Methods and compositions for RAAV production

Abstract

The disclosure relates, at least in part, to methods and compositions for improving rAAV production. The disclosure provides methods of contacting a cell culture with one or more enhancer elements to improve rAAV yield.

Description

[0001] METHODS AND COMPOSITIONS FOR RAAV PRODUCTION

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 § 119(e) of U.S. Provisional Application No. 63 / 682,073, filed on August 12, 2024, entitled “METHODS AND COMPOSITIONS FOR RAAV PRODUCTION,” and U.S. Provisional Application No. 63 / 697,392, filed on September 20, 2024, entitled “METHODS AND COMPOSITIONS FOR RAAV PRODUCTION,” the contents of each of which is hereby incorporated by reference in its entirety.

[0004] BACKGROUND

[0005] One effective way to produce viral vectors for therapeutic applications, including but not limited to gene therapy, is to use cultured cells for producing recombinant viral particles. For clinical applications, large quantities of viral particles are required, creating a need to improve quantity and quality of viral (e.g., rAAV) production.

[0006] Therefore, there is a need to improve manufacturing processes for viral vectors use in, for example, gene therapy.

[0007] SUMMARY

[0008] Aspects of the disclosure provide methods of improving rAAV production using one or more enhancer elements. In some aspects, the method comprises contacting a cell culture (e.g., a cell culture used in a transient transfection or a producer cell culture) with one or more enhancer elements, wherein the one or more enhancer elements comprise an AAV titer enhancer, nocodazole, an HD AC inhibitor, and / or DMSO, and wherein the cell culture comprises one or more recombinant nucleic acids.

[0009] In some aspects, the one or more recombinant nucleic acids comprise a recombinant adeno-associate virus (rAAV) genome. In some aspects, the rAAV genome comprises a gene of interest flanked by inverted terminal repeats (ITRs). In some aspects, the ITRs comprise AAV2 ITRs or AAV9 ITRs. In some aspects, the gene of interest encodes an antibody, an enzyme, a growth factor, a microRNA, or a hormone.

[0010] In some aspects, one of the one or more recombinant nucleic acids encodes a capsid (Cap) protein. In some aspects, the capsid protein is an AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 capsid protein or variants or chimeras or hybrids thereof.

[0011] In some aspects, one of the one or more recombinant nucleic acids encodes a Rep protein (e.g., Rep78, Rep68, Rep52, Ref40, or any combination thereof). In some aspects, the one or

[0012] #14288902v1 more recombinant nucleic acids further comprise one or more nucleic acid sequences encoding AAV helper genes, wherein optionally the AAV helper genes comprise at least one of adenovirus genes E1A, E1B, E2A, VA, and E4orf6.

[0013] In some aspects, the cell culture comprises animal cells. In some aspects, the cell culture comprises mammalian cells. In some aspects, the cell culture comprises epithelial cells. In some aspects, the cell culture comprises HEK cells. In some aspects, the cell culture comprises HEK293 cells.

[0014] In some aspects, the one or more recombinant nucleic acids comprise deoxyribonucleic acid (DNA). In some embodiments, the one or more recombinant nucleic acids comprise ribonucleic acid (RNA). In some embodiments, the one or more recombinant nucleic acids comprise one or more plasmids.

[0015] In some embodiments, the one or more recombinant nucleic acids comprise a dual plasmid transfection system. In some embodiments, the dual plasmid transfection system comprises (i) a first plasmid comprising: one or more recombinant nucleic acids encoding the Rep protein, one or more nucleic acids encoding the Cap protein and the gene of interest flanked by inverted terminal repeats (ITRs); and (ii) a second plasmid comprises: one or more nucleic acids encoding AAV helper genes.

[0016] In some aspects, the one or more recombinant nucleic acids comprise a triple plasmid transfection system. In some aspects, the triple plasmid transfection system comprises, (i) a first plasmid comprising: one or more recombinant nucleic acids encoding the Rep protein, one or more nucleic acids encoding the Cap protein; (ii) a second plasmid comprising: one or more recombinant nucleic acids encoding the gene of interest flanked by inverted terminal repeats (ITRs); and (iii) a third plasmid comprising: one or more nucleic acids encoding AAV helper genes.

[0017] In some aspects, the cell culture produces recombinant adeno-associated virus (rAAV) particles.

[0018] Aspects of the disclosure provide methods for producing recombinant adeno-associated viral (rAAV) particles, the method comprising: transient transfection of mammalian host cells using a triple or dual transfection system comprising one or more recombinant nucleic acids encoding a gene of interest flanked by AAV inverted terminal repeats (ITRs), wherein the transient transfection comprises contacting a cell culture comprising the mammalian host cells with one or more enhancer elements, wherein the one or more enhancer elements comprise an AAV titer enhancer, nocodazole, an HD AC inhibitor, and / or DMSO. In some aspects, the

[0019] #14288902v1 method further comprises contacting the cell culture comprising the mammalian host cells with a feed medium, about 4 hours to about 8 hours following the transient transfection.

[0020] In some aspects, the method comprises transient transfection of the mammalian host cells using the dual transfection system. In some embodiments, the dual plasmid transfection system comprises (i) a first plasmid comprising: one or more recombinant nucleic acids encoding an AAV Rep protein, one or more recombinant nucleic acids encoding an AAV Cap protein and the one or more recombinant nucleic acids encoding the gene of interest flanked by inverted terminal repeats (ITRs); and (ii) a second plasmid comprising: one or more recombinant nucleic acids encoding AAV helper genes.

[0021] In some aspects, the method comprises transient transfection of the mammalian host cells using the triple transfection system. In some aspects, the triple plasmid transfection system comprises, (i) a first plasmid comprising: one or more recombinant nucleic acids encoding an AAV Rep protein, one or more recombinant nucleic acids encoding an AAV Cap protein; (ii) a second plasmid comprising: the one or more recombinant nucleic acids encoding the gene of interest flanked by inverted terminal repeats (ITRs); and (iii) a third plasmid comprising: one or more recombinant nucleic acids encoding AAV helper genes.

[0022] In some aspects, the method comprises contacting a producer cell culture with one or more enhancer elements.

[0023] In some aspects, the cell culture is contacted with two or more enhancer elements. In some aspects, the cell culture is contacted with an AAV titer enhancer and an antimitotic agent, an AAV titer enhancer and an HD AC inhibitor, an AAV titer enhancer and an organic solvent, an antimitotic agent and an HD AC inhibitor, an antimitotic agent and an organic solvent, or an HD AC inhibitor and an organic solvent. In some aspects, each of the two or more enhancer elements is provided at a lower dose than when each is provided alone.

[0024] In some aspects, the cell culture is contacted with two or more enhancer elements, wherein each of the two or more enhancer elements independently comprises an AAV titer enhancer, nocodazole, an HD AC inhibitor, and / or DMSO. In some aspects, the cell culture is contacted with three or more enhancer elements, wherein each of the three or more enhancer elements independently comprises an AAV titer enhancer, nocodazole, an HD AC inhibitor, and / or DMSO. In some aspects, the cell culture is contacted with an AAV titer enhancer, nocodazole, an HD AC inhibitor, and DMSO.

[0025] In some aspects, the enhancer is added in an amount sufficient to improve rAAV production relative to a method that does not comprise contacting a cell culture with one or more enhancer elements. In some aspects, the enhancer is added in an amount sufficient to improve

[0026] #14288902v1 rAAV production by at least 3-fold relative to a method that does not comprise contacting a cell culture with one or more enhancer elements.

[0027] In some aspects, the enhancer is added in an amount sufficient to produce a higher titer of rAAV relative to a method that does not comprise contacting a cell culture with one or more enhancer elements. In some aspects, the enhancer is added in an amount sufficient to produce a titer that is at least 3-fold higher relative to a method that does not comprise contacting a cell culture with one or more enhancer elements.

