a production system for producing AAV vectors in a serum-free suspension platform and at high titers. The reagents used by this technique include a culture medium, cells, a transfection reagent, an AAV enhancer, and a lysis buffer, each of which is designed to provide maximum AAV production from a suspension culture of mammalian cells, such as HEK293 cells. The use of this new system enables delivery to a non-concentrated AAV vector of up to about 2 * 1011 viral genomes per milliliter (vg / mL).

Description

[0001] This application is a divisional application of patent application with application number 202080015703.5.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 809,407, filed on February 22, 2019. The entire contents of the foregoing application are incorporated herein by reference. Background Art

[0004] Adeno-associated virus (AAV) is a small DNA virus that infects human and some non-human primate cells. AAV is known to not cause disease and has low immunogenicity in humans. AAV vectors containing the DNA sequence of interest with little or no viral genes can be produced. These advantages have led to the use of AAV vectors in gene therapy and other clinical and research purposes.

[0005] Methods for large-scale production of AAV vectors and for generating high titers are needed. Summary of the invention

[0006] The present technology generally involves a new AAV system that produces vectors in a serum-free suspension platform and at high titers. The newly developed set of proprietary good manufacturing process (GMP) reagents used in this technology include culture media, cells, transfection reagents, AAV enhancers, and lysis buffers, each of which is designed to provide maximum AAV production from suspension cultures of mammalian cells. Using this new system, it is possible to deliver up to approximately 2×10 11

[00136] The present invention relates to non-concentrated AAV vector (that is, vector that has not been further concentrated after being harvested using the harvesting methods described herein) at a concentration of 10 viral genomes per milliliter (vg / mL).

[0007] In one aspect, a method for AAV vector production is provided herein, 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; (iv) and culturing the transfected cells in suspension culture for a period of 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 transfection step comprises contacting the cells with a transfection facilitating agent.

[0008] In embodiments, the method comprises titrating the collected AAV. In embodiments, the AAV is titrated using quantitative PCR. In embodiments, the titer of the collected AAV is at least about 2×10 10 In an embodiment, the titer of the collected AAV is between about 2×10 10 vg / mL and about 2×10 11 vg / mL.

[0009] 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.

[0010] In embodiments, the cells are cultured in a bioreactor.

[0011] In embodiments, the cells are cultured in a medium that supports the growth and expansion of HEK293 cells. In embodiments, the cells are contacted with an AAV production enhancer during culture (eg, after transfection).

[0012] In one aspect, the present invention provides 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 expansion of the HEK293 cells. In an embodiment, the AAV production system comprises a transfection reagent. In an embodiment, the AAV production system comprises a transfection facilitator. In an embodiment, the AAV production system comprises a lysis buffer. In an embodiment, the HEK293 cells are cultured at a rate of at least about 0.3×10 6 In an embodiment, the HEK293 cells are present at a density of at least about 2×10 6 In an embodiment, the HEK293 cells are present at a density of about 0.3×10 6 cells / mL and about 1×10 7 In an embodiment, the AAV vector is present at a density of at least about 2×10 cells / mL after collection. 10 The titer of the virus is present at vg / mL.

[0013] In an embodiment, the lysis buffer comprises a surfactant. In an embodiment, the surfactant is Triton-100, Triton-alter, NP-40, poloxamer 188 and NDSB-201. In an embodiment, the lysis buffer does not comprise a surfactant. In an embodiment, the lysis buffer comprises at least one of: Tris-HCl, tricine HCL, sodium citrate, sodium chloride, citric acid, EDTA, tripotassium EDTA, sodium hydroxide and sodium dihydrogen phosphate. In an embodiment, the lysis buffer comprises at least one detergent. In an embodiment, the detergent is CHAP, CHAPS, CHAPSO, big CHAP, octylthioglucoside and / or sodium deoxycholate.

[0014] In an embodiment, the AAV production enhancer comprises one or more of the following: a histone deacetylase (HDAC) inhibitor, sodium propionate, egg lecithin, lithium acetate, trichostatin hydroxyurea, nocodazole-DMSO, NaCl and caffeine. In an embodiment, the HDAC inhibitor is apicidin, belinostat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A and / or valproic acid. In an embodiment, the HDAC inhibitor is sodium butyrate, sodium phenylbutyrate, trichostatin A and / or valproic acid. In an embodiment, the AAV enhancer is added between about 0 hours and about 6 hours after transfection.

[0015] In an embodiment, the transfection reagent comprises a cationic lipid. In an embodiment, the transfection reagent further comprises a peptide. In an embodiment, the transfection facilitator comprises a cationic lipid. In an embodiment, the transfection facilitator comprises a peptide. In an embodiment, the peptide is a membrane-penetrating peptide. Non-limiting examples of membrane-penetrating peptides are provided in US9,856,496, which is incorporated herein by reference in its entirety. In an embodiment, the transfection facilitator is used at a ratio of transfection facilitator:DNA between 5:1 and about 1:5 (volume / weight).

[0016] In an embodiment, the mammalian cell is a HEK293 cell or a derivative of a HEK293 cell. In an embodiment, the HEK293 cell has been adapted for high AAV expression in an AAV vector production system. In an embodiment, the HEK293 cell can be expressed at a rate of at least 0.3×10 6In an embodiment, the HEK293 cells can be grown in suspension culture at a density of up to 1.2×10 7 In one embodiment, the cells are grown in suspension culture at a density of about 2.5×10 6 cells / mL and about 4×10 6 The cells were transfected at a cell density between 10 cells / mL.

[0017] In embodiments, no helper virus is used. In embodiments, the method comprises transfecting the cell with a packaging plasmid. In embodiments, the AAV production system comprises a packaging plasmid. In embodiments, the packaging plasmid comprises pRC and pHelper.

[0018] In embodiments, the cells are not centrifuged prior to harvesting the AAV.

[0019] In embodiments, said cells do not comprise large T antigen.

[0020] In one aspect, a kit for the production of adeno-associated virus (AAV) is provided herein. In an embodiment, the kit comprises: HEK293 cells; an enhancer; a transfection reagent comprising a cationic lipid; and a cell culture medium that supports the growth and expansion of the HEK293 cells. In an embodiment, the transfection reagent contains a cationic lipid and a peptide.

[0021] In an embodiment, the kit comprises a transfection facilitating agent. In an embodiment, the transfection facilitating agent comprises a peptide.

[0022] In an embodiment, the kit comprises a lysis buffer. In an embodiment, the lysis buffer contains at least one surfactant. In an embodiment, the surfactant is Triton-100, Triton-alter, NP-40, poloxamer 188 and / or NDSB-201. In an embodiment, the lysis buffer contains Tris-HCl, sodium citrate, TricineHCL, sodium chloride, citric acid, EDTA, EDTA tripotassium, sodium hydroxide and / or sodium dihydrogen phosphate. In an embodiment, the lysis buffer comprises at least one detergent. In an embodiment, the detergent is CHAP, CHAPS, CHAPSO, big CHAP, deoxy big CHAP, Triton X-114, octylthioglucoside and / or sodium deoxycholate. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1The effect of different conditions on the production of AAV2 is shown. AAV2 virus was produced by adherent HEK293 cells (6-well plates) transfected with polyethyleneimine (PEI) under the following three different conditions compared to System 1: Prot-1 (enhancer 1 and supplement 1); Prot-2 (supplement 1, no enhancer); Prot-3 (enhancer 1, no supplement).

[0024] Figure 2 Shown is a comparison of AAV2 production in HEK293F cells adapted to four different types of culture media.

[0025] Figure 3 A comparison of three different systems for AAV production is shown. HEK293 cells adapted to medium 4 were tested with System 1, System 2 and System 3 as detailed in the Examples.

[0026] Figure 4A-4B The growth characteristics of cloned HEK293 cells (clone 45) adapted to Medium 4 are shown. The medium supports high cell viability at high densities (approximately 11×10 6 cells / mL) of clone 45 cells ( Figure 4A ).like Figure 4B As shown in , cell aggregation is very limited at high density.

[0027] Figures 5A-5E The production of different AAV serotypes in clonal HEK293 cells is shown. Control: parental HEK293 cells in Medium 4; Expi45: clone 45 in Expi293 medium; Cl45, Cl12, Cl22 and Cl51: the indicated HEK293 clones in Medium 4.

[0028] Figure 6 A comparison of transfection reagents is shown. Transfection reagent 1 (TR1) was compared to transfection reagent 2 (TR2) for AAV2 production.

[0029] Figure 7 Shown is the effect of the timing of enhancer addition on viral titers in an AAV production system.

[0030] Figure 8 Shown are the effects of various detergents in the AAV lysis buffer on viral titers.

[0031] Fig. 9 Shown are AAV titers of extracts from cell pellets versus whole transfected cell cultures from different AAV serotypes.

[0032] Figures 10A-10BClone 45, subclonal lines C13 and C20, and LV293 cells (VPCs) are shown in Figure 2 for AAV production after transfection. Fig. 10A ) and cell viability ( Fig. 10B ) comparison.

[0033] Figures 11A-11B A comparison of clone 45 and LV293 cells (VPC) in AAV production is shown: Fig.11A The obtained viral titers (vg / mL) of different AAV serotypes were compared; Fig. 11B The infectivity of harvested AAV2 and AAV6 from each clonal cell line (as represented by % GFP in Ht1080) was compared.

[0034] Figures 12A-12B The clone 45 AAV system and the LV293-PEI system are shown in AAV6 production (vg / ml, Fig. 12A ) and AAV6 infectivity (as indicated by GFP% in Ht1080, Fig. 12B ) comparison.

[0035] Figures 13A-13B A comparison of the clone 45 AAV system and the HEK293T-PEI system in AAV production is shown: Fig.13A The obtained viral titers (vg / mL) of different AAV serotypes were compared; Fig. 13B The infectivity (as represented by % GFP in Ht1080) of the harvested AAV2, AAV6 and AAV-dj from each system was compared.

[0036] Fig.14 AAV titers in crude lysates before and after diatomaceous earth filtration are shown. DETAILED DESCRIPTION

[0037] After reading this specification, it will become apparent to those skilled in the art how to implement the present invention in various alternative embodiments and alternative applications. However, all various embodiments of the present invention will not be described herein. It will be understood that the embodiments presented herein are presented by way of example only and not limitation. Thus, this detailed description of various alternative embodiments should not be construed as limiting the scope or breadth of the present invention as set forth below.

[0038] Before disclosing and describing the present invention, it should be understood that the aspects described below are not limited to specific compositions, methods for preparing such compositions, or uses thereof, and thus these aspects can certainly vary. It should also be understood that the terms used herein are only for the purpose of describing specific aspects and are not intended to be limiting.

[0039] The detailed description of the present invention is divided into various parts only for the convenience of the reader, and the disclosure found in any part can be combined with the disclosure in another part. For the convenience of the reader, a title or sub-title may be used in the specification, which is not intended to affect the scope of the present invention.

[0040] definition

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. In this specification and the following claims, reference will be made to various terms, which shall be defined as having the following meanings:

[0042] The terms used herein are only used for the purpose of describing specific embodiments and are not intended to limit the present invention. Unless the context clearly indicates otherwise, as used herein, the singular forms "a", "an" and "the" are intended to include the plural forms.

