WO2025222013A1 - Recombinant aav production process - Google Patents

Abstract

The invention described herein provides methods and reagents, including certain endocytosis inhibitors, for enhancing AAV production / AAV yield in AAV production cells (such as HEK293 -based production cells).

Description

RECOMBINANT AAV PRODUCTION PROCESS

REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to and the benefit of the filing date of U.S. Provisional Patent Application No. 63/635,161, filed on April 17, 2024, the entire contents of which are incorporated herein by reference.

BACKGROND OF THE INVENTION

[0002] Recombinant adeno-associated virus (rAAV)-mediated gene therapy is a highly efficacious gene delivery strategy, with a promising safety profile that allows for broad clinical applications.

[0003] Due to recent success in the clinic, there is rising demand for improved rAAV production processes to increase yield, enhance quality, and reduce costs.

SUMMARY OF THE INVENTION

[0004] In one aspect, disclosed herein is a method of promoting adeno-associated virus (AAV) production / enhancing AAV yield in an AAV-production cell line, the method comprising culturing the AAV-production cell line in the presence of an endocytosis inhibitor that inhibits uptake of extracellular AAV by the production cell line.

[0005] In a related aspect, disclosed herein is a method of manufacturing adeno-associated virus (AAV), the method comprising culturing an AAV-production cell line in the presence of an endocytosis inhibitor that inhibits uptake of extracellular AAV by the production cell line.

[0006] In some embodiments, the endocytosis inhibitor inhibits clathrin-dependent endocytosis. some embodiments, the endocytosis inhibitor inhibits AAV trafficking from endoplasmic reticulum (ER) to Golgi apparatus, inhibits transportation through Golgi apparatus, and/or disrupts Golgi apparatus.

[0007] In some embodiments, the AAV-production cell line is cultured in the presence of brefeldin A, bafilomycin Al, and/or fllipin III.

[0008] In some embodiments, the AAV-production cell line is cultured in the presence of brefeldin A.

[0009] In some embodiments, the AAV-production cell line is cultured in the presence of bafilomycin AL [0010] In some embodiments, the AAV-production cell line is cultured in the presence of filipin III.

[0011] In some embodiments, the AAV-production cell line is cultured in the presence of brefeldin A and bafilomycin Al.

[0012] In some embodiments, the AAV-production cell line is cultured in the presence of brefeldin A and filipin III.

[0013] In some embodiments, the AAV-production cell line is cultured in the presence of bafilomycin Al and filipin III.

[0014] In some embodiments, the AAV-production cell line is cultured in the presence of brefeldin A, bafilomycin Al, and filipin III.

[0015] In some embodiments, the method further comprises culturing the AAV-production cell line in the presence of a cell cycle inhibitor that arrests cell cycle of the AAV-production cell line at G1 (e.g., M344 or a derivative / functional equivalent thereof) and/or G2/M (e.g., nocozale or a derivative / functional equivalent thereof).

[0016] In some embodiments, the presence of brefeldin A, bafilomycin Al, and/or filipin III reduces AAV transduction of the AAV-production cell line (e.g., a reduction of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, 99.5% as compared to a reference control).

[0017] In certain embodiments, the endocytosis inhibitor (such as brefeldin A) inhibits GEF1, and/or the endocytosis inhibitor (such as bafilomycin Al) inhibits vacuolar type (V)- ATPase.

[0018] In some embodiments, the AAV-production cell line is cultured in the presence of about 0.5 pM, 5 pM, 10 pM, or 20 pM brefeldin A.

[0019] In some embodiments, the AAV-production cell line is cultured in the presence of about 20 nM, 25 nM, 50 nM, 80 nM, 100 nM, 140 nM, 180 nM, 220 nM, 250 nM, or 300 nM bafilomycin Al.

[0020] In some embodiments, AAV production / yield is increased by at least about 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more in the presence of the endocytosis inhibitor compared to that in the absence of said endocytosis inhibitor.

[0021] In certain embodiments, AAV potency is increased by at least about 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more in the presence of the endocytosis inhibitor compared to that in the absence of said endocytosis inhibitor.

[0022] In certain embodiments, AAV produced in the presence of the endocytosis inhibitor has a level of genomic integrity about at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or higher than that produced in the absence of said endocytosis inhibitor.

[0023] In some embodiments, the AAV-production cell line is HEK293, or a derivative thereof (such as Gibco Viral Production Cells 2.0 from Thermo Fisher Scientific Inc. (VP2)). [0024] In some embodiments, the HEK293 is transfected by polynucleotides encoding an AAV Rep, an AAV Cap (e.g., AAV9, AAV8, AAVrh.74, or SLB-101), a gene of interest (e.g, a coding sequence for a functional dystrophin minigene, such as CK8-pDys) flanked by AAV ITR sequences, and helper genes necessary and sufficient for AAV packaging in the AAV production cell line.

[0025] In some embodiments, the polynucleotides comprise / consist of one, two, or three plasmids.

[0026] In some embodiments, production of AAV is in a bioreactor (e.g., a 0.5L bioreactor, a IL bioreactor, a 2L bioreactor, a 5L bioreactor, a 10L bioreactor, a 20L bioreactor, a 50L bioreactor, a 100L bioreactor, a 500L bioreactor, a lOOOL bioreactor, a ISOOL bioreactor, or a 2000L bioreactor).

[0027] In some embodiments, the method further comprises harvesting AAV. In some embodiments, harvesting AAV is performed at about 48 hrs, 72 hrs, or 96 hrs post transfection of the AAV-production cell line.

[0028] In some embodiments, the AAV-production cell line is first contacted by the endocytosis inhibitor at about 16-24 hrs (e.g., 16 hrs, 16.5 hrs, 17 hrs, 17.5 hrs, 18 hrs, 18.5 hrs, 19 hrs, 19.5 hrs, 20 hrs, 20.5 hrs, 21 hrs, 21.5 hrs, 22 hrs, 22.5 hrs, 23 hrs, 23.5 hrs, or 24 hrs) post transfecting the AAV-production cell line to initiate AAV production.

[0029] Another aspect of the invention disclosed herein is a culture for AAV production, wherein the culture comprises (1) an AAV-production cell line (e.g., HEK293 cells or derivative thereof); (2) an endocytosis inhibitor that inhibits uptake of extracellular AAV by the production cell line; and a media suitable for AAV production.

[0030] In some embodiments, the endocytosis inhibitor inhibits clathrin-dependent endocytosis.

[0031] In some embodiments, the endocytosis inhibitor inhibits AAV trafficking from endoplasmic reticulum (ER) to Golgi apparatus, inhibits transportation through Golgi apparatus, and/or disrupts Golgi apparatus.

[0032] In some embodiments, the endocytosis inhibitor inhibits GEF1 (such as brefeldin A) and/or V-ATPase (such as bafilomycin Al).

[0033] In some embodiments, the culture comprises brefeldin A, bafilomycin Al, and/or filipin III.

[0034] In some embodiments, the culture comprises brefeldin A.

[0035] In some embodiments, the culture comprises bafilomycin Al.

[0036] In some embodiments, the culture comprises fllipin III.

[0037] In some embodiments, the culture comprises brefeldin A and bafilomycin Al.

[0038] In some embodiments, the culture comprises brefeldin A and fllipin III.

[0039] In some embodiments, the culture comprises bafilomycin Al and fllipin III.

[0040] In some embodiments, the culture comprises brefeldin A, bafilomycin Al, and fllipin III.

[0041] In some embodiments, the AAV-production cell line is cultured in the presence of about 0.5 pM, 5 pM, 10 pM, or 20 pM brefeldin A.

[0042] In some embodiments, the AAV-production cell line is cultured in the presence of about 20 nM, 25 nM, 50 nM, 80 nM, 100 nM, 140 nM, 180 nM, 220 nM, 250 nM, or 300 nM bafilomycin Al.

[0043] In some embodiments, the AAV-production cell line is cultured in the presence of a cell cycle inhibitor that arrests cell cycle of the AAV-production cell line at G1 (e.g., M344 or a derivative / functional equivalent thereof) and/or G2/M (e.g., nocozale or a derivative / functional equivalent thereof).

[0044] In some embodiments, production of AAV is in a bioreactor (e.g., a 0.5L bioreactor, a IL bioreactor, a 2L bioreactor, a 5L bioreactor, a 10L bioreactor, a 20L bioreactor, a 50L bioreactor, a 100L bioreactor, a 500L bioreactor, a lOOOL bioreactor, a ISOOL bioreactor, or a 2000L bioreactor.

[0045] It should be understood that any one embodiment of the invention described herein, including those only described in the examples or claims, can be combined with any one or more other embodiments disclosed herein, unless the combination is improper or expressly disclaimed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIGs. 1 A-1B show that AAV can transduce HEK293 cells during AAV production. 2T : dual transfection (using two plasmids including one plasmid encoding helper genes sufficient to support AAV packaging in producer cells, and another plasmid encoding the RepCap and gene of interest (GOI) transcriptional cassettes). D1-D3: days 1-3. pDys: microdystrophin coding sequence as GOI. CMV: CMV promoter driving the transcription of the GOI transcriptional cassette. CK8: the muscle-specific CK8 promoter driving the transcription of the GOI transcriptional cassette.

[0047] FIG. 2A shows pDys protein expression in HEK293 cells producing AAV virus SLB101-CK8-pDys having SLB101 capsid and CK8-pDys in the GOI transcriptional cassette. RC: RepCap transcriptional cassette. 3TT: triple transfection.

[0048] FIG. 2B shows GOI (luciferase) expressed in the manufacture cells in a 50L BioReactor, as determined by expression levels of luciferase.

[0049] FIG. 3 shows that brefeldin A, bafilomycin Al, and fllipin III block transduction of AAV-SLBIOI-Luciferase on C2C12 cells (an immortalized mouse myoblast cell line).

[0050] FIGs. 4 A ( schematic and detailed views) and 4B show that crude virus (e.g., having GFP as GOI) in supernatant transduces AAV manufacture cells. Dy5: pDys.

[0051] FIGs. 5 A ( schematic and detailed views) and 5B show that brefeldin A reduces transduction in AAV manufactural cells.

[0052] FIGs. 6A and 6B show that brefeldin A increases AAV yield in HEK293 cells.

[0053] FIGs. 7A-7B show large scale (crude lysate in 500 mL shake flask) AAV production +/- brefeldin A (BrefA) at 16 hours (FIG. 7 A) or 22 hours post-transfection (FIG. 7B).

[0054] FIG. 7C shows particle distribution for AAV produced in the presence of brefeldin A or DMSO control.

[0055] FIG. 8 shows viral titers in supernatant of producer cell culture after adding brefeldin A at different time points to the culture post-transfection.

[0056] FIG. 9 shows results of brefeldin A dose response experiment.

[0057] FIG. 10 shows that brefeldin A increased Rh74_Bag3 viral particle (DT) yield by 2- fold.

[0058] FIG. 11 shows that bafilomycin Al and brefeldin A both reduced AAV transduction of the producer cells, and increased AAV yield.

[0059] FIG. 12 shows results of bafilomycin Al dose response experiment.

[0060] FIG. 13 shows that fllipin III can increase AAV yield.

[0061] FIG. 14 shows that combination of cell cycle inhibitor and brefeldin A results in synergistic effects on increasing AAV yield.

DETAILED DESCRIPTION OF THE INVENTION

[0062] The invention described herein is partially based on the discovery that recombinant AAV virus (such as the rAAV-SLBlOl virus described herein) produced in AAV packaging cells (such as the HEK293 AAV packaging cells) and secreted extracellularly can indeed transduce the AAV production cells, such as HEK293 cells (e.g., extracellular rAAV being produced in HEK293 cells can transduce the HEK293 manufacturing cells). Protein expression from rAAV-SLBlOl transgenes was observed in HEK293 cells during rAAV- SLB101 production (e.g., transduction resulting in GIO expression in the HEK293 packaging cells, as can be detected in lysates of the HEK293 packaging cells), and supernatant transfer experiments confirmed that extracellular rAAV does transduce HEK293 cells during rAAV production. Thus, loss of rAAV to such packaging cell transduction during manufacture does occur e.g., transduction reduces rAAV yield, and such loss of rAAV to transduction during manufacture can be significant).

[0063] The invention described herein is also partially based on the discovery that the addition of certain endocytosis inhibitors that inhibit uptake of extracellular AAV by the production cell line, such as brefeldin A, bafilomycin (such as bafilomycin Al), and fllipin III, into the media during AAV production / manufacture improves recombinant AAV (rAAV) yield and quality (e.g., certain endocytosis inhibitors, such as Brefeldin A and Bafilomycin and fllipin III, reduce and limit self-transduction of the packaging cells by the manufactured AAV viral particles, improving recombinant AAV yield).

[0064] Initial work using multiple AAV capsids (such as AAV6, AAV8, AAV9, AAVrh74, AAV-SLB101, and other engineered capsid) and multiple packaging cell lines (such as HEK293 cells, including Expi293 and VPC2.0 cells), surprisingly showed a large portion (e.g., about 20-60%) of the total manufactured AAV viral particles present in the extracellular culture media of the AAV packaging cells used to manufacture AAV viral particles. Such AAV viral particles, once in the extracellular culture media, can in turn transduce the AAV packaging cells via one of these endocytosis pathways, leading to expression of the gene-of- interest (GOI) encapsidated by the AAV viral particles inside the packaging cells.

[0065] Thus, experimnents were conducted demonstrating that addition of brefeldin A, bafilomycin Al, and/or fllipin III to the AAV production cell line HEK293 (which is routinely used in AAV production through the triple transfection method) during AAV manufacture surprisingly resulted in a significant reduction in the transgene protein expression in the AAV production cells. Inhibition of extracellular rAAV (e.g., rAAV- SLB101) transduction of production cells (e.g., HEK293 cells) using brefeldin A, bafilomycin Al, and/or fllipin III resulted into a significant increase in recombinant AAV (e.g., rAAV-SLBlOl) yield - e.g., 2- to 3-fold higher yield (e.g., reduced transduction translating into increased rAAV yield of 2-3 -fold). Extracellular virus (e.g., produced AAV viral particles in the extracellular space / supernatant of the AAV production cells HEK293) increased from 41% to 68% of the total virus in the case of rAAV-SLBlOl production by HEK293 cells.

[0066] Brefeldin A, ((lR,2E,6S,10E,l laS,13S,14aR)-l,13-Dihydroxy-6-methyl- l,6,7,8,9,l la,12,13,14,14a-decahydro-4H-cyclopenta[f][l]oxacyclotridecin-4-one), is a lactone antiviral produced by the fungus Penicillium brefeldianum. One activity of brefeldin A is inhibiting protein transport from the endoplasmic reticulum (ER) to the Golgi complex indirectly by preventing association of COP-I coat to the Golgi membrane and causes Golgi disassembly. Brefeldin A also inhibits clathrin-dependent endocytosis. As demonstrated herein, brefeldin A prevents AAV uptake by AAV production cells, such as HEK293 cells used in triple transfection routinely used in AAV production. While not wishing to be bound by theory, it is believed that brefeldin A inhibits AAV uptake by inhibiting endocytosis.

