Engineered cells for recombinant virus production

Abstract

The present disclosure provides, among other things, systems, engineered cell lines, and methods of making recombinant viral vectors and products. The engineered cell lines comprise one, two, three or more stably integrated genomic components, and also have improved repeatability, stability, growth and scalability compared to previously available systems and cells.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit and priority of U.S. Provisional Patent Application Nos. 63 / 414,890, filed October 10, 2022, and 63 / 478,742, filed January 6, 2023, the disclosures of each of which are incorporated herein by reference in their entirety for all purposes.

[0003] Background

[0004] Viral vectors are commonly used to deliver therapeutic genes to humans. Producing viral vectors is a complex process. For example, a variety of different problems can arise during the design and manufacturing process, including but not limited to the scalability, quality, and potency of recombinant viruses. Producing clinical - grade gene therapies remains a major obstacle to advancing the treatment of many diseases, disorders, and conditions that are otherwise unpreventable, incurable, and / or untreatable. There is a need to improve the process to achieve reproducible, consistent, and scalable manufacturing solutions and to provide commercially viable viral products that can be safely and reliably delivered to patients.

[0005] Summary of the disclosure

[0006] The present disclosure particularly provides techniques for producing virus - producing cells. As is known to those skilled in the art, even though gene therapy technologies have improved rapidly, there are still challenges associated with engineering and manufacturing cells that can reliably, consistently, and accurately achieve virus production, which are suitable for administration to a subject in need of one or more gene therapies in terms of their quality and quantity. The techniques provided herein address certain unmet needs in producing virus - producing cells and overcome certain challenges in improving the engineering and manufacturing of virus - producing cells. These engineered cells and methods for their preparation and use address production challenges and quality (such as safety) issues that are crucial for advancing gene therapies.

[0007] In some aspects, the present disclosure provides a virus - producing cell having an engineered genome that includes: a first nucleic acid comprising a first enhancer sequence and a Rep gene or a functional fragment thereof; a second nucleic acid comprising a second enhancer sequence, a first Kozak sequence, and a Cap gene or a functional fragment thereof, wherein the first Kozak sequence is an engineered Kozak sequence; and a third nucleic acid comprising a gene of interest (GOI).

[0008] In some aspects, the present disclosure provides methods for generating rAAV vectors, the rAAV vectors comprising: a first nucleic acid comprising a first enhancer sequence and a Rep gene or a functional fragment thereof; a second nucleic acid comprising a second enhancer sequence, a first Kozak sequence, and a Cap gene or a functional fragment thereof, wherein the first Kozak sequence is an engineered Kozak sequence; and a third nucleic acid comprising a gene of interest (GOI).

[0009] In some embodiments, the first enhancer sequence has at least 80% identity to any nucleic acid sequence of SEQ ID NOs: 1-10 or any functional fragment or functional derivative thereof.

[0010] In some embodiments, the second enhancer sequence has at least 80% identity to any nucleic acid sequence of SEQ ID NOs: 1-10 or any functional fragment or functional derivative thereof.

[0011] In some embodiments, the first and second enhancer sequences are the same.

[0012] In some embodiments, the first nucleic acid further comprises a second Kozak sequence, wherein the second Kozak sequence is an engineered Kozak sequence.

[0013] In some embodiments, the third nucleic acid comprises a third Kozak sequence, optionally wherein the third Kozak sequence is an engineered Kozak sequence.

[0014] In some embodiments, any one of the first, second, and / or third Kozak sequences promotes translation in insect cells and / or mammalian cells.

[0015] In some embodiments, the first and / or second Kozak sequences preferably promote translation in insect cells.

[0016] In some embodiments, the third Kozak sequence preferably promotes translation in mammalian cells.

[0017] In some embodiments, the cells are cloned.

[0018] In some embodiments, the engineered Kozak sequence of the second nucleic acid comprises a sequence having at least 80% identity to any sequence of SEQ ID NOs: 11-191 or any functional fragment or functional derivative thereof.

[0019] In some embodiments, the Kozak sequence (which is optionally engineered) of the first nucleic acid and the engineered Kozak sequence of the second nucleic acid comprise the same nucleic acid sequence.

[0020] In some embodiments, the cell is free of rhabdovirus.

[0021] In some embodiments, the GOI is flanked by a first inverted terminal repeat (ITR) sequence and a second ITR sequence.

[0022] In some embodiments, each of the first ITR sequence and the second ITR sequence has a different nucleic acid sequence.

[0023] In some embodiments, the first ITR and the second ITR are derived from a viral genome, and wherein the first ITR flanks at its 5' end and the second ITR flanks at its 3' end a total of about 500 nucleotides or less of the viral genome.

[0024] In some embodiments, the Cap gene comprises a sequence having at least 80% sequence identity to any of the nucleic acid sequences of SEQ ID NOs: 192 - 203 or any functional fragment or functional derivative thereof.

[0025] In some embodiments, the Cap gene encodes an amino acid sequence having at least about 80% sequence identity to any of the amino acid sequences of SEQ ID NOs: 204 - 209 or any functional fragment or functional derivative thereof.

[0026] In some embodiments, the first nucleic acid further comprises a promoter and optionally further comprises one or more of an intron, a microRNA, a linker, a splicing element, a polyA signal, or any combination thereof; the second nucleic acid further comprises a promoter and optionally further comprises one or more of an intron, a microRNA, a linker, a splicing element, a polyA signal, or any combination thereof; and / or the third nucleic acid further comprises a promoter and optionally further comprises one or more of an intron, a microRNA, a linker, a splicing element, a polyA signal, or any combination thereof.

[0027] In some embodiments, the promoter is selected from: (i) a constitutive promoter; (ii) an inducible promoter; (iii) a minimal promoter; and (iv) a functional derivative of any one of (i), (ii), or (iii). In some embodiments, the promoter is selected from: CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p10, p19, p40, synapsin, CaMKII, GRK1, polH, EM7, OpIE1, and any functional fragment and / or functional derivative thereof. In some embodiments, the Rep gene is derived from adeno-associated virus (AAV). In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6TM, AAV7, AAV7TM, AAV8, AAV8TM, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional fragment and / or functional derivative thereof.

[0028] In some embodiments, the Cap gene is derived from AAV. In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6TM, AAV7, AAV7TM, AAV8, AAV8TM, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivatives thereof.

[0029] In some embodiments, the first nucleic acid further comprises a first antibiotic resistance gene; the second nucleic acid further comprises a second antibiotic resistance gene; and / or the third nucleic acid further comprises a third antibiotic resistance gene. In some embodiments, each of the first and second nucleic acids comprises an antibiotic resistance gene or a functional fragment or derivative thereof. In some embodiments, each of the first and second antibiotic resistance genes comprises the same antibiotic resistance gene. In some embodiments, the third nucleic acid does not comprise an antibiotic resistance gene. In some embodiments, each of the first, second, and / or third antibiotic resistance genes is selected from: genes encoding aminoglycosides, β-lactams, macrolides, tetracyclines, or any functional fragments and / or functional derivatives thereof. In some embodiments, each of the first, second, and / or third antibiotic resistance genes is selected from: genes encoding kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, blasticidin, bleomycin, erythromycin, polymyxin B, puromycin, tetracycline, chloramphenicol, neomycin, zeocin, or any functional fragments and / or functional derivatives thereof.

[0030] In some embodiments, the first nucleic acid further comprises a first origin of replication; the second nucleic acid further comprises a second origin of replication; and / or the third nucleic acid further comprises a third origin of replication. In some embodiments, the first, second, and / or third origin of replication is selected from: pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, and any functional fragments and / or functional derivatives thereof.

[0031] In some embodiments, the first nucleic acid comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 210 or any functional fragment or functional derivative thereof. In some embodiments, the second nucleic acid comprises a nucleic acid sequence having at least 80% sequence identity with SEQ ID NO: 211-216 or any functional fragment or functional derivative thereof.

[0032] In some embodiments, the cell is a eukaryotic cell, mammalian cell, immortalized cell, insect cell, yeast cell, plant cell, fungal cell, or prokaryotic cell. In some embodiments, the cell is an A549 cell, HEK-293 cell, HEK-293T cell, BHK cell, CHO cell, HeLa cell, MRC5 cell, Sf9 cell, Sf2 cell, Sf21 cell, HighFive TM cell, Cos-1 cell, Cos-7 cell, Vero cell, BSC 1 cell, BSC 40 cell, BMT 10 cell, WI38 cell, Saos cell, C2C12 cell, L cell, HT1080 cell, HepG2 cell, Huh7 cell, K562 cell, primary cell, or any derivative thereof. In some embodiments, the cell is an Sf9 cell.

[0033] In some embodiments, the first nucleic acid, the second nucleic acid, and the third nucleic acid remain stably integrated into the genome after at least 5 passages. In some embodiments, the cell is capable of growing to at least about 1x10 7 cells / mL after at least about 24 hours.

[0034] In some embodiments, the cell is infected with a virus. In some embodiments, the virus is selected from: alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, circovirus, bocavirus, vaccinia virus, and retrovirus. In some embodiments, the virus is a wild-type virus. In some embodiments, the virus does not contain the nucleic acids required for AAV packaging.

[0035] In some embodiments, the virus has been engineered to: remove one or more endogenous genes or functions; and prevent one or more endogenous genes from producing functional gene products. In some embodiments, infection induces the expression of one or more of a first nucleic acid, a second nucleic acid, a third nucleic acid, or any combination thereof. In some embodiments, infection induces the cell to produce a recombinant virus. In some embodiments, the recombinant virus is selected from alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpes virus, poxvirus, circovirus, bocavirus, vaccinia virus, or retrovirus. In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6TM, AAV7, AAV7TM, AAV8, AAV8TM, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivatives thereof.

[0036] In some embodiments, the recombinant virus comprises a recombinant AAV (rAAV) vector. In some embodiments, the rAAV vector comprises less than 5%, 4%, 3%, 2%, or 1% contaminants from one or more non-AAV components and / or the GOI. In some embodiments, the contaminants are from alphavirus, parvovirus, baculovirus, dengue virus, lentivirus, poxvirus, circovirus, bocavirus, vaccinia virus, herpes virus, or retrovirus. In some embodiments, the contaminants are from baculovirus. In some embodiments, the contaminants are not from rhabdovirus.

[0037] In some embodiments, a plurality of cells infected according to the present disclosure produce 1×10 9 vg / L to 1×10 15Multiple cells per vg / L. In some embodiments, multiple cells infected according to the present disclosure produce 1×10 15 vg / L or more of multiple cells.

[0038] In some aspects, the present disclosure provides a recombinant virus produced by the methods provided herein. In some aspects, the present disclosure provides a composition comprising multiple virus particles produced by infecting cells as provided herein with a virus. In some embodiments, the cells are insect cells. In some embodiments, the insect cells are Sf9 cells. In some embodiments, the virus is a baculovirus. In some embodiments, the multiple virus particles comprise 1×10 9 vg / L to 1×10 15 vg / L. In some embodiments, the multiple virus particles comprise 1×10 15 vg / L or more.

[0039] In some aspects, the present disclosure provides a method of infecting cells in a subject in need thereof, the method comprising administering to the subject a composition as provided according to the present disclosure.

[0040] In some aspects, the present disclosure provides a method of treating a subject suffering from a disease, disorder or condition associated with a dysfunctional gene of interest (GOI), the method comprising administering a composition as provided herein to produce a functional gene product of the GOI and treat the disease.

[0041] In some aspects, the present disclosure provides a system for producing a recombinant virus, comprising: virus-producing cells as provided herein; and a virus for infecting the virus-producing cells, which induces the production of the recombinant virus when the virus-producing cells are infected with the virus.

[0042] In some embodiments, the cells are infected with a virus. In some embodiments, the virus is selected from: alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpes virus, poxvirus, circovirus, bocavirus, vaccinia virus, and retrovirus. In some embodiments, the virus is a wild-type virus. In some embodiments, the virus does not contain the nucleic acids required for AAV packaging. In some embodiments, the virus has been engineered to remove one or more endogenous genes or functions; and prevent one or more endogenous genes from producing a functional gene product.

[0043] In some embodiments, infection induces the expression of one or more of a first nucleic acid, a second nucleic acid, a third nucleic acid, or any combination thereof. In some embodiments, infection induces a cell to produce a recombinant virus. In some embodiments, the recombinant virus is selected from alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, circovirus, bocavirus, vaccinia virus, or retrovirus. In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6TM, AAV7, AAV7TM, AAV8, AAV8TM, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivatives thereof.

[0044] In some embodiments, the recombinant virus comprises a recombinant AAV (rAAV) vector. In some embodiments, the rAAV vector comprises less than 5%, 4%, 3%, 2%, or 1% of contaminants from one or more non-AAV components and / or GOI. In some embodiments, the contaminants are from alphavirus, parvovirus, baculovirus, dengue virus, lentivirus, poxvirus, circovirus, bocavirus, vaccinia virus, herpesvirus, or retrovirus. In some embodiments, the contaminants are from baculovirus. In some embodiments, the contaminants are not from rhabdovirus.

[0045] In some embodiments, the system produces 1x10 9 vg / L to 1x10 15 vg / L of recombinant virus. In some embodiments, the system produces 1x10 15 vg / L or more of recombinant virus.

[0046] In some aspects, the present disclosure provides a virus-producing cell having an engineered genome that includes: (a) a first nucleic acid that includes a first enhancer sequence and a Rep gene or a functional fragment thereof; (b) a second nucleic acid that includes a second enhancer sequence and a Cap gene or a functional fragment thereof; and (c) a third nucleic acid that includes a gene of interest (GOI). In some embodiments, the first enhancer sequence has at least 80% (e.g., 85%, 90%, 95%, 99%, or 100%) identity to any of the nucleic acid sequences of SEQ ID NOs: 1-10. In some embodiments, the second enhancer sequence has at least 80% (e.g., 85%, 90%, 95%, 99%, or 100%) identity to the nucleic acid sequences of SEQ ID NOs: 1-10. In some embodiments, the first and second enhancer sequences are the same. In some embodiments, the first nucleic acid further includes a first engineered Kozak sequence. In some embodiments, the first engineered Kozak sequence has at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any of the nucleic acid sequences of SEQ ID NOs: 11-191.

[0047] In some embodiments, the second nucleic acid further includes a second engineered Kozak sequence. In some embodiments, the second engineered Kozak sequence has at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to any of the nucleic acid sequences of SEQ ID NOs: 11-191. In some embodiments, the first engineered Kozak sequence and the second engineered Kozak sequence are the same.

[0048] In some embodiments, the GOI is flanked by a first inverted terminal repeat (ITR) sequence and a second ITR sequence. In some embodiments, the first ITR sequence and the second ITR sequence are the same. In some embodiments, the Cap gene includes a sequence having at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any of the nucleic acid sequences of SEQ ID NOs: 192-203. In some embodiments, the Cap gene encodes an amino acid sequence having at least about 80% (e.g., 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any of the amino acid sequences of SEQ ID NOs: 204-209.

[0049] In some embodiments, the first nucleic acid further comprises a promoter (and optionally further comprises one or more of an intron, a microRNA, a linker, a splicing element, a polyA signal, or any combination thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini-promoter, or any functional fragment and / or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p10, p19, p40, synapsin, CaMKII, GRK1, polH, EM7, OpIE1, and any functional fragment and / or functional derivative thereof.

[0050] In some embodiments, the second nucleic acid further comprises a promoter (and optionally further comprises one or more of an intron, a microRNA, a linker, a splicing element, a polyA signal, or a combination thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini-promoter, or a functional fragment and / or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p10, p19, p40, synapsin, CaMKII, GRK1, polH, EM7, OpIE1, and any functional fragment and / or functional derivative thereof. In some embodiments, the third nucleic acid further comprises a promoter (and optionally further comprises one or more of an intron, a microRNA, a linker, a splicing element, a polyA signal, or any combination thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini-promoter, or any functional fragment and / or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p10, p19, p40, synapsin, CaMKII, GRK1, polH, EM7, OpIE1, and any functional fragment and / or functional derivative thereof. In some embodiments, the Rep gene is derived from a parvovirus. In some embodiments, the parvovirus is an adeno-associated virus (AAV).In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivatives thereof. In some embodiments, the Cap gene is derived from parvovirus. In some embodiments, the parvovirus is AAV. In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivatives thereof.

[0051] In some embodiments, the first nucleic acid further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, a β-lactam, a macrolide, a tetracycline, or any derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, blasticidin, bleomycin, erythromycin, puromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, bleomycin, or any functional fragment and / or functional derivative thereof.

[0052] In some embodiments, the second nucleic acid further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, a β-lactam, a macrolide, a tetracycline, or any functional fragment and / or functional derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, blasticidin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, bleomycin, or any functional fragment and / or functional derivative thereof.

[0053] In some embodiments, the third nucleic acid further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, a β-lactam, a macrolide, a tetracycline, or any functional fragment and / or functional derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, blasticidin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, bleomycin, or any functional fragment and / or functional derivative thereof.

[0054] In some embodiments, the first nucleic acid further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, and any functional fragment and / or functional derivative thereof. In some embodiments, the first nucleic acid comprises a sequence having at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 210.

[0055] In some embodiments, the second nucleic acid further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, and any functional fragments and / or functional derivatives thereof. In some embodiments, the second nucleic acid comprises a sequence having at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any of the nucleic acid sequences of SEQ ID NOs: 211-216.

[0056] In some embodiments, the third nucleic acid further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, and any functional fragments and / or functional derivatives thereof.

[0057] In some embodiments, the cell is a eukaryotic cell, mammalian cell, immortalized cell, insect cell, yeast cell, plant cell, fungal cell, or prokaryotic cell. In some embodiments, the cell is an A549 cell, HEK-293 cell, HEK-293T cell, BHK cell, CHO cell, HeLa cell, MRC5 cell, Sf9 cell, Sf2 cell, Sf21 cell, HighFive TM cell, Cos-1 cell, Cos-7 cell, Vero cell, BSC1 cell, BSC 40 cell, BMT 10 cell, WI38 cell, Saos cell, C2C12 cell, L cell, HT1080 cell, HepG2 cell, Huh7 cell, K562 cell, primary cell, or any derivative thereof.

[0058] In some embodiments, the cell is an Sf9 cell.

[0059] In some embodiments, the first nucleic acid, the second nucleic acid, and the third nucleic acid remain stably integrated into the genome after at least 5 (e.g., 10, 20, 40) passages. In some embodiments, the cell is capable of growing to at least about 1x10 7cells / mL. In some embodiments, the cells are infected with a virus. In some embodiments, the virus is selected from: alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpes virus, poxvirus, circovirus, bocavirus, vaccinia virus, and retrovirus. In some embodiments, the virus is a wild-type virus. In some embodiments, the infection induces the expression of one or more of a first nucleic acid, a second nucleic acid, a third nucleic acid, or any combination thereof. In some embodiments, the infection induces the cells to produce a recombinant virus. In some embodiments, the recombinant virus is selected from: alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpes virus, poxvirus, circovirus, bocavirus, vaccinia virus, or retrovirus. In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivatives thereof. In some embodiments, the recombinant virus comprises a recombinant adeno-associated virus (rAAV) vector. In some embodiments, the rAAV vector comprises less than 5%, 4%, 3%, 2%, or 1% of non-AAV components and contaminants of the GOI. In some embodiments, the contaminants are from alphavirus, parvovirus, baculovirus, dengue virus, lentivirus, poxvirus, circovirus, bocavirus, vaccinia virus, herpes virus, or retrovirus. In some embodiments, the contaminants are from baculovirus.

