WO2023239627A2

WO2023239627A2 - Methods for recombinant aav production

Info

Publication number: WO2023239627A2
Application number: PCT/US2023/024392
Authority: WO
Inventors: Ping Liu; Ayda MAYER; Jie Li; Joseph M. SCARROTT; Yusuf B. JOHARI; Thilo H. POHLE; David C. James
Original assignee: Regenxbio Inc.
Priority date: 2022-06-08
Filing date: 2023-06-05
Publication date: 2023-12-14
Also published as: WO2023239627A3

Abstract

Provided herein are improved methods for producing recombinant Adeno-Associated Virus (rAAV) particles. In some embodiments, a method for producing recombinant AAV (rAAV) particles provided herein comprises culturing cells capable of producing rAAV particles in the presence of an anti-mitotic agent, e.g., nocodazole. In some embodiments, a method for producing recombinant AAV (rAAV) particles provided herein comprises culturing cells capable of producing rAAV particles in the presence of a caspase inhibitor.

Description

METHODS FOR RECOMBINANT AAV PRODUCTION

TECHNICAL FIELD

[0001] The present disclosure relates to methods of producing recombinant adcno-associatcd virus (rAAV) particles.

CROSS-REFRENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. application no. 63/350,257, filed June 8, 2022, which is incorporated herein by reference in its entirety.

BACKGROUND

[0003] Recombinant adeno-associated virus (AAV)-based vectors are currently the most widely used gene therapy products in development. The preferred use of rAAV vector systems is due, in part, to the lack of disease associated with the wild-type virus, the ability of AAV to transduce non-dividing as well as dividing cells, and the resulting long-term robust transgene expression observed in clinical trials and that indicate great potential for delivery in gene therapy indications. Additionally, different naturally occurring and recombinant rAAV vector serotypes, specifically target different tissues, organs, and cells, and help evade any pre-existing immunity to the vector, thus expanding the therapeutic applications of AAV-based gene therapies. Before replication defective virus, for example, AAV based gene therapies can be more widely adopted for late clinical stage and commercial use, new methods for large scale production of recombinant virus particles need to be developed.

[0004] Thus, there is a need in the art to improve the productivity and yield of methods for the large scale production of rAAV particles.

BRIEF SUMMARY

[0005] In one aspect, the disclosure provides a method of producing rAAV particles, comprising (a) providing a cell culture comprising a cell; (b) introducing into the cell one or more polynucleotides encoding at least one of (i) an rAAV genome to be packaged, (ii) adenovirus helper functions necessary for packaging, (iii) an AAV rep protein sufficient for packaging, and (iv) an AAV cap protein sufficient for packaging; (c) adding to the cell culture a mitotic inhibitor to a final concentration between about 1 nM and about 500 pM; and (d) maintaining the cell culture under conditions that allow production of the rAAV particles for between about 2 days and about 15 days after ((b). In some embodiments, the mitotic inhibitor is a microtubule destabilizing agent. In some embodiments, the mitotic inhibitor comprises at least one of nocodazole, vincristine, colchicine or a salt thereof. In some embodiments, the mitotic inhibitor is nocodazole. In some embodiments, the mitotic inhibitor is added after step (b). In some embodiments, the method further comprises adding to the culture a caspase inhibitor. [0006] In one aspect, the disclosure provides a method of producing rAAV particles, comprising (a) providing a cell culture comprising a cell; (b) introducing into the cell one or more polynucleotides encoding at least one of (i) an rAAV genome to be packaged, (ii) adenovirus helper functions necessary for packaging, (iii) an AAV rep protein sufficient for packaging, and (iv) an AAV cap protein sufficient for packaging; (c) adding to the cell culture a caspase inhibitor to a final concentration between about 10 nM and about 1 mM; and (d) maintaining the cell culture under conditions that allow production of the rAAV particles for between about 2 days and about 15 days after ((b). In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD-FMK, Q-VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD-Oph. In some embodiments, the caspase inhibitor is added after step (b). In some embodiments, the method further comprises adding to the culture nocodazole.

[0007] In some embodiments, a method described herein further comprises introducing into the cell one or more polynucleotides encoding (i) an rAAV genome to be packaged, (ii) adenovirus helper functions necessary for packaging, (iii) an AAV rep protein sufficient for packaging, and (iv) an AAV cap protein sufficient for packaging. In some embodiments, the cell culture is a suspension culture.

[0008] In some embodiments, a method described herein further comprises recovering the rAAV particles.

[0009] In one aspect, the disclosure provides a method for producing rAAV particles, comprising (a) providing a cell culture comprising a cell capable of producing rAAV ; (b) adding to the cell culture a mitotic inhibitor to a final concentration between about 1 nM and about 500 pM; and (c) maintaining the cell culture under conditions that allows production of the rAAV particles. In some embodiments, the mitotic inhibitor is a microtubule destabilizing agent. In some embodiments, the mitotic inhibitor comprises at least one of nocodazole, vincristine, colchicine or a salt thereof. In some embodiments, the mitotic inhibitor is nocodazole. In some embodiments, the method further comprises adding to the culture a caspase inhibitor.

[0010] In one aspect, the disclosure provides a method for producing rAAV particles, comprising (a) providing a cell culture comprising a cell capable of producing rAAV ; (b) adding to the cell culture a caspase inhibitor to a final concentration between about 10 nM and about 1 mM; and (c) maintaining the cell culture under conditions that allows production of the rAAV particles. In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD-FMK, Q-VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD- Oph In some embodiments, the method further comprises adding to the culture a mitotic inhibitor. In some embodiments, the mitotic inhibitor comprises nocodazole. [0011] In one aspect, the disclosure provides a method for producing rAAV particles, comprising culturing a cell capable of producing rAAV particles in a medium comprising between about 1 nM and about 500 pM of a mitotic inhibitor under conditions that allow the production of the rAAV particles. In some embodiments, the mitotic inhibitor is a microtubule destabilizing agent. In some embodiments, the mitotic inhibitor comprises at least one of nocodazole, vincristine, colchicine or a salt thereof. In some embodiments, the mitotic inhibitor is nocodazole. In some embodiments, the medium further comprises a caspase inhibitor.

[0012] In one aspect, the disclosure provides a method for producing rAAV particles, comprising culturing a cell capable of producing rAAV particles in a medium comprising between about 10 nM and about 1 rnM of a caspase inhibitor under conditions that allow the production of the rAAV particles. In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD-FMK, Q-VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD-Oph. In some embodiments, the medium further comprises a mitotic inhibitor. In some embodiments, the mitotic inhibitor comprises nocodazole.

[0013] In some embodiments, the cell capable of producing rAAV has been transfected with one or more polynucleotides encoding at least one of (a) an rAAV genome to be packaged, (b) adenovirus helper functions necessary for packaging, (c) an AAV rep protein sufficient for packaging, and (d) an AAV cap protein sufficient for packaging.

[0014] In some embodiments, the cell capable of producing rAAV has been transfected with one or more polynucleotides encoding (a) an rAAV genome to be packaged, (b) adenovirus helper functions necessary for packaging, (c) an AAV rep protein sufficient for packaging, and (d) an AAV cap protein sufficient for packaging.

[0015] In some embodiments, the cell culture is a suspension culture.

[0016] In some embodiments, a method described herein further comprises recovering the rAAV particles.

[0017] In one aspect, the disclosure provides a method of increasing the production of rAAV particles, comprising (a) providing a cell culture comprising a cell; (b) introducing into the cell one or more polynucleotides encoding at least one of (i) an rAAV genome to be packaged, (ii) adenovirus helper functions necessary for packaging, (iii) an AAV rep protein sufficient for packaging, and (iv) an AAV cap protein sufficient for packaging; (c) adding to the cell culture a mitotic inhibitor to a final concentration between about 1 nM and about 500 |1M; and (d) maintaining the cell culture under conditions that allow production of the rAAV particles for between about 2 days and about 15 days after ((b). In some embodiments, the mitotic inhibitor is a microtubule destabilizing agent. In some embodiments, the mitotic inhibitor comprises at least one of nocodazole, vincristine, colchicine or a salt thereof. In some embodiments, the mitotic inhibitor is nocodazole.

[0018] In one aspect, the disclosure provides a method of increasing the production of rAAV particles, comprising (a) providing a cell culture comprising a cell; (b) introducing into the cell one or more polynucleotides encoding at least one of (i) an rAAV genome to be packaged, (ii) adenovirus helper functions necessary for packaging, (iii) an AAV rep protein sufficient for packaging, and (iv) an AAV cap protein sufficient for packaging; (c) adding to the cell culture a caspase inhibitor to a final concentration between about 10 nM and about 1 mM; and (d) maintaining the cell culture under conditions that allow production of the rAAV particles for between about 2 days and about 15 days after ((b). In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD-FMK, Q-VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD-Oph.

[0019] In some embodiments, the rAAV particles comprise a capsid protein of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rhS, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 serotype. In some embodiments, the rAAV particles comprise a capsid protein of the AAV8, AAV9, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, or AAV.hu37 serotype. In some embodiments, the rAAV particles comprise a capsid protein of the AAV8 or AAV9 serotype.

[0020] In some embodiments, the genome to be packaged encodes a polypeptide or a double stranded RNA molecule. In some embodiments, the genome to be packaged encodes a microRNA.

[0021] In one aspect, the disclosure provides a composition comprising isolated rAAV particles that were produced by a method described herein.

[0022] In one aspect, the disclosure provides a composition comprising cells capable of producing rAAV particles and a cell culture medium comprising between about 1 nM and about 500 pM of a mitotic inhibitor. In some embodiments, the mitotic inhibitor is a microtubule destabilizing agent. In some embodiments, the mitotic inhibitor comprises at least one of nocodazole, vincristine, colchicine or a salt thereof. In some embodiments, the mitotic inhibitor is nocodazole.

[0023] In one aspect, the disclosure provides a composition comprising cells capable of producing rAAV par ticles and a cell culture medium comprising between about 10 nM and about 1 mM of a caspase inhibitor. In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD-FMK, Q-VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD-Oph.

[0024] Still other features and advantages of the compositions and methods described herein will become more apparent from the following detailed description when read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1. Initial screen of small molecule culture additives identifies nocodazole, M344 and z- VAD-fmk as novel putative enhancers of rAAV genomic titer. Cells were analysed 72 HPT for rAAV8 genomic titer, shown here as the fold change relative to rAAV8 producing cells containing no small molecule enhancers (Control). Nocodazole, M344, and z-VAD-fmk are highlighted (dashed boxes) (A). Mean cell volume vs genomic titer fold change is shown in (B), dashed lines indicate control values. Data shown in A are mean ± SEM for two independent experiments carried out in technical duplicate, with the exception of TUDCA, TMAO, Betaine and LiCl which are mean only data from one independent experiment carried out in technical duplicate (data from these experiments is omitted in B). NaBu - Sodium butyrate; VPA - Valproic acid; TUDCA - Tauroursodeoxy cholic acid; TMAO - Trimethylamine N-oxide; LiCl - Lithium chloride.

[0026] Figure 2. Early addition of nocodazole to rAAV8 producing cells increases genomic titer. 1AAV8 producing cells were cultured in 24-well plates and 4 pM nocodazole was added at 0, 4, 24, and 48 HPT or left untreated (Ctrl). 72 HPT cells were harvested and viable cell density (VCD) (A), viability (B), mean cell volume (C), and genomic titer expressed as a fold change relative to Ctrl (D) was measured. Data shown for VCD, viability and mean cell diameter are the mean ± S.E.M. n > 3 independent biological replicates. Data shown for genomic titer fold change are the mean ± S.E.M. n > 3 independent biological replicates. Data were analysed by one-way ANOVA followed by post-hoc Dunnett’s multiple comparisons test with respect to Ctrl. * p < 0.05, *** p < 0.001, **** p < 0.0001. [0027] Figure 3. rAAV8 producing cells treated with nocodazole arrest in G2/M phase. Flow cytometry histograms triggered against propidium iodide signal for cells show the relative abundance of cells in either G1 (blue), S (orange) or G2/M (green) phases of the cell cycle in the presence (A.) or absence (B) of 4 pM nocodazole addition at 0 HPT. Ratio of cells in G2/M phase to G1 phase at 72 HPT with 4 pM nocodazole added at 0, 4, 24, 48 HPT or untreated (Ctrl) (C). Representative fluorescent composite images of rAAV-producing cells fixed 24 HPT show staining of the fibrillar' component of the nucleolus with anti-fibrillarin (red) and nuclear DNA with DAPI (blue). Cells displaying disorganised nuclear morphology are indicated by white asterisks in nocodazole treated cultures (D) and non-treated cells (E). Images taken using 100 x oil objective. Scale bar 10 pm. Data shown in (C) are mean ± SEM for three biological replicates. [0028] Figure 4. Increased genomic titer in nocodazole and M344 treated cells is maintained in larger scale cultures and across two separate serotypes. 30 ml shake flask cultures were transfected with rAAV8 or rAAV9 producing plasmids and cultured for 5 days post-transf ection in the presence (rAAV8: O; rAAV9: □) or absence (rAAV8: •; rAAV9: ■) of 4 pM nocodazole added 4 HPT. Cultures were measured daily for VCD (A - solid lines), viability (A - dashed lines) and mean cell volume (B). Fold change analysis of genomic titer for both serotypes in the presence (+) or absence (-) of 4 pM nocodazole at day 5 (3 days post-transfection) is shown in (C). 30ml shake flask cultures were transfected with rAAV8 producing plasmids with no chemical additives (■), with the addition of 4pM nocodazole at 4 HPT (O), or with the addition of 4 pM nocodazole and 2.5 pM M344 at 4 HPT (□). Cultures were measured daily for VCD (D - solid lines) and viability (D - dashed lines). Genome titer was measured at day 5 and is shown as fold change with respect to untreated cultures (E). Data shown are the mean ± S.E.M. n = 3 independent biological replicates. Fold change between nocodazole treated and untreated cultures was analysed for each serotype separately by Student’s unpaired two-tailed t-test with respect to untreated cultures. ** p < 0.01, *** p < 0.001. Fold change between untreated and nocodazole/M344 treated cultures was analysed by one-way ANOVA with Dunnet’s post-hoc test for multiple comparisons with respect to untreated cells (nocodazole: negative/ M344: negative). * p < 0.05.

[0029] Figure 5. Multi-well plate-based screening of small molecule combinations to enhance rAAV8 titer. Cells were treated 4 HPT with various small molecules. Cells were harvested 72 HPT.

Measurements were taken at harvest of viable cell density (VCD) (A), viability (B), mean cell volume

(C), and rAAV8 genome titer as a fold change with respect to untreated cells (-/-/-) (D). Data shown are the mean ± S.E.M. n = 3 independent biological replicates. Data were analysed by one-way ANOVA followed by post-hoc Dunnett’s multiple comparisons test with respect to untreated cells (-/-/-). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0030] Figure 6. 30ml shake flask scale-up of small molecule cell culture additive combinations. 30ml shake flask cultures were transfected with rAAV8 producing plasmids with no chemical additives (■), with the addition of 4pM nocodazole at 4 HPT (O), or with the addition of 4 pM nocodazole and 2.5 pM M344 at 4 HPT (□). Cultures were measured daily for mean cell volume (A). Percentage of GFP positive cells in culture was measured at 24 HPT (B). Quantification of intact capsids at 72 HPT was carried out by rAAV8-specific capsid ELISA and data shown as fold change with respect to untreated cells (nocodazole: (-) / M344: (-)) (C). Full/empty ratio of cultures harvested at 72 HPT shown is shown in

(D). Data shown are the mean ± S.E.M. n = 3 independent biological replicates. Data was analysed by one-way ANOVA with Dunnet’s post-hoc test for multiple comparisons with respect to untreated cells (nocodazole: (-) / M344: (-)). * p < 0.05. [0031] Figure 7. Effect of Z-VAD-FMK on titers in different suspension adapted HEK293 host cell clones. Fold change (FC) of titer was calculated from titers normalized to those of Clone #1 without Z- VAD-FMK treatment.

[0032] Figure 8. Screening of caspase inhibitors for effect on rAAV production. Fold change (FC) of titer was calculated from titers normalized to those of HEK293 Clone #1 with DMSO (CTRL) treatment. [0033] Figure 9. Screening of caspase inhibitors for effect on rAAV production. Fold change (FC) of titer was calculated from titers normalized to those of HEK293 Clone #3with DMSO (CTRL) treatment. [0034] Figure 10. Screening of caspase inhibitors for effect on rAAV production. Fold change (FC) of titer was calculated from titers normalized to those of HEK293 Clone #3.7 with DMSO (CTRL) treatment.

[0035] Figure 11. Effect of caspase inhibitors on rAAV titers in shake flasks. DMSO (CTRL), 40 pM (high) or 10 pM (low) of Caspase Inhibitor 1, 40 pM (high) or 10 pM (low) of Z-VAD-FMK, 10 pM (high) or 3 pM (low) of Q-VD-OPh hydrate, 1.7 pM (high) or 400 nM (low) of Caspase-3/7 Inhibitor I were added into HEK293 Clone #1 after transient triple transfections. Fold change (FC) of titer was calculated from titers normalized to those of HEK293 Clone #3.7 with DMSO (CTRL) treatment.

[0036] Figure 12. Effect of caspase inhibitors on rAAV titers in shake flasks. DMSO (CTRL),

40 pM (high) or 10 pM (low) of Caspase Inhibitor I, 40 pM (high) or 10 pM (low) of Z-VAD-FMK, 10 pM (high) or 3 pM (low) of Q-VD-OPh hydrate, 1.7 pM (high) or 400 nM (low) of Caspase-3/7 Inhibitor I were added into HEK293 Clone #3 after transient triple transfections. Fold change (FC) of titer was calculated from titers normalized to those of HEK293 Clone #3 with DMSO (CTRL) treatment.

[0037] Figure 13. Effect of caspase inhibitors on rAAV titers in shake flasks. DMSO (CTRL),

40 pM (high) or 10 pM (low) of Caspase Inhibitor I, 40 pM (high) or 10 pM (low) of Z-VAD-FMK, 10 pM (high) or 2 pM (low) of Q-VD-OPh hydrate, 1.7 pM (high) or 400 nM (low) of Caspase-3/7 Inhibitor I were added into HEK293 Clone #3.7 after transient triple transfections. Fold change (FC) of titer was calculated from titers normalized to those of HEK293 Clone #3.7 with DMSO (CTRL) treatment.

[0038] Figure 14. Effect of Q-VD-Oph on rAAV titers. DMSO (CTRL), 10 pM (high) or 2 pM (low) of Q-VD-OPh hydrate were added into HEK293 Clone #1 (white bar), HEK293 Clone #3 (orange bar) or HEK293 Clone #3.7 (blue bar) respectively after 0 hour or 18 hours post-transfections. Fold change (FC) of titer was calculated from titers normalized to those of HEK293 Clone #1 with DMSO treatment at VCD 6.5 x 10⁶ VC/mL during triple transfection.

[0039] Figure 15. Effect of Q-VD-Oph on rAAV titers. At 0-hour post-transfection, 0 pM (blue bar) or 10 pM (orange bar) of Q-VD-OPh hydrate was added into HEK293 Clone #3 and HEK293 Clone #1 (black bar) was used as control. [0040] Figure 16. Nocodazole shows concentration dependent effects on rAAV production. Fold change (FC) of titer was calculated from titers normalized to those of the same HEK293 cell pool adapted for suspension without Nocodazole treatment.

[0041] Figure 17. Nocodazole's effect on rAAV production using different host cells. Fold change (FC) of titer was calculated from titers normalized to titers obtained using corresponding host cells with DMSO (CTRL) treatment. "saHEK293" is the HEK293 cell pool adapted for suspension.

[0042] Figure 18. Nocodazole's effect on rAAV production is altered by the helper plasmid. Fold change (FC) of titer was calculated from titers normalized to titers obtained using corresponding host cells with DMSO (CTRL) treatment. "saHEK293" is the HEK293 cell pool adapted for suspension.

[0043] Figure 19. Nocodazole's effect on rAAV production is altered by the transgene. Fold change (FC) of titer was calculated from titers normalized to titers obtained using corresponding host cells with DMSO (CTRL) treatment. "saHEK293" is the HEK293 cell pool adapted for suspension.

DETAILED DESCRIPTION

[0044] It was surprisingly found that the addition of a small molecule anti-mitotic agent (e.g., nocodazole) and/or addition of a caspase inhibitor (e.g., z-VAD-FMK and Q-VD-Oph) to a cell culture increased the production of recombinant adeno-associated virus (rAAV) production. These surprising findings were used to develop methods producing rAAV particles described herein. In some embodiments, a method described herein produces at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, or at least 100% more rAAV particles measured as GC/ml than a corresponding method without the use of mitotic inhibitor and/or caspase inhibitor.

[0045] Given the very high number of rAAV particles needed to prepare a single therapeutic unit dose, any increase in rAAV yield provides a reduction in the cost of goods per unit dose. Increased virus yield allows a corresponding reduction not only in the cost of consumables needed to produce rAAV particles, but also in the cost of capital expenditure in connection with building industrial virus purification facilities.