[0043] "Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0044] When used before numerical designations that include ranges, such as temperature, time, amount, concentration, and such others, the term "about" indicates an approximate value that can vary by (+) or (-) 10%, 5%, 1%, or any subrange or subvalue therebetween. Preferably, when used in relation to a dosage, the term "about" means that the dosage can vary by + / - 10%.

[0045] "Comprising" or "comprises" is intended to mean that compositions and methods contain the recited elements, but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" shall mean excluding other elements that have any significance for the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein will not exclude other materials or steps that do not have a substantial effect on the basic and novel characteristics of the claimed invention. "Consisting of" shall mean excluding other ingredients and substantial method steps that exceed trace elements. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0046] Adeno-associated virus (AAV) production system

[0047] As used herein, the term "cell" refers to all types of eukaryotic and prokaryotic cells. In some embodiments, the term refers to eukaryotic cells, especially mammalian cells. In certain exemplary but non-limiting embodiments, the term "cell" means human embryonic kidney (HEK) or human 293 cells or variants thereof, such as 293 (HEK293) variants that can be grown in suspension. In some embodiments, variants of 293 cells that can be grown, proliferated, and transfected in suspension culture, particularly those that can be cultured at high density (e.g., at least about 2×10 6 cells / mL, at least about 3×10 6 cells / mL or even optionally at least about 4×10 6 cells / mL or about 1.2×10 6 cells / mL).

[0048] In some embodiments, the term "high density" when used in the context of culturing cells and performing transfection workflows generally refers to a known cell line or variant of a known cell line that can be grown or cultured in an appropriate cell culture medium to a density of at least about 2×10 6 cells / mL, at least about 3×10 6 cells / mL or even optionally at least about 4×10 6 cells / mL, while still maintaining the ability to efficiently transfect and be able to transfect at high titers such as 5×10 10 The target AAV vector can be expressed in 100 viral genomes per mL (vg / mL) or more.

[0049] In certain embodiments, cells are suitable for high-density cell culture. This refers to cell lineages or (non-clone) cell groups derived from the same parental cell lineage, which have been suitable for high-density growth in high-density culture medium, while maintaining cell viability to be or higher than about 80%. By maintaining cells at high density ≥ about 40 times, 50 times, 60 times, 70 times or 80 times of continuous passage, and gradually replacing the ratio of growth medium with the required high-density culture medium, it is possible to separate or select such cells from the parental cell group. Optionally, during the process, different cell pools can be bred individually and undergo selection procedures, while synchronously assessing transfection efficiency and or AAV vector production efficiency, so that it is possible to select a cloned cell group that can be maintained and grown, efficiently transfected and express high titer AAV at high density. Known methods and techniques such as flow cytometry sorting and / or single cell cloning can be used to produce cloned cell groups. In an embodiment, flow cytometry sorting is used to separate the cell clones suitable for high-density cell culture and used in the AAV production system. In some embodiments, cell clones suitable for high-density cell culture and use in AAV production systems are obtained by single-cell cloning and confirmed as single-cell clones using known techniques such as imaging. Although it will be readily apparent to a skilled practitioner that a variety of cell types and lineages can undergo this selection procedure, it has been determined that cell lineages derived from 293 human embryonic kidney cells are particularly suitable for the selection process that adapts to high-density growth conditions. In some scenarios, cells suitable for high-density growth culture and suitable for use herein will also be capable of high-efficiency transfection and / or capable of being cultured at a rate of more than about 5×10 9 vg / mL is up to about 5×10 12 vg / mL, between about 1×10 10 vg / mL is up to about 2×10 11 vg / mL, between about 1×10 10 vg / mL is up to about 1×10 11 vg / mL, between about 8×10 10 vg / mL to about 3×10 11 vg / mL, between about 5×10 10 vg / mL to about 2×10 11 vg / mL of non-concentrated AAV vector. In some scenarios, the cells used can be cultured at a density ranging from about 1×10 6 cells / mL to about 2×10 7 cells / mL, about 1×10 6 cells / mL to about 3×10 6 cells / mL, about 2×10 6 cells / mL to about 4×10 6 cells / mL or about 2.5×106 cells / mL to about 4×10 6 In some embodiments, the cells can be cultured at a density of about 1×10 cells / mL and maintained and transfected. 6 cells / mL to about 2×10 7 cells / mL, about 1×10 6 cells / mL to about 4×10 6 cells / mL, about 1×10 6 cells / mL to about 3×10 6 cells / mL, about 1×10 6 cells / mL to about 2×10 6 The cells were transfected at a density of 10 cells / mL.

[0050] In some embodiments, the cells are grown in suspension culture. This includes cell cultures in which most or all of the cells in the culture vessel are in suspension and a few or no cells in the culture vessel are attached to a surface of the vessel or another surface within the vessel. In some embodiments, the suspension culture has ≥ about 75% of the cells in the culture vessel in suspension, not attached to a surface on or in the culture vessel. In some embodiments, the suspension culture has ≥ about 85% of the cells in the culture vessel in suspension, not attached to a surface on or in the culture vessel. In some embodiments, the suspension culture has ≥ about 95% of the cells in the culture vessel in suspension, not attached to a surface on or in the culture vessel.

[0051] The AAV production system allows 293 cells or cells derived therefrom to be expressed at approximately 0.3 × 10 6 cells / mL to about 20×10 6 In an embodiment, the cells can be grown at a density of about 0.3×10 6 cells / mL to about 12×10 6 Cells / mL are grown at a density of about 0.3×10 cells / mL and cell death is less than 20% after 5 days. In some embodiments, the 293 cells provided herein can be grown at a density of about 0.3×10 6 cells / mL to about 20×10 6 cells / mL or about 0.3×10 6 cells / mL to about 12×10 6 The 293 cells can be grown at a high density of about 0.3×10 cells / mL and the cell death is less than 20% after 6 days, 7 days or 8 days. 6 cells / mL to about 20×10 6cells / mL or about 0.3×10 6 cells / mL to about 12×10 6 The cells were grown at a high density of 10 cells / mL and the cell death was less than 10% after 5 days, 6 days, 7 days or 8 days.

[0052] Described in another way, the AAV production system provided herein allows 293 cells or cells derived therefrom to be able to produce AAV at a rate of about 0.3×10 6 cells / mL to about 20×10 6 The cells can be grown at a density of about 0.3×10 cells / mL and the cell viability in the culture is greater than 80% after 5 days. 6 cells / mL to about 12×10 6 Cells / mL can be grown at a density of about 0.3×10 cells / mL and the cell viability in the culture is greater than 80% after 5 days. In some embodiments, the 293 cells provided herein can be grown at a density of about 0.3×10 6 cells / mL to about 20×10 6 cells / mL or about 0.3×10 6 cells / mL to about 12×10 6 The 293 cells can be grown at a high density of about 0.3×10 cells / mL and the cell viability in the culture is greater than 80% after 6 days, 7 days or 8 days. 6 cells / mL to about 20×10 6 cells / mL or about 0.3×10 6 cells / mL to about 12×10 6 The cells were grown at a high density of 1,000 cells / mL and the cell viability in the culture was greater than 90% after 5 days, 6 days, 7 days or 8 days.

[0053] In some embodiments, suspension cultures of 293 cells adapted for high density as provided herein, such as clone 45, subclones of clone 45, and other clones described herein, have higher cell proliferation rates than HEK293F cells, Expi293F cells, and the like after 3 days of high density culture. TMIn some embodiments, the suspension culture of 293 cells suitable for high density as provided herein has at least 10% higher viability than the viability of HEK 293F, Expi293F or LV293 cells after 4 days, 5 days, 6 days, 7 days or 8 days of high density culture. In some embodiments, the suspension culture of 293 cells suitable for high density as provided herein has at least 10% higher viability than the viability of HEK 293F, Expi293F or LV293 cells after 3 days, 4 days, 5 days, 6 days, 7 days or 8 days of high density culture. In some embodiments, the suspension culture of 293 cells suitable for high density as provided herein has at least 20% viability higher than the viability of HEK 293F, Expi293F or LV293 cells after 3 days, 4 days, 5 days, 6 days, 7 days or 8 days of high density culture. In some embodiments, the suspension culture of 293 cells suitable for high density as provided herein has at least 25% viability higher than the viability of HEK 293F, Expi293F or LV293 cells after 3 days, 4 days, 5 days, 6 days, 7 days or 8 days of high density culture. In some embodiments, the suspension culture of 293 cells suitable for high density as provided herein has at least 30% viability higher than the viability of HEK 293F, Expi293F or LV293 cells after 3 days, 4 days, 5 days, 6 days, 7 days or 8 days of high density culture. In some embodiments, the suspension culture of 293 cells suitable for high density as provided herein has at least 40% viability higher than the viability of HEK 293F, Expi293F or LV293 cells after 3 days, 4 days, 5 days, 6 days, 7 days or 8 days of high density culture. In embodiments, the suspension culture of 293 cells suitable for high density as provided herein has about 10% to about 30% viability higher than the viability of HEK 293F, Expi293F or LV293 cells after 3 days, 4 days, 5 days, 6 days, 7 days or 8 days of high density culture. In some embodiments, the suspension culture of 293 cells suitable for high density as provided herein has about 20% to about 40% viability higher than the viability of HEK 293F, Expi293F or LV293 cells after 3 days, 4 days, 5 days, 6 days, 7 days or 8 days of high density culture.In some embodiments, a suspension culture of 293 cells adapted for high density as provided herein has about 25% to about 50% higher viability than HEK 293F, Expi293F or LV293 cells after 3 days, 4 days, 5 days, 6 days, 7 days or 8 days of high density culture.

[0054] In some embodiments, suspension cultures of 293 cells adapted for high density as provided herein, such as clone 45, subclones of clone 45, and other clones described herein, produce more than the same amount of HEK293F cells, Expi293F TM In some embodiments, the AAV titer (vg / mL) collected from suspension cultures of 293 cells adapted for high density as provided herein is 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, at least 20-fold greater than the titer collected from the same amount of HEK 293F, Expi293F, or LV293 cells. In some embodiments, the harvested AAV titer (vg / mL) produced by suspension culture of 293 cells adapted for high density as provided herein is about 2 to about 20 times, about 2 to about 5 times, about 2 to about 10 times, about 5 to about 15 times, about 5 to about 10 times, about 7 to about 20 times, about 10 to about 15 times, greater than the harvested titer of AAV vector yield or titer (vg / mL) from the same amount of HEK 293F, Expi293F or LV293 cells.

[0055] In embodiments, the cells are at very high density, e.g., greater than about 8×10 6 In an embodiment, the cells exhibit limited agglutination at greater than about 9×10 6 In an embodiment, the cells exhibit limited agglutination at a concentration of greater than about 10×10 6 In embodiments, the cells exhibit limited agglutination when grown at very high density in culture medium and under conditions as described herein.

[0056] In embodiments, the diameter of the cells is between about 15 μm and about 20 μm, such as between about 16 μm and about 19 μm or between about 16.5 μm and about 19 μm. In embodiments, the cells are at about 0.3×10 6cells / mL to about 20×10 6 The diameter of the cells is between about 15 μm and about 20 μm when grown at a density of about 1×10 cells / mL. 6 cells / mL to about 10×10 6 The diameter of the cells was between about 16 μm and about 19 μm when grown at a density of 10 cells / mL.

[0057] In embodiments, the cells are suitable for high-density growth and can be efficiently transfected and / or can be transfected at about 5×10 9 vg / mL to about 5×10 12 The 293 cells expressing AAV vectors with a yield of 10 vg / mL did not express or include the large T antigen.

