Engineered cells for recombinant virus production

Abstract

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

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 414,890, filed October 10, 2022, and U.S. Provisional Patent Application No. 63 / 478,742, filed January 6, 2023, the disclosures of each of which are incorporated herein by reference in their entirety for all purposes. [Background technology]

[0002] Viral vectors are commonly used to deliver therapeutic genes to humans. The production of viral vectors is a complex process. Several different challenges can arise in their design and manufacture, including, but not limited to, the scalability, quality, and efficacy of recombinant viruses. The production of clinical-grade gene therapies remains a major obstacle to advancing cures for many diseases, disorders, and conditions that are otherwise untreatable, incurable, and / or untreatable. Improved processes are needed to achieve reproducible, consistent, and scalable manufacturing solutions and provide commercially viable viral products that can be safely and reliably delivered to patients. Summary of the Invention

[0003] The present disclosure provides, among other things, technologies for the production of virus-producing cells. As known to those skilled in the art, even with rapid improvements in gene therapy technologies, challenges remain, for example, in engineering and manufacturing cells that reliably, consistently, and accurately achieve virus production in a quality and quantity suitable for administration to subjects in need of one or more gene therapies. The technologies provided herein address certain unmet needs in the production of virus-producing cells and overcome certain challenges for improving the engineering and manufacturing of virus-producing cells. These engineered cells and methods for making and using them address production challenges and quality (e.g., safety) concerns that are important for advancing gene therapy.

[0004] In some aspects, the disclosure provides a virus producer cell having an engineered genome 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).

[0005] In some aspects, the disclosure provides methods for generating an rAAV vector 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).

[0006] In some embodiments, the first enhancer sequence has at least 80% identity to the nucleic acid sequence of any of SEQ ID NOs: 1-10, or any functional fragment or derivative thereof.

[0007] In some embodiments, the second enhancer sequence has at least 80% identity to the nucleic acid sequence of any of SEQ ID NOs: 1-10, or any functional fragment or derivative thereof.

[0008] In some embodiments, the first enhancer sequence and the second enhancer sequence are the same.

[0009] In some embodiments, the first nucleic acid further comprises a second Kozak sequence, wherein the second Kozak sequence is an engineered Kozak sequence.

[0010] In some embodiments, the third nucleic acid comprises a third Kozak sequence, and optionally, the third Kozak sequence is an engineered Kozak sequence.

[0011] In some embodiments, any of the first, second, and / or third Kozak sequences enhances translation in insect cells and / or mammalian cells.

[0012] In some embodiments, the first and / or second Kozak sequences preferentially enhance translation in insect cells.

[0013] In some embodiments, the third Kozak sequence preferentially promotes translation in mammalian cells.

[0014] In some embodiments, the cells are clonal.

[0015] In some embodiments, the engineered Kozak sequence of the second nucleic acid comprises a sequence having at least 80% identity to any of SEQ ID NOs: 11-191, or any functional fragment or derivative thereof.

[0016] In some embodiments, the Kozak sequence of the first nucleic acid, which is optionally engineered, and the engineered Kozak sequence of the second nucleic acid comprise the same nucleic acid sequence.

[0017] In some embodiments, the cells do not contain a rhabdovirus.

[0018] In some embodiments, the GOI is flanked by a first inverted terminal repeat (ITR) sequence and a second ITR sequence.

[0019] In some embodiments, the first ITR sequence and the second ITR sequence each have a different nucleic acid sequence.

[0020] In some embodiments, the first ITR and the second ITR are derived from a viral genome, with the first ITR at its 5' end and the second ITR at its 3' end adjacent to the viral genome for a total of no more than about 500 nucleotides.

[0021] In some embodiments, the Cap gene comprises a sequence having at least 80% sequence identity to the nucleic acid sequence of any of SEQ ID NOs: 192-203, or any functional fragment or derivative thereof.

[0022] In some embodiments, the Cap gene encodes an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of any of SEQ ID NOs: 204-209, or any functional fragment or functional derivative thereof.

[0023] 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.

[0024] In some embodiments, the promoter is selected from (i) a constitutive promoter, (ii) an inducible promoter, (iii) a minipromoter, and (iv) a functional derivative of any 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 an adeno-associated virus (AAV). 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-rh 8, 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-3 B, 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.

[0025] In some embodiments, the Cap gene is derived from an AAV. In some embodiments, the AAV is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6™, AAV7, AAV7™, AAV8, 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, A selected from AV-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.

[0026] 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, the first nucleic acid and the second nucleic acid each comprise an antibiotic resistance gene or a functional fragment or derivative thereof. In some embodiments, the first antibiotic resistance gene and the second antibiotic resistance gene each comprise 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, beta-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 fragment and / or functional derivative thereof.

[0027] 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, hi some embodiments, the first, second, and / or third origin of replication are selected from pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, and any functional fragment and / or functional derivative thereof.

[0028] In some embodiments, the first nucleic acid comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 210, or to any functional fragment or derivative thereof. In some embodiments, the second nucleic acid comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NOs: 211-216, or to any functional fragment or derivative thereof.

[0029] 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, a Sf9 cell, a Sf2 cell, a Sf21 cell, a High Five™ 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, a L cell, a 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.

[0030] 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 cells are at least about 1 x 10 cells after at least about 24 hours. 7 Can be grown to 1000 cells / mL.

[0031] In some embodiments, the cells are infected with a virus. In some embodiments, the virus is selected from an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, anellovirus, bocavirus, vaccinia virus, and retrovirus. In some embodiments, the virus is a wild-type virus. In some embodiments, the virus does not contain nucleic acids necessary for AAV packaging.

[0032] In some embodiments, the virus is engineered to remove one or more endogenous genes or functions and to disrupt one or more endogenous genes for production of a functional gene product. In some embodiments, infection induces expression of one or more of the first nucleic acid, the second nucleic acid, the 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 an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, anellovirus, 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, AA V-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, AAV2tY F, 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-HSC1 2, 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.

[0033] In some embodiments, the recombinant virus comprises a recombinant AAV (rAAV) vector. In some embodiments, the rAAV vector contains less than 5%, 4%, 3%, 2%, or 1% of contaminants from one or more non-AAV components and / or GOIs. In some embodiments, the contaminants are derived from alphavirus, parvovirus, baculovirus, dengue virus, lentivirus, poxvirus, anellovirus, bocavirus, vaccinia virus, herpesvirus, or retrovirus. In some embodiments, the contaminants are derived from baculovirus. In some embodiments, the contaminants are not derived from rhabdovirus.

[0034] In some embodiments, the plurality of cells infected according to the present disclosure is 1×10 9 vg / L~1×10 15 In some embodiments, the plurality of cells infected according to the present disclosure produces a plurality of 1 x 10 vg / L. 15 This results in multiple producing more than vg / L.

[0035] 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 a plurality of viral particles produced by infecting a cell provided herein with a virus. In some embodiments, the cell is an insect cell. In some embodiments, the insect cell is an Sf9 cell. In some embodiments, the virus is a baculovirus. In some embodiments, the plurality of viral particles is greater than or equal to 1 x 10 9 vg / L~1×10 15 In some embodiments, the plurality of viral particles comprises 1 x 10 vg / L. 15 Includes vg / L or more.

[0036] In some aspects, the present disclosure provides a method of infecting a cell in a subject in need thereof, the method comprising administering to the subject a composition provided in accordance with the present disclosure.

[0037] In some aspects, the present disclosure provides methods of treating a subject having a disease, disorder, or condition associated with a dysfunctional gene of interest (GOI), comprising administering a composition provided herein to produce a functional gene product of the GOI and treat the disease.

[0038] In some aspects, the present disclosure provides a system for generating a recombinant virus, the system including a virus-producing cell provided herein and a virus for infecting the virus-producing cell, wherein the virus induces the production of the recombinant virus upon infection by the virus.

[0039] In some embodiments, the cells are infected with a virus. In some embodiments, the virus is selected from an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, anellovirus, bocavirus, vaccinia virus, and retrovirus. In some embodiments, the virus is a wild-type virus. In some embodiments, the virus does not contain nucleic acids necessary for AAV packaging. In some embodiments, the virus has been engineered to remove one or more endogenous genes or functions and to disrupt one or more endogenous genes for production of a functional gene product.

[0040] In some embodiments, the infection induces expression of one or more of the first nucleic acid, the second nucleic acid, the third nucleic acid, or any combination thereof. In some embodiments, the infection induces the cell to produce a recombinant virus. In some embodiments, the recombinant virus is selected from an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a dengue virus, a lentivirus, a herpesvirus, a poxvirus, anellovirus, a bocavirus, a vaccinia virus, or a 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, AA V-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, AAV2tY F, 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-HSC1 2, 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.

[0041] 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 GOIs. In some embodiments, the contaminants are derived from alphavirus, parvovirus, baculovirus, dengue virus, lentivirus, poxvirus, anellovirus, bocavirus, vaccinia virus, herpesvirus, or retrovirus. In some embodiments, the contaminants are derived from baculovirus. In some embodiments, the contaminants are not derived from rhabdovirus.

[0042] In some embodiments, the system 9 vg / L~1×10 15 In some embodiments, the system produces 1 x 10 vg / L of recombinant virus. 15 Produces recombinant virus at a concentration of ≥ vg / L.

[0043] In some aspects, the present disclosure provides a virus producer 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 and a Cap gene or a functional fragment thereof; and (c) a third nucleic acid comprising 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 sequence of SEQ ID NOs: 1-10. In some embodiments, the first enhancer sequence and the second enhancer sequence 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 the nucleic acid sequence of any one of SEQ ID NOs: 11-191.

[0044] 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 the nucleic acid sequence of any one of SEQ ID NOs: 11-191. In some embodiments, the first engineered Kozak sequence and the second engineered Kozak sequence are the same.

[0045] 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 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 any one 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 the nucleic acid sequence of any one of SEQ ID NOs: 204-209.

[0046] 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 minipromoter, 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.

[0047] 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 minipromoter, 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 minipromoter, or any functional fragment and / or 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 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 AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AA V-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 In some embodiments, the Cap gene is selected from 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. In some embodiments, the Cap gene is derived from a parvovirus. In some embodiments, the parvovirus is AAV.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, AA V-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 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.

[0048] In some embodiments, the first nucleic acid further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, a beta-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, zeocin, or any functional fragment and / or functional derivative thereof.

[0049] In some embodiments, the second nucleic acid further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, a beta-lactam, a macrolide, a tetracycline, or any functional fragment and / or derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, blasticidin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, zeocin, or any functional fragment and / or derivative thereof.

[0050] In some embodiments, the third nucleic acid further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, a beta-lactam, a macrolide, a tetracycline, or any functional fragment and / or derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, blasticidin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, zeocin, or any functional fragment and / or derivative thereof.

[0051] 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 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.

[0052] 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 derivative 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 the nucleic acid sequence of any one of SEQ ID NOs:211-216.

[0053] In some embodiments, the third nucleic acid further comprises an origin of replication, hi some embodiments, the origin of replication is selected from pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, F1, pUC, and any functional fragment and / or derivative thereof.

[0054] 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, a Sf9 cell, a Sf2 cell, a Sf21 cell, a High Five™ 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, a L cell, a HT1080 cell, a HepG2 cell, a Huh7 cell, a K562 cell, a primary cell, or any derivative thereof.

[0055] In some embodiments, the cells are Sf9 cells.

[0056] 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 cells are at least about 1 x 10 cells after at least about 24 hours. 7In some embodiments, the cells are infected with a virus. In some embodiments, the virus is selected from an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, anellovirus, bocavirus, vaccinia virus, and retrovirus. In some embodiments, the virus is a wild-type virus. In some embodiments, the infection induces expression of one or more of the first nucleic acid, the second nucleic acid, the 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 an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, anellovirus, 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 AV-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, A AV-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. In some embodiments, the recombinant virus comprises a recombinant adeno-associated virus (rAAV) vector.In some embodiments, the rAAV vector contains less than 5%, 4%, 3%, 2%, or 1% contaminants from non-AAV components and GOIs. In some embodiments, the contaminants are derived from alphaviruses, parvoviruses, baculoviruses, dengue viruses, lentiviruses, poxviruses, anelloviruses, bocaviruses, vaccinia viruses, herpesviruses, or retroviruses. In some embodiments, the contaminants are derived from baculoviruses.

[0057] Provided herein are methods for producing rAAV vectors, the method comprising: (a) providing a virus-producing cell having an engineered genome, the cell 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; (b) contacting the cell of step (a) with the virus; and (c) after step (b), culturing the cell to produce 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 set forth in 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 sequence of any one of SEQ ID NOs: 1-10. In some embodiments, the first enhancer sequence and the second enhancer sequence 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 the nucleic acid sequence of any one 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 the nucleic acid sequence of any one of SEQ ID NOs: 11-191. In some embodiments, the first engineered Kozak sequence and the second engineered Kozak sequence are the same. In some embodiments, the GOI is flanked by the first ITR sequence and the 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 the nucleic acid sequence of any one of SEQ ID NOs: 192203. 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 the amino acid sequence of any one 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 minipromoter, 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 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 minipromoter, or any functional fragment and / or 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 minipromoter, 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 AAV (e.g., a wild-type AAV, e.g., a rAAV. In some embodiments, the AAV is selected from the group consisting of 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, In some embodiments, the Cap gene is selected from 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. In some embodiments, the Cap gene is derived from a parvovirus. In some embodiments, the parvovirus is AAV.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, AA V-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 In some embodiments, the first nucleic acid is selected from 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. In some embodiments, the first nucleic acid further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, a beta-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, zeocin, or any functional fragment and / or 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 beta-lactam, a macrolide, a tetracycline, or any functional fragment and / or derivative thereof.In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, blasticidin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, zeocin, or any functional fragment and / or 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, beta-lactam, macrolide, tetracycline, or any functional fragment and / or derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, blasticidin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, zeocin, or any functional fragment and / or 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 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 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 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 the nucleic acid sequence of any one 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 cells are A549 cells, HEK-293 cells, HEK-293T cells, BHK cells, CHO cells, HeLa cells, MRC5 cells, Sf9 cells, Sf2 cells, Sf21 cells, High Five™ cells, Cos-1 cells, Cos-7 cells, Vero cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, WI38 cells, Saos cells, C2C12 cells, L cells, HT1080 cells, HepG2 cells, Huh7 cells, K562 cells, primary cells, or derivatives thereof. are Sf9 cells. 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 cells are at least about 1 x 10 after at least about 24 hours. 7In some embodiments, the cells are infected with a virus. In some embodiments, the virus is selected from an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, anellovirus, bocavirus, vaccinia virus, and retrovirus. In some embodiments, the virus is a wild-type virus. In some embodiments, the infection induces expression of one or more of the first nucleic acid, the second nucleic acid, the 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 an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, anellovirus, 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 AV-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, A In some embodiments, the recombinant virus comprises an rAAV vector.In some embodiments, the rAAV vector contains less than 5%, 4%, 3%, 2%, or 1% contaminants from non-AAV components and GOIs. In some embodiments, the contaminants are derived from alphaviruses, parvoviruses, baculoviruses, dengue viruses, lentiviruses, poxviruses, anelloviruses, bocaviruses, vaccinia viruses, herpesviruses, or retroviruses. In some embodiments, the contaminants are derived from baculoviruses.

[0058] In some embodiments, the present disclosure provides a recombinant virus produced by the methods of the present disclosure.

[0059] In some embodiments, the present disclosure provides a system for generating a recombinant virus, the system 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, wherein the virus induces the production of a recombinant virus upon infection by the virus.