Definitions

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. To facilitate an understanding of the disclosed methods, a number of terms and phrases are defined below.

[0047] " AAV" is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or modifications, derivatives, or pseudotypes thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. The abbreviation "rAAV" refers to recombinant adeno-associated virus. The term "AAV" includes AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, and modifications, derivatives, or pseudotypes thereof. "Primate AAV" refers to AAV that infect primates, "non-primate AAV" refers to AAV that infect non-primate mammals, "bovine AAV" refers to AAV that infect bovine mammals, etc. [0048] "Recombinant", as applied to an AAV particle means that the AAV particle is the product of one or more procedures that result in an AAV particle construct that is distinct from an AAV particle in nature.

[0049] A recombinant adeno-associated virus particle "rAAV particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide rAAV vector genome comprising a heterologous polynucleotide (i.e. a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell). The rAAV particle may be of any AAV serotype, including any modification, derivative or pseudotype (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10, or derivatives/modifications/pseudotypes thereof). Such AAV serotypes and derivatives/modifications/pseudotypes, and methods of producing such serotypes/derivatives/modifications/ pseudotypes are known in the art (see, e.g., Asokan et al., Mol. Ther. 20(4):699-708 (2012).

[0050] The rAAV particles of the disclosure may be of any serotype, or any combination of serotypes, (e.g., a population of rAAV particles that comprises two or more serotypes, e.g., comprising two or more of AAV2, AAV8, and AAV9 particles). In some embodiments, the rAAV particles are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or other AAV particles, or combinations of two or more thereof). In some embodiments, the rAAV particles are AAV8 or AAV9 particles.

[0051] In some embodiments, the rAAV particles have an AAV capsid protein of a serotype selected from the group consisting of AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16 or a derivative, modification, or pseudotype thereof. In some embodiments, the rAAV particles have an AAV capsid protein of a serotype of AAV8, AAV9, or a derivative, modification, or pseudotype thereof.

[0052] The term "cell culture," refers to cells grown adherent or in suspension, bioreactors, roller bottles, hyperstacks, microspheres, macrospheres, flasks and the like, as well as the components of the supernatant or suspension itself, including but not limited to rAAV particles, cells, cell debris, cellular contaminants, colloidal particles, biomolecules, host cell proteins, nucleic acids, and lipids, and flocculants. Large scale approaches, such as biorcactors, including suspension cultures and adherent cells growing attached to microcarriers or macrocarriers in stirred bioreactors, are also encompassed by the term "cell culture." Cell culture procedures for both large and small-scale production of proteins are encompassed by the present disclosure. In some embodiments, the term "cell culture" refers to cells grown in suspension. In some embodiments, the term "cell culture" refers to adherent cells grown attached to microcarriers or macrocarriers in stirred bioreactors. In some embodiments, the term "cell culture" refers to cells grown in a perfusion culture. In some embodiments, the term "cell culture" refers to cells grown in an alternating tangential flow (ATF) supported high-density perfusion culture.

[0053] The terms "purifying", "purification", "separate", "separating", "separation", "isolate", "isolating", or "isolation", as used herein, refer to increasing the degree of purity of a target product, e.g., rAAV particles and rAAV genome from a sample comprising the target product and one or more impurities. Typically, the degree of purity of the target product is increased by removing (completely or partially) at least one impurity from the sample. In some embodiments, the degree of purity of the rAAV in a sample is increased by removing (completely or partially) one or more impurities from the sample by using a method described herein.

[0054] " About" modifying, for example, the quantity of an ingredient in the compositions, concentration of an ingredient in the compositions, flow rate, rAAV particle yield, feed volume, salt concentration, and like values, and ranges thereof, employed in the methods provided herein, refers to variation in the numerical quantity that can occur, for example, through typical measuring and handling procedures used for making concentrates or use solutions; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or carry out the methods; and like considerations. The term "about" also encompasses amounts that differ due to aging of a composition with a particular initial concentration or mixture. The term "about" also encompasses amounts that differ due to mixing or processing a composition with a particular' initial concentration or mixture. Whether or not modified by the term "about" the claims include equivalents to the quantities. In some embodiments, the term "about" refers to ranges of approximately 10-20% greater than or less than the indicated number or range. In further embodiments, "about" refers to plus or minus 10% of the indicated number or range. For example, "about 10%" indicates a range of 9% to 11%.

[0055] As used in the present disclosure and claims, the singular forms "a", "an" and "the" include plural forms unless the context clearly dictates otherwise.

[0056] It is understood that wherever embodiments are described herein with the language "comprising" otherwise analogous embodiments described in terms of "consisting of" and/or "consisting essentially of" are also provided. It is also understood that wherever embodiments are described herein with the language "consisting essentially of" otherwise analogous embodiments described in terms of "consisting of" are also provided. [0057] The term "and/or" as used in a phrase such as "A and/or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0058] Where embodiments of the disclosure are described in terms of a Markush group or other grouping of alternatives, the disclosed method encompasses not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, and also the main group absent one or more of the group members. The disclosed methods also envisage the explicit exclusion of one or more of any of the group members in the disclosed methods.

Methods of producing a recombinant viral particle

[0059] In one aspect, the disclosure provides a method of producing rAAV particles, comprising (a) providing a cell culture comprising a cell; (b) introducing into the cell one or more polynucleotides encoding at least one of (i) an rAAV genome to be packaged, (ii) adenovirus helper functions necessary for packaging, (iii) an AAV rep protein sufficient for packaging, and (iv) an AAV cap protein sufficient for packaging; (c) adding to the cell culture a mitotic inhibitor to a final concentration between about 1 nM and about 500 pM; and (d) maintaining the cell culture under conditions that allow production of the rAAV particles for between about 2 days and about 15 days after ((b). In some embodiments, the mitotic inhibitor is a microtubule destabilizing agent. In some embodiments, the mitotic inhibitor comprises at least one of nocodazole, vincristine, colchicine or a salt thereof. In some embodiments, the mitotic inhibitor is nocodazole.

[0060] In one aspect, the disclosure provides a method for producing rAAV particles, comprising (a) providing a cell culture comprising a cell capable of producing rAAV ; (b) adding to the cell culture a mitotic inhibitor to a final concentration between about 1 nM and about 500 pM; and (c) maintaining the cell culture under conditions that allows production of the rAAV particles. In some embodiments, the mitotic inhibitor is a microtubule destabilizing agent. In some embodiments, the mitotic inhibitor comprises at least one of nocodazole, vincristine, colchicine or a salt thereof. In some embodiments, the mitotic inhibitor is nocodazole.

[0061] In one aspect, the disclosure provides a method of increasing the production of rAAV particles, comprising (a) providing a cell culture comprising a cell; (b) introducing into the cell one or more polynucleotides encoding at least one of (i) an rAAV genome to be packaged, (ii) adenovirus helper functions necessary for packaging, (iii) an AAV rep protein sufficient for packaging, and (iv) an AAV cap protein sufficient for packaging; (c) adding to the cell culture a mitotic inhibitor to a final concentration between about 1 nM and about 500 pM; and (d) maintaining the cell culture under conditions that allow production of the rAAV particles for between about 2 days and about 15 days after ((b). In some embodiments, the mitotic inhibitor is a microtubule destabilizing agent. In some embodiments, the mitotic inhibitor comprises at least one of nocodazole, vincristine, colchicine or a salt thereof. In some embodiments, the mitotic inhibitor is nocodazole.

[0062] In some embodiments, the mitotic inhibitor is capable of arresting cells within the G2/M phase of the cell cycle. The use of any mitotic inhibitor capable of arresting cells within the G2/M phase of the cell cycle known to a skilled artisan is contemplated. See, e.g., Castro-Gamero et al., Cancer. Biol. Med.Nov; 15(4): 354—374 (2018); Bates & Eastman, Br J Clin Pharmacol 83 255-268(2017), each of which are incorporated herein by reference in their entirety. In some embodiments, the mitotic inhibitor is an antimicrotubule agent. In some embodiments, the mitotic inhibitor comprises a microtubule destabilizing agent. In some embodiments, the mitotic inhibitor comprises 7- nocodazole, vincristine, Deazahypoxanthine, AD-1, Cabazitaxel, Chaicone, CI-980, Colcemid, Colchicine, Combretastatin A4, CP248, Cucurbitacin B, D-24851, Docetaxel, DTA0100, Epothilone B, Epothilone D, Ixabepilone, JAI- 51, Mebendazole, Sagopilone, ST-11, TTI-237, Vitilevuamide or a combination thereof. In some embodiments, the mitotic inhibitor is an agent modulating the activity of G2/M regulators, for example, cyclin-dependent kinases, Aurora inhibitors, PLK1, BUB, 1, and BUBR1, and surviving. In some embodiments, the mitotic inhibitor comprises a PLK-1 inhibitor, for example, BI 2536, BI 61 1, GSK461364, GW843682X, JNJ-10198409 or combinations thereof. In some embodiments, the mitotic inhibitor comprises a CDK inhibitor, for example, Abemaciclib, CVT-313, Dinaciclib, Flavopiridol, JNJ- 7706621, MK-8776, ON123300, Palbociclib or combinations thereof.

[0063] In some embodiments, the cell culture has a final mitotic inhibitor concentration between about 50 nM and about 50 pM. In some embodiments, the cell culture has a final mitotic inhibitor concentration between about 100 nM and about 20 pM. In some embodiments, the cell culture has a final mitotic inhibitor concentration between about 200 nM and about 10 pM.

[0064] In some embodiments, the cell culture has a final nocodazole concentration between about 50 nM and about 50 pM. In some embodiments, the cell culture has a final nocodazole concentration between about 100 nM and about 20 pM. In some embodiments, the cell culture has a final nocodazole concentration between about 200 nM and about 10 pM.

[0065] In some embodiments, the cell culture has a final mitotic inhibitor concentration of about 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM or 1 pM. In some embodiments, the cell culture has a final mitotic inhibitor concentration of about 0.1 pM. In some embodiments, the cell culture has a final mitotic inhibitor concentration of about 0.2 pM. In some embodiments, the cell culture has a final mitotic inhibitor concentration of about 0.3 pM. In some embodiments, the cell culture has a final mitotic inhibitor concentration of about 0.4 pM. In some embodiments, the cell culture has a final mitotic inhibitor concentration of about 0.5 pM. In some embodiments, the cell culture has a final mitotic inhibitor concentration of about 0.6 pM. In some embodiments, the cell culture has a final mitotic inhibitor concentration of about 0.7 pM. In some embodiments, the cell culture has a final mitotic inhibitor concentration of about 0.8 pM. In some embodiments, the cell culture has a final mitotic inhibitor concentration of about 0.9 pM. In some embodiments, the cell culture has a final mitotic inhibitor concentration of about 1 pM.

[0066] In some embodiments, the cell culture has a final nocodazole concentration of about 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM or 1 pM. In some embodiments, the cell culture has a final nocodazole concentration of about 0.1 pM. In some embodiments, the cell culture has a final nocodazole concentration of about 0.2 pM. In some embodiments, the cell culture has a final nocodazole concentration of about 0.3 pM. In some embodiments, the cell culture has a final nocodazole concentration of about 0.4 pM. In some embodiments, the cell culture has a final nocodazole concentration of about 0.5 pM. In some embodiments, the cell culture has a final nocodazole concentration of about 0.6 pM. In some embodiments, the cell culture has a final nocodazole concentration of about 0.7 pM. In some embodiments, the cell culture has a final nocodazole concentration of about 0.8 |1M. In some embodiments, the cell culture has a final nocodazole concentration of about 0.9 pM. In some embodiments, the cell culture has a final nocodazole concentration of about 1 pM.

[0067] In some embodiments, the mitotic inhibitor is added after step (b). In some embodiments, the mitotic inhibitor is added between about 0.5 hour and about 48 hours after step (b). In some embodiments, the mitotic inhibitor is nocodazole.

[0068] In some embodiments, the mitotic inhibitor is added less than about 48 hours, 24 hours, 12 hours, 6 hours, 3 hours, 2 hours, 1 hour or 0.5 hour after step (b). In some embodiments, the mitotic inhibitor is added less than about 48 hours after step (b). In some embodiments, the mitotic inhibitor is added less than about 24 hours after step (b). In some embodiments, the mitotic inhibitor is added less than about 12 hours after step (b). In some embodiments, the mitotic inhibitor is added less than about 6 hours after step (b). In some embodiments, the mitotic inhibitor is added less than about 3 hours after step (b). In some embodiments, the mitotic inhibitor is added less than about 2 hours after step (b). In some embodiments, the mitotic inhibitor is added less than about 1 hours after step (b). In some embodiments, the mitotic inhibitor is added less than about 0.5 hour after step (b). In some embodiments, the mitotic inhibitor is nocodazole. [0069] In some embodiments, the mitotic inhibitor is added less than about 0.5 hour, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 1 hours, or 24 hours after step (b). In some embodiments, the mitotic inhibitor is added less than about 0.5 hour after step (b). In some embodiments, the mitotic inhibitor is added less than about 1 hour after step (b). In some embodiments, the mitotic inhibitor is added less than about 2 hours after step (b). In some embodiments, the mitotic inhibitor is added less than about 3 hours after step (b). In some embodiments, the mitotic inhibitor is added less than about 6 hours after step (b). In some embodiments, the mitotic inhibitor is added less than about 9 hours after step (b). In some embodiments, the mitotic inhibitor is added less than about 12 hours after step (b). In some embodiments, the mitotic inhibitor is added less than about 24 hours after step (b). In some embodiments, the mitotic inhibitor is nocodazole.

[0070] In some embodiments, the mitotic inhibitor is added at least about 0.5 hour, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, or about 24 hours after step (b). In some embodiments, the mitotic inhibitor is added at least about 0.5 hour after step (b). In some embodiments, the mitotic inhibitor is added at least about 1 hour after step (b). In some embodiments, the mitotic inhibitor is added at least about 2 hours after step (b). In some embodiments, the mitotic inhibitor is added at least about 3 hours after step (b). In some embodiments, the mitotic inhibitor is added at least about 6 hours after step (b). In some embodiments, the mitotic inhibitor is added at least about 9 hours after step (b). In some embodiments, the mitotic inhibitor is added at least about 12 hours after step (b). In some embodiments, the mitotic inhibitor is added at least about 24 hours after step (b). In some embodiments, the mitotic inhibitor is nocodazole.

[0071] In some embodiments, the mitotic inhibitor is added about 0.5 hour, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, or about 24 hours after step (b). In some embodiments, the mitotic inhibitor is added about 0.5 hour after step (b). In some embodiments, the mitotic inhibitor is added about 1 hour after step (b). In some embodiments, the mitotic inhibitor is added about 2 hours after step (b). In some embodiments, the mitotic inhibitor is added about 3 hours after step (b). In some embodiments, the mitotic inhibitor is added about 6 hours after step (b). In some embodiments, the mitotic inhibitor is added about 9 hours after step (b). In some embodiments, the mitotic inhibitor is added about 12 hours after step (b). In some embodiments, the mitotic inhibitor is added about 24 hours after step (b). In some embodiments, the mitotic inhibitor is nocodazole.

[0072] In some embodiments, the method described herein further comprises adding to the culture a caspase inhibitor. In some embodiments, the caspase inhibitor is added after step (b). In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor is a z-VAD-FMK or Q-VD-Oph. In some embodiments, the mitotic inhibitor and the caspase inhibitor arc added at the same time. In some embodiments, the mitotic inhibitor and the caspase inhibitor are added separately in any order.

[0073] In one aspect, the disclosure provides a method of producing rAAV particles, comprising (a) providing a cell culture comprising a cell; (b) introducing into the cell one or more polynucleotides encoding at least one of (i) an rAAV genome to be packaged, (ii) adenovirus helper functions necessary for packaging, (iii) an AAV rep protein sufficient for packaging, and (iv) an AAV cap protein sufficient for packaging; (c) adding to the cell culture a caspase inhibitor to a final concentration between about 10 nM and about 1 mM; and (d) maintaining the cell culture under conditions that allow production of the rAAV particles for between about 2 days and about 15 days after ((b). In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD-FMK, Q-VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD-Oph. [0074] In one aspect, the disclosure provides a method for producing rAAV particles, comprising (a) providing a cell culture comprising a cell capable of producing rAAV ; (b) adding to the cell culture a caspase inhibitor to a final concentration between about 10 nM and about 1 mM; and (c) maintaining the cell culture under conditions that allows production of the rAAV particles. In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD-FMK, Q-VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD- Oph.

[0075] In one aspect, the disclosure provides a method of increasing the production of rAAV particles, comprising (a) providing a cell culture comprising a cell; (b) introducing into the cell one or more polynucleotides encoding at least one of (i) an rAAV genome to be packaged, (ii) adenovirus helper functions necessary for packaging, (iii) an AAV rep protein sufficient for packaging, and (iv) an AAV cap protein sufficient for packaging; (c) adding to the cell culture a caspase inhibitor to a final concentration between about 10 nM and about 1 mM; and (d) maintaining the cell culture under conditions that allow production of the rAAV particles for between about 2 days and about 15 days after ((b). In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD-FMK, Q-VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD-Oph.

[0076] In some embodiments, the cell culture has a final caspase inhibitor concentration between about 50 nM and about 100 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration between about 100 nM and about 50 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration between about 200 nM and about 20 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration between about 0.1 pM and about 1 p . In some embodiments, the cell culture has a final caspase inhibitor concentration between about 0.5 pM and about 3 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration between about 1 pM and about 5 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration between about 3 pM and about 10 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration between about 4 pM and about 20 pM. In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD- FMK, Q-VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD-Oph.

[0077] In some embodiments, the cell culture has a final caspase inhibitor concentr ation of about 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM or 1 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 0.1 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 0.2 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 0.3 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 0.4 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 0.5 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 0.6 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 0.7 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 0.8 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 0.9 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 1 pM. In some embodiments, the caspase inhibitor is a pancaspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD-FMK, Q- VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD-Oph.

[0078] In some embodiments, the cell culture has a final caspase inhibitor concentration of about 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM or 10 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 1 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 2 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 3 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 4 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 5 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 6 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 7 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 8 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 9 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 10 pM. In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD-FMK, Q-VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD-Oph.

[0079] In some embodiments, the cell culture has a final caspase inhibitor concentration of about 5 pM, 10 pM, 15 pM, 20 pM, or 25 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 5 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 10 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 15 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 20 pM. In some embodiments, the cell culture has a final caspase inhibitor concentration of about 25 pM. In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD-FMK, Q-VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD-Oph.

[0080] In some embodiments, the caspase inhibitor is added after step (b). In some embodiments, the caspase inhibitor is added between about 0.5 hour and about 48 hours after step (b).

[0081] In some embodiments, the caspase inhibitor is added less than about 48 hours, 24 hours, 12 hours, 6 hours, 3 hours, 2 hours, 1 hour or 0.5 hour after step (b). In some embodiments, the caspase inhibitor is added less than about 48 hours after step (b). In some embodiments, the caspase inhibitor is added less than about 24 hours after step (b). In some embodiments, the caspase inhibitor is added less than about 12 hours after step (b). In some embodiments, the caspase inhibitor is added less than about 6 hours after step (b). In some embodiments, the caspase inhibitor is added less than about 3 hours after step (b). In some embodiments, the caspase inhibitor is added less than about 2 hours after step (b). In some embodiments, the caspase inhibitor is added less than about 1 hours after step (b). In some embodiments, the caspase inhibitor is added less than about 0.5 hour after step (b). In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD- FMK, Q-VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD-Oph.

[0082] In some embodiments, the caspase inhibitor is added at least about 0.5 hour, hour, 2 hours, 3 hours, 6 hours or 12 hours after step (b). In some embodiments, the caspase inhibitor is added at least about 0.5 hour after step (b). In some embodiments, the caspase inhibitor is added at least about 1 hour after step (b). In some embodiments, the caspase inhibitor is added at least about 2 hours after step (b). In some embodiments, the caspase inhibitor is added at least about 3 hours after step (b). In some embodiments, the caspase inhibitor is added at least about 6 hours after step (b). In some embodiments, the caspase inhibitor is added at least about 9 hours after step (b). In some embodiments, the caspase inhibitor is added at least about 12 hours after step (b). In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD- FMK, Q-VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD-Oph. [0083] In some embodiments, the caspase inhibitor is added about 0.5 hour, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, or about 24 hours after step (b). In some embodiments, the caspase inhibitor is added about 0.5 hour after step (b). In some embodiments, the caspase inhibitor is added about 1 hour after step (b). In some embodiments, the caspase inhibitor is added about 2 hours after step (b). In some embodiments, the caspase inhibitor is added about 3 hours after step (b). In some embodiments, the caspase inhibitor is added about 6 hours after step (b). In some embodiments, the caspase inhibitor is added about 9 hours after step (b). In some embodiments, the caspase inhibitor is added about 12 hours after step (b). In some embodiments, the caspase inhibitor is added about 24 hours after step (b). In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD-FMK, Q-VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD-Oph.