[0058] A variety of cell culture media can be used to culture AAV production system cells. Researchers generally require serum-free culture media. Any culture media that supports the growth of the cells described herein can be used, including serum-free culture media. The culture media can also be protein-free.

[0059] "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 represented by the letters SFM. The phrase "protein-free" medium refers to a medium that does not contain protein (e.g., serum proteins, such as serum albumin or attachment factors, nutritional proteins, such as growth factors, or metal ion carrier proteins, such as transferrin, ceruloplasmin, etc.). In some embodiments, if a peptide is present, the peptide is a smaller peptide, such as a dipeptide or tripeptide. In some embodiments, peptides of decapeptide length or longer do not exceed about 1%, do not exceed about 0.1%, and do not exceed about 0.01% of the amino acids present in the protein-free medium.

[0060] In some embodiments, high density culture medium can be used, including any culture medium capable of maintaining the growth of mammalian cells. In some embodiments, cells are grown at a density of up to about 2×10 7 cells / mL, for example, up to about 12×10 6The high density medium used may vary between different applications and uses and may depend on the nature of the cell line being used, the nature of the transfection modality selected for transfer of the expression vector into the cells, and the amount and nature of any expression enhancers added to the system as described herein. In embodiments, the high density medium used in the systems and methods of the present invention is serum-free and protein-free. In embodiments, the cell culture medium allows the culture and growth of suspension cells up to about 2×10 7 cells / mL, for example, up to about 1.2×10 7 cells / mL or between about 2×10 6 cells / mL to about 1×10 7 In an embodiment, the medium used will enable the virus titer produced in the transient expression system to exceed at least 1×10 10 vg / mL is up to about 1×10 12 vg / mL or up to about 2×10 11 In some embodiments, the high density medium used will help cells grow at a density in the range of about 1×10 6 To about 20×10 6 cells / mL, about 1×10 6 To about 4×10 6 cells / mL or about 2.5×10 6 To about 3×10 6 The cells were transfected at a density of 10 cells / mL.

[0061] Examples of high-density culture media suitable for use herein include, but are not limited to, HuMEC basal serum-free medium, KNOCKOUT TM CTS TM XenoFREE ESC / iPSC Medium, STEMPRO TM -34SFM culture medium, STEMPRO TM NSC culture medium, ESSENTIAL TM -8 medium, medium 254, medium 106, medium 131, medium 154, medium 171, medium 171, medium 200, medium 231, HeptoZYME-SFM, human endothelial-SFM, FREESTYLE TM293 Expression Medium, Medium 154CF / PRF, Medium 154C, Medium 154CF, Medium 106, Medium 200PRF, Medium 131, Essential TM -6 culture medium, STEMPRO TM -34 culture medium, Astrocyte culture medium, AIM CTS TM 、AMINOMAX TM C-100 basal medium, AMINOMAX TM -II complete medium, CD FORTICHO TM Culture medium, CD CHO AGT culture medium, CHO-S-SFM culture medium, FREESTYLE TM CHO expression medium, CD OPTICHO TM Culture medium, CD CHO culture medium, CD DG44 culture medium, SF-900 TM Culture medium, EXPI293 TM Expression medium, LHC basal medium, LHC-8 medium, 293SFM medium, CD 293 medium, AEM growth medium, Cell culture medium, AIM Culture medium, medium, Keratinocyte-SFM medium, LHC medium, LHC-8 medium, LHC-9 medium and any derivatives or modifications thereof. In certain non-limiting embodiments, the high density medium may be CDFORTICHO TM Culture medium, CD CHO AGT culture medium, CHO-S-SFM culture medium, FREESTYLE TM CHO expression medium, CD OPTICHO TM Culture medium, CD CHO culture medium, CD DG44 culture medium, FREESTYLE TM 293 Expression Medium, EXPI293 TM Expression medium, LV-MAX TM Production medium, FREESTYLE TM F17 expression medium, DYNAMIS TM Culture medium, HEK293 medium or similar medium or a modified version thereof. The medium can be any medium suitable for (e.g., formulated for) high-density growth, propagation, transfection and maintenance of 293 cells, 293 cell variants or any other cells suitable for use in a high-density culture system.

[0062] The AAV production system also includes a transfection agent or a composition that promotes the entry of macromolecules into cells. In an embodiment, the transfection agent includes a cationic lipid. In an embodiment, the transfection agent is a cationic lipid as described in U.S. Patent No. 9,856,496, which is incorporated herein by reference in its entirety.

[0063] In some embodiments, the reagent for introducing the macromolecule into the cell may include one or more lipids that may be cationic lipids and / or neutral lipids. Preferred lipids include, but are not limited to, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), dioleoylphosphatidylcholine (DOPE), 1,2-bis(oleoyloxy)-3-(4'-trimethylammonio)propane (DOTAP), dihydroxy-dimyristyl spermine tetrahydrochloride (DHDMS), hydroxy-dimyristyl spermine tetrahydrochloride (HDMS), 1,2-dioleoyl-3-(4'-trimethylammonio)butyryl-sn-glycerol (DOTB), 1,2-dioleoyl-3-succinyl-sn-glycerocholine ester (DOSC), cholesterol-based ( 4'-trimethylammonium) butyrate (ChoTB), cetyltrimethylammonium bromide (CTAB), 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORI), 1,2-dioleoyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DOME), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), O,O'-dodecyl-N-[p(2-trimethylammonioethoxy)benzoyl]-N,N,N-trimethylammonium chloride, spermine conjugated to one or more lipids (e.g., 5-carboxysperminylglycine dioctadecylamide (DOGS), N,N I ,N II ,N III -Tetramethyl-N,N I ,N II ,N III-tetrapalmitylspermine (TM-TPS) and dipalmitoylphosphatidylethanolamine 5-carboxysperminamide (DPPES)), lipopolylysine (polylysine conjugated with DOPE), TRIS (tris(hydroxymethyl)aminomethane, tromethamine) conjugated with fatty acids (TFA) and / or peptides (such as trilysyl-alanyl-TRIS mono-, di-, tripalmitate), (3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DCChol), N-(α-trimethylammonioacetyl)-docosyl-D-glutamic acid Chloride (TMAG), dimethyl dioctadecyl ammonium bromide (DDAB), 2,3-dioleyloxy-N-[2 (spermine-carboxamido) ethyl]-N,N-dimethyl-1-propylamine-triacetic acid indium (DOSPA) and combinations thereof. Optionally, the transfection reagent may further include at least one additional helper lipid. Helper lipids are known in the art and include, but are not limited to, neutral lipids preferably selected from the group consisting of DOPE, DOPC and cholesterol. In an embodiment, the transfection reagent includes at least one cationic lipid and at least one neutral lipid.

[0064] Those skilled in the art will appreciate that certain combinations of the above-mentioned lipids have been shown to be particularly suitable for introducing nucleic acids into cells, for example, DOSPA and DOPE in a 3:1 (w / w) combination are available from Life Technologies Corporation, Carlsbad, Calif., under the trade name LIPOFECTAMINE TM DOTMA and DOPE in a 1:1 (w / w) combination are available from Thermo Fisher Scientific under the trade name 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 TM (available from Promega Corporation); LYOVEC TM (available from InvivoGen); DOTAP liposomal transfection reagent (available from Roche); Transfection reagent (available from Mirus); and insect Transfection reagent (EMD Millipore). Additional transfection reagents that can be used herein include but are not limited to 2000, 3000, which is available from Thermo Fisher Scientific; VIAFECT TM Transfection reagents, 6. Transfection reagent and HD transfection reagents, each of which is available from Promega; and TRANSFECTIN TM Lipid reagents, which are available from BioRad Laboratories, Inc.

[0065] In an embodiment, the transfection reagent is combined with a transfection facilitator. In an embodiment, the transfection facilitator comprises a peptide. In an embodiment, the transfection facilitator includes at least one peptide. In an embodiment, the at least one peptide of the transfection facilitator is a naturally occurring or non-naturally occurring membrane-penetrating peptide. In an embodiment, the at least one peptide of the transfection facilitator includes a naturally occurring or non-naturally occurring membrane-penetrating peptide sequence. Non-limiting examples of suitable membrane-penetrating peptides and peptide sequences are provided in U.S. Patent No. 9,856,496, which is incorporated herein by reference in its entirety. In an embodiment, the at least one peptide of the transfection facilitator is a fusion peptide, a cell-penetrating peptide, a nuclear localization peptide, a cell surface adhesion peptide, or a plant virus mobile peptide. In the embodiment sequence, the at least one peptide of the transfection facilitator includes a fusion peptide sequence, a cell-penetrating peptide sequence, a nuclear localization peptide sequence, a cell surface adhesion peptide sequence, or a plant virus mobile peptide sequence. Non-limiting examples of suitable fusion, cell penetration, nuclear localization, cell surface adhesion, and plant virus movement peptides and peptide sequences are provided in U.S. Patent Application Publication No. 2017 / 0253888A1, which is incorporated herein by reference in its entirety.

[0066] In an embodiment, the transfection reagent is combined with a transfection facilitator and an AAV transfer vector to form a transfection complex. In an embodiment, the transfection reagent is combined with a transfection facilitator, a rep / cap plasmid (pRC), a pHelper plasmid (encoding a helper virus component) and an AAV transfer vector to form a transfection complex.

[0067] In an embodiment, the AAV production system comprises a lysis buffer. A lysis buffer is a buffered solution for breaking up cells and releasing their contents, such as AAV vectors. In an embodiment, the lysis buffer is provided at a 5-fold concentration (five times the final concentration in contact with the cells). In an embodiment, the lysis buffer is provided at a 10-fold concentration (ten times the final concentration in contact with the cells).

[0068] In an embodiment, the lysis buffer contains a detergent. In an embodiment, the lysis buffer contains at least one detergent selected from the following: CHAP, 3-[(3-cholamidopropyl)dimethylammonium]-1-propane sulfonate (CHAPS), 3-([3-cholamidopropyl]dimethylammonium)-2-hydroxy-1-propane sulfonate (CHAPSO), N,N-bis-(3-D-glucosamidopropyl)deoxycholamide (big CHAP), octylthioglucoside (OTG) and sodium deoxycholate. In an embodiment, the lysis buffer contains a detergent selected from CHAP, CHAPS, CHAPSO, big CHAP, OTG and sodium deoxycholate. In an embodiment, the lysis buffer contains two detergents selected from CHAP, CHAPS, CHAPSO, big CHAP, OTG and sodium deoxycholate. In an embodiment, the lysis buffer contains three detergents selected from CHAP, CHAPS, CHAPSO, big CHAP, OTG and sodium deoxycholate. In an embodiment, the lysis buffer contains four or more detergents selected from CHAP, CHAPS, CHAPSO, big CHAP, OTG and sodium deoxycholate.

[0069] In embodiments, the lysis buffer comprises CHAP at a concentration (final concentration in contact with cells) between about 0.005% and about 1% (w / v). In embodiments, CHAP is present in the lysis buffer at a concentration (final concentration in contact with cells) between about 0.01% and about 1% (w / v). In embodiments, CHAP is present in the lysis buffer at a concentration (final concentration in contact with cells) between about 0.01% and about 0.8% (w / v). The concentration can be any value or subrange within the recited range, including the endpoints.