[0067] The structure of brefeldin A is below:

[0068] The bafilomycins are a family of macrolide antibiotics produced from a variety of Streptomycetes. Their chemical structure is defined by a 16-membered lactone ring scaffold. Bafilomycins exhibit a wide range of biological activity, including anti -turn or, anti-parasitic, immunosuppressant and anti-fungal activity. Bafilomycins have also been found to act as ionophores, transporting potassium K+ across biological membranes and leading to mitochondrial damage and cell death. A representative bafilomycin is bafilomycin Al ((3Z,5E,7R,8S,9S,11E,13E,15S,16R)-16- [(lS,2R,3S)-3-[(2R,4R,5S,6R)-2,4-dihydroxy-6- isopropyl-5-methyl-2-tetrahydropyranyl]-2- hydroxy-1 -methylbutyl]-8-hydroxy-3, 15- dimethoxy-5, 7, 9, 11 -tetramethyl- 1- oxacyclohexadeca-3,5,1 l,13-tetraen-2-one), a potent inhibitor of cellular autophagy. (bafilomycin Al)

[0069] Bafilomycin Al specifically targets the vacuolar-type H⁺-ATPase (V-ATPase) enzyme, a membrane-spanning proton pump that acidifies either the extracellular environment or intracellular organelles such as the lysosome of animal cells or the vacuole of plants and fungi. Thus bafilomycin Al impacts lysosomal trafficking. At higher micromolar concentrations, bafilomycin Al also acts on P-type ATPases, which have a phosphorylated transitional state. While not wishing to be bound by theory, bafilomycin Al may exerts its effect through inhibiting endocytosis / AAV endocytosis, and/or inhibiting AAV viral uncoating.

[0070] Other structurally related bafilomycins that may be used in the instant invention include: bafilomycin Bl, bafilomycin B2, bafilomycin Cl, bafilomycin C2, bafilomycin Cl amide, bafilomycin C2 amide, bafilomycin D, bafilomycin E, 9-hydroxybafilomycin D, and 29-hydroxybafilomycin D.

[0071] Filipin is a mixture of 4 chemical compounds - fllipin I (4%), II (25%), III (53%), and IV (18%) - and is thus referred to as filipin complex. It was first isolated in 1955 from the mycelium and culture filtrates of a previously unknown actinomycete, Streptomyces filipinensis, in a soil sample from the Philippine Islands. The isolate possessed potent antifungal activity, and was identified as a polyene macrolide based on its characteristic UV- Vis and IR spectra.

[0072] The major component, filipin III ((3R,4S,6S,8S,10R,12R,14R,16S,17E,19E,21E,23E, 25E,27S,28R)-4,6,8,10,12,14,16,27-Octahydroxy-3-[(lR)-l-hydroxyhexyl]-17,28-dimethyl- l-oxacyclooctacosa-17,19,21,23,25-pentaen-2-one), has the following structure: (filipin III)

[0073] The invention described herein is further partly based on the identification of the optimal timing of addition of an endocytosis inhibitor that inhibits uptake of extracellular AAV by the production cell line (e.g., brefeldin A, bafilomycin Al, and/or filipin III).

[0074] For example, optimum time for adding the endocytosis inhibitor (e.g., brefeldin A) was identified as approximately 16 to 22 h post-transfection of the packaging HEK293 cells, with an observed ~3-fold increase in rAAV-SLBlOl yield, as compared to low or no effect when administered at early or later time points.

[0075] The invention described herein is further partly based on several unexpected advantageous of including one or more of the subject endocytosis inhibitors that inhibit uptake of extracellular AAV by the production cell line.

[0076] In one embodiment, brefeldin A and bafilomycin Al were shown to have a dose- responsive effect on rAAV-SLBlOl yield, yet treatment had no impact on HEK293 cell viability or growth. Further, following iodixanol purification of rAAV produced in the presence or absence of brefeldin A, the brefeldin A-produced rAAV-SLBlOl viruses were found, based on assessment using various quality assays, to have similar if not marginally improved quality attributes (e.g., endocytosis inhibitors (such as Brefeldin A)-produced rAAV having similar or improved quality attributes).

[0077] In certain embodiments, e.g., in rAAV-SLBlOl production, the full capsid percentage of the produced viral particle (e.g., rAAV-SLBlOl) is increased with the addition of such endocytosis inhibitors such as brefeldin A.

[0078] Significantly, rAAVs (e.g., rAAV-SLBlOl) produced in the presence of the subject endocytosis inhibitors (e.g, brefeldin A) have consistently higher potency in a C2C12 assay to measure transgene expression.

[0079] The beneficial effects of including the subject endocytosis inhibitors (such as brefeldin A) are not unique to the SLB101 capsid, since brefeldin A also led to an increase in rAAV-Rh74 yield of up to 2-fold.

[0080] The beneficial effects of including the subject endocytosis inhibitors (such as brefeldin A) are further not unique to the specific HEK293 packaging cells used, since similar results were also obtained using the AAV packaging cells from a commercial AAV production vender - Forge Biologies (“Forge cells”).

[0081] Accordingly, one aspect of the disclosure herein is a method of promoting AAV production or enhancing AAV yield, wherein the method comprises culturing an AAV- production cell line in the presence of an endocytosis inhibitor that inhibits uptake of extracellular AAV by the production cell line. In some embodiments, the AAV-production cell line is cultured in the presence of brefeldin A, bafilomycin Al, and/or fllipin III.

[0082] In a related aspects, disclosed herein is a method of manufacturing AAV, the method comprising culturing an AAV-production cell line in the presence of an endocytosis inhibitor that inhibits uptake of extracellular AAV by the production cell line. In some embodiments, the AAV-production cell line is cultured in the presence of brefeldin A, bafilomycin Al, and/or fllipin III.

[0083] In some embodiments, disclosed herein is a method of promoting AAV production or enhancing AAV yield, or manufacturing AAV comprising culturing an AAV-production cell line in the presence of a bafilomycin, such as bafilomycin Al.

[0084] In some embodiments, disclosed herein is a method of promoting AAV production or enhancing AAV yield, or manufacturing AAV comprising culturing an AAV-production cell line in the presence of brefeldin A.

[0085] In some embodiments, disclosed herein is a method of promoting AAV production or enhancing AAV yield, or manufacturing AAV comprising culturing an AAV-production cell line in the presence of fllipin III.

[0086] The invention described herein is further partly based on the discovery that th beneficial effects of the subject endocytosis inhibitors (such as brefeldin A, bafilomycin Al, and/or fllipin III), can be further enhanced in a synergistic manner by certain cell-cycle inhibitors, such as inhibitors that arrest the production cells (e.g., HEK293) in G1 and/or G2/M phase(s) of the cell cycle.

[0087] Thus in some embodiments, the method further comprises culturing the AAV- production cell line in the presence of a cell cycle inhibitor that arrests cell cycle of the AAV- production cell line at G1 and/or G2/M.

[0088] In certain embodiments, the cell cycle inhibitor is an HD AC (histone deacetylase) inhibitor, such as an inhibitor for the Class I HD AC (including HDAC1, -2, -3 and -8 related to the yeast RPD3 gene); for the Class IIA HD AC (including HDAC4, -5, -7 and -9); for the Class IIB HD AC (including HDAC-6, and -10, related to the yeast Hdal gene); for the Class III HD AC (also known as the sirtuins, related to the Sir2 gene and include SIRT1-7); and/or for the Class IV HD AC (including HDAC11).

[0089] In certain embodiments, the HD AC inhibitor includes hydroxamic acids (or hydroxamates), such as trichostatin A; cyclic tetrapeptides (such as trapoxin B), and the depsipeptides; benzamides; electrophilic ketones; and the aliphatic acid compounds such as phenylbutyrate and valproic acid.

[0090] In certain embodiments, the HD AC inhibitor includes hydroxamic acids vorinostat (SAHA), belinostat (PXD101), resminostat, abexinostat, Givinostat, LAQ824, panobinostat (LBH589), benzamides : entinostat (MS-275); tacedinaline (CI994); zabadinostat; and mocetinostat (MGCD0103); nicotinamide; derivatives of NAD; dihydrocoumarin; naphthopyranone; and 2-hydroxynaphthaldehydes.

[0091] In certain embodiments, the HD AC inhibitor comprises M344 (4-(dimethylamino)-N- [7-(hydroxyamino)-7-oxoheptyl]-benzamide), which exhibits preferential Class III HD AC inhibitor activity. It shows a three-fold selectivity for inhibiting HDAC6 over HD AC 1.

[0092] In some embodiments, the cell cycle inhibitor inhits the production cells at G1 phase. [0093] In some embodiments, the cell cycle inhibitor inhits the production cells at G2/M phase. In certain embodiments, the cell cycle inhibitor comprises nocodazole (methyl [5-(2- thienylcarbonyl)-lH-benzimidazol-2-yl]carbamate). (nocodazole)

[0094] Accordingly, in some enbodiments, disclosed herein is a method of promoting AAV production or enhancing AAV yield comprising culturing an AAV-production cell line (e.g., HEK293 or a derivative thereof) in the presence of brefeldin A, bafilomycin Al, and/or filipin III, and in further combination with a cell cycle inhibitor that arrests cell cycle of the AAV-production cell line at G1 (e.g., M344) and/or G2/M e.g., nocodazole). In some embodiments, the method comprises culturing the AAV-production cell line in the presence of brefeldin A, M344, and nocodazole.

[0095] In some aspects, disclosed herein is a method of manufacturing AAV comprising culturing an AAV-production cell line in the presence of brefeldin A, bafilomycin Al, and/or filipin III and in further combination with a cell cycle inhibitor that arrests cell cycle of the AAV-production cell line at G1 (e.g, M344) and/or G2/M (e.g., nocodazole). In some embodiments, the method comprises culturing the AAV-production cell line in the presence of brefeldin A, M344, and nocodazole.

[0096] In some embodiments, the endocytosis inhibitor (e.g., brefeldin A or bafilomycin Al, and/or filipin III) reduces AAV transduction of the AAV-production cell line (e.g., a reduction of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, 99.5% as compared to a reference control). In some embodiments, the reference control is a level of AAV transduction in normal cells medium.

[0097] In some embodiments, AAV production / yield is increased by at least about 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 10 times, 20 times, 50 times, 100 times, 500 times, 1000 times or more in the presence of the endocytosis inhibitor (e.g., brefeldin A, bafilomycin Al, or filipin III) as compared to normal cells medium.

[0098] In some embodiments, the AAV-production cell line is cultured in the presence of about 0.1 pM, 0.25 pM, 0.5 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 11 pM, 12 pM, 13 pM, 14 pM, 15 pM, 16 pM, 17 pM, 18 pM, 19 pM, or 20 pM brefeldin A.

[0099] In some embodiments, the AAV-production cell line is cultured in the presence of about 0.25 pM to 25 pM, 0.5 pM to 25 pM, 0.5 pM to 20 pM, 0.25 pM to 15 pM, 0.25 pM to 10 pM, 0.5 pM to 15 pM, 0.5 to 10 pM, 0.5 pM to 8 pM, 0.5 pM to 7 pM, 1 pM to 15 pM, 1 pM to 10 pM, 1 pM to 8 pM, 2 pM to 6 pM, 4 pM to 6 pM, or 3 pM to 10 pM brefeldin A.

[0100] In some embodiments, the AAV-production cell line is cultured in the presence of about 0.5 pM, 5 pM, 10 pM, or 20 pM brefeldin A.

[0101] In some embodiments, the AAV-production cell line is cultured in the presence of about 0.5 pM brefeldin A. In some embodiments, the AAV-production cell line is cultured in the presence of about 1 pM brefeldin A. In some embodiments, the AAV-production cell line is cultured in the presence of about 2.5 pM brefeldin A. In some embodiments, the AAV- production cell line is cultured in the presence of about 5 pM brefeldin A. In some embodiments, the AAV-production cell line is cultured in the presence of about 7.5 pM brefeldin A. In some embodiments, the AAV-production cell line is cultured in the presence of about 10 pM brefeldin A. In some embodiments, the AAV-production cell line is cultured in the presence of about 12.5 pM brefeldin A. In some embodiments, the AAV-production cell line is cultured in the presence of about 15 pM brefeldin A. In some embodiments, the AAV-production cell line is cultured in the presence of about 17.5 pM brefeldin A. In some embodiments, the AAV-production cell line is cultured in the presence of about 20 pM brefeldin A.

[0102] In some embodiments, the AAV-production cell line is cultured in the presence of about 5 nM, 10 nM, 25 nM, 50 nM, 80 nM, 100 nM, 120 nM, 140 nM, 150 0M, 160 nM, 180 nM, 200 nM, 220 Nm, 240 nM, 250 Nm, 260 nM, 300 nM, 400 nM, 500 nM, 1 pM, 2.5 pM, or 5 pM bafilomycin Al.

[0103] In some embodiments, the AAV-production cell line is cultured in the presence of about 10 nM to 1 pM, 10 nM to 500 nM, 10 nM to 250 nM, or 50 nM to 250 nM bafilomycin Al.

[0104] In some embodiments, the AAV-production cell line is cultured in the presence of about 20 nM, 25 nM, 50 nM, 80 nM, 100 nM, 140 nM, 180 nM, 220 nM, 250 nM, 300 nM, 400 nM, or IpM bafilomycin Al.

[0105] In some embodiments, the AAV-production cell line is cultured in the presence of about 26.7 nM bafilomycin Al. In some embodiments, the AAV-production cell line is cultured in the presence of about 80 nM bafilomycin Al. In some embodiments, the AAV- production cell line is cultured in the presence of about 213.6 nM bafilomycin Al.

[0106] In some embodiments, the AAV-production cell line is first contacted by brefeldin A, bafilomycin Al, and/or fllipin III) at about 16-23 hrs (e.g., 16 hrs, 16.5 hrs, 17 hrs, 17.5 hrs, 18 hrs, 18.5 hrs, 19 hrs, 19.5 hrs, 20 hrs, 20.5 hrs, 21 hrs, 21.5 hrs, 22 hrs, 22.5 hrs, or 23 hrs) post transfecting the AAV-production cell line to initiate AAV production. In some embodiments, the AAV-production cell line is first contacted by the endocytosis inhibitor (e.g., brefeldin A, bafilomycin Al, and/or fllipin III) at about 16 hrs post transfecting the AAV-production cell line. In some embodiments, the AAV-production cell line is first contacted by the endocytosis inhibitor (e.g., brefeldin A, bafilomycin Al, and/or fllipin III) at about 22 hrs post transfecting the AAV-production cell line. In some embodiments, the AAV-production cell line is first contacted by the endocytosis inhibitor (e.g., brefeldin A, bafilomycin Al, and/or fllipin III) at about 23.5 hrs post transfecting the AAV-production cell line.

[0107] In some examples, the AAV produced using the methods disclosed herein has potency increased by at least about 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more in the presence of the endocytosis inhibitor (e.g., brefeldin A, bafilomycin Al, and/or fllipin III) as compared to that in the absence of said endocytosis inhibitor.

[0108] An exemplary assay of measuring AAV potency is described herein. In brief, the AAV potency assay comprises: contacting (e.g., in vitro) differentiated muscle cells (e.g., C2C12 cells) the AAV produced using the methods disclosed herein, wherein the AAV comprises a coding sequence for a functional protein, such as function dystrophin (such as a microdystrophin, e.g., pDys5); and determining transduction potency of the differentiated muscle cell by said AAV by measuring expression of the functional protein, e.g., pDys5 (e.g., using ELISA) in the differentiated muscle cells. In some examples, expression of the functional protein is determined at 3-5 days (e.g., 4 days) post infection of the differentiated muscle cells by the AAV.