[0084] In some embodiments, the caspase inhibitor is added less than about 0.5 hour, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, or 24 hours after step (b). In some embodiments, the caspase inhibitor is added less than about 0.5 hour after step (b). In some embodiments, the caspase inhibitor is added less than about 1 hour after step (b). In some embodiments, the caspase inhibitor is added less than about 2 hours after step (b). In some embodiments, the caspase inhibitor is added less than about 3 hours after step (b). In some embodiments, the caspase inhibitor is added less than about 6 hours after step (b). In some embodiments, the caspase inhibitor is added less than about 9 hours after step (b). In some embodiments, the caspase inhibitor is added less than about 12 hours after step (b). In some embodiments, the caspase inhibitor is added less than about 24 hours after step (b). In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD- FMK, Q-VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD-Oph.

[0085] In some embodiments, a method described herein further comprises adding to the culture nocodazole. In some embodiments, the nocodazole is added after step (b). In some embodiments, the caspase inhibitor and nocodazole are added at the same time. In some embodiments, the caspase inhibitor and nocodazole are added separately in any order.

[0086] In some embodiments, the cell culture is maintained for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after (b). In some embodiments, the cell culture is maintained for about 3 days after (b). In some embodiments, the cell culture is maintained for about 4 days after (b). In some embodiments, the cell culture is maintained for about 5 days after (b). In some embodiments, the cell culture is maintained for about 6 days after (b). In some embodiments, the cell culture is maintained for about 7 days after (b). In some embodiments, the mitotic inhibitor is nocodazole. In some embodiments, the caspase inhibitor comprises at least one of z-VAD-FMK, Q-VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD-Oph. [0087] In some embodiments, a method described herein further comprises introducing into the cell one or more polynucleotides encoding (i) an rAAV genome to be packaged, (ii) adenovirus helper functions necessary for packaging, (iii) an AAV rep protein sufficient for packaging, and (iv) an AAV cap protein sufficient for packaging. In some embodiments, the adenovirus helper functions comprise at least one of an adenovirus E4 gene, E2a gene, and VA gene. In some embodiments, the adenovirus helper functions comprise an adenovirus E4 gene, E2a gene, and VA gene. In some embodiments, the polynucleotide encoding the adenovirus helper functions comprises pAD Delta F6. In some embodiments, the introducing one or more polynucleotides into the cell is by transfection.

[0088] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a HEK293 cell, HEK derived cell, CHO cell, CHO derived cell, HeLa cell, SF-9 cell, BHK cell, Vero cell, CAP cell or PerC6 cell. In some embodiments, the cell is a HEK293 cell. In some embodiments, the cell culture is a suspension culture.

[0089] In some embodiments, a method described herein further comprises recovering the rAAV particles.

[0090] In some embodiments, the cell culture produces more rAAV particles measured as GC/ml than a culture in the absence of adding of the mitotic inhibitor and/or caspase inhibitor.

[0091] In some embodiments, the cell culture produces at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, or at least 100% more rAAV particles measured as GC/ml than a culture in the absence of adding of the mitotic inhibitor and/or caspase inhibitor. In some embodiments, the cell culture produces at least 10% more rAAV particles measured as GC/ml than a culture in the absence of adding of the mitotic inhibitor and/or caspase inhibitor. In some embodiments, the cell culture produces at least 20% more rAAV particles measured as GC/ml than a culture in the absence of adding of the mitotic inhibitor and/or caspase inhibitor. In some embodiments, the cell culture produces at least 30% more rAAV particles measured as GC/ml than a culture in the absence of adding of the mitotic inhibitor and/or caspase inhibitor. In some embodiments, the cell culture produces at least 50% more rAAV particles measured as GC/ml than a culture in the absence of adding of the mitotic inhibitor and/or caspase inhibitor. In some embodiments, the cell culture produces at least 75% more rAAV particles measured as GC/ml than a culture in the absence of adding of the mitotic inhibitor and/or caspase inhibitor. In some embodiments, the cell culture produces at least 100% more rAAV particles measured as GC/ml than a culture in the absence of adding of the mitotic inhibitor and/or caspase inhibitor. In some embodiments, the cell culture produces at least 125% more rAAV particles measured as GC/ml than a culture in the absence of adding of the mitotic inhibitor and/or caspase inhibitor.

[0092] In some embodiments, the cell culture has a volume between about 50 liters and about 20,000 liters. [0093] In one aspect, the disclosure provides a method for producing rAAV particles, comprising culturing a cell capable of producing rAAV particles in a medium comprising between about 1 nM and about 500 pM of a mitotic inhibitor under conditions that allow the production of the rAAV particles. In some embodiments, the mitotic inhibitor is a microtubule destabilizing agent. In some embodiments, the mitotic inhibitor comprises at least one of nocodazole, vincristine, colchicine or a salt thereof. In some embodiments, the mitotic inhibitor is nocodazole. In some embodiments, the medium has a final nocodazole concentration between about 200 nM and about 10 pM. In some embodiments, the medium further comprises a caspase inhibitor. In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor is a z-VAD-FMK or Q-VD-Oph.

[0094] In some embodiments, the mitotic inhibitor concentration is between about 50 nM and about 50 pM. In some embodiments, the mitotic inhibitor concentration is between about 100 nM and about 20 pM. In some embodiments, the mitotic inhibitor concentration is between about 200 nM and about 10 pM.

[0095] In some embodiments, the nocodazole concentration is between about 50 nM and about 50 pM. In some embodiments, the nocodazole concentration is between about 100 nM and about 20 pM. In some embodiments, the nocodazole concentration is between about 200 nM and about 10 pM.

[0096] In some embodiments, the cell culture has a final mitotic inhibitor concentration of about 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM or 1 pM. In some embodiments, the mitotic inhibitor concentration is about 0.1 pM. In some embodiments, the mitotic inhibitor concentration is about 0.2 pM. In some embodiments, the mitotic inhibitor concentration is about 0.3 pM. In some embodiments, the mitotic inhibitor concentration is about 0.4 pM. In some embodiments, the mitotic inhibitor concentration is about 0.5 pM. In some embodiments, the mitotic inhibitor concentration is about 0.6 pM. In some embodiments, the mitotic inhibitor concentration is about 0.7 pM. In some embodiments, the mitotic inhibitor concentration is about 0.8 pM. In some embodiments, the mitotic inhibitor concentration is about 0.9 pM. In some embodiments, the mitotic inhibitor concentration is about 1 pM.

[0097] In some embodiments, the mitotic inhibitor concentration is about 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM or 1 pM. In some embodiments, the mitotic inhibitor concentration is about 0.1 pM. In some embodiments, the mitotic inhibitor concentration is about 0.2 pM. In some embodiments, the mitotic inhibitor concentration is about 0.3 pM. In some embodiments, the mitotic inhibitor concentration is about 0.4 pM. In some embodiments, the mitotic inhibitor concentration is about 0.5 pM. In some embodiments, the mitotic inhibitor concentration is about 0.6 pM. In some embodiments, the mitotic inhibitor concentration is about 0.7 pM. In some embodiments, the mitotic inhibitor concentration is about 0.8 pM. In some embodiments, the mitotic inhibitor concentration is about 0.9 pM. In some embodiments, the mitotic inhibitor concentration is about 1 pM.

[0098] In one aspect, the disclosure provides a method for producing rAAV particles, comprising culturing a cell capable of producing rAAV particles in a medium comprising between about 10 nM and about 1 mM of a caspase inhibitor under conditions that allow the production of the rAAV particles. In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD-FMK, Q-VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD-Oph. In some embodiments, the medium has a caspase inhibitor concentration between about 200 nM and about 20 pM. In some embodiments, the medium further comprises a mitotic inhibitor. In some embodiments, the mitotic inhibitor comprises nocodazole.

[0099] In some embodiments, the caspase inhibitor concentration is between about 50 nM and about 100 pM. In some embodiments, the caspase inhibitor concentration is between about 100 nM and about 50 pM. In some embodiments, the caspase inhibitor concentration is between about 200 nM and about 20 pM. In some embodiments, the caspase inhibitor concentration is between about 0.1 pM and about 1 pM. In some embodiments, the caspase inhibitor concentration is between about 0.5 pM and about 3 pM. In some embodiments, the caspase inhibitor concentration is between about 1 pM and about 5 pM. In some embodiments, the caspase inhibitor concentration is between about 3 pM and about 10 pM. In some embodiments, the caspase inhibitor concentration is between about 4 pM and about 20 pM. In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD-FMK, Q-VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD-Oph.

[00100] In some embodiments, the caspase inhibitor concentration is about 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM or 1 pM. In some embodiments, the caspase inhibitor concentration is about 0.1 pM. In some embodiments, the caspase inhibitor concentration is about 0.2 pM. In some embodiments, the caspase inhibitor concentration is about 0.3 pM. In some embodiments, the caspase inhibitor concentration is about 0.4 pM. In some embodiments, the caspase inhibitor concentration is about 0.5 pM. In some embodiments, the caspase inhibitor concentration is about 0.6 pM. In some embodiments, the caspase inhibitor concentration is about 0.7 pM. In some embodiments, the caspase inhibitor concentration is about 0.8 pM. In some embodiments, the caspase inhibitor concentration is about 0.9 pM. In some embodiments, the caspase inhibitor concentration is about 1 pM. In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD-FMK, Q-VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD-Oph.

[00101] In some embodiments, the caspase inhibitor concentration is about 1 pM , 2 pM , 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM or 10 pM. In some embodiments, the caspase inhibitor concentration is about 1 pM. In some embodiments, the caspase inhibitor concentration is about 2 pM. In some embodiments, the caspase inhibitor concentration is about 3 pM. In some embodiments, the caspase inhibitor concentration is about 4 pM. In some embodiments, the caspase inhibitor concentration is about 5 pM. In some embodiments, the caspase inhibitor concentration is about 6 pM. In some embodiments, the caspase inhibitor concentration is about 7 pM. In some embodiments, the caspase inhibitor concentration is about 8 pM. In some embodiments, the caspase inhibitor concentration is about 9 pM. In some embodiments, the caspase inhibitor concentration is about 10 pM. In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD-FMK, Q-VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD- Oph.

[00102] In some embodiments, the caspase inhibitor concentration is about 5 pM, 10 pM, 15 pM, 20 pM, or 25 pM. In some embodiments, the caspase inhibitor concentration is about 5 pM. In some embodiments, the caspase inhibitor concentration is about 10 pM. In some embodiments, the caspase inhibitor concentration is about 15 pM. In some embodiments, the caspase inhibitor concentration is about 20 pM. In some embodiments, the caspase inhibitor concentration is about 25 pM. In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD-FMK, Q-VD-Oph or a salt thereof. In some embodiments, the caspase inhibitor is Q-VD-Oph.

[00103] In some embodiments, the cell capable of producing rAAV comprises one or more polynucleotides encoding at least one of (a) an rAAV genome to be packaged, (b) adenovirus helper functions necessary for packaging, (c) an AAV rep protein sufficient for packaging, and (d) an AAV cap protein sufficient for packaging. In some embodiments, the cell capable of producing rAAV comprises one or more polynucleotides encoding (a) an rAAV genome to be packaged, (b) adenovirus helper functions necessary for packaging, (c) an AAV rep protein sufficient for packaging, and (d) an AAV cap protein sufficient for packaging.

[00104] In some embodiments, the cell capable of producing rAAV has been transfected with one or more polynucleotides encoding at least one of (a) an rAAV genome to be packaged, (b) adenovirus helper functions necessary for packaging, (c) an AAV rep protein sufficient for packaging, and (d) an AAV cap protein sufficient for packaging. In some embodiments, the cell capable of producing rAAV has been transfected with one or more polynucleotides encoding (a) an rAAV genome to be packaged, (b) adenovirus helper functions necessary for packaging, (c) an AAV rep protein sufficient for packaging, and (d) an AAV cap protein sufficient for packaging.

[00105] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is a HEK293 cell, HEK derived cell, CHO cell, CHO derived cell, HeLa cell, SF-9 cell, BHK cell, Vero cell, CAP cell or PerC6 cell. In some embodiments, the cell is a HEK293 cell. In some embodiments, the cell culture is a suspension culture.

[00106] In some embodiments, the culturing under conditions that allow production of the rAAV particles is for between about 2 days and about 10 days. In some embodiments, the culturing under conditions that allow production of the rAAV particles is for between about 5 days and 14 days.

[00107] In some embodiments, the maintaining the cell culture or culturing under conditions that allow production of the rAAV particles is for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days.

[00108] In some embodiments, the maintaining the cell culture or culturing under conditions that allow production of the rAAV particles is for about 3 days. In some embodiments, the maintaining the cell culture or culturing under conditions that allow production of the rAAV particles is for about 4 days. In some embodiments, the maintaining the cell culture or culturing under conditions that allow production of the rAAV particles is for about 5 days. In some embodiments, the maintaining the cell culture or culturing under conditions that allow production of the rAAV particles is for about 6 days. In some embodiments, the maintaining the cell culture or culturing under conditions that allow production of the rAAV particles is for about 7 days. In some embodiments, the maintaining the cell culture or culturing under conditions that allow production of the rAAV particles is for about 8 days.

[00109] In some embodiments, a method described herein further comprises recovering the rAAV particles.

[00110] In some embodiments, the cell culture produces more rAAV particles measured as GC/ml than a culture in the absence of mitotic inhibitor and/or caspase inhibitor.

[00111] In some embodiments, the cell culture produces at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, or at least 100% more rAAV particles measured as GC/ml than a culture in the absence of mitotic inhibitor and/or caspase inhibitor. In some embodiments, the cell culture produces at least 10% more rAAV particles measured as GC/ml than a culture in the absence of mitotic inhibitor and/or caspase inhibitor. In some embodiments, the cell culture produces at least 20% more rAAV particles measured as GC/ml than a culture in the absence of mitotic inhibitor and/or caspase inhibitor. In some embodiments, the cell culture produces at least 30% more rAAV particles measured as GC/ml than a culture in the absence of mitotic inhibitor and/or caspase inhibitor. In some embodiments, the cell culture produces at least 50% more rAAV particles measured as GC/ml than a culture in the absence of mitotic inhibitor and/or caspase inhibitor. In some embodiments, the cell culture produces at least 75% more rAAV particles measured as GC/ml than a culture in the absence of mitotic inhibitor and/or caspase inhibitor. In some embodiments, the cell culture produces at least 100% more rAAV particles measured as GC/ml than a culture in the absence of mitotic inhibitor and/or caspase inhibitor. In some embodiments, the cell culture produces at least 125% more rAAV particles measured as GC/ml than a culture in the absence of mitotic inhibitor and/or caspase inhibitor.

[00112] In some embodiments, the cell culture has a volume between about 50 liters and about 20,000 liters.

[00113] In some embodiments, the rAAV particles comprise a capsid protein of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 serotype.

[00114] In some embodiments, the rAAV particles comprise a capsid protein of the AAV8, AAV9, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, or AAV.hu37 serotype.

[00115] In some embodiments, the rAAV particles comprise a capsid protein of the AAV8 or AAV9 serotype.

[00116] In some embodiments, the rAAV particle comprises a transgene encoding a gene product. In some embodiments, the gene product is a polypeptide or a double stranded RNA molecule. In some embodiments, the gene product is a polypeptide. In some embodiments, the transgene encodes an antibody or antigen-binding fragment thereof, fusion protein, Fc-fusion polypeptide, immunoadhesin, immunoglobulin, engineered protein, protein fragment or enzyme. In some embodiments, the transgene comprises a regulatory element operatively connected to a polynucleotide encoding the gene product. [00117] In some embodiments, the genome to be packaged encodes a polypeptide or a double stranded RNA molecule. In some embodiments, the genome to be packaged encodes a polypeptide. In some embodiments, the genome to be packaged encodes an anti-VEGF Fab, anti-kallikrein antibody, anti-TNF antibody, microdystrophin, minidystrophin, iduronidase (IDU(A), iduronate 2-sulfatase (IDS), low- density lipoprotein receptor (LDLR), tripeptidyl peptidase 1 (TPP1), or non-membrane associated splice variant of VEGF receptor 1 (sFlt-1). In some embodiments, the genome to be packaged encodes an gamma-sarcoglycan, Rab Escort Protein 1 (REP1/CHM), retinoid isomcrohydrolasc (RPE65), cyclic nucleotide gated channel alpha 3 (CNGA3), cyclic nucleotide gated channel beta 3 (CNGB3), aromatic L- amino acid decarboxylase (AAD(C), lysosome-associated membrane protein 2 isoform B (LAMP2(B), Factor VIII, Factor IX, retinitis pigmentosa GTPase regulator (RPGR), retinoschisin (RSI), sarcoplasmic reticulum calcium ATPase (SERCA2(a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamic acid decarboxylase (GA(D), Glial cell line-derived neurotrophic factor (GDN(F), aquaporin 1 (AQP1), dystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pas(e), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1), arylsulfatase B (ARS(B), N-acetyl-alpha-glucosaminidase (NAGLU), alpha-glucosidase (GA(A), alphagalactosidase (GL(A), beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha 1-antitrypsin (AAT), phosphodiesterase 6B (PDE6(B), ornithine carbamoyltransferase 90T(C), survival motor neuron (SMN1), survival motor neuron (SMN2), neurturin (NRTN), Neurotrophin-3 (NT-3/NTF3), porphobilinogen deaminase (PBG(D), nerve growth factor (NG(F), mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPC(A), dysferlin, MER proto-oncogene, tyrosine kinase (MERTK), cystic fibrosis transmembrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgGl) Fc fusion. In some embodiments, the genome to be packaged encodes a dystrophin or a microdystrophin. In some embodiments, the genome to be packaged encodes a microRNA.

[00118] In one aspect, the disclosure provides a composition comprising isolated rAAV particles that were produced by a method described herein.

[00119] In one aspect, the disclosure provides a composition comprising cells capable of producing rAAV particles and a cell culture medium comprising between about 1 nM and about 500 |1M of a mitotic inhibitor. In some embodiments, the mitotic inhibitor is a microtubule destabilizing agent. In some embodiments, the mitotic inhibitor comprises at least one of nocodazole, vincristine, colchicine or a salt thereof. In some embodiments, the mitotic inhibitor is nocodazole.

[00120] In one aspect, the disclosure provides a composition comprising cells capable of producing rAAV particles and a cell culture medium comprising between about 10 nM and about 1 mM of a caspase inhibitor. In some embodiments, the caspase inhibitor is a pan-caspase inhibitor. In some embodiments, the caspase inhibitor comprises at least one of z-VAD-FMK, Q-VD-Oph or a salt thereof.

[00121] In some embodiments, the caspase inhibitor is Q-VD-Oph.

[00122] In some embodiments, the rAAV particles comprise a capsid protein of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 serotype. In some embodiments, the rAAV particles comprise a capsid protein of the AAV8, AAV9, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, or AAV.hu37 serotype. In some embodiments, the rAAV particles comprise a capsid protein of the AAV8 serotype. In some embodiments, the rAAV particles comprise a capsid protein of the AAV9 serotype.

[00123] In some embodiments, a method described herein increases production of rAAV particles while maintaining or improving the quality attributes of the rAAV particles and compositions comprising thereof. In some embodiments, the quality of rAAV particles and compositions comprising thereof is assessed by determining the concentration of rAAV particles (e.g., GC/ml), the percentage of particles comprising a copy of the rAAV genome; the ratio of particles without a genome, infectivity of the rAAV particles, stability of rAAV particles, concentration of residual host cell proteins, or concentration of residual host cell nucleic acids (e.g., host cell genomic DNA, plasmid encoding rep and cap genes, plasmid encoding helper functions, plasmid encoding rAAV genome). In some embodiments, the quality of rAAV particles produced by a method described herein or compositions comprising thereof is the same as that of rAAV particles or compositions produced by a reference method that does not use a mitotic inhibitor and/or a caspase inhibitor. In some embodiments, the quality of rAAV particles produced by a method described herein or compositions comprising thereof is better than the quality of rAAV particles or compositions produced by a reference method that does not use a mitotic inhibitor and/or a caspase inhibitor.

[00124] Numerous cell culture based systems are known in the art for production of rAAV particles, any of which can be used to practice a method described herein. rAAV production cultures for the production of rAAV virus particles require; (1) suitable host cells, including, for example, human-derived cell lines such as HeLa, A549, or HEK293 cells and their derivatives (HEK293T cells, HEK293F cells), or mammalian cell lines such as Vero, CAP® cells, CHO cells or CHO-derived cells; (2) suitable helper virus function, provided by wild type or mutant adenovirus (such as temperature sensitive adenovirus), herpes virus, baculovirus, or a plasmid construct providing helper functions; (3) AAV rep and cap genes and gene products; (4) a transgene (such as a therapeutic transgene) flanked by AAV ITR sequences; and (5) suitable media and media components to support rAAV production.

[00125] A skilled artisan is aware of the numerous methods by which AAV rep and cap genes, AAV helper genes (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene), and rAAV genomes (comprising one or more genes of interest flanked by inverted terminal repeats (ITRs)) can be introduced into cells to produce or package rAAV. The phrase “adenovirus helper functions” refers to a number of viral helper genes expressed in a cell (as RNA or protein) such that the AAV grows efficiently in the cell. The skilled artisan understands that helper viruses, including adenovirus and herpes simplex virus (HSV), promote AAV replication and certain genes have been identified that provide the essential functions, e.g. the helper may induce changes to the cellular environment that facilitate such AAV gene expression and replication. In some embodiments, of a method described herein, AAV rep and cap genes, helper genes, and rAAV genomes are introduced into cells by transfection of one or more plasmid vectors encoding the AAV rep and cap genes, helper genes, and rAAV genome.