[0070] In embodiments, the lysis buffer comprises CHAPS at a concentration (final concentration in contact with cells) between about 0.005% and about 1% (w / v). In embodiments, CHAPS is present in the lysis buffer at a concentration (final concentration in contact with cells) between about 0.01% and about 1% (w / v). In embodiments, CHAPS is present in the lysis buffer at a concentration (final concentration in contact with cells) between about 0.01% and about 0.8% (w / v). The concentration can be any value or subrange within the recited range, including the endpoints.

[0071] In embodiments, the lysis buffer comprises CHAPSO at a concentration (final concentration in contact with cells) between about 0.005% and about 1% (w / v). In embodiments, CHAPSO is present in the lysis buffer at a concentration (final concentration in contact with cells) between about 0.01% and about 1% (w / v). In embodiments, CHAPSO is present in the lysis buffer at a concentration (final concentration in contact with cells) between about 0.01% and about 0.8% (w / v). The concentration can be any value or subrange within the recited range, including the endpoints.

[0072] In embodiments, the lysis buffer comprises large CHAP at a concentration (final concentration in contact with cells) between about 0.005% and about 1% (w / v). In embodiments, large CHAP is present in the lysis buffer at a concentration (final concentration in contact with cells) between about 0.01% and about 1% (w / v). In embodiments, large CHAP is present in the lysis buffer at a concentration (final concentration in contact with cells) between about 0.01% and about 0.8% (w / v). The concentration can be any value or subrange within the recited range, including the endpoints.

[0073] In embodiments, the lysis buffer comprises OTG at a concentration (final concentration in contact with cells) between about 0.005% and about 1% (w / v). In embodiments, OTG is present in the lysis buffer at a concentration (final concentration in contact with cells) between about 0.01% and about 1% (w / v). In embodiments, OTG is present in the lysis buffer at a concentration (final concentration in contact with cells) between about 0.01% and about 0.8% (w / v). The concentration can be any value or sub-range within the recited range, including the endpoints.

[0074] In embodiments, the lysis buffer comprises sodium deoxycholate at a concentration (final concentration in contact with cells) between about 0.005% and about 1% (w / v). In embodiments, sodium deoxycholate is present in the lysis buffer at a concentration (final concentration in contact with cells) between about 0.01% and about 1% (w / v). In embodiments, sodium deoxycholate is present in the lysis buffer at a concentration (final concentration in contact with cells) between about 0.01% and about 0.8% (w / v). The concentration can be any value or subrange within the recited range, including the endpoints.

[0075] In an embodiment, the lysis buffer contains at least one surfactant selected from the group consisting of Triton-100, Triton-alter, NP40 and Poloxamer 188 (copolymer of polyoxyethylene and polyoxypropylene; F-68). In an embodiment, the lysis buffer contains a surfactant selected from Triton-100, Triton-alter, NP40 and poloxamer 188. In an embodiment, the lysis buffer contains two surfactants selected from Triton-100, Triton-alter, NP40 and poloxamer 188. In an embodiment, the lysis buffer contains three surfactants selected from Triton-100, Triton-alter, NP40 and poloxamer 188. In an embodiment, the lysis buffer contains four surfactants selected from Triton-100, Triton-alter, NP40 and poloxamer 188. Poloxamer 188 has the following formula (I):

[0076]

[0077] In embodiments, the lysis buffer contains Triton-100 or Triton-alter at a concentration (final concentration in contact with cells) between about 0.01% and about 0.1% (w / v). In embodiments, the lysis buffer contains Triton-100 or Triton-alter at a concentration (final concentration in contact with cells) between about 0.05% and about 0.1% (w / v). In embodiments, the lysis buffer contains Triton-100 or Triton-alter at a concentration (final concentration in contact with cells) between about 0.01% and about 0.05% (w / v). The concentration can be any value or sub-range within the recited range, including the endpoints.

[0078] In embodiments, the lysis buffer contains 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 contains 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 contains 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 contains 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 contains 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 contains 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 contains NP40 at a concentration (final concentration in contact with cells) between about 0.05% and about 0.3% (w / v). In embodiments, the lysis buffer contains NP40 at a concentration (final concentration in contact with cells) between about 0.05% and about 0.2% (w / v). In embodiments, the lysis buffer contains NP40 at a concentration (final concentration in contact with cells) between about 0.05% and about 0.1% (w / v). The concentration can be any value or sub-range within the recited range, including the endpoints.

[0079] In embodiments, the lysis buffer contains a concentration (final concentration in contact with cells) of poloxamer 188 between about 0.08% and about 0.2% (w / v). In embodiments, the lysis buffer contains a concentration (final concentration in contact with cells) of poloxamer 188 between about 0.09% and about 0.2% (w / v). In embodiments, the lysis buffer contains a concentration (final concentration in contact with cells) of poloxamer 188 between about 0.1% and about 0.2% (w / v). In embodiments, the lysis buffer contains a concentration (final concentration in contact with cells) of poloxamer 188 between about 0.08% and about 0.15% (w / v). In embodiments, the lysis buffer contains a concentration (final concentration in contact with cells) of poloxamer 188 between about 0.08% and about 0.1% (w / v). The concentration can be any value or sub-range within the recited range, including the endpoints.

[0080] In an embodiment, the lysis buffer contains at least one salt. In an embodiment, the salt is sodium citrate, sodium chloride, potassium chloride, ammonium sulfate, ammonium phosphate and / or sodium phosphate (e.g., sodium dihydrogen phosphate, disodium phosphate, trisodium phosphate).

[0081] In an embodiment, the lysis buffer contains a sodium citrate at a concentration (final concentration in contact with cells) between about 1mM and about 1000mM. In an embodiment, the lysis buffer contains a sodium citrate at a concentration (final concentration in contact with cells) between about 1mM and about 500mM. In an embodiment, the lysis buffer contains a sodium citrate at a concentration (final concentration in contact with cells) between about 1mM and about 400mM. In an embodiment, the lysis buffer contains a sodium citrate at a concentration (final concentration in contact with cells) between about 1mM and about 300mM. In an embodiment, the lysis buffer contains a sodium citrate at a concentration (final concentration in contact with cells) between about 1mM and about 200mM. In an embodiment, the lysis buffer contains a sodium citrate at a concentration (final concentration in contact with cells) between about 1mM and about 100mM. In an embodiment, the lysis buffer contains a sodium citrate at a concentration (final concentration in contact with cells) between about 10mM and about 1000mM. In an embodiment, the lysis buffer contains a sodium citrate concentration (final concentration in contact with cells) between about 10mM and about 500mM. In an embodiment, the lysis buffer contains a sodium citrate concentration (final concentration in contact with cells) between about 10mM and about 400mM. In an embodiment, the lysis buffer contains a sodium citrate concentration (final concentration in contact with cells) between about 10mM and about 300mM. In an embodiment, the lysis buffer contains a sodium citrate concentration (final concentration in contact with cells) between about 10mM and about 200mM. In an embodiment, the lysis buffer contains a sodium citrate concentration (final concentration in contact with cells) between about 10mM and about 100mM. Concentration can be any value or sub-range within the described range, including endpoints.

[0082] In an embodiment, the lysis buffer contains a sodium chloride concentration (final concentration in contact with cells) between about 1mM and about 1000mM. In an embodiment, the lysis buffer contains a sodium chloride concentration (final concentration in contact with cells) between about 1mM and about 500mM. In an embodiment, the lysis buffer contains a sodium chloride concentration (final concentration in contact with cells) between about 1mM and about 400mM. In an embodiment, the lysis buffer contains a sodium chloride concentration (final concentration in contact with cells) between about 1mM and about 300mM. In an embodiment, the lysis buffer contains a sodium chloride concentration (final concentration in contact with cells) between about 1mM and about 200mM. In an embodiment, the lysis buffer contains a sodium chloride concentration (final concentration in contact with cells) between about 1mM and about 100mM. In an embodiment, the lysis buffer contains a sodium chloride concentration (final concentration in contact with cells) between about 10mM and about 1000mM. In an embodiment, the lysis buffer contains a sodium chloride concentration (final concentration in contact with the cell) between about 10mM and about 500mM. In an embodiment, the lysis buffer contains a sodium chloride concentration (final concentration in contact with the cell) between about 10mM and about 400mM. In an embodiment, the lysis buffer contains a sodium chloride concentration (final concentration in contact with the cell) between about 10mM and about 300mM. In an embodiment, the lysis buffer contains a sodium chloride concentration (final concentration in contact with the cell) between about 10mM and about 200mM. In an embodiment, the lysis buffer contains a sodium chloride concentration (final concentration in contact with the cell) between about 10mM and about 100mM. Concentration can be any value or sub-range within the described range, including endpoints.

[0083] In embodiments, the lysis buffer contains ammonium phosphate at a concentration (final concentration in contact with cells) between about 0.5mM and about 500mM. In embodiments, the lysis buffer contains ammonium phosphate at a concentration (final concentration in contact with cells) between about 0.5mM and about 250mM. In embodiments, the lysis buffer contains ammonium phosphate at a concentration (final concentration in contact with cells) between about 0.5mM and about 100mM. In embodiments, the lysis buffer contains ammonium phosphate at a concentration (final concentration in contact with cells) between about 0.5mM and about 50mM. In embodiments, the lysis buffer contains ammonium phosphate at a concentration (final concentration in contact with cells) between about 0.5mM and about 10mM. In embodiments, the lysis buffer contains ammonium phosphate at a concentration (final concentration in contact with cells) between about 1mM and about 500mM. In embodiments, the lysis buffer contains ammonium phosphate at a concentration (final concentration in contact with cells) between about 1mM and about 250mM. In embodiments, the lysis buffer contains ammonium phosphate at a concentration (final concentration in contact with cells) between about 1 mM and about 100 mM. In embodiments, the lysis buffer contains ammonium phosphate at a concentration (final concentration in contact with cells) between about 1 mM and about 50 mM. In embodiments, the lysis buffer contains ammonium phosphate at a concentration (final concentration in contact with cells) between about 1 mM and about 10 mM. The concentration can be any value or subrange within the recited range, including the endpoints.

[0084] In an embodiment, the lysis buffer contains a sodium phosphate (e.g., sodium dihydrogen phosphate, disodium phosphate, trisodium phosphate) at a concentration (final concentration in contact with cells) between about 0.5mM and about 500mM. In an embodiment, the lysis buffer contains a sodium phosphate (e.g., sodium dihydrogen phosphate, disodium phosphate, trisodium phosphate) at a concentration (final concentration in contact with cells) between about 0.5mM and about 250mM. In an embodiment, the lysis buffer contains a sodium phosphate (e.g., sodium dihydrogen phosphate, disodium phosphate, trisodium phosphate) at a concentration (final concentration in contact with cells) between about 0.5mM and about 100mM. In an embodiment, the lysis buffer contains a sodium phosphate (e.g., sodium dihydrogen phosphate, disodium phosphate, trisodium phosphate) at a concentration (final concentration in contact with cells) between about 0.5mM and about 50mM. In an embodiment, the lysis buffer contains a sodium phosphate (e.g., sodium dihydrogen phosphate, disodium phosphate, trisodium phosphate) with a concentration (final concentration in contact with cells) between about 0.5mM and about 10mM. In an embodiment, the lysis buffer contains a sodium phosphate (e.g., sodium dihydrogen phosphate, disodium phosphate, trisodium phosphate) with a concentration (final concentration in contact with cells) between about 1mM and about 500mM. In an embodiment, the lysis buffer contains a sodium phosphate (e.g., sodium dihydrogen phosphate, disodium phosphate, trisodium phosphate) with a concentration (final concentration in contact with cells) between about 1mM and about 250mM. In an embodiment, the lysis buffer contains a sodium phosphate (e.g., sodium dihydrogen phosphate, disodium phosphate, trisodium phosphate) with a concentration (final concentration in contact with cells) between about 1mM and about 100mM. In an embodiment, the lysis buffer contains a sodium phosphate (e.g., sodium dihydrogen phosphate, disodium phosphate, trisodium phosphate) with a concentration (final concentration in contact with cells) between about 1mM and about 50mM. In an embodiment, the lysis buffer contains sodium phosphate (e.g., sodium dihydrogen phosphate, disodium phosphate, trisodium phosphate) at a concentration (final concentration in contact with cells) between about 1 mM and about 10 mM. The concentration can be any value or sub-range within the recited range, including the endpoints.