[0028] In some aspects, the enhancer is added in an amount sufficient to produce a greater percentage of full capsids relative to a method that does not comprise contacting a cell culture with one or more enhancer elements. In some aspects, the enhancer is added in an amount sufficient to produce a percentage of full capsids that is at least 3-fold higher relative to a method that does not comprise contacting a cell culture with one or more enhancer elements.

[0029] In some aspects, the method further comprises isolating rAAV from the cell culture. In some aspects, the rAAV comprises a titer of at least about 3E11 vg / mL.

[0030] BRIEF DESCRIPTION OF DRAWINGS

[0031] FIG. 1 shows the titer of rAAV produced by control cells, cells treated with IX titer enhancer, and cells treated with 1.5X titer enhancer. Results show that titer enhancer increases vector genomes (vg) / mL at lower cell density.

[0032] FIG. 2 shows the titer of rAAV produced by cells treated with titer enhancer relative to control cells at higher cell density (about 6 million cells / mL cell density). Titer was measured for each of four non-limiting examples of transgene constructs. Across transgene constructs, the titer enhancer increased vector genomes / mL produced. For each transgene, the left bar represents the control cells and the right bar represents the cells treated with the titer enhancer.

[0033] FIGs. 3A-3B show the percentage of particles with a full capsid (FIG. 3A) and partially full capsids (FIG. 3B) using four different transgenes. For each transgene, the left bar represents the control cells and the right bar represents the cells treated with the titer enhancer.

[0034] FIGs. 4A-4B show the titer of rAAV produced by control cells and cells treated with one or more additives. FIG. 4A shows titer of rAAV produced with a construct containing transgene 1, and FIG. 4B shows titer of rAAV produced with a construct containing transgene 3.

[0035] FIGs. 5A-5B show diagrams representing the manufacturing process timeline. FIG. 5A shows the progression of manufacturing steps, including addition of additives (e.g., enhancer elements). FIG. 5B shows the timeline of full capsid generation.

[0036] #14288902v1 FIG. 6 shows improvement in rAAV productivity at varied concentrations of each enhancer element. Additive 1 is an AAV titer enhancer (Minis Bio). Additive 2 is nocodazole. Additive 3 is M344. Additive 4 is DMSO.

[0037] FIG. 7 shows improvement in rAAV productivity for each enhancer element in larger culture volumes (AM250).

[0038] FIGs. 8A-8B show improvement of rAAV productivity in the presence of IX additive 1 (AAV titer enhancer (Mirus Bio)), in an intensified cell culture process (FIG. 8A) and across varied capsid and transgene combinations (FIG. 8B).

[0039] FIGs. 9A-9C show fold change in rAAV productivity (FIG. 9A), percent full capsids (“full vectors”) (FIG. 9B), and percent partially full capsids (“partial vectors”) ( (FIG. 9C) in AM250 and 3L cultures with and without IX additive 1 (AAV titer enhancer (Mirus Bio)).

[0040] FIGs. 10A-10C show fold change in packaging efficiency at harvest (FIG. 10A), total genomes at 24 hours post-transfection (FIG. 10B), and total genomes at 48 hours (FIG. IOC) in AM250 and 3L cultures with and without IX additive 1 (AAV titer enhancer (Mirus Bio)).

[0041] FIGs. 11A-11D show characteristics of cells treated with 0.7X or IX doses of additive 1 (AAV titer enhancer (Mirus Bio)) (FIGs. 11A-11C) or additive 2 (nocodazole) (FIG. 11D) over 3 days. As shown in FIG. 11A, cells treated with 0.7X or IX doses of additive 1 grow more slowly than control cells. As shown in FIG. 11B, cells treated with 0.7X or IX doses of additive 1 are larger than control cells. As shown in FIG. 11C, more cells treated with 0.7X or IX doses of additive 1 are synced in G2 phase relative to control cells. As shown in FIG. 11D more cells treated with IX dos, 2.5X, or 5X doses of additive 2 are synced in G2 phase relative to control cells.

[0042] DETAILED DESCRIPTION

[0043] The present disclosure provides methods and compositions for cell culture production of rAAV. Aspects of the present disclosure provide contacting a cell culture (e.g., cell culture media) with one or more enhancer elements.

[0044] As described herein, it was surprisingly found that the addition of one or more enhancer elements to the cell culture (e.g., cell culture media) led to improved rAAV properties, such as yield, titer, full and partial capsid production, and larger scale production. Therefore, in some embodiments, methods and compositions described herein are useful for increasing rAAV yield. In some embodiments, methods and compositions described herein are useful for increasing the percentage of full capsids produced. In some embodiments, methods and compositions described

[0045] #14288902v1 herein are useful for large scale production and reducing the cost of producing rAAV for therapeutic applications.

[0046] Enhancer Elements

[0047] In some aspects, the disclosure provides methods of contacting a cell culture (e.g., cell culture media) with one or more enhancer elements (“enhancers”). In some aspects, an enhancer element slows the growth rate of cells in the cell culture. In some aspects, an enhancer element synchronizes the cells in the cell culture such that they are substantially all in the same growth phase. In some aspects, the cells in the cell culture treated with an enhancer element are substantially all synchronized in G2 phase. In some aspects, an enhancer element increases cell size. In some aspects, an enhancer element increases cell viability. In some aspects, an enhancer element does two or more of: slows cell growth, increases cell size, synchronizes cells in G2, and increases viability of the cells.

[0048] In some aspects, the enhancer element is an AAV titer enhancer (e.g., a titer enhancer sold by Minis Bio under the name RevIT titer enhancer; Reese et al., Cytotherapy, 2024, 26(6):S145-S146). In some embodiments, the AAV titer enhancer is a small molecule, such as those disclosed in WO 2024 / 229131, incorporated by reference to the extent it relates to small moelcules capable of increasing AAV production. In some embodiments, the AAV titer enhancer comprises a polypeptide or polynucleotide sequence encoding, derived from, or functionally related to Rev-responsive elements (RREs) (e.g., HIV-1 RREs) or similar regulatory motifs that facilitate nuclear export or post-transcriptional processing of AAV-related transcripts. In some embodiments, the AAV titer enhancer additionally or alternatively comprises export-enhancing RNA motifs (e.g., sequences that recruit host cell RNA export machinery (such as the CRMl / exportin 1 pathway)). Examples of RNA export-enhancing elements include constitutive transport elements (CTEs) (for example, from Mason-Pfizer Monkey Virus (MPMV)) and post-transcriptional regulatory elements (for example, from woodchuck hepatitis virus, WPRE)). In some embodiments, the AAV titer enhancer additionally or alternatively comprises synthetic non-coding sequences (e.g., engineered to mimic or enhance the function of RREs). In some embodiments, a synthetic non-coding sequence is useful to improve expression in a specific type of cell (e.g., a human producer cell, such as HEK293). In some embodiments, the AAV titer enhancer additionally or alternatively comprises a transacting protein component (e.g., a Rev protein, such as HIV-1 Rev protein). In some aspects, the AAV titer enhancer synchronizes cells in G2 phase. In some aspects, the AAV titer enhancer slows growth of cells in the cell culture and / or increases the size of the cells in the cell culture.

[0049] #14288902v1 In some embodiments, the AAV titer enhancer is present in the cell culture at a concentration ranging from about 10 pL to about 100 p L per 20 mL of cell culture, including, for example, concentrations of about 10 pL, 20 pL, 30 pL, 40 pL, 50 pL, 60 pL, 75 pL, or 100 pL per 20 mL (e.g., about 20 pL to about 80 pL per 20 mL, about 25 pL to about 75 pL, about 30 pL to about 70 pL, about 40 pL to about 60 pL, or about 25 pL to about 50 pL per 20 mL of cell culture media). In some embodiments, the AAV titer enhancer is present in the cell culture media at a concentration of about 30 pL per 20 mL. In some embodiments, the AAV titer enhancer is present in the cell culture media at a concentration of about 45 pL per 20 mL.