[0060] The present disclosure provides methods for generating rAAV vectors, including: (a) providing a virus-producing cell having an engineered genome, wherein the cell comprises: (i) a first nucleic acid comprising a first enhancer sequence and a Rep gene or a functional fragment thereof; (ii) a second nucleic acid comprising a second enhancer sequence and a Cap gene or a functional fragment thereof; and (iii) a third nucleic acid comprising a GOI; (b) contacting the cell of step (a) with a virus; and (c) culturing the cell after step (b) to produce a recombinant virus comprising the rAAV vector. In some embodiments, the first enhancer sequence has at least 80% (e.g., 85%, 90%, 95%, 99% or 100%) identity to any of the nucleic acid sequences of SEQ ID NOs: 1-10. In some embodiments, the second enhancer sequence has at least 80% (e.g., 85%, 90%, 95%, 99% or 100%) identity to any of the nucleic acid sequences of SEQ ID NOs: 1-10. In some embodiments, the first and second enhancer sequences are the same. In some embodiments, the first nucleic acid further comprises a first engineered Kozak sequence. In some embodiments, the first engineered Kozak sequence has at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identity to any of the nucleic acid sequences of SEQ ID NOs: 11-191. In some embodiments, the second nucleic acid further comprises a second engineered Kozak sequence. In some embodiments, the second engineered Kozak sequence has at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identity to any of the nucleic acid sequences of SEQ ID NOs: 11-191. In some embodiments, the first and second engineered Kozak sequences are the same. In some embodiments, the GOI is flanked by a first ITR sequence and a second ITR sequence. In some embodiments, the first ITR sequence and the second ITR sequence are the same. In some embodiments, the Cap gene comprises a sequence having at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any of the nucleic acid sequences of SEQ ID NOs: 192-203. In some embodiments, the Cap gene encodes an amino acid sequence having at least about 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any of the amino acid sequences of SEQ ID NOs: 204-209. In some embodiments, the first nucleic acid further comprises a promoter (and optionally further comprises one or more of an intron, a microRNA, a linker, a splicing element, a polyA signal, or any combination thereof).In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini-promoter, or any functional fragment and / or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p10, p19, p40, synapsin, CaMKII, GRK1, polH, EM7, OpIE1, and any functional fragment and / or functional derivative thereof. In some embodiments, the second nucleic acid further comprises a promoter (and optionally further comprises one or more of an intron, a microRNA, a linker, a splicing element, a polyA signal, or any combination thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini-promoter, or any functional fragment and / or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p10, p19, p40, synapsin, CaMKII, GRK1, polH, EM7, OpIE1, and any functional fragment and / or functional derivative thereof. In some embodiments, the third nucleic acid further comprises a promoter (and optionally further comprises one or more of an intron, a microRNA, a linker, a splicing element, a polyA signal, or any combination thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini-promoter, or any functional fragment and / or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p10, p19, p40, synapsin, CaMKII, GRK1, polH, EM7, OpIE1, and any functional fragment and / or functional derivative thereof. In some embodiments, the Rep gene is derived from parvovirus. In some embodiments, the parvovirus is AAV (such as wild-type AAV; such as rAAV).In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivatives thereof. In some embodiments, the Cap gene is derived from parvovirus. In some embodiments, the parvovirus is AAV. In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivatives thereof.In some embodiments, the first nucleic acid further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, β-lactam, macrolide, tetracycline, or any functional fragment and / or functional derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, blasticidin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, bleomycin, or any functional fragment and / or functional derivative thereof. In some embodiments, the second nucleic acid further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, β-lactam, macrolide, tetracycline, or any functional fragment and / or functional derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, blasticidin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, bleomycin, or any functional fragment and / or functional derivative thereof. In some embodiments, the third nucleic acid further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, β-lactam, macrolide, tetracycline, or any functional fragment and / or functional derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, blasticidin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, bleomycin, or any functional fragment and / or functional derivative thereof. In some embodiments, the first nucleic acid further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, and any functional fragment and / or functional derivative thereof. In some embodiments, the second nucleic acid further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, and any functional fragment and / or functional derivative thereof. In some embodiments, the third nucleic acid further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, and any functional fragment and / or functional derivative thereof. In some embodiments, the first nucleic acid comprises a sequence having at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 210.In some embodiments, the second nucleic acid comprises a sequence having at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any of the nucleic acid sequences of SEQ ID NOs: 211-216. In some embodiments, the cell is a eukaryotic cell, mammalian cell, immortalized cell, insect cell, yeast cell, plant cell, fungal cell or prokaryotic cell. In some embodiments, the cell is an A549 cell, HEK-293 cell, HEK-293T cell, BHK cell, CHO cell, HeLa cell, MRC5 cell, Sf9 cell, Sf2 cell, Sf21 cell, High Five. TM cell, Cos-1 cell, Cos-7 cell, Vero cell, BSC1 cell, BSC40 cell, BMT10 cell, WI38 cell, Saos cell, C2C12 cell, L cell, HT1080 cell, HepG2 cell, Huh7 cell, K562 cell, primary cell or a derivative thereof. In some embodiments, the cell is an Sf9 cell. In some embodiments, the first nucleic acid, the second nucleic acid and the third nucleic acid remain stably integrated in the genome after at least 5 (e.g., 10, 20, 40) passages. In some embodiments, the cell is capable of growing to at least about 1x10 7cells / mL. In some embodiments, the cells are infected with a virus. In some embodiments, the virus is selected from: alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpes virus, poxvirus, circovirus, bocavirus, vaccinia virus, and retrovirus. In some embodiments, the virus is a wild-type virus. In some embodiments, the infection induces the expression of one or more of a first nucleic acid, a second nucleic acid, a third nucleic acid, or any combination thereof. In some embodiments, the infection induces the cells to produce a recombinant virus. In some embodiments, the recombinant virus is selected from alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpes virus, poxvirus, circovirus, bocavirus, vaccinia virus, or retrovirus. In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivatives thereof. In some embodiments, the recombinant virus comprises an rAAV vector. In some embodiments, the rAAV vector comprises less than 5%, 4%, 3%, 2%, or 1% non-AAV components and contaminants of the GOI. In some embodiments, the contaminants are from alphavirus, parvovirus, baculovirus, dengue virus, lentivirus, poxvirus, circovirus, bocavirus, vaccinia virus, herpes virus, or retrovirus. In some embodiments, the contaminants are from baculovirus.

[0061] In some embodiments, the present disclosure provides a recombinant virus produced by the methods of the present disclosure.

[0062] In some embodiments, the present disclosure provides a system for generating a recombinant virus, comprising: (a) a virus-producing cell having an engineered genome comprising: (i) a first nucleic acid comprising a first enhancer sequence and a Rep gene or a functional fragment thereof; (ii) a second nucleic acid comprising a second enhancer sequence and a Cap gene or a functional fragment thereof; and (iii) a third nucleic acid comprising a GOI; and (b) a virus for infecting the virus-producing cell, which induces the production of the recombinant virus when the virus-producing cell is infected with the virus.

[0063] In some embodiments, the first enhancer sequence has at least 80% (e.g., 85%, 90%, 95%, 99% or 100%) identity to any of the nucleic acid sequences of SEQ ID NOs: 1-10. In some embodiments, the second enhancer sequence has at least 80% (e.g., 85%, 90%, 95%, 99% or 100%) identity to the nucleic acid sequences of SEQ ID NOs: 1-10. In some embodiments, the first and second enhancer sequences are the same.

[0064] In some embodiments, the first nucleic acid further comprises a first engineered Kozak sequence. In some embodiments, the first engineered Kozak sequence has at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identity to any of the nucleic acid sequences of SEQ ID NOs: 2-42. In some embodiments, the second nucleic acid further comprises a second engineered Kozak sequence. In some embodiments, the second engineered Kozak sequence has at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identity to any of the nucleic acid sequences of SEQ ID NOs: 2-42. In some embodiments, the first engineered Kozak sequence and the second engineered Kozak sequence are the same.

[0065] In some embodiments, the GOI is flanked by a first ITR sequence and a second ITR sequence. In some embodiments, the first ITR sequence and the second ITR sequence are the same. In some embodiments, the Cap gene comprises a sequence having at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any of the nucleic acid sequences of SEQ ID NOs: 192-203. In some embodiments, the Cap gene encodes an amino acid sequence having at least about 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any of the amino acid sequences of SEQ ID NOs: 204-209.

[0066] In some embodiments, the first nucleic acid further comprises a promoter (and optionally further comprises one or more of an intron, a microRNA, a linker, a splicing element, a polyA signal, or any combination thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini-promoter, or any functional fragment and / or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p10, p19, p40, synapsin, CaMKII, GRK1, polH, EM7, OpIE1, and any functional fragment and / or functional derivative thereof. In some embodiments, the second nucleic acid further comprises a promoter (and optionally further comprises one or more of an intron, a microRNA, a linker, a splicing element, a polyA signal, or a combination thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini-promoter, or any functional fragment and / or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p10, p19, p40, synapsin, CaMKII, GRK1, polH, EM7, OpIE1, and any functional fragment and / or functional derivative thereof. In some embodiments, the third nucleic acid further comprises a promoter (and optionally further comprises one or more of an intron, a microRNA, a linker, a splicing element, a polyA signal, or any combination thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini-promoter, or any functional fragment and / or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p10, p19, p40, synapsin, CaMKII, GRK1, polH, EM7, OpIE1, and any functional fragment and / or functional derivative thereof. In some embodiments, the Rep gene is derived from a parvovirus. In some embodiments, the parvovirus is AAV.In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivatives thereof. In some embodiments, the Cap gene is derived from parvovirus. In some embodiments, the parvovirus is AAV. In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivatives thereof.In some embodiments, the first nucleic acid further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, a β-lactam, a macrolide, a tetracycline, or any functional fragment and / or functional derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, blasticidin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, bleomycin, or any functional fragment and / or functional derivative thereof. In some embodiments, the second nucleic acid further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, a β-lactam, a macrolide, a tetracycline, or any derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, bleomycin, or any functional fragment and / or functional derivative thereof. In some embodiments, the third nucleic acid further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, a β-lactam, a macrolide, a tetracycline, or any functional fragment and / or functional derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, blasticidin, bleomycin, erythromycin, puromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, bleomycin, or any functional fragment and / or functional derivative thereof.

[0067] In some embodiments, the first nucleic acid further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, and any functional fragments and / or functional derivatives thereof. In some embodiments, the second nucleic acid further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, and any functional fragments and / or functional derivatives thereof. In some embodiments, the third nucleic acid further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, and any functional fragments and / or functional derivatives thereof. In some embodiments, the first nucleic acid comprises a sequence having at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with the nucleic acid sequence of SEQ ID NO: 210. In some embodiments, the second nucleic acid comprises a sequence having at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity with any of the nucleic acid sequences of SEQ ID NOs: 211-216. In some embodiments, the cell is a eukaryotic cell, a mammalian cell, an immortalized cell, an insect cell, a yeast cell, a plant cell, a fungal cell or a prokaryotic cell. In some embodiments, the cell is an A549 cell, a HEK-293 cell, a HEK-293T cell, a BHK cell, a CHO cell, a HeLa cell, an MRC5 cell, an Sf9 cell, an Sf2 cell, an Sf21 cell, a High Five TM cell, a Cos-1 cell, a Cos-7 cell, a Vero cell, a BSC1 cell, a BSC40 cell, a BMT10 cell, a WI38 cell, a Saos cell, a C2C12 cell, an L cell, an HT1080 cell, a HepG2 cell, a Huh7 cell, a K562 cell, a primary cell or any derivative thereof. In some embodiments, the cell is an Sf9 cell.

[0068] In some embodiments, the first nucleic acid, the second nucleic acid and the third nucleic acid are stably integrated into the genome after at least 5 passages (e.g., 10, 20, 40 passages).

[0069] In some embodiments, the cell is capable of growing to at least about 1x10 after at least about 24 hours 7cells / mL. In some embodiments, the cells are infected with a virus. In some embodiments, the virus is selected from: alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpes virus, poxvirus, circovirus, bocavirus, vaccinia virus, and retrovirus. In some embodiments, the virus is a wild-type virus. In some embodiments, the infection induces the expression of one or more of a first nucleic acid, a second nucleic acid, a third nucleic acid, or a combination thereof. In some embodiments, the infection induces the cells to produce a recombinant virus. In some embodiments, the recombinant virus is selected from: alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpes virus, poxvirus, circovirus, bocavirus, vaccinia virus, or retrovirus. In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivatives thereof. In some embodiments, the recombinant virus comprises an rAAV vector.

[0070] In some embodiments, the rAAV vector comprises less than 5%, 4%, 3%, 2%, or 1% of non-AAV components and contaminants of the GOI. In some embodiments, the contaminants are from alphavirus, parvovirus, baculovirus, dengue virus, lentivirus, poxvirus, circovirus, bocavirus, vaccinia virus, herpes virus, or retrovirus. In some embodiments, the contaminants are from baculovirus. Brief Description of the Drawings

[0072] The present disclosure is more fully understood with reference to the following drawings.

[0073] Figure 1A Shows the exemplary nucleic acid construct design for exemplary Rep and Cap proteins.

[0074] Figure 1B Shows the Western blot of adeno-associated virus (AAV) serotype 1 (AAV1) and AAV5 capsid proteins. Figure 1C Shows the affinity-purified capsid proteins from AAV7, AAV8, and AAV9.

[0075] Figures 2A - 2E Is a schematic diagram showing an exemplary method for producing recombinant virus, which starts with inoculating and selecting clonal cells ( Figure 2A and Figure 2B respectively), the clonal cells grow into a monolayer and are infected with an exemplary non-recombinant virus to produce exemplary recombinant virus particles containing the gene of interest (GOI) ( Figure 2C ), and finally the culture medium containing excess components is discarded ( Figure 2D ), and the recombinant virus is harvested from the remaining cell monolayer, including finally separating the virus from cell debris ( Figure 2E ). Abbreviations: BEV, baculovirus; GOI, gene of interest; PCR, polymerase chain reaction; WT, wild type.

[0076] Figure 3 Shows the results of clone screening to determine high-yield clones by yield (vg / mL).

[0077] Figure 4 Is a graph showing the quantified differences in scale time represented by passage number (x-axis) and cells / mL (y-axis) in traditional and optimized methods.

[0078] Figures 5A - 5D Shows the measurement results of the amounts of host cell proteins and activated virus present during the production process and before and after purification, indicating that both host cell proteins and activated virus impurities are reduced during the purification process. Figure 5A and Figure 5B Are gel diagrams showing the reduction of impurities during the purification process. Figure 5C Is a bar graph showing the host cell proteins (HCP) detected in fractions at different purification steps in ng / mL. Figure 5D Is a bar graph showing the concentrations (in ng / mL) of Sf9 and baculovirus (BEV) DNA in the eluates purified by AAVX and AEX columns. Under AAVX and AEX conditions, Sf9 = left column, while BEV = right column.

[0079] Figure 6It is a graph showing a stability study of measuring the volume (vg / L) or titer per unit (vg / cell) of an exemplary production clone prepared according to the present disclosure over 20 passages.

[0080] Figure 7 It is a graph showing the growth and infection kinetics measured over time in terms of cell density (cells / mL) and viability (%).

[0081] Figure 8 It is a bar graph showing a comparison of genomic sequencing analysis of Sf9 and BEV in purified Sf9AAV.

[0082] Figure 9 It is a schematic diagram showing an exemplary nucleic acid cassette containing a gene of interest (GOI) and inverted terminal repeats (ITR) before and after restriction digest.

[0083] Figure 10 It is a bar graph showing the enzyme levels in the cerebella of AAV-treated mice with wild-type (WT; circles), knockout (KO; squares), and AAVs produced using HEK293 cells (triangles) or Sf9 production cells (diamonds) prepared according to the present disclosure.

[0084] Figure 11A It is a bar graph showing the biodistribution of vector genomes in target tissues of an exemplary AAV7 serotype gene therapy prepared using an exemplary Sf9 production cell line of the present disclosure (diamonds) vs. a standard exemplary HEK293 cell platform (triangles). Figure 11B It is a graph showing the efficacy of prolonging survival after administration of AAV therapy produced in HEK293 or Sf9 cells according to the present disclosure.

[0085] Figure 12A It is a bar graph showing the enzyme levels in the forebrains of wild-type (WT; circles), knockout (KO; squares), and AAV-treated mice with AAVs produced using HEK293 cells (triangles) or Sf9 production cells (diamonds) prepared according to the present disclosure. Figure 12B It is a bar graph showing the enzyme levels in the hindbrains of wild-type (WT; circles), knockout (KO; squares), and AAV-treated mice with AAVs encoding an enzyme produced using HEK293 cells (triangles) or Sf9 production cells (diamonds) prepared according to the present disclosure. Figure 12C It is a graph showing the enzyme levels in the hindbrains of wild-type (WT; circles), knockout (KO; squares), and AAV-treated mice with AAVs produced using HEK293 cells (triangles) or Sf9 production cells (diamonds) prepared according to the present disclosure.

[0086] Figure 13 are images showing exemplary biodistribution of AAV in brain tissue following cisterna magna (ICM) administration (arrows indicate locations of detected AAV; locations are described and quantification is shown in Table 3).

[0087] Detailed Description

[0088] Viral vectors are commonly used to deliver therapeutic genes to humans. The production of viral vectors is a complex process. For example, a variety of different issues can arise during the design and manufacturing process, including but not limited to scalability, quality, and potency of the recombinant virus. The production of clinical-grade gene therapies remains a major obstacle to advancing cures for many diseases, disorders, and conditions that are otherwise unpreventable, uncurable, and / or untreatable. Process improvements are needed to achieve reproducible, consistent, scalable manufacturing solutions and provide commercially viable viral products that can be safely and reliably delivered to patients.

[0089] The present disclosure provides insights into systems having certain components that produce and provide commercially relevant or scalable quantities of clinical or clinically relevant quality reproducible, stable viral products.

[0090] Among other things, this paper also provides methods for using systems, cell lines, manufacturing methods, and feature combinations that result in products that are more reproducible, more scalable, and more reliable in clinical products. The disclosed system includes an engineered virus production cell line that results in low-cost, robust, and highly reproducible large-scale viral product manufacturing. Such virus production cell lines employ an innovative combination of stably integrated and inducible components in an engineered cell system.

[0091] definition

[0092] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the claimed subject matter belongs. Generally, the terms and techniques related to immunology, oncology, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein are those well known and commonly used in the art. Units of measurement not otherwise defined conform to the International System of Units (SI), NIST Special Publication 330, 2019 edition.

[0093] As used herein, unless the context clearly indicates otherwise, all numerical values or numerical ranges include the integers within such ranges or covering such ranges, as well as fractions of the numerical values or integers within such ranges or covering such ranges. Thus, for example, when referring to a range of 90 - 100%, it includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so on. In another example, a range of 1 - 5,000 times includes 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19- or 20 times, etc., as well as 1.1-, 1.2-, 1.3-, 1.4- or 1.5 times, etc., 2.1-, 2.2-, 2.3-, 2.4- or 2.5 times, etc., and so on.

[0094] The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit any embodiment. As used herein, the singular forms "a", "an" and "the" are also intended to include the plural forms unless the context clearly indicates otherwise.

[0095] It will further be understood that when used herein, the terms "comprises" and / or "comprising" specify the presence of the stated features, integers, steps, operations, elements and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof. "Consisting essentially of" means that at least each of the stated features, integers, steps, operations, elements and / or components is present, but does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof.

[0096] As used herein, the term "and / or" includes any combination and all combinations of one or more of the related listed items.