[0085] In an embodiment, the lysis buffer contains a chelating agent. In an embodiment, the chelating agent is ethylenediaminetetraacetic acid (EDTA), tripotassium EDTA and / or ethylene glycol tetraacetic acid (EGTA).

[0086] In an embodiment, the lysis buffer contains EDTA at a concentration (final concentration in contact with cells) between about 0.1mM and about 50mM. In an embodiment, the lysis buffer contains EDTA at a concentration (final concentration in contact with cells) between about 0.1mM and about 40mM. In an embodiment, the lysis buffer contains EDTA at a concentration (final concentration in contact with cells) between about 0.1mM and about 30mM. In an embodiment, the lysis buffer contains EDTA at a concentration (final concentration in contact with cells) between about 0.1mM and about 20mM. In an embodiment, the lysis buffer contains EDTA at a concentration (final concentration in contact with cells) between about 0.1mM and about 10mM. In an embodiment, the lysis buffer contains EDTA at a concentration (final concentration in contact with cells) between about 0.1mM and about 5mM. In an embodiment, the lysis buffer contains EDTA at a concentration (final concentration in contact with cells) between about 1mM and about 50mM. In an embodiment, the lysis buffer contains EDTA at a concentration (final concentration in contact with cells) between about 1mM and about 40mM. In an embodiment, the lysis buffer contains EDTA at a concentration (final concentration in contact with cells) between about 1mM and about 30mM. In an embodiment, the lysis buffer contains EDTA at a concentration (final concentration in contact with cells) between about 1mM and about 20mM. In an embodiment, the lysis buffer contains EDTA at a concentration (final concentration in contact with cells) between about 1mM and about 10mM. In an embodiment, the lysis buffer contains EDTA at a concentration (final concentration in contact with cells) between about 1mM and about 5mM. Concentration can be any value or subrange within the recited range, including endpoints.

[0087] In an embodiment, the lysis buffer contains a concentration (final concentration in contact with cells) of EDTA tripotassium between about 0.1mM and about 50mM. In an embodiment, the lysis buffer contains a concentration (final concentration in contact with cells) of EDTA tripotassium between about 0.1mM and about 40mM. In an embodiment, the lysis buffer contains a concentration (final concentration in contact with cells) of EDTA tripotassium between about 0.1mM and about 30mM. In an embodiment, the lysis buffer contains a concentration (final concentration in contact with cells) of EDTA tripotassium between about 0.1mM and about 20mM. In an embodiment, the lysis buffer contains a concentration (final concentration in contact with cells) of EDTA tripotassium between about 0.1mM and about 10mM. In an embodiment, the lysis buffer contains a concentration (final concentration in contact with cells) of EDTA tripotassium between about 0.1mM and about 5mM. In an embodiment, the lysis buffer contains a concentration (final concentration in contact with cells) of EDTA tripotassium between about 1mM and about 50mM. In an embodiment, the lysis buffer contains a concentration (final concentration in contact with cells) of EDTA tripotassium between about 1mM and about 40mM. In an embodiment, the lysis buffer contains a concentration (final concentration in contact with cells) of EDTA tripotassium between about 1mM and about 30mM. In an embodiment, the lysis buffer contains a concentration (final concentration in contact with cells) of EDTA tripotassium between about 1mM and about 20mM. In an embodiment, the lysis buffer contains a concentration (final concentration in contact with cells) of EDTA tripotassium between about 1mM and about 10mM. In an embodiment, the lysis buffer contains a concentration (final concentration in contact with cells) of EDTA tripotassium between about 1mM and about 5mM. Concentration can be any value or sub-range within the described range, including endpoints.

[0088] In an embodiment, the lysis buffer contains EGTA at a concentration (final concentration in contact with cells) between about 0.1mM and about 50mM. In an embodiment, the lysis buffer contains EGTA at a concentration (final concentration in contact with cells) between about 0.1mM and about 40mM. In an embodiment, the lysis buffer contains EGTA at a concentration (final concentration in contact with cells) between about 0.1mM and about 30mM. In an embodiment, the lysis buffer contains EGTA at a concentration (final concentration in contact with cells) between about 0.1mM and about 20mM. In an embodiment, the lysis buffer contains EGTA at a concentration (final concentration in contact with cells) between about 0.1mM and about 10mM. In an embodiment, the lysis buffer contains EGTA at a concentration (final concentration in contact with cells) between about 0.1mM and about 5mM. In an embodiment, the lysis buffer contains EGTA at a concentration (final concentration in contact with cells) between about 1mM and about 50mM. In an embodiment, the lysis buffer contains EGTA at a concentration (final concentration in contact with the cells) between about 1mM and about 40mM. In an embodiment, the lysis buffer contains EGTA at a concentration (final concentration in contact with the cells) between about 1mM and about 30mM. In an embodiment, the lysis buffer contains EGTA at a concentration (final concentration in contact with the cells) between about 1mM and about 20mM. In an embodiment, the lysis buffer contains EGTA at a concentration (final concentration in contact with the cells) between about 1mM and about 10mM. In an embodiment, the lysis buffer contains EGTA at a concentration (final concentration in contact with the cells) between about 1mM and about 5mM. Concentration can be any value or subrange within the recited range, including endpoints.

[0089] In an embodiment, the lysis buffer contains at least one additional compound. For example, the lysis buffer can contain 3-(1-pyridyl)propane sulfonate (NDSB 201; non-detergent sulfobetaine 201). In an embodiment, the lysis buffer contains Tris-HCl. In an embodiment, the lysis buffer contains citric acid. In an embodiment, the lysis buffer contains sodium hydroxide (NaOH).

[0090] In embodiments, the lysis buffer contains NDSB-201 at a concentration (final concentration in contact with cells) between about 0.5M and about 1M. In embodiments, the lysis buffer contains NDSB-201 at a concentration (final concentration in contact with cells) between about 0.6M and about 1M. In embodiments, the lysis buffer contains NDSB-201 at a concentration (final concentration in contact with cells) between about 0.7M and about 1M. In embodiments, the lysis buffer contains NDSB-201 at a concentration (final concentration in contact with cells) between about 0.8M and about 1M. In embodiments, the lysis buffer contains NDSB-201 at a concentration (final concentration in contact with cells) between about 0.9M and about 1M. In embodiments, the lysis buffer contains NDSB-201 at a concentration (final concentration in contact with cells) between about 0.5M and about 0.9M. In embodiments, the lysis buffer contains NDSB-201 at a concentration (final concentration in contact with cells) between about 0.5 M and about 0.8 M. In embodiments, the lysis buffer contains NDSB-201 at a concentration (final concentration in contact with cells) between about 0.5 M and about 0.7 M. In embodiments, the lysis buffer contains NDSB-201 at a concentration (final concentration in contact with cells) between about 0.5 M and about 0.6 M. The concentration can be any value or sub-range within the recited range, including the endpoints.

[0091] In embodiments, the lysis buffer contains Tris-HCl at a concentration (final concentration in contact with cells) between about 5mM and about 20mM. In embodiments, the lysis buffer contains Tris-HCl at a concentration (final concentration in contact with cells) between about 6mM and about 20mM. In embodiments, the lysis buffer contains Tris-HCl at a concentration (final concentration in contact with cells) between about 8mM and about 20mM. In embodiments, the lysis buffer contains Tris-HCl at a concentration (final concentration in contact with cells) between about 10mM and about 20mM. In embodiments, the lysis buffer contains Tris-HCl at a concentration (final concentration in contact with cells) between about 12mM and about 20mM. In embodiments, the lysis buffer contains Tris-HCl at a concentration (final concentration in contact with cells) between about 14mM and about 20mM. In embodiments, the lysis buffer contains Tris-HCl at a concentration (final concentration in contact with cells) between about 15mM and about 20mM. In embodiments, the lysis buffer contains Tris-HCl at a concentration (final concentration in contact with cells) between about 10 mM and about 20 mM. In embodiments, the lysis buffer contains Tris-HCl at a concentration (final concentration in contact with cells) between about 12 mM and about 20 mM. In embodiments, the lysis buffer contains Tris-HCl at a concentration (final concentration in contact with cells) between about 15 mM and about 20 mM. The concentration can be any value or sub-range within the recited range, including the endpoints.

[0092] In embodiments, the lysis buffer contains a concentration (final concentration in contact with cells) of citric acid between about 20mM and about 100mM. In embodiments, the lysis buffer contains a concentration (final concentration in contact with cells) of citric acid between about 40mM and about 100mM. In embodiments, the lysis buffer contains a concentration (final concentration in contact with cells) of citric acid between about 50mM and about 100mM. In embodiments, the lysis buffer contains a concentration (final concentration in contact with cells) of citric acid between about 60mM and about 100mM. In embodiments, the lysis buffer contains a concentration (final concentration in contact with cells) of citric acid between about 80mM and about 100mM. In embodiments, the lysis buffer contains a concentration (final concentration in contact with cells) of citric acid between about 20mM and about 80mM. In embodiments, the lysis buffer contains a concentration (final concentration in contact with cells) of citric acid between about 20mM and about 60mM. In an embodiment, the lysis buffer contains citric acid at a concentration (final concentration in contact with cells) between about 20 mM and about 40 mM. The concentration can be any value or sub-range within the recited range, including the endpoints.

[0093] In embodiments, the lysis buffer contains NaOH at a concentration (final concentration in contact with cells) between about 1mM and about 50mM. In embodiments, the lysis buffer contains NaOH at a concentration (final concentration in contact with cells) between about 10mM and about 50mM. In embodiments, the lysis buffer contains NaOH at a concentration (final concentration in contact with cells) between about 20mM and about 50mM. In embodiments, the lysis buffer contains NaOH at a concentration (final concentration in contact with cells) between about 30mM and about 50mM. In embodiments, the lysis buffer contains NaOH at a concentration (final concentration in contact with cells) between about 40mM and about 50mM. In embodiments, the lysis buffer contains NaOH at a concentration (final concentration in contact with cells) between about 1mM and about 40mM. In embodiments, the lysis buffer contains NaOH at a concentration (final concentration in contact with cells) between about 1mM and about 30mM. In embodiments, the lysis buffer contains NaOH at a concentration (final concentration in contact with cells) between about 1 mM and about 20 mM. In embodiments, the lysis buffer contains NaOH at a concentration (final concentration in contact with cells) between about 1 mM and about 10 mM. The concentration can be any value or sub-range within the recited range, including the endpoints.