[0109] In some embodiments, infection of the differentiated muscle cells (e.g., C2C12 cells) by AAV produced in presence of the endocytosis inhibitor (e.g., brefeldin A or bafilomycin Al, or filipin III) results increased expression of a functional protein (e.g., pDys5) that is at least about 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more than a reference control. In some embodiments, the reference control is expression levels of the functional protein (e.g., pDys5) in the differentiated muscle cells (e.g., C2C12 cells) infected by AAV produced in normal medium.

[0110] In certain embodiments, AAV produced in the presence of the endocytosis inhibitor (e.g., brefeldin A, bafilomycin Al, and/or fllipin III) has a level of genomic integrity about at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or higher than that produced in the absence of the endocytosis inhibitor.

[OHl] Genomic integrity of the packaged GOI inside the AAV viral particles can be meaured using any of the conventional techniques known in the art. In certain embodiments, genomic integrity is measured by determining the percentage of AAV viral particles having near full-length GOI DNA or AAV vector genome in the viral particle. This can be measured by using, e.g., ddPCR or similar techniques, to determine the presence of DNA sequences at both ends of the AAV vector genome, such as the promoter or 5’ UTR encoding sequences at one end, and the 3 ’ UTR encoding sequences at the other end. Viral particles having only sequences at one but not both ends of the AAV vector genome are considered lacking of genomic integrity.

[0112] In some embodiments, the AAV production line is HEK293, or a derivative thereof. [0113] The HEK293 cell line is an immortalized (by sheared adenovirus 5 (Ad5)) human embryonic kidney cell line widely used in research and commertial production of proteins as well as AAV viral particles. It is a robust and fast-growing cell line with numerous derivatives, such as HEK293S, HEK293T, HEK293F, HEK293FT, HEK293FTM, HEK293SG, HEK293SGGD, HEK293H, HEK293E, HEK293MSR and HEK293A.

[0114] In certain embodiments, the HEK293 is transfected by polynucleotides encoding an AAV Rep, an AAV Cap (e.g., AAV9, AAV8, AAVrh.74, or SLB-101 or other engineered capsids), a gene of interest (e.g., a coding sequence for a functional dystrophin minigene, such as CK8-pDys) flanked by AAV ITR sequences, and helper genes necessary and sufficient for AAV packaging in the AAV production cell line.

[0115] In certain embodiments, the polynucleotides comprise / consist of one, two, or three plasmids.

[0116] In some embodiments, production of AAV is in a bioreactor (e.g., a 0.5L bioreactor, a IL bioreactor, a 2L bioreactor, a 5L bioreactor, a 10L bioreactor, a 20L bioreactor, a 50L bioreactor, a 100L bioreactor, a 500L bioreactor, a lOOOL bioreactor, a 1500L bioreactor, or a 2000L bioreactor).

[0117] In some embodiments, the method of promoting AAV production / enhancing AAV yield disclosed herein further comprises harvesting AAV. In some embodiments, harvesting AAV is performed at about 48 hrs, 72 hrs, or 96 hrs post transfection of the AAV-production cell line.

[0118] In certain embodiments, the method further comprising contacting the AAV packaging cells (e.g., HEK293 cells) with a second agent to further enhance AAV production yield. In certain embodiments, the second agent comprises an anti-mitotic agent (such as one that arrests the packaging cells at G2/M phase, e.g., nocodazole) and/or a selective HD AC inhibitor (such as M344).

[0119] Additional detailed aspects of the invention are further described in the sections below.

Dual Transfection AAV Vector

[0120] In some examples, the method disclosed herein comprises culturing an AAV- production cell line in the presence of an endocytosis inhibitor, wherein the AAV-production cell line is transfected by polynucleotides encoding an AAV Rep, an AAV Cap (e.g., AAV9, AAV8, AAVrh.74, or SLB-101), a gene of interest (e.g., a coding sequence for a functional dystrophin minigene, such as CK8-pDys) flanked by AAV ITR sequences, and helper genes necessary and sufficient for AAV packaging in the AAV production cell line.

[0121] In some embodiments, the polynucleotides comprise / consist of one plasmid, two plasmids (dual transfection (DT) vector system), or three plasmids (triple transfection (TT) vector system).

[0122] Conventional AAV production systems utilize three plasmid vectors (the so-called triple transfection vector system): one with a GOI (such as coding sequence for a microdystrophin (pDys) or other functional GOI, or for a reporter gene such as luciferase or GFP) flanked by AAV ITR sequences, for packaging into the AAV viral particles; another with an expression cassette encoding the AAV Rep and Cap proteins (including wild type capsids, AAV-SLB101 capsid, and other engineered capsid) useful for AAV packaging; and yet another provides the useful helper genes from other viruses (such as adenovirus, herpesvirus, or papillomavirus) for productive AAV life cycle.

[0123] The recombinant dual transfection vectors described herein can improve the conventional AAV production system by inserting both the GOI cassette and the rep-cap expression cassette into a single plasmid, to create the dual transfection vector (e.g., plasmid) of the invention that can be used in double transfection with the helper plasmid (e.g., the same helper plasmid used in the triple transfection method described herein).

[0124] In some embodiments, the polynucleotides used for the methods described herein comprise / consist of a vector plasmid and a helper plasmid, wherein the vector plasmid comprises a GO I, a rep gene encoding functional Rep proteins, and a cap gene encoding functional Cap proteins. In some embodiments, the vector plasmid does not comprise a polynucleotide sequence encoding a helper gene.

[0125] As used herein, the term “plasmid” includes a nucleic acid molecule that can replicate independently of a cell chromosome. The term “plasmid” is intended to include circular nucleic acid molecules and linear nucleic acid molecules. Furthermore, the term “plasmid” is intended to include bacterial plasmids, cosmids, minicircles (Nehlsen etal., Gene Ther. Mol. Biol., 10: 233-244, 2006; and Kay et al., Nature Biotechnology, 28: 1287-1289, 2010) and ministrings (Nafissi et al., Mol Ther Nucleic Acids, 3:el65, 2014). In certain embodiments, the plasmid is a circular nucleic acid (DNA) molecule. In certain embodiments, the plasmid is a nucleic acid molecule that is of bacterial origin.

[0126] In some embodiments, the GOI is within a pro- AAV cassette comprising the GOI operably linked to a promoter.

[0127] In certain embodiments, the rDNA vector comprises two or more copies of the pro- AAV cassette. In certain embodiments, all copies of the pro-AAV cassette comprise the same GOI. In certain embodiments, at least two of the pro-AAV cassettes comprise different GOI. The latter embodiment can be useful, for example, if AAV vectors are used to deliver different parts of the same functional assembly, such as a CRISPR/Cas effector enzyme and a coding sequence for one or more guide RNAs.

[0128] In certain embodiments, the pro-AAV cassette further comprises: (1) an enhancer that promotes the transcription of the GOI from the promoter; (2) a 5’ UTR; (3) a Kozak sequence; (4) a heterologous intron that promotes transcription and/or translation of the GOI; (5) a 3’ UTR; (6) a WPRE sequence; and/or (7) a polyA signal sequence.

[0129] In some embodiments, the coding sequence for the AAV Rep and the coding sequence for the AAV Cap are within a RepCap cassette comprising an operably-linked RepCap promoter. In certain embodiments, the operably-linked RepCap promoter comprises the AAV P5 promoter.

[0130] In some embodiments, the RepCap cassette and the pro-AAV cassette are immediately adjacent to each other (e.g., with substantially no intervening polynucleotide sequence).

[0131] In some other embodiments, the RepCap cassette and the pro-AAV cassette are not immediately adjacent to each other.

[0132] In some embodiments, the RepCap cassette and the pro-AAV cassette have the same transcription direction.

[0133] In some other embodiments, the RepCap cassette and the pro-AAV cassette have opposite transcription directions.

[0134] In some embodiments, the pro-AAV cassette is upstream of the RepCap cassette. [0135] In some other embodiments, the pro-AAV cassette is downstream of the RepCap cassette.

[0136] In some embodiments, the GOI is in front of (z.e., upstream or more 5’ to) the coding sequence for the AAV Rep. In some embodiments, the GOI is in front of the coding sequence for the AAV Cap. In some embodiments, the GOI is in front of the coding sequences for both the AAV Rep and AAV Cap.

[0137] In certain embodiments, the coding sequence for the AAV Rep is under the transcriptional control of an AAV P5 promoter positioned downstream or 3’ to the GOI. [0138] In some embodiments, the expression of the AAV Rep in a production cell comprising AAV helper genes is sufficient to package an AAV vector genome (vg) comprising the GOI flanked by the 5’- and 3-ITR sequences into an AAV capsid comprising the AAV Cap.

[0139] In some embodiments, the recombinant DNA vector (dual transfection vector) further comprises a bacterial replication Ori gene, a selection marker (such as an antibiotic resistance gene, e.g., KanR or AmpR) under the transcriptional control of a selection marker promoter, such as a bacterial promoter.

[0140] In certain embodiments, the GOI is a functional equivalent of dystrophin e.g., a dystrophin minigene encoding a functional micro-dystrophin protein).

[0141] In certain embodiments, the GOI includes a gene responsible for / defective in LGMD2E (limb-girdle muscular dystrophy type 2E), LGMD2D (limb-girdle muscular dystrophy type 2D), LGMD2C (limb-girdle muscular dystrophy type 2C), LGMD2B (limbgirdle muscular dystrophy type 2B), LGMD2L (limb-girdle muscular dystrophy type 2L), LGMD2I (limb-girdle muscular dystrophy type 21), or a gene or coding sequence for NAGLU (a-N-acetylglucosaminidase, for Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IIEB)), sulfamidase or SGSH (for mucopolysaccharidosis type IIIA or MPS IIIA), Factor IX, Factor VIII, Myotubularin 1 (MTM1), Survival of Motor Neuron (SMN, for spinal muscular atrophy or SMA), GalNAc transferase GALGT2, calpain-3 (CAPN-3), acid alpha-glucosidase (GAA, for Pompe disease), alpha-galactosidase A or GLA (for Fabry disease), glucocerebrosidase, dystrophin or microdystrophin.

[0142] In certain embodiments, the GOI is a microdystrophin gene (e.g., one described in US7,906,l l l; US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; WO2016115543; WO 2023/018854, or US 10, 166,272, each one incorporated herein by reference). In certain embodiments, the GOI is a microdystrophin gene having the nucleotide sequence of SEQ ID NO: 1 of WO 2023/018854 (incorporated herein by reference).

[0143] In certain embodiments, the microdystrophin gene comprises a coding sequence for R16 and R17 spectrin-like repeats for the full-length dystrophin protein (such as one described in US7,892,824).

[0144] In certain embodiments, the microdystrophin gene comprises a coding sequence for the Rl, R16, R17, R23, and R24 spectrin-like repeats of the full-length dystrophin protein (such as the microdystrophin gene described in PCT/US2016/013733).

[0145] Diseases or conditions having a potential to benefit from the rAAV produced by the dual transfection vector of the invention include: Huntington’s disease, X-linked myotubular myopathy (XLMTM), Acid maltase deficiency (e.g., Pompe disease), Spinal Muscular Atrophy (SMA), Myasthenia Gravis (MG), Amyotrophic lateral sclerosis (ALS), Friedreich’s ataxia, Mitochondrial myopathy, Muscular dystrophies (Duchenne’s muscular dystrophy, Myotonic dystrophy, Becker muscular dystrophy (BMD), Limb-girdle muscular dystrophy (LGMD), Facioscapulohumeral muscular dystrophy (FSH), Congenital muscular dystrophy (CDM), Oculopharyngeal muscular dystrophy (OPMD), Distal muscular dystrophy, Emery- Dreifuss muscular dystrophy (EDMD), Mucopolysaccharidoses (MPS), Metachromatic leukodystrophy (MLD), Batten Disease, Rett Syndrome, Krabbe Disease, Canavan disease, X-Linked Retinoschisis, Achromatopsia (CNGB3 and CNGA3), X-Linked Retinitis Pigmentosa, Age-Related Macular Degeneration, neovascularized macular degeneration, Pompe, Fabry’s disease, MPS I, II, IIIA, IIIB, Gaucher’s disease, Dannon Disease, AlAt Deficiency, Friedreich ataxia, Wilson’s Disease, Batten Disease (CLN1, CLN3, CLN6, CLN8), Wolman Disease, Tay-Sachs, Niemann-Lick Type C, CDKL5 deficiency Disorder, B-thalassemia, Sickle cell disease.

[0146] In certain embodiments, diseases or conditions having a potential to benefit from the rAAV produced by the dual transfection vector of the invention may include: Becker muscular dystrophy (BMD), Congenital muscular dystrophies (CMD), Bethlem CMD, Fukuyama CMD, Muscle-eye-brain diseases (MEBs), Rigid spine syndromes, Ullrich CMD, Walker-Warburg syndromes (WWS), Duchenne muscular dystrophy (DMD), Emery- Dreifuss muscular dystrophy (EDMD), Facioscapulohumeral muscular dystrophy (FSHD), Limb-girdle muscular dystrophies (LGMD), Myotonic dystrophy (DM), Oculopharyngeal muscular dystrophy (OPMD), Motor neuron diseases including ALS (amyotrophic lateral sclerosis), Spinal -bulbar muscular atrophy (SBMA), Spinal muscular atrophy (SMA).

[0147] In certain embodiments, the GOI is a coding sequence for BAG3 (BCL-2-associated athanogene 3). BAG3 has been implicated in selected macroautophagy (aggrephagy), wherein aggregated proteins are degraded. Under stress conditions and during normal cellular aging, BAG3 acts with other molecular chaperones HSP70 and HSPB8, along with ubiquitin receptor p62/SQSTMl to target aggregated proteins for autophagic degradation. Loss of function of BAG3 can disrupt cellular clearing of protein aggregates which may lead to physiological complications and dysfunction. BAG3 mediated clearance is involved in many cellular processes which require the clearance of aggregate or aggregate prone proteins, and may be associated with age-related neurodegenerative disorders, like Alzheimer’s disease (marked by tau- protein), Huntington’s disease (involving mutated huntingtin/polyQ proteins), and amyotrophic lateral sclerosis (mutated SOD1). Additionally, BAG3 has been shown to play a role in a variety of other disease states, including cancer and myopathies.

BAG3 mutations in cardiomyopathy may significantly increase burdens associated with heart disease and increase severe cardiac events.

[0148] In certain embodiments, the coding sequence for BAG3 is a human coding sequence for BAG3. In some embodiments, the human BAG3 coding sequence is codon-optimized for expression in human cells, such as human cardiac muscle cells. In some embodiments, the coding sequence for BAG3 is CpG depleted. In certain embodiments, the coding sequence for BAG3 is any one of the BAG3 constructs in WO2023108159A1 (incorporated herein by reference).

[0149] In some embodiments, the amino acid sequence of the BAG3 encoded by the coding sequence for BAG3 is at least about 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to one or a combination of the amino acid sequences set forth as SEQ ID NOs: 23-26, 101, or 83-85 of WO2023108159A1 (incorporated herein by reference). In some embodiments, the amino acid sequence of the BAG3 encoded by the coding sequence for BAG3 is at least about 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequences set forth as SEQ ID NOs: 23-26 of WO2023108159A1 (incorporated herein by reference) arranged in sequence. In some embodiments, the amino acid sequence of the BAG3 encoded by the coding sequence for BAG3 is at least about 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequences set forth as SEQ ID NO: 101 of WO2023108159A1 (incorporated herein by reference). In some embodiments, the amino acid sequence of the BAG3 encoded by the coding sequence for BAG3 is at least about 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequences set forth as SEQ ID NOs: 83-85 of WO2023108159A1 (incorporated herein by reference) arranged in sequence.