[00126] Molecular biology techniques to develop plasmid or viral vectors encoding the AAV rep and cap genes, helper genes, and/or rAAV genome are commonly known in the art. In some embodiments, AAV rep and cap genes are encoded by one plasmid vector. In some embodiments, AAV helper genes (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene) are encoded by one plasmid vector. In some embodiments, the Ela gene or Elb gene is stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transfection by one viral vector. In some embodiments, the Ela gene and Elb gene are stably expressed by the host cell, and the E4 gene, E2a gene, and VA gene are introduced into the cell by transfection by one plasmid vector. In some embodiments, one or more helper genes are stably expressed by the host cell, and one or more helper genes are introduced into the cell by transfection by one plasmid vector. In some embodiments, the helper genes are stably expressed by the host cell. In some embodiments, AAV rep and cap genes are encoded by one viral vector. In some embodiments, AAV helper genes (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene) are encoded by one viral vector. In some embodiments, the Ela gene or Elb gene is stably expressed by the host cell, and the remaining AAV helper genes are introduced into the cell by transfection by one viral vector. In some embodiments, the Ela gene and Elb gene are stably expressed by the host cell, and the E4 gene, E2a gene, and VA gene are introduced into the cell by transfection by one viral vector. In some embodiments, one or more helper genes are stably expressed by the host cell, and one or more helper genes are introduced into the cell by transfection by one viral vector. In some embodiments, the AAV rep and cap genes, the adenovirus helper functions necessary for packaging, and the rAAV genome to be packaged are introduced to the cells by transfection with one or more polynucleotides, e.g., vectors. In some embodiments, a method described herein comprises transfecting the cells with a mixture of three polynucleotides: one encoding the cap and rep genes, one encoding adenovirus helper functions necessary for packaging (e.g., adenovirus Ela gene, Elb gene, E4 gene, E2a gene, and VA gene), and one encoding the rAAV genome to be packaged. In some embodiments, the AAV cap gene is an AAV8 or AAV9 cap gene. In some embodiments, the AAV cap gene is an AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.PHB, or AAV.7m8 cap gene. In some embodiments, the AAV cap gene encodes a capsid protein with high sequence homology to AAV8 or AAV9 such as, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37. In some embodiments, the vector encoding the rAAV genome to be packaged comprises a gene of interest flanked by AAV ITRs. In some embodiments, the AAV ITRs are from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rhS, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other AAV serotype.

[00127] Any combination of vectors can be used to introduce AAV rep and cap genes, AAV helper genes, and rAAV genome to a cell in which rAAV particles are to be produced or packaged. In some embodiments, of a method described herein, a first plasmid vector encoding an rAAV genome comprising a gene of interest flanked by AAV inverted terminal repeats (ITRs), a second vector encoding AAV rep and cap genes, and a third vector encoding helper genes can be used. In some embodiments, a mixture of the three vectors is co-transfected into a cell.

[00128] In some embodiments, a combination of transfection and infection is used by using both plasmid vectors as well as viral vectors.

[00129] In some embodiments, one or more of rep and cap genes, and AAV helper genes are constitutively expressed by the cells and does not need to be transfected or transduced into the cells. In some embodiments, the cell constitutively expresses rep and/or cap genes. In some embodiments, the cell constitutively expresses one or more AAV helper genes. In some embodiments, the cell constitutively expresses Ela. In some embodiments, the cell comprises a stable transgene encoding the rAAV genome. [00130] In some embodiments, AAV rep, cap, and helper genes (e.g., Ela gene, Elb gene, E4 gene, E2a gene, or VA gene) can be of any AAV serotype. Similarly, AAV ITRs can also be of any AAV serotype. For example, in some embodiments, AAV ITRs are from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhl O, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1 , AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other AAV serotypes (e.g., a hybrid serotype harboring sequences from more than one serotype). In some embodiments, AAV cap gene is from AAV9 or AAV8 cap gene. In some embodiments, an AAV cap gene is from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rhS, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other AAV serotypes (e.g., a hybrid serotype harboring sequences from more than one serotype). In some embodiments, AAV rep and cap genes for the production of a rAAV particle is from different serotypes. For example, the rep gene is from AAV2 whereas the cap gene is from AAV9.

[00131] Any suitable media known in the art can be used for the production of recombinant virus particles (e.g., rAAV particles) according to a method described herein. These media include, without limitation, media produced by Hy clone Laboratories and JRH including Modified Eagle Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), and Sf-900 II SFM media as described in U.S. Pat. No. 6,723,551, which is incorporated herein by reference in its entirety. In some embodiments, the medium comprises Dynamis™ Medium, FreeStyle™ 293 Expression Medium, or Expi293™ Expression Medium from Invitrogen/ ThermoFisher. In some embodiments, the medium comprises Dynamis™ Medium. In some embodiments, a method described herein uses a cell culture comprising a serum-free medium, an animal-component free medium, or a chemically defined medium. In some embodiments, the medium is an animal-component free medium. In some embodiments, the medium comprises serum. In some embodiments, the medium comprises fetal bovine serum. In some embodiments, the medium is a glutamine-free medium. In some embodiments, the medium comprises glutamine. In some embodiments, the medium is supplemented with one or more of nutrients, salts, buffering agents, and additives (e.g., antifoam agent). In some embodiments, the medium is supplemented with glutamine. In some embodiments, the medium is supplemented with serum. In some embodiments, the medium is supplemented with fetal bovine serum. In some embodiments, the medium is supplemented with poloxamer, e.g., Kolliphor® P 188 Bio. In some embodiments, a medium is a base medium. In some embodiments, the medium is a feed medium.

[00132J Recombinant virus (e.g., rAAV) production cultures can routinely be grown under a variety of conditions (over a wide temperature range, for varying lengths of time, and the like) suitable to the particular host cell being utilized. As is known in the art, virus production cultures include suspension- adapted host cells such as HeLa cells, HEK293 cells, HEK293 derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CAP® cells, CHO cells, CHO-K1 cells, CHO derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK- 21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells and SF-9 cells which can be cultured in a variety of ways including, for example, spinner flasks, stirred tank biorcactors, and disposable systems such as the Wave bag system. Numerous suspension cultures are known in the art for production of rAAV particles, including for example, the cultures disclosed in U.S. Patent Nos. 6,995,006, 9,783,826, and in U.S. Pat. Appl. Pub. No. 20120122155, each of which is incorporated herein by reference in its entirety.

[00133] Any cell or cell line that is known in the art to produce a recombinant virus particles (e.g., rAAV particles) can be used in any one of the methods described herein. In some embodiments, a method of producing recombinant virus particles (e.g., rAAV particles) or increasing the production of recombinant virus particles (e.g., a rAAV particles) described herein uses HeLa cells, HEK293 cells, HEK293 derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CAP® cells, CHO cells, CHO- K1 cells, CHO derived cells, EB66 cells, LLC-MK cells, MDCK cells, RAF cells, RK cells, TCMK-1 cells, PK15 cells, BHK cells, BHK-21 cells, NS-1 cells, BHK cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells or SF-9 cells. In some embodiments, a method described herein uses mammalian cells. In some embodiments, a method described herein uses insect cells, e.g., SF-9 cells. In some embodiments, a method described herein uses cells adapted for growth in suspension culture. In some embodiments, a method described herein uses HEK293 cells adapted for growth in suspension culture. In some embodiments, a method described herein uses CAP® cells. CAP® cells are an immortalized human amniocyte cell line. See, e.g., Zeh et al., PLoS One., 14(8): e0221679 (2019). In some embodiments, a method described herein uses CAP® cells comprising a polynucleotide encoding helper functions for the production of AAV. In some embodiments, a method described herein uses a subclone of CAP® cells comprising a polynucleotide encoding helper functions for the production of AAV. In some embodiments, a method described herein uses ELEVECTA® Alpha cells. In some embodiments, a method described herein uses HEK293 derived suspension cells. In some embodiments, the HEK293 derived cells are Clone #1, Clone #2.1, Clone #2.2, Clone #2.3, Clone #3, Clone #3.1, Clone #3.2, Clone #3.3, Clone #3.4, Clone #3.5, Clone #3.6, or Clone #3.7.

[00134] In some embodiments, a cell culture described herein is a suspension culture. In some embodiments, a large scale suspension cell culture described herein comprises HEK293 cells adapted for growth in suspension culture. In some embodiments, a cell culture described herein comprises a serum- free medium, an animal-component free medium, or a chemically defined medium. In some embodiments, a cell culture described herein comprises a serum-free medium. In some embodiments, suspension-adapted cells are cultured in a shaker flask, a spinner flask, a cell bag, or a bioreactor.

[00135] In some embodiments, a cell culture described herein comprises a serum-free medium, an animalcomponent free medium, or a chemically defined medium. In some embodiments, a cell culture described herein comprises a serum-free medium.

[00136] In some embodiments, a large scale suspension culture cell culture described herein comprises a high density cell culture. In some embodiments, the culture has a total cell density of between about lxl0E+06 cells/ml and about 30xl0E+06 cells/ml. In some embodiments, more than about 50% of the cells are viable cells. In some embodiments, the cells are HeLa cells, HEK293 cells, HEK293 derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CAP® cells, or SF-9 cells. In further embodiments, the cells are HEK293 cells. In further embodiments, the cells are HEK293 derived cells. In further embodiments, the HEK293 derived cells are Clone #1, Clone #2.1, Clone #2.2, Clone #2.3, Clone #3, Clone #3.1, Clone #3.2, Clone #3.3, Clone #3.4, Clone #3.5, Clone #3.6, or Clone #3.7.

[00137] Methods described herein can be used in the production of rAAV particles comprising a capsid protein from any AAV capsid serotype. In some embodiments, the rAAV particles comprise a capsid protein from an AAV capsid serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles comprise a capsid protein that is a derivative, modification, or pseudotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 capsid protein.

[00138] In some embodiments, the rAAV particles comprise a capsid protein from an AAV capsid serotype selected from AAV8 and AAV9. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV9.

[00139] In some embodiments, the rAAV particles comprise a capsid protein from an AAV capsid serotype selected from the group consisting of AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.PHB, and AAV.7m8. In some embodiments, the rAAV particles comprise a capsid protein with high sequence homology to AAV8 or AAV9 such as, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[00140] In some embodiments, the rAAV particles comprise a capsid protein that is a derivative, modification, or pseudotype of AAV8 or AAV9 capsid protein. In some embodiments, the rAAV particles comprise a capsid protein that has an AAV8 capsid protein at least 80% or more identical, c.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of AAV8 capsid protein.

[00141] In some embodiments, the rAAV particles comprise a capsid protein that is a derivative, modification, or pseudotype of AAV9 capsid protein. In some embodiments, rAAV particles comprise a capsid protein that has an AAV9 capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of AAV9 capsid protein.

[00142] In some embodiments, the rAAV particles comprise a capsid protein that has at least 80% or more identity, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identity, to the VP1, VP2 and/or VP3 sequence of AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.PHB, or AAV.7m8 capsid protein. In some embodiments, the rAAV particles comprise a capsid protein that has at least 80% or more identity, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identity, to the VP1, VP2 and/or VP3 sequence of an AAV capsid protein with high sequence homology to AAV8 or AAV9 such as, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, and AAV.hu37.

[00143] In additional embodiments, the rAAV particles comprise a mosaic capsid. In additional embodiments, the rAAV particles comprise a pseudotyped rAAV particle. In additional embodiments, the rAAV particles comprise a capsid containing a capsid protein chimera of two or more AAV capsid serotypes. rAAV Particles

[00144] The provided methods are suitable for use in the production of any isolated recombinant AAV particles. As such, the rAAV can be of any serotype, modification, or derivative, known in the art, or any combination thereof (e.g., a population of rAAV particles that comprises two or more serotypes, e.g., comprising two or more of rAAV2, rAAV8, and rAAV9 particles) known in the art. In some embodiments, the rAAV particles are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7,AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or other rAAV particles, or combinations of two or more thereof.

[00145] In some embodiments, rAAV particles have a capsid protein from an AAV serotype selected from AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.AncSO, AAV.Anc8OL65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LKO3, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC1O , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 or a derivative, modification, or pseudotype thereof. In some embodiments, rAAV particles comprise a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to e.g., VP1, VP2 and/or VP3 sequence of an AAV capsid serotype selected from AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, rAAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16. [00146] In some embodiments, rAAV particles comprise a capsid protein from an AAV capsid serotype selected from AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.AncSO, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16, or a derivative, modification, or pseudotype thereof. In some embodiments, rAAV particles comprise a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to e.g., VP1, VP2 and/or VP3 sequence of an AAV capsid serotype selected from AAV1 , AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV.rhS, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.AncSO, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

[00147] In some embodiments, rAAV particles comprise the capsid of Anc80 or Anc80L65, as described in Zinn et al., 2015, Cell Rep. 12(6): 1056-1068, which is incorporated by reference in its entirety. In certain embodiments, the rAAV particles comprise the capsid with one of the following amino acid insertions: LGETTRP or LALGETTRP, as described in United States Patent Nos. 9,193,956; 9458517; and 9,587,282 and US patent application publication no. 2016/0376323, each of which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise the capsid of AAV.7m8, as described in United States Patent Nos. 9,193,956; 9,458,517; and 9,587,282 and US patent application publication no. 2016/0376323, each of which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in United States Patent No. 9,585,971, such as AAVPHP.B. In some embodiments, rAAV particles comprise any AAV capsid disclosed in United States Patent No. 9,840,719 and WO 2015/013313, such as AAV.Rh74 and RHM4-1, each of which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in WO 2014/172669, such as AAV rh.74, which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise the capsid of AAV2/5, as described in Georgiadis et al., 2016, Gene Therapy 23: 857-862 and Georgiadis et al., 2018, Gene Therapy 25: 450, each of which is incorporated by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in WO 2017/070491, such as AAV2tYF, which is incorporated herein by reference in its entirety. In some embodiments, rAAV particles comprise the capsids of AAVLK03 or AAV3B, as described in Puzzo et al., 2017, Sci. Transl. Med. 29(9): 418, which is incorporated by reference in its entirety. In some embodiments, rAAV particles comprise any AAV capsid disclosed in US Pat Nos. 8,628,966; US 8,927,514; US 9,923,120 and WO 2016/049230, such as HSC1, HSC2, HSC3, HSC4, HSC5, HSC6, HSC7, HSC8, HSC9, HSC10 , HSC11, HSC12, HSC13, HSC14, HSC15, or HSC16, each of which is incorporated by reference in its entirety. [00148] In some embodiments, rAAV particles comprise an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; US patent application publication nos. 2015/0374803; 2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024; 2017/0051257; and International Patent Application Nos. PCT/US2015/034799; PCT/EP2015/053335. In some embodiments, rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in any of the following patents and patent applications, each of which is incorporated herein by reference in its entirety: United States Patent Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; US patent application publication nos. 2015/0374803; 2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024; 2017/0051257; and International Patent Application Nos. PCT/US2015/034799; PCT/EP2015/053335. [00149] In some embodiments, rAAV particles have a capsid protein disclosed in Inti. Appl. Publ. No. WO 2003/052051 (see, e.g., SEQ ID NO: 2), WO 2005/033321 (see, e.g., SEQ ID NOs: 123 and 88), WO 03/042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97), WO 2006/068888 (see, e.g., SEQ ID NOs: 1 and 3- 6), WO 2006/110689, (see, e.g., SEQ ID NOs: 5-38) W02009/104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31), W0 2010/127097 (see, e.g., SEQ ID NOs: 5-38), and WO 2015/191508 (see, e.g., SEQ ID NOs: 80-294), and U.S. Appl. Publ. No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5-10), the contents of each of which is herein incorporated by reference in its entirety. In some embodiments, rAAV particles have a capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of an AAV capsid disclosed in Inti. Appl. Publ. No. WO 2003/052051 (see, e.g., SEQ ID NO: 2), WO 2005/033321 (see, e.g., SEQ ID NOs: 123 and 88), WO 03/042397 (see, e.g., SEQ ID NOs: 2, 81, 85, and 97), WO 2006/068888 (see, e.g., SEQ ID NOs: 1 and 3-6), WO 2006/110689 (see, e.g., SEQ ID NOs: 5-38) W02009/104964 (see, e.g., SEQ ID NOs: 1-5, 7, 9, 20, 22, 24 and 31), W0 2010/127097 (see, e.g., SEQ ID NOs: 5-38), and WO 2015/191508 (see, e.g., SEQ ID NOs: 80-294), and U.S. Appl. Publ. No. 20150023924 (see, e.g., SEQ ID NOs: 1, 5-10).

[00150] Nucleic acid sequences of AAV based viral vectors and methods of making recombinant AAV and AAV capsids are taught, for example, in United States Patent Nos. 7,282,199; 7,906,111; 8,524,446; 8,999,678; 8,628,966; 8,927,514; 8,734,809; US 9,284,357; 9,409,953; 9,169,299; 9,193,956; 9458517; and 9,587,282; US patent application publication nos. 2015/0374803; 2015/0126588; 2017/0067908; 2013/0224836; 2016/0215024; 2017/0051257; International Patent Application Nos.

PCT/US2015/034799; PCT/EP2015/053335; WO 2003/052051, WO 2005/033321, WO 03/042397, WO 2006/068888, WO 2006/110689, W02009/104964, W02010/127097, and WO 2015/191508, and U.S. Appl. Publ. No. 20150023924.

[00151] The provided methods are suitable for use in the production of recombinant AAV encoding a transgene. In certain embodiments, the transgene is from Tables 1 A-1 C. In some embodiments, the rAAV genome comprises a vector comprising the following components: (1) AAV inverted terminal repeats that flank an expression cassette; (2) regulatory control elements, such as a) promoter /enhancers, b) a polyA signal, and c) optionally an intron; and (3) nucleic acid sequences coding for a transgene. In other embodiments for expressing an intact or substantially intact monoclonal antibody (mAb), the rAAV genome comprises a vector comprising the following components: (1) AAV inverted terminal repeats that flank an expression cassette; (2) regulatory control elements, such as a) promoter /enhancers, b) a polyA signal, and c) optionally an intron; and (3) nucleic acid sequences coding for the light chain Fab and heavy chain Fab of the antibody, or at least the heavy chain or light chain Fab, and optionally a heavy chain Fc region. In still other embodiments for expressing an intact or substantially intact mAb, the rAAV genome comprises a vector comprising the following components: (1) AAV inverted terminal repeats that flank an expression cassette; (2) regulatory control elements, such as a) promoter /enhancers, b) a polyA signal, and c) optionally an intron; and (3) nucleic acid sequences coding for the heavy chain Fab of an anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA- 651, ), anti-ALKl (e.g., ascrinvacumab), anti-C5 (e.g., tesidolumab and eculizumab), anti-CD105 (e.g., carotuximab), anti-CClQ (e.g., ANX-007), anti-TNFa (e.g., adalimumab, infliximab, and golimumab), anti-RGMa (e.g., elezanumab), anti-TTR (e.g., NI-301 and PRX-004), anti-CTGF (e.g., pamrevlumab), anti-IL6R (e.g., satralizumab and sarilumab), anti-IL4R (e.g., dupilumab), anti-IL17A (e.g., ixekizumab and secukinumab), anti- IL-5 (e.g., mepolizumab), anti-IL12/IL23 (e.g., ustekinumab), anti-CD19 (e.g., inebilizumab), anti-ITGF7 mAb (e.g., etrolizumab), anti-SOST mAb (e.g., romosozumab), anti-pKal mAb (e.g., lanadelumab), anti-ITGA4 (e.g., natalizumab), anti-ITGA4B7 (e.g., vedolizumab), anti-BLyS (e.g., belimumab), anti-PD-1 (e.g., nivolumab and pembrolizumab), anti-RANKL (e.g., densomab), anti- PCSK9 (e.g., alirocumab and evolocumab), anti-ANGPTL3 (e.g., evinacumab*), anti-OxPL (e.g., E06), anti-fD (e.g., lampalizumab), or anti-MMP9 (e.g., andecaliximab); optionally an Fc polypeptide of the same isotype as the native form of the therapeutic antibody, such as an IgG isotype amino acid sequence IgGl, IgG2 or IgG4 or modified Fc thereof; and the light chain of an anti-VEGF (e.g., sevacizumab, ranibizumab, bevacizumab, and brolucizumab), anti-EpoR (e.g., LKA-651, ), anti-ALKl (e.g., ascrinvacumab), anti-C5 (e.g., tesidolumab and eculizumab), anti-CD105 or anti-ENG (e.g., carotuximab), anti-CClQ (e.g., ANX-007), anti-TNFa (e.g., adalimumab, infliximab, and golimumab), anti-RGMa (e.g., elezanumab), anti-TTR (e.g., NI-301 and PRX-004), anti-CTGF (e.g., pamrevlumab), anti-IL6R (e.g., satralizumab and sarilumab), anti-IL4R (e.g., dupilumab), anti-IL17A (e.g., ixekizumab and secukinumab), anti- IL-5 (e.g., mepolizumab), anti-IL12/IL23 (e.g., ustekinumab), anti-CD19 (e.g., inebilizumab), anti-ITGF7 mAb (e.g., etrolizumab), anti-SOST mAb (e.g., romosozumab), anti-pKal mAb (e.g., lanadelumab), anti-ITGA4 (e.g., natalizumab), anti-ITGA4B7 (e.g., vedolizumab), anti-BLyS (e.g., belimumab), anti-PD-1 (e.g., nivolumab and pembrolizumab), anti-RANKL (e.g., densomab), anti- PCSK9 (e.g., alirocumab and evolocumab), anti-ANGPTL3 (e.g., evinacumab), anti-OxPL (e.g., E06), anti-fD (e.g., lampalizumab), or anti-MMP9 (e.g., andecaliximab); wherein the heavy chain (Fab and optionally Fc region) and the light chain are separated by a self-cleaving furin (F)/F2A or flexible linker, ensuring expression of equal amounts of the heavy and the light chain polypeptides.