[0097] Unless otherwise specified or obvious from the context, as used herein, the term "about" when referring to a number or a numerical range should be understood to mean the stated number and plus or minus 10% of that number, or 10% below the listed lower limit and 10% above the listed upper limit of the range.

[0098] As used herein, the term "adeno-associated virus vector" or "AAV vector" refers to a vector derived from an adeno-associated virus selected from the following: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16 and any functional derivatives thereof. The AAV vector can be a mammalian (e.g., human, e.g., non-human primate) virus. The AAV vector can be an avian (AAAV) virus. In some embodiments, the AAV is a mammalian (e.g., human, e.g., non-human primate) AAV or an avian AAV (AAAV); that is, in some embodiments, the starting and / or engineered AAV is or is derived from a virus capable of infecting a mammalian or avian organism. The AAV vector can have one or more AAV wild-type genes deleted in whole or in part, e.g., the Rep and / or Cap genes, but retain functional flanking inverted terminal repeat (ITR) sequences. The functional ITR sequences facilitate the rescue, replication, and packaging of AAV virions. The AAV vector can contain a single-stranded (ss) or self-complementary (sc) genome. Thus, the AAV vector is defined herein as containing at least those cis sequences (e.g., functional ITRs) required for viral replication and packaging. The ITRs need not be wild-type polynucleotide sequences and are altered in some embodiments. For example, by insertion, deletion, or replacement of nucleotides, provided that the sequence provides functional rescue, replication, and packaging.

[0099] As used herein, the term "adeno-associated virus inverted terminal repeat" or "AAV ITR" refers to the regions flanking each end of the AAV genome, which together function in cis as an origin of DNA replication and as a packaging signal for the virus. Together with the AAV Rep coding region, AAV ITRs can also efficiently excise and integrate a polynucleotide sequence inserted between two flanking ITRs into the mammalian, avian, or insect genome. Typically, wild-type AAV ITRs contain 145 bases, but the ITRs can contain fewer or greater numbers of bases. As used herein, "AAV ITR" does not necessarily contain a wild-type polynucleotide sequence, and in some embodiments, the wild-type polynucleotide sequence is altered. For example, by insertion, deletion, or replacement of nucleotides. Additionally, the AAV ITR is derived from any one of several AAV serotypes selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivatives thereof. In addition, the 5' and 3' ITRs flanking a selected polynucleotide sequence in an AAV vector do not need to be the same or derived from the same AAV serotype or isolate, so long as they function as intended. For example, to achieve a desired therapeutic or genome editing effect.

[0100] As used herein, the term "engineered Kozak sequence" describes a reference Kozak sequence that has been modified (e.g., as compared to a reference or endogenous Kozak sequence) by one or more nucleotide alterations to alter (e.g., increase or decrease) the translation of a downstream gene product. A Kozak sequence as used herein is a sequence that encodes a translation start site, and an engineered Kozak sequence alters translation relative to the translation that occurs in the absence of the engineered Kozak sequence. The translational change that occurs with an engineered Kozak sequence can be an increase or a decrease in a particular downstream gene product. For example, an engineered Kozak sequence as provided herein is capable of incorporating a higher level of VP1 into an AAV capsid as compared to a reference Kozak sequence (e.g., prior to engineering).

[0101] As used herein, the term “functional derivative” of a biomolecule (such as a polynucleotide, such as a polypeptide, such as a viral particle (such as an AAV particle)) refers to a biomolecule that has been modified relative to a reference biomolecule, where the resulting biomolecule does not necessarily contain the same sequence (such as a nucleic acid sequence, an amino acid sequence), but has at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the activity of the reference biomolecule in a suitable assay. When the biomolecule is a polynucleotide or polypeptide, it is expected that the polynucleotide or polypeptide has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (but not necessarily contiguous portions of the sequence) of the nucleic acid or amino acid sequence of the reference biomolecule and has at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity. For example, a functional derivative of a polypeptide can be a polypeptide that contains one or more amino acid modifications relative to the reference biomolecule and retains certain functions, such as binding to a specific receptor, but the binding can be stronger, equivalent, or weaker than the reference biomolecule as long as the binding of the functional derivative achieves at least a portion of the activity of the reference biomolecule. A functional derivative of a polynucleotide can be a nucleic acid sequence that contains one or more nucleotide modifications but still encodes a protein or protein fragment that has the same or similar function as the protein encoded by the reference biomolecule, or has the same or similar function as the reference biomacromolecule (such as as a regulatory element, such as a promoter or enhancer). In addition, such a biomolecule or its functional derivative can be an “independent” functional unit or system (such as a modified or variant AAV) and / or part of a larger system, such as, for example, a vector (such as a gene cassette encoding an antibiotic resistance gene or a functional portion thereof; such as a gene cassette encoding a specific Kozak sequence, etc.).

[0102] As used herein, the term "functional fragment" of a biomolecule, such as a polynucleotide or polypeptide, refers to a fragment (i.e., shorter and / or smaller) of a reference biomolecule that has the same or similar functional activity as the reference biomolecule. The expected similar functional activity can be greater than, approximately equal to, or less than the functional activity of the reference biomolecule, as long as the functional fragment achieves at least a portion of the activity of the reference biomolecule. When the reference biomolecule is a polypeptide, it is expected that the polypeptide fragment retains at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the activity of the reference polypeptide in a suitable assay. For example, when the reference biomolecule is a polypeptide, it is expected that the polypeptide fragment can be a polypeptide that is shorter and / or smaller than the reference polypeptide by cleavage or other modification, but still retains the functional activity of the reference polypeptide, such as binding to a specific receptor; when the reference biomolecule is a polynucleotide, it is expected that the polynucleotide fragment retains a portion of the activity of the reference polynucleotide. For example, in the case of a polynucleotide encoding a protein, it is expected that the protein encoded by the polynucleotide fragment has at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the activity of the protein encoded by the reference polynucleotide in a suitable assay. In the case of a polynucleotide acting as a regulatory element (e.g., a promoter or enhancer), it is expected that the polynucleotide fragment has at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the activity of the reference polynucleotide in a suitable assay.

[0103] As used herein, the term "parvovirus" encompasses the family Parvoviridae, which includes, but is not limited to, the autonomous parvoviruses and the dependoviruses. Autonomous parvoviruses include, for example, the genus Bocavirus, the genus dependovirus, the genus erythrovirus, the genus Amdovirus, the genus Parvovirus, the genus Densovirus, the genus iterovirus, the genus containvirus, the genus Avarporvovirus, the genus Copiparvovirus, the genus Protoparvovirus, the genus tetrapivorvirus, the genus Ambidensovirus, the genus brevinnovovirus, the genus hepdensovirus, and the genus pendensovirus. Exemplary autonomous parvoviruses include, but are not limited to, porcine parvovirus, murine parvovirus, canine parvovirus, mink enteritis virus, bovine parvovirus, chicken parvovirus, feline panleukopenia virus (feline panleukosis virus), feline parvovirus, goose parvovirus, H1 parvovirus, Muscovy duck parvovirus, snake parvovirus, and B19 virus.

[0104] As used herein, the terms "percent identity", "% identity", or "sequence identity" refer to the degree to which two sequences (e.g., nucleotide sequences, such as DNA, RNA, etc., e.g., polypeptide sequences) have the same residues at the same positions in an alignment. For example, "a nucleotide sequence has X% identity to SEQ ID NO:Y" refers to the percent identity ratio of the nucleotide sequence to SEQ ID NO:Y, and is elaborated as X% of the residues in the nucleotide sequence are the same as the corresponding residues of the sequence disclosed in SEQ ID NO:Y. A sequence having X% identity to a reference sequence may contain more nucleotide or amino acid residues than specified in the reference sequence, but must contain the sequence corresponding to the reference sequence. In most cases, the sequences under discussion will contain the sequence corresponding to all specified reference sequences. Typically, such calculations are performed using computer programs. Exemplary programs for comparing and aligning sequence pairs include ALIGN, FASTA, gapped BLAST, BLASTP, BLASTN, or GCG.

[0105] As used herein, the term "plasmid" refers to an extrachromosomal element that carries genes that can replicate independently of the cell chromosome. A plasmid can be in the form of a circular double-stranded DNA molecule. Such elements can include autonomous replication sequences, genomic integration sequences, phage or nucleotide sequences, and linear, circular or supercoiled, or single-stranded or double-stranded DNA or RNA of any origin.

[0106] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to nucleotide chains of any length and include DNA and RNA. In some embodiments, the nucleotides are deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate incorporated into the chain by DNA or RNA polymerase. Polynucleotides can contain modified nucleotides such as methylated nucleotides and their analogs. If present, the modification is imparted to the nucleotide structure either before or after strand assembly. In some embodiments, the nucleotide sequence is interrupted by non-nucleotide components. In some embodiments, the polynucleotide is further modified after polymerization, such as by binding to a labeling component. Other types of modifications include, for example, "caps", substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as modifications with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates) and charged linkages (e.g., phosphorothioates, dithiophosphates), modifications containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine), modifications with intercalators (e.g., acridine, psoralen), modifications containing chelating agents (e.g., metals, radioactive metals, boron, oxidative metals), modifications containing alkylating agents, modifications with modified linkages (e.g., α-anomeric nucleic acids), and unmodified forms of polynucleotides. In some embodiments, any hydroxyl group normally present in the sugar is substituted, e.g., with a phosphonate group, a phosphate group, protected with a standard protecting group, or activated to prepare an additional linkage with an additional nucleotide or to bind to a solid support. In some embodiments, the 5' and 3' terminal OHs are phosphorylated or substituted with an amine or an organic capping group of 1 to 20 carbon atoms. Other hydroxyl groups can also be derivatized as standard protecting groups. In some embodiments, the polynucleotide also contains analogs in the form of ribose or deoxyribose, including, for example, 2'-O-methyl ribose, 2'-O-allyl ribose, 2'-fluoro ribose or 2'-azido ribose, carbocyclic sugar analogs, α- or β-anomeric sugars, epimeric sugars (such as arabinose, xylose or lyxose), pyranoses, furanoses, sedoheptuloses, acyclic analogs, and abasic nucleoside analogs such as methyl nucleosides. In some embodiments, one or more phosphodiester bonds are replaced by alternative linking groups. These alternative linking groups can include, but are not limited to, embodiments where the phosphodiester is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NRi ("amidate"), P(O)R, P(O)OR', CO or CH2 ("acetal"), where each R or R' is independently H or a substituted or unsubstituted alkyl (1-20 C), optionally containing an ether (-O-) bond, aryl, alkenyl, cycloalkyl, cycloalkenyl or arylalkynyl. The linkages in the polynucleotide need not all be the same.The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0107] As used herein, the terms "promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence that controls the expression of a coding sequence or functional RNA. Typically, the coding sequence is located 3' to the promoter sequence. A promoter may be derived, for example, from a native gene, comprise different elements derived from different promoters found in nature, and / or be a synthetic DNA fragment. In some embodiments, different promoters direct the expression of a gene in different tissues or cell types, or at different developmental stages, or in response to different environmental or physiological conditions or inducer molecules. In most cases, a promoter that causes a gene to be expressed in most cell types is generally referred to as a "constitutive promoter". A promoter that causes gene expression in a particular cell and tissue type is generally referred to as a "cell-specific promoter" or a "tissue-specific promoter", respectively. A promoter that causes gene expression at a particular stage of development or cell differentiation is generally referred to as a "development-specific promoter" or a "cell differentiation-specific promoter". A promoter that is induced and causes gene expression after exposure or treatment of a cell with an agent, biomolecule, chemical, ligand, light, etc. that induces the promoter is generally referred to as an "inducible promoter" or a "regulatable promoter". In some embodiments, it is further recognized that different lengths of DNA fragments have the same promoter activity since the exact boundaries of regulatory sequences have not been fully determined in most cases.

[0108] As used herein, a "recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector derived from AAV and comprising one or more heterologous sequences (i.e., nucleic acid sequences not of AAV origin) flanked by at least one AAV ITR. In some embodiments, such rAAV vectors replicate and package into virus particles when present in a host cell that has a suitable helper plasmid or virus (or expresses suitable helper functions) and expresses the AAV Rep and Cap gene products (i.e., the AAV Rep and Cap proteins). When an rAAV vector is incorporated into a larger polynucleotide (e.g., a chromosome or other vector such as a plasmid used for cloning or transfection), the rAAV vector is then referred to as a "pro-vector" and is "rescued" by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions.

[0109] As used herein, the terms "gene of interest (GOI)" and "transgene" refer to a polynucleotide that is introduced into a cell and is capable of being transcribed by the cell into RNA and optionally translated into protein and / or expressed under appropriate conditions. A gene of interest or transgene may confer desired properties on the cell into which it is introduced or otherwise result in a desired therapeutic or diagnostic outcome.

[0110] As used herein, the term "prevent" means to stop or reduce the occurrence of something, at least for a period of time, e.g., for example, to prevent the appearance of at least one symptom of a disease, to prevent the production of a specific gene product, or to reduce the amount of gene product produced by a specific gene.

[0111] As used herein, the phrase "pharmaceutically acceptable" means a compound, material, composition, and / or dosage form within the scope of reasonable medical judgment that is suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0112] As used herein, the phrase "pharmaceutically acceptable carrier" means a reagent (e.g., excipient, carrier, buffer, etc.) suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, absorption delaying agents, etc. that are compatible with the administration of a drug. Standard pharmaceutical carriers can include, for example, phosphate buffered saline solutions, water, emulsions (e.g., such as oil / water or water / oil emulsions), and various types of wetting agents. The composition can also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rd Edition, 2020).

[0113] As used herein, "treat", "treating", and "treatment" mean treating a disease, disorder, or symptom or manifestation in such a subject (e.g., a human). This includes preventing a disease or disorder; inhibiting a disease, disorder, etc., i.e., slowing or stopping its progression or development; and ameliorating a disease, disorder, etc., i.e., causing the regression of the disease state.

[0114] As used herein, "subject" and "patient" mean an organism treated with a composition prepared according to the present disclosure and / or the methods provided herein. Such organisms preferably include, but are not limited to, mammals (e.g., mice, monkeys, horses, cows, pigs, canines, felines, etc.), and more preferably include humans.

[0115] As used herein, the term "vector" includes nucleic acid vectors, e.g., DNA vectors (such as plasmids), RNA vectors, or another suitable replicon (such as viral vectors). A variety of vectors have been developed for delivering polynucleotides encoding exogenous polynucleotides or proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are disclosed in, e.g., WO 1994 / 011026; which is incorporated herein by reference in its entirety as it relates to vectors suitable for expressing a nucleic acid molecule of interest. Expression vectors suitable for the compositions and methods described herein contain polynucleotide sequences and, e.g., additional sequence elements for expressing heterologous nucleic acid material (such as nucleic acid molecules) in cells. Certain vectors for expressing the nucleic acid molecules provided herein may include plasmids containing regulatory sequences (such as promoter and enhancer regions) that can direct and / or otherwise affect gene transcription. In some embodiments, the promoter can be a compact bidirectional promoter which, in some embodiments, does not contain an enhancer. Other useful vectors for expressing the nucleic acid molecules disclosed herein contain polynucleotide sequences that enhance the translation rate of these polynucleotides or improve the stability or nuclear export of the RNA produced by gene transcription. These sequence elements include, e.g., 5' and 3' untranslated regions, internal ribosome entry sites (IRES), and polyadenylation signals (polyA) to direct efficient transcription of the genes carried on the expression vector.

[0116] Production cell system

[0117] Producing viral products for clinical populations requires addressing and often overcoming multiple challenges related to manufacturing and commercial viability. Among other things, the present disclosure provides innovative strategies for producing stable and inducible production cell lines that can be used to manufacture viral products. These strategies involve, for the first time, combining three exogenous cellular components stably integrated into a host cell, and then infecting with an inducible virus to produce recombinant viral products that can be used for commercial and / or clinically relevant applications.

[0118] In some embodiments, the present disclosure provides engineered viral production cell lines comprising an inducible promoter, an enhancer motif, and an engineered Kozak sequence. In some such embodiments, these elements are stably integrated into the genome of the host production cell line. Also provided herein are viral production cell lines comprising all of the nucleic acids required to encode the components necessary for recombinant virus production, wherein the nucleic acids are stably integrated and include, but are not limited to, Rep, Cap, and the gene of interest (GOI) (e.g., a nucleic acid encoding at least one Rep protein, a nucleic acid encoding at least one Cap protein, and a nucleic acid encoding at least one GOI).

[0119] In some aspects, the present disclosure provides systems, engineered cell lines, and methods for manufacturing viral vectors and products. While any suitable virus is contemplated, by way of example only, adeno-associated virus (AAV) has been frequently used in recombinant viral vector systems for therapeutic delivery. Wild-type AAV is a small, non-enveloped human parvovirus that can infect humans but is non-pathogenic to humans. The wild-type AAV genome contains two open reading frames, Rep and Cap, flanked by two inverted terminal repeats (ITRs). These ITRs base pair to allow synthesis of complementary DNA strands. Rep and Cap are translated to produce multiple different proteins (Rep78, Rep68, Rep52, Rep40 - required for the AAV life cycle; VP1, VP2, VP3 - capsid proteins).

[0120] Among other things, the present disclosure provides sequences (e.g., nucleic acid sequences, e.g., amino acid sequences) of one or more of the components provided herein. For example, nucleic acid sequences can be provided using combinations of A, G, C, and / or T; and / or A, G, C, and / or U. That is, in a given context, in some embodiments, the polynucleotides provided herein can comprise one or more A, G, C, and / or T nucleobases. In some embodiments, the polynucleotides provided herein can comprise one or more A, G, C, and / or U nucleobases. As will be understood by those skilled in the art, in a given context, "T" or "U" in a given polynucleotide indicates that they will be considered interchangeable where appropriate. For example, in a given appropriate context, a polynucleotide having the sequence CAGTTTATGGT can also be understood to be CAGUUUAUGGU and function as CAGUUUAUGGU, and as will be understood by those skilled in the art, can be used interchangeably in appropriate circumstances and / or contexts (e.g., DNA / cDNA as compared to, e.g., mRNA / RNA). It should be understood that throughout the specification (e.g., as provided in the sequence listing in SEQ ID NOs: 1-203 or 210-216), in each case where a polynucleotide comprises, consists essentially of, or consists of a nucleotide sequence comprising one or more thymine nucleobases ("T"), another polynucleotide comprising, consisting essentially of, or consisting of the same nucleotide sequence comprising uracil nucleobase ("U") in place of thymine (T) is also contemplated; or, in each case where a polynucleotide comprises, consists essentially of, or consists of a nucleotide sequence comprising one or more uracil nucleobases ("U"), another polynucleotide comprising, consisting essentially of, or consisting of the same nucleotide sequence comprising thymine nucleobase ("T") in place of uracil ("U") is also contemplated. Thus, for any sequence provided herein, including any of the sequences shown in SEQ ID NOs: 1-191 or 210-216, any sequence comprising "T" should also be understood to contemplate a polynucleotide comprising "U" and be used in appropriate circumstances, in a given context and / or situation in accordance with the techniques provided herein.

[0121] The generation of recombinant adeno-associated virus (rAAV) vectors involves providing an AAV transfer plasmid in which the transgene is placed between two ITRs, and Rep and Cap are supplied in trans. The transfer plasmid is then provided to a cell (e.g., HEK-293 cells) along with Rep, Cap, and additional helper plasmids to produce recombinant virus comprising the rAAV vector.