[0150] In certain embodiments, the coding sequence for BAG3 is operatively linked to a promoter and/or an enhancer element, such as a CMV promoter / enhancer, or a cardiac muscle-specific promoter/enhancer, such as a MHCK7 promoter/enhancer. In certain embodiments, the promoter is CBA (Chicken P-Actin). In some embodiments, the promoter is CMV or mini CMV. In some embodiments, the promoter is CK8. In some embodiments, the promoter is MHCK7. In some embodiments, the promoter is CMV, mini-CMV, HSV, TK, RSV, SV40, MMTV, Ad E1A, CBA, CK8, MHCK7, Desmin (optionally mDES), and combinations thereof.

[0151] In certain embodiments, the coding sequence for BAG3 is used / encompassed in AAV viral particles for treating dilated cardiomyopathy.

[0152] In certain embodiments, the GOI is a coding sequence for cardiac troponin T (TNNT2). TNNT2 is a component of the troponin complex within the thin filament of the sarcomere which allows actomyosin interaction and contraction to occur in response to Ca2+. Mutations in or perturbations in the function of TNNT2 are causative of hypertrophic (HCM) and dilated cardiomyopathy (DCM).

[0153] In certain embodiments, the coding sequence for TNNT2 is a human coding sequence for TNNT2. In some embodiments, the human TNNT2 coding sequence is codon-optimized for expression in human cells, such as human cardiac muscle cells. In some embodiments, the coding sequence for TNNT2 is CpG depleted. In certain embodiments, the coding sequence for TNNT2 is any one of the TNNT2 constructs in WO2023178339A2 (incorporated herein by reference).

[0154] In certain embodiments, the amino acid sequence of TNNT2 encoded by the coding sequence for TNNT2 is at least about 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence set forth as SEQ ID NO: 32-46 of WO2023178339A2 (incorporated herein by reference) arranged in sequence. In certain embodiments, the amino acid sequence of the TNNT2 encoded by the coding sequence for TNNT2 is any one of the synthetic TNNT2 sequences in Table 1 of WO2023178339A2 (incorporated herein by reference). [0155] In certain embodiments, the coding sequence for TNNT2 is operatively linked to a promoter and/or an enhancer element. In certain embodiments, the promoter is a cardiac specific promoter. In some embodiments, the promoter is TNNT2. In some embodiments, the promoter is MHCK9. In some embodiments, the promoter is MHCK7. In some embodiments, the promoter is CBA (Chicken P- Actin). In some embodiments, the promoter is CMV or mini CMV. In some embodiments, the promoter is a Desmin promoter.

[0156] In certain embodiments, the coding sequence for TNNT2 is used / encompassed in AAV viral particles for treating dilated cardiomyopathy or hypertrophic cardiomyopathy. [0157] In certain embodiments, the GOI is a coding sequence for frataxin (FXN), which mutation is linked to Friedreich’s ataxia. More specifically, Friedreich’s ataxia is caused by a mutation in the FXN gene which encodes frataxin. Individuals who inherit two defective copies of the gene will develop this disease. Although rare, Friedreich’s ataxia is the most common form of hereditary ataxia in the United States, affecting about 1 in every 50,000 people. There is currently no approved cure for Friedreich’s ataxia.

[0158] In certain embodiments, the coding sequence for frataxin / FXN is a human coding sequence for frataxin / FXN. In some embodiments, the human frataxin / FXN coding sequence is codon-optimized for expression in human cells, such as human cardiac and/or CNS tissues / cells. In some embodiments, the coding sequence for frataxin / FXN is CpG depleted. In certain embodiments, the coding sequence for frataxin / FXN is any one of the frataxin / FXN constructs in WO2022147575A1 (incorporated herein by reference).

[0159] In certain embodiments, the coding sequence for frataxin / FXN has at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 of WO2022147575A1 (incorporated herein by reference). In certain embodiments, the coding sequence for frataxin / FXN encodes human frataxin comprising the amino acid sequence of SEQ ID NO: 3 of WO2022147575A1 (incorporated herein by reference). In certain embodiments, the coding sequence for frataxin / FXN exhibits enhanced expression of human frataxin as compared to a polynucleotide encoding frataxin but not having the same sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 of WO2022147575A1. In certain embodiments, the coding sequence for frataxin / FXN further comprises an untranslated region (UTR) that imparts regulatory control on expression of the human frataxin encoded by the polynucleotide. The UTR region may comprise a sequence that comprises at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 6 of WO2022147575A1 (incorporated herein by reference). In certain embodiments, the amino acid sequence of the frataxin / FXN, and/or the coding sequence for frataxin / FXN is any one disclosed in WO2022147575A1 (incorporated herein by reference).

[0160] In certain embodiments, the coding sequence for frataxin / FXN further comprises a promoter driving expression of the frataxin. In certain embodiments, the promoter is a cardiac-specific promoter. For example, promoter is a cardiac-restricted promoter selected from cardiac troponin C, cardiac troponin I, and cardiac troponin T (cTnT). In some embodiments, the promoter is a muscle-specific promoter, such as desmin. In some embodiments, the promoter is a CNS-specific promoter, such as the synapsin (SYN) promoter. In certain embodiments, the promoter comprises a cytomegalovirus (CMV) enhancer element functionally coupled to a chicken beta actin (CBA) promoter. In certain embodiments, the CMV enhancer element comprises a sequence that comprises at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 4 of WO2022147575A1 (incorporated herein by reference). In certain embodiments, the CBA promoter comprises a sequence that comprises at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 5 of WO2022147575A1 (incorporated herein by reference).

[0161] In certain embodiments, the coding sequence for frataxin / FXN is used / encompassed in AAV (e.g., AAV9 or AAVrh74) viral particles for treating Friedreich’s ataxia. In certain embodiments, the treatment may further comprise administering an immunomodulatory regimen comprising an antibody directed against a cancer marker (such as CD20) and an additional agent (such as an mTOR pathway inhibitor).

[0162] In certain embodiments, the GOI is a coding sequence for RNA binding motif protein 20 (RBM20). Mutations in or perturbations in the function of RBM20 are known to be causative of DCM (Dilated Cardiomyopathy). RBM20 is a major regulator of heart- specific alternative splicing of the TTN gene, which is found to be most frequently mutated in patients with idiopathic DCM (approximately 20- 25%). The TTN gene has the largest number of exons (364 in humans) and titin, a sarcomeric protein encoded by the TTN gene, is the largest known protein in mammals. In an RBM20 mutant rat strain lacking nearly all the RBM20 exons, the shortest cardiac titin isoform N2B is not expressed. Therefore, RBM20 is a key regulator of TTN pre-mRNA processing in the heart and may cause DCM phenotypes through altered splicing of the RBM20-regulated genes. Missense mutations in a highly conserved RSRSP stretch, within an arginine/serine (RS)-rich region and not in the RNA binding domains are the most frequent disease alleles.

[0163] In certain embodiments, the coding sequence for RBM20 is a human coding sequence for RBM20. In some embodiments, the human RBM20 coding sequence is codon-optimized for expression in human cells, such as human cardiac and/or CNS tissues / cells. In some embodiments, the coding sequence for RBM20 is CpG depleted. In certain embodiments, the coding sequence for RBM20 is any one of the RBM20 constructs in WO2023178337A2 (incorporated herein by reference).

[0164] In certain embodiments, the coding sequence for RBM20 is at least about 85% sequence identity to SEQ ID NO: 5 of WO2023178337A2 (incorporated herein by reference). [0165] In certain embodiments, the coding sequence for RBM20 comprises or is operatively linked to a promoter. In certain embodiments, the promoter comprises a cardiac specific promoter, such as a TNNT2 promoter, a MHCK9 promoter, a CB A (Chicken beta- Actin) promoter or a truncated chicken beta-actin (smCB A) promoter, and combinations thereof. In certain embodiments, the promoter is a CMV or mini-CMV promoter. In some embodiments, the promoter is a Desmin promoter. In some embodiments, the promoter is a muscle creatine kinase (MCK) promoter. In some embodiments, the promoter is a MHCK7 promoter. In some embodiments, the promoter is a MHCK9 promoter. In certain embodiments, the promoter is a constitutive viral promoter, which may include the Herpes Simplex virus (HSV) promoter, the thymidine kinase (TK) promoter, the Rous Sarcoma Virus (RSV) promoter, the Simian Virus 40 (SV40) promoter, the Mouse Mammary Tumor Virus (MMTV) promoter, the Ad El A promoter and the cytomegalovirus (CMV) promoters. In certain embodiments, the promoter comprises a nucleic acid sequence having at least about 85% sequence identity (e.g., identical) to the sequence of SEQ ID NO: 2 or 16 of WO2023178337A2 (incorporated herein by reference).

[0166] In certain embodiments, the coding sequence for RBM20 is used / encompassed in AAV (e.g., AAV9, AAVrh74, or AAVrhlO) viral particles for treating dilated cardiomyopathy. In certain embodiments, the treatment may further comprise administering an immunomodulatory regimen comprising an antibody directed against a cancer marker (such as CD20) and an additional agent (such as an mTOR pathway inhibitor). In certain embodiments, the coding sequence for RBM20 is in an expression cassette that further comprises a silencing element, wherein the coding sequence for RBM20 and the silencing element are each operably linked to a promoter and optionally an enhancer element. The silencing elements can be used to epigenetically silence genes at both the post-transcriptional level or the pre-transcriptional level. In some embodiments, the silencing element is a short hairpin RNA (shRNA). In some embodiments, the silencing element is an siRNA. In a nonlimiting example, epigenetic modulation of gene expression by siRNA silencing elements can result from siRNA mediated modification of chromatin structure or methylation pattern to alter gene expression.

[0167] In certain embodiments, the GOI is a coding sequence for MYBPC3. The MYBPC3 gene provides instructions for making cardiac myosin binding protein C (cardiac MyBP-C). Cardiac MyBP-C is found in cardiac muscle cells, where it plays a role in sarcomere contraction. Muscle contraction depends heavily on the activity of sarcomeres resident in myocytes. Mutations in MYBPC3 are common causes of familial hypertrophic cardiomyopathy, accounting for up to 30% of all cases. Though some individuals have no obvious health effects, all affected individuals possess an increased risk of heart failure and sudden death. MYBPC3 mutations generally present phenotypically shorter or otherwise altered MyBP-C proteins. Reduced MyBPC in the sarcomere disrupts myosin conformations, which may contribute to various cardiac disease states.

[0168] In certain embodiments, the coding sequence for MYBPC3 is a human coding sequence for MYBPC3. In some embodiments, the human MYBPC3 coding sequence is codon-optimized for expression in human cells, such as human cardiac and/or CNS tissues / cells. In some embodiments, the coding sequence for MYBPC3 is CpG depleted. In certain embodiments, the coding sequence for MYBPC3 is any one of the MYBPC3 constructs in WO2023108157A1 (incorporated herein by reference).

[0169] In certain embodiments, the coding sequence for MYBPC3 is at least about 85% sequence identity to the sequence of SEQ ID NO: 9, 29 or 43 of WO2023108157A1 (incorporated herein by reference).

[0170] In certain embodiments, the coding sequence for MYBPC3 comprises or is operatively linked to a promoter. In certain embodiments, the promoter comprises a cardiac specific promoter. In certain embodiments, the promoter is selected from the group consisting of: CMV, mini-CMV, CBA, HSV, TK, RSV, SV40, MMTV, Ad El A, and combinations thereof, and wherein the cardiac specific enhancer or regulatory element comprises an alphaMHC enhancer. In certain embodiments, the MYBPC3 promoter sequence has at least about 85% sequence identity to the sequence of SEQ ID NO: 5, 24, or 39 of WO2023108157A1 (incorporated herein by reference).

[0171] In certain embodiments, the coding sequence for MYBPC3 is used / encompassed in AAV (e.g., AAV9, AAVrh74, or AAVrhlO) viral particles for treating hypertrophic cardiomyopathy.

[0172] In certain embodiments, the GOI is a coding sequence for calsequestrin 2 (CASQ2), the mutation of which is responsible for the primary inherited arrhythmia syndrome CPVT (catecholaminergic polymorphic ventricular tachycardia). CASQ2 is a calcium-binding protein which, through its role in Ca2+ regulation, is integral to excitation-contraction coupling in the heart and in regulating the rate of heart beats. CPVT is a rare, serious and life-threatening disease which primarily manifests in children in the first and second decades of life, with the mean onset of CPVT symptoms being between seven and twelve years. CPVT is an inherited cardiac arrhythmia syndrome characterized by adrenergically induced polymorphic arrythmias in the presence of a normal resting sinus rhythm and a structurally normal heart. It is estimated that the prevalence of CPVT is 1 per 10,000 persons. CPVT manifestations typically involve syncope, cardiac arrest and/or sudden cardiac death. The most common symptoms/signs include syncope (52-100%), cardiac arrest (8-48%), seizurelike events (40%), and hypoxic-ischemic encephalopathy (20%). CPVT is a significant cause of sudden death at a young age and mortality is high (up to 50%). Data from the recent Pediatric and Congenital Electrophysiology Society CPVT registry suggest that three of every four children with CPVT present with life-threatening symptoms, which often occur during resting wakeful activities highlighting the unpredictable nature of CPVT. CPVT may be caused by a gain-of-function mutation in the ryanodine receptor 2 (encoded by the RYR2 gene), which is referred to as CPVT-1, as well as a loss-of-function mutations in the calsequestrin 2, or CASQ2, gene, which is referred to as CPVT-2.

[0173] In certain embodiments, the coding sequence for CASQ2 is a human coding sequence for CASQ2. In some embodiments, the human CASQ2 coding sequence is codon-optimized for expression in human cells, such as human cardiac cells. In some embodiments, the coding sequence for CASQ2 is CpG depleted. In certain embodiments, the coding sequence for CASQ2 is any one of the CASQ2 constructs in US20170360957A1 (incorporated herein by reference).

[0174] In certain embodiments, the coding sequence for CASQ2 encodes a protein that is at least about 85%, 90%, 95% or higher sequence identity to the sequence of SEQ ID NO: 2 of US20170360957A1 (incorporated herein by reference).

[0175] In certain embodiments, the coding sequence for CASQ2 comprises or is operatively linked to a promoter. In certain embodiments, the promoter comprises a cardiac specific promoter, such as a TNNT2 promoter, a MHCK9 promoter, a CB A (Chicken beta- Actin) promoter or a truncated chicken beta-actin (smCB A) promoter, and combinations thereof. In certain embodiments, the promoter is a CMV or mini-CMV promoter. In some embodiments, the promoter is a Desmin promoter. In some embodiments, the promoter is a muscle creatine kinase (MCK) promoter. In some embodiments, the promoter is a MHCK7 promoter. In some embodiments, the promoter is a MHCK9 promoter. In certain embodiments, the promoter is a constitutive viral promoter, which may include the Herpes Simplex virus (HSV) promoter, the thymidine kinase (TK) promoter, the Rous Sarcoma Virus (RSV) promoter, the Simian Virus 40 (SV40) promoter, the Mouse Mammary Tumor Virus (MMTV) promoter, the Ad El A promoter and the cytomegalovirus (CMV) promoters. In certain embodiments, the promoter comprises a nucleic acid sequence having at least about 85% sequence identity (e.g., identical) to the sequence of SEQ ID NO: 2 or 16 of WO2023178337A2 (incorporated herein by reference).