Table 1A

Table IB

Table 1C

[00152] In some embodiments, the rAAV particles are rAAV viral vectors encoding an anti-VEGF Fab. In specific embodiments, the rAAV particles are rAAV8-based viral vectors encoding an anti-VEGF Fab. In more specific embodiments, the rAAV particles are rAAV8-based viral vectors encoding ranibizumab. In some embodiments, the rAAV particles are rAAV viral vectors encoding iduronidase (IDUA). In specific embodiments, the rAAV particles are rAAV9-based viral vectors encoding IDUA. In some embodiments, the rAAV particles are rAAV viral vectors encoding iduronate 2-sulfatase (IDS). In specific embodiments, the rAAV particles are rAAV9-based viral vectors encoding IDS. In some embodiments, the rAAV particles are rAAV viral vectors encoding a low-density lipoprotein receptor (LDLR). In specific embodiments, the rAAV particles are rAAV8-based viral vectors encoding LDLR. In some embodiments, the rAAV particles are rAAV viral vectors encoding tripeptidyl peptidase 1 (TPP1) protein. In specific embodiments, the rAAV particles are rAAV9-based viral vectors encoding TPP1. In some embodiments, the rAAV particles are rAAV viral vectors encoding non-membrane associated splice variant of VEGF receptor 1 (sFlt-1). In some embodiments, the rAAV particles are rAAV viral vectors encoding gamma-sarcoglycan, Rab Escort Protein 1 (REP1/CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide gated channel alpha 3 (CNGA3), cyclic nucleotide gated channel beta 3 (CNGB3), aromatic L-amino acid decarboxylase (AADC), lysosome-associated membrane protein 2 isoform B (LAMP2B), Factor VIII, Factor IX, retinitis pigmentosa GTPase regulator (RPGR), retinoschisin (RSI), sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamic acid decarboxylase (GAD), Glial cell line-derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, microdystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6- phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1), arylsulfatase B (ARSB), N-acetyl-alpha-glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase (GLA), beta-galactosidase (GLB1), lipoprotein lipase (LPL), alpha 1- antitrypsin (AAT), phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9OTC), survival motor neuron (SMN1 ), survival motor neuron (SMN2), neurturin (NRTN), Neurotrophin-3 (NT-3/NTF3), porphobilinogen deaminase (PBGD), nerve growth factor (NGF), mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dysferlin, MER proto-oncogene, tyrosine kinase (MERTK), cystic fibrosis transmembrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR)-immunoglobulin (IgGl) Fc fusion.

[00153] In additional embodiments, rAAV particles comprise a pseudotyped AAV capsid. In some embodiments, the pseudotyped AAV capsids are rAAV2/8 or rAAV2/9 pseudotyped AAV capsids. Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g. , Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et al., J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001). [00154] In additional embodiments, rAAV particles comprise a capsid containing a capsid protein chimeric of two or more AAV capsid serotypes. In some embodiments, the capsid protein is a chimeric of 2 or more AAV capsid proteins from AAV serotypes selected from AAV1, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

[00155] In certain embodiments, a single-stranded AAV (ssAAV) can be used. In certain embodiments, a self-complementary vector, e.g., scAAV, can be used (see, e.g., Wu, 2007, Human Gene Therapy, 18(2): 171 -82, McCarty et al, 2001, Gene Therapy, Vol. 8, Number 16, Pages 1248-1254; and U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety).

[00156] In some embodiments, the rAAV particles comprise a capsid protein from an AAV capsid serotype selected from AAV8 or AAV9. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV8. In some embodiments, the rAAV particles have an AAV capsid serotype of AAV9. [00157] In some embodiments, the rAAV particles comprise a capsid protein that is a derivative, modification, or pseudotype of AAV8 or AAV9 capsid protein. In some embodiments, the rAAV particles comprise a capsid protein that has an AAV8 capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of AAV8 capsid protein.

[00158] In some embodiments, the rAAV particles comprise a capsid protein that is a derivative, modification, or pseudotype of AAV9 capsid protein. In some embodiments, the rAAV particles comprise a capsid protein that has an AAV9 capsid protein at least 80% or more identical, e.g., 85%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc., i.e. up to 100% identical, to the VP1, VP2 and/or VP3 sequence of AAV9 capsid protein.

[00159] In additional embodiments, the rAAV particles comprise a mosaic capsid. Mosaic AAV particles are composed of a mixture of viral capsid proteins from different serotypes of AAV. In some embodiments, the rAAV particles comprise a mosaic capsid containing capsid proteins of a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles comprise a mosaic capsid containing capsid proteins of a serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.8, AAVrh.10, AAVhu.37, AAVrh.20, and AAVrh.74.

[00160] In additional embodiments, the rAAV particles comprise a pseudotyped rAAV particle. In some embodiments, the pseudotyped rAAV particle comprises (a) a nucleic acid vector comprising AAV ITRs and (b) a capsid comprised of capsid proteins derived from AAVx (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV21YF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16). In additional embodiments, the rAAV particles comprise a pseudotyped rAAV particle comprised of a capsid protein of an AAV serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVrh.8, and AAVrh.10, AAVhu.37, AAVrh.20, and AAVrh.74. In additional embodiments, the rAAV particles comprise a pseudotyped rAAV particle containing AAV8 capsid protein. In additional embodiments, the rAAV particles comprise a pseudotyped rAAV particle is comprised of AAV9 capsid protein. In some embodiments, the pseudotyped rAAV8 or rAAV9 particles are rAAV2/8 or rAAV2/9 pseudotyped particles. Methods for producing and using pseudotyped rAAV particles are known in the art (see, e.g., Duan et al., J. Virol., 75:7662-7671 (2001); Halbert et ah, J. Virol., 74:1524-1532 (2000); Zolotukhin et al., Methods 28:158-167 (2002); and Auricchio et al., Hum. Molec. Genet. 10:3075-3081, (2001).

[00161] In additional embodiments, the rAAV particles comprise a capsid containing a capsid protein chimeric of two or more AAV capsid serotypes. In some embodiments, the rAAV particles comprise an AAV capsid protein chimeric of AAV8 capsid protein and one or more AAV capsid proteins from an AAV serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles comprise an AAV capsid protein chimeric of AAV8 capsid protein and one or more AAV capsid proteins from an AAV serotype selected from AAV1, AAV2, AAV5, AAV6, AAV7, AAV9, AAV10, rAAVrhlO, AAVrh.8, AAVrh.lO, AAVhu.37, AAVrh.20, and AAVrh.74. In some embodiments, the rAAV particles comprise an AAV capsid protein chimeric of AAV9 capsid protein the capsid protein of one or more AAV capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the rAAV particles comprise an AAV capsid protein chimeric of AAV9 capsid protein the capsid protein of one or more AAV capsid serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, AAVrh.lO, AAVhu.37, AAVrh.20, and AAVrh.74.

Methods for Isolating RAAV particles

[00162] In some embodiments, the disclosure provides methods for producing recombinant adeno- associated virus (rAAV) particles, comprising isolating rAAV particles from a feed comprising an impurity (for example, rAAV production culture). In some embodiments, a method for producing recombinant adeno-associated virus (rAAV) particles described herein comprises (a) isolating rAAV particles from a feed comprising an impurity (for example, rAAV production culture), and (b) formulating the isolated rAAV particles to produce the formulation.

[00163] In some embodiments, the disclosure further provides methods for producing a pharmaceutical unit dosage of a formulation comprising isolated recombinant adeno-associated virus (rAAV) particles, comprising isolating rAAV particles from a feed comprising an impurity (for example, rAAV production culture), and formulating the isolated rAAV particles.

[00164] Isolated rAAV particles can be isolated using methods known in the ait. In some embodiments, methods of isolating rAAV particles comprises downstream processing such as, for example, harvest of a cell culture, clarification of the harvested cell culture (e.g., by centrifugation or depth filtration), 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. In some embodiments, downstream processing includes at least 2, at least 3, at least 4, at least 5 or at least 6 of: harvest of a cell culture, clarification of the harvested cell culture (e.g., by centrifugation or depth filtration), tangential flow filtration, affinity chromatography, anion exchange chromatography, cation exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, and sterile filtration. In some embodiments, downstream processing comprises harvest of a cell culture, clarification of the harvested cell culture (e.g., by depth filtration), sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, downstream processing comprises clarification of a harvested cell culture, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, downstream processing comprises clarification of a harvested cell culture by depth filtration, sterile filtration, tangential flow filtration, affinity chromatography, and anion exchange chromatography. In some embodiments, clarification of the harvested cell culture comprises sterile filtration. In some embodiments, downstream processing does not include centrifugation. In some embodiments, the rAAV particles comprise a capsid protein of the AAV8 serotype. In some embodiments, the rAAV particles comprise a capsid protein of the AAV9 serotype.

[00165] In some embodiments, a method of isolating rAAV particles produced according to a method described herein comprises harvest of a cell culture, clarification of the harvested cell culture (e.g., by depth filtration), a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration. In some embodiments, a method of isolating rAAV particles described herein comprises harvest of a cell culture, clarification of the harvested cell culture (e.g., by depth filtration), a first sterile filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a tangential flow filtration, and a second sterile filtration. In some embodiments, a method of isolating rAAV particles produced according to a method described herein comprises clarification of a harvested cell culture, a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration. In some embodiments, a method of isolating rAAV particles described herein comprises clarification of a harvested cell culture, a first sterile filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography using a quaternary amine ligand), tangential flow filtration, and a second sterile filtration. In some embodiments, a method of isolating rAAV particles produced according to a method described herein comprises clarification of a harvested cell culture by depth filtration, a first sterile filtration, a first tangential flow filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography using a quaternary amine ligand), a second tangential flow filtration, and a second sterile filtration. In some embodiments, a method of isolating rAAV particles described herein comprises clarification of a harvested cell culture by depth filtration, a first sterile filtration, affinity chromatography, anion exchange chromatography (e.g., monolith anion exchange chromatography or AEX chromatography using a quaternary amine ligand), tangential flow filtration, and a second sterile filtration. In some embodiments, the method does not include centrifugation. In some embodiments, clarification of the harvested cell culture comprises sterile filtration. In some embodiments, the rAAV particles comprise a capsid protein of the AAV8 serotype. In some embodiments, the rAAV particles comprise a capsid protein of the AAV9 serotype.

[00166] Numerous methods are known in the art for production of rAAV particles, including transfection, stable cell line production, and infectious hybrid virus production systems which include adenovirus- AAV hybrids, herpesvirus- AAV hybrids and baculovirus-AAV hybrids. rAAV production cultures for the production of rAAV virus particles all require; (1) suitable host cells, including, for example, human- derived cell lines such as HeLa, A549, or HEK293 cells and their derivatives (HEK293T cells, HEK293F cells), mammalian cell lines such as Vero, or insect-derived cell lines such as SF-9 in the case of baculovirus production systems; (2) suitable helper virus function, provided by wild type or mutant adenovirus (such as temperature sensitive adenovirus), herpes virus, baculovirus, or a plasmid construct providing helper functions; (3) AAV rep and cap genes and gene products; (4) a transgene (such as a therapeutic transgene) flanked by AAV ITR sequences; and (5) suitable media and media components to support rAAV production. In some embodiments, the suitable helper virus function is provided by a recombinant polynucleotide described herein or a plasmid described herein. Suitable media known in the art may be used for the production of rAAV vectors. These media include, without limitation, media produced by Hyclone Laboratories and JRH including Modified Eagle Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), and Sf-900 II SFM media as described in U.S. Pat. No. 6,723,551, which is incorporated herein by reference in its entirety.

[00167] rAAV production cultures can routinely be grown under a variety of conditions (over a wide temperature range, for varying lengths of time, and the like) suitable to the particular host cell being utilized. As is known in the art, rAAV production cultures include attachment-dependent cultures which can be cultured in suitable attachment-dependent vessels such as, for example, roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors. rAAV vector production cultures may also include suspension-adapted host cells such as HeLa cells, HEK293 cells, HEK293 derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CAP® cells, CHO cells, CHO-K1 cells, CHO derived cells, EB66 cells, BSC cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WL38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells or SF-9 cells which can be cultured in a variety of ways including, for example, spinner flasks, stirred tank biorcactors, and disposable systems such as the Wave bag system. In some embodiments, the cells are HEK293 cells. In some embodiments, the cells are HEK293 cells adapted for growth in suspension culture. Numerous suspension cultures are known in the art for production of rAAV particles, including for example, the cultures disclosed in U.S. Patent Nos. 6,995,006, 9,783,826, and in U.S. Pat. Appl. Pub. No. 20120122155, each of which is incorporated herein by reference in its entirety. In some embodiments, the cells are CAP® cells. CAP® cells are an immortalised human amniocyte cell line. See, e.g., Zeh et al., PLoS One., 14(8): e0221679 (2019). In some embodiments, the cells are CAP® cells comprising a polynucleotide encoding helper functions for the production of AAV. In some embodiments, the cells are a subclone of CAP® cells comprising a polynucleotide encoding helper functions for the production of AAV. In some embodiments, the cells are ELEVECTA® Alpha cells.

[00168] In some embodiments, the rAAV production culture comprises a high density cell culture. In some embodiments, the culture has a total cell density of between about lxl0E+06 cells/ml and about 30xl0E+06 cells/ml. In some embodiments, more than about 50% of the cells arc viable cells. In some embodiments, the cells are HeLa cells, HEK293 cells, HEK293 derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CAP® cells, or SF-9 cells. In further embodiments, the cells are HEK293 cells. In further embodiments, the cells are HEK293 cells adapted for growth in suspension culture. [00169] In additional embodiments of the provided method the rAAV production culture comprises a suspension culture comprising rAAV particles. Numerous suspension cultures are known in the art for production of rAAV particles, including for example, the cultures disclosed in U.S. Patent Nos. 6,995,006, 9,783,826, and in U.S. Pat. Appl. Pub. No. 20120122155, each of which is incorporated herein by reference in its entirety. In some embodiments, the suspension culture comprises a culture of mammalian cells or insect cells. In some embodiments, the suspension culture comprises a culture of HeLa cells, HEK293 cells, HEK293 derived cells (e.g., HEK293T cells, HEK293F cells), Vero cells, CAP® cells, CHO cells, CHO-K1 cells, CHO derived cells, EB66 cells, BSC cells, HepG2 cells, LLC- MK cells, CV-1 cells, COS cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 3T3 cells, 293 cells, RK cells, Per.C6 cells, chicken embryo cells or SF-9 cells. In some embodiments, the suspension culture comprises a culture of HEK293 cells. In some embodiments, the suspension culture comprises CAP® cells. CAP® cells are an immortalized human amniocyte cell line. See, e.g., Zeh et al., PLoS One., 14(8): e0221679 (2019). In some embodiments, the suspension culture comprises CAP® cells comprising a polynucleotide encoding helper functions for the production of AAV. In some embodiments, the suspension culture comprises a subclone of CAP® cells comprising a polynucleotide encoding helper functions for the production of AAV. In some embodiments, the suspension culture comprises ELEVECTA® Alpha cells. In further embodiments, the suspension culture comprises HEK293 derived cells. In further embodiments, the HEK293 derived suspension cells are Clone #1, Clone #2.1, Clone #2.2, Clone #2.3, Clone #3, Clone #3.1, Clone #3.2, Clone #3.3, Clone #3.4, Clone #3.5, Clone #3.6, or Clone #3.7.

[00170] In some embodiments, methods for the production of rAAV particles encompasses providing a cell culture comprising a cell capable of producing rAAV ; adding to the cell culture a histone deacetylase (HD AC) inhibitor to a final concentration between about 0.1 mM and about 20 mM; and maintaining the cell culture under conditions that allows production of the rAAV particles. In some embodiments, the HD AC inhibitor comprises a short-chain fatty acid or salt thereof. In some embodiments, the HD AC inhibitor comprises butyrate (e.g., sodium butyrate), valproate (e.g., sodium valproate), propionate (e.g., sodium propionate), or a combination thereof.

[00171] In some embodiments, rAAV particles are produced as disclosed in WO 2020/033842, which is incorporated herein by reference in its entirety.

[00172] Recombinant AAV particles can be harvested from rAAV production cultures by harvest of the production culture comprising host cells or by harvest of the spent media from the production culture, provided the cells are cultured under conditions known in the art to cause release of rAAV particles into the media from intact host cells. Recombinant AAV particles can also be harvested from rAAV production cultures by lysis of the host cells of the production culture. Suitable methods of lysing cells are also known in the art and include for example multiple freeze/thaw cycles, sonication, microfluidization, and treatment with chemicals, such as detergents and/or proteases.

[00173] At harvest, rAAV production cultures can contain one or more of the following: (1) host cell proteins; (2) host cell DNA; (3) plasmid DNA; (4) helper virus; (5) helper virus proteins; (6) helper virus DNA; and (7) media components including, for example, serum proteins, amino acids, transferrins and other low molecular' weight proteins. rAAV production cultures can further contain product-related impurities, for example, inactive vector forms, empty viral capsids, aggregated viral particles or capsids, mis-folded viral capsids, degraded viral particle.

[00174] In some embodiments, the rAAV production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is clarified by filtration through a series of depth filters. Clarification can also be achieved by a variety of other standard techniques known in the art, such as, centrifugation or filtration through any cellulose acetate filter of 0.2 nun or greater pore size known in the art. In some embodiments, clarification of the harvested cell culture comprises sterile filtration. In some embodiments, the production culture harvest is clarified by centrifugation. In some embodiments, clarification of the production culture harvest does not included centrifugation.

[00175] In some embodiments, harvested cell culture is clarified using filtration. In some embodiments, clarification of the harvested cell culture comprises depth filtration. In some embodiments, clarification of the harvested cell culture further comprises depth filtration and sterile filtration. In some embodiments, harvested cell culture is clarified using a filter train comprising one or more different filtration media. In some embodiments, the filter train comprises a depth filtration media. In some embodiments, the filter train comprises one or more depth filtration media. In some embodiments, the filter train comprises two depth filtration media. In some embodiments, the filter train comprises a sterile filtration media. In some embodiments, the filter train comprises 2 depth filtration media and a sterile filtration media. In some embodiments, the depth filter media is a porous depth filter. In some embodiments, the filter train comprises Clarisolve® 20MS, Millistak+® COHC, and a sterilizing grade filter media. In some embodiments, the filter train comprises Clarisolve® 20MS, Millistak+® COHC, and Sartopore® 2 XLG 0.2 pm. In some embodiments, the harvested cell culture is pretreated before contacting it with the depth filter. In some embodiments, the pretreating comprises adding a salt to the harvested cell culture. In some embodiments, the pretreating comprises adding a chemical flocculent to the harvested cell culture. In some embodiments, the harvested cell culture is not pre-treated before contacting it with the depth filter. [00176] In some embodiments, the production culture harvest is clarified by filtration are disclosed in WO 2019/212921, which is incorporated herein by reference in its entirety.

[00177] In some embodiments, the rAAV production culture harvest is treated with a nuclease (e.g., Benzonase®) or endonuclease (e.g., endonuclease from Serratia marcescens) to digest high molecular weight DNA present in the production culture. The nuclease or endonuclease digestion can routinely be performed under standard conditions known in the art. For example, nuclease digestion is performed at a final concentration of 1-2.5 units/ml of Benzonase® at a temperature ranging from ambient to 37°C for a period of 30 minutes to several hours.