[0060] In some embodiments, the first enhancer sequence has at least 80% (e.g., 85%, 90%, 95%, 99%, or 100%) identity to the nucleic acid sequence of any 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 sequence of SEQ ID NOs: 1-10. In some embodiments, the first enhancer sequence and the second enhancer sequence are the same.

[0061] 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 the nucleic acid sequence of any one 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 the nucleic acid sequence of any one of SEQ ID NOs: 2-42. In some embodiments, the first engineered Kozak sequence and the second engineered Kozak sequence are the same.

[0062] 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 the nucleic acid sequence of any one 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 the amino acid sequence of any one of SEQ ID NOs: 204-209.

[0063] 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 minipromoter, 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 minipromoter, or any functional fragment and / or 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 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 minipromoter, or any functional fragment and / or 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 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 AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AA V-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 In some embodiments, the Cap gene is selected from 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. In some embodiments, the Cap gene is derived from a parvovirus. In some embodiments, the parvovirus is AAV.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, AA V-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 In some embodiments, the first nucleic acid is selected from 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. In some embodiments, the first nucleic acid further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, a beta-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, zeocin, 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 beta-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, zeocin, or any functional fragment and / or 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, beta-lactam, macrolide, tetracycline, or any functional fragment and / or derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, blasticidin, bleomycin, erythromycin, puromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, zeocin, or any functional fragment and / or derivative thereof.

[0064] 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 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 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 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 the nucleic acid sequence of any one 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 cells are A549 cells, HEK-293 cells, HEK-293T cells, BHK cells, CHO cells, HeLa cells, MRC5 cells, Sf9 cells, Sf2 cells, Sf21 cells, High Five™ cells, Cos-1 cells, Cos-7 cells, Vero cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, WI38 cells, Saos cells, C2C12 cells, L cells, HT1080 cells, HepG2 cells, Huh7 cells, K562 cells, primary cells, or any derivatives thereof. In some embodiments, the cells are Sf9 cells.

[0065] 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 (eg, 10, 20, 40) passages.

[0066] In some embodiments, the cells are at least about 1 x 10 after at least about 24 hours. 7In some embodiments, the cells are infected with a virus. In some embodiments, the virus is selected from an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, anellovirus, bocavirus, vaccinia virus, and retrovirus. In some embodiments, the virus is a wild-type virus. In some embodiments, the infection induces expression of one or more of the first nucleic acid, the second nucleic acid, the 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 an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, anellovirus, 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 AV-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, A In some embodiments, the recombinant virus comprises an rAAV vector.

[0067] In some embodiments, the rAAV vector contains less than 5%, 4%, 3%, 2%, or 1% contaminants from non-AAV components and GOIs. In some embodiments, the contaminants are derived from alphaviruses, parvoviruses, baculoviruses, dengue viruses, lentiviruses, poxviruses, anelloviruses, bocaviruses, vaccinia viruses, herpesviruses, or retroviruses. In some embodiments, the contaminants are derived from baculoviruses. [Brief explanation of the drawings]

[0068] The present disclosure is more fully understood with reference to the following drawings. [Figure 1A] 1 shows exemplary nucleic acid construct designs for exemplary Rep and Cap proteins. [Figure 1B] 1 shows a Western blot of capsid proteins in adeno-associated virus (AAV) serotype 1 (AAV1) and AAV5. [Figure 1C] Affinity-purified capsid proteins from AAV7, AAV8, and AAV9 are shown. [Figure 2A] Schematic diagram showing an exemplary method for producing recombinant virus, beginning with seeding and selection of clonal cells (Figures 2A and 2B, respectively), growing the clonal cells into a monolayer, and infecting them with an exemplary non-recombinant virus to produce exemplary recombinant virus particles containing a gene of interest (GOI) (Figure 2C), and concluding with discarding the medium containing excess components (Figure 2D) and recovering the recombinant virus from the remaining cell monolayer, including final separation of the virus from cellular debris (Figure 2E). Abbreviations: BEV, baculovirus; GOI, gene of interest; PCR, polymerase chain reaction; WT, wild type. [Figure 2B]Schematic diagram showing an exemplary method for producing recombinant virus, beginning with seeding and selection of clonal cells (Figures 2A and 2B, respectively), growing the clonal cells into a monolayer, and infecting them with an exemplary non-recombinant virus to produce exemplary recombinant virus particles containing a gene of interest (GOI) (Figure 2C), and concluding with discarding the medium containing excess components (Figure 2D) and recovering the recombinant virus from the remaining cell monolayer, including final separation of the virus from cellular debris (Figure 2E). Abbreviations: BEV, baculovirus; GOI, gene of interest; PCR, polymerase chain reaction; WT, wild type. [Figure 2C] Schematic diagram showing an exemplary method for producing recombinant virus, beginning with seeding and selection of clonal cells (Figures 2A and 2B, respectively), growing the clonal cells into a monolayer, and infecting them with an exemplary non-recombinant virus to produce exemplary recombinant virus particles containing a gene of interest (GOI) (Figure 2C), and concluding with discarding the medium containing excess components (Figure 2D) and recovering the recombinant virus from the remaining cell monolayer, including final separation of the virus from cellular debris (Figure 2E). Abbreviations: BEV, baculovirus; GOI, gene of interest; PCR, polymerase chain reaction; WT, wild type. [Figure 2D] Schematic diagram showing an exemplary method for producing recombinant virus, beginning with seeding and selection of clonal cells (Figures 2A and 2B, respectively), growing the clonal cells into a monolayer, and infecting them with an exemplary non-recombinant virus to produce exemplary recombinant virus particles containing a gene of interest (GOI) (Figure 2C), and concluding with discarding the medium containing excess components (Figure 2D) and recovering the recombinant virus from the remaining cell monolayer, including final separation of the virus from cellular debris (Figure 2E). Abbreviations: BEV, baculovirus; GOI, gene of interest; PCR, polymerase chain reaction; WT, wild type. [Figure 2E]Schematic diagram showing an exemplary method for producing recombinant virus, beginning with seeding and selection of clonal cells (Figures 2A and 2B, respectively), growing the clonal cells into a monolayer, and infecting them with an exemplary non-recombinant virus to produce exemplary recombinant virus particles containing a gene of interest (GOI) (Figure 2C), and concluding with discarding the medium containing excess components (Figure 2D) and recovering the recombinant virus from the remaining cell monolayer, including final separation of the virus from cellular debris (Figure 2E). Abbreviations: BEV, baculovirus; GOI, gene of interest; PCR, polymerase chain reaction; WT, wild type. [Figure 3] Shown are the results of clone screening to identify top producing clones as determined by yield (vg / mL). [Figure 4] FIG. 1 is a graph showing the quantified difference in scale time shown as passage number (x-axis) and cells / mL (y-axis) for conventional and optimized methods. [Figure 5A] Measurements of the amount of host cell proteins and activated virus present throughout production and pre- and post-purification demonstrate that both host cell protein and activated virus impurities decreased over the course of purification. Figures 5A and 5B are gels showing the reduction in impurities over the course of purification. Figure 5C is a bar graph showing the host cell proteins (HCPs) detected in fractions from various purification steps in ng / mL. Figure 5D is a bar graph showing the DNA concentrations of Sf9 and baculovirus (BEV) DNA in ng / mL in the eluates from AAVX and AEX column purifications. Sf9 = left bar and BEV = right bar for AAVX and AEX conditions. [Figure 5B]Measurements of the amount of host cell proteins and activated virus present throughout production and pre- and post-purification demonstrate that both host cell protein and activated virus impurities decreased over the course of purification. Figures 5A and 5B are gels showing the reduction in impurities over the course of purification. Figure 5C is a bar graph showing the host cell proteins (HCPs) detected in fractions from various purification steps in ng / mL. Figure 5D is a bar graph showing the DNA concentrations of Sf9 and baculovirus (BEV) DNA in ng / mL in the eluates from AAVX and AEX column purifications. Sf9 = left bar and BEV = right bar for AAVX and AEX conditions. [Figure 5C] Measurements of the amount of host cell proteins and activated virus present throughout production and pre- and post-purification demonstrate that both host cell protein and activated virus impurities decreased over the course of purification. Figures 5A and 5B are gels showing the reduction in impurities over the course of purification. Figure 5C is a bar graph showing the host cell proteins (HCPs) detected in fractions from various purification steps in ng / mL. Figure 5D is a bar graph showing the DNA concentrations of Sf9 and baculovirus (BEV) DNA in ng / mL in the eluates from AAVX and AEX column purifications. Sf9 = left bar and BEV = right bar for AAVX and AEX conditions. [Figure 5D]Measurements of the amount of host cell proteins and activated virus present throughout production and pre- and post-purification demonstrate that both host cell protein and activated virus impurities decreased over the course of purification. Figures 5A and 5B are gels showing the reduction in impurities over the course of purification. Figure 5C is a bar graph showing the host cell proteins (HCPs) detected in fractions from various purification steps in ng / mL. Figure 5D is a bar graph showing the DNA concentrations of Sf9 and baculovirus (BEV) DNA in ng / mL in the eluates from AAVX and AEX column purifications. Sf9 = left bar and BEV = right bar for AAVX and AEX conditions. [Figure 6] 1 is a graph showing a stability study measuring volume (vg / L) or unit (vg / cell) titer over 20 passages using exemplary producer clones generated according to the present disclosure. [Figure 7] Graphs showing growth and infection kinetics as measured by cell density (cells / mL) and viability (%) over time. [Figure 8] 1 is a bar graph showing a comparison of the sequencing analysis of Sf9 and BEV genomes in purified Sf9 AAV. [Figure 9] FIG. 1 is a schematic diagram showing an exemplary nucleic acid cassette containing a gene of interest (GOI) and inverted terminal repeats (ITRs) before and after restriction digestion. [Figure 10] 1 is a bar graph showing enzyme levels in the cerebellum of wild-type (WT; circles), knockout (KO; squares), and AAV-treated mice using AAV produced using HEK293 cells (triangles) or Sf9 producer cells (diamonds) generated according to the present disclosure. [Figure 11A] 1 is a bar graph showing the biodistribution of vector genomes in target tissues of an exemplary AAV7 serotype gene therapy performed using an exemplary Sf9 producer cell line of the present disclosure (diamonds) versus a standard exemplary HEK293 cell platform (triangles). [Figure 11B]1 is a graph showing the efficacy of AAV therapeutics produced in HEK293 or Sf9 cells according to the present disclosure in extending survival after administration. [Figure 12A] 1 is a bar graph showing enzyme levels in the forebrains of wild-type (WT; circles), knockout (KO; squares), and AAV-treated mice using AAV produced using HEK293 cells or Sf9 producer cells generated according to the present disclosure. [Figure 12B] 1 is a bar graph showing enzyme levels in the hindbrain of wild-type (WT; circles), knockout (KO; squares), and AAV-treated mice using AAV encoding the enzyme produced using HEK293 cells (triangles) or Sf9 producer cells (diamonds) generated according to the present disclosure. [Figure 12C] Graph showing enzyme levels in the hindbrain of wild-type (WT; circles), knockout (KO; squares), and AAV-treated mice using AAV produced using HEK293 cells (triangles) or Sf9 producer cells (diamonds) generated according to the present disclosure. [Figure 13] 1 shows an image showing exemplary biodistribution of AAV in brain tissue after intracisternal (intra-cisterna magna, ICM) administration (arrows indicate the location of detected AAV; Table 3 describes the location and provides quantification). DETAILED DESCRIPTION OF THE INVENTION

[0069] Viral vectors are commonly used to deliver therapeutic genes to humans. The production of viral vectors is a complex process. Several different challenges can arise in their design and manufacture, including, but not limited to, the scalability, quality, and efficacy of recombinant viruses. The production of clinical-grade gene therapies remains a major obstacle to advancing cures for many diseases, disorders, and conditions that are otherwise untreatable, incurable, and / or untreatable. Improved processes are needed to achieve reproducible, consistent, and scalable manufacturing solutions and provide commercially viable viral products that can be safely and reliably delivered to patients.

[0070] The present disclosure provides insight that a system with certain components produces and provides reproducible and stable viral products of clinical quality or clinically relevant quality in commercially relevant or scalable quantities.

[0071] Among other things, provided herein are systems, cell lines, manufacturing methods, and methods of using combinations of features that result in more reproducible, scalable, and reliable products for use in clinical products. The disclosed systems include engineered virus-producing cell lines that result in low-cost, robust, and highly reproducible large-scale viral product manufacturing. Such virus producer cell lines use an innovative combination of stably integrated inducible components in an engineered cell system.

[0072] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. Generally, the nomenclature utilized in connection with, 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 are in accordance with the International System of Units (SI), NIST Special Publication 330, 2019 edition.

[0073] As used herein, all numerical values ​​or ranges include whole integers within or inclusive of such ranges, and fractions of values ​​or integers within or inclusive of ranges, unless the context clearly dictates otherwise. Thus, for example, a reference to a range of 90 to 100% 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. In another example, reference to a range of 1 to 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.

[0074] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of any embodiments. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0075] It will be further understood that, as used herein, the terms "comprises" and / or "comprising" specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. "Consisting essentially of" specifies the presence of at least each stated feature, integer, step, operation, element, and / or component, but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0076] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0077] Unless specifically stated otherwise or clear from the context, as used herein, the term "about" with respect to a number or range of numbers is understood to mean the stated number and that number plus or minus 10%, or 10% below the recited lower limit and 10% above the recited upper limit with respect to the recited values ​​for the range.

[0078] As used herein, the term "adeno-associated viral vector" or "AAV vector" refers to any of a wide variety of vectors, including, but not limited to, 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 vectors refer to vectors derived from adeno-associated viruses selected from the group consisting of 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 may be a mammalian (e.g., human, e.g., non-human primate) virus. The AAV vector may be an avian (AAAV) virus. In some embodiments, the AAV is mammalian (e.g., human, e.g., non-human primate) AAV or avian AAV (AAAV), i.e., in some embodiments, the starting and / or engineered AAV is or is derived from a virus capable of infecting a mammalian or avian organism. AAV vectors can have one or more of the AAV wild-type genes (e.g., the Rep and / or Cap genes) deleted in whole or in part, but retain functional flanking inverted terminal repeat (ITR) sequences. Functional ITR sequences facilitate rescue, replication, and packaging of AAV virions. AAV vectors can comprise a single-stranded (ss) or self-complementary (sc) genome.Thus, an AAV vector is defined herein to contain at least those sequences required in cis for viral replication and packaging (e.g., functional ITRs). The ITRs need not be wild-type polynucleotide sequences, and in some embodiments are modified, e.g., by nucleotide insertion, deletion, or substitution, so long as the sequences provide for functional rescue, replication, and packaging.

[0079] 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 that function together in cis as an origin of DNA replication and as a viral packaging signal. The AAV ITRs, together with the AAV Rep coding region, can also provide for efficient excision and integration of a polynucleotide sequence inserted between the two flanking ITRs into a mammalian, avian, or insect genome. Typically, a wild-type AAV ITR contains 145 bases, although ITRs may contain fewer or more bases. As used herein, "AAV ITR" does not necessarily include the wild-type polynucleotide sequence, which in some embodiments is modified, for example, by the insertion, deletion, or substitution of nucleotides. Furthermore, AAV ITR 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 The polynucleotide sequence may be derived from any of several AAV serotypes selected from 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. Furthermore, the 5' and 3' ITRs flanking the selected polynucleotide sequence in the AAV vector need not be identical or derived from the same AAV serotype or isolate, so long as they function as intended, e.g., to enable a desired therapeutic or genome editing effect.