[0176] In certain embodiments, diseases or conditions having a potential to benefit from the rAAV produced by the dual transfection vector of the invention may include ion channel diseases, which are typically marked by muscular weakness, absent muscle tone, or episodic muscle paralysis. They include Andersen-Tawil syndrome, Hyperkalemic periodic paralysis, Hypokalemic periodic paralysis, Myotonia congenita, Becker myotonia, Thomsen myotonia, Paramyotonia congenita, Potassium-aggravated myotonia.

[0177] In certain embodiments, diseases or conditions having a potential to benefit from the rAAV produced by the dual transfection vector of the invention may include mitochondrial diseases, which occur when structures that produce energy for a cell malfunction. Such diseases include: Friedreich’s ataxia (FA), Mitochondrial myopathies, Kearns-Sayre syndrome (KSS), Leigh syndrome (subacute necrotizing encephalomyopathy), Mitochondrial DNA depletion syndromes, Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), Myoclonus epilepsy with ragged red fibers (MERRF), Neuropathy, ataxia and retinitis pigmentosa (NARP), Pearson syndrome, Progressive external opthalmoplegia (PEO).

[0178] In certain embodiments, diseases or conditions having a potential to benefit from the rAAV produced by the dual transfection vector of the invention may include myopathies, which is a disease of muscle in which the muscle fibers do not function properly, resulting in muscular weakness. Myopathies include: Cap myopathies, Centronuclear myopathies, Congenital myopathies with fiber type disproportion, Core myopathies, Central core disease, Multiminicore myopathies, Myosin storage myopathies, Myotubular myopathy, Nemaline myopathies, Distal myopathies, GNE myopathy/Nonaka myopathy/hereditary inclusion-body myopathy (HIBM), Laing distal myopathy, Markesberg-Griggs late-onset distal myopathy, Miyoshi myopathy, Udd myopathy/tibial muscular dystrophy, Vocal cord and pharyngeal distal myopathy, Welander distal myopathy, Endocrine myopathies, Hyperthyroid myopathy, Hypothyroid myopathy, Inflammatory myopathies, Dermatomyositis, Inclusion-body myositis, Polymyositis, Metabolic myopathies, Acid maltase deficiency (AMD, Pompe disease), Carnitine deficiency, Carnitine palmityl transferase deficiency, Debrancher enzyme deficiency (Cori disease, Forbes disease), Lactate dehydrogenase deficiency, Myoadenylate deaminase deficiency, Phosphofructokinase deficiency (Tarui disease), Phosphoglycerate kinase deficiency, Phosphoglycerate mutase deficiency, Phosphorylase deficiency (McArdle disease), Myofibrillar myopathies (MFM), Scapuloperoneal myopathy.

[0179] In certain embodiments, diseases or conditions having a potential to benefit from the rAAV produced by the dual transfection vector of the invention may include neuromuscular junction diseases, which result from the destruction, malfunction or absence of one or more key proteins involved in the transmission of signals between muscles and nerves. Such diseases include: Congenital myasthenic syndromes (CMS), Lambert-Eaton myasthenic syndrome (LEMS), Myasthenia gravis (MG).

[0180] In certain embodiments, diseases or conditions having a potential to benefit from the rAAV produced by the dual transfection vector of the invention may include peripheral nerve diseases, in which the motor and sensory nerves that connect the brain and spinal cord to the rest of the body are affected, causing impaired sensations, movement or other functions.

Such diseases include: Charcot-Marie-Tooth disease (CMT), Giant axonal neuropathy (GAN), muscle wasting in cachexia and aging.

[0181] In certain embodiments, the 5’ and 3’ AAV ITR sequences flanking said GOI are both from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, or AAV-DJ.

[0182] In certain embodiments, the tropism of the AAV includes skeletal muscle (such as AAV1, AAV6, AAV7, AAV8, or AAV9, or a derivative thereof, preferably AAV9 or a derivative thereof, more preferably, SLB-101).

[0183] In certain embodiments, the GOI coding sequence comprises a polyA signal sequence or polyadenylation site.

[0184] In certain embodiments, the polyadenylation site is a bovine growth hormone (bGH) polyadenylation site.

[0185] In certain embodiments, the polyadenylation site or poly(A) signal sequence is from other suitable sources, e.g., synthetic sequences or sequences from other eukaryotic genes or viruses.

[0186] In certain embodiments, the GOI coding sequence is partially or fully codon- optimized for expression in a mammalian host cell. For example, the most 3’ 300-350 nucleotides of the coding sequence may be codon-optimized for expression in the mammalian host cell.

[0187] In certain embodiments, the AAV ITR, the AAV Rep, and the AAV Cap are from the same or different AAVs.

[0188] In certain embodiments, the AAV ITR is AAV2 ITR, the AAV Rep is Rep2 from AAV2, and the AAV Cap is Cap9 from AAV9 or a derivative thereof (such as a spectrum or transcription derivative thereof).

[0189] In certain embodiments, the coding sequence for AAV Rep and Cap proteins is under the transcriptional control of a promoter, such as an AAV p5 promoter, a modified p5 promoter lacking RBE (Rep-Binding Element), an HPV P97 promoter containing a REP binding site and a transcription start site-localized YY1 binding site, or a ubiquitous promoter (such as CMV promoter, EFla promoter, CAG promoter, or CB promoter).

[0190] In certain embodiments, the modified P5 promoter is a recombinant P5 promoter. In some embodiments, the modified P5 promoter comprises a REP binding site and a transcription start site-localized Ying-Yang 1 (YY1) binding site. In some embodiments, the modified P5 promoter comprises an exogenous spacer sequence inserted between the REP binding site and YY1 binding site. In some embodiments, the spacer is 5 nucleotides to 100 nucleotides in length (e.g., about 5 nt).

[0191] In certain embodiments, the modified P5 promoter is a recombinant P5 promoter comprising a REP binding site and a transcription start site-localized YY1 binding site, and wherein said modified P5 promoter comprises an exogenous spacer sequence inserted between the REP binding site and YY1 binding site; optionally, the spacer is 5 nucleotides to 100 nucleotides in length e.g., about 5 nt).

[0192] In certain embodiments, the AAV helper genes comprise adenoviral, herpesviral, or papillomaviral genes useful for AAV packaging (such as El A, E1B, E2A, E4 and VA RNA), optionally operably linked to a promoter as one transcriptional unit.

[0193] In certain embodiments, the helper plasmid comprises a helper virus gene that sufficiently supports AAV packaging.

[0194] In certain embodiments, the helper virus gene comprises: (i) an adenovirus gene, optionally an Adenovirus 5 or Adenovirus 2 gene; and/or (ii) a VA nucleic acid encoding functional VA RNA I and II, an E2A gene encoding a functional E2A protein, and an E4 gene encoding a functional E4 protein.

[0195] In some embodiments, the methods or culture systems disclosed herein comprise using a HSV-1 -based vector or Baculovirus-based vector for AAV production. Rep and Cap Genes

[0196] In certain embodiments, the coding sequence for the AAV Rep and Cap proteins, and the GOI flanked by AAV ITR sequences, are integrated into a single dual transfection (DT) vector of the invention, e.g., a DT plasmid.

[0197] The Rep proteins are important for replication and packaging, while the capsid proteins are assembled to create the protein shell of the AAV, or AAV capsid, which form the outer capsid shell that protects the viral genome, as well as being actively involved in cell binding and internalization. Alternative splicing and alternate initiation codons and promoters result in the generation of four different Rep proteins (Rep78, Rep68, Rep52, and Rep40) from a single open reading frame and the generation of three capsid proteins (VP; VP1/VP2/VP3) from a single open reading frame. While not wishing to be bound by theory, the AAV capsid protein typically comprises a molar ratio of 1 : 1 : 10 of VP1 :VP2:VP3. As used herein, an “AAV serotype” is defined primarily by the AAV capsid. In some instances, the ITRs are also specifically described by the AAV serotype.

[0198] In certain embodiments, the AAV ITR, the AAV Rep, and the AAV Cap are from the same or different AAV serotypes.

[0199] In some embodiments, the AAV ITR, the AAV Rep, and the AAV Cap are from AAVPHP.B (PHP.B), AAVPHP.A (PHP. A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1-35, AAVPHP.B2 (PHP.B2), AAVPHP.B3 (PHP.B3), AAVPHP.N/PHP.B-DGT, AAVPHP.B-EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B- DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B-SNP(3), AAVPHP.B-SNP, AAVPHP.B-QGT, AAVPHP.B-NQT, AAVPHP.B- EGS, AAVPHP.B-SGN, AAVPHP.B-EGT, AAVPHP.B-DST, AAVPHP.B-DST, AAVPHP.B-STP, AAVPHP.B-PQP, AAVPHP.B-SQP, AAVPHP.B-QLP, AAVPHP.B- TMP, AAVPHP.B-TTP, AAVPHP.S/G2A12, AAVG2A15/G2A3 (G2A3), AAVG2B4 (G2B4), AAVG2B5 (G2B5), PHP.S, AAV1, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV5, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9, AAV9 K449R, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV10, AAV11, AAV12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-lb, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAVl-7/rh.48, AAVl-8/rh.49, AAV2- 15/rh.62, AAV2-3/rh.61, AAV2-4/rh.5O, AAV2-5/rh.51, AAV3.1/hu.6, AAV3.1/hu.9, AAV3-9/rh.52, AAV3-1 l/rh.53, AAV4-8/r 11.64, AAV4-9/rh.54, AAV4-19/rh.55, AAV5- 3/rh.57, AAV5-22/rh.58, AAV7.3/hu.7, AAV16.8/hu.lO, AAV16.12/hu.l l, AAV29.3/bb. l, AAV29.5/bb.2, AAV106.1/hu.37, AAV114.3/hu.4O, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54, AAV145.6/hu.55, AAV161.1O/hu.6O, AAV161.6/hu.61, AAV33.12/hu.l7, AAV33.4/hu.l5, AAV33.8/hu. l6, AAV52/hu.l9, AAV52.1/hu.2O, AAV58.2/hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAV A3.7, AAVC1, AAVC2, AAVC5, AAV-DJ, AAV-DJ8, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi. l, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVLK03, AAVH-l/hu.l, AAVH-5/hu.3, AAVLG-10/rh.40, AAVLG-4/rh.38, AAVLG-9/hu.39, AAVN721-8/rh.43, AAVCh.5, AAVCh.5Rl, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5Rl, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu.l, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.l l, AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.l 8, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44Rl, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48Rl, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14/9, AAVhu.t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.l3R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.61, AAVrh.64, AAVrh.64Rl, AAVrh.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVrh8R, AAVrh8R A586R mutant, AAVrh8R R533 A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhEl.l, AAVhErl.5, AAVhER1.14, AAVhErl.8, AAVhErl.16, AAVhErl.18, AAVhErl.35, AAVhErl.7, AAVhErl.36, AAVhEr2.29, AAVhEr2.4, AAVhEr2.16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhER1.23, AAVhEr3.1, AAV2.5T , AAV-PAEC, AAV-LK01, AAV-LK02, AAV-LKO3, AAV-LK04, AAV-LKO5, AAV-LK06, AAV-LK07, AAV-LKO8, AAV-LK09, AAV-LK1O, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC11, AAV-PAEC 12, AAV-2-pre-miRNA-101 , AAV-8h, AAV-8b, AAV-h, AAV-b, AAV SM 10-2 , AAV Shuffle 100-1 , AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100-2, AAV SM 10-1, AAV SM 10-8 , AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.5O, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.l l, AAVhu.53, AAV4-8/rh.64, AAVLG-9/hu.39, AAV54.5/hu.23, AAV54.2/hu.22, AAV54.7/hu.24, AAV54.1/hu.21, AAV54.4R/hu.27, AAV46.2/hu.28, AAV46.6/hu.29, AAV128.1/hu.43, true type AAV (ttAAV), UPENN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7.1, AAV CBr-7.10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-El, AAV CBr- E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6.1, AAV CHt-6.10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-Pl, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-Bl, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-Hl, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd- H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-Fl, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1, AAV CLvl-1, AAV Clvl-10, AAV CLvl-2, AAV CLv-12, AAV CLvl-3, AAV CLv-13, AAV CLvl-4, AAV Civ 1-7, AAV Civ 1-8, AAV Civ 1-9, AAV CLv- 2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-Dl, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-El, AAV CLv-Kl, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-Ml, AAV CLv-Ml 1, AAV CLv-M2, AAV CLv-M5, AAV CLv- M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-Rl, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-11, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8.10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1/HSC1, AAVF11/HSC11, AAVF12/HSC12, AAVF13/HSC13, AAVF14/HSC14, AAVF15/HSC15, AAVF16/HSC16, AAVF17/HSC17, AAVF2/HSC2, AAVF3/HSC3, AAVF4/HSC4, AAVF5/HSC5, AAVF6/HSC6, AAVF7/HSC7, AAVF8/HSC8, and/or AAVF9/HSC9 and variants thereof. [0200] In certain embodiments, the AAV Cap is a derivative of wild-type AAV9. In certain embodiments, the derivative comprises an insertion of a short peptide (e.g., 3, 4, 5, 6, 7, 8, or 9 residues) in-between residues 588 and 589 of the wild-type AAV9 capsid VP1. In certain embodiments, the insertion comprises, consists essentially of, or consists of RGDLGLS into residues 588 and 589 of wild-type AAV9 Capsid VP1.

[0201] In certain embodiments, the AAV Cap has a VP1 capsid sequence is SLB-101 (see SEQ ID NO: 14 and FIG. 6 of WO2021/072197, incorporated herein by reference), in which RGDLGLS is inserted between residues 588 and 589 of wild-type AAV9 VP1:

MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGN GLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVF QAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTES VPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTS TRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNN WGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPF PADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHS QSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVST TVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLI FGKQGTGRDNV DADKVMI TNEEE I KT TNPVATE S YGQVATNHQSAQKGDLGLS QAQTGWVQNQG I LPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSF ITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYL TRNL

[0202] In certain embodiments, the rDNA vector / plasmid of the invention comprises a cap gene promoter. The cap gene promoter may be operably linked to a cap gene. In certain embodiments, the cap gene promoter is a native cap gene promoter.

[0203] The native cap gene (z.e., the cap gene of a wild type AAV) is operably linked to a p40 promoter, a p5 promoter and a pl9 promoter. Thus, in one embodiment, the rDNA vector / plasmid of the invention comprises a cap gene promoter such as an AAV p40 promoter, a p5 promoter, and/or a pl9 promoter. [0204] The native p40 promoter is contained within the native rep gene. In some embodiments, the p40 promoter has a sequence of or at least 95%, at least 98%, or 99% identical to that of AAV2. In some embodiments, the at least one cap gene promoter is comprised in a promoter region comprising a p40 promoter, a p5 promoter and a pl9 promoter.

[0205] The native p5 promoter is upstream of the native rep gene. In some embodiments, the p5 promoter has a sequence of or at least 95%, at least 98%, or at least 99% identical to that of AAV2.

[0206] The pl9 promoter is contained within the native rep gene. In some embodiments, the pl9 promoter has a sequence of or at least 95%, at least 98%, or at least 99% identical to that of AAV2.

[0207] In some embodiments, the AAV ITR, the AAV Rep and the AAV Cap are from the same AAV. In some other embodiments, the AAV ITR, the AAV Rep and the AAV Cap are from different AAVs. For example, the AAV Rep may be from AAV2, and the AAV Cap may be from AAV9 or a derivative thereof such as the aforementioned wt AAV9 derivatives, including SLB-101 (see SEQ ID NO: 14 and FIG. 6 of WO2021/072197, incorporated herein by reference).