[0050] In some aspects, the enhancer element is an antimitotic agent (e.g., nocodazole). In some embodiments, the antimitotic agent is selected from the group consisting of: taxanes (e.g., paclitaxel and docetaxel), epothilones (e.g., epothilone B, ixabepilone, BMS-310705, sagopilone (ZK-EPO), and epothilone D ), vinca alkaloids (e.g., vinblastine, vindesin, vinorelbin, and vincristine), and analogs and derivatives thereof. In some embodiments, the enhancer element is nocodazole. In some embodiments, the antimitotic agent (e.g., nocodazole) is present in the cell culture at a concentration ranging from approximately 100 ng / mL to approximately 1500 ng / mL. In some embodiments, the concentration of the antimitotic agent (e.g., nocodazole) in the cell culture media is about 100 ng / mL to about 1250 ng / mL, about 250 ng / mL to about 1000 ng / mL, about 500 ng / mL to about 1500 ng / mL, about 250 ng / mL to about 750 ng / mL, or about 600 ng / mL to about 1300 ng / mL. In some embodiments, the antimitotic agent (e.g., nocodazole) is present in the cell culture media at a concentration of about 125 ng / mL, 250 ng / mL, 500 ng / mL, 626 ng / mL, 750 ng / mL, 1000 ng / mL, 1250 ng / mL, or 1500 ng / mL. In some embodiments, the antimitotic agent (e.g., nocodazole) is present in the cell culture media at a concentration of about 125 ng / mL. In some embodiments, the antimitotic agent (e.g., nocodazole) is present in the cell culture media at a concentration of about 250 ng / mL. In some embodiments, the antimitotic agent (e.g., nocodazole) is present in the cell culture media at a concentration of about 625 ng / mL. In some embodiments, the antimitotic agent (e.g., nocodazole) is present in the cell culture media at a concentration of about 1250 ng / mL.

[0051] In some aspects, the enhancer element is a histone deacetylase (HD AC) inhibitor. In some embodiments, the HD AC inhibitor is a hydroxamic acid (e.g., vironstat, belinostat, and panobinostat), short-chain fatty acid (e.g., valproic acid), benzamide (e.g., entinostat, compounds having structures similar to that of benzamides), cyclic peptide (e.g., romidepsin), or a sirtuin inhibitor (e.g., SIRT1, SIRT2). (In some embodiments, the HD AC inhibitor is M344 (4- (dimethylamino)-N-(7-(hydroxyamino)-7-oxoheptyl)benzamide). Examples of HD AC inhibitors are provided, for example, in Pu et al., Experimental Hematology & Oncology, 13, 45 (2024);

[0052] #14288902v1 Kim & Bae, Am J Transl Res. 2010 Dec 26;3(2):166-179. See also, WO 212 / 106343 and WO 2006 / 120456, each incorporated by reference to the extent it relates to HD AC inhibitors. In some embodiments, the HD AC inhibitor (e.g., M344) is present in the cell culture media at a concentration ranging from approximately 100 ng / mL to approximately 1,500 ng / mL. In certain embodiments, the HD AC inhibitor (e.g., M344) is present in the cell culture media at a concentration of about 200 ng / mL to about 1,200 ng / mL, about 225 ng / mL to about 1,125 ng / mL, about 300 ng / mL to about 900 ng / mL, about 400 ng / mL to about 1,300 ng / mL, or about 500 ng / mL to about 1,000 ng / mL. In some embodiments, the HD AC inhibitor (e.g., M344) is present in the cell culture media at a concentration of about 225 ng / mL, 500 ng / mL, 675 ng / mL, 1,000 ng / mL, 1,125 ng / mL, or 1,500 ng / mL.

[0053] In some aspects, the enhancer element is an organic solvent (e.g., dimethyl sulfoxide, DMSO). In some embodiments, the organic solvent in DMSO. In some embodiments, the organic solvent (e.g., DMSO) is present in the cell culture media at a concentration ranging from about 1% to about 20% (v / v). The term “NIN” refers to volume per volume and indicates the volume of a solute (e.g., organic solvent) expressed as a percentage of the total volume of the solution or mixture (e.g., cell culture media). For example, a 2% (v / v) concentration means 2 milliliters of the solvent are present in every 100 milliliters of the total solution. In some embodiments, the concentration of organic solvent (e.g., DMSO) is about 1% to about 15%, about 2% to about 18%, about 5% to about 12%, or about 8% to about 20% (v / v). In some embodiments, the organic solvent (e.g., DMSO) is present in the cell culture media at a concentration of about 1%, 2%, 5%, 10%, 15%, or 20% (v / v). In some embodiments, the organic solvent (e.g., DMSO) is present in the cell culture media at a concentration of 2% (v / v).

[0054] In some aspects, the enhancer element is provided in an amount sufficient to improve rAAV production relative to a method that does not comprise contacting a cell culture with one or more enhancer elements (a “control method”). In some aspects, rAAV production is improved by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, or 15-fold relative to a control method.

[0055] In some aspects, the enhancer element is provided in an amount sufficient to produce a higher titer of rAAV relative to a method that does not comprise contacting a cell culture with one or more enhancer elements (a “control method”). In some aspects, rAAV titer is increased by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, or 15-fold relative to a control method.

[0056] In some aspects, the enhancer is provided in an amount sufficient to produce a greater percentage of full capsids relative to a method that does not comprise contacting a cell culture

[0057] #14288902v1 with one or more enhancer elements (a “control method”). In some aspects, the percentage of full rAAV capsids is improved at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, or 15 -fold relative to a control method. In some aspects, the percentage of full capsids is increased by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control method.

[0058] In some aspects, the cell culture (e.g., cell culture media) is contacted with two or more enhancer elements. In some aspects, the cell culture is contacted with an AAV titer enhancer and an antimitotic agent, an AAV titer enhancer and an HD AC inhibitor, an AAV titer enhancer and an organic solvent, an antimitotic agent and an HD AC inhibitor, an antimitotic agent and an organic solvent, or an HD AC inhibitor and an organic solvent. In some aspects, each of the two or more enhancer elements is provided at a lower dose than when each is provided alone.

[0059] In some aspects, the cell culture is contacted with two or more enhancer elements. In some aspects, the cell culture is contacted with an AAV titer enhancer and nocodazole, an AAV titer enhancer and an HDAC inhibitor (e.g., M344), an AAV titer enhancer and DMSO, nocodazole and an HDAC inhibitor (e.g., M344), nocodazole and DMSO, or an HDAC inhibitor (e.g., M344) and DMSO. In some aspects, each of the two or more enhancer elements is provided at a lower dose than when each is provided alone.

[0060] In some aspects, the cell culture is contacted with three or more enhancer elements e.g., the cell culture is contacted with an antimitotic agent, an HDAC inhibitor, and an organic solvent). In some aspects, the cell culture is contacted with four or more enhancer elements. In some aspects, the cell culture is contacted with five or more enhancer elements. In some aspects, the cell culture is contacted with six or more enhancer elements.

[0061] In some aspects, the two or more enhancer elements are provided in an amount sufficient to improve rAAV production relative to a method that does not comprise contacting a cell culture with one or more enhancer elements (a “control method”). In some aspects, rAAV production is improved by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 11 -fold, 12-fold, 13 -fold, 14-fold, or 15-fold relative to a control method.

[0062] In some aspects, the two or more enhancer elements s are provided in an amount sufficient to produce a higher titer of rAAV relative to a method that does not comprise contacting a cell culture with one or more enhancer elements (a “control method”). In some aspects, rAAV titer is increased by at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, or 15 -fold relative to a control method.

[0063] #14288902v1 In some aspects, the two or more enhancer elements are provided in an amount sufficient to produce a greater percentage of full capsids relative to a method that does not comprise contacting a cell culture with one or more enhancer elements (a “control method”). In some aspects, the percentage of full rAAV capsids is improved at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, or 15-fold relative to a control method. In some aspects, the percentage of full capsids is increased by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control method.

[0064] Methods

[0065] Aspects of the disclosure provide methods for producing recombinant adeno-associated viral (rAAV) particles. Aspects of the disclosure provide methods of preparing a cell culture (including cell culture media) for rAAV production.