[00178] Sterile filtration encompasses filtration using a sterilizing grade filter media. In some embodiments, the sterilizing grade filter media is a 0.2 or 0.22 pm pore filter. In some embodiments, the sterilizing grade filter media comprises polyethersulfone (PES). In some embodiments, the sterilizing grade filter media comprises poly vinylidene fluoride (PVDF). In some embodiments, the sterilizing grade filter media has a hydrophilic heterogeneous double layer design. In some embodiments, the sterilizing grade filter media has a hydrophilic heterogeneous double layer design of a 0.8 pm pre-filter and 0.2 pm final filter membrane. In some embodiments, the sterilizing grade filter media has a hydrophilic heterogeneous double layer design of a 1.2 pm pre-filter and 0.2 pm final filter membrane. In some embodiments, the sterilizing grade filter media is a 0.2 or 0.22 pm pore filter. In further embodiments, the sterilizing grade filter media is a 0.2 pm pore filter. In some embodiments, the sterilizing grade filter media is a Sartopore® 2 XLG 0.2 pm, Durapore™ PVDF Membranes 0.45pm, or Sartoguard® PES 1.2 pm + 0.2 pm nominal pore size combination. In some embodiments, the sterilizing grade filter media is a Sartopore® 2 XLG 0.2 pm. [00179] In some embodiments, the clarified feed is concentrated via tangential flow filtration ("TFF") before being applied to a chromatographic medium, for example, affinity chromatography medium. Large scale concentration of viruses using TFF ultrafiltration has been described by Paul et al., Human Gene Therapy 4:609-615 (1993). TFF concentration of the clarified feed enables a technically manageable volume of clarified feed to be subjected to chromatography and allows for more reasonable sizing of columns without the need for lengthy recirculation times. In some embodiments, the clarified feed is concentrated between at least two-fold and at least ten-fold. In some embodiments, the clarified feed is concentrated between at least ten-fold and at least twenty-fold. In some embodiments, the clarified feed is concentrated between at least twenty-fold and at least fifty-fold. In some embodiments, the clarified feed is concentrated about twenty-fold. One of ordinary skill in the art will also recognize that TFF can also be used to remove small molecule impurities (e.g., cell culture contaminants comprising media components, serum albumin, or other serum proteins) form the clarified feed via diafiltration. In some embodiments, the clarified feed is subjected to diafiltration to remove small molecule impurities. In some embodiments, the diafiltration comprises the use of between about 3 and about 10 diafiltration volume of buffer. In some embodiments, the diafiltration comprises the use of about 5 diafiltration volume of buffer. One of ordinary skill in the ait will also recognize that TFF can also be used at any step in the purification process where it is desirable to exchange buffers before performing the next step in the purification process. In some embodiments, the methods for isolating rAAV from the clarified feed described herein comprise the use of TFF to exchange buffers.

[00180] Affinity chromatography can be used to isolate rAAV particles from a composition. In some embodiments, affinity chromatography is used to isolate rAAV particles from the clarified feed. In some embodiments, affinity chromatography is used to isolate rAAV particles from the clarified feed that has been subjected to tangential flow filtration. Suitable affinity chromatography media are known in the art and include without limitation, AVB Sepharose™, POROS™ CaptureSelect™ AAVX affinity resin, POROS™ CaptureSelect™ AAV9 affinity resin, and POROS™ CaptureSelect™ AAV8 affinity resin. In some embodiments, the affinity chromatography media is POROS™ CaptureSelect™ AAV9 affinity resin. In some embodiments, the affinity chromatography media is POROS™ CaptureSelect™ AAV8 affinity resin. In some embodiments, the affinity chromatography media is POROS™ CaptureSelect™ AAVX affinity resin.

[00181] Anion exchange chromatography can be used to isolate rAAV particles from a composition. In some embodiments, anion exchange chromatography is used after affinity chromatography as a final concentration and polish step. Suitable anion exchange chromatography media are known in the art and include without limitation, UNOsphcrc™ Q (Biorad, Hercules, Calif.), and N-chargcd amino or imino resins such as e.g., POROS™ 50 PI, or any DEAE, TMAE, tertiary or quaternary amine, or PEI-based resins known in the art (U.S. Pat. No. 6,989,264; Brument et al., Mol. Therapy 6(5):678-686 (2002); Gao et al., Hum. Gene Therapy 11:2079-2091 (2000)). In some embodiments, the anion exchange chromatography media comprises a quaternary amine. In some embodiments, the anion exchange media is a monolith anion exchange chromatography resin. In some embodiments, the monolith anion exchange chromatography media comprises glycidylmethacrylate-ethylenedimethacrylate or styrenedi vinylbenzene polymers. In some embodiments, the monolith anion exchange chromatography media is selected from the group consisting of CIMmultus™ QA-1 Advanced Composite Column (Quaternary amine), CIMmultus™ DEAE-1 Advanced Composite Column (Diethylamino), CIM® QA Disk (Quaternary amine), CIM® DEAE, and CIM® EDA Disk (Ethylene diamino). In some embodiments, the monolith anion exchange chromatography media is CIMmultus™ QA-1 Advanced Composite Column (Quaternary amine). In some embodiments, the monolith anion exchange chromatography media is CIM® QA Disk (Quaternary amine). In some embodiments, the anion exchange chromatography media is CIM QA (BIA Separations, Slovenia). In some embodiments, the anion exchange chromatography media is BIA CIM® QA-80 (Column volume is 80mL). One of ordinary skill in the art can appreciate that wash buffers of suitable ionic strength can be identified such that the rAAV remains bound to the resin while impurities, including without limitation impurities which may be introduced by upstream purification steps are stripped away.

[00182] In some embodiments, anion exchange chromatography is performed according to a method disclosed in WO 2019/241535, which is incorporated herein by reference in its entirety.

[00183] In some embodiments, a method of isolating rAAV particles comprises determining the vector genome titer, capsid titer, and/or the ratio of full to empty capsids in a composition comprising the isolated rAAV particles. In some embodiments, the vector genome titer is determined by quantitative PCR (qPCR) or digital PCR (dPCR) or droplet digital PCR (ddPCR). In some embodiments, the capsid titer is determined by serotype-specific ELISA. In some embodiments, the ratio of full to empty capsids is determined by Analytical Ultracentrifugation (AUC) or Transmission Electron Microscopy (TEM). [00184] In some embodiments, the vector genome titer, capsid titer, and/or the ratio of full to empty capsids is determined by spectrophotometry, for example, by measuring the absorbance of the composition at 260 nm; and measuring the absorbance of the composition at 280 nm. In some embodiments, the rAAV particles are not denatured prior to measuring the absorbance of the composition. In some embodiments, the rAAV particles are denatured prior to measuring the absorbance of the composition. In some embodiments, the absorbance of the composition at 260 nm and 280 nm is determined using a spectrophotometer. In some embodiments, the absorbance of the composition at 260 nm and 280 nm is determined using a HPLC. In some embodiments, the absorbance is peak absorbance. Several methods for measuring the absorbance of a composition at 260 nm and 280 nm are known in the art. Methods of determining vector genome titer and capsid titer of a composition comprising the isolated recombinant rAAV particles are disclosed in WO 2019/212922, which is incorporated herein by reference in its entirety.

[00185] In additional embodiments the disclosure provides compositions comprising isolated rAAV particles produced according to a method described herein. In some embodiment, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[00186] As used herein the term "pharmaceutically acceptable means a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, which is suitable for one or more routes of administration, in vivo delivery or contact. A "pharmaceutically acceptable” composition is a material that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject without causing substantial undesirable biological effects. Thus, such a pharmaceutical composition may be used, for example in administering rAAV isolated according to the disclosed methods to a subject. Such compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules and crystals. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions. Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as set forth herein or known to one of skill in the ait. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes. Pharmaceutical compositions and delivery systems appropriate for rAAV particles and methods and uses of the invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20th ed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12th ed., Merck Publishing Group, Whitehouse, N.J.; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 11th ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford, N.Y., pp. 253-315).

[00187] In some embodiments, the composition is a pharmaceutical unit dose. A "unit dose” refers to a physically discrete unit suited as a unitary dosage for the subject to be treated; each unit containing a predetermined quantity optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filling agent) which, when administered in one or more doses, is calculated to produce a desired effect (e.g., prophylactic or therapeutic effect). Unit dose forms may be within, for example, ampules and vials, which may include a liquid composition, or a composition in a freeze-dried or lyophilized state; a sterile liquid carrier, for example, can be added prior to administration or delivery in vivo. Individual unit dose forms can be included in multi-dose kits or containers. Recombinant vector (e.g., AAV) sequences, plasmids, vector genomes, and recombinant virus particles, and pharmaceutical compositions thereof can be packaged in single or multiple unit dose form for ease of administration and uniformity of dosage. In some embodiments, the composition comprises rAAV particles comprising an AAV capsid protein from an AAV capsid serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rhS, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16. In some embodiments, the AAV capsid serotype is AAV8. In some embodiments, the AAV capsid serotype is AAV9.

EXAMPLES

Example 1. Screening of chemical additives to increase crude viral titer.

[00188] rAAV products can be characterised with reference to several critical quality attributes (CQAs) such as genomic titer, full/empty capsid ratio, and capsid content. Gimpel, A. L. et al. Mol. Ther. - Methods Clin. Dev. 20, 740-754 (2021); Wright, J. F. Biotechnol. J. 16, 1-5 (2021). These attributes strongly affect the ability of the final vector product to perform as intended in a clinical and/or research context. Analytical characterisation of relevant CQAs is therefore an important tool in both upstream and downstream process development. In this study, two CQAs (genomic titer and full/empty capsid ratio) have been assessed after addition of small molecule modulators of rAAV production.

[00189] The biological processes underpinning AAV production are reliant on a complex network of molecular interactions involving host-cell proteins, transiently expressed helper and viral genes, and the AAV viral genome itself (reviewed in Maurer, A. C. & Weitzman, M. D. Hum. Gene Ther. 31, 499-511 (2020); Meier, A. F. et al. Viruses 12, 8-12 (2020); Sha, S. et al. Biotechnol. Adv. 49, 107764 (2021)). Small molecule enhancers of recombinant protein production have demonstrated efficacy in a wide variety of mammalian cell lines and the addition of chemicals as diverse as sodium chloride, sodium butyrate, and soy peptones have been shown in previous studies to improve rAAV production yields. Johari, Y. et al. C. Biotechnol. Bioeng. 112, 2527-2542 (2015); Gimpel, A. L. et al. Mol. Ther. - Methods Clin. Dev. 20, 740-754 (2021); Wright, J. F. Biotechnol. J. 16, 1-5 (2021); Zhao, H. et al. Mol. Ther. - Methods Clin. Dev. 18, 312-320 (2020); Yu, C. et al. Mol. Ther. - Methods Clin. Dev. 21, 1-13 (2021); Hildinger, M. et al. Biotechnol. Lett. 29, 1713-1721 (2007). Targeting of discreet pathways involved in the replication, packaging, and trafficking of viral particles by small molecule cell culture additives therefore offers a simple and cost-effective way of increasing viral titer and reducing overall production costs.

[00190] With this in mind, a panel of putative small molecule culture additives was chosen based on either their reported propensity to increase recombinant protein expression in mammalian cell systems, or their reported positive effects on rAAV transduction efficiency. Due to the diverse mechanisms involved in AAV vector production, the panel featured chemicals that exert their influence on recombinant protein expression via a broad range of functional mechanisms. Such functional classes included histone deacetylase inhibitors (HDACi), chemical chaperones, inhibitors of proteasomal function, cell cycle modulators, and caspase inhibitors.

[00191] Chemical additives were introduced to rAAV8 producing suspension HEK293 cells at 24 HPT. Each additive was used at three different concentrations; low, medium, high - values taken from previous uses in literature and different for each additive. See Table 2 for concentration and chemical data. Total cell culture was harvested at 72 HPT, cells were chemically lysed and the rAAV8 genomic titer in the crude supernatant was measured by ddPCR. Crude genomic titer in cells containing chemical additives was compared to untreated rAAV 8 producing cells to determine positive changes to titer mediated by the small molecules. Several chemicals displayed a clear concentration-dependent impact on genomic titer, both positively (M344, an HDACi; z-VAD-fmk, a pan-caspase inhibitor; and nocodazole, a mitotic spindle poison and cell cycle modulator) and negatively (all three proteasomal inhibitors: ONX0912, MLN9708, zLLL/MG132) (Fig. 1A). Of note, nocodazole treatment increased mean cell volume at all three concentrations, an attribute that appears to correlate positively with measured genomic titer (Fig. IB).

Table 2. Product information and final concentrations of chemical additives in media for initial screening.

Example 2. Nocodazole addition at early timepoints after triple-transfection results in increased rAAV8 genomic titer.

[00192] Nocodazole is an anti-mitotic agent, used as both a chemotherapeutic and as a common agent of cell cycle synchronisation. Blajeski, A. L. et al. J. Clin. Invest. 110, 91-99 (2002); Beswick, R. W. et al. Leuk. Res. 30, 427-436 (2006); Uetake, Y. & Sluder, G. Curr. Biol. 17, 2081-2086 (2007). Nocodazole exerts its effect by reversibly inhibiting the polymerisation of P-tubulin, destabilising microtubules and preventing the formation of mitotic spindles, thus arresting cells within the G2/M phase of the cell cycle. Cells treated with nocodazole typically enter mitosis but are unable to progress through cytokinesis, leading to either apoptosis in cells remaining in arrested mitosis for an extended period of time, or subsequent “mitotic slippage” into G0/G1 phase followed by apoptosis. Uetake, Y. & Sluder, G. Curr. Biol. 17, 2081-2086 (2007); Vasquez, R. J. et al. Mol. Biol. Cell 8, 973-985 (1997); Quignon, F. et al. Oncogene 26, 165-172 (2007). In addition to its use as a cell synchronisation agent, nocodazole has previously been shown to increase recombinant protein expression in a transient mammalian cell system. Tait, A. S. et al. Biotechnol. Bioeng. 88, 707-721 (2004).

[00193] Initial multi-well plate screening showed that 4 pM nocodazole added to culture media 24 HPT resulted in a modest, yet not statistically significant, 1.3-fold increase in rAAV8 genomic titer at 72 HPT compared to untreated control cells (Fig. 1A - high dose nocodazole). Addition of 4 pM nocodazole at 0 and 4 HPT resulted in a considerable reduction in cell density at 72 HPT of 73.8% and 63.75% respectively, compared to rAAV8 producing cells without nocodazole (Ctrl) (Fig. 2A). Also apparent at early addition timepoints were lower cell viabilities (yet not significantly lower) and a significantly higher mean cell volume compared to cultures not treated with nocodazole (Fig 2B & C). Previous work by Tait et al., (2004) observed increased cell size in nocodazole treated Chinese hamster ovary (CHO) cells producing a recombinant monoclonal antibody. Most strikingly, addition of nocodazole 4 HPT resulted in a >2-fold increase in rAAV8 genomic titer at harvest compared to control cells (Fig. 2D).

[00194] A previous study determined that nocodazole increased replication of wildtype AAV DNA, yet did not result in a greater number of AAV particles. Ogston, P. et al. J. Virol. 74, 3494-3504 (2000). Contrary to this, a modest increase in genomic titer of 1.3-fold was seen at the same timepoint, with the sensitivity of ddPCR most likely contributing to our ability to detect this. Also observed was an increase in total rAAV particles as quantified by AAV8-specific capsid ELISA (data not shown). Of note is that the addition of nocodazole was restricted to 24 hrs post-infection of cells by wildtype AAV - therefore cells were treated at later timepoints than what is shown here to be optimal in an rAAV production context.

Example 3. rAAV producing cultures treated with nocodazole have an increased proportion of cells in G2/M phase.

[00195] As previously noted, the effect of nocodazole on cell cycle progression is well characterised and has been used extensively in research to arrest cells in G2/M phase. Flow cytometry was carried out to determine the cell cycle status of rAAV8-producing cultures treated with nocodazole. Cells treated at the point of transfection were found to be arrested in G2/M phase after DNA content analysis by propidium iodide staining, with almost twice the number of cells in G2/M phase compared to G1 phase at 72 HPT (Fig. 3A). In stark contrast, untreated cells producing rAAV8 were found to be almost entirely in either G1 or S phase at the same timepoint (Fig. 3B). The ratio of cells in G2/M : G1 phase was found to decrease the later nocodazole was added (Fig. 3C). Interesting to note is the observation that a significant number of cells remain arrested in G2/M phase at 72 HPT when nocodazole is added as late as 24 hrs after transfection, yet the positive effect on rAAV8 genomic titer is substantially lessened compared to earlier treatment. This would suggest a critical temporal component to the addition of nocodazole, such that later additions to cell culture are sub-optimal in terms of producing high rAAV titers.

[00196] To further confirm the cell cycle status of nocodazole treated cells, immunocytochemical visualisation of cells was carried out. Cells transiting mitosis during the normal cell cycle progression exhibit characteristic morphological changes associated with discreet mitotic phases, and the process of cell division necessitates widespread disorganisation of the cellular environment. The nucleolus is a dynamic compartment within the nucleus which undergoes extensive remodelling during cell cycle progression, particularly during mitosis. Hernandez-Verdun, D. Nucleus 2, 189-194 (2011). Previous immunocytochemical examination of nucleolar localisation throughout normal cell cycle progression shows a distinctive dispersal of nucleolar protein staining throughout the cytoplasm during prometaphase and metaphase - concomitant with the breakdown of the nuclear membrane during mitosis. Ma, N. et al. J. Cell Sci. 120, 2091-2105 (2007); Amin, M. A. et al. Biochem. Biophys. Res. Commim. 360, 320-326 (2007); Stenstrdm, L. et al. Mol. Syst. Biol. 16, 1-16 (2020). Nucleolar proteins in cells in interphase and prophase are typically found located within the nucleus itself. Of note, the nucleolus is considered to be a likely site for AAV capsid assembly and Rep-mediated loading of AAV genome into capsids as well as being linked more generally to viral replication in other human viruses. Wistuba, A. et al. J. Virol. 71, 1341-1352 (1997); Qiu, J. & Brown, K. E. Virology 257, 373-382 (1999); Bevington, J. M. et al. Virology 357, 102-113 (2007); Sonntag, F. et al. Proc. Natl. Acad. Sci. U. S. A. 107, 10220-10225 (2010); Greco, A. Rev. Med. Virol. 19, 201-214 (2009).

[00197] Cells from rAAV8 producing cultures - both with and without nocodazole addition - were harvested and fixed at 24 HPT. Cells were stained with a nucleolar marker (fibrillarin - a protein component of the nucleolus associated with ribosomal RNA processing), and DAPI (a marker of AT-rich nuclear DNA), and widefield fluorescent microscopy used to visualise the phenotypic changes induced by nocodazole addition. Amin, M. A. et al. Biochem. Biophys. Res. Commun. 360, 320-326 (2007). Cells treated with nocodazole exhibited a greater proportion of cells displaying a disorganised nuclear phenotype - with fibrillarin distributed broadly throughout the cytoplasm, and with condensed nuclear DNA (Fig. 3D) - strongly indicative of cells either progressing through mitosis or arrested within G2/M phase. In contrast, the majority of untreated cells displayed highly localised or punctate staining of fibrillarin within the area of nuclear DNA staining (Fig. 3E), suggestive of a majority of cells in interphase or prophase. The reduction in cell proliferation, decreased viability and enlarged cell volume observed in nocodazole -treated rAAV8 producing cultures relative to untreated rAAV8 producing cultures can therefore be confidently attributed to the extended arrest of cells in G2/M phase, later progressing to apoptosis.

Example 4. Nocodazole addition improves rAAV genomic titer in shake flask cultures and is not serotype dependent.

[00198] To investigate the effect of nocodazole addition in a system more representative of large scale rAAV production, culture volume was scaled up from the initial multi-well plate culture volume of 0.7 ml to 30 ml culture volume in shake flasks. To rule out a serotype-dependent effect of nocodazole addition, plasmids for both rAAV8 and rAAV9 serotypes were transfected in separate shake flask experiments and VCD, viability, and mean cell volume measured daily, in cultures with and without the addition of 4 pM nocodazole. Genomic titer was measured from 1 - 5 days post-transfection. All measured parameters of nocodazole induced cell cycle arrest (reduced VCD, decreased viability, increased mean cell volume) and increased genomic titer were consistent with those seen at smaller culture volumes (Fig. 4A & B). Comparable to multi-well plate-based cultures, mean genomic titer was increased by up to 2.5-fold in nocodazole-treated cultures compared to untreated cultures (Fig. 4C). There were also no observed significant differences in the above-described measurements between cultures producing rAAV8 or rAAV9, suggesting a broad applicability for nocodazole addition in rAAV manufacturing.

Example 5. Combined use of small molecules in shake flask cell culture has an additive effect on rAAV production.