[0208] In certain embodiments, the AAV ITR is AAV2 ITR, the AAV Rep is Rep2 from AAV2, and the AAV Cap is Cap9 from AAV9 (or a derivative thereof).

[0209] In certain embodiments, the coding sequence for AAV Rep protein encodes a wildtype Rep 40, Rep 52, Rep 68 and/or Rep 78 of an AAV, such as AAV2. In certain embodiments, these Rep proteins are transcribed from one or more of the rep promoters p5, pl9 and/or p40.

GOI in rAAV and Treatable Diseases

[0210] The system and method of the invention can be used to produce recombinant AAV vectors carrying a GOI flanked by AAV ITR sequences.

[0211] In certain embodiments, the rDNA vector (plasmid) of the invention comprises ITR sequences derived from AAV1, AAV2, AAV4 and/or AAV6. In certain embodiments, the ITR sequences are AAV2 ITR sequences.

[0212] As used herein, “gene of interest” or GOI or goi generally refers to a nucleic acid or polynucleotide sequence, such as a gene, an open reading frame (ORF), or a coding sequence for protein or RNA, such as non-coding RNA that includes siRNA, miRNA, shRNA, antisense RNA or a precursor thereof. However, in certain circumstances or context, the term GOI also loosely refers to a protein (encoded by the GOI), or a disease or indication that can be remedied by the GOI, or a disease or indication can be (but is not necessarily) caused by loss of function of the GOI.

[0213] For example, and merely to illustrate, the gene GALGT2 encodes the protein GalNAc transferase (P-l,4-N-acetylgalactosamine galactosyltransferase), which is an enzyme that transfers a complex sugar molecule onto a few specific proteins, including dystroglycan. Under normal circumstances, GalNAc transferase is found only at the neuromuscular junction (NMJ), where some components of the dystroglycan-associated protein complex are different than elsewhere in muscle. Importantly, at the NMJ, utrophin is present instead of dystrophin. In the mdx mouse model of muscular dystrophy, viral gene transfer of GALGT2 results in expression of GalNAc transferase across the entire muscle membrane, instead of only at the normal expression domain of the NMJ, as well as upregulation of utrophin across the entire muscle fiber. In the mdx mouse, this expression can correct muscle functional deficits to the same degree as does microdystrophin gene expression. Furthermore, overexpression of GALGT2 corrects muscle pathology in mouse models of other muscular dystrophies, including LGMD2A and congenital muscular dystrophy (MDC1 A). Thus, GALGT2 is a GOI for treating muscular dystrophy such as DMD, BMD, LGMD2A and MDC1 A, even though GALGT2 is not necessarily defective per se in the patient in need of treatment.

[0214] In another example, Sarcolipin (SLN) inhibits the sarco/endoplasmic reticulum (SR) Ca²⁺ ATPase (SERCA), and is abnormally elevated in the muscle of DMD patients and animal models such as the mdx mouse model of DMD. Reducing SLN levels by AAV9- mediated RNA interference ameliorates dystrophic pathology in the severe dystrophin/utrophin double mutant (mdx:utr ) mouse model of DMD, including attenuation of muscle pathology and improvement of diaphragm, skeletal muscle and cardiac function. Thus, the coding sequence for SLN RNAi is a GOI that remedies DMD.

[0215] Thus the GOI can be a gene (or protein) that, when expressed, replaces a mutated, damaged, or inactive gene or protein. The GOI can be a gene (or protein) that, when expressed, assists an already functioning process that can benefit from further modification for therapy in a disease, disorder, or dysfunction. The GOI can be a gene (or protein) that, when expressed, assists a dysfunctional process that can benefit from further modification for therapy in a disease, disorder, or dysfunction. A GOI nucleic acid sequence can be DNA, RNA, or synthetic nucleic acid molecule. The GOI can be a protein, an enzyme, a structural protein, a functional protein, or an adaptable protein based on cell function(s). The GOI can provide therapeutic benefit or a treatment modality for a disease, disorder, or dysfunction. [0216] In certain embodiments, the recombinant DNA vector of the invention comprises more than one copy of the GOI. In some embodiments, the plasmid of the invention comprises more than one copy of the GOI. In certain embodiments, the multiple GOI copies are the same. In some other embodiments, the multiple GOI are different.

[0217] In certain other embodiments, the GOI may be a CRISPR/Cas effector enzyme, such as a Class 2, Type II, IV, V, or VI effector enzyme, including CRISPR-Cas9, Cas 12, Cas 13, etc. In certain embodiments, the GOI may be a TALEN, or other genetic based gene editing protein that functions upon intracellular delivery for their intended activity, such as gene editing or gene knockout in a target cell, tissue, or organism / individual.

[0218] In certain embodiments, CRISPR/Cas effector enzyme lacks endonuclease activity (dCas, such as dCas9).

[0219] In certain embodiments, the Cas or dCas is further fused to a base editor, such as a cytosine base editor (CBE, e.g., APOBEC, BE1, BE2, BE3, Target-AID base editor, SaBE3, BE3 PAM variants, BE3 editing window variants, AID, CDA1, APOBEC3G, HF-BE3, BE4, BE4max, and AncBE4max), an adenine base editor (ABE, e.g., ABE7.10, ABE 6.3, ABE7.8, ABE7.9, ABEmax, ABE8e(TadA-8e V106W), ABE8 and variants thereof), or a dual base editor (SPACE, A&C-BEmax).

[0220] Representative (non-limiting) gene of interest (GOI) may include: a gene responsible for / defective in LGMD2E (limb-girdle muscular dystrophy type 2E), LGMD2D (limb-girdle muscular dystrophy type 2D), LGMD2C (limb-girdle muscular dystrophy type 2C), LGMD2B (limb-girdle muscular dystrophy type 2B), LGMD2L (limb-girdle muscular dystrophy type 2L), LGMD2I (limb-girdle muscular dystrophy type 21), or a gene or coding sequence for NAGLU (a-N-acetylglucosaminidase, for Sanfilippo syndrome or mucopolysaccharidosis type IIIB (MPS IIIB)), sulfamidase or SGSH (for mucopolysaccharidosis type IIIA or MPS IIIA), Factor IX, Factor VIII, Myotubularin 1 (MTM1), Survival of Motor Neuron (SMN, for spinal muscular atrophy or SMA), GalNAc transferase GALGT2, calpain-3 (CAPN-3), acid alpha-glucosidase (GAA, for Pompe disease), alpha-galactosidase A or GLA (for Fabry disease), glucocerebrosidase, dystrophin or microdystrophin.

[0221] In certain embodiments, the GOI is a microdystrophin gene.

[0222] In certain embodiments, the microdystrophin gene is any such one described in the following patents: US7,906,l l l; US7,001,761; US7,510,867; US6, 869,777; US8,501,920; US7,892,824; WO2016115543; WO 2023/018854, or US 10, 166,272, each one incorporated herein by reference). In certain embodiments, the GOI is a microdystrophin gene having the nucleotide sequence of SEQ ID NO: 1 of WO 2023/018854 (incorporated herein by reference). In certain embodiments, the microdystrophin gene is capable of being packaged into a rAAV virion, e.g., no more than about 4.7 kb in size.

[0223] In certain embodiments, the microdystrophin gene contains within its coding sequence spectrin-like repeats R16 and R17 that are capable of restoring nitric oxide synthase (nNOS) activity to the sarcolemma (such as those described in US7,892,824).

[0224] In certain embodiments, the microdystrophin gene comprises a coding sequence for the Rl, R16, R17, R23, and R24 spectrin-like repeats (i.e., SRI, SR16, SR17, SR23, and SR24, respectively) of the full-length dystrophin protein, such as one described in PCT/US2016/013733 (incorporated herein by reference). In certain embodiments, the microdystrophin gene does not encode any other spectrin repeats of the full-length dystrophin protein, other than SRI, SR16, SR17, SR23, and SR24.

Production Cell

[0225] In certain embodiments, the production cell is a HEK293 cell (such as an Expi293F cell), or cells derived from HEK293 (e.g., a VP2 cell), a Vero cell, an HUH7 cell, a HepG2 cell, a HeLa cell, an A549 cell, a BHK cell, or an insect cell (such as Sf9).

[0226] One aspect of the invention comprises a production cell comprising the dual recombinant DNA vector (dual transfection vector) as described herein.

[0227] In general, a production cell of the invention is capable or suitable for the production of rAAV. The production cell is typically derived from a eukaryotic cell line, such as a vertebrate cell line, including a mammalian cell line (e.g., a human cell line).

[0228] In certain embodiments, the production cell is a cell selected from the group consisting of a HEK293T cell, a HEK293 cell, a HEK293EBNA cell, a CAP cell, a CAP-T cell, an AGE1.CR cell, a PerC6 cell, a C139 cell, an EB66 cell, a BHK cell, a COS cell, a Vero cell, a Hela cell, and an A549 cell.

[0229] In certain embodiments, the production cell is selected from the group consisting of a HEK293T cell, a HEK293 cell, a HEK293EBNA cell, a CAP cell, a CAP-T cell, an AGE1.CR cell, a PerC6 cell, a C139 cell, and an EB66 cell.

[0230] In certain embodiments, the production cell is selected from the group consisting of a HEK293T cell, a HEK293 cell, and a HEK293EBNA cell.

[0231] In certain embodiments, the production cell is a HEK293T cell or a cell derived from HEK293.

[0232] In certain embodiments, the production cell is HEK293S, HEK293T, HEK293F, HEK293FT, HEK293FTM, HEK293SG, HEK293SGGD, HEK293H, HEK293E, HEK293MSR or HEK293 A.

[0233] In certain embodiments, the production cell is a cell that expresses a functional adenoviral E1A/B protein. For example, the production cell may comprise a chromosome comprising a gene encoding a functional adenoviral El A/B protein.

[0234] In certain embodiments, the production cell is derived from a vertebrate, such as human, monkey, bovine, porcine, equine and other equids, canine, feline, ovine, goat, murine, rat, rabbit, mink, opossum, camel and other cameloids, chicken and other avian, armadillo, frog, or reptile, or derived from an insect cell.

[0235] In certain embodiments, the production cell is a cell line suitable for AAV packaging, such as Expi cells, HEK293 cells (or a derivative thereof such as VP2 cells).

[0236] In certain embodiments, the production cell is a HEK293 cell (such as an Expi293F cell), a HeLa cell, an A549 cell, a BHK cell, or an insect cell (such as Sf9).

[0237] In certain embodiments, the production cell is HEK293 (human embryonic kidney), which can be grown using standard tissue culture media such as DMEM complemented with L-Gln, 5-10% fetal bovine serum (FBS), and 1% penicillin-streptomycin.

[0238] In some embodiments, the HEK293 cells are grown on a solid support, including tissue culture plates, dishes, flasks, and bottles. For growing adherent HEK293 cells, the percentage of FBS can be reduced during rAAV production in order to limit contamination by animal-derived components.

[0239] In some embodiments, the HEK293 cells are adapted to grow in suspension.

[0240] In certain embodiments, the production cell is a Vero cell, such as a Vero75.4 or V75 cell described herein. Such cells may grow on a solid support, including tissue culture plates, dishes, flasks, bottles, and microcarrier that allows the adherent Vero cells to grow in suspension-like conditions.

[0241] In certain embodiments, the production cell is a BHK (baby hamster kidney) cell, such as BHK21 or sBHK27. In certain embodiments, the BHK cells are adapted to grow in serum-free suspension.

[0242] In certain embodiments, the production cell is a HEK293 cell. In certain embodiments, the HEK293 cell is adapted for growth in serum-free media (such as F 17 or Expi293 media) and in suspension, thus is amenable for large scale growth in a bioreactor, for example, Grieger et al. (Mol. Ther. 24:287-297, 2016, incorporated herein by reference). [0243] In certain embodiments, the HEK293 cell is a HEK293T cell which expresses SV40 T antigen (the temperature sensitive allele tsA1609) and the neomycin/geneticin-resistance gene.

[0244] In certain embodiments, for production of rAAV particles, the production cell comprises helper virus proteins useful (e.g., required) for AAV packaging.

[0245] In some embodiments, the coding sequences of the helper virus proteins are introduced into the production cell in a plasmid through transfection. In some other embodiments, the coding sequences of the helper virus proteins are integrated into the production cell genome.

[0246] In certain embodiments, the production cell of the invention may be adapted for use in producing recombinant AAV vectors encoding a GOI, which may be used in gene therapy, such as inthe section entitled “Recombinant AAV Production” below. In such embodiments, one or more rAAV production cell lines may be infected by the vectors disclosed herein, such as vectors or plasmids encoding AAV Rep and Cap proteins.

[0247] In certain embodiments, such producer / production cell line for rAAV production is a HeLa- or A549-derived cell line transfected with the DT vector / plasmid of the invention, optionally containing a drug selection marker.

[0248] In certain embodiments, such producer cell line for rAAV production is a Vero cell.

[0249] In certain embodiments, such producer cell line for rAAV production is a BHK cell.

[0250] In certain embodiments, such producer cell line for rAAV production is a HEK293 cell.

[0251] In certain embodiments, such producer / production cell line for rAAV production comprises a DT vector / vector of the invention comprising the GOI flanked by the AAV ITR sequences. The GOI can be any one of the GOI described herein useful for gene therapy, such as a dystrophin minigene or a microdystrophin gene described in US7, 906,111;

US7,001,761; US7,510,867; US6,869,777; US8,501,920; US7,892,824; W02020/086844; or US10,166,272, WO 2023/018854, or in PCT/US2016/013733 (each incorporated herein by reference).

[0252] For example, PCT/US2016/013733 (WO2016/115543 A2) describes a microdystrophin gene operatively connected to a regulatory cassette, wherein the micro-dystrophin gene encodes a protein comprising: an amino-terminal actin-binding domain; a P- dystroglycan binding domain; and a spectrin-like repeat domain, comprising at least four spectrin-like repeats, wherein two of the at least four spectrin-like repeats comprise a neuronal nitric oxide synthase binding domain. In certain embodiments, the at least four spectrin-like repeats include spectrin-like repeat 1 (SRI), spectrin-like repeat 16 (SRI 6), spectrin-like repeat 17 (SRI 7), and spectrin-like repeat 24 (SR24). In certain embodiments, the protein encoded by the micro-dystrophin gene further comprises at least a portion of a hinge domain, such as at least one of a Hinge 1 domain, a Hinge 2 domain, a Hinge 3 domain, a Hinge 4 domain, and a hinge-like domain. In certain embodiments, the microdystrophin gene comprises, in N- to C-terminal order: a Hinge 1 domain (Hl); a spectrin-like repeat 1 (SRI); a spectrin-like repeat 16 (SR16); a spectrin-like repeat 17 (SR17); a spectrinlike repeat 24 (SR24); and a Hinge 4 domain (H4). In certain embodiments, Hl is directly coupled to SRI. In certain embodiments, SRI is directly coupled to SR16. In certain embodiments, SRI 6 is directly coupled to SRI 7. In certain embodiments, SRI 7 is directly coupled to SR24. In certain embodiments, SR24 is directly coupled to the H4. In certain embodiments, the protein encoded by the micro-dystrophin gene further comprises between SRI and SRI 6, in N- to C-terminal order, a spectrin-like repeat 2 (SR2) and a spectrin-like repeat 3 (SR3). In certain embodiments, SRI is directly coupled to SR2 and SR2 is further coupled to SR3. In certain embodiments, Hl is directly coupled to SRI, SRI is directly coupled to SR16, SR16 is directly coupled to SR17, SR17 is directly coupled to SR23, SR23 is directly coupled to SR24, and SR24 is directly coupled to H4.

