ENGINEERED CELLS FOR RECOMBINANT VIRUS PRODUCTION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application Nos. 63/414,890, filed on October 10, 2022, and 63/478,742, filed on January 6, 2023, the disclosures of each of which is incorporated by reference herein in its entirety for all purposes.

BACKGROUND

[0002] Viral vectors are commonly used to deliver therapeutic genes to humans. Production of viral vectors is a complex process. For example, several different issues can arise with design and manufacturing, including but not limited to scalability, quality, and potency of the recombinant viruses. Production of clinical grade gene therapies remains a major hurdle in advancing cures for a number of otherwise unpreventable, incurable and/or unbeatable diseases, disorders, and conditions. Improved processes are needed to achieve reproducible, consistent, scalable manufacturing solutions and provide commercially viable viral products that can be safely and reliably delivered to patients.

SUMMARY OF THE DISCLOSURE

[0003] The present disclosure provides, among other things, technologies for production of virus-producing cells. As is known to those of skill in the art, even as gene therapy technologies have rapidly improved, there are still challenges present related to engineering and manufacturing cells that reliably, consistently, and accurately achieve viral production at qualities and quantities suitable for, e.g., administration to subjects in need of one or more gene therapies. Technologies provided herein address certain unmet needs in production of virus-producing cells and overcome certain challenges to improve engineering and manufacturing of virus-producing cells. These engineered cells and methods of making and using them solve production challenges and quality (e.g., safety) concerns that are critical to advancing gene therapies.

[0002] In some aspects, the present disclosure provides a virus-producing cell, the virusproducing cell having an engineered genome comprising a first nucleic acid comprising a first enhancer sequence and a Rep gene or functional fragment thereof; a second nucleic acid comprising a second enhancer sequence, a first Kozak sequence, and a Cap gene or functional fragment thereof, wherein the first Kozak sequence is an engineered Kozak sequence; and a third nucleic acid comprising a gene-of-interest (GOI).

[0003] In some aspects, the present disclosure provides method for generating an rAAV vector, comprising: a first nucleic acid comprising a first enhancer sequence and a Rep gene or functional fragment thereof; a second nucleic acid comprising a second enhancer sequence, a first Kozak sequence, and a Cap gene or functional fragment thereof, wherein the first Kozak sequence is an engineered Kozak sequence; and a third nucleic acid comprising a gene-of-interest (GOI).

[0004] 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 functional derivative thereof.

[0005] 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 functional derivative thereof.

[0006] In some embodiments, the first and the second enhancer sequences are the same.

[0007] In some embodiments, the first nucleic acid further comprises a second Kozak sequence, wherein the second Kozak sequence is an engineered Kozak sequence.

[0008] In some embodiments, the third nucleic acid comprises a third Kozak sequence, optionally wherein the third Kozak sequence is an engineered Kozak sequence.

[0009] In some embodiments, any of the first, second, and/or third Kozak sequences facilitates translation in an insect cell and/or a mammalian cell.

[0010] In some embodiments, the first and/or second Kozak sequence preferentially facilitate translation in an insect cell.

[0011] In some embodiments, the third Kozak sequence preferentially facilitates translation in a mammalian cell.

[0012] In some embodiments, the cell is clonal.

[0013] In some embodiments, the engineered Kozak sequence of the second nucleic acid comprises a sequence with at least 80% identity to that of any of SEQ ID NOs: 11-191 or any functional fragment or functional derivative thereof.

[0014] 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.

[0015] In some embodiments, the cell is rhabdovirus-free. [0016] In some embodiments, the GOI is flanked by a first by a first inverted terminal repeat (ITR) sequence and a second ITR sequence.

[0017] In some embodiments, each of the first ITR sequence and the second ITR sequences has a different nucleic acid sequence.

[0018] In some embodiments, the first ITR and the second ITR are derived from a viral genome and wherein the first ITR is flanked on its 5' end and the second ITR flanked on its 3' end by a total of about 500 nucleotides or less of viral genome.