[00199] After the successful translation of nocodazole addition from a positive hit in the initial multi-well plate-based screening assay to shake flask culture, the ability of other small molecule additives identified in the initial screen to further improve rAAV titer was tested. M344 is a synthetic analogue of the antifungal drug Trichostatin A and an inhibitor of Class I and IIB histone deacetylases. It has been used to reduce tumorigenesis in vitro, as treatments for spinal muscular atrophy and Alzheimer’s disease in vivo and recently as an enhancer of recombinant protein expression in mammalian cell culture. Li, X. & Chen, B. Am. J. Biomed. Sci. 1, 352-363 (2009); Riessland, M. et al. Hum. Genet. 120, 101-110 (2006);

Volmar, C. H. et al. Proc. Natl. Acad. Sci. U. S. A. 114, E9135-E9144 (2017), Chang, J. et al. Molecules 25, 353 (2020). Z-VAD-fmk is a pan-caspase inhibitor which can act to reduce the accumulation of apoptotic cells in culture. Li, X. etal. Front. Immunol. 10, 1824 (2019). M344 and z-VAD-fmk were added individually to rAAV8 producing cultures at an early timepoint of addition (4 HPT) to see if this could, as in the case of nocodazole, further improve on the modest titer increase seen at 24 HPT addition. As no further titer improvement was observed for either chemical at early addition timepoints (data not shown), the effect of adding each chemical to rAAV producing cultures together with nocodazole at 4 HPT in a multi-well plate format was subsequently investigated. Interestingly, the combination of 2.5 pM M344 and 4 pM nocodazole was found to produce an additive effect, increasing crude rAAV genomic titer 2.6-fold compared to untreated cultures, an improvement on nocodazole alone (2-fold increase compared to untreated) (Fig. 5D). The opposite effect was observed in cultures containing 5 pM z-VAD- fmk and 4 pM nocodazole, where the addition of the pan-caspase inhibitor attenuated the increase in genomic titer observed in nocodazole-alone containing cultures, reducing it from a 2-fold improvement to a non-significant increase of 1.3-fold (Fig. 5D). The inclusion of z-VAD-fmk in the initial screening experiment was predicated on its anti-caspase activity, based on a hypothesis that caspase mediated apoptosis - linked to rAAV production and expression of rAAV Rep proteins - would negatively affect final crude titer. Saudan, P. et al. EMBO J. 19, 4351-4361 (2000); Berthet, C. et al. Proc. Natl. Acad. Sci. U. S. A. 102, 13634-13639 (2005). Additionally, caspase-mediated apoptosis induced by nocodazole - mediated cell cycle cdysregulation would further increase cell death within the production cultures. An observed increase in cell viability and VCD in cultures treated with nocodazole/z-VAD-fmk relative to untreated, nocodazole treated or nocodazole/M344 treated cells suggests that apoptosis was reduced (Fig. 5A & B) but with an unexpected reduction in crude genomic titer (Fig. 5D). The observed reduction may be a consequence of off-target induction of autophagy via inactivation of n-glycanasc 1 (NGLY1) that has been shown to occur in HEK293 cells treated with z-VAD-fmk. Needs, S. H. et al. FEBS J. 1-17 doi:10.1111/febs.16345 (2022). While the role of autophagy in rAAV production is undetermined, inhibition of autophagy has been shown to increase recombinant protein expression in CHO cells and unintentional upregulation of autophagy may result in a reduction in viral component proteins necessary for production of high viral titers. Jardon, M. A. et al. Biotechnol. Bioeng. 109, 1228-1238 (2012); Nasseri, S. S. et al. Biochem. Eng. J. 91, 37-45 (2014). Interestingly, the highest mean cell volume, which appeared to correlate positively with genomic titer, was measured in cells treated with both nocodazole and z-VAD-fmk (Fig. 6C). This may be a result of reduced apoptotic cell death allowing cell size to increase but with the caveat that any benefit gained from this from a production perspective is attenuated by the potential negative off-target effects of z-VAD-fmk.

[00200] As the combination of nocodazole and M344 produced an improvement on nocodazole alone in the screening assay, this combination of small molecules was next scaled up to 30 ml shake flask cultures. Measurements of VCD, viability and mean cell volume closely replicated those observed in the screening assay (Fig. 4D & Fig. 6A), albeit with an improvement in genomic titer with nocodazole/M344 from a 2.6-fold increase in the screening assay to a 3-fold increase compared to untreated cells in larger volume cultures (Fig. 5E). Fluorescent imaging and automated quantification of GFP positive cells from 1AAV8 producing cultures at 24 HPT shows an approximately 2-fold increase in the percentage of GFP positive cells in nocodazole/M344 treated cultures compared to untreated or nocodazole alone (Fig. 6B), suggesting that the increase in rAAV8 titer with the addition of M344 is driven by a mechanism separate or complementary to that of nocodazole. AAV8 capsid specific ELISA analysis of capsid titer showed a significant increase in intact capsids after addition of nocodazole (4.3-fold increase) and nocodazole/M344 (11.3-fold increase) compared to untreated controls (Fig. 6C). The resulting analysis of full/empty capsid ratio shows a decreased percentage of full capsids compared to total capsids in cultures containing the small molecule modulators of rAAV titer, likely due to the very high capsid titers resulting from small molecule addition (Fig. 6D).

[00201] Improving the yield of intact, genome-containing rAAV particles during vector production is a critical step to reducing overall product costs. Shown here is a simple method by which viral vector titer, a fundamental CQA of viral vector production, may be quickly improved in an established and previously optimised suspension system.

Example 6. Nocodazole addition improves rAAV genomic titer.

[00202] Small molecule enhancers of recombinant protein expression have been extensively used across a wide range of mammalian cell systems to improve transient production performance. Johari, Y. et al. C. Biotechnol. Bioeng. 112, 2527—2542 (2015); Yang, W. C. et al. Mol. Biotechnol. 56, 421-428 (2014); Allen, M. J. et al. Biotechnol. Bioeng. 100, 1193—1204 (2008); Tait, A. S. et al. Biotechnol. Bioeng. 88, 707-721 (2004); Meyer, H. J. et al. Biotechnol. Prog. 33, 1579-1588 (2017); Chang, J. et al. Molecules 25, 353 (2020); Johari, Y. B. et al. Biotechnol. Bioeng. 118, 1013-1021 (2021). The ease of use and low cost of small molecule enhancers (particularly for chemicals with pronounced biological activity at low dosages) makes them an attractive solution to improving rAAV yield. As discussed herein, an easy to execute multi-well plate-based screening assay of common small molecule enhancers of recombinant protein expression was employed to increase total rAAV vector yield by > 3-fold compared to untreated cultures. Also shown is that positive hits in low culture screening assays are scalable and consistent at larger culture volumes, display additive interactions with other positive hits, and that even modest improvements at the screening level can be optimised to produce significantly improved vector yields at scale.

[00203] Investigation of the mechanisms behind the increase in titer point to the arrest of rAAV producing cells in mitosis soon after PEl-mediated transient transfection of rAAV producing plasmids. Prior studies have shown a strong preference for wildtype AAV replication within the G2/M phase of the cell cycle. Franzoso, F. D. et al. J. Virol. 91, 1-19 (2017); Raj, K. et al. Nature 412, 914—917 (2001). Nocodazole has been shown to significantly improve transient transfection efficiency in both CHO cells and in a HEK293 suspension process for rAAV production which may stem from increased nuclear' permissibility of transfection complexes due to the breakdown of the nuclear membrane during mitosis. Tait, A. S. et al. Biotechnol. Bioeng. 88, 707-721 (2004); Feng, L. et al. Biotechnol. Appl. Biochem. 50, 121 (2008). Measurement in this study of GFP-positive cells at 24 HPT did not show an increase in cells treated with nocodazole alone, but the combined addition of nocodazole and a positive regulator of transcription (M344) had a significant effect on GFP expression. Feng et al., (2008) found that a brief severe hypothermic shock (1 hour at 4 °C) followed by a recovery to 37 °C prior to transfection, increased the proportion of HEK293 cells in G2/M phase and improved transfection efficiency. A reduction in cell proliferation caused by nocodazole addition may also benefit viral production by reducing plasmid copy number dilution and maintaining mRNA transcript levels. Meyer, H. J. et al. Biotechnol. Prog. 33, 1579— 1588 (2017). A recent study utilising a CRISPR-mediated genome wide screening strategy identified two target genes (ITPRIP and SKA2) that when modulated in cells increased rAAV genome titer and improved full/empty capsid ratios, with both target genes (strongly, in the case of SKA2) associated with cell cycle modulation. Barnes, C. R. et al. Mol. Ther. - Nucleic Acids 26, 94-103 (2021). Further to this, a proteomic study of HEK cells during AAV5 production highlighted a number of proteins involved in cell cycle and proliferation as being strongly downregulated during production. Strasser, L. et al. Int. J. Mol. Sci. 22 (2021). This apparent link between the cell cycle and AAV production, and the abundance of cell cycle modulating molecules, necessitates further investigation into the use of cell cycle modulators for both improving rAAV production yields and ultimately improving our understanding of the underlying biological processes governing rAAV production. The apparent important temporal aspect of cell cycle regulation within the production process may also provide avenues for non-chemical interventions to improve vector yield as this relationship becomes better understood.

[00204] Due to the robustness of the results between small-scale plate-based cultures and larger scale shake flasks, it is believed that the screening process described here could be further scaled down and automated to increase throughput, especially with the availability of instrumentation that can rapidly and accurately dispense very small volumes of small molecules into culture. As minimal volumes are required for ddPCR analysis of genomic titer, identification of novel enhancers of rAAV could be rapidly incorporated into existing rAAV production platforms with minimal changes to existing protocols.

[00205] As demonstrated here, an increase in overall vector yield may come with the disadvantage of reduced full/empty capsid ratios - another important CQA of vector production. While strategies to remove empty capsids from final vector preparations are improving, it would be advantageous to be able to better manipulate capsid loading to improve product quality. While no improvements in vector quality here is shown herein, it is believed that use of titer boosting molecules in parallel with available process optimisation techniques such as time of plasmid addition or staggered transfections of rep/cap/ITR/helper plasmids, optimised culture conditions, or inducible expression of viral vector components may well overcome these limitations.

[00206] In summary, shown here is that the use of readily available small molecule enhancers can significantly improve rAAV production yield. Also shown here is that small molecule enhancers of rAAV production are amenable to optimisation in an existing suspension HEK293 cell system and that positive hits from initial small-scale screening of enhancer molecules can translate to larger scale production platforms. Also shown is that increased titer resulting from nocodazole treatment is consistent across two different serotypes, suggesting broad applicability in rAAV manufacturing.

Example 7. Materials and Methods.

[00207] Cell culture. Suspension adapted Human Embryonic Kidney 293 (HEK293) cells were provided by REGENXB1O. Cells were cultured in nutrient supplemented Dynamis Medium (Life Technologies). Cells were maintained in an orbital shaking incubator (Infors) at 30ml culture volumes in 125 ml Erlenmeyer flasks (Corning) at 37°C, 5% CO2 and 85% humidity, with agitation at 140 rpm. Smaller scale cultures were grown in either 50 ml TubeSpin bioreactor tubes (10 ml culture volume) (TPP) or non-tissue culture treated shallow-well 24-well plates (0.7 ml culture volume/well, 240 rpm shaking) (Corning) utilising the Deutz system. Routine cell density and cell viability measurements were performed on a ViCell automated cell counter (Beckman-Coulter). Measurements of density and viability and determination of mean cell volume during rAAV production runs were performed using the Norma HT system (Iprasense) utilising 3 pl total cell culture and 20 pm slide chambers. Mean cell volume (V) was calculated as V=4/3 7rr^A3 where r = measured cell diameter/2.

[00208] rAAV vector production in suspension adapted HEK293 cells. Cells were triple transfected using PEIPro (Polyplus) and plasmids supplied by REGENXBIO. Helper genes were expressed from pAD Delta F6. Rep/Cap genes for rAAV8 and rAAV9 were expressed from pAAV2/8KanRGX and pAAV2/9KanRGX respectively. The inverted terminal repeat (ITR) plasmid carrying a reporter gene expression cassette was pAAV_CAG_GFP. For rAAV production, plasmids and PEIPro were separately added to Dynamis Media and then combined and incubated at room temperature for 10 minutes. After incubation, the DNA:PEI polyplex transfection mixture was added to cells at 10% working culture volume. For analysis, 450 pl of total cell culture was added to 50 pl lOx cell lysis buffer (5% Tween-20, 3 M NaCl, 10 mM MgC12) containing lx complete™, EDTA-free Protease Inhibitor Cocktail (Roche) and incubated for 1 hr at 37 °C with gentle agitation. Samples were briefly centrifuged at 12,000 RPM to remove cell debris and the resulting supernatant used to determine viral genome and capsid titer.

[00209] Crude lysate rAAV titer analysis. Genome titer was quantified by digital droplet PCR (ddPCR). 5 pl of post-lysis supernatant from total cell culture was treated with DNAse I (Roche) to remove residual plasmid DNA. DNAse I-treated samples were diluted 1000- or 10,000-fold in PCR dilution buffer (GeneAmp™ PCR Buffer 1 (Thermo Scientific), 0.02% UltraPure^1M Salmon Sperm DNA Solution (Invitrogen), 0.1% Pluronic™ F-68 Non-ionic Surfactant). Droplet formation and subsequent post-PCR droplet analysis was performed using the QX200 system (Bio-Rad), with absolute quantification of AAV genome copies/pl determined using the Quantasoft analysis software (Bio-Rad). Genome detection was achieved using primers and a FAM-labelled probe targeting the PolyA sequence of the pAAV-CAG-GFP plasmid. Capsid titer quantification was performed using the AAV8 titration ELISA (Progen) from total cell lysis supernatant diluted in lx ASSB assay buffer (Progen).

[00210] Cell culture chemical additive screening in multi-well plates. Chemical additives for screening were diluted in either sterile water or dimethyl sulfoxide (DMSO) where appropriate. High, medium, and low concentrations for each chemical were determined based on available literature pertaining to their observed effects on recombinant protein expression. Screening was performed in shallow well 24- well plates. Cells were grown to a density of 4 x 10⁶ viable cells/ml in a suitable culture volume. The cell culture was triple-transfected with AAV-producing plasmids as a pool and the transfected cells were transferred to 24 well plates. Chemical additives were added 24 hours posttransfection (HPT) in duplicate wells for the initial screen and harvested for viral genome titer analysis by ddPCR at 72 HPT. [00211] DNA content analysis by flow cytometry. 1 x 10⁶ total cells were harvested 72 HPT and fixed in 70% ethanol at 4°C for 30 minutes, with gentle vortexing to prevent clumping. Ethanol was removed and the cells washed twice in lx PBS. Fixed cells were treated with 100 pl RNAse A (100 pg/ml in PBS; Qiagen) for 5 mins at RT and the DNA stained with the subsequent addition of 400 pl propidium iodide (PI) (50 pg/ml in PBS; Thermo Scientific) at room temperature for a minimum of 30 minutes. PI- stained cells were analysed by flow cytometry using an LSRII instrument (BD Biosciences). Gated single cell populations were detected based on PI signal and the resulting histograms analysed using FlowJo software to determine the relative distribution of cells within the cell cycle (G1/S/G2-M) phases.

[00212] Immunocytochemistry. rAAV producing cells were harvested 24 hours after nocodazole addition. 1 x 10⁶ cells were spun down at 250g for 5 minutes, culture media was removed, and cells resuspended in 1ml PBS. Resuspended cells were incubated with no agitation for 30 minutes at room temperature and allowed to adhere by gravity sedimentation to lysine-coated glass coverslips in 24-well plates. After 30 minutes, PBS and unadhered cells were removed by gentle aspiration. Adhered cells were fixed by 10-minute incubation at room temperature with 0.5ml 4% paraformaldehyde in PBS. Fixed cells were washed with PBS before permeabilization with 0.5ml 0.5% Triton-XlOO for 10 minutes.

Permeabilised cells were blocked by 10% normal goat serum (Life Technologies; #016201) in PBS for 30 mins. Cells were then incubated with anti-Fibrillarin antibody (Abeam; ab5821, 0.1 pg/ml in blocking buffer) for 1 hour at room temperature before incubation with goat anti-Rabbit Alexa-594 conjugated secondary antibody (Abeam; ab 150080, 1:1000 dilution in blocking buffer) for 45 minutes at room temperature. Coverslips were transferred to a glass slide and mounted with Fluoroshield mounting media containing DAPI (Sigma- Aldrich; F6507) for visualisation of nuclei. Slides were imaged using a Nikon Eclipse Ti fluorescent microscope and images adjusted for brightness and contrast using ImageJ software.

Example 8. Use of caspase inhibitors in rAAV Production.

[00213] The production of rAAV in a HEK293 suspension cell culture has been described previously (Joshua C. Grieger, et al. Mol Ther. 24(2):287-297 (2016)). Three essential elements are delivered into HEK293 producer cells through transient transfection: 1) helper virus proteins, for example, as a helper plasmid; 2) AAV rep and cap genes; and 3) a gene of interest flanked by AAV ITR sequence.

[00214] Many factors have impact on rAAV production, such as the cell line, cell density, culture medium, cell condition, harvest time, total amount of DNA used in the transfection, and optimal plasmid ratios of the three plasmids during transfection (Joshua C. Grieger et al, Mol Ther. 24(2): 287-297 (2016); Huiren Zhao, et al, Mol Ther Methods Clin Dev. 18:312-320 (2020)). One of the factors impacting rAAV production is the decreased viability of HEK293 cells post-transfection. Several factors were attributed to the decrease in viability of post-transfected cells: 1) occurrence of apoptosis following plasmid DNA uptake (L H Li, et al, Exp Cell Res .1999 Dec 15;253(2):541-50 (1999)); 2) Endoplasmic Reticulum (ER) stress and immune defense induced by the viruses (Shanshan Li, et al, Crit Rev Microbiol. 2015 Jun;41(2): 150-64 (2015); and 3) mitochondrial dysfunction and oxidative damage induced by reactive oxygen species (ROS) at high cell density condition (Ja Hyun Koo, et al, Cell Metab. 28(2): 196-206 (2018)). Described herein are the effects of caspase inhibitors on rAAV titers improvement through regulation of apoptosis.

Table 3. Select caspase inhibitors

[00215] Z-VAD-FMK significantly improves rAAV titers produced by both HEK293 Clone #1 and HEK293 Clone #3 derived clones. Fig. 7. Fold change (FC) of titer was calculated from titers normalized to those of HEK293 Clone #1 without Z-VAD-FMK treatment. Z-VAD-FMK is a pancaspase inhibitor compound that prevents apoptosis. Z-VAD-FMK, a widely used broad spectrum caspase inhibitor, has been shown to inhibit enzymes other than caspases. Z-VAD-FMK can also inhibit T celldependent inflammation as well (Akiko Iwata, et al, J Immunol. 170(6)13386-91 (2003)).

[00216] To further delineate the specificity of caspase inhibitors on rAAV titer improvement, different kinds of caspase inhibitors were chosen for evaluation, including 2 of the broad-spectrum caspase inhibitors (Z-VAD-FMK and Q-VD-OPh), and 6 of the specific caspase inhibitors (Caspase 3 inhibitor I, Caspase 1/4 inhibitor, Caspase 6 inhibitor I, Caspase 3/7 inhibitor, Caspase 8 inhibitor II & Caspase 9 inhibitor I). The first step of specificity screening was performed in three cell clones (HEK293 Clone #1, HEK293 Clone #3 & HEK293 Clone #3.7) cultured in 24-deep wells. Data shown in Figs. 8-10 demonstrated that the three cell clones’ production titer can be improved by means of 4 caspase inhibitors including Caspase 1/4 inhibitor, Caspase 3/7 inhibitor, Z-VAD-FMK, and Q-VD-OPh. The improvement on titers by adding all 4 of caspase inhibitors was also confirmed when three clones were cultured in shake flask. On the other hand, as shown in Figs 8-10 and Figs. 11-13, specific caspase inhibitors had less effect on titer enhancement in comparison to pan-caspase inhibitors. For HEK293 Clone #3, Q-VD-OPh can significantly improve titers even at lower concentrations (2 pM), while the FMK-based caspase inhibitor only works functionally at higher doses (10 pM). For HEK293 Clone #3.7 , similar effect on titer improvement was also noted during the addition of Q-VD-OPh.

[00217] Both Z-VAD-FMK and Q-VD-OPh are potent pan-caspase inhibitors that protect cells from caspase-dependent apoptosis in many different cell types. Q-VD-OPh has better aqueous stability, cell permeability, and higher efficacy than FMK-based caspase inhibitors and displays no cytotoxic effects when incorporated alone.

[00218] To develop a process for rAAV production comprising Q-VD-OPh addition into transfected cell culture, the design of experiments (DoE) approach was applied using three major factors. These factors were dose concentrations, incubation time and cell densities. Some of the data obtained is shown in Fig. 14. The data were analyzed using JMP Statistical Software. The RSquare for the fitting model was 0.956 indicating a good fit. The prediction profiler was also used to identify conditions that maximizing rAAV titers. The predicted optimal condition is to use HEK293 Clone #3 with addition of 10 pM Q-VD-OPh at 0-hour post-transfection. The predicted optimal process was performed in a 3L bioreactor. A process using HEK293 Clone #1 without Q-VD-OPh was used as control. Fold change (FC) of titer was calculated from titers normalized to titers of control (HEK293 Clone#l). The absolute titers can reach the values which is close to the predicted maximal titers. The close match between the predicted titer and actual titer indicated the reliability of the fitting model. As shown in Fig.15, the addition of Q-VD-OPh can increase the titer by -30% compared to the titer produced by HEK293 Clone#3 without inhibitor treatment, which is consistent with the data in shake flask results, indicating it is scalable in large-scale manufacture.. Example 9. Nocodazole has concentration dependent effect on rAAV Production.

[00219] The effects of different concentrations nocodazole on rAAV titer were tested. As shown in Fig.