[0122] The present disclosure provides the surprising finding that stable integration of Rep, Cap, and ITR-GOI constructs into a host cell does not render the host cell genome unstable. Moreover, the quality of the viral product is not reduced. Among other things, this is achieved by using engineered Kozak sequences and / or enhancer sequences. For example, it is contemplated that toxicity in a host cell can be modulated by modulating Rep levels. Alternatively or additionally, in some embodiments, a concatamerization strategy is used (e.g., generating constructs of Rep or Cap constructs prior to stable integration into the cellular genome). In some embodiments, the concatamers of the present disclosure comprise at least two monomers. In some embodiments, the concatamer comprises three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more monomers. The concatamer can be of any size that comprises at least two monomers and is still small enough to be isolated and purified using standard techniques. For example, in some embodiments, such concatamers can be purified using standard gel purification methods known to those of skill in the art.

[0123] The techniques provided herein, such as methods or systems, achieve stable expression of Rep and Cap by providing engineered Kozak sequences and / or enhancer sequences, and / or optionally combining constructs in concatamers prior to contacting the cells in which the constructs are to be stably integrated. These methods improve parameters such as stability and reproducibility, accelerate growth, improve the production efficiency of viral products, and the like.

[0124] In some embodiments, engineered cells (e.g., viral production cells having stably integrated Rep, Cap, and ITR-GOI) are capable of rapid growth. In some embodiments, the cells are capable of growing to at least about 1 x 10 7 cells / mL in at least about 24 hours (e.g., at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 hours). In some embodiments, the cells are capable of growing to at least about 1 - 2 x 10 7 cells / mL in at least about 24 hours (e.g., at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 hours). As a non-limiting example, the doubling time of Sf9 cells in suspension culture can be between about 18 - 30 hours, while it is at most greater than 72 hours during single cell isolation.

[0125] In some embodiments, the cells are capable of growing to at least about 1 x 10 9 - 1 x 10 15cells / mL. In some embodiments, the cells are able to grow to at least about 1 x 10 15 cells / mL or more after at least about 24 hours (e.g., at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 hours). In some embodiments, the cells double after 0, 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 hours or longer. In some embodiments, the doubling time in suspension culture is between about 10 - 40 hours, about 15 - 35 hours, about 18 - 30 hours, about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours. In some embodiments, the doubling time of the clonal cells is longer than the doubling time of the suspension culture. In some such embodiments, the doubling time range can be from about 20 hours to about 84 hours; from about 24 hours to about 72 hours; from about 48 hours to about 72 hours; about 24, 48 or 72 hours. In some such embodiments, the rapid growth of single cells facilitates or provides the ability to select cells free of rhabdovirus.

[0126] In some embodiments, cells with the Rep, Cap, and ITR - GOI constructs stably integrated into the host cell do not cause host cell genomic instability. This can be confirmed by using sequencing analysis (e.g., next - generation sequencing) to show a low or no percentage of reads / sequences mapping to host - or vector - related nucleic acids. For example, in some embodiments, the sequencing analysis shows a low percentage of reads / sequences mapping to the Sf9 or baculovirus genome, which supports minimal packaging of Sf9 and baculovirus DNA in the assembled capsid. The low baculovirus packaging amount confirms that the stable integration of the three constructs is a reliable, reproducible, safe, and improved method compared to cells integrating only one or two components. The low amount of the Sf9 genome, considering the stable integration of the Cap, Rep, and GOI - ITR constructs into the cells, is a particularly surprising finding because those skilled in the art would expect that integrating ITR - GOI into cells with Cap and Rep would result in a greater amount of host cell components (e.g., genomic DNA) being packaged into the final viral product.

[0127] Without being bound by theory, it is expected that stable genomic integration will help to induce the production of AAV with high volumetric titers by active infection using recombinant baculoviruses that do not contain AAV elements, thereby alleviating the BEV genomic stability issues during scale-up, allowing for the generation of larger, more replicable, and / or healthier numbers of cells and amounts of viral products according to the present disclosure. The systems disclosed herein are applicable to a variety of viruses. In some embodiments, the virus is AAV. In some embodiments, the systems provided herein are adapted to AAV capsid serotypes and are effective at packaging both self-complementary and single-stranded AAV genomes.

[0128] The systems of the present disclosure can be used to produce engineered production cell line populations. In some embodiments, such cell lines can be produced by rapidly screening image-verified clonal production cell populations. In some embodiments, these populations are identified as rhabdovirus-free candidates. In some embodiments, such populations are capable of exceeding E5 AAV gc / cell or E14 AAVgc / L. In some embodiments, the cell lines produced by the systems disclosed herein produce virus in a replicable and consistent manner after multiple (e.g., 5, 10, 15, 20, 25, etc.) passages after resuscitation. In some such embodiments, the capabilities of the system support the linear scalability of the system, as opposed to the non-linear scalability of many other previously described methods. It is expected that these successes will be achieved because the techniques of the present disclosure provide and allow for stable integration of Rep, Cap, and / or GOI, thereby preventing loss of potency of these genes, as well as regulating the expression of Rep, Cap, or both, and preventing cytotoxicity. In contrast, previously described methods are often hampered by complications such as, for example, Rep toxicity or the absence of one or more components required for the cells to produce viral products. Importantly, the viruses produced using the engineered cell lines provided herein are at least as potent and efficacious as viruses derived from other systems. Additionally, in some embodiments, the engineered cells are insect cells, and the viruses produced using them are at least as potent and efficacious in vivo as viruses derived from mammalian-derived counterparts.

[0129] In some embodiments, the present disclosure provides an improved cell system, the improvement including a single host cell that contains at least three components stably integrated into the host cell genome, and an inducing virus that induces the host cell to start producing a recombinant virus (e.g., AAV). The host cell containing these three stably integrated components includes at least: a first nucleic acid that contains a first enhancer sequence and a Rep gene or a functional fragment thereof; a second nucleic acid that contains a second enhancer sequence and a Cap gene or a functional fragment thereof; and a third nucleic acid that contains a (GOI). In some such embodiments, any of the nucleic acids further contains an enhancer sequence and / or an engineered Kozak sequence. After being stably integrated into the host cell genome, when the host cell contacts the inducing virus, the inducing virus activates the integrated components, thereby producing a recombinant virus containing the GOI.

[0130] Engineered cell

[0131] In some aspects, the present disclosure provides virus-producing cells. In some embodiments, the virus-producing cells have an engineered genome containing stably integrated components. In some such embodiments, the components include: (a) a first nucleic acid that contains a first enhancer sequence and a Rep gene or a functional fragment or functional derivative thereof; (b) a second nucleic acid that contains a second enhancer sequence and a Cap gene or a functional fragment or functional derivative thereof; and (c) a third nucleic acid that contains a GOI.

[0132] A virus-producing cell can be any type of cell that can be used for the production of recombinant viruses. In some embodiments, the cell is a eukaryotic cell, a mammalian cell, an immortalized cell, an insect cell, a yeast cell, a plant cell, a fungal cell, or a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell (e.g., a plant cell, an animal cell, a protist cell, or a fungal cell), a mammalian cell (a Chinese hamster ovary (CHO) cell, a baby hamster kidney (BHK) cell, a human embryonic kidney (HEK) cell, a mouse myeloma (NS0) cell, or a human retinal cell), an immortalized cell (e.g., a HeLa cell, a COS cell, a HEK-293T cell, an MDCK cell, a 3T3 cell, a PC12 cell, a Huh7 cell, a HepG2 cell, a K562 cell, an N2a cell, or a SY5Y cell), an insect cell (e.g., a Spodoptera frugiperda cell, a Trichoplusia ni cell, a Drosophila melanogaster cell, a Drosophila Schneider cell, an S2 cell, an S21 cell, or a Heliothis virescens cell), a yeast cell (e.g., a Saccharomyces cerevisiae cell, a Cryptococcus cell, or a Candida cell), a plant cell (e.g., a parenchyma cell, a collenchyma cell, or a sclerenchyma cell), a fungal cell (e.g., a Saccharomyces cerevisiae cell, a Cryptococcus cell, or a Candida cell), or a prokaryotic cell (such as an Escherichia coli cell, a Streptococcus cell, a Streptomyces soil bacterium cell, or an archaeal cell).

[0133] In some embodiments, the cell is from a cell line. In some embodiments, the cell is a primary cell.

[0134] In some embodiments, the cell is an A549 cell, a HEK-293 cell, a HEK-293T cell, a BHK cell, a CHO cell, a HeLa cell, an MRC5 cell, an Sf2 cell, an Sf9 cell, an Sf2 cell, a High Five TM cell, an Sf21 cell, a BTI-Tn-5B1-4 cell, a Cos-1 cell, a Cos-7 cell, a Vero cell, a BSC 1 cell, a BSC 40 cell, a BMT 10 cell, a WI38 cell, a Saos cell, a C2C12 cell, an L cell, an HT1080 cell, a HepG2 cell, a Huh7 cell, a K562 cell, or any derivative thereof. In some embodiments, the cell is an Sf9 cell.

[0135] In some embodiments, the present disclosure provides improved engineered cells, the improvement including combining at least three stably integrated components into a single host cell, the components including at least: a first nucleic acid comprising a first enhancer sequence and a Rep gene or any functional derivative or functional fragment thereof; a second nucleic acid comprising a second enhancer sequence and a Cap gene or any functional derivative or functional fragment thereof; and a third nucleic acid comprising a GOI. In some such embodiments, any of the nucleic acids further comprises an enhancer sequence and / or a Kozak sequence, wherein, in some embodiments, the Kozak sequence is an engineered Kozak sequence.

[0136] In some embodiments, the cell may optionally further comprise a helper virus or any functional derivative or fragment thereof and / or a helper plasmid and / or any functional derivative or fragment thereof. In some such embodiments, such helper plasmids, derivatives or fragments may be part of one or more constructs or components (e.g., part of a component comprising Rep and / or Cap, etc.). In some such embodiments, the helper is an AAV helper.

[0137] In some embodiments, the stable integration of one or more components into the host cell genome is random with respect to the location within the host cell genome. In some embodiments, the stable integration of one or more components into the genome is site-specific and / or targeted to one or more specific locations in the host cell genome.

[0138] After stable integration into the host cell genome, the host cell is subsequently contacted with an inducing virus, which activates the integrated components to produce a recombinant virus comprising the GOI. Compared to the previously described engineered cells, this engineered cell produces more virus, faster, and more reproducibly.

[0139] Rep and Rep constructs

[0140] Parvoviruses (including AAV) contain a Rep gene that encodes four proteins required for viral genome replication and packaging. These four proteins are Rep78, Rep68, Rep52, and Rep40, respectively. In some embodiments, a viral production cell having the engineered genome described herein comprises a first nucleic acid comprising a Rep gene or any functional derivative or functional fragment thereof. In some embodiments, the Rep gene encodes a protein selected from Rep78, Rep68, Rep52, Rep40, and any functional derivative or functional fragment thereof.

[0141] In some embodiments, the Rep gene or a functional derivative or functional fragment thereof is derived from a parvovirus. In some embodiments, the parvovirus is AAV. In some embodiments, the Rep gene or a functional derivative or functional fragment thereof is derived from AAV. In some embodiments, the AAV is mammalian (e.g., human, e.g., non-human primate) AAV or avian AAV (AAAV); that is, in some embodiments, the starting and / or engineered AAV is or is derived from a virus capable of infecting mammalian or avian organisms. In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16 and any functional derivatives thereof.

[0142] In some embodiments, the first nucleic acid comprising the Rep gene or a functional fragment thereof further comprises a promoter (and optionally, further comprises one or more of an intron, microRNA, linker, splicing element, polyA signal, or any combination thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini-promoter, or any functional fragment and / or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p10, p19, p40, synapsin, CaMKII, GRK1, polH, EM7, OpIE1 and any functional fragments and / or functional derivatives thereof.

[0143] In some embodiments, the first nucleic acid comprising a functional fragment of its Rep gene further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, β-lactam, macrolide, tetracycline, or any functional fragment and / or functional derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, blasticidin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, bleomycin, or any functional fragment and / or functional derivative thereof.

[0144] In some embodiments, the first nucleic acid comprising the Rep gene or a functional fragment thereof further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, and any functional fragment and / or functional derivative thereof.

[0145] In some embodiments, the first nucleic acid comprising the Rep gene or a functional fragment thereof comprises a sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 210 or any functional fragment and / or functional derivative thereof, such as the portion of SEQ ID NO: 210 encoding the Rep protein or a functional portion thereof. In some embodiments, the first nucleic acid comprising the Rep gene or a functional fragment thereof comprises a sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more, or 100%) sequence identity to the sequence encoding the Rep gene or a functional derivative or functional fragment thereof generated using the nucleic acid sequence of SEQ ID NO: 210.

[0146] Cap and Cap constructs

[0147] The capsid protein of AAV forms the outer non-nucleic acid portion of the virion and is encoded by the AAV Cap gene. The Cap gene encodes three structural proteins: VP1, VP2, and VP3, all of which are translated from the same mRNA. These three proteins are produced in different amounts and have different sizes: VP1 is approximately 87 kDa; VP2 is approximately 72 kDa; and VP3 is approximately 63 kDa. VP3 is produced in the highest amount of these three, and VP1 and VP2 are produced in lower amounts (relative to the total amount of capsid protein). The AAV capsid typically contains its VP1, VP2, and VP3 proteins in a ratio of 1:1:10. As known to those skilled in the art, in some embodiments, there may be other proteins translated from the Cap transcript, depending on the reading frame during translation (e.g., assembly activating protein (AAP), e.g., membrane-associated auxiliary protein (MAAP)).

[0148] In some embodiments, the virus-producing cell having the engineered genome described herein comprises a second nucleic acid comprising the Cap gene or any functional derivative or functional fragment thereof.

[0149] In some embodiments, the Cap gene or its functional derivatives or functional fragments are derived from parvovirus. In some embodiments, the parvovirus is AAV. In some embodiments, the AAV is mammalian (e.g., human, e.g., non-human primate) AAV or AAVV; that is, in some embodiments, the starting and / or engineered AAV is or is derived from a virus capable of infecting mammalian or avian organisms. In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16 and any of its functional derivatives.

[0150] In some embodiments, the Cap gene or a functional fragment thereof encodes one or more proteins selected from the group consisting of VP1, VP2, VP3, MAAP, AAP, and any functional fragments and / or functional derivatives thereof. In some embodiments, the Cap gene or a functional derivative or functional fragment thereof comprises a sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%) sequence identity to any of the nucleic acid sequences of SEQ ID NOs: 192 - 203. In some embodiments, the amino acid sequence encoded by the Cap gene or a functional derivative or functional fragment thereof has at least about 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%) sequence identity to any of the amino acid sequences of SEQ ID NOs: 204 - 209. In some embodiments, the nucleic acid sequence of SEQ ID NOs: 192 or 193 encodes the amino acid sequence of SEQ ID NO: 204. In some embodiments, the nucleic acid sequence of SEQ ID NOs: 194 or 195 encodes the amino acid sequence of SEQ ID NO: 205. In some embodiments, the nucleic acid sequence of SEQ ID NOs: 196 or 197 encodes the amino acid sequence of SEQ ID NO: 206. In some embodiments, the nucleic acid sequence of SEQ ID NOs: 198 or 199 encodes the amino acid sequence of SEQ ID NO: 207. In some embodiments, the nucleic acid sequence of SEQ ID NOs: 200 or 201 encodes the amino acid sequence of SEQ ID NO: 208. In some embodiments, the nucleic acid sequence of SEQ ID NOs: 202 or 203 encodes the amino acid sequence of SEQ ID NO: 209.

[0151] In some embodiments, the second nucleic acid comprising the Cap gene or its functional derivative or functional fragment further comprises a promoter (and optionally further comprises one or more of an intron, a microRNA, a linker, a splicing element, a polyA signal, or any combination thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini-promoter, or any functional fragment and / or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p10, p19, p40, synapsin, CaMKII, GRK1, polH, EM7, OpIE1, and any functional fragment and / or functional derivative thereof. As will be understood by one of ordinary skill in the art, in a given context, certain promoters may be classified as early promoters or late promoters, and those skilled in the art know how and when to use such promoters to effect the expression of a component.

[0152] In some embodiments, the second nucleic acid comprising the Cap gene or its functional fragment further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, a β-lactam, a macrolide, a tetracycline, or any functional fragment and / or functional derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, blasticidin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, bleomycin, or any functional fragment and / or functional derivative thereof.

[0153] In some embodiments, the second nucleic acid comprising the Cap gene or its functional fragment further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, and any functional fragment and / or functional derivative thereof.

[0154] In some embodiments, the second nucleic acid comprises a sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more, or 100%) sequence identity to any of the nucleic acid sequences of SEQ ID NOs: 211-216. In some embodiments, the first nucleic acid comprising the Cap gene or a functional fragment thereof comprises a sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more, or 100%) sequence identity to any of the nucleic acid sequences of SEQ ID NOs: 211-216 or any of its functional fragments and / or functional derivatives (such as, for example, any part of SEQ ID NOs: 211-216 encoding the Cap protein or a functional portion thereof). In some embodiments, the second nucleic acid comprising the Cap gene or a functional fragment thereof comprises a sequence having at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more, or 100%) sequence identity to the sequence encoding the Cap gene or its functional derivative or functional fragment generated using any of the nucleic acid sequences of SEQ ID NOs: 211-216.

[0155] Enhancer sequence

[0156] Enhancers are small molecules used to enhance the viral transduction process and increase the expression of target genes. Enhancer sequences for improving the viral transduction process are provided herein in some embodiments. In some embodiments, the virus-producing cell comprises a first nucleic acid containing a first enhancer sequence and a second nucleic acid containing a second enhancer sequence.

[0157] In some embodiments, the first and second enhancer sequences are the same. In some embodiments, the first and second enhancer sequences are different. In some embodiments, the first enhancer sequence is a homologous region (hr) enhancer sequence. In some embodiments, the second enhancer sequence is an hr enhancer sequence. In some embodiments, the enhancer is or comprises a sequence from Autographa Californica Nucleopolyhedrovirus (NCBI Taxonomy ID 46015).

[0158] In some embodiments, the first enhancer sequence has at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%) identity to any nucleic acid sequence of SEQ ID NOs: 1-10 or a functional fragment or functional derivative thereof.

[0159] In some embodiments, the second enhancer sequence has at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%) identity to any nucleic acid sequence of SEQ ID NOs: 1-10 or a functional fragment or functional derivative thereof.

[0160] In some embodiments, the first enhancer sequence is derived from alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, circovirus, bocavirus, vaccinia virus, or retrovirus. In some embodiments, the first enhancer sequence is derived from baculovirus. In some embodiments, the AAV is mammalian (e.g., human, e.g., non-human primate) AAV or avian AAV (AAAV); that is, in some embodiments, the starting and / or engineered AAV is or is derived from a virus capable of infecting mammalian or avian organisms. In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivatives thereof. In some embodiments, the first enhancer sequence is derived from baculovirus.

[0161] In some embodiments, the second enhancer sequence is derived from alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpes virus, poxvirus, circovirus, bocavirus, vaccinia virus, or retrovirus. In some embodiments, the second enhancer sequence is derived from baculovirus. In some embodiments, the AAV is mammalian (e.g., human, e.g., non-human primate) AAV or avian AAV (AAAV); that is, in some embodiments, the starting and / or engineered AAV is or is derived from a virus capable of infecting mammalian or avian organisms. In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivatives thereof. In some embodiments, the second enhancer sequence is derived from baculovirus.