[0019] 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 functional derivative thereof.

[0020] 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.

[0021] 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.

[0022] In some embodiments, 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). In some embodiments, the promoter is selected from: CMV, CBA, EFla, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, plO, pl9, p40, Synapsin, CaMKII, GRK1, polH, EM7, OpIEl, 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 selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6TM, AAV7, AAV7TM, AAV8, AAV8TM, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV- rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV- HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV- HSC15, AAV-TT, AAV-DJ, AAV-DJ/8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional fragment and/or functional derivative thereof. [0023] In some embodiments, the Cap gene is derived from an AAV. In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV6TM, AAV7, AAV7TM, AAV8, AAV8TM, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV- hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV- HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ/8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivative thereof.

[0024] In some embodiments, the first nucleic acid further comprises a first antibiotic resistance gene; the second nucleic acid further comprises a second antibiotic resistance gene; and/or the third nucleic acid further comprises a third antibiotic resistance gene. In some embodiments, each of the first and the second nucleic acids comprise an antibiotic resistance gene or functional fragment or derivative thereof. In some embodiments, each of the first and the second antibiotic resistance genes 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: a gene encoding an aminoglycoside, a beta-lactam, a macrolide, a tetracycline, or any functional fragment and/or functional derivative thereof. In some embodiments, each of the first, second, and/or third antibiotic resistance genes is selected from: a gene 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.

[0025] In some embodiments, the first nucleic acid further comprises a first origin of replication; the second nucleic acid further comprises a second origin of replication; and/or the third nucleic acid further comprises a third origin of replication. In some embodiments, the first, second, and/or third origin of replication is selected from: pMBl, pBR322, ColEl, R6K, pl 5 A, pSClOl, ColE2, Fl, pUC, and any functional fragment and/or functional derivative thereof.

[0026] In some embodiments, the first nucleic acid comprises a nucleic acid sequence having at least 80% sequence identity to that of SEQ ID NO: 210 or to any functional fragment or functional derivative thereof. In some embodiments, the second nucleic acid comprises a nucleic acid sequence having at least 80% sequence identity to that of SEQ ID NOs: 211 — 216 or to any functional fragment or functional derivative thereof.

[0027] In some embodiments, the cell is a eukaryotic cell, a mammalian cell, an immortalized cell, an insect cell, a yeast cell, a plant cell, a fungal cell, or a prokaryotic cell. In some embodiments, the cell is an A549 cell, a HEK-293 cell, a HEK-293T cell, a BHK cell, a CHO cell, a HeLa cell, an MRC5 cell, an Sf9 cell, an Sf2 cell, an Sf21 cell, a High Five™ cell, a Cos-1 cell, a Cos-7 cell, a Vero cell, a BSC 1 cell, a BSC 40 cell, a BMT 10 cell, a WI38 cell, a Saos cell, a C2C12 cell, an L cell, an HT1080 cell, a HepG2 cell, a Huh7 cell, a K562 cell, a primary cell, or any derivative thereof. In some embodiments, the cell is an Sf9 cell.

[0028] In some embodiments, the first nucleic acid, the second nucleic acid, and the third nucleic acid remain stably integrated in the genome after at least 5 passages. In some embodiments, the cell is able to grow to at least about 1 x 10 ⁷ cells/mL after at least about 24 hours.

[0029] In some embodiments, the cell is infected with a virus. In some embodiments, the virus is selected from: an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a Dengue virus, a lentivirus, a herpesvirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, and a retrovirus. In some embodiments, the virus is a wild-type virus. In some embodiments, the virus does not comprise a nucleic acid necessary for AAV packaging. [0030] In some embodiments, the virus has been engineered to: remove one or more endogenous genes or functions; and prevent one or more endogenous genes for production a functional gene product. 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 combinations 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, an 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, AAV-rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV- 2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV- HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ/8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivative thereof.