[0080] As used herein, the term "engineered Kozak sequence" describes a reference Kozak sequence that has been modified (e.g., compared to a reference or endogenous Kozak sequence) by one or more nucleotide changes to alter (e.g., increase or decrease) translation of a downstream gene product. As used herein, a Kozak sequence is a sequence that encodes a translation start site, and the engineered Kozak sequence alters translation compared to translation that occurs in the presence of an unengineered Kozak sequence. The change in translation that occurs with the engineered Kozak sequence can be an increase or decrease in a particular downstream gene product. For example, the engineered Kozak sequences provided herein can incorporate higher levels of VP1 into AAV capsids compared to the reference Kozak sequence (e.g., before engineering).

[0081] As used herein, the term "functional derivative" of a biomolecule (e.g., a polynucleotide, e.g., a polypeptide, e.g., a viral particle (e.g., an AAV particle)) refers to a biomolecule that has been modified relative to a reference biomolecule, such that the resulting biomolecule does not necessarily contain the same sequence (e.g., nucleic acid sequence, 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. Where the biomolecule is a polynucleotide or polypeptide, it is contemplated that the polynucleotide or polypeptide comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to 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 over a contiguous portion) the nucleic acid or amino acid sequence of the reference biomolecule. For example, a functional derivative of a polypeptide may be a polypeptide that contains one or more amino acid modifications compared to a reference biomolecule and retains a certain function, such as binding to a particular receptor, but the binding may be stronger, equivalent, or weaker than that of the reference biomolecule, so 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 may be a nucleic acid sequence that contains one or more nucleotide modifications but still encodes a protein or protein fragment that functions the same or similarly as a protein encoded by the reference biomolecule, or has the same or similar function (e.g., as a regulatory element, e.g., promoter or enhancer) as the reference biomolecule.Furthermore, such biomolecules or functional derivatives thereof may be "free-standing" functional units or systems (e.g., modified or variant AAVs) and / or parts of larger systems, such as vectors (e.g., gene cassettes encoding antibiotic resistance genes or functional portions thereof, e.g., gene cassettes encoding specific Kozak sequences, etc.).

[0082] As used herein, the term "functional fragment" of a biomolecule (e.g., 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. It is contemplated that the similar functional activity may be greater than, approximately equal to, or less than the functional activity of the reference biomolecule, so 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 contemplated 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, if the reference biomolecule is a polypeptide, it is contemplated that a polypeptide fragment can be a polypeptide that has been truncated or otherwise modified to be shorter and / or smaller than the reference polypeptide, but still retains the functional activity of the reference polypeptide, such as binding to a particular receptor, and if the reference biomolecule is a polynucleotide, it is contemplated that a polynucleotide fragment retains some of the same activity of the reference polynucleotide. For example, in the case of a polynucleotide that encodes a protein, it is contemplated that the polynucleotide fragment encodes a protein that 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 polynucleotides that act as regulatory elements (e.g., promoters or enhancers), it is contemplated that the polynucleotide fragment will have at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the activity as a reference polynucleotide in a suitable assay.

[0083] As used herein, the term "parvovirus" encompasses the family Parvoviridae, including, but not limited to, the self-replicating Parvovirus and virus-dependent genera. Autonomous parvoviruses include, for example, members of the genera Bocavirus, Dependovirus, Erythrovirus, Amdovirus, Parvovirus, Densovirus, Ierovirus, Cottravirus, Avarporvovirus, Copiparvovirus, Protoparvovirus, Tetraparvovirus, Ambientesovirus, Brevicula, brevinnovovirus, hepatodensovirus, and pendensovirus. Exemplary autonomous parvoviruses include, but are not limited to, porcine parvovirus, mouse parvovirus, canine parvovirus, mink enterovirus, bovine parvovirus, chicken parvovirus, feline panleukopenia virus (feline panleukosis virus), feline parvovirus, goose parvovirus, H1 parvovirus, Muscovy duck parvovirus, snake parvovirus, and B19 virus.

[0084] As used herein, the terms "percent identity," "% identity," or "sequence identity" refer to the degree to which two sequences (e.g., nucleotide sequences, e.g., DNA, RNA, etc., e.g., polypeptide sequences) have the same residues at the same positions in an alignment. For example, "a nucleotide sequence is X% identical to SEQ ID NO: Y" refers to the % identity of a nucleotide sequence to SEQ ID NO: Y, where X% of the residues in the nucleotide sequence are recited as being identical to the corresponding residues in the sequence disclosed in SEQ ID NO: Y. A sequence said to be X% identical to a reference sequence may contain more nucleotides or amino acid residues than specified in the reference sequence, but must include sequence that corresponds to the reference sequence. In most cases, the sequence in question includes sequence that corresponds to all of the specified reference sequence. Generally, computer programs are used for such calculations. Exemplary programs for comparing and aligning pairs of sequences include ALIGN, FASTA, gapped BLAST, BLASTP, BLASTN, or GCG.

[0085] As used herein, the term "plasmid" refers to an extrachromosomal element carrying genes that can replicate independently of the chromosomes of a cell. Plasmids may be in the form of circular double-stranded DNA molecules. Such elements may include autonomously replicating sequences, genome-integrating sequences, phage or nucleotide sequences, as well as linear, circular or supercoiled, single-stranded or double-stranded DNA or RNA of any origin.

[0086] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably herein and refer to a chain of nucleotides of any length, including DNA and RNA. In some embodiments, a nucleotide is a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base, and / or its analog, or any substrate that can be incorporated into a chain by a DNA or RNA polymerase. A polynucleotide may include modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure are imparted before or after assembly of the chain. In some embodiments, the sequence of nucleotides is interrupted by non-nucleotide components. In some embodiments, a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps," substitution of one or more of the naturally occurring nucleotides with analogs, internucleotide modifications, such as modifications with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates) and modifications with charged linkages (e.g., phosphorothioates, phosphorodithioates), 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 chelators (e.g., metals, radioactive metals, boron, metal oxides), modifications containing alkylating agents, modifications with modified linkages (e.g., alpha-anomeric nucleic acids), and the like, as well as unmodified forms of polynucleotides. In some embodiments, any of the hydroxyl groups normally present on the sugar are replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare further linkages to additional nucleotides, or conjugated to a solid support. In some embodiments, the 5' and 3' terminal OH are phosphorylated or substituted with amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls can also be derivatized to standard protecting groups.In some embodiments, polynucleotides also contain analogous forms of ribose or deoxyribose sugars, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro-, or 2'-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose sugars, furanose sugars, sedoheptulose, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. In some embodiments, one or more phosphodiester linkages are replaced by alternative linking groups. These alternative linking groups may include, but are not limited to, embodiments in which phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NRi ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal") (each R or R' is independently H or substituted or unsubstituted alkyl (1-20C), optionally including an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl). Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[0087] 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. Promoters can be derived, for example, from a native gene or can be composed of different elements from different promoters found in nature and / or synthetic DNA segments. In some embodiments, different promoters direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions or inducers. A promoter that causes a gene to be expressed in most cell types most of the time is generally referred to as a "constitutive promoter." A promoter that causes a gene to be expressed in specific cell and tissue types is generally referred to as a "cell-specific promoter" or "tissue-specific promoter," respectively. A promoter that causes a gene to be expressed at a specific stage of development or cell differentiation is generally referred to as a "development-specific promoter" or "cell differentiation-specific promoter." Promoters that induce and result in expression of a gene after cells are exposed to or treated with a promoter-inducing drug, biomolecule, chemical, ligand, light, etc. are commonly referred to as "inducible promoters" or "regulatable promoters." In some embodiments, it is further recognized that because the exact boundaries of regulatory sequences are in most cases not completely defined, DNA fragments of different lengths may have the same promoter activity.

[0088] As used herein, "recombinant AAV vector (rAAV vector)" refers to a polynucleotide vector derived from AAV and containing one or more heterologous sequences (i.e., nucleic acid sequences not of AAV origin) flanking at least one AAV ITR. In some embodiments, such rAAV vectors are replicated and packaged into viral 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., AAV Rep and Cap proteins). When an rAAV vector is integrated into a larger polynucleotide (e.g., into a chromosome or into another vector, such as a plasmid used for cloning or transfection), the rAAV vector is referred to as a "provector" that is "rescued" by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions.

[0089] As used herein, the terms "gene of interest (GOI)" and "transgene" refer to a polynucleotide that can be introduced into a cell and transcribed by the cell into RNA, optionally translated into protein, and / or expressed under appropriate conditions. A gene of interest or transgene may confer a desired characteristic on the cell into which it is introduced, or may otherwise bring about a desired therapeutic or diagnostic result.

[0090] As used herein, the term "prevent" means to stop or reduce something from happening, at least for a period of time, such as, for example, stopping at least one symptom of a disease from appearing, stopping the production of a particular gene product, or reducing the amount of a gene product produced by a particular gene.

[0091] As used herein, the phrase "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit-risk ratio.

[0092] As used herein, the phrase "pharmaceutically acceptable carrier" refers to an agent (e.g., an excipient, carrier, buffer, etc.) that is suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit-to-risk ratio. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. Typical pharmaceutical carriers may include, for example, phosphate-buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rd ed. 2020).

[0093] As used herein, "treat," "treating," and "treatment" refer to the treatment of a disease, disorder, or symptom or sign thereof in a subject, e.g., a human. This includes preventing the disease or disorder, inhibiting the disease, disorder, etc. (i.e., slowing or halting its progression or onset), and alleviating the disease, disorder, etc. (i.e., causing regression of the disease state).

[0094] As used herein, "subject" and "patient" refer to an organism treated by compositions made in accordance with the present disclosure and / or methods provided herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, etc.), and more preferably include humans.

[0095] As used herein, the term "vector" includes nucleic acid vectors, e.g., DNA vectors such as plasmids, RNA vectors, or other suitable replicons (e.g., viral vectors). Various vectors have been developed for delivering exogenous polynucleotides or polynucleotides encoding proteins into prokaryotic or eukaryotic cells. Examples of such expression vectors are disclosed, for example, in WO 1994 / 011026, which is incorporated herein by reference, as it relates to vectors suitable for expressing nucleic acid molecules of interest. Expression vectors suitable for use in the compositions and methods described herein contain a polynucleotide sequence as well as additional sequence elements used, for example, for expression of heterologous nucleic acid material (e.g., nucleic acid molecules) in cells. Certain vectors used for expression of the nucleic acid molecules provided herein can include plasmids containing regulatory sequences, such as promoter and enhancer regions, capable of directing and / or otherwise affecting gene transcription. In some embodiments, the promoter may be a compact bidirectional promoter and, in some embodiments, does not contain an enhancer. Other useful vectors for expressing the nucleic acid molecule drugs disclosed herein contain polynucleotide sequences that increase the translation rate of these polynucleotides or improve the stability or nuclear export of the RNA resulting from gene transcription. These sequence elements include, for example, 5' and 3' untranslated regions, internal ribosomal entry sites (IRES), and polyadenylation signals (polyA) to direct efficient transcription of the genes carried on the expression vector.

[0096] Producer cell lines Producing viral products for use in clinical populations requires addressing, and often overcoming, several challenges related to manufacturing and commercial viability. Among other things, the present disclosure provides innovative strategies for generating stable and inducible producer cell lines that can be used in the manufacture of viral products. These strategies involve, for the first time, combining three exogenous cellular components that are stably integrated into host cells and then infected with an inducing virus to produce a recombinant viral product that can be used for commercial and / or clinically relevant purposes.

[0097] In some embodiments, the present disclosure provides engineered viral producer 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 producer cell line. Also provided herein are viral producer cell lines comprising all the nucleic acids required to encode the components required for recombinant viral production, where the nucleic acids are stably integrated and include, but are not limited to, Rep, Cap, and a 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).

[0098] In some aspects, the present disclosure provides systems, engineered cell lines, and methods for producing viral vectors and viral products. While the use of any suitable virus is contemplated, adeno-associated virus (AAV), by way of example, is frequently used in recombinant viral vector systems for therapeutic drug delivery. Wild-type AAV is a small, non-enveloped human parvovirus that is infectious but 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 ITR base pairs allow for the synthesis of complementary DNA strands. Rep and Cap are translated to produce several different proteins (Rep78, Rep68, Rep52, Rep40—required for the AAV life cycle; VP1, VP2, VP3—capsid proteins).

[0099] Among other things, the present disclosure provides sequences (e.g., nucleic acid sequences, e.g., amino acid sequences) for one or more 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, a polynucleotide provided herein can include one or more A, G, C, and / or T nucleobases. In some embodiments, a polynucleotide provided herein can include one or more A, G, C, and / or U nucleobases. As will be understood by one of skill in the art, in a given context, the designation of either "T" or "U" in a given polynucleotide is considered interchangeable under appropriate circumstances. For example, a polynucleotide having a sequence of CAGTTTATGGT can also be understood, in a given appropriate context, to be and function as CAGUUUAUGGU, and can be used interchangeably under appropriate circumstances and / or context, as will be understood by one of skill in the art (e.g., DNA / cDNA versus, e.g., mRNA / RNA). It should be understood that throughout the description (e.g., as provided in the Sequence Listing, in SEQ ID NOS: 1-203 or 210-216), in each instance where a polynucleotide comprises, consists essentially of, or consists of a nucleotide sequence that includes one or more thymine nucleobases ("T"), another polynucleotide that comprises, consists essentially of, or consists of the same nucleotide sequence that includes uracil nucleobases ("U") in place of thymine (T) is also contemplated, or alternatively, in each instance where a polynucleotide comprises, consists essentially of, or consists of a nucleotide sequence that includes one or more uracil nucleobases ("U"), another polynucleotide that comprises, consists essentially of, or consists of the same nucleotide sequence that includes thymine nucleobases ("T") in place of uracil (U) is also contemplated.Thus, for any sequence provided herein, including those set forth in any of SEQ ID NOS: 1-191 or 210-216, it should be understood that any sequence containing a "T" also contemplates a polynucleotide containing a "U" and may be used in accordance with the teachings provided herein, as appropriate in a given context and / or situation.

[0100] The generation of recombinant adeno-associated virus (rAAV) vectors involves providing an AAV transfer plasmid in which a transgene is placed between two ITRs and Rep and Cap are supplied in trans. The transfer plasmid, along with Rep, Cap, and additional helper plasmids, is then provided to cells (e.g., HEK-293 cells) to produce recombinant virus containing the rAAV vector.

[0101] The present disclosure provides the surprising discovery that stable integration of Rep, Cap, and ITR-GOI constructs into host cells does not destabilize the host cell genome. Furthermore, the quality of the viral product is not compromised. This is achieved, among other things, by using engineered Kozak and / or enhancer sequences. For example, it is contemplated that toxicity can be regulated in host cells by modulating Rep levels. Alternatively or additionally, in some embodiments, a concatemerization strategy is used (e.g., generating concatemers of Rep or Cap constructs prior to stable integration into the cellular genome). In some embodiments, concatemers of the present disclosure comprise at least two monomers. In some embodiments, concatemers comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more monomers. Concatemers can be of any size, including at least two monomers, and are small enough to be separated and purified using standard techniques. For example, in some embodiments, such concatemers may be purified using standard gel purification methods known to those of skill in the art.

[0102] The techniques provided herein, such as methods or systems, achieve stable expression of Rep and Cap by providing engineered Kozak and / or enhancer sequences prior to contacting the construct with cells into which it will stably integrate, and / or optionally in combination with concatemerization of the construct. These approaches improve parameters such as stabilization and reproducibility, accelerate growth, and improve the efficiency of viral product production.