[0094] The AAV production system also includes an AAV production enhancer (AAV enhancer). The AAV enhancer includes one or more of the following: a histone deacetylase (HDAC) inhibitor, sodium propionate, sodium butyrate, theobromine, and caffeine.

[0095] In some embodiments, the HDAC inhibitor is selected from apixidin, belinostat, CI-994, CRA-024781, curcumin, panobinostat, sodium butyrate, sodium phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A and valproic acid. In an embodiment, the HDAC inhibitor is sodium butyrate, sodium phenylbutyrate, trichostatin A and / or valproic acid.

[0096] In an embodiment, sodium propionate and / or HDAC inhibitor is provided in the form of water. In an embodiment, caffeine is added to the cell culture expression medium such as Expi293 TM Supplied in expression medium.

[0097] In embodiments, sodium propionate is included at a concentration of about 1mM to 50mM (final concentration in contact with cells). In embodiments, sodium propionate is included at about 1mM to 40mM. In embodiments, sodium propionate is included at about 1mM to 30mM. In embodiments, sodium propionate is included at about 1mM to 20mM. In embodiments, sodium propionate is included at about 1mM to 10mM. In embodiments, sodium propionate is included at about 1mM to 5mM. In embodiments, sodium propionate is included at about 2mM to 30mM. In embodiments, sodium propionate is included at about 2mM to 20mM. In embodiments, sodium propionate is included at about 2mM to 10mM. In embodiments, sodium propionate is included at about 2mM to 5mM. The concentration can be any value or sub-range within the recited range, including the endpoints.

[0098] In an embodiment, the HDAC inhibitor is included in a concentration of about 0.1mM to about 100mM (final concentration in contact with cells). In an embodiment, the HDAC inhibitor is included in a concentration of about 0.1mM to about 75mM. In an embodiment, the HDAC inhibitor is included in a concentration of about 0.1mM to about 50mM. In an embodiment, the HDAC inhibitor is included in a concentration of about 0.1mM to about 25mM. In an embodiment, the HDAC inhibitor is included in a concentration of about 0.1mM to about 10mM. In an embodiment, the HDAC inhibitor is included in a concentration of about 0.1mM to about 9mM. In an embodiment, the HDAC inhibitor is included in a concentration of about 0.1mM to about 8mM. In an embodiment, the HDAC inhibitor is included in a concentration of about 0.1mM to about 7mM. In an embodiment, the HDAC inhibitor is included in a concentration of about 0.1mM to about 6mM. In an embodiment, the HDAC inhibitor is included in a concentration of about 0.1mM to about 5mM. In an embodiment, the HDAC inhibitor is included in a concentration of about 1mM to about 100mM. In an embodiment, the HDAC inhibitor is included in a concentration of about 1mM to about 50mM. In an embodiment, the HDAC inhibitor is included in a concentration of about 1mM to about 25mM. In an embodiment, the HDAC inhibitor is included in a concentration of about 1mM to about 10mM. In an embodiment, the HDAC inhibitor is included in a concentration of about 1mM to about 9mM. In an embodiment, the HDAC inhibitor is included in a concentration of about 1mM to about 8mM. In an embodiment, the HDAC inhibitor is included in a concentration of about 1mM to about 7mM. In an embodiment, the HDAC inhibitor is included in a concentration of about 1mM to about 6mM. In an embodiment, the HDAC inhibitor is included in a concentration of about 1mM to about 5mM. The concentration can be any value or sub-range within the described range, including endpoints.

[0099] In embodiments, caffeine is included at a concentration of about 0.1 mM to about 50 mM (final concentration in contact with cells). In embodiments, caffeine is included at a concentration of about 0.1 mM to about 25 mM. In embodiments, caffeine is included at a concentration of about 0.1 mM to about 15 mM. In embodiments, caffeine is included at a concentration of about 0.1 mM to about 10 mM. In embodiments, caffeine is included at a concentration of about 0.1 mM to about 9 mM. In embodiments, caffeine is included at a concentration of about 0.1 mM to about 8 mM. In embodiments, caffeine is included at a concentration of about 0.1 mM to about 7 mM. In embodiments, caffeine is included at a concentration of about 0.1 mM to about 6 mM. In embodiments, caffeine is included 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 sub-range within the recited range, including the endpoints.

[0100] In some embodiments, the AAV enhancer is added at one or more time points, such as at the time of transfection (about 0 hours) until about 48 hours after transfection. The AAV enhancer can be added from about 1 hour to about 16 hours after 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 from about 1 hour to about 16 hours after transfection. In some embodiments, the AAV enhancer can be added about 4 to 5 hours after transfection. In some embodiments, the AAV enhancer can be added about 0 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 16 hours, 24 hours, 36 hours or 48 hours after transfection. The AAV enhancer can be added at any time (or sub-range) within the described range including the endpoints.

[0101] 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. In order to properly package AAV, packaging plasmids can be used. 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 any from any serotype AAV including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, DJ or DJ / 8. Packaging plasmids encoding AAV rep and cap genes are commonly 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, for example, from Cell Biolabs, Inc., Vector Biolabs, Addgene, Clontech, and Thermo Fisher Scientific.

[0102] In an embodiment, a helper virus (e.g., from an adenovirus or herpes virus) component is required for the normal function of the AAV production system. The helper virus component can be present on a plasmid (and is often referred to as a pAAV-Helper or pHelper plasmid) or otherwise present in the cell. The helper virus component includes, but is not limited to, E1A, E1B, E2A, E4, and / or VA.

[0103] Provided herein is a kit for the production of adeno-associated virus (AAV). In an embodiment, the kit comprises: 293 cells suitable for high-density suspension culture; AAV production enhancer; transfection reagent, the transfection reagent comprising a cationic lipid; and a cell culture medium, the cell culture medium supporting the growth and expansion of the 293 cells. In an embodiment, the transfection reagent contains a cationic lipid and a peptide. In an embodiment, the transfection reagent comprises at least one cationic lipid and at least one neutral lipid. In an embodiment, the AAV production enhancer comprises one or more of the following: HDAC inhibitors, sodium propionate, sodium butyrate, theobromine and caffeine. In an embodiment, the 293 cells do not include large T antigens.

[0104] In an embodiment, the kit comprises a transfection facilitating agent. In an embodiment, the transfection facilitating agent comprises a peptide. In an embodiment, the transfection facilitating agent comprises a membrane permeable peptide.

[0105] In an embodiment, the kit comprises a lysis buffer. In an embodiment, the lysis buffer contains at least one surfactant. In an embodiment, the surfactant is Triton-100, Triton-alter, NP-40, poloxamer 188 and / or NDSB-201. In an embodiment, the lysis buffer contains Tris-HCl, sodium citrate, TricineHCL, sodium chloride, citric acid, EDTA, EDTA tripotassium, sodium hydroxide and / or sodium dihydrogen phosphate. In an embodiment, the lysis buffer comprises at least one detergent. In an embodiment, the detergent is CHAP, CHAPS, CHAPSO, big CHAP, deoxy big CHAP, Triton X-114, octylthioglucoside and / or sodium deoxycholate.

[0106] Methods for using adeno-associated virus (AAV) production systems

[0107] The AAV production system as described herein can be used to produce AAV vectors. In embodiments, the AAV vectors are produced at high titers.

[0108] In one aspect, a method for AAV vector production is provided, the method 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 period of time sufficient to express 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 transfection step comprises contacting the cells with a transfection facilitating agent. In embodiments, the cells are contacted with an enhancing agent after transfection.

[0109] 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.

[0110] In other embodiments, the transfection facilitator is combined with the AAV transfer vector to form a DNA / transfection facilitator mixture, and then the transfection reagent is combined with the DNA / transfection facilitator mixture before being added to the cells. In an embodiment, the transfection facilitator is combined with the AAV transfer vector, the pRep / Cap plasmid and the pHelper plasmid to form a DNA / transfection facilitator mixture, and then the transfection reagent is combined with the DNA / transfection facilitator mixture before being added to the cells. In some embodiments, the DNA and the transfection facilitator are combined in a tube, the transfection reagent is diluted into the culture medium in the second tube, and then the diluted transfection reagent is added to the DNA / transfection facilitator mixture to form a DNA / transfection facilitator / transfection reagent complex. In other embodiments, the DNA and the transfection facilitator are combined in a tube, and then the transfection reagent is added to the same tube to form a DNA / transfection facilitator / transfection reagent complex. In other embodiments, the DNA and the transfection reagent are combined in a tube, and then the transfection facilitating agent is added to the same tube to form a DNA / transfection facilitating agent / transfection reagent complex.

[0111] In an embodiment, the transfection facilitator is used at a ratio of transfection facilitator:DNA between 5:1 and about 1:5 (volume / weight). In an embodiment, the transfection reagent is combined with a transfection facilitator, a rep / cap plasmid (pRC), a pHelper plasmid (encoding a helper virus component), and an AAV transfer vector to form a transfection complex.

[0112] In embodiments, the AAV is harvested using a lysis buffer. In embodiments, the cells are not centrifuged prior to harvesting the AAV. In embodiments, the lysis buffer is added directly to the transfected cell culture (e.g., cells and culture medium).

[0113] In an embodiment, a crude culture lysate containing AAV is filtered prior to downstream processing such as nuclease treatment and purification. In an embodiment, the crude lysate is mixed with diatomaceous earth, and the mixture is then passed through a filter, such as 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. Such a filtration step of the crude AAV lysate, for example with diatomaceous earth, cellulose, or equivalent, reduces the number of filters required and reduces the filtration and processing time of the AAV lysate prior to the purification process.

[0114] In embodiments, the cells are cultured in a bioreactor. In embodiments, the cells are cultured in a flask.

[0115] In embodiments, the method comprises titrating the collected AAV. The AAV may be titrated using any method. In embodiments, the AAV is titrated using polymerase chain reaction (PCR). In embodiments, the AAV is titrated using quantitative PCR (qPCR). In embodiments, the AAV is titrated using digital droplet PCR. In embodiments, the AAV is titrated using ELISA. In embodiments, a viral titer kit such as QUICKTITER is used. TM The AAV is titrated using an AAV quantitation kit (Cell Labs, Inc.); see also U.S. Pat. No. 6,841,357, which is incorporated herein by reference in its entirety. In an embodiment, the AAV is titrated by determining the concentration of viral particles that can transduce cells (infectivity titer), such as by a cell transduction assay. In an embodiment, the AAV is titrated using a DNA dot blot.

[0116] In an embodiment, the titer of the collected AAV is at least about 1×10 10 In an embodiment, the titer of the collected AAV is at least about 2×10 10 vg / mL. In an embodiment, the titer of the collected AAV is at least about 3×10 10 vg / mL. In an embodiment, the titer of the collected AAV is at least about 4×10 10 In an embodiment, the titer of the collected AAV is at least about 5×10 10 In an embodiment, the titer of the collected AAV is at least about 6×10 10 In an embodiment, the titer of the collected AAV is at least about 7×10 10 vg / mL. In an embodiment, the titer of the collected AAV is at least about 8×10 10 vg / mL. In an embodiment, the titer of the collected AAV is at least about 9×10 10 In an embodiment, the titer of the collected AAV is at least about 1×10 11 In an embodiment, the titer of the collected AAV is at least about 2×10 11 vg / mL. In an embodiment, the titer of the collected AAV is at least about 3×10 11 vg / mL. In an embodiment, the titer of the collected AAV is at least about 4×10 11 In an embodiment, the titer of the collected AAV is at least about 5×1011 In an embodiment, the titer of the collected AAV is at least about 6×10 11 vg / mL. In an embodiment, the titer of the collected AAV is at least about 7×10 11 vg / mL. In an embodiment, the titer of the collected AAV is at least about 8×10 11 vg / mL. In an embodiment, the titer of the collected AAV is at least about 9×10 11 vg / mL.