[0066] In some aspects, the method comprises transient transfection of mammalian host cells (e.g., human host cells). In some aspects, transfection comprises contacting a cell culture with one or more nucleic acids, one or more enhancer elements, and one or more transfection reagents. In some aspects, the one or more recombinant nucleic acids comprise deoxyribonucleic acid (DNA). In some embodiments, the one or more recombinant nucleic acids comprise ribonucleic acid (RNA). In some embodiments, the one or more recombinant nucleic acids comprise one or more plasmids.

[0067] In some aspects, transient transfection comprises a triple or dual transfection system. As used herein, a dual transfection system refers to the introduction of two different plasmids into a host cell simultaneously. Each plasmid carriers different genetic elements needed to produce the desired product (e.g., AAV vectors). For example, one plasmid might contain the rep / cap genes (which encode viral replication and capsid proteins), and the other plasmid might carry the vector genome (the therapeutic gene flanked by viral inverted terminal repeats). The host cell machinery expresses these components to produce recombinant virus particles. In some aspects, a dual plasmid transfection system comprises (i) a first plasmid comprising: one or more recombinant nucleic acids encoding an AAV Rep protein, one or more recombinant nucleic acids encoding an AAV Cap protein and the one or more recombinant nucleic acids encoding the gene of interest flanked by inverted terminal repeats (ITRs); and (ii) a second plasmid comprising: one or more recombinant nucleic acids encoding AAV helper genes. As used herein, a triple transfection system refers to the introduction of three different plasmids into a host cell simultaneously. In some aspects, the triple plasmid transfection system comprises, (i) a

[0068] #14288902v1 first plasmid comprising: one or more recombinant nucleic acids encoding an AAV Rep protein, one or more recombinant nucleic acids encoding an AAV Cap protein; (ii) a second plasmid comprising: the one or more recombinant nucleic acids encoding the gene of interest flanked by inverted terminal repeats (ITRs); and (iii) a third plasmid comprising: one or more recombinant nucleic acids encoding AAV helper genes. Examples of AAV helper genes include those genes that provide necessary helper functions from adenovirus or herpesvirus genes (e.g., E2A, E4, and VA RNA). They typically support viral replication but are not present in the final vector.

[0069] In some aspects, the transfection system comprises one or more recombinant nucleic acids encoding a gene of interest flanked by AAV inverted terminal repeats (ITRs). In some embodiments, the ITRs are AAV2 or AAV9 ITRs. In some embodiments, the ITRs are synthetic or modified ITRs.

[0070] In some aspects, the transient transfection comprises contacting a cell culture comprising the mammalian host cells with one or more enhancer elements. In some aspects, the cell culture is contacted with the one or more enhancer elements at the same time as the one or more recombinant nucleic acids. In some embodiments, the cell culture is contacted with the one or more enhancer elements at a different time as the one or more recombinant nucleic acids (e.g., before or after).

[0071] In some aspects, the method further comprises contacting the cell culture comprising the mammalian host cells with a feed medium, about 4 hours to about 8 hours (e.g., 4, 5, 6, 7, 8 or more hours) following the transient transfection.

[0072] In some aspects, the cell culture produces recombinant adeno-associated virus (rAAV) particles.

[0073] In some aspects, the method further comprises isolating rAAV particles from the cell culture. In some aspects, isolating rAAV from the cell culture comprises harvesting the cell culture. In some aspects, isolating rAAV from the cell culture comprises lysing the cells comprising the cell culture. In some aspects, isolating rAAV from the cell culture comprises removing rAAV from the cell culture medium.

[0074] In some aspects, the titer of rAAV isolated from the cell culture is at least lxl0Al 1, 2xlOAl l, 3xl0Al l, 4xlOAl l, 5xl0Al l, 6xlOAl l, 7xlOAl l, 8xl0Al l, 9xlOAl l, lxlOA12,

[0075] 2xlOA12, 3xlOA12, 4 xlOA12, 5xlOA12, 6xlOA12 vg / mL.

[0076] In some embodiments, the method is carried out at a room temperature. In some embodiments, the method is carried out at 10-40 °C, for example 15-35 °C, 15-20 °C, 20-25 °C, or 25-30 °C (e.g., about 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C,

[0077] 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C,

[0078] #14288902v1 36°C, 37°C, 38°C, 39°C, or 40°C). In some embodiments, one or more steps of the method are carried out at room temperature. In some embodiments, one or more steps of the method are carried out at 10-40 °C, for example 15-35 °C, 15-20 °C, 20-25 °C, or 25-30 °C (e.g., about 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C).

[0079] In some embodiments, pH of the medium is monitored. In some embodiments, the method is carried out at a pH of between 6.9 and 7.5, between 7 and 7.4, between 7.1 and 7.3, between 6.9 and 7.2, or between 7 and 7.3. In some embodiments, the pH is about 7. In some embodiments, the pH of the medium is measured for one or more steps of the method. In some embodiments, one or more steps of the method are carried out at a pH of between 6.9 and 7.5, between 7 and 7.4, between 7.1 and 7.3, between 6.9 and 7.2, or between 7 and 7.3. In some embodiments, the pH is about 7. rAAV Production

[0080] Recombinant adeno-associated virus (rAAV) vectors are useful in gene therapy to deliver therapeutic genes to patient cells and tissue. An rAAV particle typically comprises a recombinant nucleic acid encapsidated within rAAV capsid proteins to form an rAAV particle that can be administered to a subject. The recombinant nucleic acid (e.g., recombinant AAV genome) typically includes a heterologous gene of interest (e.g., encoding a therapeutic nucleic acid and / or protein) flanked by AAV inverted terminal repeat (ITR) sequences. In some embodiments, the rAAV capsid proteins can be naturally occurring capsids of different AAV serotypes. For example, different AAV serotypes have different tissue tropisms and can be used to target different tissue types and associated diseases. In some embodiments, the rAAV capsid proteins include one or more amino acid substitutions relative to naturally occurring capsid proteins.

[0081] Different manufacturing techniques can be used to produce rAAV particles. Typically, rAAV particles are assembled in a host cell culture (e.g., in a bioreactor or other cell culture vessel). In some embodiments, the cell culture is maintained or grown using standard methods (e.g., in a suspension culture, or on plates). The assembled rAAV is then isolated from the cell culture. In some embodiments, the cell culture comprises mammalian cells, insect cells or cells of another cell type.

[0082] Manufacturing rAAV, in some aspects, comprises using a cell culture to produce and assemble the parts of rAAV particles. Without intending to be bound by theory, producing

[0083] #14288902v1 rAAV particles comprises i) expression of Rep and Cap genes, ii) providing a recombinant genome, typically comprising a gene of interest, and iii) providing the additional functions, sometime referred to as helper functions, required for rAAV replication. In some embodiments, the cell culture comprises cells that have genes encoding Rep and Cap integrated into the cells’ genome. In some embodiments, the cell culture comprises cells that have been stably transfected with a plasmid comprising the genes encoding Rep and Cap. In some embodiments, the cells further comprise a plasmid containing an rAAV genome. Without intending to be bound by theory, these cells are typically infected with a helper virus that provides additional factors necessary for rAAV replication. In some embodiments, the helper virus is an adenovirus, such as Ad5. In some embodiments, the cell culture comprises cells that are transiently transfected with plasmids encoding Rep, Cap, an rAAV genome, and helper functions. In some embodiments, two plasmids are used (double transfection). In some embodiments, three plasmids are used (triple transfection). Providing the helper virus, or transfecting with all necessary components, causes the cells to begin producing rAAV.

[0084] Aspects of the disclosure relate, at least in part, to methods and compositions for culturing a cell culture under conditions suitable for rAAV production (“production conditions”). As described herein, production conditions are conditions under which expression of one or more genes required for rAAV production, packaging, and / or growth is promoted (e.g., by addition of a helper virus, induction of an inducible promoter, or transfection with one or more vectors encoding one or more genes required for rAAV production, packaging, and / or growth).