[0162] Kozak sequence

[0163] The Kozak sequence is a nucleic acid motif that functions as a protein translation initiation site in most eukaryotic mRNA transcripts. In some embodiments herein, engineered Kozak sequences are provided that can improve the expression of viral proteins involved in the production of recombinant viruses. Such sequences can be selected to promote preferential translation in insect cells relative to mammalian cells. Preferential translation means that, for example, the quantity and / or quality of the downstream gene product is improved in one cell type or condition compared to another cell type or condition (e.g., in insect cells compared to mammalian cells). Preferential translation does not mean that a given engineered Kozak sequence will not promote translation in another cell system (e.g., mammalian cells), rather, preferential translation refers to a design that is engineered and / or optimized to function in insect cells, and different designs that are preferentially translated in mammalian cells may function in insect cells but not as well. Various engineered Kozak sequences are known in the art and can be used alone, in combination, and / or further modified according to the present disclosure (see, e.g., WO 2017 / 181162; Viruses 2023, 15, 1983).

[0164] In some embodiments, the Kozak sequence is used in a nucleic acid containing a rep coding sequence, and the Kozak sequence is compatible with both mammalian and insect cell systems. In some embodiments, the Kozak sequence preferentially promotes translation in insect cells. In some embodiments, the Kozak sequence promotes preferential translation in insect cells compared to, for example, mammalian cells.

[0165] In some embodiments, the Kozak sequence is used in a nucleic acid containing a cap coding sequence, and the Kozak sequence is compatible with both mammalian and insect cell systems. In some embodiments, the Kozak sequence preferentially promotes translation in insect cells. In some embodiments, the Kozak sequence promotes preferential translation in insect cells compared to, for example, mammalian cells.

[0166] In some embodiments, the Kozak sequence is used in a nucleic acid containing a GOI and optionally an ITR. In some such embodiments, the Kozak sequence is compatible with both mammalian and insect cell systems. In some embodiments, the Kozak sequence preferentially promotes translation in mammalian cells. In some embodiments, the Kozak sequence promotes preferential translation in mammalian cells compared to, for example, insect cells. In some embodiments, the GOI is not translated in insect cells. In some embodiments, the GOI is translated in insect cells but is removed during the manufacturing process along with any other contaminants (such as contaminants from insect cells, etc.).

[0167] In some embodiments, engineered Kozak sequences are used to increase or improve the expression of a particular protein (e.g., Cap protein, such as VP1, etc.). In some embodiments, engineered Kozak sequences are used to regulate (e.g., attenuate, reduce, alter, etc.) the expression of a particular protein (e.g., Rep protein). In some embodiments, the Kozak sequences of the present disclosure comprise, consist essentially of, or consist of four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen. In some embodiments, the Kozak sequence comprises a sequence of ten nucleotide residues, wherein six nucleotide residues are located at the 5' end of the start codon sequence (e.g., ATG / AUG), and one nucleotide residue is located at the 3' end of the start sequence (e.g., G). In some embodiments, the Kozak sequence consists essentially of six nucleotide residues at the 5' end of the start codon sequence (e.g., ATG / AUG) and one nucleotide residue at the 3' end of the start sequence (e.g., G). In some embodiments, the Kozak sequence is selected from any of SEQ ID NOs: 11-191. In some embodiments, the Kozak sequence is selected based on the serotype and the sequence required to maintain the open reading frame of a given capsid (see, e.g., Table 1).

[0168] As will be understood by those skilled in the art, when generating comparative products using different platforms such as mammalian cells (e.g., HEK293 cells), it is not necessarily required to use such engineered Kozak sequences optimized for insect cells. That is, in some embodiments, when generating viral products (e.g., rAAV containing a gene of interest) in insect (e.g., Sf9) and mammalian (e.g., HEK293) systems for purposes of comparison, the mammalian system will use endogenous rep / cap sequences, while the insect system will use sequences containing engineered Kozak sequences (see, e.g., Table 1; see also SEQ ID NOs: 193, 195, 197, 199, 201, and 203, each of which contains an engineered Kozak sequence that preferentially translates the capsid in insect cells).

[0169] Preferred engineered Kozak sequences according to the present disclosure are those that can produce higher levels of VP1 protein compared to non-engineered Kozak sequences. That is, in some embodiments, when a Kozak sequence that preferentially translates in a mammalian system is transferred to an insect system, it results in a decrease in the amount of certain proteins (such as, for example, VP1). A decrease in the amount of such structural proteins results in a decrease in the quality and quantity of the viral capsid, and thus a decrease in the overall quality and quantity of the therapeutic agent.

[0170] In some embodiments, the virus-producing cell comprises a first nucleic acid, which further comprises a first Kozak sequence.

[0171] In some embodiments, such virus-producing cells comprise a first engineered Kozak sequence. In some embodiments, the first engineered Kozak sequence has at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more, or 100%) identity to any of the nucleic acid sequences of SEQ ID NOs: 11-191 or a functional fragment and / or functional derivative thereof.

[0172] In some embodiments, the virus-producing cells comprise a second nucleic acid, which further comprises a second engineered Kozak sequence. In some embodiments, the second engineered Kozak sequence has at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more, or 100%) identity to any of the nucleic acid sequences of SEQ ID NOs: 11-191 or a functional fragment and / or functional derivative thereof. In some embodiments, the first and second engineered Kozak sequences are the same. In some embodiments, the first and second engineered Kozak sequences are different. In some embodiments, the first Kozak sequence is unengineered and the second Kozak sequence is engineered.

[0173] In some embodiments, the Kozak sequence comprises or consists essentially of a polynucleotide sequence comprising five terminal residues as shown by AXGYY, where X = U or T, and Y = A, G, C, or T / U. In some embodiments, the Kozak sequence can be a "leaky" Kozak sequence and comprises the formula ACGYY, where the ACG region can result in "readthrough" such that the ribosome does not bind as frequently and / or strongly as when a Kozak sequence comprising the sequence ATGYY is present, where ATG confers a "stronger" Kozak sequence. In some embodiments, the Kozak sequence comprises a polynucleotide having a sequence comprising five terminal residues as shown by AXGGC (SEQ ID NO:217), where X = U or T. In some embodiments, constructs comprising, consisting essentially of, or consisting of a Kozak sequence comprising "AXGGC" may be desirable compared to a Kozak sequence comprising AXGYY. In some embodiments, the Kozak sequence comprises or consists essentially of a polynucleotide sequence comprising five terminal residues as shown by AXGYY, where X = U or T, and Y = A, G, C, or T / U. In some embodiments, an engineered Kozak sequence has the formula XXXXXXATGYY, where X is any nucleotide and Y is G or C. In some embodiments, an engineered Kozak sequence has the formula XXXXXXATGXX, where X is A, G, C, or T / U. In some embodiments, the Kozak sequence is not a suboptimal sequence, such as a sequence comprising a "leaky" Kozak (such as ACG).

[0174] Without being bound by theory, in some such embodiments, in a construct encoding a polypeptide, such a sequence as shown in the terminal portion of the Kozak sequence is capable of preserving the amino acid sequence of one or more polypeptides (e.g., Rep, e.g., Cap, e.g., AAV Rep, e.g., AAV Cap, etc.). In some embodiments, the Kozak sequence is modified to encode a non-wild-type amino acid sequence, such as non-wild-type Rep and / or non-wild-type Cap (e.g., e.g., by altering the second amino acid encoded by Kozak). In some embodiments, the strategies provided by the present disclosure more faithfully recapitulate and / or preserve certain features of AAV, for example, by not altering the amino acids near the start codon of a given construct (e.g., Rep construct, e.g., Cap construct, etc.).

[0175] In some embodiments, as an alternative or supplement to the Kozak sequence (i.e., optionally modified if present), the construct can include one or more artificial introns. As is known to those skilled in the art (see, e.g., Mol Ther. May 2008; 16(5):924-30; U.S. Patent No. 8,945,918), one or more artificial intron sequences can be used to drive gene expression of one or more viral (e.g., AAV) components (such as the rep and cap genes) in insect cells. In such a method, the artificial intron is arranged in a nucleic acid sequence that includes, for example, an artificial intron that also includes an insect cell (e.g., Sf9) promoter (e.g., polH, such as p10, etc.). Such an arrangement can be organized such that the artificial intron facilitates the translation of genes having overlapping open reading frames (such as rep and cap), such that different splice forms of the protein are translated when the intron is present compared to when the intron is spliced out (e.g., when present, VP1 of cap is translated, and when spliced out, VP2 and VP3 are translated).

[0176] In some embodiments, the present disclosure contemplates that engineered Kozak sequences are preferably used to regulate Cap expression compared to artificial introns.

[0177] Depending on the AAV serotype used, certain engineered Kozak sequences may be preferred. Table 1 shows exemplary engineered Kozak sequences used in certain AAV serotypes.

[0178] Table 1. Exemplary Engineered Kozak Sequences and AAV Serotypes

[0179]

[0180] Target gene

[0181] In some embodiments, virus-producing cells having the engineered genomes described herein contain a third nucleic acid comprising the GOI. In some embodiments, the GOI is flanked by a first ITR sequence and a second ITR sequence. In some embodiments, the first ITR and / or the second ITR sequence is a wild-type ITR sequence. In some embodiments, the first and / or second ITR sequences are modified relative to the wild-type sequence. In some embodiments, the first and / or second ITR sequences are derived from the same serotype. In some embodiments, the first and / or second ITR sequences are derived from different serotypes. In some embodiments, the first ITR sequence and the second ITR sequence are the same. In some embodiments, the first ITR sequence and the second ITR sequence are different. In some embodiments, the first ITR and the second ITR sequences are asymmetric. For example, in some embodiments, the first ITR may be longer than the second ITR, and vice versa. In some embodiments, one or both of the ITRs can be a "mutant" ITR (e.g., modified relative to the ITRs found in nature, such as deletion of certain portions, such as "trs" mutant ITRs, etc.).

[0182] In some embodiments, the third nucleic acid comprising the GOI further comprises a promoter (and optionally further comprises one or more of an intron, microRNA, linker, splicing element, polyA signal, or any combination thereof). In some embodiments, the promoter is selected from constitutive promoters, inducible promoters, mini-promoters, or functional fragments and / or functional derivatives thereof. In some embodiments, the promoter is selected from: CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p10, p19, p40, synapsin, CaMKII, GRK1, polH, EM7, OpIE1, and any functional fragments and / or functional derivatives thereof.

[0183] The present disclosure contemplates that in some embodiments, it may be preferable to reduce the amount of host DNA excised from the plasmid genome upon excision of the ITR-GOI cassette. As provided herein, the present disclosure contemplates that a way to reduce the excess plasmid / prokaryotic DNA around the ITR-GOI cassette is to use a restriction enzyme digest to remove the host DNA flanking the ITR-GOI-ITR cassette (see, for example Figure 13 ). Figure 13Depicts a simplified diagram of a cassette in which two ITRs (each of which can be or not be a mutated or modified ITR and can be the same or different) flank the GOI. When the plasmid containing the construct is linearized into monomers, the enzyme is also used to digest away additional genomic sequences outside of the ITR-GOI-ITR cassette. In some embodiments, the total amount of plasmid / prokaryotic DNA of the outer segment of the ITR (the side not containing the GOI) contains less than about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 nucleotides. The amount of DNA on either ITR flank (outside of the GOI) does not have to be of the same length - for example, in some embodiments, the DNA on one ITR flank may be longer or shorter than the DNA on the other ITR flank. In some embodiments, the flank DNA on the 5' end of the GOI cassette may be about 100, 200, 300, 400, 500, 600, or 700 nucleotides, and / or the flank DNA on the 3' end of the GOI cassette may be about 50, 60, 70, 80, 100, 200, 300, 400, 500, 750, 1000, 1250, 1500, or 1750 nucleotides. Having less flank DNA (e.g., from plasmid / prokaryotic sources) may be desirable for downstream purification processes and clinical manufacturing processes.

[0184] Induced virus

[0185] In some embodiments, virus-producing cells are described herein, wherein the cells are infected with a virus to induce the expression of first, second, and / or third nucleic acids stably integrated into the host virus-producing cells. In some embodiments, the virus is selected from alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpes virus, poxvirus, circovirus, bocavirus, vaccinia virus, or retrovirus. In some embodiments, the virus is a wild-type virus. In some embodiments, the wild-type virus is a wild-type baculovirus. In some embodiments, a wild-type virus refers to a virus that is different from the virus found in nature but is not a recombinant virus and / or does not contain recombinant components (such as GOI or ITR, etc.). In some embodiments, a wild-type virus is a virus that does not contain any foreign genes and / or foreign sequences (such as GOI, such as ITR). In some embodiments, a wild-type virus is a baculovirus that contains foreign genes (such as antibiotic resistance genes, detectable marker genes, etc.) but does not contain genes designed to be inserted into engineered cells (such as GOI, such as ITR). In some embodiments, a wild-type virus is a baculovirus engineered to remove certain endogenous functions (such as enzyme activity / protease function), for example, by removing or inactivating certain proteases (such as ChIA or vCath). In some embodiments, the virus can be recombinant but does not contain any heterologous or foreign components designed to be inserted into cells, such as, for example, AAV elements for insertion into host cells (such as insect cells). In some embodiments, the virus is not a recombinant virus.

[0186] In some embodiments, infection induces the expression of one or more stably integrated nucleic acid sequences described herein. In some embodiments, infection produces a recombinant virus. In some embodiments, the recombinant virus is selected from: alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, circovirus, bocavirus, vaccinia virus, and retrovirus. In some embodiments, the AAV is mammalian (e.g., human, e.g., non-human primate) AAV or avian AAV (AAAV); that is, in some embodiments, the starting and / or engineered AAV is or is derived from a virus capable of infecting a mammalian or avian organism. In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivatives thereof.

[0187] In some embodiments, the virus-producing cells described herein comprise rAAV vectors produced with improved quality (e.g., reduced contaminants, improved potency, etc.). In some embodiments, the rAAV vector comprises less than 5%, 4%, 3%, 2%, or 1% contaminants from non-AAV components and the GOI. In some embodiments, the contaminants are from alphavirus, parvovirus, baculovirus, dengue virus, lentivirus, poxvirus, circovirus, bocavirus, vaccinia virus, herpesvirus, or retrovirus. In some embodiments, the contaminants are from baculovirus. In some embodiments, the contaminants may be from host cell genomic components (e.g., insect cell genomic contaminants).

[0188] Manufacturing method

[0189] Methods for generating recombinant viruses, such as rAAV vectors for gene therapy, face difficulties in large-scale manufacturing and production. Specifically, problems arise due to low vector concentration, oncogenic host cell DNA, and difficulty in purifying rAAV vectors from cells. Thus, improved strategies are needed to develop large-scale manufacturing solutions and provide commercially viable AAV products for a large clinical population. In certain embodiments, provided herein are virus-producing cells with a stably integrated genome that contains the components required for rAAV production. These cells can also contain components designed to operate in insect cells, such as hr enhancer elements and promoters (e.g., polH, p10, etc.). That is, as provided herein, among other things, the present disclosure provides virus cells in which all components for preparing a recombinant gene therapy (e.g., rAAV) are added (by transformation) to the genome of a single cell (e.g., Sf9 cell), and no component of the therapeutic agent (e.g., ITR-GOI) is introduced via, for example, a recombinant baculovirus. Instead, all components are present in the virus cell prior to infection such that baculovirus infection (e.g., with wild-type baculovirus or other baculovirus that does not contain any ITR or GOI components) is all that is required to induce the production of recombinant virus (e.g., rAAV).

[0190] In some embodiments, provided herein is further a method for producing a recombinant virus comprising an rAAV vector using the virus-producing cells described herein, which method requires fewer steps (e.g., multiple transfections) and results in improved scalability, quality, and potency.

[0191] Also provided herein is a method for generating an rAAV vector, which comprises: (a) providing a virus-producing cell with an engineered genome, wherein the cell comprises or consists essentially of: (i) a first nucleic acid comprising a first enhancer sequence and a Rep gene or a functional fragment thereof; (ii) a second nucleic acid comprising a second enhancer sequence and a Cap gene or a functional fragment thereof; and (iii) a third nucleic acid comprising a GOI; (b) contacting the cell of step (a) with a virus; and (c) culturing the cell after step (b) to produce a recombinant virus comprising an rAAV vector.

[0192] In some embodiments, the virus-producing cell comprises one or more of a first nucleic acid, a second nucleic acid, a third nucleic acid, or a combination thereof stably integrated into the cell genome. In some embodiments, the virus-producing cell comprises a first nucleic acid, a second nucleic acid, and a third nucleic acid stably integrated into the cell genome. In some embodiments, the virus-producing cell is inducible.

[0193] In some embodiments, virus-producing cells are selected based on productivity by infecting each clonal population with a recombinant viral expression vector (e.g., BEV) that does not contain any AAV elements. In some embodiments, clones are screened under antibiotic-free and / or serum-free conditions. Infection can initiate the expression of the AAV Rep and AAV Cap genes that integrate into the genome. Expression of these genes can rescue the integrated AAV genome and package it into assembled AAV capsids. After the production phase, AAV can subsequently be harvested from the cell monolayer by freeze-thaw and nuclease treatment (or in some embodiments, from suspension cultures by salt and / or detergent lysis), and the resulting AAV content produced by each clonal population is separate and can optionally be quantified by PCR. (See Figures 2A - 2E for a schematic overview).

[0194] In some embodiments, the clones are subsequently expanded to a production bioreactor to produce recombinant viral vectors. In some embodiments, the recombinant viral vectors (e.g., rAAV) are subsequently harvested, any active virus is inactivated (e.g., by heating) and / or removed (e.g., with enzymes and / or physical separation), and the virus particles are purified. In some embodiments, the recombinant viral vectors are purified and formulated. In some embodiments, the contents of the virus particles are released and further processed (e.g., processed, e.g., purified).

[0195] In some embodiments, a suitable culture medium is used to produce the recombinant vector. These culture media include, but are not limited to, media suitable for the cell type (e.g., mammalian, insect, etc.), such as, for example, media produced by Hyclone Laboratories and JRH, which include Modified Eagle Medium (MEM), Roswell Park Memorial Institute (RPMI) 1640, Eagle's Minimum Essential Medium (EMEM), Dulbecco's Modified Eagle Medium (DMEM), ExpiSf-CD Medium (Thermo Fisher Scientific), Sf-900 II (Thermo Fisher Scientific), Sf-900 III (Thermo Fisher Scientific), ESF-AF (Expression Systems), IS SfInsectACF (FUJIFILM Irvine Scientific), 4Cell Insect Medium (Sartorius), Hyclone SFX (Cytiva Life Sciences), EX-Cell (Sigma Aldrich), and / or custom formulations, particularly with respect to custom culture medium formulations for the production of recombinant vectors.

[0196] In some embodiments, the suitable production medium of the present disclosure is supplemented with a serum or serum-derived recombinant protein at a level of 0.5 - 20 (v / v or w / v). In some embodiments, the vector is produced under serum-free conditions, which are also referred to as media free of animal-derived products. In some embodiments, the medium can be chemically defined. In some embodiments, commercial or custom media are designed to support the production of the vector, including, but not limited to, supplementation with glucose, vitamins, amino acids, and / or growth factors to increase the titer and / or yield of the vector in the production culture.

[0197] Vector production cultures include a variety of conditions suitable for the particular host cell utilized (e.g., over a wide temperature range, for different lengths of time, etc.). Vector production cultures include adherent-dependent cultures that are cultured in suitable adherent-dependent vessels such as, for example, plates, flasks, cell stacks, roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors. In some embodiments, the vector production culture includes suspension-adapted host cells such as HeLa, HEK-293, and SF-9 cells, which are cultured in a variety of ways including, for example, roller bottles, stirred-tank bioreactors, single-use bioreactors such as Cytiva Xcellerex and Sartorius, and disposable systems such as Wave bag systems.