[0117] In an embodiment, the titer of the collected AAV is between about 1×10 10 vg / mL and about 1×10 12 In an embodiment, the titer of the collected AAV is between about 2×10 10 vg / mL and about 1×10 12 In an embodiment, the titer of the collected AAV is between about 3×10 10 vg / mL and about 1×10 12 In an embodiment, the titer of the collected AAV is between about 4×10 10 vg / mL and about 1×10 12 In an embodiment, the titer of the collected AAV is between about 5×10 10 vg / mL and about 1×10 12 In an embodiment, the titer of the collected AAV is between about 6×10 10 vg / mL and about 1×10 12 In an embodiment, the titer of the collected AAV is between about 7×10 10 vg / mL and about 1×10 12 In an embodiment, the titer of the collected AAV is between about 8×10 10 vg / mL and about 1×10 12 In an embodiment, the titer of the collected AAV is between about 9×10 10 vg / mL and about 1×10 12 In an embodiment, the titer of the collected AAV is between about 1×10 11 vg / mL and about 1×10 12 In an embodiment, the titer of the collected AAV is between about 2×10 11 vg / mL and about 1×10 12In an embodiment, the titer of the collected AAV is between about 3×10 11 vg / mL and about 1×10 12 In an embodiment, the titer of the collected AAV is between about 4×10 11 vg / mL and about 1×10 12 In an embodiment, the titer of the collected AAV is between about 5×10 11 vg / mL and about 1×10 12 In an embodiment, the titer of the collected AAV is between about 2×10 10 vg / mL and about 9×10 11 In an embodiment, the titer of the collected AAV is between about 2×10 10 vg / mL and about 8×10 11 In an embodiment, the titer of the collected AAV is between about 2×10 10 vg / mL and about 7×10 11 In an embodiment, the titer of the collected AAV is between about 2×10 10 vg / mL and about 6×10 11 In an embodiment, the titer of the collected AAV is between about 2×10 10 vg / mL and about 5×10 11 In an embodiment, the titer of the collected AAV is between about 2×10 10 vg / mL and about 4×10 11 In an embodiment, the titer of the collected AAV is between about 2×10 10 vg / mL and about 3×10 11 In an embodiment, the titer of the collected AAV is between about 2×10 10 vg / mL and about 2×10 11 In an embodiment, the titer of the collected AAV is between about 2×10 10 vg / mL and about 1×10 11 The titer can be any value or subrange within the recited range, including the endpoints.

[0118] The cells may be cultured in any volume of cell culture medium that supports cell growth and AAV production. In an embodiment, the cells are cultured in a volume of about 15 milliliters (mL) to about 200 liters (L). In an embodiment, the cells are cultured in a volume of about 30 mL to about 200 L. In an embodiment, the cells are cultured in a volume of about 50 mL to about 200 L. In an embodiment, the cells are cultured in a volume of about 100 mL to about 200 L. In an embodiment, the cells are cultured in a volume of about 500 mL to about 200 L. In an embodiment, the cells are cultured in a volume of about 1 L to about 200 L. In an embodiment, the cells are cultured in a volume of about 10 L to about 200 L. In an embodiment, the cells are cultured in a volume of about 15 mL to about 100 L. In an embodiment, the cells are cultured in a volume of about 15 mL to about 50 L. In an embodiment, the cells are cultured in a volume of about 15 mL to about 20 L. In an embodiment, 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 mL. 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.

[0119] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes thereto will inspire those skilled in the art and will be included within the spirit and purview of the present application and the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0120] Examples

[0121] Those skilled in the art will appreciate 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 such descriptions.

[0122] Example 1: Effect of culture conditions on virus titer

[0123] Adherent HEK293T cells and HEK293F cells were cultured in an incubator at 37°C, 8% CO2, and 80% humidity. AAV transfer vectors, pAAV-RC, and pAAV-Helper were used in 6-well plates at approximately 4 × 10 6Cells were transfected at a density of 10 cells / mL: Prot-1 (Enhancer 1 and Supplement 1); Prot-2 (Supplement 1, no Enhancer); Prot-3 (Enhancer 1, no Supplement). System 1 contains medium that supports the growth and proliferation of HEK293 cells (LV-MAX TM Production medium, GIBCO TM , Thermo Fisher Scientific, Catalog No. A3583401), LV-MAX TM Transfection reagent, LV-MAX TM Supplements and LV-MAX TM Enhancer (GIBCO TM , Thermo Fisher Scientific, Catalog No. A35348). AAV was extracted by adding 200 μL of 5X AAV lysis buffer to 800 μL of transfected cell culture, mixing well, and incubating at room temperature for 30 minutes. After incubation, the tube was inverted to completely lyse the cells and then spun at maximum speed in a benchtop centrifuge for 10 minutes. The supernatant containing crude AAV was collected.

[0124] AAV was titrated by qPCR with primers and probes against ITR2. Briefly, extracts were treated with DNase and proteinase K. Extracts were diluted 1:50 in water and subjected to qPCR and compared to a standard curve (digested AAV plasmid).

[0125] Figure 1 Results are provided in. For HEK293F cells, without LV-MAX TM Supplementation of system 1 produced the highest AAV titers.

[0126] Example 2: Effect of culture medium on virus titer

[0127] HEK293F cells were established in four different types of commercially available culture media that support HEK293F cells in suspension culture. The cells were frozen in the culture media to which they were adapted 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 compounded with an AAV transfection reagent in an Opti-MEM complex solution buffer at a ratio of 1:4w / w and with a transfection facilitator at a ratio of 2:1 (v / w), and then incubated at room temperature for 10 minutes. After incubation, the DNA / transfection reagent complex mixture was directly added to the prepared cell culture. AAV2 production occurred at the 6th cell passage in all four cell systems / culture medium types.

[0128] Figure 2It is shown that HEK293F cells adapted to Medium 4 produced more AAV2 compared to the other media tested.

[0129] Example 3: Characteristics of cells adapted to medium 4

[0130] 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 the following systems: System 1: HEK293F cells in the presence of LV-MAX transfection reagent, enhancer, and supplements (see Example 1); System 2: HEK293F cells using a different transfection reagent (Transfection Reagent 2) in the presence of LV-MAX enhancer and supplements; and System 3: HEK293 cells adapted to Medium 4 using Transfection Reagent 2 and LV-MAX enhancer without supplements. Virus titers were highest in HEK293 cells adapted to Medium 4 using System 3 for all AAV serotypes ( Figure 3 ). Transfection reagent 2 is a cationic lipid transfection reagent comprising a peptide-containing transfection facilitating agent (as described herein).

[0131] Example 4: Clone 45 cells

[0132] The growth characteristics of a clonal population of HEK293 cells (clone 45) adapted to Medium 4 were evaluated. At passage 5, clone 45 cells were split to 0.3 × 10 6 The cell density was 10 viable cells / mL. Cell density and cell viability were collected every day for 9 days. The growth curve is shown in Figure 4A Medium 4 supports up to approximately 11 × 10 6 The high cell density of 10 cells / mL and clone 45 cells in high-density culture showed high cell viability. Figure 4B As shown in , clone 45 cells showed very little agglutination even at high density.

[0133] Clone 45 cells and three other medium 4-adapted clones were evaluated for their ability to produce acceptable titers of different AAV serotypes in medium 4. TM Expression medium (Expi45; GIBCO TM, Thermo Fisher Scientific, Catalog No. A1435101) or Medium 4 (Cl45) and transfected with AAV2, AAV6, AAV-dj, AAV8 or AAV9 vectors of interest encoding a transgene for GFP. Similarly, clone lines Clone 12 (Cl12), Clone 22 (Cl22) and Clone 51 (Cl51) adapted to Medium 4 were grown in Medium 4 and transfected with vectors of five AAV serotypes. Transfection was 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 clones adapted to Medium 4. The titers of AAV produced were determined using qPCR using AAV-GFP primers and probes as described elsewhere herein. For each serotype, Clone 45 cells grown in Medium 4 produced between approximately 1×10 11 vg / mL and about 2×10 11 vg / mL between high cell titers ( Figures 5A-5E ).

[0134] The effects of different transfection reagents on viral production in clone 45 cells were also evaluated. TM Cells were transfected with either transfection reagent (TR1) or transfection reagent 2 (TR2). Under these conditions, TM Compared with transfection reagent 2, transfection reagent 2 mediated plasmid delivery with more AAV virus produced ( Figure 6 ).

[0135] Example 5: Adding enhancers to improve virus production

[0136] At 0, 4.5, 8, 15.5, 24, or 28 hours after transfection, clone 45 cells in medium 4 were transfected with AAV plasmid using transfection reagent 2 with or without the addition of enhancers. Addition of enhancers, particularly between 0 and 15.5 hours after transfection, increased AAV production ( Figure 7 ). The enhancer contains HDAC inhibitor, sodium propionate, sodium butyrate and caffeine.

[0137] Example 6: Screening of lysis buffer reagents

[0138] use A design of experiments (DOE) platform was used to design screening experiments using four core chemicals as potential detergents in the lysis buffer: Triton-alter, CHAPSO, Big CHAP, and NDSB-201.

[0139] The DOE platform allows researchers to change multiple parameters simultaneously, rather than changing each in the parameters individually, and then consider each optimization parameter of the entire optimized formulation. When the second-order effect between the parameters can affect the result, the DOE platform changes all candidate parameters simultaneously, allowing more effective and accurate results. Experiments using the DOE platform also require less operation and are more economical than traditional experimental methods. See Kauffman et al., "Optimization of Lipid Nanoparticle Formulations for mRNA Delivery in Vivo with Fractional Factorial and Definitive Screening Designs", Nano Letters 15: 7300-7306 (2015) and supplementary materials for theoretical discussions on the DOE platform.

[0140] Triton-alter, CHAPSO, big CHAP, and NDSB-201 were evaluated at different concentrations as indicated in Table 1. Percentages are provided in units of weight per volume (w / v).

[0141] Table 1: The amount of each detergent used.

[0142]

[0143]

[0144] The results were analyzed using Jmp software to determine the impact of detergents during the cell lysis process. Figure 8 Results for three different AAV serotypes are presented in .

[0145] The lysis buffer was also tested on AAV extracts from transfected cell cultures versus cell pellets. Clone 45 cells were transfected with AAV plasmids using transfection reagent 2 and enhancer. AAV was extracted 70-72 hours after transfection by adding 1x lysis buffer to the cell pellet or 5x lysis buffer to the whole cell culture (transfected cells and culture medium). Fig. 9 The AAV titers generated are provided in .

[0146] Example 7: First AAV production protocol

[0147] Follow the guidelines for suspension culture of AAV producer cells below. Grow cells according to standard AAV suspension cell culture protocols. When the cells reach a density of approximately 3 × 10 6to 6.5×10 6 When the number of viable cells / mL reaches 0.3×10, the cells are usually subcultured every 3-4 days. After about 3 or 4 days of culture, the cells divide into 0.3×10 6 to 0.6×10 6 Cells were counted daily at approximately the same time each day to monitor cell growth. During cell culture, an orbital shaker (19 mm orbital diameter) was used at approximately 125 rpm for 125 mL to 1 L shake flasks. The incubator was set to approximately 37° C., approximately 8% CO 2 , and approximately 75%-80% humidity.