[0031] In some embodiments, the recombinant virus comprises a recombinant AAV (rAAV) vector. In some embodiments, the rAAV vector comprises less than 5%, 4%, 3%, 2%, or 1% contaminants from one or more non-AAV components and/or a GOI. In some embodiments, the contaminants are from an alphavirus, a parvovirus, a baculovirus, a Dengue virus, a lentivirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, a herpesvirus, or a retrovirus. In some embodiments, the contaminants are from a baculovirus. In some embodiments, the contaminants are not from a rhabdovirus.

[0032] In some embodiments, a plurality of cells infected in accordance with the present disclosure results in a plurality that produces 1X10 ⁹ vg/L to IxlO ¹⁵ vg/L. In some embodiments, a plurality of cells infected in accordance with the present disclosure results in a plurality that produces IxlO ¹⁵ vg/L or greater.

[0033] In some aspects, the present disclosure provides a recombinant virus produced by methods provided herein. In some aspects, the present disclosure provides a composition comprising a plurality of viral particles produced by infecting a cell as 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 comprises 1X10 ⁹ vg/L to IxlO ¹⁵ vg/L. In some embodiments, the plurality of viral particles comprises IxlO ¹⁵ vg/L or greater.

[0034] In some aspects, the present disclosure provides a method of infecting cells in a subject in need thereof, the method comprising administering to the subject a composition as provided in accordance with the present disclosure.

[0035] In some aspects, the present disclosure provides a method of treating a subject having a disease, disorder, or condition associated with a dysfunctional gene of interest (GOI), the method comprising administering a composition as provided herein to produce a functional gene product of the GOI and treat the disease.

[0036] In some aspects, the present disclosure provides a system of generating a recombinant virus comprising: a virus-producing cell as provided herein; and a virus for infecting the virus-producing cell, which when infected with the virus induces the production of the recombinant virus.

[0037] In some embodiments, the cell is infected with a virus. In some embodiments, the virus is selected from: an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a Dengue virus, a lentivirus, a herpesvirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, and a retrovirus. In some embodiments, the virus is a wild-type virus. In some embodiments, the virus does not comprise a nucleic acid necessary for AAV packaging. In some embodiments, the virus has been engineered to remove one or more endogenous genes or functions; and prevent one or more endogenous genes for production a functional gene product.

[0038] 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 combinations 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, an 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, AAV-rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV- HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ/8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivative thereof.

[0039] In some embodiments, the recombinant virus comprises a recombinant AAV (rAAV) vector. In some embodiments, the rAAV vector comprises less than 5%, 4%, 3%, 2%, or 1% contaminants from one or more non-AAV components and/or a GOI. In some embodiments, the contaminants are from an alphavirus, a parvovirus, a baculovirus, a Dengue virus, a lentivirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, a herpesvirus, or a retrovirus. In some embodiments, the contaminants are from a baculovirus. In some embodiments, the contaminants are not from a rhabdovirus.

[0040] In some embodiments, the system produces 1 x 10 ⁹ vg/L to 1 x 10 ¹⁵ vg/L of recombinant virus. In some embodiments, the system produces IxlO ¹⁵ vg/L or greater of recombinant virus.

[0041] In some aspects, the present disclosure provides virus-producing cells, the virusproducing cell having an engineered genome comprising: (a) a first nucleic acid comprising a first enhancer sequence and a Rep gene or functional fragment thereof; (b) a second nucleic acid comprising a second enhancer sequence and a Cap gene or functional fragment thereof; and (c) a third nucleic acid comprising a gene-of-interest (GO I). 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 and the second enhancer sequences are the same. In some embodiments, the first nucleic acid further comprises a first engineered Kozak sequence. In some embodiments, the first engineered Kozak sequence has at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the nucleic acid sequence of any one of SEQ ID NOs: 11- 191.

[0042] 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 and the second engineered Kozak sequences are the same.

[0043] 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 sequences 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 amino acid sequence of any one of SEQ ID NOs: 204-209.

[0044] 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 combinations thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini promoter, or any functional fragment and/or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EFla, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, plO, pl9, p40, Synapsin, CaMKII, GRK1, polH, EM7, OpIEl, and any functional fragment and/or functional derivative thereof.