[0198] In some embodiments, the viral particles of the present disclosure are harvested from the vector production culture by lysing the host cells of the production culture or by harvesting spent medium from the production culture, provided that the cells are cultured under conditions that result in the release of the viral particles from the intact cells into the medium. Suitable methods for lysing cells include, for example, multiple freeze-thaw cycles, sonication, microfluidization, and treatment with chemicals such as detergents and / or proteases.

[0199] In further embodiments, the viral particles are purified. As used herein, the term "purified" includes a preparation of viral particles that is free of at least some of the other components in which the viral particles are naturally found or were initially present when prepared. Thus, for example, in some embodiments, the viral particles are prepared by enriching and isolating the viral particles from a source mixture such as a culture lysate or production culture supernatant using purification techniques. In some embodiments, enrichment is measured in a variety of ways such as, for example, by the ratio of DNase-resistant particles (DRP) or genomic copies (gc) present in solution, or by infectivity, or relative to a second potential interfering substance present in the source mixture, the second potential interfering substance such as contaminants including production culture contaminants or process contaminants including helper viruses, medium components, etc.

[0200] In some embodiments, the vector production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is clarified by filtration through a series of depth filters including a sterilizing grade DOHC Millipore Millistak+HC Pod filter, a sterilizing grade A1HC Millipore Millistak+HC Pod filter, and a 0.2 μm Opticap XL 10 Millipore Express SHC hydrophilic membrane filter. Clarification can also be achieved by a variety of other standard techniques such as centrifugation or filtration through any cellulose acetate filter with a pore size of 0.2 μm or greater.

[0201] In some embodiments, the vector production culture harvest is further treated with to digest any high molecular weight DNA present in the production culture. In some embodiments, the digestion is carried out under standard conditions including, for example, a temperature range from room temperature to 37 °C for a period of 30 minutes to several hours, at a final concentration of at least 1 - 2.5 units / ml (and in some embodiments, up to 50 units / ml).

[0202] In some embodiments, one or more of the following exemplary purification steps are used to isolate or purify virus particles: freeze-thaw; equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) for concentrating virus particles; capture of the vector by hydroxyapatite chromatography; helper virus heat inactivation; capture of the vector by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and capture of the vector by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. In some embodiments, these steps can be used alone, in various combinations, or in different orders. In some embodiments, the method includes all steps and optionally in the order described below.

[0203] In some embodiments, methods of generating recombinant vectors (e.g., rAAV) include providing a stable integration viral production cell with an auxiliary plasmid. In some embodiments, cells are transfected with an auxiliary plasmid that provides auxiliary functions for AAV. In some embodiments, the auxiliary plasmid provides adenovirus functions, including but not limited to E1A, E1B, E4, and E2A. In some embodiments, the auxiliary plasmid provides other viral functions, including but not limited to VA RNA, Gag, Pol, Tat, Rev, Env, and VSV-G. In some embodiments, the adenovirus gene sequences providing these functions are obtained from any known adenovirus serotype, such as serotypes 2, 3, 4, 7, 12, and 40, and further include any currently identified human types. In some embodiments, these methods involve transfecting cells with a vector expressing one or more genes required for AAV replication, AAV gene transcription, and / or AAV packaging.

[0204] The present disclosure also provides methods for generating recombinant vectors, wherein the method includes providing a viral production cell containing an engineered genome under the control of a promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, these cells are viral production cells containing selected components under the control of a constitutive promoter and other selected components under the control of one or more inducible promoters. For example, the generated viral production cells contain E1 auxiliary functions under the control of a constitutive promoter, but contain Rep and / or Cap proteins under the control of one or more inducible promoters.

[0205] Products produced using the platforms provided herein (e.g., in Sf9 cells) can also be compared with products produced using standard platforms (e.g., mammalian cell systems (e.g., HEK293 cells)). In such systems, appropriate rep and cap genes can be transfected into mammalian cells on a single plasmid (e.g., pRep / cap) or using a single plasmid, each plasmid encoding the rep and cap genes. Whether on a single plasmid or more than one plasmid, the transfected material contains sequences for producing a viral product (e.g., rAAV) containing a gene of interest. Typically, the gene of interest can be introduced into mammalian cells via a separate plasmid containing the ITR-GOI component (e.g., in addition to one or more plasmids encoding rep and / or cap), such that when the cells containing ITR-GOI express the rep and cap components, they produce a capsidated viral gene product containing the gene of interest.

[0206] General methods for transfecting, transforming, and infecting mammalian cells to produce virus (e.g., AAV)-based gene therapies are known in the art. As will be apparent to those skilled in the art, depending on the circumstances, the specific rep and cap genes and their regulatory sequences of the target serotype will be endogenous sequences of the specific serotype or sequences corresponding to the specific capsid (e.g., AAV6TM, AAV7TM, AAV8TM, etc.). Such plasmids will be plasmids that facilitate expression in the mammalian system and generally do not include engineered Kozaks such as those used in the insect cell system disclosed herein. The general principles of recombinant AAV production are reviewed, for example, in Carter, 1992, Current Opinions in Biotechnology, 1533 - 539; and Muzyczka, 1992, Curr. Topics in Microbial. and Immunol., 158:97 - 129. Various methods are also described in: Ratschin et al., Mol, Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA. 81:6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al., (1989, J. Virol., 633822 - 3828): U.S. Pat. No. 5,173,414: WO 95 / 13365 and corresponding U.S. Pat. No. 5,658,776; WO 95 / 13392; WO96 / 17947; PCT / US98 / 18600; WO 97 / 09441 (PCT / US96.4423); WO 97 / 08298 (PCT / US96 / 13872); WO 97 / 21825 (PCT / US96 / 20777): WO 97 / 06243 (PCT / FR96 / 01064); WO99 / 11764; Perrin et al., (1995) Vaccine 13:1244 - 1250; Paul et al., (1993) Human Gene Therapy 4:609 - 615; Clark et al., (1996) Gene Therapy 3:1124 - 1132; U.S. Pat. Nos. 5,786,211; 5,871,982; and 6,258,595.

[0207] The methods disclosed herein result in improved manufacture of recombinant viral vectors, including improved quantity of recombinant virus, more efficient and faster production times, and higher reproducibility and scalability, without compromising the efficacy of the recombinant viral products produced.

[0208] In some embodiments, the present disclosure provides an improved method for manufacturing a recombinant viral product, the improvement comprising combining three stably integrated components into a single host cell, wherein the three stably integrated components comprise: a first nucleic acid comprising a first enhancer sequence and a Rep gene or a fragment thereof; a second nucleic acid comprising a second enhancer sequence and a Cap gene or a fragment thereof; and a third nucleic acid comprising a GOI. In some such embodiments, any of the nucleic acids further comprises an enhancer sequence and / or an engineered Kozak sequence. After stable integration into the host cell genome, the host cell is contacted with an inducing virus, thereby activating the integrated components and resulting in the production of a recombinant virus comprising the GOI.

[0209] For example, additional details regarding the preparation and use of recombinant viruses containing a gene of interest (including AAV) can be found in PCT Publication Nos. WO 2010 / 114948 and WO 2017 / 181162.

[0210] Throughout the specification, when a composition is described as having, including, or comprising a particular component, or when a process and method are described as having, including, or comprising a particular step, furthermore, and even if not explicitly recited therein, it is contemplated that there are compositions of the present disclosure consisting essentially of or consisting of the components, and / or there are processes and methods according to the present disclosure consisting essentially of or consisting of the processing steps, and these steps can occur in any order unless so specified.

[0211] Any and all examples or exemplary language used herein, such as "such as" or "including", are merely intended to better illustrate the present disclosure and do not, unless otherwise stated, constitute a limitation on the scope of the present disclosure. No language in the specification should be construed as indicating that any unrecited element is essential for the practice of any embodiment of the present disclosure.

[0212] When an element or component is considered to be included in and / or selected from a list of such elements or components, it should be understood that the element or component can be any one of the elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0213] In addition, it should be understood that the elements and / or features of the compositions or methods provided herein can be combined in a variety of ways without departing from the spirit and scope of anything (whether expressly or implicitly) disclosed herein. For example, when referring to a particular compound, unless the context otherwise dictates, that compound can be used in various embodiments of the compositions and / or methods of the present disclosure. In other words, within the present application, the embodiments have been described and depicted in a manner that enables the writing and drawing of a clear and concise application, but it is intended and will be understood that the embodiments can be combined or separated differently without departing from the present disclosure. For example, it will be understood that all of the features described and depicted herein can be applied to all aspects of any invention provided, described, and / or depicted herein.

[0214] It should be understood that the order of steps or the order of performing certain operations is not important, so long as what is disclosed and / or claimed remains operable regardless of the order. In addition, two or more steps or operations can be carried out simultaneously.

[0215] The present disclosure provides multiple aspects and embodiments of one or more inventions, which specifically contemplate any and all combinations and permutations of the aspects and embodiments disclosed herein.

[0216] Pharmaceutical composition

[0217] Once generated, the recombinant AAV particles provided herein can be formulated into a pharmaceutical composition.

[0218] For therapeutic use, a composition comprising a recombinant virus provided herein is combined with a pharmaceutically acceptable carrier. A variety of carriers (e.g., diluents, excipients, etc.) for formulating and preparing pharmaceutical compositions are known and / or readily available to those skilled in the art. Depending on the circumstances, the carrier can include a liquid (e.g., a sterile liquid) or a solid. The carrier can be selected from or comprise the following substances that are compatible with drug administration: water, aqueous solvents, non-aqueous solvents, dispersion media, surfactants, antioxidants, buffers, adjuvants, tonicity agents, stabilizers, bulking agents, lyoprotectants, metal ions, chelating agents, isosmotic agents, and absorption retardants, among others. The use of such media and reagents for pharmaceutically active substances is known in the art. Generally, the carrier is approved by the U.S. Food and Drug Administration (FDA) and complies with the standards of the United States Pharmacopeia (USP), European Pharmacopeia (EP), British Pharmacopeia, and / or other international pharmacopeias. Formulations suitable for the present disclosure can be found in, for example, Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rd Edition, 2020). For a brief review of drug delivery methods, see, for example, Langer (1990) SCIENCE 249:1527-1533. The resulting pharmaceutical composition is suitable for administration to a subject (such as an animal, such as a mammal, such as a human).

[0219] The pharmaceutical composition may contain formulation materials for regulating, maintaining or preserving, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or permeability of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrin); proteins (such as serum albumin, gelatin or immunoglobulins); coloring agents, flavoring agents and diluents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerol, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as Pluronics, polyethylene glycol (PEG), sorbitan esters, polysorbates (such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapol)); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol sorbitol); delivery carriers; diluents; excipients and / or pharmaceutical aids (see, for example, Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rd Edition, 2020)).

[0220] In certain embodiments, the pharmaceutical composition may contain a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled-delivery vehicles, such as liposome carriers, bioerodible microparticles or porous beads, and depot injection, are also known to those skilled in the art. Sustained-release formulations may include, for example, porous polymer microparticles or semipermeable polymer matrices in the form of shaped articles such as membranes or microcapsules. Sustained-release matrices may include polyesters, hydrogels, polylactic acid, copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, poly(2-hydroxyethyl-methacrylate), ethylene-vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained-release compositions may also contain liposomes, which can be prepared by any of several methods known in the art.

[0221] Optionally, the pharmaceutical composition may contain nanoparticles or lipid droplets, such as polymeric nanoparticles, liposomes, or micelles (see Anselmo et al., (2016) B IOENG .T RANSL .M ED .1:10-29).

[0222] The pharmaceutical composition containing the rAAV of the present disclosure may be in unit dosage form and may be prepared by any suitable method. The formulation of the pharmaceutical composition should be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intraperitoneal, intradermal, inhalation, transdermal, intracerebroventricular (ICV), intracerebral parenchyma, intracisternal magna (ICM), intrathecal, intradural, etc.

[0223] The available formulations can be prepared by methods known in the pharmaceutical art. See, for example, Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rd Edition, 2020). Components of formulations suitable for parenteral administration include sterile diluents such as water for injection, normal saline, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetate, citrate, or phosphate; and agents for adjusting tonicity such as sodium chloride or dextrose.

[0224] Suitable carriers will be known to those skilled in the art. For example, for intravenous administration, suitable carriers include normal saline, bacteriostatic water, polyethoxylated castor oil, or phosphate buffered saline (PBS). The carrier should be stable under manufacturing and storage conditions and should prevent the growth of microorganisms. The carrier can be a solvent or a dispersion medium that contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In the case of injection into the central nervous system (e.g., ICV, ICM, intracerebral, intrathecal, etc.), the carrier will be appropriately adjusted.

[0225] The pharmaceutical formulation is preferably sterile. The formulation or its components can be sterilized, for example, by appropriate methods that maintain the activity and stability of the GOI encoded therein. Sterilization can be accomplished by any suitable method, such as filtration through a sterile filter membrane. If the composition is lyophilized, filter sterilization can be performed before or after lyophilization and reconstitution.

[0226] Depending on the drug substance and the formulation, when the dosage form is liquid or solid, the resulting dosage form can be stable for an extended period, such as 1 month, 3 months, 6 months, 1 year, 2 years, 3 years, or longer. The formulation can be stable at room temperature or higher temperatures. The dosage form is expected to be stable in PBS under ambient conditions. Alternatively, the dosage form can be frozen (e.g., liquid or lyophilized) and stable at an appropriate temperature, such as, for example, -20°C, -80°C). As appropriate, the dosage form can be formulated as a unit dose, which can include, for example, a specific vg / L as provided herein.

[0227] The compositions described herein can be administered locally or systemically. It is expected that the compositions described herein can be administered by parenteral administration. In some embodiments, this administration is preferably directly into the central nervous system. Formulations for injection administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. In certain embodiments, the pharmaceutical composition can be administered subcutaneously or can be administered intravenously, such as via intravenous infusion. In certain embodiments, it is expected that the gene therapy disclosed herein can be administered by systemic administration.

[0228] The dosage will depend on variables such as the type and extent of the disease or indication to be treated, the overall health of the patient, the in vivo potency of the active ingredient, and any toxicity issues, the pharmaceutical formulation, and the route of administration. The initial dose can be increased beyond the upper limit in order to rapidly achieve the desired blood level or tissue level. Alternatively, the initial dose can be less than the optimal dose, and the daily dose can be gradually increased during the course of treatment. Human doses can be optimized, for example, in conventional Phase I dose escalation studies. The dosing frequency may vary depending on factors such as the route of administration, the dose, and the disease, disorder, or condition being treated. Exemplary dosing frequencies are once daily, once weekly, and once every two weeks.

[0229] Use and treatment methods

[0230] The rAAV provided herein can be used in a variety of different methods. For example, rAAV can be used in methods for treating diseases, disorders or conditions associated with a dysfunctional target gene. The method includes contacting cells in a subject in need thereof, wherein the composition comprises a nucleic acid encoding a target gene, which when expressed will treat a disease, disorder or condition associated with the dysfunctional GOI.

[0231] In some embodiments, the disease, disorder or condition is associated with a dysfunctional gene that expresses or affects the function of one or more cells of the central nervous system and / or the peripheral nervous system.

[0232] It is contemplated that the treatment can be accomplished using rAAV alone, as a monotherapy or as part of a combination therapy. The combination therapy can include one or more additional drugs or treatment methods known to those skilled in the art for treating inflammatory and / or autoimmune diseases and that may have been previously used, are ongoing, or are added to the treatment of a subject in need thereof.

[0233] For example, a subject can be evaluated by a healthcare provider before, during and / or after treatment with the compositions provided herein. Based on the evaluation results, the treatment can be continued or stopped, the treatment frequency or dose can be changed, or the patient can be treated with a different gene therapy. According to the dosage regimens described herein, a composition comprising a gene therapy as provided herein can be administered to a subject in discrete time periods, including optionally until the disease, disorder or condition is treated.

[0234] Incorporation by reference

[0235] All publications and patents (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.) cited throughout this specification, whether above or below, are hereby incorporated by reference in their entirety for all purposes. To the extent that the material incorporated by reference conflicts or is inconsistent with this specification, this specification will supersede any such material.

[0236] Equivalents

[0237] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Accordingly, the foregoing embodiments are to be considered illustrative in all respects of the invention described herein rather than limiting. Thus, the scope of the invention is indicated by the appended claims rather than the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein. Examples

[0238] The following are examples of specific implementation schemes for implementing the present disclosure. These examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way.

[0239] Example 1. Production of AAV using an engineered cell line with stably integrated exogenous viral components

[0240] This example generally describes the production of AAV using the engineered cell line described herein. An engineered cell line was produced to stably integrate AAV components into Sf9 cells. The engineered cell contains the AAV Cap component, the AAV Rep component, and the GOI, which are stably integrated into the genome of the cell. The Cap, Rep, and GOI components were integrated into the genome by applying an engineered construct. Multiple AAV serotypes were tested.

[0241] The Kozak-optimized AAV capsid sequence, the AAV Rep sequence, and the GOI were stably integrated into Sf9 cells by random integration and antibiotic selection, generating a heterogeneous library of stably transformed Sf9 production cells. This is further described in Example 2.

[0242] For each serotype, the Kozak sequence was first screened by SDS-PAGE and Western blotting to obtain a series of capsid stoichiometries. The major candidate sequences incorporating enhanced VP1 into the AAV capsid were cloned into a cell line integration vector together with enhancer elements.

[0243] Clonal Sf9 cell lines were derived from the heterogeneous library by methods of single-cell seeding and whole-well imaging. Fast-growing clones showing favorable growth kinetics were scaled up for productivity screening. This is further described in Example 3.

[0244] The clones were screened for productivity based on infecting with baculovirus and harvesting the AAV produced by each clone. The clones producing the highest amount of AAV were further scaled up to analyze product attributes and improve production parameters.

[0245] Infection parameters were determined using a small stirred-tank bioreactor. This production scale was sufficient to examine the process and product critical quality attributes of AAV derived from each clonal cell line.

[0246] The AAVs produced using the engineered Sf9 production cell line platform and the mammalian HEK293 triple transfection platform were purified and evaluated head-to-head in a rodent model. This will be further described in Example 4.

[0247] Example 2: Isolation and characterization of Sf9 production cell lines

[0248] As described in Example 1, Sf9 production cell lines were designed to improve productivity and potency. These production cells were engineered to produce AAV using homologous region (hr) enhancer elements and engineered Kozak sequences. An exemplary construct design is as Figure 1A shown. The hr elements used in this example are native to the baculovirus genome, where they act as transcriptional enhancers adjacent to transgenes. This mechanism was retained and incorporated into the Sf9 cell genome in the form of DNA concatemers encoding AAV Rep and AAV Cap proteins. Engineered Kozak sequences were screened and compared to determine their ability to incorporate higher levels of VP1 into the AAV capsid. These higher levels of VP1 may contribute to a higher likelihood of endosomal escape and increased capsid potency. As Figure 1B (western blot showing AAV1 and AAV5 capsids) and Figure 1C (affinity-purified AAV7, AAV8, and AAV9 capsids) demonstrate, this engineering approach works across AAV serotypes.

[0249] Clonal populations of production cells were generated by single cell seeding and whole-well imaging. Random integration of DNA concatemers and linearized ITR-flanked transgene constructs was performed by DNA transfection into Sf9 cells and antibiotic selection of stable transformants. The scale of the heterogeneous library of stably transformed Sf9-AAV production cell lines was expanded to facilitate initial testing of the ability to produce and purify AAV products using these engineered cells. Clonal populations of these Sf9-AAV production cell lines were isolated by single cell seeding and whole-well imaging. Validated clonal populations were monitored over a period of 3 - 4 weeks before transfer to 96-well plates for head-to-head screening.