[0085] Transient transfection

[0086] Transient transfection is one way of introducing heterologous genetic material to cells of interest. As used herein, “transfection” means nucleic acid transfection. Briefly, transfection comprises contacting a cell culture with one or more nucleic acids and a transfection reagent, also called a “transfecting agent.”

[0087] In some embodiments, a transfection reagent comprises a commercially available transfection reagent. In some embodiments, a transfection reagent comprises a chemical transfection reagent. In some embodiments, a transfection reagent comprises a liposomal-based transfection reagent. In some embodiments, a transfection reagent comprises a liposome. In some embodiments, the liposome is a positively charged or cationic liposome. In some embodiments, a transfection reagent comprises a non-liposomal-based transfection reagent. In

[0088] #14288902v1 some embodiments, a transfection reagent comprises a calcium phosphate, dendrimer, polymer, nanoparticle, or non-liposomal lipid. In some embodiments, a transfection reagent comprises lipofectamine or a variant thereof. In some embodiments, transfection is carried out by electroporation nucleofection which is a method that uses a combination of electrical and chemical factors, using a device (e.g., Nucleofector™ by Lonza). Transfection reagents include, but are not limited to, polyethylenimine (PEI), animal-free transfection reagents (e.g., such as those sold under the trademark FectoVIR® (polyplus)), pure lipid or high-lipid based transfection agents (such as those sold as: Nanofectamine (GE Healthcare), Oligofectamine (Invitrogen), RNAiMAX (Invitrogen), siPORT (ThermoFisher), DharmaFECT (Dharmacon), Endofectin@MAX (GeneCopoeia), Escort IV Liposome (Sigma- Aldrich)), and mixed lipid and / or non-lipid based transfection reagents (such as those sold as Arrest-In (Dharmacon), TurboFect (Thermo), Effectene (Qiagen), Attractene (Qiagen), PolyFect (Qiagen), SuperFect (Qiagen), ExpressFect (Thomas), Genejammer (Stratagene), FuGENE (Promega), INTERFERin (Polyplus), NanoFectin (System Biosciences), X-tremeGENE (Roche), Xfect (ClonTech), Escort IV (Sigma- Aldrich), and N-TER (Sigma- Aldrich)). In some embodiments, the transfection reagent is FECTOVIR®.

[0089] In some embodiments, the cell culture is contacted with one or more nucleic acids, optionally in the presence of an transfection reagent. In some embodiments, the one or more nucleic acids are recombinant nucleic acids. In some embodiments, the one or more recombinant nucleic acids comprise deoxyribonucleic acid (DNA) in some embodiments, the one or more recombinant nucleic acids comprise ribonucleic acid (RNA).

[0090] In some embodiments, the one or more recombinant nucleic acids comprise one or more plasmids. In some embodiments, the one or more recombinant nucleic acids are provided as one or more plasmids. In some embodiments, the plasmids comprise further polynucleotide sequences, such as one or more replication elements or regulatory elements, such as promoters and / or transcriptional control elements. In some embodiments, the one or more nucleic acids comprise two plasmids. In some embodiments, the two plasmids comprise a dual transfection system. In some embodiments, the one or more nucleic acids comprise three plasmids. In some embodiments, the three plasmids comprise a triple transfection system. In some embodiments, multiple molar ratios of plasmids are used. In some embodiments, in the dual transfection system, one plasmid comprises a gene of interest flanked by ITRs, nucleic acid sequences for encoding AAV Rep and Cap proteins, and a second plasmid comprises nucleic acid sequences encoding AAV helper functions, such as a pAdhelper plasmid comprising nucleic acid

[0091] #14288902v1 sequences for helper genes e.g., E1A, E1B, E2A, VA, and E4orf6 functions. In some embodiments, switching from a three plasmid system (also referred to herein as a “triple transfection plasmid system”) to a dual plasmid system increases AAV particle production by at least 2 fold. In some embodiments, AAV particle production is increased by about 3 fold by switching from a triple transfection plasmid system to a dual transfection plasmid system.

[0092] In some embodiments, the one or more recombinant nucleic acids encode a recombinant AAV (rAAV) genome. In some embodiments, the rAAV genome comprises a gene of interest flanked by inverted terminal repeats (ITRs). In some embodiments, the ITRs comprise AAV2 ITRs or AAV9 ITRs. In some embodiments, the gene of interest encodes an antibody, enzyme, protein, growth factor, miRNA, or hormone. In some embodiments, the one or more recombinant nucleic acids encode a capsid (Cap) protein. In some embodiments, the capsid protein is an AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV 11, or AAV 12 capsid protein, or a variant thereof. In some embodiments, the one or more recombinant nucleic acids also encode a Rep protein.

[0093] Producer cells

[0094] In some embodiments, a cell culture associated with the disclosure comprises producer cells. In some embodiments, a producer cell stably expresses rep and cap genes suitable for rAAV packaging. In some embodiments, a producer cell further comprises an rAAV genome. In some embodiments, the rAAV genome comprises a therapeutic gene.

[0095] In some embodiments, the cell culture (e.g., producer cells of the cell culture) comprises HeLa cells. In some embodiments, the cell culture comprises human embryonic kidney (HEK) cells. In some embodiments, the cell culture comprises Chinese hamster ovary (CHO) cells. In some embodiments, the cell culture comprises Vero cells. In some embodiments, the cell culture comprises Madin-Darby canine kidney (MDCK) cells. In some embodiments, the cell culture comprises baby hamster kidney (BHK) cells. In some embodiments, the cell culture comprises A549 cells. In some embodiments, the cell culture comprises amniocyte cells.

[0096] In some embodiments, the producer cell culture is infected with a helper virus. In some embodiments, the helper virus is an adenovirus. In some embodiments, the helper virus is Ad5. In some embodiments, the producer cell culture does not require helper virus.

[0097] #14288902v1 Cell Culture Media

[0098] Aspects of the present disclosure provide methods of preparing a cell culture for rAAV production and contacting a cell culture with one or more enhancer elements, optionally wherein the cell culture comprises cell culture media (medium).

[0099] In some aspects, the cell culture is provided in a medium. In some aspects, the cell culture is incubated in a medium during rAAV production. In some aspects, the medium is an appropriate medium for cell growth, infection, transfection, and / or rAAV production in cells (for example, mammalian cells). In some aspects, the medium is a medium that supports cell growth (e.g., high-density cell growth). In some aspects, the medium provides one or more nutrients. In some aspects, the medium is a feed medium. In some aspects, the medium comprises a complex medium. In some aspects, the medium is a defined medium. In some aspects, the medium comprises Minimal Essential Medium (MEM), Eagle’s Minimum Essential Medium (EMEM), Dulbecco’s Modified Eagle’s Medium (DMEM), LV-MAX media (Gibco), EX-Cell media, or RPMI media. In some aspects, the medium is a serum-free medium. In some embodiments, the medium is supplemented with serum. In some aspects, the medium is an appropriate medium for cell growth, infection, transfection, and / or rAAV production in HEK293 cells. In some aspects, the medium is an appropriate medium for cell growth, infection, transfection, and / or rAAV production in HeLa cells. In some aspects, the medium is an appropriate medium for AAV production.

[0100] In some embodiments, the cell culture media (medium) comprises a balanced formulation of carbon and nitrogen sources, essential inorganic salts, trace elements, vitamins, buffering agents, and optional supplements such as lipids or growth factors. The medium may be used in batch, fed-batch, or perfusion-based processes and is suitable for use in adherent or suspension-adapted cells, such as HEK293 or HeLa cells.

[0101] In some embodiments, the cell culture medium includes one or more carbon sources selected from glucose, galactose, fructose, mannose, glycerol, or their derivatives. In some embodiments, the one or more carbon sources are present in the medium in a concentration range of about 0.5 g / L to 10 g / L (e.g., 1 g / L to 8 g / L; 2 g / L to 7 g / L; 3 g / L to 6 g / L, or 4 g / L to 5 g / L; about 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L or more).