[0045] 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 combinations thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini promoter, or a functional fragment and/or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EFla, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, plO, pl9, p40, Synapsin, CaMKII, GRK1, polH, EM7, OpIEl, 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 combinations thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini promoter, or any functional fragment and/or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EFla, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, plO, pl9, p40, Synapsin, CaMKII, GRK1, polH, EM7, OpIEl, 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 a adeno-associated virus (AAV). In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV- 7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV- HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV- HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ/8, AAV-Myo, AAV-NP40, AAV- NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivative thereof. In some embodiments, the Cap gene is derived from a parvovirus. In some embodiments, the parvovirus is an AAV. In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV- rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV- PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV- HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ/8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV- NP66, AAV-HSC16, and any functional derivative thereof.

[0046] 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.

[0047] 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 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.

[0048] 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 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.

[0049] In some embodiments, the first nucleic acid further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMBl, pBR322, ColEl, R6K, pl 5 A, pSClOl, ColE2, Fl, pUC, and any functional fragment and/or functional derivative thereof. In some embodiments, the first nucleic acid comprises a sequence having at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 210.

[0050] In some embodiments, the second nucleic acid further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMBl, pBR322, ColEl, R6K, pl5A, pSClOl, ColE2, Fl, pUC, and any functional fragment and/or functional 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.

[0051] In some embodiments, the third nucleic acid further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMBl, pBR322, ColEl, R6K, pl5A, pSClOl, ColE2, Fl, pUC, and any functional fragment and/or functional derivative thereof.

[0052] 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, an HEK-293 cell, an HEK-293T cell, a BHK cell, a CHO cell, a HeLa cell, an MRC5 cell, an Sf9 cell, an Sf2 cell, an Sf21 cell, a High Five™ cell, a Cos-1 cell, a Cos-7 cell, a Vero cell, a BSC 1 cell, a BSC 40 cell, a BMT 10 cell, a WI38 cell, a Saos cell, a C2C12 cell, an L cell, an HT1080 cell, a HepG2 cell, a Huh7 cell, a K562 cell, a primary cell, or any derivative thereof.

[0053] In some embodiments, the cell is an Sf9 cell.

[0054] In some embodiments, the first nucleic acid, the second nucleic acid, and the third nucleic acid remain stably integrated in the genome after at least 5 (e.g., 10, 20, 40) passages. In some embodiments, the cell is able to grow to at least about 1 x 10 ⁷ cells/mL after at least about 24 hours. In some embodiments, the cell is infected with a virus. In some embodiments, the virus is selected from: an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a Dengue virus, a lentivirus, a herpesvirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, and a 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 combinations 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, an anellovirus, a bocavirus, a vaccinia virus, or a 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-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV- PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV- HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ/8, AAV-Myo, AAV-NP40, AAV-NP59, AAV- NP22, AAV-NP66, AAV-HSC16, and any functional derivative thereof. In some embodiments, the recombinant virus comprises a recombinant adeno-associated viral (rAAV) vector. In some embodiments, the rAAV vector comprises less than 5%, 4%, 3%, 2%, or 1% contaminants from non- AAV components and a GOI. In some embodiments, the contaminants are from an alphavirus, a parvovirus, a baculovirus, a Dengue virus, a lentivirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, a herpesvirus, or a retrovirus. In some embodiments, the contaminants are from a baculovirus.