[0250] The production cell lines produced as described herein were subjected to productivity-based screening by infecting each clonal population with a recombinant baculovirus expression vector (BEV) that does not contain any AAV elements. As Figures 2A - 2E shown, clones were seeded into well plates ( Figure 2A ) and allowed to grow into confluent monolayers ( Figure 2B and 2C ). The plates were imaged prior to infection to determine confluency. The BEV contains a visual reporter for easy detection and also does not contain naturally occurring proteases (e.g., VCath, ChIA). Visualization allows purification (prior to infection) as well as monitoring of the infection rate (e.g., monitoring how many cells show the detectable marker). In most cases, 90% - 95% of the cells fluoresce. Clones were screened under antibiotic and serum-free conditions, reflecting the conditions of a mature manufacturing process.

[0251] After performing a baseline measurement of confluence to estimate cell number, each monolayer consisting of cells from a monoclonal was infected with a "WT" BEV (meaning it has no AAV elements such as ITRs and / or GOIs). Figure 2C ) Infection triggered the expression of the genomically integrated AAV Rep and AAV Cap genes and induced AAV production. Figure 2C )

[0252] Expression of these genes led to the rescue of the integrated AAV genome and the packaging of the gene of interest (GOI) into assembled AAV capsids.

[0253] After the production phase, AAV was harvested from the cell monolayer using freeze-thaw and nuclease treatment. Briefly, the medium (including any BEV and any unpackaged components) was discarded, Figure 2D ) and AAV was isolated from the cell monolayer (by freeze / thaw cycles and nuclease treatment), and subsequently the cell debris was separated / clarified from the AAV, Figure 2E ) thereby separating the supernatant containing BEV and AAV from the cell monolayer. As shown in the schematic of the right panel, the resulting AAV content produced by each clone population was quantified by PCR. Figure 2E

[0254] To identify high-producing clones, Sf9 AAV production candidates were screened based on the amount of AAV produced by the production candidates. Prior to optimization, the adherent clone screening assay was improved to select production clones with a yield >E14 vg / L in a more mature suspension-based manufacturing process. That is, although the screening was performed in adherent cultures, clones with yields in the E10 vg / mL range were generated during the screening assay, and prior to optimization, in a more mature suspension-based manufacturing process, the yields were typically in the high E13 - low E14 vg / L range. The identified top producers (see Figure 3 ) were scaled up and further analyzed for product analysis and production optimization.

[0255] An amplification method was developed to reduce the number of passages required to convert cells from 96-well adherent cultures to suspension. This greatly accelerated cell growth and reduced the time required to establish a suspension-ready research cell bank, as can be seen in Figure 4 . Using this amplification method to accelerate growth resulted in doubling the cell number in half the time (i.e., a 4-fold increase). The process from transfection, clone screening, AAV product analysis, and expansion of the RCB bank takes approximately 3 months.

[0256] Example 3. Sf9 Quality Analysis

[0257] ​This example describes the quality of Sf9 cells produced according to the systems, methods, and compositions (e.g., engineered cells) provided herein.

[0258] Sf9-derived AAV was purified by affinity capture, and the results showed that the stoichiometry of the capsid was maintained and the level of VP1 incorporation into the purified capsid was increased. An anion exchange method has been developed for enriching genome-containing capsids, and the current enrichment rate is 70%-80%, depending on the serotype. The downstream purification method removed host cell proteins in addition to removing host cell and baculovirus DNA impurities from the final AAV preparation.

[0259] Figures 5A - 5D Measurements of the amounts of host cell proteins (HCP) and activated virus present during production and before and after purification were shown, demonstrating that both host cell proteins and activated virus impurities were reduced during purification, and the increased stoichiometry did not affect the ability to purify recombinant virus from host cells or other contaminants from viruses involved in manufacturing (e.g., activated baculovirus). Figure 5A and 5B are gels showing the reduction of impurities during purification. Figure 5C is a bar graph showing the HCP (in ng / mL) detected in fractions of various purification steps. Figure 5D is a bar graph showing the DNA concentrations of Sf9 and BEV DNA detected in the eluates from AAVX and AEX column purifications; the left bar on each of AAVX and AEX is from Sf9 DNA measurements, while the right bar is from BEV DNA measurements. These data confirm that the final products provided by the systems, methods, and cells herein have at least the same biophysical and biochemical properties as the final products traditionally obtained using other (e.g., HEK293) cell systems, and existing equipment (e.g., AEX columns, affinity purification columns, etc.) can be successfully used to purify viruses produced using the methods provided herein. Since Sf9 cells are a heterogeneous population of rhabdovirus-positive and rhabdovirus-negative cells, the production cell line was screened for the presence of rhabdovirus. During single cell isolation and clone screening, Sf9 AAV production cell lines free of rhabdovirus were identified, thus eliminating the presence of rhabdovirus during the manufacturing process, as seen in Table 2.

[0260] Table 2. Rhabdovirus status of AAV production cell lines

[0261]

[0262]

[0263] The Sf9 AAV production cell line exhibits robust manufacturing capabilities. Stability studies were conducted by reviving exemplary production clones. In this example, the exemplary production clones are independent research cell banks of Sf9 AAV5 production clones. The revived cultures underwent an additional 20 passages, where a subset of the cultures was infected at passages 5, 10, 15, and 20. AAV was harvested from each clone at each infection time point and quantitatively analyzed by droplet digital PCR (ddPCR). As Figure 6 shown, no drift in titer (vg / L) or unit (vg / cell) was observed during the 20 passages, indicating stable integration of the AAV production components.

[0264] The screening of the Sf9 AAV production cell line was based on growth and infection kinetics similar to traditional IC-BEV manufacturing methods. During the growth and infection phases, the cell density (cells / mL) and percentage of viability were measured over time. As Figure 7 shown, the stable integration of the genetic components does not affect the doubling ability (24 - 30 hours), high-density growth ability (>1x10 7 cells / mL ) of Sf9 cells, or alter their infection kinetics in response to baculovirus infection.

[0265] The post-translational modifications of AAV capsids produced on the Sf9 and HEK293 platforms (further described in Example 4) were analyzed by LCMS (see Table 3). As previously reported, alternative patterns of post-translational modifications were observed between the manufacturing methods. Next-generation Illumina sequencing was performed on purified Sf9 AAV to quantify any contaminants from the Sf9 or baculovirus genomes. As Figure 8 shown and presented in Table 3, the percentage of reads / sequences mapped to the Sf9 or baculovirus genomes is very low, which supports minimal packaging of Sf9 and baculovirus DNA in the assembled capsids. The low packaging amount of baculovirus confirms that the stable integration of the three constructs is a reliable, reproducible, safe, and improved method compared to cells integrating only one or two components. The low amount of the Sf9 genome is a particularly surprising finding considering that the cells stably integrate the Cap, Rep, and GOI-ITR constructs, as those skilled in the art would expect that integrating ITR-GOI into cells with Cap and Rep would result in a greater packaging amount of the host cell genome.

[0266] Table 3. LCMS analysis of post-translational modifications

[0267]

[0268]

[0269] ----- = No modification detected

[0270] Example 4. Virus-producing Sf9 cells with engineered genomes

[0271] This example generally describes a method for preparing virus-producing cells using an engineered genome.

[0272] First, a plasmid containing an antibiotic resistance gene (e.g., puromycin, e.g., blasticidin), an enhancer (e.g., an hr element such as any one of SEQ ID NOs: 1-10 or a functional fragment or functional derivative thereof), an engineered Kozak sequence (which can optionally be engineered and selected from any one of SEQ ID NOs: 1-191 or any functional fragment or functional derivative thereof in a construct containing a Cap sequence), a promoter (e.g., an inducible promoter), and a Rep sequence (encoded by a plasmid such as SEQ ID NO: 210) or a Cap sequence (such as a nucleic acid (SEQ ID NOs: 192-203) or a plasmid (SEQ ID NOs: 211-261) encoding a protein of any amino acid (SEQ ID NOs: 204-209)) is linearized at unique IIS-type restriction enzyme sites to generate DNA monomers.

[0273] As exemplified herein, for virus gene therapy products produced by Sf9, the plasmid for each rep and cap sequence contains the enhancer sequence of SEQ ID NO:1, a promoter (polH or p10), and an antibiotic selection cassette encoding blasticidin. The rep gene contains the rep sequence in the plasmid of SEQ ID NO:201, and each cap protein is selected by serotype and according to the sequence shown in any of SEQ ID NOs: 192-216, and the serotypes are: (AAV1: SEQ ID NO:192 (nucleic acid without engineered Kozak), SEQ ID NO:193 (nucleic acid with engineered Kozak for insect cell platform), SEQ ID NO:204 (amino acid sequence of the capsid protein); and SEQ ID NO:211 (plasmid); AAV5: SEQ ID NO:194 (nucleic acid without engineered Kozak), SEQ ID NO:195 (nucleic acid with engineered Kozak for insect cell platform), SEQ ID NO:205 (amino acid sequence of the capsid protein); and SEQ ID NO:212 (plasmid); AAV7: SEQ ID NO:196 (nucleic acid without engineered Kozak), SEQ ID NO:197 (nucleic acid with engineered Kozak for insect cell platform), SEQ ID NO:206 (amino acid sequence of the capsid protein); and SEQ ID NO:213 (plasmid); AAV7TM: SEQ ID NO:198 (nucleic acid without engineered Kozak), SEQ ID NO:199 (nucleic acid with engineered Kozak for insect cell platform), SEQ ID NO:207 (amino acid sequence of the capsid protein); and SEQ ID NO:214 (plasmid); AAV8TM: SEQ ID NO:200 (nucleic acid without engineered Kozak), SEQ ID NO:201 (nucleic acid with engineered Kozak for insect cell platform), SEQ ID NO:208 (amino acid sequence of the capsid protein); and SEQ ID NO:215 (plasmid); AAV9: SEQ ID NO:202 (nucleic acid without engineered Kozak), SEQ ID NO:203 (engineered Kozak nucleic acid for insect cell platform), SEQ ID NO:209 (amino acid sequence of the capsid protein); and SEQ ID NO:216 (plasmid)).The Cap gene transferred into the Sf9 genome further contains an engineered Kozak sequence selected from SEQ ID NO: 11 or 13 (which is also represented in the capsid sequences of SEQ ID NO: 193 (AAV1), 195 (AAV5), 197 (AAV7), 199 (AAV7TM), 201 (AAV8TM), and 203 (AAV9)).

[0274] For the insect cell platform provided herein, the DNA of each nucleic acid sequence (rep and cap) is purified and then assembled into monomers in a head-to-tail orientation by a ligation method to form high-molecular-weight concatemers. These high-molecular-weight concatemers are then further purified and quantified for transfection.

[0275] The GOI is linearized into monomers using a restriction enzyme ( Figure 9 ). Briefly, plasmid DNA containing a restriction enzyme site and engineered inverted terminal repeats flanking the nucleotide sequence encoding the gene of interest is subjected to restriction enzyme digestion. This restriction enzyme digestion process removes unwanted sequences such as antibiotic resistance markers used for propagation in bacteria. These GOI monomers (GOI flanked by ITRs) are purified and prepared for transfection to integrate them into the Sf9 genome. Restriction enzyme digestion removes a large number of plasmid DNA elements / prokaryotic DNA sequences, resulting in a total length of approximately 1700 base pairs of flanking genomic DNA.

[0276] Before transfection into Sf9 cells, the DNA was analyzed to calculate the DNA volume of each component to be transfected into Sf9 cells. At the time of transfection, Sf9 cells were first seeded at approximately 80% confluence. Sf9 cells were transfected according to the manufacturing protocol and subjected to antibiotic resistance 48 hours after transfection. Sf9 cells were transfected with DNA or sham. Cell viability was detected 48 hours after transfection. The cells were alive and able to produce AAV, which was purified and administered to mouse test subjects as shown in Example 5.

[0277] To compare with the gene therapy products produced in Sf9 cells, AAV virus particles were also produced using the HEK293 system. Using standard transfection methods known to those skilled in the art, HEK293 cells producing virus were generated using the following methods: (a) (i) endogenous rep / cap genes for each target serotype (such as AAV1, 5, 7, 8, and 9), or (ii) appropriately modified variant capsid protein sequences different from the endogenous AAV sequences (such as AAV7TM, such as AAV8TM), where the rep / cap genes are expressed on the same plasmid; and (b) the ITR-GOI nucleic acid exemplified in Example 5. The production of recombinant virus (encapsidated GOI) was induced by infecting HEK293 cells.

[0278] Example 5. In Vivo Head-to-Head Comparison of Sf9-Derived AAV vs. HEK293-Derived AAV

[0279] This example compared AAVs derived from Sf9 and HEK293 host cells. The AAV produced using the Sf9 system prepared according to the disclosure of the present invention showed similar therapeutic effects compared to the AAV prepared using HEK293 cells.

[0280] AAV5 (SEQ ID NO: 194 for mammalian (HEK) cells, SEQ ID NO: 195 for insect (Sf9) cells) encoding a single-stranded GOI (the GOI encodes an exemplary enzyme) was generated using the Sf9 platform according to the present disclosure or using the standard HEK293 platform described in Example 4 and administered via intracisternal (ICM) injection. Enzyme levels in the cerebella of wild-type, knockout, and Sf9 AAV- or HEK293 AAV-treated mice were measured. As Figure 10 shown, a head-to-head comparison of AAVs produced from Sf9 cells and HEK293 cells prepared as provided herein found no significant difference in the resulting enzyme activity levels measured in the cerebella of animals treated with capsids made using different platforms (HEK293 cells vs. Sf9 cells prepared according to the present disclosure), and the enzyme levels in each AAV treatment group were higher than those of wild-type and knockout animals in the exemplary mouse model.

[0281] Manufacture self-complementary AAV encoding an exemplary GOI, a variant of AAV7 (SEQ ID NO: 198 for mammalian (HEK) cells or SEQ ID NO: 199 for insect (Sf9) cells) using the Sf9 production cell line of the present disclosure, and compare it with the HEK293-produced AAV described in Example 4 in an exemplary disease mouse model. Head-to-head comparison of Sf9-produced AAV (containing the GOI) with AAV (containing the GOI) using the HEK293 platform showed equal biodistribution of the vector genome in the target tissue ( Figure 11A ), and efficacy in prolonging the survival of the disease model ( Figure 11B ).

[0282] As described in Example 4, use the Sf9 production cell line prepared according to the present disclosure and the Sf9 production cell line prepared using the standard HEK293 platform to manufacture a variant of AAV8 (SEQ ID NO: 200 for mammalian (HEK) cells or SEQ ID NO: 201 for insect (Sf9) cells) encoding an exemplary therapeutic GOI sequence. The AAV gene therapy products manufactured on each platform were administered to neonatal mice at the same dose. Ten weeks after administration, tissues were harvested and the resulting enzyme activity was analyzed. As shown by the enzyme levels measured in the forebrain ( Figure 12A ), hindbrain ( Figure 12B ), and cerebellum ( Figure 12C ), no difference in enzyme levels was observed after treatment with vectors (Sf9 or HEK) prepared by different manufacturing methods, or the result was that there was also no difference in the biodistribution and expression of the therapeutic transgene (not shown).

[0283] Importantly, these data also indicate that the Sf9 production system is suitable for self-complementary (see, for example, Figure 11A and 11B ) and single-stranded AAV (see, for example, Figure 12A and 12B ).

[0284] AAV9 encoding a GFP reporter gene construct (SEQ ID NO: 202 for mammalian (HEK) cells or SEQ ID NO: 203 for insect (Sf9) cells) was manufactured on the Sf9 insect cell and HEK293 mammalian cell platforms as described in Example 4. AAVs derived from three independent Sf9 clones were compared with materials derived from triple transfection of HEK293 cells. After administration into the cisterna magna (ICM) of the cerebellum, the samples were blinded and analyzed by a neuropathologist. There were no significant differences between the biodistribution profiles of AAV vectors derived from the Sf9 platform or the mammalian HEK293 platform (see Figure 13 , arrow and Table 4).

[0285] In each serotype and for each GOI, there is no difference between AAV gene therapy products produced using Sf9 insect cells as provided herein and products produced using a HEK293-based mammalian cell platform. Additionally, the insect cell-based therapy is scalable and reproducible, thus providing commercially useful amounts of AAV particles suitable for gene therapy.

[0286] These results provide multiple examples demonstrating that such insect cell-based methods can be used for various serotypes and GOIs. Thus, such insect cell methods are capable of producing virus-based gene therapies that are at least as good as mammalian cell systems, while also providing multiple advantages over such HEK293 / mammalian cell platforms, which can be used to improve the production of recombinant gene therapy products.

[0287] Table 4. Biodistribution of Sf9 vs. HEK293

[0288]

[0289]

[0290] Sequence Listing

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

Claims

1. A virus-producing cell, the virus-producing cell having an engineered genome, comprising: a) A first nucleic acid comprising a first enhancer sequence and a Rep gene or a functional fragment thereof; b) A second nucleic acid comprising a second enhancer sequence, a first Kozak sequence, and a Cap gene or a functional fragment thereof, wherein the first Kozak sequence is an engineered Kozak sequence; and c) A third nucleic acid comprising a gene of interest (GOI).

2. The cell according to claim 1, wherein the first enhancer sequence has at least 80% identity with any one of the nucleic acid sequences of SEQ ID NOs: 1-10 or any functional fragment or functional derivative thereof.

3. The cell according to claim 1 or 2, wherein the second enhancer sequence has at least 80% identity with any one of the nucleic acid sequences of SEQ ID NOs: 1-10 or any functional fragment or functional derivative thereof.

4. The cell according to any one of claims 1-3, wherein the first and second enhancer sequences are the same.

5. The cell according to any one of the preceding claims, wherein the first nucleic acid further comprises a second Kozak sequence, wherein the second Kozak sequence is an engineered Kozak sequence.

6. The cell according to any one of the preceding claims, wherein the third nucleic acid comprises a third Kozak sequence, optionally wherein the third Kozak sequence is an engineered Kozak sequence.

7. The cell according to any one of the preceding claims, wherein any one of the first, second, and / or third Kozak sequences promotes translation in insect cells and / or mammalian cells.

8. The cell according to claim 7, wherein the first and / or second Kozak sequence preferably promotes translation in insect cells.

9. The cell according to claim 7 or 8, wherein the third Kozak sequence preferably promotes translation in mammalian cells.

10. The cell according to any one of the preceding claims, wherein the cell is cloned.

11. The cell according to any one of claims 1-11, wherein the engineered Kozak sequence of the second nucleic acid comprises a sequence having at least 80% identity with any one of the sequences of SEQ ID NOs: 11-191 or any functional fragment or functional derivative thereof.

12. The cell according to any one of the preceding claims, wherein optionally the Kozak sequence of the first nucleic acid engineered to be the same as the engineered Kozak sequence of the second nucleic acid.

13. The cell according to any one of the preceding claims, wherein the cell is free of rhabdovirus.

14. The cell according to any one of the preceding claims, wherein the GOI is flanked by a first inverted terminal repeat (ITR) sequence and a second ITR sequence.

15. The cell according to claim 13, wherein each of the first ITR sequence and the second ITR sequence has a different nucleic acid sequence.

16. The cell according to claim 15 or 16, wherein the first ITR and the second ITR are derived from a viral genome, and wherein the first ITR is flanked at its 5' end and the second ITR is flanked at its 3' end by a total of about 500 nucleotides or less of the viral genome.