16, addition of 0.4 microM nocodazole significantly increased rAAV titer. The experiment used a plasmid comprising a rAAV genome encoding a GFP reporter gene, a plasmid encoding Rep and AAV8 Cap genes, and the pAD Delta F6 helper plasmid. rAAV particles were produced in a 125 ml shake flask comprising a suspension adapted HEK293 cell culture. 6 microM, 4 microM or 0.4 microM nocodazole was added to the culture following transfection. A control culture with no nocodazole was also included. Fold change (FC) of titer was calculated from titers normalized to those obtained without nocodazole treatment. Nocodazole showed concentration dependent effects on rAAV production, wherein the addition of 0.4 microM nocodazole produced a -30% increase in rAAV titer.

[00220] As shown in Fig. 17, nocodazole's effect on rAAV titer was host clone dependent. The rAAV particles produced were the same as in Fig. 16. rAAV particles were produced in a 125 ml shake flask. In addition to the suspension adapted HEK293 cell culture, the experiment shown in Fig. 17 used 2 isolated clonal cell lines as host cells. In addition to testing the effect of 4microM and 0.4 microM nocodazole, the experiment also included cultures comprising 10 microM Q-VD-Oph caspase inhibitor, or a combination of nocodazole and the caspase inhibitor. Fold change (FC) of titer was calculated from titers normalized to titers obtained using corresponding host cells with DMSO (CTRL) treatment.

Incubation with 0.4 microM nocodazole significantly improved rAAV production, albeit the increase in rAAV titer was host cell dependent. The combination of nocodazole and Q-VD-Oph improved rAAV titer to a much greater degree than either agent alone.

[00221] As shown in Fig. 18, nocodazole's effect on rAAV titer was affected by the helper plasmid. The experiment used an improved helper plasmid, which can induce a higher rAAV titer than the pAD Delta F6 helper plasmid used in Figs. 16 and 17. Fold change (FC) of titer was calculated from titers normalized to titers obtained using corresponding host cells with H2O (CTRL) treatment. Incubation with 0.4 microM nocodazole improved rAAV production, albeit the relative increase in rAAV titer was generally lower than what was observed using the pAD Delta F6 helper plasmid. Considering that the improved helper plasmid can induce a higher rAAV titer than the pAD Delta F6 helper plasmid, it is believed that the apparently lower improvement in titer with nocodazole treatment was due to the increased production of rAAV particles by the control reactions.

[00222] As shown in Fig. 19, nocodazole's effect on rAAV titer was affected by the rAAV genome. The experiment used a plasmid comprising an rAAV genome comprising a different promoter driving the same reporter gene than the one used in Figs. 16-18. Fold change (FC) of titer was calculated from titers normalized to titers obtained using corresponding host cells with DMSO (CTRL) treatment. Incubation with 0.4 microM nocodazole or with nocodazole and Q-VD-Oph improved rAAV production, albeit the relative increase was host cell dependent and the relative increase was lower for some host cells than what was seen in Fig. 17.

[00223] While the disclosed methods have been described in connection with what is presently considered to be the most practical and preferred embodiments, it is to be understood that the methods encompassed by the disclosure are not to be limited to the disclosed embodiments, but on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[00224] All publications, patents, patent applications, internet sites, and accession numbers/database sequences including both polynucleotide and polypeptide sequences cited herein are hereby incorporated by reference herein in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession number/database sequence were specifically and individually indicated to be so incorporated by reference.

Claims

CLAIMS What is claimed is:

1. A method of producing rAAV particles, comprising a) providing a cell culture comprising a cell; b) introducing into the cell one or more polynucleotides encoding at least one of i. an rAAV genome to be packaged, ii. adenovirus helper functions necessary for packaging, iii. an AAV rep protein sufficient for packaging, and iv. an AAV cap protein sufficient for packaging; c) adding to the cell culture a mitotic inhibitor to a final concentration between about 1 nM and about 500 pM; and d) maintaining the cell culture under conditions that allow production of the rAAV particles for between about 2 days and about 15 days after (b).

2. The method of claim 1, wherein the mitotic inhibitor is a microtubule destabilizing agent.

3. The method of claim 1 or claim 2, wherein the mitotic inhibitor comprises at least one of nocodazole, vincristine, colchicine or a salt thereof.

4. The method of claim 3, wherein the mitotic inhibitor is nocodazole.

5. The method of claim 4, wherein the cell culture has a final nocodazole concentration between about 50 nM and about 50 pM.

6. The method of claim 4, wherein the cell culture has a final nocodazole concentration between about 100 nM and about 20 pM.

7. The method of claim 4, wherein the cell culture has a final nocodazole concentration between about 200 nM and about 10 pM.

8. The method of claim 4, wherein the cell culture has a final nocodazole concentration of about 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM or 1 pM.

9. The method of claim 4, wherein the cell culture has a final nocodazole concentration of about 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM or 10 pM.

10. The method of any one of claims 1 to 9, wherein the mitotic inhibitor is added after step b).

11. The method of claim 10, wherein the mitotic inhibitor is added less than about 48 hours, 24 hours,

12 hours, 6 hours, 3 hours, 2 hours, 1 hour or 0.5 hour after step b).

12. The method of claim 10, wherein the mitotic inhibitor is added between about 0.5 hour and about 48 hours after step b). The method of claim 10, wherein the mitotic inhibitor is added at least about 0.5 hour, 1 hour, 2 hours, 3 hours, 6 hours or 12 hours after step b). The method of claim 10, wherein the mitotic inhibitor is added about 0.5 hour, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, or about 24 hours after step b). The method of any one of claims 1 to 14, further comprising adding to the culture a caspase inhibitor. The method of claim 15, wherein the caspase inhibitor is added after step b). The method of claim 15 or claim 16, wherein the caspase inhibitor is a pan-caspase inhibitor. The method of claim 15 or claim 16, wherein the caspase inhibitor is a z-VAD-FMK or Q-VD- Oph. The method of any one of claims 15 to 18, wherein the mitotic inhibitor and the caspase inhibitor are added at the same time. The method of any one of claims 15 to 18, wherein the mitotic inhibitor and the caspase inhibitor are added separately in any order. A method of producing rAAV particles, comprising a) providing a cell culture comprising a cell; b) introducing into the cell one or more polynucleotides encoding at least one of i. an rAAV genome to be packaged, ii. adenovirus helper functions necessary for packaging, iii. an AAV rep protein sufficient for packaging, and iv. an AAV cap protein sufficient for packaging; c) adding to the cell culture a caspase inhibitor to a final concentration between about 10 nM and about 1 mM; and d) maintaining the cell culture under conditions that allow production of the rAAV particles for between about 2 days and about 15 days after (b). The method of claim 21, wherein the caspase inhibitor is a pan-caspase inhibitor. The method of claim 21 or claim 22, wherein the caspase inhibitor comprises at least one of z- VAD-FMK, Q-VD-Oph or a salt thereof. The method of claim 23, wherein the caspase inhibitor is Q-VD-Oph. The method of claim 23 or 24, wherein the cell culture has a final caspase inhibitor concentration between about 50 nM and about 100 pM. The method of claim 23 or 24, wherein the cell culture has a final caspase inhibitor concentration between about 100 nM and about 50 pM. The method of claim 23 or 24, wherein the cell culture has a final caspase inhibitor concentration between about 200 nM and about 20 pM. The method of claim 23 or 24, wherein the cell culture has a final caspase inhibitor concentration of about 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM or 1 pM. The method of claim 23 or 24, wherein the cell culture has a final caspase inhibitor concentration of about 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM or 10 pM. The method of claim 23 or 24, wherein the cell culture has a final caspase inhibitor concentration of about 5 pM, 10 pM, 15 pM, 20 pM, or 25 pM. The method of any one of claims 21 to 30, wherein the caspase inhibitor is added after step b). The method of claim 31 , wherein the caspase inhibitor is added less than about 48 hours, 24 hours, 12 hours, 6 hours, 3 hours, 2 hours, 1 hour or 0.5 hour after step b). The method of claim 31, wherein the caspase inhibitor is added between about 0.5 hour and about 48 hours after step b). The method of claim 31, wherein the caspase inhibitor is added at least about 0.5 hour, 1 hour, 2 hours, 3 hours, 6 hours or 12 hours after step b). The method of claim 31, wherein the caspase inhibitor is added about 0.5 hour, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, or about 24 hours after step b). The method of any one of claims 21 to 35, further comprising adding to the culture nocodazole. The method of claim 36, wherein the nocodazole is added after step b). The method of claim 36 or claim 37, wherein the caspase inhibitor and nocodazole are added at the same time. The method of claim 36 or claim 37, wherein the caspase inhibitor and nocodazole are added separately in any order. The method of any one of claims 1 to 39, wherein the cell culture is maintained for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days after b). The method of claim 40, wherein the cell culture is maintained for about 5 days after b). The method of any one of claims 1 to 41, comprising introducing into the cell one or more polynucleotides encoding i. an rAAV genome to be packaged, ii. adenovirus helper functions necessary for packaging, iii. an AAV rep protein sufficient for packaging, and iv. an AAV cap protein sufficient for packaging. The method of any one of claims 1 to 42, wherein the adenovirus helper functions comprise at least one of an adenovirus E4 gene, E2a gene, and VA gene. The method of any one of claims 1 to 42, wherein the adenovirus helper functions comprise an adenovirus E4 gene, E2a gene, and VA gene. The method of claim 44, wherein the polynucleotide encoding the adenovirus helper functions comprises pAD Delta F6. The method of any one of claims 1 to 45, wherein the introducing one or more polynucleotides into the cell is by transfection. The method of any one of claims 1 to 46, wherein the cell is a mammalian cell. The method of any one of claims 1 to 46, wherein the cell is an insect cell. The method of any one of claims 1 to 46, wherein the cell is a HEK293 cell, HEK derived cell, CHO cell, CHO derived cell, HeLa cell, SF-9 cell, BHK cell, Vero cell, CAP cell or PerC6 cell. The method of any one of claims 1 to 46, wherein the cell is a HEK293 cell. The method of any one of claims 1 to 50, wherein the cell culture is a suspension culture. The method of any one of claims 1 to 51, further comprising recovering the rAAV particles. The method of any one of claims 1 to 52, wherein the cell culture produces more rAAV particles measured as GC/ml than a culture in the absence of adding of the mitotic inhibitor and/or caspase inhibitor. The method of any one of claims 1 to 52, wherein the cell culture produces at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, or at least 100% more rAAV particles measured as GC/ml than a culture in the absence of adding of the mitotic inhibitor and/or caspase inhibitor. The method of any one of claims 1 to 35, wherein the cell culture has a volume between about 50 liters and about 20,000 liters. A method for producing rAAV particles, comprising a) providing a cell culture comprising a cell capable of producing rAAV ; b) adding to the cell culture a mitotic inhibitor to a final concentration between about 1 nM and about 500 pM; and c) maintaining the cell culture under conditions that allows production of the rAAV particles. The method of claim 56, wherein the mitotic inhibitor is a microtubule destabilizing agent. The method of claim 56 or claim 57, wherein the mitotic inhibitor comprises at least one of nocodazole, vincristine, colchicine or a salt thereof. The method of claim 58, wherein the mitotic inhibitor is nocodazole. The method of claim 59, wherein the cell culture has a final nocodazole concentration between about 200 nM and about 10 pM. The method of any one of claims 56 to 60, further comprising adding to the culture a caspase inhibitor. A method for producing rAAV particles, comprising a) providing a cell culture comprising a cell capable of producing rAAV ; b) adding to the cell culture a caspase inhibitor to a final concentration between about 10 nM and about 1 mM; and c) maintaining the cell culture under conditions that allows production of the rAAV particles. The method of claim 62, wherein the caspase inhibitor is a pan-caspase inhibitor. The method of claim 62 or claim 63, wherein the caspase inhibitor comprises at least one of z- VAD-FMK, Q-VD-Oph or a salt thereof. The method of claim 64, wherein the caspase inhibitor is Q-VD-Oph. The method of claim 59, wherein the cell culture has a final caspase inhibitor concentration between about 200 nM and about 20 pM. The method of any one of claims 62 to 66, further comprising adding to the culture a mitotic inhibitor. The method of claim 67, wherein the mitotic inhibitor comprises nocodazole. A method for producing rAAV particles, comprising culturing a cell capable of producing rAAV particles in a medium comprising between about 1 nM and about 500 pM of a mitotic inhibitor under conditions that allow the production of the rAAV particles. The method of claim 69, wherein the mitotic inhibitor is a microtubule destabilizing agent. The method of claim 69 or claim 70, wherein the mitotic inhibitor comprises at least one of nocodazole, vincristine, colchicine or a salt thereof. The method of claim 71, wherein the mitotic inhibitor is nocodazole. The method of claim 72, wherein the medium has a final nocodazole concentration between about 200 nM and about 10 pM. The method of any one of claims 69 to 73, wherein the medium further comprises a caspase inhibitor. A method for producing rAAV particles, comprising culturing a cell capable of producing rAAV particles in a medium comprising between about 10 nM and about 1 mM of a caspase inhibitor under conditions that allow the production of the rAAV particles. The method of claim 75, wherein the caspase inhibitor is a pan-caspase inhibitor. The method of claim 75 or claim 76, wherein the caspase inhibitor comprises at least one of z- VAD-FMK, Q-VD-Oph or a salt thereof. The method of claim 77, wherein the caspase inhibitor is Q-VD-Oph. The method of claim 77 or claim 78, wherein the medium has a final caspase inhibitor concentration between about 200 nM and about 20 pM. The method of any one of claims 75 to 78, wherein the medium further comprises a mitotic inhibitor. The method of claim 80, wherein the mitotic inhibitor comprises nocodazole. The method of any one of claims 56 to 81, wherein the cell capable of producing rAAV has been transfected with one or more polynucleotides encoding at least one of a) an rAAV genome to be packaged, b) adenovirus helper functions necessary for packaging, c) an AAV rep protein sufficient for packaging, and d) an AAV cap protein sufficient for packaging. The method of any one of claims 56 to 81, wherein the cell capable of producing rAAV has been transfected with one or more polynucleotides encoding a) an rAAV genome to be packaged, b) adenovirus helper functions necessary for packaging, c) an AAV rep protein sufficient for packaging, and d) an AAV cap protein sufficient for packaging. The method of any one of claims 56 to 83, wherein the cell is a mammalian cell or an insect cell. The method of any one of claims 56 to 83, wherein the cell is a HEK293 cell, HeLa cell, SF-9 cell, BHK cell, Vero cell, CAP cell or PerC6 cell, optionally wherein the cell is a HEK293 cell. The method of any one of claims 56 to 85, wherein the cell culture is a suspension culture. The method of any one of claims 77 to 86, wherein the culturing under conditions that allow production of the rAAV particles is for between about 2 days and about 10 days or between about 5 days and 14 days. The method of any one of claims 56 to 87, wherein the maintaining the cell culture or culturing under conditions that allow production of the rAAV particles is for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. The method of claim 88, wherein the maintaining the cell culture or culturing under conditions that allow production of the rAAV particles is for about 5 days. The method of any one of claims 56 to 89, further comprising recovering the rAAV particles. The method of any one of claims 56 to 90, wherein the cell culture produces more rAAV particles measured as GC/ml than a culture in the absence of mitotic inhibitor and/or caspase inhibitor. The method of any one of claims 56 to 90, wherein the cell culture produces at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, or at least 100% more rAAV particles measured as GC/ml than a culture in the absence of mitotic inhibitor and/or caspase inhibitor. A method of increasing the production of rAAV particles, comprising a) providing a cell culture comprising a cell; b) introducing into the cell one or more polynucleotides encoding at least one of i. an rAAV genome to be packaged, ii. adenovirus helper functions necessary for packaging, iii. an AAV rep protein sufficient for packaging, and iv. an AAV cap protein sufficient for packaging; c) adding to the cell culture a mitotic inhibitor to a final concentration between about 1 nM and about 500 pM; and d) maintaining the cell culture under conditions that allow production of the rAAV particles for between about 2 days and about 15 days after (b). The method of claim 93, wherein the mitotic inhibitor is a microtubule destabilizing agent. The method of claim 93 or claim 94, wherein the mitotic inhibitor comprises at least one of nocodazole, vincristine, colchicine or a salt thereof. The method of claim 95, wherein the mitotic inhibitor is nocodazole. A method of increasing the production of rAAV particles, comprising a) providing a cell culture comprising a cell; b) intr oducing into the cell one or more polynucleotides encoding at least one of i. an rAAV genome to be packaged, ii. adenovirus helper functions necessary for packaging, iii. an AAV rep protein sufficient for packaging, and iv. an AAV cap protein sufficient for packaging; c) adding to the cell culture a caspase inhibitor to a final concentration between about 10 nM and about 1 mM; and d) maintaining the cell culture under conditions that allow production of the rAAV particles for between about 2 days and about 15 days after (b). The method of claim 97, wherein the caspase inhibitor is a pan-caspase inhibitor. The method of claim 97 or claim 98, wherein the caspase inhibitor comprises at least one of z- VAD-FMK, Q-VD-Oph or a salt thereof. . The method of claim 99, wherein the caspase inhibitor is Q-VD-Oph.

. The method of any one of claims 1 to 100, wherein the rAAV particles comprise a capsid protein of the AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 and AAV16, AAV.rh8, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.hu32, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16 serotype. . The method of any one of claims 1 to 100, wherein the rAAV particles comprise a capsid protein of the AAV8, AAV9, AAV.rhlO, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, or AAV.hu37 serotype. . The method of any one of claims 1 to 100, wherein the rAAV particles comprise a capsid protein of the AAV8 or AAV9 serotype. . The method of any one of claims 1 to 13, wherein the genome to be packaged encodes a polypeptide or a double stranded RNA molecule. . The method of claim 104, wherein the genome to be packaged encodes a polypeptide.. The method of claim 105, wherein the genome to be packaged encodes an anti-VEGF Fab, anti-kallikrein antibody, anti-TNF antibody, microdystrophin, minidystrophin, iduronidase (IDUA), iduronate 2-sulfatase (IDS), low-density lipoprotein receptor (LDLR), tripeptidyl peptidase 1 (TPP1), or non-membrane associated splice variant of VEGF receptor 1 (sFlt-1).. The method of claim 105, wherein the genome to be packaged encodes an gamma- sarcoglycan, Rab Escort Protein 1 (REP1/CHM), retinoid isomerohydrolase (RPE65), cyclic nucleotide gated channel alpha 3 (CNGA3), cyclic nucleotide gated channel beta 3 (CNGB3), aromatic L-amino acid decarboxylase (AADC), lysosome-associated membrane protein 2 isoform B (LAMP2B), Factor VIII, Factor IX, retinitis pigmentosa GTPase regulator (RPGR), retinoschisin (RSI), sarcoplasmic reticulum calcium ATPase (SERCA2a), aflibercept, battenin (CLN3), transmembrane ER protein (CLN6), glutamic acid decarboxylase (GAD), Glial cell line- derived neurotrophic factor (GDNF), aquaporin 1 (AQP1), dystrophin, myotubularin 1 (MTM1), follistatin (FST), glucose-6-phosphatase (G6Pase), apolipoprotein A2 (APOA2), uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1), arylsulfatase B (ARSB), N-acetyl-alpha- glucosaminidase (NAGLU), alpha-glucosidase (GAA), alpha-galactosidase (GLA), betagalactosidase (GLB1), lipoprotein lipase (LPL), alpha 1-antitrypsin (AAT), phosphodiesterase 6B (PDE6B), ornithine carbamoyltransferase 9OTC), survival motor neuron (SMN1), survival motor neuron (SMN2), neurturin (NRTN), Neurotrophin-3 (NT-3/NTF3), porphobilinogen deaminase

19 (PBGD), nerve growth factor (NGF), mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 4 (MT-ND4), protective protein cathepsin A (PPCA), dysferlin, MER proto-oncogene, tyrosine kinase (MERTK), cystic fibrosis transmembrane conductance regulator (CFTR), or tumor necrosis factor receptor (TNFR) -immunoglobulin (IgGl) Fc fusion.. The method of claim 105, wherein the genome to be packaged encodes a dystrophin or a microdystrophin. . The method of claim 104, wherein the genome to be packaged encodes a microRNA.. A composition comprising isolated rAAV particles that were produced by the method of any one of claims 1 to 109. . A composition comprising cells capable of producing rAAV particles and a cell culture medium comprising between about 1 nM and about 500 pM of a mitotic inhibitor. . The composition of claim 111, wherein the mitotic inhibitor is a microtubule destabilizing agent. . The composition of claim 111 or claim 112, wherein the mitotic inhibitor comprises at least one of nocodazole, vincristine, colchicine or a salt thereof. . The composition of claim 113, wherein the mitotic inhibitor is nocodazole. . A composition comprising cells capable of producing rAAV particles and a cell culture medium comprising between about 10 nM and about 1 mM of a caspase inhibitor. . The method of claim 115, wherein the caspase inhibitor is a pan-caspase inhibitor.. The method of claim 115 or claim 116, wherein the caspase inhibitor comprises at least one of z-VAD-FMK, Q-VD-Oph or a salt thereof. . The method of claim 117, wherein the caspase inhibitor is Q-VD-Oph.