[0055] Provided herein are methods for generating an rAAV vector, comprising: (a) providing a virus-producing cell having an engineered genome, wherein the cell comprises: (i) a first nucleic acid comprising a first enhancer sequence and a Rep gene or functional fragment thereof; (ii) a second nucleic acid comprising a second enhancer sequence and a Cap gene or functional fragment thereof; and iii. a third nucleic acid comprising a GOI; (b) contacting the cell of step (a) with a virus; and (c) after step (b), culturing the cells to produce a recombinant virus comprising the rAAV vector. In some embodiments, the first enhancer sequence has at least 80% (e.g., 85%, 90%, 95%, 99%, or 100%) identity to 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 any of SEQ ID NOs: 1-10. In some embodiments, the first and the second enhancer sequences are the same. In some embodiments, the first nucleic acid further comprises a first engineered Kozak sequence. In some embodiments, the first engineered Kozak sequence has at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to 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 and the second engineered Kozak sequences are the same. In some embodiments, the GOI is flanked by a first ITR sequence and a second ITR sequence. In some embodiments, the first ITR sequence and the second ITR sequences 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 combinations thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini promoter, or any functional fragment and/or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EFla, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, plO, pl 9, p40, Synapsin, CaMKII, GRK1, polH, EM7, OpIEl, 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 combinations thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini promoter, or any functional fragment and/or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EFla, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, plO, pl9, p40, Synapsin, CaMKII, GRK1, polH, EM7, OpIEl, 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 combinations thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini promoter, or any functional fragment and/or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EFla, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, plO, pl9, p40, Synapsin, CaMKII, GRK1, polH, EM7, OpIEl, 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., an rAAV. In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV- 2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV- HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ/8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivative thereof. In some embodiments, the Cap gene is derived from a parvovirus. In some embodiments, the parvovirus is an AAV. In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rhlO, AAV- rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV- HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ/8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivative thereof. In some embodiments, the first nucleic acid further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, a betalactam, 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 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 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 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 first nucleic acid further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMBl, pBR322, ColEl, R6K, pl5A, pSClOl, ColE2, Fl, pUC, and any functional fragment and/or functional derivative thereof. In some embodiments, the second nucleic acid further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMBl, pBR322, ColEl, R6K, pl5A, pSClOl, ColE2, Fl, pUC, and any functional fragment and/or functional derivative thereof. In some embodiments, the third nucleic acid further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMBl, pBR322, ColEl, R6K, pl5A, pSClOl, ColE2, Fl, pUC, and any functional fragment and/or functional derivative thereof. In some embodiments, the first nucleic acid comprises a sequence having at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 210. In some embodiments, the second nucleic acid comprises a sequence having at least 80% e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to 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 cell is an A549 cell, an HEK-293 cell, an HEK-293T cell, a BHK cell, a CHO cell, a HeLa cell, an MRC5 cell, an Sf9 cell, an Sf2 cell, an Sf21 cell, a High Five™ cell, a Cos-1 cell, a Cos-7 cell, a Vero cell, a BSC 1 cell, a BSC 40 cell, a BMT 10 cell, a WI38 cell, a Saos cell, a C2C12 cell, an L cell, an HT1080 cell, a HepG2 cell, a Huh7 cell, a K562 cell, a primary cell, or a derivative thereof. In some embodiments, the cell is an Sf9 cell. In some embodiments, the first nucleic acid, the second nucleic acid, and the third nucleic acid remain stably integrated in the genome after at least 5 (e.g., 10, 20, 40) passages. In some embodiments, the cell is able to grow to at least about 1 x 10 ⁷ cells/mL after at least about 24 hours. In some embodiments, the cell is infected with a virus. In some embodiments, the virus is selected from: an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a Dengue virus, a lentivirus, a herpesvirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, and a 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 combinations 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, an anellovirus, a bocavirus, a vaccinia virus, or a 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-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV- Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV- HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ/8, AAV- Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivative thereof. In some embodiments, the recombinant virus comprises a rAAV vector. In some embodiments, the rAAV vector comprises less than 5%, 4%, 3%, 2%, or 1% contaminants from non- AAV components and a GOI. In some embodiments, the contaminants are from an alphavirus, a parvovirus, a baculovirus, a Dengue virus, a lentivirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, a herpesvirus, or a retrovirus. In some embodiments, the contaminants are from a baculovirus.

[0056] In some embodiments, the present disclosure provides recombinant viruses produced by methods of the present disclosure.