[0103] In some embodiments, engineered cells (e.g., virus-producing cells stably integrated with Rep, Cap, and ITR-GOI) can rapidly proliferate. In some embodiments, the cells can reach a population of at least about 1 x 10 after at least about 24 hours (e.g., at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 hours). 7 In some embodiments, the cells can be expanded to at least about 1-2 x 10 cells / mL after at least about 24 hours (e.g., at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 hours). 7 As a non-limiting example, the doubling time of Sf9 cells can be approximately 18-30 hours in suspension culture and up to greater than 72 hours during single cell isolation.

[0104] In some embodiments, the cells are at least about 1 x 10 cells after at least about 24 hours (e.g., at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 hours). 9 ~1×10 15 In some embodiments, the cells can be expanded to at least about 1 x 10 cells / mL after at least about 24 hours (e.g., at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 hours). 15In 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 more. In some embodiments, the doubling time of a suspension culture is 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 clonal cells is longer than the doubling time of suspension cultures. In some such embodiments, the doubling time can range from about 20 hours to about 84 hours, from about 24 hours to about 72 hours, from about 48 hours to about 72 hours, or about 24, 48, or 72 hours. In some such embodiments, rapid growth from a single cell facilitates or provides the ability to select cells that are free of rhabdovirus.

[0105] In some embodiments, cells in which the Rep, Cap, and ITR-GOI constructs have stably integrated into the host cell do not destabilize the host cell genome. This can be confirmed by using sequencing analysis (e.g., next-generation sequencing) to demonstrate a low or no percentage of reads / sequences mapping to host- or vector-related nucleic acids. For example, in some embodiments, sequencing analysis shows a low percentage of reads / sequences mapping to the Sf9 or baculovirus genome, supporting minimal packaging of Sf9 and baculovirus DNA in assembled capsids. The low amount of baculovirus packaging confirms that stable integration of the three constructs is a reliable, reproducible, safe, and improved approach compared to cells that integrate only one or two components. The small amount of Sf9 genome is a surprising finding, especially considering that the cells stably integrate the Cap, Rep, and GOI-ITR constructs, since one skilled in the art would expect that integration of the ITR-GOI into cells with Cap and Rep would result in larger amounts of host cell components (e.g., genomic DNA) being packaged into the final viral product.

[0106] Without being bound by theory, it is contemplated that stable genome integration facilitates induction of AAV production at high titers by active infection using recombinant baculovirus lacking AAV elements that alleviate issues of BEV genome stability during scale-up, allowing for higher, more reproducible, and / or healthier cell numbers and viral product yields produced in accordance with the present disclosure. The systems disclosed herein are adaptable to multiple viruses. In some embodiments, the virus is AAV. In some embodiments, the systems provided herein are adaptable to AAV capsid serotypes and efficiently package self-complementary and single-stranded AAV genomes.

[0107] The system of the present disclosure can be used to produce a population of engineered producer cell lines. In some embodiments, such cell lines can be produced by rapidly screening image-verified clonal producer cell populations. In some embodiments, the population is identified as a candidate that does not contain rhabdovirus. In some such embodiments, such a population can exceed E5 AAV gc / cell or E14 AAV gc / L. In some embodiments, the system disclosed herein produces cell lines that produce virus in a reproducible and consistent manner over many (e.g., 5, 10, 15, 20, 25, etc.) passages after thawing. In some such embodiments, the system's capabilities support linear scalability, in contrast to the nonlinear scalability of many other previously described approaches. It is contemplated that these successes are achieved because the technology of the present disclosure causes and allows Rep, Cap, and / or GOI to be stably integrated, preventing the loss of efficacy of these genes, and regulating the expression of Rep, Cap, or both, preventing cellular toxicity. In contrast, previously described processes are often hampered by complications (e.g., Rep toxicity or loss of one or more components required for cells to produce viral products). Importantly, viruses produced using engineered cell lines as provided herein are at least as potent and effective as viruses derived using other systems. Furthermore, in some embodiments, the engineered cells are insect cells, and viruses produced therewith are at least as potent and effective in vivo as viruses derived from their mammalian-derived counterparts.

[0108] In some embodiments, the present disclosure provides improved cell systems, the improvement comprising a single host cell comprising at least three components stably integrated into the host cell's genome and an inducer virus that induces the host cell to begin producing recombinant virus (e.g., AAV). The host cell comprising the three stably integrated components comprises at least 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 and a Cap gene or a functional fragment thereof, and a third nucleic acid comprising a GOI. In some such embodiments, any one of the nucleic acids further comprises an enhancer sequence and / or an engineered Kozak sequence. Upon stable integration into the host cell genome, when the host cell is contacted with the inducer virus, the inducer virus activates the integrated components, resulting in the production of recombinant virus comprising the GOI.

[0109] Engineered cells In some aspects, the disclosure provides a virus-producing cell. In some embodiments, the virus-producing cell has an engineered genome comprising stably integrated components. In some such embodiments, the components comprise: (a) a first nucleic acid comprising a first enhancer sequence and a Rep gene or a functional fragment or derivative thereof; (b) a second nucleic acid comprising a second enhancer sequence and a Cap gene or a functional fragment or derivative thereof; and (c) a third nucleic acid comprising a GOI.

[0110] The virus-producing cell can be any type of cell that can be used for recombinant virus production, hi 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 (e.g., a Chinese hamster ovary (CHO) cell, a baby hamster kidney (BHK) cell, a human embryo 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 The cell may be a Saccharomyces cerevisiae cell, a Cryptococcus cell, or a Candida cell), a plant cell (e.g., a parenchyma cell, a sclerenchyma 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 (e.g., an E. coli cell, a streptococcus bacterial cell, a streptomyces soil bacterial cell, or an archaeal cell).

[0111] In some embodiments, the cells are from a cell line. In some embodiments, the cells are primary cells.

[0112] In some embodiments, the cells are A549 cells, HEK-293 cells, HEK-293T cells, BHK cells, CHO cells, HeLa cells, MRC5 cells, Sf2 cells, Sf9 cells, Sf2 cells, High Five™ cells, Sf21 cells, BTI-Tn-5B1-4 cells, Cos-1 cells, Cos-7 cells, Vero cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, WI38 cells, Saos cells, C2C12 cells, L cells, HT1080 cells, HepG2 cells, Huh7 cells, K562 cells, or any derivatives thereof. In some embodiments, the cells are Sf9 cells.

[0113] In some embodiments, the present disclosure provides improved engineered cells, the improvement comprising combining in a single host cell at least three stably integrated components comprising at least a first nucleic acid comprising a first enhancer sequence and a Rep gene or any functional derivative or fragment thereof, a second nucleic acid comprising a second enhancer sequence and a Cap gene or any functional derivative or fragment thereof, and a third nucleic acid comprising a GOI. In some such embodiments, any one of the nucleic acids further comprises an enhancer sequence and / or a Kozak sequence, and in some embodiments, the Kozak sequence is an engineered Kozak sequence.

[0114] In some embodiments, the cells may optionally further comprise a helper virus or a functional derivative or fragment thereof, and / or a helper plasmid and / or a functional derivative or fragment thereof. In some such embodiments, such a helper plasmid, derivative, or fragment may be part of one or more constructs or components (e.g., part of a component including Rep and / or Cap, etc.). In some such embodiments, the helper is an AAV helper.

[0115] In some embodiments, stable integration of one or more components into the genome of the host cell is random with respect to location within the host cell genome, hi some embodiments, stable integration of one or more components into the genome is site-specific and / or directed to one or more particular locations in the host cell genome.

[0116] Upon stable integration into the host cell genome, the host cells are then contacted with an inducer virus that activates the integrated components, resulting in the production of recombinant virus containing the GOI. The engineered cells produce more virus, more rapidly, and more reproducibly than previously described engineered cells.

[0117] Rep and Rep constructs Parvoviruses, including AAV, contain a Rep gene that encodes four proteins necessary for replication and packaging of the viral genome. The four proteins are Rep78, Rep68, Rep52, and Rep40. A virus-producing cell having an engineered genome described herein, in some embodiments, comprises a first nucleic acid comprising a Rep gene or any functional derivative or fragment thereof. In some embodiments, the Rep gene encodes a protein selected from Rep78, Rep68, Rep52, Rep40, and any functional derivative or fragment thereof.

[0118] 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 an AAV. In some embodiments, the AAV is a mammalian (e.g., human, e.g., non-human primate) AAV or an avian AAV (AAAV), i.e., in some embodiments, the starting and / or engineered AAV is or is derived from a virus that is capable of infecting a mammalian or avian organism. 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, AA V-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 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.

[0119] In some embodiments, the first nucleic acid comprising a Rep gene or a functional fragment thereof 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 minipromoter, 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.

[0120] In some embodiments, the first nucleic acid comprising a Rep gene or a functional fragment thereof further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, a beta-lactam, a macrolide, a tetracycline, or any functional fragment and / or derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, blasticidin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, zeocin, or any functional fragment and / or derivative thereof.

[0121] In some embodiments, the first nucleic acid comprising a Rep gene or a functional fragment thereof further comprises an origin of replication, hi 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.

[0122] In some embodiments, the first nucleic acid comprising a 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 a portion of SEQ ID NO: 210 that encodes a 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 a sequence encoding the rep gene or a functional derivative or functional fragment thereof produced using the nucleic acid sequence of SEQ ID NO: 210.

[0123] Cap and Cap constructs The capsid proteins of AAV constitute the external, non-nucleic acid portion of the virion and are 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 the three products, while VP1 and VP2 are produced in lower amounts (relative to the total amount of capsid proteins). AAV capsids generally contain their VP1, VP2, and VP3 proteins in a ratio of VP1:VP2:VP3 = 1:1:10. As known to those of skill 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 proteins (AAPs), e.g., membrane-associated accessory proteins (MAAPs)).

[0124] Virus-producing cells with engineered genomes described herein, in some embodiments, comprise a second nucleic acid comprising a Cap gene or any functional derivative or fragment thereof.

[0125] In some embodiments, the Cap gene, or a functional derivative or fragment thereof, is derived from a 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 AAAV, i.e., 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 AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AA V-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 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.

[0126] In some embodiments, the Cap gene or functional fragment thereof encodes a protein selected from one or more of the group consisting of VP1, VP2, VP3, MAAP, AAP, and any functional fragment and / or functional derivative thereof. In some embodiments, the Cap gene or 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 the nucleic acid sequence of any one of SEQ ID NOs: 192-203. In some embodiments, the Cap gene, or a functional derivative or functional fragment thereof, encodes an amino acid sequence having 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 the amino acid sequence of any one of SEQ ID NOs: 204-209. In some embodiments, the nucleic acid sequence of SEQ ID NO: 192 or 193 encodes the amino acid sequence of SEQ ID NO: 204. In some embodiments, the nucleic acid sequence of SEQ ID NO: 194 or 195 encodes the amino acid sequence of SEQ ID NO: 205. In some embodiments, the nucleic acid sequence of SEQ ID NO: 196 or 197 encodes the amino acid sequence of SEQ ID NO: 206. In some embodiments, the nucleic acid sequence of SEQ ID NO: 198 or 199 encodes the amino acid sequence of SEQ ID NO: 207. In some embodiments, the nucleic acid sequence of SEQ ID NO: 200 or 201 encodes the amino acid sequence of SEQ ID NO: 208. In some embodiments, the nucleic acid sequence of SEQ ID NO:202 or 203 encodes the amino acid sequence of SEQ ID NO:209.

[0127] In some embodiments, the second nucleic acid comprising the Cap gene or a functional derivative or fragment thereof 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 minipromoter, or any functional fragment and / or 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 derivative thereof. As will be understood by those skilled in the art, in a given context, certain promoters can be classified as early promoters or late promoters, and those skilled in the art will know how and when to use such promoters to achieve expression of a component.

[0128] In some embodiments, the second nucleic acid comprising a Cap gene or a functional fragment thereof further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, a beta-lactam, a macrolide, a tetracycline, or any functional fragment and / or derivative thereof. In some embodiments, the antibiotic resistance gene encodes kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, blasticidin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, zeocin, or any functional fragment and / or derivative thereof.

[0129] In some embodiments, the second nucleic acid comprising a Cap gene or a functional fragment thereof further comprises an origin of replication, hi 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.

[0130] 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 the nucleic acid sequence of any one of SEQ ID NOs: 211-216. In some embodiments, the first nucleic acid comprising a 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 nucleic acid sequence of any one of SEQ ID NOs: 211-216 or any functional fragment and / or functional derivative thereof, such as any portion of SEQ ID NOs: 211-216 that encodes a Cap protein or a functional portion thereof. In some embodiments, the second nucleic acid comprising a 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 a sequence encoding a Cap gene or a functional derivative or functional fragment thereof produced using the nucleic acid sequence of any of SEQ ID NOs: 211-216.

[0131] Enhancer sequence Enhancers are small molecules used to enhance viral transduction processes and increase target gene expression. In some embodiments, the present disclosure provides enhancer sequences for improving viral transduction processes. In some embodiments, virus-producing cells comprise a first nucleic acid comprising a first enhancer sequence and a second nucleic acid comprising a second enhancer sequence.

[0132] In some embodiments, the first enhancer sequence and the second enhancer sequence are the same. In some embodiments, the first enhancer sequence and the second enhancer sequence are different. In some embodiments, the first enhancer sequence is a homologous region (hr) enhancer sequence. In some embodiments, the second enhancer sequence is a hr enhancer sequence. In some embodiments, the enhancer is or comprises a sequence derived from Autographa californica nuclear polyhedrosis virus (NCBI Taxonomy ID 46015).

[0133] 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 of the nucleic acid sequences of SEQ ID NOs: 1-10, or to a functional fragment or functional derivative thereof.

[0134] 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 a nucleic acid sequence of any of SEQ ID NOs: 1-10, or to a functional fragment or functional derivative thereof.

[0135] In some embodiments, the first enhancer sequence is derived from an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, anellovirus, bocavirus, vaccinia virus, or retrovirus. In some embodiments, the first enhancer sequence is derived from a baculovirus. In some embodiments, the AAV is mammalian (e.g., human, e.g., non-human primate) AAV or avian AAV (AAAV), i.e., 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 AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AA V-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 In some embodiments, the first enhancer sequence is selected from 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. In some embodiments, the first enhancer sequence is derived from a baculovirus.

[0136] In some embodiments, the second enhancer sequence is derived from an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, anellovirus, bocavirus, vaccinia virus, or retrovirus. In some embodiments, the second enhancer sequence is derived from a baculovirus. In some embodiments, the AAV is mammalian (e.g., human, e.g., non-human primate) AAV or avian AAV (AAAV), i.e., 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 AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AA V-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 In some embodiments, the second enhancer sequence is selected from 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. In some embodiments, the second enhancer sequence is derived from a baculovirus.

[0137] Kozak sequence A Kozak sequence is a nucleic acid motif that functions as a protein translation initiation site in most eukaryotic mRNA transcripts. In some embodiments, engineered Kozak sequences are provided herein that improve the expression of viral proteins involved in recombinant virus production. Such sequences can be selected to promote preferential translation in insect cells relative to translation in mammalian cells. Preferential translation refers, for example, to improved quantity and / or quality of a downstream gene product in one cell type or condition compared to another (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 designed and / or optimized to function in insect cells, whereas a different design that translates preferentially in mammalian cells may function in insect cells, but not as well. A variety of engineered Kozak sequences are known in the art and can be used alone, in combination, and / or further modified in accordance with the present disclosure (see, e.g., WO 2017 / 181162; Viruses 2023, 15, 1983).

[0138] In some embodiments, a 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 preferentially promotes translation in insect cells, e.g., compared to mammalian cells.

[0139] In some embodiments, a Kozak sequence is used in a nucleic acid comprising 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 preferentially promotes translation in insect cells, e.g., compared to mammalian cells.

[0140] In some embodiments, a Kozak sequence is used in a nucleic acid comprising a GOI and, optionally, ITRs. 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, e.g., compared to 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 from, e.g., insect cells.