[0253] In certain embodiments, the regulatory cassette is selected from the group consisting of a CK8 promoter and a cardiac troponin T (cTnT) promoter. In certain embodiments, the protein encoded by the micro-dystrophin gene has between five spectrin-like repeats and eight spectrin-like repeats. In certain embodiments, the protein encoded by the microdystrophin gene has at least 80% or 90% sequence identity to the amino acid sequence of SEQ ID NO: 4 or 5 in WO2016/115543 A2 (incorporated herein by reference).

[0254] In certain embodiments, the GOI is a microdystrophin gene having the nucleotide sequence of SEQ ID NO: 1 of WO 2023/018854 (incorporated herein by reference).

EXAMPLES

Example 1. Inhibiting Endocytosis in AA V Production Cells Enhances AA V Yield [0255] The instant study demonstrates that, during rAAV manufacture by transient transfection in shake flasks, extracellular rAAV being produced in producer HEK293 cells (or derivative cells) can transduce the HEK293 cells in culture. Meanwhile, adding certain endocytosis inhibitors, such as brefeldin A, bafilomycin Al, or fllipin III a antiviral compound, to HEK293 cells during manufacture resulted in a significant reduction in rAAV transduction of the producer cells and transgene protein expression in the producer cells, and an increase in rAAV yield of up to 3-fold.

[0256] It was discovered that when a representative AAV construct - SLBlOl-pDys (which encodes a microdystrophin (pDys) gene as the gene of interest, and which has a capsid named AAV-SLB101 or “SLB101” for short) or SLBIOI-Luc (which encodes a luciferase as the gene of interest) - was transfected into HEK293 cells and samples were harvested at 72 hours post-transfection in shake flask culture, about 50% of the total manufactured AAV was secreted to cell culture medium at day 4 post transfection. This was also observed in large scale AAV culture - when triple transfection was performed with SLBlOl-luc plasmid into HEK293 cells in a 50 L BioReactor, and when samples were harvested at 72 and 92 hours post transfection, around 30% and 90% of total AAV was detected in medium at 72 hours and 96 hours post transfection, respectively. These results demonstrated secretion of a large portion (e.g., about 20-60%) of the total manufactured AAV viral particles into culture media during AAV manufacture.

[0257] The following experiment demonstrates that the AAV viral particles secreted into the culture media transduce the producer HEK293 cells while the cells are producing AAV. Specifically, HEK293 cells were transiently transfected by plasmids necessary to produce the SLBlOl-pDys viral particles (e.g., via the traditional triple transfection, or via dual transfection using two plasmids, one plasmid encoding the RepCap genes including the SLB101 capsid, as well as the microdystrophin gene of interest, and another plasmid encoding the helper genes required for AAV viral production). Luciferase-encoding virus having the same SLB101 capsid (e.g., SLBIOI-CMV-Luciferase or SLB101-CK8-Luciferase, with the luciferase coding sequence under the transcriptional control of the CMV promoter and the muscle-specific CK8 promoter, respectively) was then added to the culture at 24 hours post transfection. The luciferase-encoding virus served as a surrogate for the HEK293 produced SLBlOl-pDys viral particles since infection of (and the resulting detectable luciferase expression in) the HEK293 cells by the former would be indicative of infection of (and the resulting pDys expression in) the HEK293 cells by the latter.

[0258] A luciferase assay was conducted at 48 hours post transfection (i.e., day 1 post transduction by the Luciferase-encoding virus) and 96 hours post transfection (i.e., day 3 post transduction by the Luciferase-encoding virus) (FIG. 1A). The results (FIG. IB) showed that SLBIOI-CMV-Luciferase AAV transduced HEK293 cells and expressed the encoded luciferase (and by inference, the HEK293 -produced SLBlOl-pDys viral particles transduced the same HEK293 cells and expressed the encoded pDys gene) while the HEK293 cells were making SLBlOl-pDys AAV. As a control, the SLB101-CK8-Luciferase AAV likely also transduced HEK293 cells in suspension while the cells were making SLBlOl-pDys AAV, but only background level of luciferase was expressed likely because the muscle-specific CK8 promoter was not particularly active in the HEK293 cells (see the much lower luciferase activity when using the muscle-specific CK8 promoter versus using the strong CMV promoter to drive luciferase expression in HEK293 cells). These data provide evidence that AAV produced by the producer cells and secreted into the culture media indeed transduced the producer cells that were actively manufacturing additional AAV virus.

[0259] The following experiment directly demonstrated expression of the pDys gene carried by the HEK293 -produced SLBlOl-pDys viral particles in the HEK293 producer cells.

[0260] To measure AAV protein expression in the transfected producer cells, HEK293 cells were transfected with individual or all three plasmids (TT3) required for AAV production, and expression of the encoded pDys and other AAV proteins were determined by Western blot. As expected, the AAV capsid VP proteins (e.g., AAV-SLB101) were detected by the Bl Ab only in cells transfected with the Cap gene - i.e., by all three plasmids (TT3). Rep proteins were detected by the IF 11 Ab only in cells transfected with the Rep gene - i.e., the single RepCap (RC) plasmid or the TT3 plasmids. Significant pDys protein expression was observed in cells transfected with three plasmids, whereas only low levels of pDys expression was detected in cells transfected with the GOI plasmid only (pDys transgene plasmid) or dual transfection of the GOI plasmid with either the RC plasmid or the Helper plasmid. This data was expected because HEK293 cells transfected by all three plasmids were expected to produce large amounts of AAV viral particles carrying the pDys gene, at least some of the viral particle-carried pDys gene would be expressed in the HEK293 cells at least partially due to transduction of the HEK293 cells by the produced viral particles (see above). Meanwhile, HEK293 cells transfected only by the single GOI plasmid carrying the pDys gene were only expected to express low level of pDys due to the lower level of pDys gene and the relative ineffectiveness of the CK8 promoter in the HEK293 cells.

[0261] In a separate experiment, HEK293 cells were transfected with one, two, or three plasmids required for AAV production, and pDys and AAV proteins levels were determined by Western blot at 24 hours and 48 hours post transfection (FIG. 2A). AAV VP proteins were detected in cells transfected with RC plasmids (which comprise the Rep and Cap genes) + Helper plasmids or 3TT plasmids. Rep proteins were detected in cells transfected with RC plasmids in the single, dual, and triple transfection group. Notably, significant levels of pDys protein were observed in cells transfected with three plasmids, whereas low pDys expression was detected in cells transfected with GOI alone or GOI + RC (without helper plasmid). Thus, the Helper plasmid increased Rep and pDys protein expression. Further, high pDys expression in TT3 HEK293 cells was likely due to transduction of HEK293 cells by SLB101- CK8-pDys AAV particles.

[0262] The advantageous effects of brefeldin A were also not unique to the specific HEK293 packaging cells used. Similar results were also obtained using the AAV packaging cells from a commercial AAV production vender - Forge Biologies, which employs its proprietary HEK293 suspension Ignition Cells™ (“Forge cells”) for large scale cGMP AAV manufacture.

[0263] Specifically, the effects of brefeldin A were verified in 50 L scale BioReactor using Forge cells. Transgene expression during AAV production and extracellular virus was detected in 50L BioReactor, as shown in FIG. 2B. AAV was essentially not detected about 48 hrs post transfection, but 35% and 90% of the AAV were detected in supernatant (“super”) at about 72 hrs and 96 hours, respectively.

[0264] Next, a panel of endocytosis/cell entry inhibitors were tested for inhibition of SLBIOI-CMV-Luc transduction in C2C12 cells - a mouse myoblast cell line that can differentiate rapidly to form contractile myotubes and producing characteristic muscle proteins. The C2C12 wild-type cells were seeded in 24-well plates overnight, and differentiated for 4 days, before the endocytosis inhibitor and SLBIOI-CMV-Luc virus was added to each well at 1E4 vg/cell (1 x 10⁴ vector genome/cell). The transduced cells were incubated for 2 more days, before Luminescent Assay (Promega) was used to measure luciferase activity.

[0265] brefeldin A, bafilomycin Al, and fllipin III were found to reduce AAV transduction of manufactural cells (FIG. 3). Complete inhibition of SLB101 transduction was observed with bafilomycin Al and brefeldin A, in that undetectable luciferase activity was measured (FIG. 3). brefeldin A was selected as an exemplary inhibitor of AAV transduction in the subsequent experiments.

[0266] The next experiment also provided direct evidence that crude virus in supernatant can transduce AVV manufacturing cells. HEK293 producer cells were transfected with triple transfection plasmids to generate GFP-encoding viruses. Then one third of the supernatant was collected on day 1, day 2, and day 3 post transfection. The crude GFP virus-containing supernatant was then added to AAV-dy5 manufactural cells (producing SLBlOl-pDys). Twenty-four hours after adding the GFP crude viruses, SLBlOl-pDys manufactural cells were harvest for FACS analysis to measure the percentage of GFP positive cells (FIG. 4A). Treatment with the day 1 (DI) supernatant resulted in about 2% of the manufactural cells being GFP positive. This level significantly increased to about 32% and 34% in manufactural cells that were treated with day 2 (D2) and day 3 (D3) supernatant, respectively. These data demonstrate that crude virus in supernatant can transduce manufactural cells (FIG. 4B). [0267] Next, HEK293 cells were transfected with triple transfection plasmids to generate GFP viruses, and the supernatant was collected on day 1 and day 2 post transfection. The day 1 and day 2 GFP virus-containing supernatant was then added to AAV-dy5 (SLBlOl-pDys) manufactural cells that were treated with DMSO or brefeldin A at 16 hours post transfection. Cells were harvested at 48 hours post transduction (i.e., 48 hours after adding the GFP virus supernatant) for FACS analysis to measure GFP positive cells (FIG. 5A). Because day 1 (DI) GFP virus supernatant had a low amount of virus, low levels of GFP positive cells were detected in the AAV-dy5 manufactural cells, indicating low levels of transduction by the virus. Such levels of GFP positive cells significantly increased to about 59% in manufactural cells treated with day 2 (D2) supernatant (FIG. 5B). Importantly, brefeldin A significantly reduced the levels of GFP positive cells treated with both day 1 (DI) and day 2 (D2) supernatant, indicating that brefeldin A reduced transduction levels in both groups of manufactural cells.

[0268] The following experiment demonstrates that inhibition of transduction of AAV production cells by the produced AAV viruses increased AAV yield. Specifically, brefeldin A was added to the producer HEK293 cells at about 16 hours post transfection, and AAV titer was measured by ddPCR at 72 hours (D3) and 96 hours (D4) post transfection (FIG. 6A). A significant increase in AAV yield was detected in manufactural HEK293 cells treated with brefeldin A in comparison with DMSO control at both time points post transfection (FIG. 6B, top panel). This increase in AAV yield was observed in whole cell lysate (or “total lysate”) and supernatant (or “extracellular”) (FIG. 6B, top panel). Moreover, treatment with brefeldin A reduced pDys expression in cells at 48 hours post transfection (FIG. 6B, lower panel). These data indicate that brefeldin A increased AAV yield by ~2 fold, and reduced pDys expression in HEK293 cells (e.g., self-transduction of manufacturing HEK293 cells by SLBlOl-pDys). Thus, increase in AAV yield can be achieved through reduction of AAV transduction of the producer cells.

[0269] The above finding was repeated and confirmed in a scale-up experiment performed in 500 mL shake flask - brefeldin A can also increase AAV yield in large scale culture (FIG. 7). Specifically, AAV was produced in F17 medium using VP2 cells transfected with the GOI/RepCap plasmid and a suitable helper plasmid i.e., dual transfection), at an GOI/RepCap:Helper (w/w) ratio of 1 : 1, and DNA amount of 0.75 pg/cell. The FECTOVIR®- AAV transfection reagent (Polyplus, NY) was used for large scale AAV manufacturing. Brefeldin A was added at 16 hours or 22 hours post transfection, at a concentration of 10 pM in the culture. Western blot was performed at 2 days post transfection. Increased AAV titer was observed both in whole cell lysate (wcl) (or “total lysate”) and supernatant (z.e., media) (or “extracellular”) treated with brefeldin A at 16 hours (FIG. 7A) as compared with DMSO control. This increase was more significant in supernatant, with a 3.5-fold increase in supernatant, versus a 2.9-fold increase in whole cell lysate (wcl). Similar increases in AAV yield were observed in in cell pellet and supernatant treated with brefeldin A at 22 hours post transfection (FIG. 7B). A significant reduction in pDys expression (resulting from selftransduction of manufacturing HEK293 cells by SLBlOl-pDys) was observed in cells treated with brefeldin A as compared to that treated with DMSO control. These results indicate that brefeldin A significantly increased AAV titer in large scale cultures (e.g., by 2-3 fold). Further, brefeldin A apparently altered AAV viral distribution in the 500 mL scale production, resulting in more (-70% increase from 41% to 68%) particles in supernatant when brefeldin A was added (FIG. 7C). Similar to the observation in in 50 L scale BioReactor AAV culture, where brefeldin A altered AAV distribution, resulting in increased percentages of the virus in supernatant compared to cells. While not wishing to be bound by any particular theory, the increased percentage of extracellular AAV in the media was may have been achieved through inhibition of AAV endocytosis / transduction of the HEK293 packaging cells.

[0270] AAV produced in large scale was purified for measurement of viral yield. AAV from 500 mL shake flask cultures was purified by iodixanol gradient centrifugation at day 4 post transfection, brefeldin A increased total AAV yield in crude lysate by about 2.9-fold relative to DMSO control. AAV was then purified by iodixanol gradient centrifugation for measurement of viral yield. Surprisingly, iodixanol-purified AAV from lysate treated with brefeldin A were 7-fold higher in titer than DMSO control. The purified AAV viral particles were also used to transduce AD cells to test additional quality attributes of the viral particles manufactured in the presence brefeldin A. Results are shown in Table 1.

Table 1. brefeldin A improves AAV quality.

[0271] For example, Table 1 shows higher levels of AAV genomic integrity were observed when the AAV viral particles were produced in the presence of brefeldin A, compared to AAV viral particles produced in the presence of DMSO as control. Both batches of viral particles were produced on a 500 mL scale. Genomic integrity of AAV produced in the presence of brefeldin A was higher than that produced in the presence of DMSO, when purified whole viral particles, raw whole cell lysate (wcl raw), and raw supernatant (super raw) were used for the comparisons.

[0272] In another set of experiments summarized in Table 1, results demonstrate that AAV viral particles produced in the presence of brefeldin A were more potent that those produced in the presence of DMSO, in terms of GOI (e.g., the microdystrophin gene pD5 or dy5) titer and potency.

[0273] Briefly, to carry out the potency assay, C2C12 cells were transduced with AAV-dy5 (SLBlOl-pDys) produced in the presence of brefeldin A or DMSO. Four days post transduction, cells were collected and the amount of pDys expression was measured using an ELISA-based assay. Higher uDys expression was detected in C2C12 cells transduced with AAV generated using brefeldin A as compared to DMSO control.

[0274] The results of the potency assay are also summarized below, showing on average of about 1.6 fold increase in potency when brefeldin A was added instead of DMSO. See Table 2.

Table 2. Potency assay.