[0057] In some embodiments, the present disclosure provides systems of generating a recombinant virus comprising: (a) a virus-producing cell, the virus-producing cell having an engineered genome comprising: (i) a first nucleic acid comprising a first enhancer sequence and a Rep gene or functional fragment thereof; (ii) a second nucleic acid comprising a second enhancer sequence and a Cap gene or functional fragment thereof; and (iii) a third nucleic acid comprising a GOI; and (b) a virus for infecting the virus-producing cell, which when infected with the virus induces the production of the recombinant virus.

[0058] 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 and the second enhancer sequences are the same.

[0059] 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 and the second engineered Kozak sequences are the same.

[0060] 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 sequences 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.

[0061] 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 combinations thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini promoter, or any functional fragment and/or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EFla, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, plO, pl9, p40, Synapsin, CaMKII, GRK1, polH, EM7, OpIEl, 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 combinations thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini promoter, or any functional fragment and/or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EFla, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, plO, pl 9, p40, Synapsin, CaMKII, GRK1, polH, EM7, OpIEl, 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 combinations thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini promoter, or any functional fragment and/or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EFla, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, plO, pl 9, p40, Synapsin, CaMKII, GRK1, polH, EM7, OpIEl, 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. In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rhlO, AAV- rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV- HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ/8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivative thereof. In some embodiments, the Cap gene is derived from a parvovirus. In some embodiments, the parvovirus is an AAV. In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV- rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV- PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV- HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ/8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV- NP66, AAV-HSC16, and any functional derivative thereof. 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 functional derivative thereof. In some embodiments, the third nucleic acid further comprises an antibiotic resistance gene. In some embodiments, the antibiotic resistance gene encodes an aminoglycoside, a 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, puromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, zeocin, or any functional fragment and/or functional derivative thereof.

[0062] In some embodiments, the first nucleic acid further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMBl, pBR322, ColEl, R6K, pl5A, pSClOl, ColE2, Fl, pUC, and any functional fragment and/or functional derivative thereof. In some embodiments, the second nucleic acid further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMBl, pBR322, ColEl, R6K, pl5A, pSClOl, ColE2, Fl, pUC, and any functional fragment and/or functional derivative thereof. In some embodiments, the third nucleic acid further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMBl, pBR322, ColEl, R6K, p!5A, pSClOl, ColE2, Fl, pUC, and any functional fragment and/or functional derivative thereof. In some embodiments, the first nucleic acid comprises a sequence having at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 210. In some embodiments, the second nucleic acid comprises a sequence having at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to 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 cell is a A549 cell, a HEK-293 cell, a HEK-293T cell, a BHK cell, a CHO cell, a HeLa cell, an MRC5 cell, an Sf9, an Sf2 cell, an 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, an L cell, an HT1080 cell, a HepG2 cell, a Huh7 cell, a K562 cell, a primary cell, or any derivative thereof. In some embodiments, the cell is an Sf9 cell.

[0063] In some embodiments, the first nucleic acid, the second nucleic acid, and the third nucleic acid remain stably integrated in the genome after at least 5 e.g., 10, 20, 40) passages. [0064] In some embodiments, the cell is able to grow to at least about 1 x 10 ⁷ cells/mL after at least about 24 hours. In some embodiments, the cell is infected with a virus. In some embodiments, the virus is selected from: an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a Dengue virus, a lentivirus, a herpesvirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, and a 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 combinations 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, an anellovirus, a bocavirus, a vaccinia virus, or a 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-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV- PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV- HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ/8, AAV-Myo, AAV-NP40, AAV-NP59, AAV- NP22, AAV-NP66, AAV-HSC16, and any functional derivative thereof. In some embodiments, the recombinant virus comprises a rAAV vector.

[0065] In some embodiments, the rAAV vector comprises less than 5%, 4%, 3%, 2%, or 1% contaminants from non- AAV components and a GOI. In some embodiments, the contaminants are from an alphavirus, a parvovirus, a baculovirus, a Dengue virus, a lentivirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, a herpesvirus, or a retrovirus. In some embodiments, the contaminants are from a baculovirus.