[0141] In some embodiments, engineered Kozak sequences are used to increase or improve expression of a particular protein (e.g., a Cap protein, such as VP1). In some embodiments, engineered Kozak sequences are used to modulate (e.g., attenuate, reduce, alter, etc.) expression of a particular protein (e.g., a Rep protein). In some embodiments, a Kozak sequence of the present disclosure comprises, consists essentially of, or consists of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotide residues. In some embodiments, a Kozak sequence comprises a 10-nucleotide residue sequence having six of those nucleotide residues (e.g., ATG / AUG) 5' to the start codon sequence and one nucleotide residue (e.g., G) 3' to the start sequence. In some embodiments, a Kozak sequence consists essentially of six nucleotide residues (e.g., ATG / AUG) 5' to the start codon sequence and one nucleotide residue (e.g., G) 3' to the start sequence. 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).

[0142] As will be appreciated by those skilled in the art, when generating comparative products using a different platform, such as mammalian cells (e.g., HEK293 cells), it is not necessary to use such engineered Kozak sequences optimized for insect cells. That is, in some embodiments, when generating a viral product (e.g., an rAAV containing a gene of interest) in an insect system (e.g., Sf9) and a mammalian system (e.g., HEK293), for example, for comparison with each other, the mammalian system uses endogenous rep / cap sequences and the insect system uses 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 engineered Kozak sequences that preferentially translate capsid in insect cells).

[0143] Preferred engineered Kozak sequences according to the present disclosure are those that can result in higher levels of VP1 protein compared to non-engineered Kozak sequences. That is, in some embodiments, Kozak sequences that are compatible with preferential translation in mammalian systems can result in lower amounts of certain proteins (e.g., VP1) when transferred to an insect system. Lower amounts of such structural proteins reduce the quality and quantity of the viral capsid and, therefore, the overall quality and quantity of the therapeutic agent.

[0144] The virus-producing cell, in some embodiments, comprises a first nucleic acid that further comprises a first Kozak sequence.

[0145] In some embodiments, such virus-producing cells comprise a first engineered Kozak sequence that 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 the nucleic acid sequence of any one of SEQ ID NOs: 11-191, or to a functional fragment and / or functional derivative thereof.

[0146] The virus-producing cell, in some embodiments, comprises 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 the nucleic acid sequence of any one of SEQ ID NOs: 11-191, or to a functional fragment and / or functional derivative thereof. In some embodiments, the first engineered Kozak sequence and the second engineered Kozak sequence are the same. In some embodiments, the first engineered Kozak sequence and the second engineered Kozak sequence are different. In some embodiments, the first Kozak sequence is unengineered and the second Kozak sequence is engineered.

[0147] In some embodiments, the Kozak sequence comprises or consists essentially of a polynucleotide whose sequence comprises the five terminal residues written as AXGYY, where X=U or T and Y=A, G, C, or T / U. In some embodiments, the Kozak sequence may be a "leaky" Kozak sequence and may comprise the formula ACGYY, where the ACG region may provide "readthrough" so that ribosomes do not bind as frequently and / or strongly as in the presence of a Kozak sequence comprising the sequence ATGYY, and the ATG confers a "stronger" Kozak sequence. In some embodiments, the Kozak sequence comprises a polynucleotide having a sequence comprising the five terminal residues written as 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 relative to a Kozak sequence comprising AXGYY. In some embodiments, the Kozak sequence comprises or consists essentially of a polynucleotide that is a sequence including the five terminal residues written as AXGYY, where X=U or T and Y=A, G, C, or T / U. In some embodiments, the engineered Kozak sequence has a formula comprising XXXXXXATGYY, where X is any nucleotide and Y is G or C. In some embodiments, the engineered Kozak sequence has a formula comprising XXXXXXATGXX, where X is A, G, C, or T / U. In some embodiments, the Kozak sequence is not a suboptimal sequence, such as one that includes a "leaky" Kozak sequence such as ACG.

[0148] Without being bound by theory, in some such embodiments, such sequences set forth at the terminal portions of Kozak sequences in constructs encoding polypeptides can preserve 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 (e.g., by altering the second amino acid encoded by Kozak, etc.) to encode a non-wild-type amino acid sequence, e.g., a non-wild-type Rep and / or a non-wild-type Cap. In some embodiments, strategies provided by the present disclosure more faithfully recapitulate and / or preserve certain characteristics of AAV, for example, by not altering amino acids near the start codon of a given construct (e.g., a Rep construct, e.g., a Cap construct, etc.).

[0149] In some embodiments, instead of or in addition to a Kozak sequence (if present, optionally modified), the construct may contain one or more artificial introns. As known to those skilled in the art (see, e.g., Mol Ther. 2008 May;16(5):924-30; U.S. Pat. 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 an approach, the artificial intron is placed in a nucleic acid sequence that also contains an insect cell (e.g., Sf9) promoter (e.g., polH, e.g., p10). This arrangement can be configured so that the artificial intron promotes translation of genes with overlapping open reading frames (e.g., rep and cap), resulting in different splice forms of proteins being translated when the intron is present compared to when it is spliced ​​out (e.g., VP1 of cap is translated when present, and VP2 and VP3 are translated when spliced ​​out).

[0150] In some embodiments, the present disclosure contemplates that an engineered Kozak sequence is preferable for regulating Cap expression compared to an artificial intron.

[0151] Certain engineered Kozak sequences may be preferred depending on the AAV serotype used. Exemplary engineered Kozak sequences for use in certain AAV serotypes are shown in Table 1.

[0152] [Table 1]

[0153] Gene of interest In some embodiments, the virus producer cells having an engineered genome described herein comprise a third nucleic acid comprising a 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 second ITR sequence are wild-type ITR sequences. In some embodiments, the first and / or second ITR sequence are modified compared to the wild-type sequence. In some embodiments, the first and / or second ITR sequence are derived from the same serotype. In some embodiments, the first and / or second ITR sequence are derived from different serotypes. In some embodiments, the first and second ITR sequences are the same. In some embodiments, the first and second ITR sequences are different. In some embodiments, the first and second ITR sequences are asymmetric. For example, in some embodiments, the first ITR may be longer than the second ITR, or vice versa. In some embodiments, one or both ITRs may be "mutant" ITRs (e.g., modified relative to the naturally occurring ITR, such as to delete a particular portion, such as a "trs" mutant ITR).

[0154] In some embodiments, the third nucleic acid comprising the GOI 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 minipromoter, 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.

[0155] The present disclosure contemplates that, in some embodiments, it may be preferable to reduce the amount of host DNA excised from the plasmid genome in which the ITR-GOI cassette is excised. As provided herein, the present disclosure contemplates that a method for reducing excess plasmid / prokaryotic DNA surrounding the ITR-GOI cassette is to use restriction enzyme digests to remove host DNA flanking the ITR-GOI-ITR cassette (see, e.g., Figure 13). Figure 13 shows a simplified diagram of a cassette in which two ITRs (each of which may or may not be mutant or modified ITRs, and which may be the same or different) flank the GOI. When a plasmid containing this construct is linearized to monomers, enzymes are also used to digest away additional genomic sequences outside the ITR-GOI-ITR cassette. In some embodiments, the total amount of plasmid / prokaryotic DNA on the outer segments of the ITRs (the side not containing the GOI) comprises 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 flanking either ITR (outside the GOI) need not be the same length; for example, in some embodiments, the DNA flanking one ITR may be longer or shorter than the DNA flanking the other ITR. In some embodiments, the flanking DNA on the 5' end of the GOI cassette may be about 100, 200, 300, 400, 500, 600, or 700 nucleotides and / or the flanking DNA on the 3' end of the GOI cassette may be about 50, 60, 70, 80, 100, 200, 300, 400, 500, 750, 100, 1250, 1500, or 1750 nucleotides. Less flanking DNA (e.g., from plasmid / prokaryotic sources) may be desirable for downstream purification and clinical manufacturing processes.

[0156] Inducing virus In some embodiments, a virus-producing cell is described herein, wherein the cell is infected with a virus to induce expression of a first, second, and / or third nucleic acid stably integrated into the host virus-producing cell. In some embodiments, the virus is selected from an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, anellovirus, 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 differs from a virus found in nature, but is not a recombinant virus and / or does not contain recombinant components such as a GOI or ITRs. In some embodiments, a wild-type virus is a virus that does not contain an exogenous gene and / or exogenous sequence (e.g., a GOI, e.g., an ITR). In some embodiments, the wild-type virus is a baculovirus that includes an exogenous gene (e.g., an antibiotic resistance gene, a detectable marker gene, etc.), but does not include a gene (e.g., a GOI, e.g., ITR) that is designed to be inserted into the engineered cell. In some embodiments, the wild-type virus is a baculovirus that has been engineered to remove certain endogenous functions, such as enzyme activity / protease function, for example, by removing or rendering non-functional certain proteases, such as ChIA or vCath. In some embodiments, the virus may be recombinant but lack any heterologous or exogenous components that are designed to be inserted into a cell, such as, for example, AAV elements for insertion into a host cell, such as an insect cell. In some embodiments, the virus is not a recombinant virus.

[0157] In some embodiments, the infection induces expression of one or more stably integrated nucleic acid sequences described herein. In some embodiments, the infection produces a recombinant virus. In some embodiments, the recombinant virus is selected from an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, anellovirus, bocavirus, vaccinia virus, and retrovirus. In some embodiments, the AAV is a mammalian (e.g., human, e.g., non-human primate) AAV or an avian AAV (AAAV), i.e., 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 AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rh10, AA V-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 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.

[0158] The virus-producing cells described herein, in some embodiments, comprise rAAV vectors produced with improved quality (e.g., reduced contaminants, improved potency, etc.). In some embodiments, the rAAV vectors contain less than 5%, 4%, 3%, 2%, or 1% contaminants from non-AAV components and GOIs. In some embodiments, the contaminants are derived from alphaviruses, parvoviruses, baculoviruses, dengue viruses, lentiviruses, poxviruses, anelloviruses, bocaviruses, vaccinia viruses, herpesviruses, or retroviruses. In some embodiments, the contaminants are derived from baculoviruses. In some embodiments, the contaminants may be derived from host cell genomic components (e.g., insect cell genomic contaminants).

[0159] Manufacturing method Processes for generating recombinant viruses, including recombinant viral vectors such as rAAV vectors used in gene therapy, face challenges in large-scale manufacturing and production. Specifically, problems arise due to low vector concentration, oncogenic host cell DNA, and the difficulty of purifying rAAV vectors from cells. Therefore, improved strategies are needed to develop large-scale manufacturing solutions and provide commercially viable AAV products to large clinical populations. Provided herein, in certain embodiments, are virus-producing cells with stably integrated genomes containing the components necessary for rAAV production. The 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, the present disclosure provides, among other things, viral cells in which all components for generating recombinant gene therapy (e.g., rAAV) are added (via transformation) to the genome of a single cell (e.g., Sf9 cell) and none of the therapeutic components (e.g., ITR-GOI) are introduced, for example, via a recombinant baculovirus. Rather, all components are present in the viral cells 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 production of recombinant virus (e.g., rAAV).

[0160] Further provided herein, in some embodiments, are methods for producing recombinant viruses comprising rAAV vectors using the virus-producing cells described herein, which require fewer steps (e.g., multiple transfections) and result in improved scalability, quality, and efficacy.

[0161] Also provided herein is a method for producing an rAAV vector, the method comprising: (a) providing a virus-producing cell having an engineered genome, the cell comprising or consisting 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 virus; and (c) after step (b), culturing the cell to produce recombinant virus comprising the rAAV vector.

[0162] 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 genome of the cell. 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 genome of the cell. In some embodiments, the virus-producing cell is inducible.

[0163] In some embodiments, virus-producing cells are selected for productivity by infecting each clonal population with a recombinant viral expression vector (e.g., BEV) lacking any AAV elements. In some embodiments, clones are screened under antibiotic and / or serum-free conditions. Infection can initiate expression of the AAV Rep and AAV Cap genes integrated into the genome. Expression of these genes can rescue the integrated AAV genome and its packaging into assembled AAV capsids. Following the production phase, AAV can then be recovered from the cell monolayer by freeze-thawing 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 can be separated and, optionally, quantified by PCR. (See Figures 2A-2E for a schematic diagram.)

[0164] In some embodiments, the clones are then expanded into production bioreactors to produce the recombinant viral vector. In some embodiments, the recombinant viral vector (e.g., rAAV) is then harvested, any active virus is inactivated (e.g., by heat) and / or removed (e.g., by enzymes and / or physical separation), and the viral particles are purified. In some embodiments, the recombinant viral vector is purified and formulated. In some embodiments, the contents of the viral particles are released and further processed (e.g., treated, e.g., purified).

[0165] In some embodiments, a suitable medium is used for the production of the recombinant vector. These media include, but are not limited to, media appropriate for the cell type (e.g., mammalian, insect, etc.), such as, for example, Modified Eagle Medium (MEM), Roswell Park Memorial Institute (RPMI) 1640, Eagle's Minimal 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 Sf Insect ACF (FUJIFILM Irvine Scientific), 4 Cell Insect Media (Sartorius), Hyclone SFX (Cytiva Life Sciences), EX-Cell (Sigma Aldrich), and / or media produced by Hyclone Laboratories and JRH, including custom formulations, particularly for custom media formulations for use in the production of recombinant vectors.

[0166] In some embodiments, suitable production culture media of the present disclosure are supplemented with serum or serum-derived recombinant proteins at levels of 0.5-20 (v / v or w / v). In some embodiments, vectors are produced under serum-free conditions, also referred to as animal-derived product-free media. In some embodiments, media may be chemically defined. In some embodiments, commercially available or custom media are designed to support vector production, including, but not limited to, supplementation with glucose, vitamins, amino acids, and / or growth factors to increase vector titer and / or yield in the production culture.

[0167] Vector production cultures include a variety of conditions (e.g., over a wide temperature range, for various lengths of time, etc.) that are suitable for the particular host cells utilized. Vector production cultures include attachment-dependent cultures cultured in suitable attachment-dependent vessels such as plates, flasks, cell stacks, roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors. In some embodiments, vector production cultures include suspension-adapted host cells such as HeLa, HEK-293, and SF-9 cells cultured in a variety of ways, including spinner flasks, stirred-tank bioreactors, single-use bioreactors such as Cytiva Xcellerex and Sartorius, and disposable systems such as the Wave bag system.

[0168] In some embodiments, viral particles of the present disclosure are recovered from vector production cultures by lysis of host cells of the production culture or by collection of spent medium from the production culture, provided that the cells are cultured under conditions that cause release of viral particles from 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.

[0169] In further embodiments, the viral particles are purified. As used herein, the term "purified" includes preparations of viral particles that lack at least some of the other components present in the viral particles when they are naturally occurring or initially prepared. Thus, for example, in some embodiments, isolated viral particles are prepared using a purification technique to enrich them from a source mixture, such as a culture lysate or production culture supernatant. In some embodiments, enrichment is measured in various ways, such as by the percentage of DNase-resistant particles (DRP) or genome copies (gc) present in the solution, or by infectivity, or measured with respect to a second potentially interfering substance present in the source mixture, such as a production culture contaminant or in-process contaminant, including helper virus, media components, etc.

[0170] In some embodiments, vector production culture harvests are clarified to remove host cell debris. In some embodiments, production culture harvests are clarified by filtration through a series of depth filters, including, for example, a Grade DOHC Millipore Millistak+HC Pod Filter, a Grade A1HC Millipore Millistak+HC Pod Filter, and a 0.2 μm Filter 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 larger.