[0102] In some embodiments, the nitrogen source may include amino acids (e.g., glutamine, asparagine, arginine), peptides, or inorganic nitrogen such as ammonium salts (e.g., ammonium chloride or ammonium sulfate), present in total concentrations ranging from approximately 0.1 g / L to 5 g / L (e.g., 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L or

[0103] #14288902v1 more). In some embodiments, glutamine or a stabilized dipeptide form (e.g., alanyl-glutamine) may be included at 2-6 mM (e.g., 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, or more) to support both energy metabolism and protein biosynthesis.

[0104] The cell culture media (medium), in some embodiments, comprises inorganic salts. Inorganic salts are provided, in some embodiments, to maintain osmolarity, membrane stability, and ionic balance. Examples of inorganic salts include, but are not limited to, sodium chloride (e.g., 1-8 g / L), potassium chloride (e.g., 0.1-1.0 g / L), calcium chloride (e.g., 0.05-0.5 g / L), magnesium sulfate (e.g., 0.05-0.5 g / L), and sodium phosphate buffers (e.g., 0.5-2.0 g / L). The pH of the cell culture medium (media) is maintained, in some embodiments, in the range of about 6.8 to 7.6 (e.g., 7.0-7.4), using a combination of HEPES (e.g., 10-25 mM), sodium bicarbonate (e.g., 1-3 g / L), or other suitable buffering agents. The osmolality of the cell culture medium, in some embodiments, is adjusted to support cell viability and viral vector production, and may range from 280 to 350 mOsm / kg, depending on the specific cell line and process conditions. In some embodiments, the osmolality is maintained at a value selected from 280, 290, 300, 310, 320, 330, 340, or 350 mOsm / kg. Osmolality may be adjusted using combinations of sodium chloride, potassium chloride, or other osmotically active components, and may be monitored throughout the culture process to ensure consistency and reproducibility.

[0105] Cell Culture

[0106] In some aspects, the present disclosure relates to methods of scaling up and / or increasing rAAV production by a cell culture.

[0107] In some aspects, the cell culture comprises animal cells. In some aspects, the cell culture comprises mammalian cells. In some embodiments, the cell culture comprises HeLa cells. In some embodiments, the cell culture comprises human embryonic kidney (HEK) cells. In some embodiments, the cell culture comprises Chinese hamster ovary (CHO) cells. In some embodiments, the cell culture comprises Vero cells. In some embodiments, the cell culture comprises Madin-Darby canine kidney (MDCK) cells. In some embodiments, the cell culture comprises baby hamster kidney (BHK) cells. In some embodiments, the cell culture comprises A549 cells. In some embodiments, the cell culture comprises amniocyte cells.

[0108] In some embodiments, the cell culture produces adeno-associated virus (AAV) particles. In some embodiments, the cell culture produces recombinant AAV (rAAV) particles. In some embodiments, the presently described methods are combined with other methods to increase production of rAAV particles. In some embodiments, combining the presently-described

[0109] #14288902v1 methods with additional improvements in, for example feeding methods, leads to a 10-fold increase in AAV particle production or productivity. In some embodiments, successful scale-up of the entire manufacturing method is demonstrated from 250 mL reactors to 500 L bioreactors. In some embodiments, the presently disclosed method improves the production of rAAV particles relative to a control method (e.g., a method in which an enhancer element is not used). In some embodiments, the presently disclosed method increases production of rAAV particles relative to a control method. In some embodiments, the method of this application increases production of rAAV particles by 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 -fold, 12 fold, 13 fold, 14 fold, or 15 fold relative to a control method.

[0110] In some embodiments, the method of this application improves the percent of full capsids produced relative to a control method. In some embodiments, the method of this application increases production of full capsids by 1.5 fold, 2 fold, 3 fold, 4 fold, or 5 fold (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) relative to a control method.

[0111] In some embodiments, the method of this application improves packaging efficiency at harvest relative to a control method. In some embodiments, the method of this application increases packaging efficiency by 1.5 fold, 2 fold, 3 fold, 4 fold, or 5 fold e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) relative to a control method. In some embodiments, packaging efficiency is improved due to higher capsids with total genomes in an early phase of rAAV production. In some embodiments, the method of this application increases total genomes at 24 and / or 48 hour timepoints (e.g., 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 hours, or more). In some embodiments, the method of this application increases total genomes at 24 and / or 48 hour timepoints by 1.25 fold, 1.5 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 11 fold, 12 fold, 13 fold, 14 fold, 15 fold, 16 fold, 17 fold, 18 fold, 19 fold, 20 fold, 21 fold, 22 fold, 23 fold, 24 fold, or 25 fold relative to a control method.

[0112] In some embodiments, the method of this application reduces the time required to produce levels of rAAV for harvest relative to a control method. In some embodiments, the method reduces the time by 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 24 hours, 28 hours, or more. In some embodiments, the method reduces the time by 1 day.

[0113] #14288902v1 Recombinant A A Vs

[0114] Naturally occurring AAV capsid proteins can be used to produce rAAVs for gene therapy. Different naturally occurring AAVs have different characteristics (including for example different tissue tropisms) and can be used for different indications. AAVs are highly prevalent within the human population (see Gao, G., et al., Clades of Adeno-associated viruses are widely disseminated in human tissues J Virol. 2004. 78(12): p. 6381-8, and Boutin. S., et al., Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population, implications forgone therapy using AAV vectors. Hum Gene Ther. 2010. 21(6): p. 704-12) and are useful as viral vectors. Many serotypes exist, each with different tropism for tissue types (see Zincarelli, C., et al., Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther, 2008. 16(6): p. 1073-80), which allows specific tissues to be preferentially targeted with appropriate pseudotyping. Some serotypes, such as serotypes 8, 9, and rhlO, transduce the mammalian body. See Zincarelli, C., et al. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther, 2008. 16(6): p. 1073-80, Inagaki, K., et al., Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8. Mol Ther, 2006. 14(1): p. 45-53, Keeler, A. M., et al., Long-term correction of very long-chain acyl-coA dehydrogenase deficiency in mice using AAV9 gene therapy. Mol Ther, 2012. 20(6): p. 1131-8, Gray, S. J., et al., Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates. Mol Ther, 2011. 19(6): p. 1058-69, Okada, H., et al., Robust Long-term Transduction of Common Marmoset Neuromuscular Tissue With rAAVl and rAAV9. Mol Ther Nucleic Acids, 2013. 2: p. e95, and Foust, K. D., et al., Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol, 2009. 27(1): p. 59-65. AAV9 has been demonstrated to cross the blood-brain barrier (see Foust, K. D., et al., Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol, 2009. 27(1): p. 59-65, and Rahim, A. A., et al., Intravenous administration of AAV2 / 9 to the fetal and neonatal mouse leads to differential targeting of CNS cell types and extensive transduction of the nervous system. FASEB J, 2011. 25(10): p. 3505-18) that is inaccessible to many viral vectors and biologies. Certain AAVs have a payload of 4.7-5.0 kb (including viral inverted terminal repeats (ITRs), which are required in cis for viral packaging). See Wu, Z., H. Yang, and P. Colosi, Effect of genome size on AAV vector packaging. Mol Ther, 2010. 18(1): p. 80-6 and Dong, J. Y., P. D. Fan, and R. A. Frizzell, Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum Gene Ther, 1996. 7(17): p. 2101-12.

[0115] #14288902v1 In some embodiments, rAAVs can include one or more variant AAV capsid proteins having one or more amino acid substitutions relative to a naturally occurring AAV capsid protein.

[0116] Accordingly, in some embodiments, the rAAV particles comprise AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAV12 capsid proteins, or amino acid sequence variants thereof, or chimeras, or hybrids thereof.