BRIEF DESCRIPTION OF THE DRAWINGS

[0066] The disclosure is more completely understood with reference to the following drawings.

[0067] FIG. 1A shows exemplary nucleic acid construct designs for exemplary Rep and Cap proteins.

[0068] FIG. IB shows a Western blot of capsid proteins in adeno-associated virus (AAV) serotype 1 (AAV1) and AAV5. FIG. 1C shows affinity purified capsid proteins from AAV7, AAV8, and AAV9.

[0069] FIGS. 2A-2E are schematic illustrations showing an exemplary method for producing recombinant virus beginning from seeding and selecting a clonal cell (FIG. 2A and FIG. 2B, respectively) grown into a monolayer and infected with an exemplary nonrecombinant virus to produce exemplary recombinant viral particles comprising a gene-of- interest (GOI) (FIG. 2C) and ending by discarding the media comprising excess components (FIG. 2D) and harvesting the recombinant virus from the remaining cell monolayer, including a final separation of virus from cell debris (FIG. 2E). Abbreviations: BEV, baculovirus; GOI, gene-of-interest; PCR, polymerase chain reaction; WT, wild-type.

[0070] FIG. 3 shows results of a clone screen to identify top producing clones as determined by yield (vg/mL).

[0071] FIG. 4 is a graph showing quantified differences in scale times shown as number of passages (x-axis) and cells/mL (y-axis) in traditional and optimized methods.

[0072] FIGS. 5A-5D show measurements of amount of host-cell-protein and activating virus present over the course of production and pre- and post-purification, demonstrating that both host cell proteins and activating virus impurities were reduced over the course of purification. FIG. 5A and FIG. 5B are gels showing that impurities were reduced over the course of purification. FIG. 5C is a bar graph showing host cell protein (HCP) in ng/mL detected in fractions from various purification steps. FIG. 5D is a bar graph showing DNA concentration in ng/mL of Sf9 and baculovirus (BEV) DNA in eluates from AAVX and AEX column purifications. Sf9 = left bars and BEV = right bars on AAVX and AEX conditions. [0073] FIG. 6 is a graph showing a stability study measuring volumetric (vg/L) or unit (vg/cell) titer over 20 passages using exemplary producer clones made in accordance with the present disclosure.

[0074] FIG. 7 is a graph showing growth and infection kinetics as measured by cell density (cells/mL) and viability (%) over time.

[0075] FIG. 8 is a bar chart showing a comparison of sequencing analysis of Sf9 and BEV genomes in purified Sf9 AAVs.

[0076] FIG. 9 is a schematic showing an exemplary nucleic acid cassette comprising a gene of interest (GOI) and inverted terminal repeats (ITR) before and after restriction digestion.

[0077] FIG. 10 is a bar chart showing enzyme levels in the cerebella of wild-type (WT; circles), knock-out (KO; squares), and AAV-treated mice using AAVs produced with HEK293 cells (triangles) or with Sf9 producer cells (diamonds) made in accordance with the present disclosure.

[0078] FIG. 11A is a bar chart showing biodistribution of vector genomes in target tissues of an exemplary AAV7 serotype gene therapy made using an exemplary Sf9 producer cells line (diamonds) of the present disclosure versus a standard exemplary HEK293 cell platform (triangles). FIG. 1 IB is a graph showing efficacy in extension of survival after administration of AAV therapeutics produced in HEK293 or in Sf9 cells in accordance with the present disclosure.