[0171] In some embodiments, the vector production culture harvest is further treated with Benzonase® to digest any high molecular weight DNA present in the production culture. In some embodiments, Benzonase® digestion is carried out under standard conditions, e.g., with a final concentration of Benzonase® of at least 1-2.5 units / mL (in some embodiments, up to 50 units / mL), at temperatures ranging from ambient to 37°C, for a period of 30 minutes to several hours.

[0172] In some embodiments, viral particles are isolated or purified using one or more of the following exemplary purification steps: freeze-thaw; equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) to concentrate viral particles; vector capture by apatite chromatography; heat inactivation of helper virus; vector capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and vector capture by anion exchange chromatography, cation exchange chromatography, or affinity chromatography. In some embodiments, these steps are used alone, in various combinations, or in different orders. In some embodiments, the method includes all of the steps, optionally in the order listed below.

[0173] In some embodiments, methods for generating recombinant vectors (e.g., rAAV) involve providing stably integrated virus-producing cells containing a helper plasmid. In some embodiments, the cells are transfected with a helper plasmid that provides helper functions for AAV. In some embodiments, the helper plasmid provides adenoviral functions, including, but not limited to, E1A, E1B, E4, and E2A. In some embodiments, the helper plasmid provides other viral functions, including, but not limited to, VA RNA, Gag, Pol, Tat, Rev, Env, and VSV-G. The sequences of the adenoviral genes that provide these functions are, in some embodiments, derived from any known adenoviral serotype (e.g., serotypes 2, 3, 4, 7, 12, and 40), further including any of the currently identified human types. In some embodiments, the methods involve transfecting the cells with a vector that expresses one or more genes required for AAV replication, AAV gene transcription, and / or AAV packaging.

[0174] The present disclosure also provides methods for generating recombinant vectors, comprising providing a virus producer cell comprising 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, the cell is a virus producer cell comprising selected components under the control of a constitutive promoter and other selected components under the control of one or more inducible promoters. For example, virus producer cells are generated that comprise E1 helper functions under the control of a constitutive promoter, but Rep and / or Cap proteins under the control of one or more inducible promoters.

[0175] Products produced using the platforms provided herein, such as Sf9 cells, can be compared to those produced using standard platforms, such as mammalian cell lines (e.g., HEK293 cells). In such systems, appropriate rep and cap genes can be transfected into mammalian cells either on a single plasmid (e.g., pRep / Cap) or using individual plasmids, each encoding a rep and cap gene. Whether on a single plasmid or on two or more plasmids, the transfected material contains sequences for producing a viral product (e.g., rAAV) containing the gene of interest. Generally, the gene of interest can be introduced into a mammal via a separate plasmid (e.g., in addition to one or more plasmids encoding rep and / or cap) containing the ITR-GOI components, such that when the ITR-GOI-containing cells express the rep and cap components, they produce an encapsidated viral gene product containing the gene of interest.

[0176] General approaches for transfecting, transforming, and infecting mammalian cells to provide virus (e.g., AAV)-based gene therapy are known in the art. As will be apparent to those skilled in the art, depending on the context, the specific rep and cap genes and their regulatory sequences for the serotype of interest will either be endogenous sequences for the particular serotype or sequences corresponding to a particular capsid (e.g., AAV6™, AAV7™, AAV8™, etc.). Such plasmids facilitate expression in mammalian systems and generally do not contain engineered Kozak vectors, such as those used in the insect cell systems disclosed herein. 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 approaches have also been proposed by 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. al., J. Virol., 62:1963 (1988); and Lebkowski et al. 1988 Mol. Cell. Biol., 7: 349 (1988). Samulski et al. 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; WO 96 / 17947; International Application PCT / US98 / 18600; WO 97 / 09441 (International Application PCT / US96.4423); WO 97 / 08298 (International Application PCT / US96 / 13872); WO 97 / 21825 (International Application PCT / US96 / 20777); WO 97 / 06243 (International Application PCT / FR96 / 01064); WO 99 / 11764; Perrin et al. 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. Patent Nos. 5,786,211; 5,871,982; and 6,258,595.

[0177] The methods disclosed herein result in improvements in recombinant viral vector production, including improved yields of recombinant virus, more efficient and faster production times, and greater reproducibility and scalability without any decrease in the efficacy of the recombinant virus produced.

[0178] In some embodiments, the present disclosure provides an improvement in the production of recombinant viral products, the improvement comprising combining three stably integrated components into a single host cell, the three stably integrated components comprising 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 one of the nucleic acids further comprises an enhancer sequence and / or an engineered Kozak sequence. Upon stable integration into the host cell genome, the host cell is contacted with an inducer virus that activates the integrated components, resulting in the production of a recombinant virus comprising the GOI.

[0179] Other details for making and using recombinant viruses containing genes of interest, including AAV, can be found, for example, in PCT Publication Nos. WO 2010 / 114948 and WO 2017 / 181162.

[0180] Throughout the description, when compositions are described as having, including, or comprising certain components, or when processes and methods are described as having, including, or comprising certain steps, it is further contemplated, even if not explicitly stated, that there are compositions of the present disclosure that consist essentially of or consist of the recited components, and / or that there are processes and methods according to the present disclosure that consist essentially of or consist of the recited processing steps, which may occur in any order unless so specified.

[0181] Any and all examples, or the use of exemplary language herein, such as "such as" or "including," is intended merely to better describe the disclosure and does not impose limitations on the scope of the disclosure unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of any embodiment of the disclosure.

[0182] When an element or component is said to be included in and / or selected from a list of described elements or components, it is to be understood that the element or component can be any one of the described elements or components, or the element or component can be selected from a group consisting of two or more of the described elements or components.

[0183] Furthermore, it should be understood that elements and / or features of the compositions or methods provided herein, whether expressly or implicitly stated herein, can be combined in various ways without departing from the spirit and scope of what is disclosed. For example, when a particular compound is referenced, that compound can be used in various embodiments of the compositions and / or methods of the present disclosure, unless otherwise understood from the context. In other words, within this application, embodiments have been described and illustrated to enable a clear and concise application to be written and drawn, but it is intended and understood that the embodiments can be combined or separated in various ways without departing from the present disclosure. For example, it should be understood that all features described and illustrated herein may be applicable to all aspects of any invention provided, described, and / or illustrated herein.

[0184] It should be understood that the order of steps or order of performing certain actions is immaterial so long as what is disclosed and / or claimed remains operable regardless of order. Moreover, two or more steps or actions may be conducted simultaneously.

[0185] The present disclosure provides multiple aspects and embodiments of one or more inventions, which are specifically contemplated in any and all combinations and permutations of the aspects and embodiments disclosed herein.

[0186] Pharmaceutical Composition Once produced, the recombinant AAV particles provided herein can be formulated into pharmaceutical compositions.

[0187] For therapeutic use, compositions containing the recombinant viruses provided herein are combined with a pharmaceutically acceptable carrier. Various carriers (e.g., diluents, excipients, etc.) used in formulating and preparing pharmaceutical compositions are known to those skilled in the art and / or readily available. Depending on the situation, the carrier may comprise a liquid (e.g., a sterile liquid) or a solid. The carrier may be selected from or contain water, aqueous solvents, non-aqueous solvents, dispersion media, surfactants, antioxidants, buffers, adjuvants, isotonicity agents, stabilizers, bulking agents, lyoprotectants, metal ions, chelating agents, isotonicity agents, and absorption delaying agents, etc., that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art. Typically, the carrier is approved by the United States Food and Drug Administration and meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or other International Pharmacopoeias. Formulations suitable for use in the present disclosure are described, for example, in Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23 rded. 2020). For a brief review of drug delivery methods, see, e.g., Langer (1990) SCIENCE 249:1527-1533. The resulting pharmaceutical composition is suitable for administration to a subject (e.g., an animal, e.g., a mammal, e.g., a human).

[0188] Pharmaceutical compositions may contain formulation materials to, for example, modify, maintain, or preserve the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. In such embodiments, suitable formulation materials include 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 borate, bicarbonate, Tris-HCl, citrate, phosphate, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (ethylenediamine tetraacetic acid, acid, EDTA, etc.); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); bulking agents; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin), proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; 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 glycerin, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as Pluronic, polyethylene glycol, glycol, PEG), sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal, etc.; stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium chloride or potassium chloride, mannitol sorbitol, etc.); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants (e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (2013) 23:199-200). rded.2020)), but are not limited to these.

[0189] In certain embodiments, the pharmaceutical composition may contain a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled-delivery means (e.g., liposome carriers, biodegradable microparticles or porous beads, and depot injections) are also known to those skilled in the art. Sustained-release preparations may include, for example, porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained-release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2-hydroxyethyl-inethacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained-release compositions may also include liposomes, which can be prepared by any of several methods known in the art.

[0190] Optionally, the pharmaceutical composition may contain nanoparticles, or lipid droplets, such as polymeric nanoparticles, liposomes, or micelles (see Anselmo et al. (2016) BIOENG.TRANSL.MED. 1:10-29).

[0191] Pharmaceutical compositions containing the rAAV of the present disclosure may be provided in unit dosage form and can be prepared by any suitable method. Pharmaceutical compositions should be formulated to be compatible with their intended route of administration. Examples of routes of administration include intravenous (IV), intraperitoneal, intradermal, inhalation, transdermal, intracerebroventricular (ICV), intraparenchymal, intracisternal (ICM), intrathecal, and intradural.

[0192] Useful formulations can be prepared by methods known in the pharmaceutical arts, see, for example, Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (2003). rded. 2020). Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA, and buffers such as acetates, citrates, or phosphates; and agents for adjusting tonicity such as sodium chloride or dextrose.

[0193] Suitable carriers are known to those skilled in the art. For example, for intravenous administration, suitable carriers include physiological saline, bacteriostatic water, polyethoxylated castor oil, or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage and preserved against microorganisms. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. For injection into the central nervous system (e.g., ICV, ICM, intraparenchymal, intrathecal, etc.), the carrier will be appropriately adjusted.

[0194] Pharmaceutical formulations are preferably sterile. The formulation or its components can be sterilized by, for example, a method appropriate to maintain the activity and stability of the GOI encoded therein. Sterilization can be achieved by any suitable method (e.g., filtration through a sterile filtration membrane). If the composition is lyophilized, sterile filtration can be performed before or after lyophilization and reconstitution.

[0195] When the dosage form is liquid or solid, depending on the drug substance and formulation, the resulting dosage form may be stable for extended periods, such as 1 month, 3 months, 6 months, 1 year, 2 years, 3 years, or more. The formulation may be stable at room temperature or above. It is contemplated that the dosage form is stable in PBS at ambient conditions. Alternatively, the dosage form is frozen (e.g., liquid or lyophilized) and stable at an appropriate temperature, such as -20°C, -80°C, etc.

[0196] Optionally, the dosage form can be formulated as a unit dose that can contain, for example, a particular vg / L as provided herein.

[0197] The compositions described herein can be administered locally or systemically. It is contemplated that the compositions described herein can be administered parenterally. In some embodiments, such administration is preferably performed directly into the central nervous system. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. In certain embodiments, the pharmaceutical compositions can be administered subcutaneously or intravenously, for example, via intravenous infusion. In certain embodiments, it is contemplated that the gene therapy methods disclosed herein can be administered systemically.

[0198] The amount administered depends on variables such as the type and severity of the disease or symptom being treated, the patient's overall health, the in vivo efficacy of the active ingredient and any toxicity concerns, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased above the upper level to rapidly achieve the desired blood or tissue level. Alternatively, the initial dosage may be lower than the optimal amount, and the daily dosage may be gradually increased over the course of treatment. The human dosage can be optimized, for example, in a conventional Phase I dose-escalation study. The frequency of administration can vary depending on factors such as the route of administration, the dosage, and the disease, disorder, or condition being treated. Exemplary administration frequencies are once daily, once weekly, and once every two weeks.

[0199] Methods of Use and Treatment The rAAV provided herein can be used in a variety of different approaches. For example, the rAAV can be used in a method for treating a disease, disorder, or condition associated with a dysfunctional gene of interest. The method includes contacting cells in a subject in need thereof, and the composition includes a nucleic acid encoding a gene of interest, which, when expressed, will treat a disease, disorder, or condition associated with a dysfunctional GOI.

[0200] In some embodiments, the disease, disorder, or condition is associated with a dysfunctional gene that is expressed in or affects one or more cells of the central and / or peripheral nervous system.

[0201] It is contemplated that therapy may be achieved using rAAV alone, as a monotherapy, or as part of a combination therapy, which may include one or more additional agents or therapeutic approaches known to those skilled in the art for treating inflammatory and / or autoimmune diseases, and which may have been previously used, may already be ongoing, or may be added to a treatment for a subject in need thereof.

[0202] The subject can be evaluated, for example, by a healthcare provider, before, during, and / or after treatment with the compositions provided herein. Depending on the results of the evaluation, treatment may be continued or discontinued, the frequency or dosage of treatment may be modified, or the patient may be treated with a different gene therapy. The subject can be administered compositions comprising a gene therapy provided herein, optionally for separate periods of time according to the dosage paradigms described herein, including until the disease, disorder, or condition is treated.

[0203] Incorporation by Reference All publications and patents (including all patents, patent applications, scientific publications, manufacturer's specifications, instruction manuals, etc.) cited throughout the body of this specification, whether supra or infra, are hereby incorporated by reference in their entirety for all purposes. To the extent that the material incorporated by reference contradicts or is inconsistent with this specification, the present specification will take precedence over any such material.

[0204] equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments, therefore, are to be considered in all respects as illustrative rather than limiting of the invention described herein. The scope of the invention is, therefore, 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. [Example]

[0205] Below are examples of specific embodiments for carrying out the present disclosure. The examples are provided for illustrative purposes only and are not intended to limit the scope of the disclosure in any way.

[0206] Example 1. Production of AAV using engineered cell lines with stably integrated exogenous viral components This example generally describes the production of AAV using the engineered cell lines described herein. Engineered cell lines were generated to stably integrate AAV components into Sf9 cells. The engineered cells contained AAV Cap components, AAV Rep components, and a GOI stably integrated into the cell's genome. The Cap, Rep, and GOI components were integrated into the genome using engineered constructs. Various AAV serotypes were tested.

[0207] The Kozak-optimized AAV capsid sequence and AAV Rep sequence and GOI were stably integrated into Sf9 cells by random integration and antibiotic selection to generate a heterogeneous pool of stably transformed Sf9 producer cells, as further described in Example 2.

[0208] For each serotype, Kozak sequences were first screened by SDS-PAGE and Western blot to obtain a range of capsid stoichiometries. The primary candidate sequences that enhanced VP1 incorporation into the AAV capsid were cloned together with enhancer elements into a cell line that integrates the vector.

[0209] Clonal Sf9 cell lines were derived from the heterogeneous pool by a process of single-cell seeding and whole-well imaging. Rapidly growing clones exhibiting favorable growth kinetics were scaled up for productivity screening, which is further described in Example 3.

[0210] Clones were selected for productivity by infecting them with baculovirus and harvesting the AAV produced by each clone. The clone that produced the highest amount of AAV was further scaled up to analyze product attributes and improve production parameters.

[0211] Infection parameters were determined using small-scale stirred-tank bioreactors, a production scale sufficient to examine process and key product quality attributes of AAV derived from each clonal cell line.

[0212] AAV produced using the engineered Sf9 producer cell line platform and the mammalian HEK293 triple transfection platform was purified and evaluated head-to-head in rodent models, as further described in Example 4.