[0275] Brefeldin A reduced P5-promoter-associated mispackaging during AAV production as summarized in Table 1. Specifically, mispackaging in 500 mL production was measured by determining the ratio of AAV viral particles having P5 promoter DNA (that was mispackaged into AAV viral particles) and those having the GOI (e.g., pD5 or dy5). The resulting P5/dy5 ratio reflected the extent of mispackaging. GOI titers and P5 : GOI as a percentage / ratio were both measured by ddPCR when the DT plasmid system having a P5 promoter for the RepCap transcriptional cassette was used for viral production.

Mispackaging of P5 promoter sequence, in relation to the correct GOI sequence, was significantly reduced (roughly 40-50% reduction) in the brefeldin A sample as compared to the DMSO control.

[0276] A similar experiment was conducted measuring mispackaging of residue host packaging cell (e.g., HEK293) DNA in relation to the correct GOI sequence (e.g., dy5).

Table 1 shows that residual host HEK293 DNA in the brefeldin A sample was much less than that in the DMSO sampleA time series analysis was performed to show that the optimal timing to add brefeldin A to the HEK293 production cell line was roughly between 22-25 hrs, or about 23.5 hrs post transfection. Specifically, brefeldin A (or DMSO as control) were added to AAV production culture at different time points (2 hrs, 7 hrs, 11.5 hrs, 15 hrs, 18 hrs, 21 hrs, 20 hrs, 22 hrs, 23.5 hrs, 25.5 hrs, and 28.5 hrs) post transfection of the HEK293 cells by dual transfection (DT). Adding brefeldin A to HEK293 cells resulted in increased AAV titer in supernatant at all time points compared to DMSO control (see FIG. 8). Adding brefeldin A between about 22 to 23.5 hours post transfection apparently resulted in the highest yield increase of virus in supernatant, while adding at or after 25.5 hrs post transfection performed worse than adding at 22 hrs. Thus, the optimal timing of brefeldin A addition appeared to be between 16 to 25 hrs (e.g., between 18-24 hrs, between 22-24 hrs, or about 23.5 hrs) post-transfection of the HEK293 production cells, with about 75% to ~3-fold increase in rAAV yield in supernatant compared to DMSO control. Less benefit was realized when brefeldin A was administered during transfection or at much earlier or much later time points.

[0277] It was also found that adding brefeldin A at about 22 hours post-transfection significantly increased AAV yield in large scale production (e.g., 500 mL). Brefeldin A (or DMSO control) was added to transfected HEK239 cells cultured in 500 mL shake flask, and AAV yield was measured on day 3 post transfection in supernatant, cell pellet (whole cell lysate), and virus purified by iodixanol gradient centrifugation. About 1.6-fold increase in AAV titer was observed in whole cell lysate treated with brefeldin A relative to DMSO control. Consistent with prior experiments, this increase was more significant in supernatant of brefeldin A treatment group, by about 2.7-fold more than DMSO control. Brefeldin A treatment increased the purified virus titer by 1.93-fold relative to DMSO control. Further, significant reduction in pDys expression was observed in cells treated with brefeldin A as compared to DMSO control.

[0278] These data together show that addition of brefeldin A at about 22 h post-transfection increases AAV titer at 500 mL scale of HEK293 cells.

[0279] The purified AAV was subjected to quality attribute testing and genomic integrity analysis.

[0280] Based on ELISA essay, brefeldin A treatment resulted in increased AAV capsid levels in supernatant at Day 3 post transfection of the production cell line - by about 3.44-fold higher than that of DMSO control, consistent with the prior observation that brefeldin A treatment led to increased AAV viral particles in the supernatant.

[0281] Table 1 shows that the cell pellet of the DMSO treated group showed slightly higher AAV capsid levels than that of brefeldin A treated group, though this increased AAV capsid level was accompanied by slightly lower GOI (e.g., dy5) titers than that of the brefeldin treatment group. This result indicated that there were more empty capsids in the pellet of the DMSO control group, than in the brefeldin A treatment group. Thus, brefeldin A treatment also reduced the empty AAV viral particles without packaged DNA (e.g., GOI).

[0282] Genomic integrity analysis (Table 1) showed that pellet AAV produced in the presence of brefeldin A also had better genomic integrity than that of DMSO control. [0283] Potency assay analysis of AAV produced in the presence of brefeldin A revealed that such viral particles had higher potency than those produced in the absence of brefeldin A (Tabe 1). Higher pDys expression in C2C12 cells was observed from AAV produced in the presence of brefeldin A vs DMSO control. The potency is the highest in AAV generated from the brefeldin A pellet.

[0284] These data highlight that loss of rAAV to transduction occurs during manufacture, and that addition of brefeldin A inhibits transduction and consequently improves rAAV yield and quality.

[0285] Further experiments identified the optimal dose / concentration for brefeldin A in enhancing AAV production in HEK293 cells, based on a brefeldin A dose-response curve. Dose response analysis showed that between 5-10 pM of brefeldin A significantly improved viral yields in HEK293 culture (FIG. 9).

[0286] The effects of brefeldin A to increase AAV yield were not specific for the SLB-101 capsid. In HKE293 cells transfected with the Rh74_Bag3 capsid using the AAV dual transfection system, brefeldin A was added to HEK293 cells at about 22 hours post transfection of HEK293 at 50 mL shake flask scale (FIG. 10). Supernatant and pellet of the transfected HEK293 cells were harvested at day 3 post transfection for subsequent analysis. About 2-fold (1.9-fold) increase in Rh74-Bag3 viral particle was observed in whole cell lysate of brefeldin A treatment group (the bar labled as “Rh74-BAG3” in FIG. 10) as comparison to DMSO control (the bar labled as “DMSO” in FIG. 10), confirming that brefeldin A increased AAV yield in HEK293 cells when a different AAV capsid was used. Addition of Brefeldin A resulted in increased AAV yield for all AAV serotypes and transgenes tested.

[0287] In addition to brefeldin A, another two endocytosis inhibitors - bafilomycin Al (FIGs. 11 and 12) and filipin III (FIG. 13) - were also shown to significantly enhance AAV yield. Bafilomycin Al inhibits endosome acidification. Adding bafilomycin Al (either 0.08 or 0.16 pM) to 50 mL shake flask HEK293 cell culture transfected by dual plasmids (DT) for production of SLB101-pDys5 resulted in reduced levels of pDys5 expression e.g., due to self-transduction by SLBlOl-pDys of the manufacturing / packaging HEK293 cells) in whole cell lysate (wcl) 48 hrs post transfection (ptf) (see FIG. 11, left panel), and increased yield of AAV in supernatant (see FIG. 11, right panel and FIG. 12). filipin III was shown to increase AAV yield (FIG. 13). These improvements in AAV yield are comparable to results of brefeldin A at 10 pM.

[0288] This provides additional evidence that inhibition of endocytosis in HEK293 production cells improves AAV yield.

[0289] Cell cycle inhibitors that were used in combination with brefeldin A further increases AAV yield (Fig. 14). Nocodazole can arrest cell cycle in G2 and/or M phase, and M344, which is a histone deacetylase inhibitor, can arrest cell cycle in G1 phase. Nocodazole (4.0 pM) and M344 (2.5 pM) were added to culture at 4 hours post transfection and brefeldin A was added at 22 hours post transfection. Nocodazole, M344, and brefeldin A in combination resulted in an increase in AAV yield by 4.6 folds as comparison to DMSO control. Such synergistic increases were unexpected, given that addition of nocodazole and M344 had no significant impact on AAV production, and adding brefeldin A only led to a 1.6-fold increase in AAV yield as comparison to DMSO control. This provides evidence that combination treatment of brefeldin A and cell cycle inhibitors, such as nocodazole and M344, can further improve AAV production from AAV-transfected production cells.

Example 2 Exemplary Procedures and Methods

Transfection and sampling

[0290] About 24 hours prior to transfection, HEK293 cells (Expi293™ working cell bank) were inoculated in 50 mL fresh Freestyle™ F17 media (supplemented with 4 mM Glutamax, 0.1% F68) in a shake flask at 1.5* 10⁶ (1.5E6) cells/mL concentration. On the day of transfection, the target cell density is 2.5E6 - 3.5E6 cells/mL. A plasmid cocktail was prepared by adding plasmids to 15 mL of un-supplemented Freestyle™ F17 media in a tube, mixing, adding PEI (polyethylenimine) transfection reagent, incubating the mixture for 5 minutes, and mix the cocktail again by inverting the tube. Each plasmid cocktail was added to the cells in each shake flask. The cells and plasmid cocktail were gently mixed and then incubated at 110 RPM, 5% CO2 and 37°C. About 2-20 hours post transfection, the plasmids were neutralized with 50 pL of Gibco™ anti-clumping agent.

[0291] Samples were collected from the flasks at 2-4 days post transfection, and the cells were lysed by Triton-X treatment. After centrifugation, the supernatant of the centrifuged lysed cells was collected and stored at -80 °C for ddPCR analysis.

AAV9 production using dual transfection system [0292] Dual plasmid comprising RepCap genes for AAV9 and a microdystrophin variant as the gene-of interest (GOI) were transfected into HEK293 cells with helper plasmid at a Dual: Helper (w/w) ratio of 1 : 1 or 1.5: 1.

Claims

WHAT IS CLAIMED IS

1. A method of promoting adeno-associated virus (AAV) production / enhancing AAV yield in an AAV-production cell line, the method comprising culturing the AAV- production cell line in the presence of an endocytosis inhibitor that inhibits uptake of extracellular AAV by the production cell line.

2. A method of manufacturing adeno-associated virus (AAV), the method comprising culturing an AAV-production cell line in the presence of an endocytosis inhibitor that inhibits uptake of extracellular AAV by the production cell line.

3. The method of claim 1 or 2, wherein the AAV-production cell line is cultured in the presence of brefeldin A, bafilomycin Al, and/or fllipin III.

4. The method of any one of claims 1-3, wherein the AAV-production cell line is cultured in the presence of brefeldin A.

5. The method of any one of claims 1-3, wherein the AAV-production cell line is cultured in the presence of bafilomycin Al.

6. The method of any one of claims 1-5, further comprising culturing the AAV- production cell line in the presence of a cell cycle inhibitor that arrests cell cycle of the AAV-production cell line at G1 and/or G2/M.

7. The method of claim 6, wherein the cell cycle inhibitor that arrests cell cycle of the AAV-production cell line at G1 comprises M344.

8. The method of claim 6, wherein the cell cycle inhibitor that arrests cell cycle of the AAV-production cell line at G2/M comprises nocodazole.

9. The method of any one of claims 1-8, wherein the AAV-production cell line is cultured in the presence of brefeldin A and bafilomycin Al.

10. The method of any one of claims 3-9, wherein the presence of brefeldin A, bafilomycin Al, and/or fl lipin III reduces AAV transduction of the AAV-production cell line (e.g., a reduction of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, 99.5% as compared to a reference control).

11. The method of any one of claims 1-10, wherein the AAV-production cell line is cultured in the presence of about 0.5 pM, 5 pM, 10 pM, or 20 pM brefeldin A.

12. The method of any one of claims 1-11, wherein the AAV-production cell line is cultured in the presence of about 20 nM, 25 nM, 50 nM, 80 nM, 100 nM, 140 nM, 180 nM, 220 nM, 250 nM, or 300 nM bafilomycin Al.

13. The method of any one of claims 1-12, wherein AAV production / yield is increased by at least about 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more in the presence of the endocytosis inhibitor compared to that in the absence of the endocytosis inhibitor.

14. The method of any one of claims 1-13, wherein AAV potency is increased by at least about 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more in the presence of the endocytosis inhibitor compared to that in the absence of the endocytosis inhibitor.

15. The method of any one of claims 1-14, wherein AAV produced in the presence of the endocytosis inhibitor has a level of genomic integrity about at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or higher than that produced in the absence of the endocytosis inhibitor.

16. The method of any one of claims 1-15, wherein the AAV-production cell line is HEK293, or a derivative thereof (such as VP2 from Thermo Fisher).

17. The method of claim 16, wherein said HEK293 is transfected by polynucleotides encoding an AAV Rep, an AAV Cap (e.g., AAV9, AAV8, AAVrh.74, or SLB-101), a gene of interest (e.g, a coding sequence for a functional dystrophin minigene, such as CK8-pDys) flanked by AAV ITR sequences, and helper genes necessary and sufficient for AAV packaging in said AAV production cell line.

18. The method of claim 17, wherein the polynucleotides comprise / consist of one, two, or three plasmids.

19. The method of any one of claims 1-18, wherein production of AAV is in a bioreactor (e.g, a 0.5L bioreactor, IL bioreactor, 2L bioreactor, 5L bioreactor, 10L bioreactor, 20L bioreactor, 50L bioreactor, 100L bioreactor, 500L bioreactor, lOOOL bioreactor, ISOOL bioreactor, or 2000L bioreactor).

20. The method of any one of claims 1-19, further comprising harvesting AAV.

21. The method of claim 20, wherein harvesting AAV is performed at about 48 hrs, 72 hrs, or 96 hrs post transfection of the AAV-production cell line.

22. The method of any one of claims 1-21, wherein the AAV-production cell line is first contacted by brefeldin A and/or bafilomycin Al at about 16-24 hrs (e.g., 16 hrs, 16.5 hrs, 17 hrs, 17.5 hrs, 18 hrs, 18.5 hrs, 19 hrs, 19.5 hrs, 20 hrs, 20.5 hrs, 21 hrs, 21.5 hrs, 22 hrs, 22.5 hrs, 23 hrs, 23.5 hrs, or 24 hrs) post transfecting the AAV-production cell line to initiate AAV production.

23. A culture for AAV production, comprising:

(1) an AAV-production cell line (e.g., HEK293 cells);

(2) an endocytosis inhibitor that inhibits uptake of extracellular AAV by the production cell line; and,

(3) a medium suitable for AAV production.

24. The culture of claim 23, wherein the culture comprises brefeldin A, bafilomycin Al, and/or fllipin III.

25. The culture of claim 23 or 24, wherein the culture comprises brefeldin A.

26. The culture of any one of claims 23-25, wherein the culture comprises bafilomycin Al.

27. The culture of any one of claims 23-26, wherein the culture comprises brefeldin A and bafilomycin Al.

28. The culture of any one of claims 23-27, wherein the AAV-production cell line is cultured in the presence of about 0.5 pM, 5 pM, 10 pM, 20 pM, or 5-10 pM of brefeldin A.

29. The culture of any one of claims 23-28, wherein the AAV-production cell line is cultured in the presence of about 20 nM, 25 nM, 50 nM, 80 nM, 100 nM, 140 nM, 180 nM, 220 nM, 250 nM, or 300 nM bafilomycin Al.

30. The culture of any one of claims 23-29, wherein the AAV-production cell line is cultured in the presence of a cell cycle inhibitor that arrests cell cycle of the AAV- production cell line at G1 and/or G2/M.

31. The culture of claim 30, wherein the cell cycle inhibitor that arrests cell cycle of the AAV-production cell line at G1 comprises M344.

32. The method of claim 30, wherein the cell cycle inhibitor that arrests cell cycle of the AAV-production cell line at G2/M comprises nocodazole.

33. The culture of any one of claims 23-32, wherein production of AAV is in a bioreactor (e.g., a 0.5L bioreactor, IL bioreactor, 2L bioreactor, 5L bioreactor, 10L bioreactor, 20L bioreactor, 50L bioreactor, 100L bioreactor, 500L bioreactor, lOOOL bioreactor, ISOOL bioreactor, or 2000L bioreactor).