17. The cell according to any one of the preceding claims, wherein the Cap gene comprises a sequence having at least 80% sequence identity to any of the nucleic acid sequences of SEQ ID NOs: 192 - 203 or any functional fragment or functional derivative thereof.

18. The cell according to any one of the preceding claims, wherein the Cap gene encodes an amino acid sequence having at least about 80% sequence identity to any of the amino acid sequences of SEQ ID NOs: 204 - 209 or any functional fragment or functional derivative thereof.

19. The cell according to any one of the preceding claims, wherein: a) the first nucleic acid further comprises a promoter and optionally further comprises one or more of an intron, a microRNA, a linker, a splicing element, a polyA signal, or any combination thereof; b) the second nucleic acid further comprises a promoter and optionally further comprises one or more of an intron, a microRNA, a linker, a splicing element, a polyA signal, or any combination thereof; and / or c) the third nucleic acid further comprises a promoter and optionally further comprises one or more of an intron, a microRNA, a linker, a splicing element, a polyA signal, or any combination thereof.

20. The cell according to claim 19, wherein the promoter is selected from: (i) a constitutive promoter; (ii) an inducible promoter; (iii) a mini - promoter; and (iv) a functional derivative of any of (i), (ii), or (iii).

21. The cell according to claim 19 or 20, wherein the promoter is selected from: CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p10, p19, p40, synapsin, CaMKII, GRK1, polH, EM7, OpIE1, and any functional fragment and / or functional derivative thereof.

22. The cell according to any one of the preceding claims, wherein the Rep gene is derived from adeno - associated virus (AAV).

23. The cell according to claim 22, wherein the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6TM, AAV7, AAV7TM, AAV8, AAV8TM, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16 and any functional fragment and / or functional derivative thereof.

24. The cell according to any one of the preceding claims, wherein the Cap gene is derived from AAV.

25. The cell according to claim 24, wherein the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6TM, AAV7, AAV7TM, AAV8, AAV8TM, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16 and any functional derivatives thereof.

26. The cell according to any one of the preceding claims, wherein: a) the first nucleic acid further comprises a first antibiotic resistance gene; b) the second nucleic acid further comprises a second antibiotic resistance gene; and / or c) the third nucleic acid further comprises a third antibiotic resistance gene.

27. The cell according to any one of the preceding claims, wherein each of the first and second nucleic acids comprises an antibiotic resistance gene or a functional fragment or derivative thereof.

28. The cell according to claim 26 or 27, wherein each of the first and second antibiotic resistance genes comprises the same antibiotic resistance gene.

29. The cell according to any one of the preceding claims, wherein the third nucleic acid does not comprise an antibiotic resistance gene.

30. The cell according to any one of claims 26-29, wherein each of the first, second and / or third antibiotic resistance genes is selected from: genes encoding aminoglycoside, β-lactam, macrolide, tetracycline or any functional fragment and / or functional derivative thereof.

31. The cell according to any one of claims 26-30, wherein each of the first, second and / or third antibiotic resistance genes is selected from: genes encoding kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, blasticidin, bleomycin, erythromycin, polymyxin B, puromycin, tetracycline, chloramphenicol, neomycin, bleomycin or any functional fragment and / or functional derivative thereof.

32. The cell according to any one of the preceding claims, wherein: a) the first nucleic acid further comprises a first origin of replication; b) the second nucleic acid further comprises a second origin of replication; and / or c) the third nucleic acid further comprises a third origin of replication.

33. The cell according to claim 32, wherein the first, second, and / or third origin of replication is selected from: pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, and any functional fragment and / or functional derivative thereof.

34. The cell according to any one of the preceding claims, wherein the first nucleic acid comprises a nucleic acid sequence having at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 210 or any functional fragment or functional derivative thereof.

35. The cell according to any one of the preceding claims, wherein the second nucleic acid comprises a nucleic acid sequence having at least 80% sequence identity with the nucleic acid sequences of SEQ ID NOs: 211–216 or any functional fragment or functional derivative thereof.

36. The cell according to any one of the preceding claims, wherein the cell is a eukaryotic cell, mammalian cell, immortalized cell, insect cell, yeast cell, plant cell, fungal cell, or prokaryotic cell.

37. The cell according to any one of the preceding claims, wherein the cell is an A549 cell, HEK-293 cell, HEK-293T cell, BHK cell, CHO cell, HeLa cell, MRC5 cell, Sf9 cell, Sf2 cell, Sf21 cell, HighFive TM cell, Cos-1 cell, Cos-7 cell, Vero cell, BSC1 cell, BSC40 cell, BMT10 cell, WI38 cell, Saos cell, C2C12 cell, L cell, HT1080 cell, HepG2 cell, Huh7 cell, K562 cell, a primary cell or any derivative thereof.

38. The cell according to any one of the preceding claims, wherein the cell is an Sf9 cell.

39. The cell according to any one of the preceding claims, wherein the first nucleic acid, second nucleic acid, and third nucleic acid are stably integrated into the genome after at least 5 passages.

40. The cell according to any one of the preceding claims, wherein the cell is capable of growing to at least about 1 x 10 7 cells / mL after at least about 24 hours.

41. The cell according to any one of the preceding claims, wherein the cell is infected with a virus.

42. The cell according to claim 41, wherein the virus is selected from: alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, circovirus, bocavirus, vaccinia virus, and retrovirus.

43. The cell according to claim 41 or 42, wherein the virus is a wild-type virus.

44. The cell according to claim 41 or 42, wherein the virus does not contain the nucleic acids necessary for AAV packaging.

45. The cell according to claim 41 or 42, wherein the virus is engineered to: a) remove one or more endogenous genes or functions; and b) prevent one or more endogenous genes from producing functional gene products.

46. The cell according to claim 41 or 42, wherein the infection induces the expression of one or more of the first nucleic acid, the second nucleic acid, the third nucleic acid, or any combination thereof.

47. The cell according to claim 41 or 42, wherein the infection induces the cell to produce a recombinant virus.

48. The cell according to claim 47, wherein the recombinant virus is selected from alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, circovirus, bocavirus, vaccinia virus, or retrovirus.

49. The cell according to claim 48, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6TM, AAV7, AAV7TM, AAV8, AAV8TM, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16 and any functional derivative thereof.

50. The cell according to claim 47, wherein the recombinant virus comprises a recombinant AAV (rAAV) vector.

51. The cell according to claim 50, wherein the rAAV vector comprises less than 5%, 4%, 3%, 2% or 1% of contaminants from one or more non-AAV components and / or the GOI.

52. The cell according to claim 51, wherein the contaminants are from alphavirus, parvovirus, baculovirus, dengue virus, lentivirus, poxvirus, circovirus, bocavirus, vaccinia virus, herpes virus or retrovirus.

53. The cell according to claim 52, wherein the contaminants are from baculovirus.

54. The cell according to claims 51-53, wherein the contaminants are not from rhabdovirus.

55. A plurality of cells according to any one of claims 41 - 55, wherein said infection results in the production of from 1X10 9 vg / L to 1x10 15 vg / L of the plurality of cells.

56. A plurality of cells according to any one of claims 41 - 55, wherein said infection results in the production of 1 x 10 15 vg / L or more of the plurality of cells.

57. A method for generating an rAAV vector, comprising: a) a first nucleic acid comprising a first enhancer sequence and a Rep gene or a functional fragment thereof; b) a second nucleic acid comprising a second enhancer sequence, a first Kozak sequence and a Cap gene or a functional fragment thereof, wherein the first Kozak sequence is an engineered Kozak sequence; and c) a third nucleic acid comprising a gene of interest (GOI).

58. The method according to claim 57, wherein the first enhancer sequence has at least 80% identity with any nucleic acid sequence of SEQ ID Nos: 1-10 or any functional fragment or functional derivative thereof.

59. The method according to claim 57 or 58, wherein the second enhancer sequence has at least 80% identity with any of the nucleic acid sequences of SEQ ID Nos: 1-10 or any functional fragment or functional derivative thereof.

60. The method according to any one of claims 57-59, wherein the first and second enhancer sequences are the same.

61. The method according to any one of the preceding claims, wherein the first nucleic acid further comprises a second Kozak sequence, wherein the second Kozak sequence is an engineered Kozak sequence.

62. The method according to any one of the preceding claims, wherein the third nucleic acid comprises a third Kozak sequence, optionally wherein the third Kozak sequence is an engineered Kozak sequence.

63. The method according to any one of the preceding claims, wherein any one of the first, second, and / or third Kozak sequences promotes translation in insect cells and / or mammalian cells.

64. The method according to claim 63, wherein the first and / or second Kozak sequences preferably promote translation in insect cells.

65. The method according to claim 63 or 64, wherein the third Kozak sequence preferably promotes translation in mammalian cells.

66. The method according to any one of the preceding claims, wherein the cell is cloned.

67. The method according to any one of claims 57-66, wherein the engineered Kozak sequence of the second nucleic acid comprises a sequence having at least 80% identity with any of the sequences of SEQ ID NOs: 11-191 or any functional fragment or functional derivative thereof.

68. The method according to any one of the preceding claims, wherein optionally the Kozak sequence of the first nucleic acid engineered to be the same as the engineered Kozak sequence of the second nucleic acid.

69. The method according to any one of the preceding claims, wherein the cell is free of rhabdovirus.

70. The method according to any one of the preceding claims, wherein the GOI is flanked by a first inverted terminal repeat (ITR) sequence and a second ITR sequence.

71. The method according to claim 70, wherein each of the first ITR sequence and the second ITR sequence has a different nucleic acid sequence.

72. The method according to claim 70 or 71, wherein the first ITR and the second ITR are derived from a viral genome, and wherein the first ITR flanks at its 5' end and the second ITR flanks at its 3' end a total of about 500 nucleotides or less of the viral genome.

73. The method according to any one of the preceding claims, wherein the Cap gene comprises a sequence having at least 80% sequence identity with any of the nucleic acid sequences of SEQ ID NOs: 192-203 or any functional fragment or functional derivative thereof.

74. The method according to any one of the preceding claims, wherein the Cap gene encodes an amino acid sequence having at least about 80% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 204-209 or any functional fragment or functional derivative thereof.

75. The method according to any one of the preceding claims, wherein: a) the first nucleic acid further comprises a promoter and optionally further comprises one or more of an intron, a microRNA, a linker, a splicing element, a polyA signal or any combination thereof; b) the second nucleic acid further comprises a promoter and optionally further comprises one or more of an intron, a microRNA, a linker, a splicing element, a polyA signal or any combination thereof; and / or c) the third nucleic acid further comprises a promoter and optionally further comprises one or more of an intron, a microRNA, a linker, a splicing element, a polyA signal or any combination thereof.

76. The method according to claim 75, wherein the promoter is selected from: (i) a constitutive promoter; (ii) an inducible promoter; (iii) a mini-promoter; and (iv) a functional derivative of any one of (i), (ii) or (iii).

77. The method according to claim 75 or 76, wherein the promoter is selected from: CMV, CBA, EF1a, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, p10, p19, p40, synapsin, CaMKII, GRK1, polH, EM7, OpIE1 and any functional fragment and / or functional derivative thereof.

78. The method according to any one of the preceding claims, wherein the Rep gene is derived from adeno-associated virus (AAV).

79. The method according to claim 78, wherein the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6TM, AAV7, AAV7TM, AAV8, AAV8TM, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16 and any functional fragment and / or functional derivative thereof.

80. The method according to any one of the preceding claims, wherein the Cap gene is derived from AAV.

81. The method according to claim 80, wherein the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6TM, AAV7, AAV7TM, AAV8, AAV8TM, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16 and any functional derivatives thereof.

82. The method according to any one of the preceding claims, wherein: a) the first nucleic acid further comprises a first antibiotic resistance gene; b) the second nucleic acid further comprises a second antibiotic resistance gene; and / or c) the third nucleic acid further comprises a third antibiotic resistance gene.

83. The method according to any one of the preceding claims, wherein each of the first and second nucleic acids comprises an antibiotic resistance gene or a functional fragment or derivative thereof.

84. The method according to claim 82 or 83, wherein each of the first and second antibiotic resistance genes comprises the same antibiotic resistance gene.

85. The method according to any one of the preceding claims, wherein the third nucleic acid does not comprise an antibiotic resistance gene.

86. The method according to any one of claims 82-85, wherein each of the first, second and / or third antibiotic resistance genes is selected from: genes encoding aminoglycosides, β-lactams, macrolides, tetracyclines or any functional fragments and / or functional derivatives thereof.

87. The method according to any one of claims 82-86, wherein each of the first, second and / or third antibiotic resistance genes is selected from: genes encoding kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, blasticidin, bleomycin, erythromycin, polymyxin B, puromycin, tetracycline, chloramphenicol, neomycin, bleomycin or any functional fragments and / or functional derivatives thereof.

88. The method according to any one of the preceding claims, wherein: a) the first nucleic acid further comprises a first origin of replication; b) the second nucleic acid further comprises a second origin of replication; and / or c) the third nucleic acid further comprises a third origin of replication.

89. The method according to claim 88, wherein the first, second and / or third origin of replication is selected from: pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC and any functional fragments and / or functional derivatives thereof.

90. The method according to any one of the preceding claims, wherein the first nucleic acid comprises a nucleic acid sequence having at least 80% sequence identity with the nucleic acid sequence of SEQ ID NO: 210 or any functional fragment or functional derivative thereof.

91. The method according to any one of the preceding claims, wherein the second nucleic acid comprises a nucleic acid sequence having at least 80% sequence identity with the nucleic acid sequences of SEQ ID NOs: 211 - 216 or any functional fragment or functional derivative thereof.

92. The method according to any one of the preceding claims, wherein the cell is a eukaryotic cell, mammalian cell, immortalized cell, insect cell, yeast cell, plant cell, fungal cell or prokaryotic cell.

93. The method according to any one of the preceding claims, wherein the cell is an A549 cell, HEK-293 cell, HEK-293T cell, BHK cell, CHO cell, HeLa cell, MRC5 cell, Sf9 cell, Sf2 cell, Sf21 cell, HighFive TM cell, Cos-1 cell, Cos-7 cell, Vero cell, BSC1 cell, BSC40 cell, BMT10 cell, WI38 cell, Saos cell, C2C12 cell, L cell, HT1080 cell, HepG2 cell, Huh7 cell, K562 cell, a primary cell or any derivative thereof.

94. The method according to any one of the preceding claims, wherein the cell is an Sf9 cell.

95. The method according to any one of the preceding claims, wherein the first nucleic acid, the second nucleic acid and the third nucleic acid are stably integrated into the genome after at least 5 passages.

96. The method according to any one of the preceding claims, wherein the cells are capable of growing to at least about 1 x 10 7 cells / mL after at least about 24 hours.

97. The method according to any one of the preceding claims, wherein the cell is infected with a virus.

98. The method according to claim 97, wherein the virus is selected from: alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, circovirus, bocavirus, vaccinia virus and retrovirus.

99. The method according to claim 97 or 98, wherein the virus is a wild-type virus.

100. The method according to claim 97 or 98, wherein the virus does not contain the nucleic acids necessary for AAV packaging.

101. The method according to claim 97 or 98, wherein the virus is engineered to: a) remove one or more endogenous genes or functions; and b) prevent one or more endogenous genes from producing functional gene products.

102. The method according to claim 97 or 98, wherein the infection induces the expression of one or more of the first nucleic acid, the second nucleic acid, the third nucleic acid or any combination thereof.

103. The method according to claim 97 or 98, wherein the infection induces the cell to produce a recombinant virus.

104. The method according to claim 103, wherein the recombinant virus is selected from alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, circovirus, bocavirus, vaccinia virus or retrovirus.

105. The method according to claim 104, wherein When the recombinant virus is AAV, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6TM, AAV7, AAV7TM, AAV8, AAV8TM, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16 and any functional derivatives thereof.

106. The method according to claim 105, wherein the recombinant virus comprises a recombinant AAV (rAAV) vector.

107. The method according to claim 106, wherein the rAAV vector comprises less than 5%, 4%, 3%, 2% or 1% of contaminants from one or more non-AAV components and / or the GOI.

108. The method according to claim 107, wherein the contaminants are from alphavirus, parvovirus, baculovirus, dengue virus, lentivirus, poxvirus, circovirus, bocavirus, vaccinia virus, herpes virus or retrovirus.

109. The method according to claim 108, wherein the contaminants are from baculovirus.

110. The method according to claims 107-109, wherein the contaminants are not from rhabdovirus.

111. A recombinant virus produced by the method according to any one of claims 57-110.

112. A composition comprising a plurality of viral particles produced by infecting the cells according to any one of claims 1-40 with a virus.

113. The composition according to claim 112, wherein the cells are insect cells.

114. The composition according to claim 113, wherein the insect cells are Sf9 cells.

115. The composition according to claim 112, wherein the virus is baculovirus.

116. The composition according to claim 112, wherein the plurality of viral particles comprises 1×10 9 vg / L to 1×10 15 vg / L.

117. The composition according to claim 112, wherein the plurality of viral particles comprises 1×10 15 vg / L or more.

118. A method of infecting cells in a subject in need thereof, the method comprising administering to the subject the composition of any one of claims 112-117.

119. A method of treating a subject suffering from a disease, disorder or condition associated with a dysfunctional gene of interest (GOI), the method comprising administering the composition of any one of claims 112-118 to produce a functional gene product of the GOI and treat the disease.

120. A system for producing a recombinant virus, comprising: a) a virus-producing cell of any one of claims 1-40; and b) a virus for infecting the virus-producing cell, which induces the production of a recombinant virus when the cell is infected with the virus.

121. The system according to any one of the preceding claims, wherein the cell is infected with the virus.

122. The system according to claim 121, wherein the virus is selected from: alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpes virus, poxvirus, circovirus, bocavirus, vaccinia virus and retrovirus.

123. The system according to claim 121 or 122, wherein the virus is a wild-type virus.

124. The system according to claim 121 or 122, wherein the virus does not contain the nucleic acids necessary for AAV packaging.

125. The system according to claim 121 or 122, wherein the virus is engineered to: a) remove one or more endogenous genes or functions; and b) prevent one or more endogenous genes from producing a functional gene product.

126. The system according to claim 121 or 122, wherein the infection induces the expression of one or more of the first nucleic acid, the second nucleic acid, the third nucleic acid or any combination thereof.

127. The system according to claim 121 or 122, wherein the infection induces the cell to produce a recombinant virus.

128. The system according to claim 127, wherein the recombinant virus is selected from alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpes virus, poxvirus, circovirus, bocavirus, vaccinia virus or retrovirus.

129. The system according to claim 128, wherein the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6TM, AAV7, AAV7TM, AAV8, AAV8TM, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-1, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ / 8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16 and any functional derivatives thereof.

130. The system according to claim 127, wherein the recombinant virus comprises a recombinant AAV (rAAV) vector.

131. The system according to claim 130, wherein the rAAV vector contains less than 5%, 4%, 3%, 2% or 1% of contaminants from one or more non-AAV components and / or the GOI.

132. The system according to claim 131, wherein the contaminants are from alphavirus, parvovirus, baculovirus, dengue virus, lentivirus, poxvirus, circovirus, bocavirus, vaccinia virus, herpes virus or retrovirus.

133. The system according to claim 132, wherein the contaminants are from baculovirus.

134. The system according to any one of claims 130-132, wherein the contaminants are not from rhabdovirus.

135. The system according to any one of claims 120-134, which generates recombinant virus at 1x10 9 vg / L to 1x10 15 vg / L.

136. The system according to any one of claims 120-134, which produces 1 x 10 15 vg / L or more of recombinant virus.

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