[0148] Reagents and Materials:

[0149] 125mL, 250mL and 1L polycarbonate, disposable, sterile, upwardly vented, unbaffled Erlenmeyer flasks 50mL sterile conical tubes

[0150] Opti-MEM I Medium

[0151] Clone 45 cells

[0152] AAV293 cell culture medium

[0153] AAV transfection reagents and transfection facilitators

[0154] AAV Enhancer

[0155] 5X AAV Lysis Buffer

[0156] For example, if the cells divide to 0.55 × 10 on Friday morning 6 If the cell count is 4.0 × 10 cells / mL, they can be cultured in a 1 L flask in about 300 mL of medium for 3 days. For example, on Monday morning, cells are prepared by counting the cells and a cell density of 4.0 × 10 6 Cells can be diluted to about 3.0×10 cells / mL in fresh warm culture medium. 6 cells / mL and culture for an additional 24 hours (approximately).

[0157] For example, transfection can be performed on Tuesday. Cells can be counted and diluted to between approximately 2.5×10 in 30 mL of cell culture medium in a 125 mL flask. 6 cells / mL and about 4×10 6 Table 2 provides the amount of each plasmid at various ratios. Table 3 provides additional transfection guidelines.

[0158] Table 2: Preparation of different DNA ratios

[0159]

[0160] Table 3: DNA / transfection reagent complex preparation

[0161]

[0162] Prepare DNA / transfection reagent as follows. Label two tubes Tube 1 and Tube 2. In Tube 1: Place 4.5 mL of OPTI-MEM TM I medium and 135 μg of DNA (at the ratio indicated in Table 2), and add 270 μL of transfection facilitator. In tube 2: 4.5 mL of OPTI-MEM TM I medium was mixed with 540 μL of transfection reagent and incubated at room temperature for 1 minute. Tubes 1 and 2 were fully combined by adding the solution of tube 2 to tube 1 and mixing, then incubated at room temperature for 10 minutes. Approximately 3.2 mL of DNA / AAV transfection reagent complex was added to each flask of cells. AAV enhancer was added at 1% v / v per flask at the time of transfection.

[0163] Collect AAV 70-72 hours after transfection, for example Friday morning. Alternatively, the transfected cell culture can be stored at -80°C (800 μL samples should be stored separately at -80°C for titration). Add 5xAAV lysis buffer at a dilution of 1:5 (200 μL lysis buffer per 800 μL transfected cell culture sample), pipette up and down and vortex to mix. Incubate the sample for about 30 minutes and then invert by hand 25-30 times. Once the culture solution becomes clear and a large piece of cell debris is observed, the cells will be completely lysed.

[0164] The lysed cells are centrifuged for 10 minutes at 4° C. (maximum speed in a tabletop centrifuge). The supernatant containing the crude AAV is transferred to a new tube and stored at 4° C. The samples are titered, for example, by qPCR.

[0165] Example 8: Measurement of AAV titers from production in Example 7

[0166] The supernatant from Example 7 was mixed thoroughly and 100 μL of the crude AAV sample was aliquoted into each of 2 wells of a 96-well round-bottom plate with a lid. The sample was digested with DNase I by adding 2 μL of the 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 incubated at 37°C for 60 minutes, 95°C for 20 minutes, and then at 4°C.

[0167] DNase treated samples were digested with proteinase K by adding 19 μL 2x PK buffer and 20 μg proteinase K to each sample and incubating at 60°C for 60 minutes, 95°C for 10 minutes and then at 4°C.

[0168] After proteinase K digestion, samples were diluted 1:50 in water. Quantitative PCR (qPCR) was run using primers specific for the AAV-GFP gene and a labeled probe.

[0169] Generate a standard curve using linearized AAV transfer plasmid. For pAAV-GFP, linearize the plasmid by digestion with HindIII or BamHI and then determine the DNA concentration. Linearization can be determined by loading the cut and uncut plasmids onto a 0.8% agarose gel and observing the resulting bands after electrophoresis. The uncut plasmid should appear as a smear and the cut plasmid should be a large band of approximately 5 kb.

[0170] QPCR was performed in 384-well qPCR sample plates using 2X EXPRESS qPCR Supermix and premixed ROX (Thermo Fisher Scientific) according to the manufacturer's instructions. In brief, 3 μL of diluted samples (or standard curve) were combined with 7.5 μL 2x Supermix, 0.11 μL AAV-GFP probe (FAM / TAMRA), 1.13 μL GFP-specific primers (mixed with 10 μM forward and reverse primers) and 3.26 μL water. Samples were run in a qPCR machine using the following cycle program: 50°C for 2 minutes (UDG incubation); 95°C for 2 minutes; 40 cycles: 95°C for 15 seconds, 60°C for 1 minute.

[0171] Example 9: AAV-GFP virus infectivity test protocol

[0172] The ability of AAV to infect target cells (eg, Ht1080 or HEK293) can be tested. Approximately 4 hours prior to infection, cells are seeded at a density of 7000 cells / well in 100 μL of culture medium in 96-well plates.

[0173] Cells were infected by adding 1 μL of crude AAV preparation to each well. Cells were incubated for approximately 3 days. For AAVs containing a gene that expresses green fluorescent protein (GFP), flow cytometry was run to measure the percentage of cells expressing GFP.

[0174] Example 10: Comparison of AAV production systems

[0175] AAV vector production from clone 45 and from two subclonal lines derived from clone 45 was compared to AAV vector production from a HEK293F derivative LV293 (LV-MAX Virus Producing Cells (VPCs), 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 with Transfection Reagent 2 in the presence of a transfection facilitator as described above. Approximately 72 hours after transfection, cultures were determined for AAV titer (by qPCR for GFP (Example 8)) and cell viability. AAV titer (by qPCR for GFP (Example 8)) and cell viability were compared to cultures grown with LV293 (VPCs, Fig. 10A ) clone 45 and subclonal lines C13 and C20 produced significantly higher AAV8 titers (ranging from approximately 1.5 × 10 11 To about 2.5×10 11 vg / mL). In addition, compared with LV293 (VPC, Fig. 10B ) cultures, AAV production cultures produced with clones 45, C13, and C20 had significantly higher cell viability compared to cultures produced with clones 45, C13, and C20.

[0176] Vector production between various AAV serotypes in clone 45 cells was compared with vector production in LV293 (VPC) cells. Cells were grown according to the suspension cell culture protocol for AAV as described above and diluted (at 3×10 6 Clone 45 was diluted at 2.5 × 10 6 Cells were transfected with pAAV-GFP, pAAV-Helper, and pAAV-RC for AAV2, AAV6, AAV8, and AAV9. Transfection was performed with Transfection Reagent 2 with a transfection facilitator as described above. AAV was harvested from the culture approximately 72 hours after transfection. AAV titers were determined by qPCR for GFP and by the infectivity test protocol (Example 9). Exemplary results are shown in FIG11 . Fig.11A ) between serotypes, the AAV production system using clone 45 as producer cells produced significantly higher viral titers (ranging from approximately 1.3 × 10 11 To about 2.15×10 11 vg / mL). Fig. 11B As shown in , the AAV production system using clone 45 also resulted in greater infectivity of AAV2 and AAV6 compared to LV293 VPC cells.

[0177] The AAV vector production system provided herein was compared to two production systems based on polyethyleneimine (PEI). 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) PEI-GFP was used without an enhancer. TM (Polyplus transfection) transfected LV293 cells and (2) transfected clone 45 cells with transfection reagent 2 in the presence of transfection facilitators and AAV production enhancers as described above. After the culture period, AAV was harvested and titers were determined by qPCR methods for GFP and by an infectivity test protocol. Exemplary results are shown in Figure 12. Compared to LV293 with the PEIpro system, the AAV production system provided herein resulted in significantly higher AAV6 titers ( Fig. 12A ) and infectious ( Fig. 12B ).

[0178] In another analysis, vector production between various AAV serotypes was compared using the following: (1) HEK293T adherent cells with PEI-MAX transfection reagent (Polysciences, Inc.) and Clone 45 cells with Transfection Reagent 2 and Transfection Helper as described above. Cells were transfected with pAAV-GFP, pAAV-Helper, and pAAV-RC for AAV2, AAV6, AAV8, AAV9, and AAV-dj. After the incubation period, AAVs were harvested and titers were determined by qPCR methods for GFP and by an infectivity test protocol. Exemplary results are shown in Fig.13A Compared to HEK293T using the PEI-MAX system, the AAV production system provided herein produced significantly higher AAV titers among 4 of the 5 serotypes tested ( Fig.13A ).like Fig. 13B As shown in , the AAV production system provided herein also results in greater infectivity of AAV 2 and AAV6 compared to using the PEI-MAX system.

[0179] Example 11: Post-collection processing

[0180] After transfection, virus production and cell lysis, the crude AAV lysate was filtered using diatomaceous earth before downstream processing. Two samples from the same AAV culture were lysed by freeze-thaw method (in the absence of lysis buffer) or by adding 10X AAV lysis buffer and incubating for 30 minutes (as described above) to form a crude AAV lysate. Immediately after lysis, diatomaceous earth (DE) was mixed with the crude AAV lysate and the mixture was passed through a 2 micron filter. Different amounts of DE per mL of lysate were tested, including 0.5g DE:30mL lysate and 1g DE:100mL lysate. Before and after DE filtration, samples were collected from each lysate to determine the recovery of AAV after the filtration step. GFP qPCR was used to determine the AAV titer from the sample, and exemplary results are shown in Fig.14 The AAV lysis buffer method for forming a crude lysate resulted in a higher recovery of AAV titers during DE filtration, whereas the freeze-thaw cell lysis method resulted in a significantly lower recovery. Fig.14 As shown in, for example, approximately 100% titer recovery was obtained with lysis buffer, compared to approximately 10% recovery with freeze-thaw. The single DE filtration step reduces the number of filters required and greatly reduces the filtration and processing time of AAV lysates prior to downstream processing such as nuclease treatment and purification.

Claims

1. A method for producing an adeno-associated virus (AAV) vector, the method comprising: (a) growing mammalian cells in suspension culture; (b) transfecting the mammalian cell with the AAV transfer vector using a transfection reagent; (c) contacting the transfected cells with an enhancing agent; (d) culturing the transfected cells in suspension culture for a period of time sufficient to express the AAV vector, Thereby, a transfected AAV cell culture is generated; (e) harvesting AAV from the transfected AAV cell culture.

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

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

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

5. The method according to any one of claims 2 to 4, wherein the lysis buffer comprises at least one detergent selected from the group consisting of CHAP, CHAPS, CHAPSO, big CHAP, octylthioglucoside and sodium deoxycholate.

6. The method according to any one of claims 1 to 3, wherein the enhancing agent comprises one or more of the following: a histone deacetylase (HDAC) inhibitor, sodium propionate and caffeine.

7. The method of claim 6, 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.

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

9. The method of any one of the preceding claims, wherein the enhancer is added between about 0 hours and about 12 hours after transfection.

10. The method of any one of the preceding claims, wherein the transfection reagent comprises a cationic lipid.

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