[0117] In some embodiments, the rAAV particles comprise an rAAV genome. In some embodiments, an rAAV genome comprises a gene of interest flanked by inverted terminal repeats (ITRs). In some embodiments, a gene of interest comprises a therapeutic molecule, such as a therapeutic protein or therapeutic RNA. In some embodiments, a therapeutic molecule is an antibody, a protein, a peptide, an enzyme, or a ribozyme. In some embodiments, a gene of interest encodes a detectable molecule (e.g., for the purpose to conducting research studies). In some embodiments, the detectable molecule is a fluorescent protein, a bioluminescent protein, or a protein that provides color (e.g., P-galactosidase, P-lactamases, P-glucuronidase, or spheriodenone). In some embodiments, a detectable molecule is a fluorescent, bioluminescent, enzymatic protein, or functional peptide or functional polypeptide thereof. In some embodiments, a gene of interest encodes a therapeutic protein or therapeutic RNA. In some embodiments, a therapeutic gene encodes an antibody, a peptibody, a growth factor, a clotting factor, a hormone, a membrane protein, a cytokine, a chemokine, an activating or inhibitory peptide acting on cell surface receptors or ion channels, a cell-permeant peptide targeting intracellular processes, a thrombolytic, an enzyme, a bone morphogenetic proteins, a nuclease or other protein used for gene editing, an Fc-fusion protein, an anticoagulant, a nuclease, guide RNA, or other nucleic acid or protein for gene editing.

[0118] Downstream Processing

[0119] In some embodiments, rAAV is isolated from the cell culture and prepared (e.g., purified and / or sterilized) for delivery to a human subject (e.g., to deliver a therapeutic gene to the subject to assist in the treatment of a disease or condition).

[0120] In some embodiments, the rAAV is harvested from the cell culture. In some embodiments, the harvested rAAV product is clarified, for example, by centrifugation or with depth filtration.

[0121] #14288902v1 In some embodiments, rAAV isolation comprises a further clarification and / or purification step. In some embodiments, rAAV isolation comprises a flocculation step. In some embodiments, a lysis agent, for example a detergent, can be used along with the flocculation agent.

[0122] In some embodiments, rAAV particles are further purified, for example using one or more affinity, ion exchange chromatography, and / or hydrophobic interaction chromatography steps, e.g., after clarification of an rAAV preparation. In some embodiments, the rAAV is harvested, clarified, and then further purified using tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, sterile filtration, or any combination(s) thereof.

[0123] In some embodiments, rAAV particles (e.g., after one or more purification steps) are added to a pharmaceutically acceptable solution. The term “pharmaceutically acceptable solution” refers to a diluent, adjuvant, excipient, or vehicle with which the rAAV particle is administered to a subject.

[0124] The disclosure also provides compositions comprising rAAV particles, and methods of administering the rAAV particles to a subject (e.g., a human subject having a condition that the therapeutic RNA and / or protein can help treat). In some embodiments, the rAAV particles (e.g., pharmaceutical compositions comprising the rAAV particles and a pharmaceutically acceptable solution) are administered subcutaneously, intraocularly, intravitreally, subretinally, parenterally, intravenously (IV), intracerebro-ventricularly, intramuscularly, intrathecally (IT), intracistemally, orally, intraperitoneally, by oral or nasal inhalation, or by direct injection to one or more cells, tissues, or organs by direct injection. In some embodiments, the administration is a route suitable for systemic delivery, such as by intravenous injection.

[0125] These and other aspects are illustrated by the following non-limiting examples.

[0126] EXAMPLES

[0127] Example 1. Enhancer elements improve rAAV yield

[0128] Cells were grown using standard methods in preparation for rAAV production. On Day 0 (approximately 24 hours prior to transfection), cells were split to half the target transfection density. On Day 1, before the transfection, a volume of cell culture media equal to 10% of the total cell culture volume was mixed with DNA (0.5ug DNA for each million cells / volume) and an enhancer element or sham. The media, DNA, and enhancer combination was mixed and

[0129] #14288902v1 transfection reagent (FectoVir) was added, maintaining a ratio of lug DNA to luL transfection reagent. The solution was then mixed and incubated for 20 minutes. After 20 minutes, the mixture was added to cells prepared for transfection.

[0130] Nocodazole (additive 2) was provided at 225 ng / mL. DMSO was provided at 2% (additive 4). An HD AC inhibitor (M344) (additive 3) was provided at 225 ng / mL. The AAV titer enhancer (additive 1) was provided at IX or 1.5X.

[0131] Titer was measured by ddPCR at harvest for each of the tested conditions. Results are shown in FIG. 1 for a shake flask scale experiment comparing dose of AAV titer enhancer, and FIG. 2 for a scaled-up experiment comparing the effect of AAV titer enhancer across different transgene constructs. The effect of single enhancer elements relative to combinations of enhancer elements is shown in FIGs. 4A-4B for two transgene constructs.

[0132] Percent full and partially full capsids was also determined. Results are shown in FIGs. 3A-3B.

[0133] Varied concentrations of each enhancer were further tested at low density. Experiments were carried out as described above. Nocodazole (additive 2) was provided at 0.5X, IX, 2.5X, and 5X, with IX being equivalent to 250 ng / mL. DMSO was provided at 2% (additive 4). An HD AC inhibitor (M344) (additive 3) was provided at IX, 3X, and 5X, with IX being 225 ng / mL. The AAV titer enhancer (additive 1) was provided at 0.7X or IX. At a dose of IX, 30 uL of additive 1 were added per 20 mL of cell culture. Results are shown in FIG. 6.

[0134] Example 2. Enhancer elements improve rAAV productivity in larger scale

[0135] The enhancer elements described in Example 1 were tested in Amber 250 (AM250) scale cultures with low cell density (approximately 3 million cells / mL) at the time of transfection. The concentration of each enhancer element that showed the greatest improvement in titer in smaller scale (see Example 1) was used in the scaled- up experiment. As shown in FIG. 7, enhancer elements increased rAAV titer in larger scale cultures, similar to smaller-scale experiments.

[0136] Example 3. AAV titer enhancer improves productivity in intensified process for several rAAV constructs

[0137] The effectiveness of the AAV titer enhancer (additive 1) was tested with an intensified production process. The intensified production process comprised an additional feed at 24 hours post-transfection and higher cell density (approximately 6 million cells / mL). The intensified process was tested with and without the AAV titer enhancer. The titers from the intensified process was further compared with production in a low density culture without the AAV titer

[0138] #14288902v1 enhancer. As shown in FIG. 8A, the AAV titer enhancer increased productivity in the intensified process relative to the same conditions without the AAV titer enhancer or a low density culture.

[0139] The intensified process supplemented with the AAV titer enhancer was tested in with four different AAV constructs, each comprising a different capsid and a different transgene. As shown in FIG. 8B, the AAV titer enhancer increased productivity in each case.

[0140] The effect of additive on each combination of capsid and transgene was tested at AM250 and 3L scale. FIGs. 9A-9C show fold change in rAAV productivity (FIG. 9A), percent full capsids (FIG. 9B), and percent partially full capsids (FIG. 9C) in AM250 and 3L cultures with and without IX additive 1 (AAV titer enhancer (Minis Bio)). The results show no significant negative impact on AAV quality when using additive 1.

[0141] Example 4. AAV titer enhancer increases packaging efficiency for several rAAV constructs

[0142] Cells were used for rAAV production as described above. Packaging efficiency and total genomes present at 24 and 48 hours were measured. Results are shown in FIGs. 10A-10C.

[0143] FIGs. 10A-10C show fold change in packaging efficiency at harvest (FIG. 10A), total genomes at 24 hours post-transfection (FIG. 10B), and total genomes at 48 hours posttransfection (FIG. 10C) in AM250 and 3L cultures with and without IX additive 1 (AAV titer enhancer (Mirus Bio)). Additive 1 showed an increase in packaging efficiency at harvest.