[0213] Example 2: Isolation and characterization of Sf9 producer cell lines As described in Example 1, the Sf9 producer cell line was engineered to increase productivity and potency. The producer cells were engineered to produce AAV using the homology region (hr) enhancer element and an engineered Kozak sequence. An exemplary construct design is shown in Figure 1A. The hr element used in this example is native to the baculovirus genome and acts as a transcriptional enhancer for the adjacent transgene. This mechanism was preserved and integrated into the Sf9 cell genome in the form of DNA concatemers encoding the AAV Rep and AAV Cap proteins. The engineered Kozak sequences were screened and compared to determine their ability to incorporate higher levels of VP1 into AAV capsids. These higher VP1 levels may promote increased endosomal escape probability and increased capsid potency. This engineering approach works across AAV serotypes, as demonstrated in Figure 1B (Western blots showing AAV1 and AAV5 capsids) and Figure 1C (affinity-purified AAV7, AAV8, and AAV9 capsids).

[0214] Clonal populations of cells were generated by single-cell seeding and whole-well imaging. Random integration of DNA concatemers and linearized ITR-flanked gene-of-interest constructs was achieved by transfection of DNA into Sf9 cells and antibiotic selection of stable transformants. To facilitate initial testing of the ability to produce and purify AAV products using these engineered cells, a heterogeneous pool of stably transformed Sf9 AAV producer cell lines was scaled up. Clonal populations of these Sf9 AAV producer cell lines were isolated by single-cell seeding and whole-well imaging. Validated clonal populations were monitored over a 3-4 week period and then transferred to 96-well plates for head-to-head screening.

[0215] Producer cell lines generated as described herein were selected for productivity by infecting each clonal population with a recombinant baculovirus expression vector (BEV) lacking any AAV elements. Clones were seeded into well plates (Figure 2A) and grown to confluent monolayers (Figures 2B and 2C), as shown in Figures 2A-2E. Plates were imaged prior to infection to determine confluency. BEV contained a visible reporter to facilitate detection and lacked naturally occurring proteases (e.g., VCath, ChIA). Visualization allowed for purification (before infection) and monitoring of infection rates (e.g., by monitoring how many cells displayed detectable label). In most cases, 90–95% of cells were fluorescent. Clones were screened in antibiotic- and serum-free conditions (mirroring those of the mature manufacturing process).

[0216] Following a baseline measurement of confluency to estimate cell number, each monolayer consisting of cells derived from a single clone was infected with "WT" BEV, which means that it lacked any AAV elements, such as the ITRs and / or GOI (Figure 2C). Infection initiated expression of the AAV Rep and AAV Cap genes integrated into the genome, inducing AAV production (Figure 2C).

[0217] Expression of these genes resulted in rescue of the integrated AAV genome and packaging of the gene of interest (GOI) into assembled AAV capsids.

[0218] Following the production phase, AAV was recovered from the cell monolayer using freeze-thaw and nuclease treatment. Briefly, the medium (containing 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). Cell debris was subsequently separated / clarified from the AAV (Figure 2E), thereby separating the supernatant containing BEV and AAV from the cell monolayer. The resulting AAV content produced by each clonal population was quantified by PCR, as shown in the schematic diagram in Figure 2E, right panel.

[0219] To identify high-producing clones, clonal Sf9 AAV producer candidates were selected based on the amount of AAV they produced. The adherent clone screening assay was refined to select producer clones that yielded >E14 vg / L in a more mature suspension-based manufacturing process before optimization. That is, although screening was performed in adherent cultures, clones producing in the E10 vg / mL range during the screening assay typically yielded in the high E13 to low E14 vg / L range in a more mature suspension-based manufacturing process before optimization. The top producers identified (see Figure 3) were scaled up and further analyzed for product analysis and production optimization.

[0220] A scale-up process was developed to reduce the number of passages required to transition cells from 96-well adherent cultures to suspension. As seen in Figure 4, this significantly accelerated growth and reduced the time to establish a suspension-ready research cell bank. Accelerated growth using the scale-up process resulted in a doubling of cell numbers in half the time (i.e., a four-fold increase). The scale-up process, from transfection, clone selection, AAV product analysis, and banking of the RCB, took approximately three months.

[0221] Example 3: Sf9 quality analysis This example illustrates the quality of Sf9 cells produced according to the systems, methods, and compositions (e.g., engineered cells) provided herein.

[0222] Purification of Sf9-derived AAV by affinity capture demonstrated preservation of capsid stoichiometry and enhanced levels of VP1 incorporation into purified capsids. Anion exchange methods were developed to enrich genome-containing capsids, currently yielding concentrations of 70%–80% depending on the serotype. Downstream purification methods removed host cell proteins in addition to host cell and baculovirus DNA impurities in the final AAV preparation.

[0223] Figures 5A-5D show measurements of the amount of host cell proteins (HCPs) and activated virus present throughout the production process and before and after purification. Both host cell proteins and activated virus impurities decreased throughout the purification process, demonstrating that the enhanced stoichiometry did not affect the ability to purify recombinant virus from host cells or other potential contaminants from the viruses involved in production (e.g., activated baculovirus). Figures 5A and 5B are gels showing the reduction in impurities over the purification process. Figure 5C is a bar graph showing the HCPs detected in fractions from various purification steps in ng / mL. 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 for AAVX and AEX is from Sf9 DNA measurement, and the right bar is from BEV DNA measurement. These data confirm that the system, method and cell provided herein provide a final product with biophysical and biochemical properties that are at least the same as those that have been obtained previously using other (e.g., HEK293) based cell systems, and that existing equipment (e.g., AEX column, affinity purification column, etc.) can be successfully used to purify the virus produced using the method provided herein.Because Sf9 cell is a heterogeneous population of rhabdovirus-positive and rhabdovirus-negative cells, producer cell line was screened for the presence of rhabdovirus.During single cell isolation and clonal selection, rhabdovirus-free Sf9 AAV producer cell line was identified, and as shown in Table 2, the presence of rhabdovirus was eliminated in the production process.

[0224] [Table 2]

[0225] The Sf9 AAV producer cell line demonstrates robust manufacturing capabilities. Stability studies were performed by thawing an exemplary producer clone. In this example, the exemplary producer clone was a free research cell bank of the Sf9 AAV5 producer clone. The thawed cultures were passaged for an additional 20 passages, and subsets of the cultures were infected at passages 5, 10, 15, and 20. AAV was harvested from each clone at each infection time point and quantified by droplet digital PCR (ddPCR). As shown in Figure 6, no drift in volume (vg / L) or unit (vg / cell) titer was observed over the 20 passages, indicating stable integration of the AAV production components.

[0226] The Sf9 AAV producer cell line was selected based on growth and infection kinetics similar to conventional IC-BEV production methods. Cell density (cells / mL) and percent viability were measured over time during the growth and infection phases. Stable integration of the genetic components was confirmed by the doubling ability of Sf9 cells (24-30 hours), high density (>1 x 10), and viability as shown in Figure 7. 7 The results showed that the β-lactamase inhibitor β-lactamase (β-lactamase inhibitor β-lactamase) did not affect the proliferation of the β-lactamase inhibitor β-lactamase (β-lactamase inhibitor ...

[0227] AAV capsids produced on both the Sf9 and HEK293 platforms (further described in Example 4) were analyzed for post-translational modifications by LCMS (see Table 3). As previously reported, alternative patterns of post-translational modifications were observed between production methods. Next-generation Illumina sequencing of purified Sf9 AAV was performed to quantify any contaminants from the Sf9 or baculovirus genome. As illustrated in Figure 8 and shown in Table 3, the percentage of reads / sequences mapping to the Sf9 or baculovirus genome was very low, supporting minimal packaging of Sf9 and baculovirus DNA in assembled capsids. The low amount of baculovirus packaging confirms that stable integration of the three constructs is reliable, reproducible, safe, and an improved approach compared to cells that integrate only one or two components. One skilled in the art would expect that integrating the ITR-GOI into cells with Cap and Rep would result in increased amounts of host cell genome packaging, so the small amount of Sf9 genome is a surprising finding, especially considering that the cells stably integrate the Cap, Rep, and GOI-ITR constructs.

[0228] [Table 3] ----=No modifications detected

[0229] Example 4. Virus-producing Sf9 cells with engineered genomes This example generally describes a process for generating virus-producing cells with engineered genomes.

[0230] First, a plasmid containing an antibiotic resistance gene (e.g., puromycin, e.g., blasticidin), an enhancer (e.g., an hr element such as comprising any of SEQ ID NOS: 1-10 or a functional fragment or derivative thereof), an engineered Kozak sequence (which may optionally be engineered and selected from any of SEQ ID NOS: 1-191 or any functional fragment or derivative thereof in a construct comprising a Cap sequence), a promoter (e.g., an inducible promoter), and a Cap sequence such as nucleic acids (SEQ ID NOS: 192-203) or plasmids (SEQ ID NOS: 211-261) encoding a protein of either a Rep sequence (encoded by a plasmid such as SEQ ID NOS: 210) or amino acids (SEQ ID NOS: 204-209) can be linearized at a unique Type IIS restriction enzyme site to generate DNA monomers.

[0231] As exemplified herein, for Sf9-produced viral gene therapy products, the plasmids for each of the rep and cap sequences contained the enhancer sequence of SEQ ID NO: 1, a promoter (polH ​​or p10), and an antibiotic selection cassette encoding blasticidin. The rep gene comprises the rep sequence in the plasmid of SEQ ID NO:201, and the cap protein is sequenced as follows, depending on the serotype: AAV1: SEQ ID NO:192 (nucleic acid without engineered Kozak), SEQ ID NO:193 (nucleic acid with engineered Kozak used in an 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 used in an 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 used in an insect cell platform), SEQ ID NO:206 (amino acid sequence of the capsid protein); and SEQ ID NO:213 (plasmid). AAV7™: SEQ ID NO: 198 (nucleic acid without engineered Kozak), SEQ ID NO: 199 (nucleic acid with engineered Kozak used in an insect cell platform), SEQ ID NO: 207 (amino acid sequence of the capsid protein); and SEQ ID NO: 214 (plasmid); AAV8™: SEQ ID NO: 200 (nucleic acid without engineered Kozak), SEQ ID NO: 201 (nucleic acid with engineered Kozak used in an 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 (nucleic acid with engineered Kozak used in an insect cell platform), SEQ ID NO: 209 (amino acid sequence of the capsid protein); and SEQ ID NO: 216 (plasmid), and according to the sequences set forth in any of SEQ ID NOs: 192 to 216.The Cap gene transformed into the Sf9 genome also contained an engineered Kozak sequence selected from SEQ ID NO: 11 or 13 (also represented in the capsid sequences of SEQ ID NOs: 193 (AAV1), 195 (AAV5), 197 (AAV7), 199 (AAV7™), 201 (AAV8™), and 203 (AAV9)).

[0232] For the insect cell platform provided herein, the DNA of each nucleic acid (rep and cap) was purified and then assembled into monomers together by a process of ligation into very high molecular weight concatemers in a head-to-tail orientation. These high molecular weight concatemers were then further purified and quantified for transfection.

[0233] The GOI was linearized into monomers using restriction enzymes (Figure 9). Briefly, restriction digestion was performed on plasmid DNA containing restriction endonuclease sites and engineered inverted terminal repeats flanking the nucleotide sequence encoding the gene of interest. This restriction digest process removes undesired sequences (e.g., antibiotic resistance markers used for propagation in bacteria). These GOI monomers (containing the GOI flanked by ITRs) were purified and prepared for transfection to integrate them into the Sf9 genome. Restriction digestion removed a substantial amount of plasmid DNA elements / prokaryotic DNA sequences, such that the total length of the flanking genomic DNA was approximately 1,700 base pairs.

[0234] Prior to transfection into Sf9 cells, DNA was analyzed to calculate the amount of DNA for each component to be transfected into Sf9 cells. For transfection, Sf9 cells were initially seeded at approximately 80% confluence. Sf9 cells were transfected according to the manufacturer's protocol and subjected to antibiotic resistance 48 hours post-transfection. Sf9 cells were transfected with either DNA or mock. Cells were then assayed for viability 48 hours post-transfection. Cells were viable and capable of producing purified AAV, which was administered to mouse test subjects as described in Example 5.

[0235] For comparison with gene therapy products produced in Sf9 cells, AAV particles were also produced using the HEK293 system. Virus-producing HEK293 cells were generated using standard transfection methods known to those skilled in the art with: (a) (i) the endogenous rep / cap genes for each serotype of interest (e.g., AAV1, 5, 7, 8, and 9) or (ii) appropriately modified sequences (rep / cap genes expressed on the same plasmid) that differ from the endogenous AAV sequence for variant capsid proteins (e.g., for AAV7™, for AAV8™), and (b) an ITR-GOI nucleic acid, as exemplified in Example 5. Recombinant virus production (of the encapsidated GOI) was induced via infection of HEK293 cells.

[0236] Example 5. In vivo head-to-head comparison of Sf9-derived AAV and HEK293-derived AAV This example compares AAV derived from Sf9 and HEK293 host cells. AAV produced using the Sf9 system engineered according to the present disclosure demonstrated similar therapeutic efficacy compared to AAV produced using HEK293 cells.

[0237] AAV5 (SEQ ID NO: 194 for mammalian (HEK) cells or SEQ ID NO: 195 for insect (Sf9) cells) encoding a single-chain GOI encoding an exemplary enzyme was generated using an Sf9 platform according to the present disclosure or a standard HEK293 platform, as described in Example 4, and administered via intracisternal membrane (ICM) injection. Enzyme levels were measured in the cerebellum of wild-type, knockout, and Sf9 AAV- or HEK293 AAV-treated mice. As shown in Figure 10, a head-to-head comparison of AAV produced by Sf9 cells generated as provided herein with AAV produced by HEK293 cells revealed no significant difference in the resulting levels of enzyme activity measured in the cerebellum of animals treated with capsids produced using the different platforms (HEK293 cells and Sf9 cells generated according to the present disclosure), with enzyme levels in each AAV-treated group being higher than those of wild-type and knockout animals in the exemplary mouse model.

[0238] A 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), was produced using the Sf9 producer cell line of the present disclosure and compared to HEK293-produced AAV described in Example 4 in an exemplary mouse model of disease. A head-to-head comparison of Sf9-produced AAV (containing the GOI) and AAV (containing the GOI) using the HEK293 platform demonstrated equal biodistribution of the vector genome in target tissues ( FIG. 11A ) and efficacy in extending survival in the disease model ( FIG. 11B ).

[0239] AAV8 variants encoding exemplary therapeutic GOI sequences (SEQ ID NO: 200 for mammalian (HEK) cells or SEQ ID NO: 201 for insect (Sf9) cells) were produced using both the Sf9 producer cell line generated according to the present disclosure and a standard HEK293 platform, as described in Example 4. Equal doses of AAV gene therapy products produced on each platform were administered to neonatal mice. Ten weeks after administration, tissues were harvested and analyzed for the resulting enzyme activity. No differences were observed in enzyme levels as a result of treatment with vectors generated by the different production methods (Sf9 or HEK), or in the biodistribution and expression of the therapeutic transgene (not shown), as demonstrated by the enzyme levels measured in the forebrain ( FIG. 12A ), hindbrain ( FIG. 12B ), and cerebellum ( FIG. 12C ).

[0240] Importantly, these data also demonstrate that the Sf9 producer strain is suitable for both self-complementary AAV (see, e.g., Figures 11A and 11B) and single-stranded AAV (see, e.g., Figures 12A and 12B).

[0241] AAV9 (SEQ ID NO: 202 for mammalian (HEK) cells or SEQ ID NO: 203 for insect (Sf9) cells) encoding a GFP reporter construct was produced in both Sf9 insect cells and HEK293 mammalian platforms, as described in Example 4. AAV derived from three independent Sf9 clones was compared to material derived from a triple transfection of HEK293 cells. After intracisternal administration (ICM), samples were analyzed blinded by a neurohistologist. No appreciable differences were observed between the biodistribution profiles of AAV vectors derived from either the Sf9 or mammalian HEK293 platforms (see Figure 13, arrows, and Table 4).

[0242] For each serotype and each GOI, the AAV gene therapy products produced using Sf9 insect cells provided herein did not show any difference from those produced using HEK293-based mammalian cell platforms. Furthermore, insect cell-based therapeutics are scalable and reproducible, and therefore can provide commercially useful quantities of AAV particles suitable for gene therapy.

[0243] These results provide various examples demonstrating that such insect cell-based approaches can be used with a variety of serotypes and GOIs. Thus, while such insect cell approaches can deliver viral-based gene therapy at least as well as mammalian cell systems, they also offer several advantages over such HEK293 / mammalian cell platforms that can be used to improve the production of recombinant gene therapy products.

[0244] [Table 4]

[0245] [Table 5-1]

[0246] [Table 5-2]

[0247] [Table 5-3]

[0248] [Table 5-4]

[0249] [Table 5-5]

[0250] Table 5-6

[0251] Table 5-7

[0252] Table 5-8

[0253] Table 5-9

[0254] Table 5-10

[0255] Table 5-11

[0256] Table 5-12

[0257] Table 5-13

[0258] Table 5-14

[0259] Table 5-15

[0260] Table 5-16

[0261] Table 5-17

[0262] Table 5-18

[0263] Table 5-19

[0264] Table 5-20

[0265] Table 5-21

[0266] Table 5-22

[0267] Table 5-23

[0268] Table 5-24

[0269] Table 5-25

[0270] Table 5-26

[0271] Table 5-27

[0272] Table 5-28

[0273] Table 5-29

[0274] Table 5-30

[0275] Table 5-31

[0276] Table 5-32

[0277] Table 5-33

[0278] Table 5-34

[0279] Table 5-35

[0280] Table 5-36

[0281] Table 5-37

[0282] Table 5-38

[0283] Table 5-39

[0284] Table 5-40

[0285] Table 5-41

[0286] Table 5-42

Claims

1. A virus-producing cell, (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); and A virus-producing cell having an engineered genome, comprising:

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

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

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

5. The cell of any one of claims 1 to 4, wherein the first nucleic acid further comprises a second Kozak sequence, and the second Kozak sequence is an engineered Kozak sequence.

6. 6. The cell of any one of claims 1 to 5, wherein the third nucleic acid comprises a third Kozak sequence, and optionally the third Kozak sequence is an engineered Kozak sequence.

7. The cell of any one of claims 1 to 6, wherein any of the first, second, and / or third Kozak sequences enhances translation in insect cells and / or mammalian cells.

8. The cell of claim 7 , wherein the first and / or second Kozak sequences preferentially promote translation in insect cells.

9. 9. The cell of claim 7 or 8, wherein the third Kozak sequence preferentially promotes translation in mammalian cells.

10. The cell according to any one of claims 1 to 9, wherein the cell is a clone.

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

12. 12. The cell of any one of claims 1 to 11, wherein the Kozak sequence of the first nucleic acid, which is optionally engineered, and the engineered Kozak sequence of the second nucleic acid comprise the same nucleic acid sequence.

13. The cell of any one of claims 1 to 12, wherein the cell does not contain a rhabdovirus.

14. The cell of any one of claims 1 to 13, wherein the GOI is flanked by a first inverted terminal repeat (ITR) sequence and a second ITR sequence.

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

16. The cell of claim 15 or 16, wherein the first ITR and the second ITR are derived from a viral genome, and the first ITR is adjacent to the viral genome at its 5' end and the second ITR is adjacent to the viral genome at its 3' end, totaling no more than about 500 nucleotides.

17. 17. The cell according to any one of claims 1 to 16, wherein the Cap gene comprises a sequence having at least 80% sequence identity to the nucleic acid sequence of any of SEQ ID NOs: 192 to 203 or any functional fragment or functional derivative thereof.

18. 18. The cell of any one of claims 1 to 17, wherein the Cap gene encodes an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of any of SEQ ID NOs: 204 to 209, or any functional fragment or functional derivative thereof.

19. (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. 20. The cell of 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. 21. The cell of 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 of any one of claims 1 to 21, wherein the Rep gene is derived from an adeno-associated virus (AAV).

23. 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 23. The cell of claim 22, selected from 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 fragments and / or functional derivatives thereof.

24. The cell according to any one of claims 1 to 23, wherein the Cap gene is derived from AAV.

25. 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, A AV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC1 2. The cell of claim 24, wherein the cell is selected from 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.

26. (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. 27. The cell of any one of claims 1 to 26, wherein the first nucleic acid and the second nucleic acid each comprise an antibiotic resistance gene or a functional fragment or derivative thereof.

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

29. The cell of any one of claims 1 to 28, wherein the third nucleic acid does not contain an antibiotic resistance gene.

30. 30. The cell of any one of claims 26 to 29, wherein each of the first, second, and / or third antibiotic resistance genes is selected from genes encoding aminoglycosides, beta-lactams, macrolides, tetracyclines, or any functional fragments and / or functional derivatives thereof.

31. 31. The cell of any one of claims 26 to 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, zeocin, or any functional fragment and / or functional derivative thereof.

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

34. 34. The cell of any one of claims 1 to 33, wherein the first nucleic acid comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 210, or to any functional fragment or functional derivative thereof.

35. 35. The cell of any one of claims 1 to 34, wherein the second nucleic acid comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NOs: 211-216, or to any functional fragment or functional derivative thereof.

36. 36. The cell of any one of claims 1 to 35, wherein 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.

37. 37. The cell of any one of claims 1 to 36, wherein 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, a Sf9 cell, a Sf2 cell, a Sf21 cell, a High Five™ 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, a L cell, a HT1080 cell, a HepG2 cell, a Huh7 cell, a K562 cell, a primary cell, or any derivative thereof.

38. The cell of any one of claims 1 to 37, wherein the cell is an Sf9 cell.

39. 39. The cell of any one of claims 1 to 38, wherein the first nucleic acid, the second nucleic acid, and the third nucleic acid remain stably integrated into the genome after at least five passages.

40. The cells are at least about 1 x 10 cells after at least about 24 hours ７ The cell according to any one of claims 1 to 39, which is capable of growing to 1000 cells / mL.

41. The cell of any one of claims 1 to 40, wherein the cell is infected with a virus.

42. 42. The cell of claim 41, wherein the virus is selected from an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a dengue virus, a lentivirus, a herpesvirus, a poxvirus, anellovirus, a bocavirus, a vaccinia virus, and a retrovirus.

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

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

45. The virus (a) removing one or more endogenous genes or functions; and 43. The cell of claim 41 or 42, wherein (b) the cell has been engineered to disrupt one or more endogenous genes for production of a functional gene product.

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

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

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

49. 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-HS The cell of claim 48, which is selected from the group consisting of 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. 48. The cell of claim 47, wherein the recombinant virus comprises a recombinant AAV (rAAV) vector.

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

52. 52. The cell of claim 51, wherein the contaminant is derived from an alphavirus, parvovirus, baculovirus, dengue virus, lentivirus, poxvirus, anellovirus, bocavirus, vaccinia virus, herpesvirus, or retrovirus.

53. 53. The cell of claim 52, wherein the contaminant is derived from a baculovirus.

54. The cell according to any one of claims 51 to 53, wherein the contaminant is not derived from a rhabdovirus.

55. The infection is 1 x 10 ９ vg / L ~ 1 x 10 １５ 56. The cell of any one of claims 41 to 55, wherein the cell produces a plurality of vg / L.

56. The infection is 1 x 10 １５ 56. The cell of any one of claims 41 to 55, wherein the cell produces a plurality of ATP producing ATP in an amount of 1000 kJ / L or more.

57. (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); and A method for producing an rAAV vector, comprising:

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

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

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

61. 61. The method of any one of claims 57 to 60, wherein the first nucleic acid further comprises a second Kozak sequence, and the second Kozak sequence is an engineered Kozak sequence.

62. 62. The method of any one of claims 57-61, wherein the third nucleic acid comprises a third Kozak sequence, and optionally the third Kozak sequence is an engineered Kozak sequence.

63. 63. The method of any one of claims 57 to 62, wherein any of the first, second, and / or third Kozak sequences enhances translation in insect cells and / or mammalian cells.

64. 64. The method of claim 63, wherein the first and / or second Kozak sequences preferentially enhance translation in insect cells.

65. 65. The method of claim 63 or 64, wherein the third Kozak sequence preferentially promotes translation in mammalian cells.

66. 66. The method of any one of claims 57 to 65, wherein the cell is clonal.

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

68. 68. The method of any one of claims 57 to 67, wherein the Kozak sequence of the first nucleic acid, which is optionally engineered, and the engineered Kozak sequence of the second nucleic acid comprise the same nucleic acid sequence.

69. 69. The method of any one of claims 57 to 68, wherein the cells are free of rhabdovirus.

70. 70. The method of any one of claims 57 to 69, wherein the GOI is flanked by a first inverted terminal repeat (ITR) sequence and a second ITR sequence.

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

72. 72. The method of claim 70 or 71, wherein the first ITR and the second ITR are derived from a viral genome, and the first ITR flanks the viral genome at its 5' end and the second ITR flanks the viral genome at its 3' end, totaling no more than about 500 nucleotides.

73. 73. The method of any one of claims 57 to 72, wherein the Cap gene comprises a sequence having at least 80% sequence identity to the nucleic acid sequence of any of SEQ ID NOs: 192 to 203 or any functional fragment or functional derivative thereof.

74. 74. The method of any one of claims 57 to 73, wherein the Cap gene encodes an amino acid sequence having at least about 80% sequence identity to the amino acid sequence of any of SEQ ID NOs: 204 to 209, or any functional fragment or functional derivative thereof.

75. (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 75. The method of any one of claims 57-74, wherein (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. 76. The method of 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 of (i), (ii), or (iii).

77. 77. The method of 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. 78. The method of any one of claims 57 to 77, wherein the Rep gene is derived from an adeno-associated virus (AAV).

79. 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 79. The method of claim 78, wherein the antibody is selected from 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. 80. The method of any one of claims 57 to 79, wherein the Cap gene is derived from AAV.

81. 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, A AV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC1 2, 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.

82. 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 The method of any one of claims 57 to 81, wherein c) the third nucleic acid further comprises a third antibiotic resistance gene.

83. 83. The method of any one of claims 57 to 82, wherein the first nucleic acid and the second nucleic acid each comprise an antibiotic resistance gene or a functional fragment or derivative thereof.

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

85. 85. The method of any one of claims 57 to 84, wherein the third nucleic acid does not comprise an antibiotic resistance gene.

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

87. 87. The method of any one of claims 82 to 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, zeocin, or any functional fragment and / or functional derivative thereof.

88. (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. 89. The method of claim 88, wherein the first, second, and / or third origin of replication is selected from pMB1, pBR322, ColE1, R6K, p15A, pSC101, ColE2, Fl, pUC, and any functional fragment and / or functional derivative thereof.

90. 90. The method of any one of claims 57 to 89, wherein the first nucleic acid comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 210, or to any functional fragment or derivative thereof.

91. 91. The method of any one of claims 57 to 90, wherein the second nucleic acid comprises a nucleic acid sequence having at least 80% sequence identity to a nucleic acid sequence of SEQ ID NOs: 211 to 216, or to any functional fragment or functional derivative thereof.

92. 92. The method of any one of claims 57 to 91, wherein 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.

93. 93. The method of any one of claims 57 to 92, wherein 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, a Sf9 cell, a Sf2 cell, a Sf21 cell, a High Five™ 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, a L cell, a HT1080 cell, a HepG2 cell, a Huh7 cell, a K562 cell, a primary cell, or any derivative thereof.

94. 94. The method of any one of claims 57 to 93, wherein the cell is an Sf9 cell.

95. 95. The method of any one of claims 57 to 94, wherein the first nucleic acid, the second nucleic acid, and the third nucleic acid remain stably integrated into the genome after at least five passages.

96. The cells are at least about 1 x 10 cells after at least about 24 hours ７ The method of any one of claims 57 to 95, wherein the method is capable of growing to 1000 cells / mL.

97. 97. The method of any one of claims 57 to 96, wherein the cells are infected with a virus.

98. 98. The method of claim 97, wherein the virus is selected from an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a dengue virus, a lentivirus, a herpesvirus, a poxvirus, anellovirus, a bocavirus, a vaccinia virus, and a retrovirus.

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

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

101. The virus a) removing one or more endogenous genes or functions; and 99. The method of claim 97 or 98, wherein b) one or more endogenous genes have been engineered to prevent them from producing a functional gene product.

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

103. 99. The method of claim 97 or 98, wherein the infection induces the cells to produce recombinant virus.

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

105. 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, AA V14, 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-HS C12, 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.

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

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

108. 108. The method of claim 107, wherein the contaminant is derived from an alphavirus, parvovirus, baculovirus, dengue virus, lentivirus, poxvirus, anellovirus, bocavirus, vaccinia virus, herpesvirus, or retrovirus.

109. 109. The method of claim 108, wherein the contaminant is derived from a baculovirus.

110. The method of any one of claims 107 to 109, wherein the contaminant is not from a rhabdovirus.

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

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

113. 113. The composition of claim 112, wherein the cell is an insect cell.

114. 114. The composition of claim 113, wherein the insect cell is an Sf9 cell.

115. 113. The composition of claim 112, wherein the virus is a baculovirus.

116. The plurality of virus particles is 1 x 10 ９ vg / L ~ 1 x 10 １５ 113. The composition of claim 112, comprising:

117. The plurality of virus particles is 1 x 10 １５ 113. The composition of claim 112, comprising at least 1000 mg / L of Calcium.

118. 118. A method of infecting a cell in a subject in need thereof, comprising administering to said subject a composition according to claims 112-117.

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

120. 1. A system for producing a recombinant virus, comprising: a) a virus-producing cell according to any one of claims 1 to 40; b) a virus for infecting the virus-producing cells, the virus inducing production of the recombinant virus when the virus infects the virus-producing cells.

121. The system of claim 120, wherein the cells are infected with a virus.

122. 122. The system of claim 121, wherein the virus is selected from an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a dengue virus, a lentivirus, a herpesvirus, a poxvirus, anellovirus, a bocavirus, a vaccinia virus, and a retrovirus.

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

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

125. The virus (a) removing one or more endogenous genes or functions; and (b) the system of claim 121 or 122, which has been engineered to disrupt one or more endogenous genes for production of a functional gene product.

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

127. 123. The system of claim 121 or 122, wherein the infection induces the cells to produce recombinant virus.

128. 128. The system of claim 127, wherein the recombinant virus is selected from an alphavirus, parvovirus, adenovirus, AAV, baculovirus, dengue virus, lentivirus, herpesvirus, poxvirus, anellovirus, bocavirus, vaccinia virus, or retrovirus.

129. 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-HSC 12, 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.

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

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

132. 132. The system of claim 131, wherein the contaminant is derived from an alphavirus, parvovirus, baculovirus, dengue virus, lentivirus, poxvirus, anellovirus, bocavirus, vaccinia virus, herpesvirus, or retrovirus.

133. The system of claim 132, wherein the contaminant is derived from a baculovirus.

134. The system of any one of claims 130 to 132, wherein the contaminant is not derived from a rhabdovirus.

135. 1 x 10 ９ vg / L ~ 1 x 10 １５ The system according to any one of claims 120 to 134, which produces a recombinant virus of vg / L.

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

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