[0144] Example 5. Cellular pathways influenced by additives

[0145] Characteristics of cells treated with additive 1 and additive 2 were measured to determine the effect of each additive on cells. Results are shown in FIGs. 11A-11D. As shown in FIG. 11A, cells treated with 0.7X or IX doses of additive 1 grow more slowly than control cells, as measured by a cell counter over time. As shown in FIG. 11B, cells treated with 0.7X or IX doses of additive 1 are larger than control cells, as measured by a cell counter that quantifies cell diameter via imaging. As shown in FIG. 11C, more cells treated with 0.7X or IX doses of additive 1 are synced in G2 phase relative to control cells, as measured by NucleoCounter. NucleoCounter stained nuclear DNA and measured fluorescence. Double fluorescence indicates twice as much DNA, which indicates the cell is in G2 phase. As shown in FIG. 11D more cells treated with IX, 2.5X, or 5X doses of additive 2 are synced in G2 phase relative to control cells.

[0146] #14288902v1 EQUIVALENTS

[0147] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0148] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0149] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0150] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0151] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in

[0152] #14288902v1 conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0153] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0154] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0155] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0156] #14288902v1 In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that embodiments described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative embodiments, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the disclosure describes “a composition comprising A and B”, the disclosure also contemplates the alternative embodiments “a composition consisting of A and B” and “a composition consisting essentially of A and B”.

[0157] #14288902v1

Claims

CLAIMSWhat is claimed is:

1. A method for producing recombinant adeno-associated viral (rAAV) particles, the method comprising: transiently transfecting mammalian host cells using a triple or dual transfection system comprising one or more recombinant nucleic acids encoding a gene of interest flanked by AAV inverted terminal repeats (ITRs), wherein the transient transfection comprises contacting a cell culture comprising the mammalian host cells with one or more enhancer elements, wherein the one or more enhancer elements comprise an AAV titer enhancer, nocodazole, an HD AC inhibitor, and / or DMSO.

2. A method comprising contacting a cell culture with one or more enhancer elements, wherein the one or more enhancer elements comprise an AAV titer enhancer, nocodazole, an HD AC inhibitor, and / or DMSO, and wherein the cell culture comprises one or more recombinant nucleic acids.

3. The method of claim 1 or claim 2, wherein the one or more recombinant nucleic acids comprise a recombinant adeno-associate virus (rAAV) genome.

4. The method of claim 3, wherein the rAAV genome comprises a gene of interest flanked by inverted terminal repeats (ITRs).

5. The method of claim 4, wherein the ITRs comprise AAV2 ITRs or AAV9 ITRs.

6. The method of claim 4 or 5, wherein the gene of interest encodes an antibody, an enzyme, a growth factor, a microRNA, or a hormone.

7. The method of any one of claims 1-6, wherein one of the one or more recombinant nucleic acids encodes a capsid (Cap) protein.

8. The method of claim 7, wherein the capsid protein is an AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10 capsid protein or variants or chimeras or hybrids thereof.#14288902v19. The method of any one of claims 1-8, wherein one of the one or more recombinant nucleic acids encodes a Rep protein, optionally wherein the Rep protein is Rep78, Rep68,Rep52, Ref40, or any combination thereof.

10. The method of any one of claims 1-9, wherein the one or more recombinant nucleic acids further comprise one or more nucleic acid sequences encoding AAV helper genes, wherein optionally the AAV helper genes comprise at least one of adenovirus genes E1A, E1B, E2A, VA, and E4orf6.

11. The method of any one of claims 1-10, wherein the cell culture comprises epithelial cells.

12. The method of any one of claims 1-11, wherein the cell culture comprises HEK cells.

13. The method of any one of claims 1-12, wherein the cell culture comprises HEK293 cells.

14. The method of any one of claims 1-13, wherein the one or more recombinant nucleic acids comprise deoxyribonucleic acid (DNA).

15. The method of any one of claims 1-13, wherein the one or more recombinant nucleic acids comprise ribonucleic acid (RNA).

16. The method of any one of claims 1-14, wherein the one or more recombinant nucleic acids comprise one or more plasmids.

17. The method of claim 16, wherein the one or more recombinant nucleic acids comprise a dual plasmid transfection system.

18. The method of claim 17, wherein the dual plasmid transfection system comprises (i) a first plasmid comprising: one or more recombinant nucleic acids encoding the Rep protein, one or more nucleic acids encoding the Cap protein and the gene of interest flanked by inverted terminal repeats (ITRs); and (ii) a second plasmid comprises: one or more nucleic acids encoding AAV helper genes.#14288902v119. The method of claim 16, wherein the one or more recombinant nucleic acids comprise a triple plasmid transfection system.

20. The method of claim 19, wherein the triple plasmid transfection system comprises, (i) a first plasmid comprising: one or more recombinant nucleic acids encoding the Rep protein, one or more nucleic acids encoding the Cap protein; (ii) a second plasmid comprising: one or more recombinant nucleic acids encoding the gene of interest flanked by inverted terminal repeats (ITRs); and (iii) a third plasmid comprising: one or more nucleic acids encoding AAV helper genes.

21. The method of any one of claims 1-20, wherein the cell culture produces recombinant adeno-associated virus (rAAV) particles.

22. The method of any one of claims 1-21, further comprising contacting the cell culture comprising the mammalian host cells with a feed medium, about 4 hours to about 8 hours following the transient transfection.

23. The method of any one of claims 1-22, comprising transient transfection of the mammalian host cells using the dual transfection system.

24. The method of claim 23, wherein the dual plasmid transfection system comprises (i) a first plasmid comprising: one or more recombinant nucleic acids encoding an AAV Rep protein, one or more recombinant nucleic acids encoding an AAV Cap protein and the one or more recombinant nucleic acids encoding the gene of interest flanked by inverted terminal repeats (ITRs); and (ii) a second plasmid comprising: one or more recombinant nucleic acids encoding AAV helper genes.

25. The method of any one of claims 1-22, comprising transient transfection of the mammalian host cells using the triple transfection system.

26. The method of claim 25, wherein the triple plasmid transfection system comprises, (i) a first plasmid comprising: one or more recombinant nucleic acids encoding an AAV Rep protein, one or more recombinant nucleic acids encoding an AAV Cap protein; (ii) a second plasmid comprising: the one or more recombinant nucleic acids encoding the gene of interest flanked by#14288902v1inverted terminal repeats (ITRs); and (iii) a third plasmid comprising: one or more recombinant nucleic acids encoding AAV helper genes.

27. The method of any one of claims 1-26, wherein the cell culture is contacted with two or more enhancer elements, wherein each of the two or more enhancer elements independently comprises an AAV titer enhancer, nocodazole, an HD AC inhibitor, and / or DMSO.

28. The method of any one of claims 1-27, wherein the enhancer is added in an amount sufficient to improve rAAV production relative to a method that does not comprise contacting a cell culture with one or more enhancer elements.

29. The method of any one of claims 1-28, wherein the enhancer is added in an amount sufficient to improve rAAV production by at least 3-fold relative to a method that does not comprise contacting a cell culture with one or more enhancer elements.

30. The method of any one of claims 1-29, wherein the enhancer is added in an amount sufficient to produce a higher titer of rAAV relative to a method that does not comprise contacting a cell culture with one or more enhancer elements.

31. The method of any one of claims 1-30, wherein the enhancer is added in an amount sufficient to produce a titer that is at least 3-fold higher relative to a method that does not comprise contacting a cell culture with one or more enhancer elements.

32. The method of any one of claims 1-31, wherein the enhancer is added in an amount sufficient to produce a greater percentage of full capsids relative to a method that does not comprise contacting a cell culture with one or more enhancer elements.

33. The method of any one of claims 1-32, wherein the enhancer is added in an amount sufficient to produce a percentage of full capsids that is at least 3-fold higher relative to a method that does not comprise contacting a cell culture with one or more enhancer elements.

34. The method of any one of the previous claims, wherein the method further comprises isolating rAAV from the cell culture.#14288902v135. The method of claim 34, wherein the rAAV comprises a titer of at least about 3xlOAl 1 vg / mL.#14288902v1

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