[0079] FIG. 12A is a bar chart showing enzyme levels in the forebrains of wild-type (WT; circles), knock-out (KO; squares), and AAV-treated mice using AAVs produced with HEK293 cells or with Sf9 producer cells made in accordance with the present disclosure. FIG. 12B is a bar chart showing enzyme levels in the hindbrains of wild-type (WT; circles), knock-out (KO; squares), and AAV-treated mice using enzyme-encoding AAVs produced with HEK293 cells (triangles) or with Sf9 producer cells (diamonds)made in accordance with the present disclosure. FIG. 12C is a graph showing enzyme levels in the hindbrains of wildtype (WT; circles), knock-out (KO; squares), and AAV-treated mice using AAVs produced with HEK293 cells (triangles) or with Sf9 producer cells (diamonds) made in accordance with the present disclosure. [0080] FIG. 13 is an image showing exemplary biodistribution of AAV in brain tissue after intra-cistema magna (ICM) administration (arrows indicate locations of detected AAV;

Table 3 describes locations and shows quantification).

DETAILED DESCRIPTION

[0081] Viral vectors are commonly used to deliver therapeutic genes to humans. Production of viral vectors is a complex process. For example, several different issues can arise with design and manufacturing, including but not limited to scalability, quality, and potency of the recombinant viruses. Production of clinical grade gene therapies remains a major hurdle in advancing cures for a number of otherwise unpreventable, incurable and/or untreatable diseases, disorders, and conditions. Improved processes are needed to achieve reproducible, consistent, scalable manufacturing solutions and provide commercially viable viral products that can be safely and reliably delivered to patients.

[0082] The present disclosure provides the insight that systems having certain components produce and provide reproducible, stable viral products of clinical or clinically- relevant quality in commercially relevant or scalable quantities.

[0083] Among other things, provided herein are systems, cell lines, methods of manufacturing, and methods of use of a combination of features that result in products that are more reproducible, scalable, and reliable for use in clinical products. Systems of the present disclosure include engineered virus-producing cell lines that result in low-cost, robust, and highly reproducible large-scale viral product manufacturing. Such viral producer cell lines use innovative combinations of stably-integrated and inducible components in an engineered cell system.

Definitions

[0084] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. Generally, nomenclatures utilized in connection with, and techniques of, 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 measure not otherwise defined accord with The International System of Units (SI), NIST Special Publication 330, 2019 edition.

[0085] As used herein, all numerical values or numerical ranges comprise whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90-100%, comprises 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. In another example, reference to a range of 1-5,000-fold comprises 1-, 2-, 3-, 4- , 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, or 20-fold, etc., as well as 1.1-, 1.2-, 1.3-, 1.4-, or 1.5-fold, etc., 2.1-, 2.2-, 2.3-, 2.4-, or 2.5-fold, etc., and so forth. [0086] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms “a,” “an,” and “the” are intended to comprise the plural forms as well, unless the context clearly indicates otherwise.

[0087] It will be further understood that the terms “comprises” and/or “comprising,” when used herein, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. “Consisting essentially of’ specifies the presence of at least each of the stated features, integers, steps, operations, elements, and/or components, but does not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

[0088] As used herein, the term “and/or” comprises any and all combinations of one or more of the associated listed items.

[0089] Unless specifically stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers ±10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

[0090] As used herein, the terms “adeno-associated virus vector” or “AAV vector” refer to a vector derived from an adeno-associated virus selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV- 2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV- HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ/8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivative thereof. An AAV vector may be a mammalian (e.g., human, e.g., non-human primate) virus. An AAV vector may be an avian (AAAV) virus. In some embodiments, the AAV is a mammalian (e.g., human, e.g., non-human primate) AAV or an avian AAV (AAAV); that is, in some embodiments, the starting and/or engineered AAV is or is derived from a virus capable of infecting a mammalian or avian organism. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, e.g., the Rep and/or Cap genes, but retain functional flanking inverted terminal repeat (ITR) sequences. Functional ITR sequences promote the rescue, replication, and packaging of the AAV virion. An AAV vector may comprise a single stranded (ss) or self-complementary (sc) genome. Thus, an AAV vector is defined herein to comprise at least those sequences required in cis for replication and packaging (e.g., functional ITRs) of the virus. ITRs do not need to be the wild-type polynucleotide sequences and, in some embodiments, are altered, e.g., by the insertion, deletion, or substitution of nucleotides, so long as the sequences provide for functional rescue, replication, and packaging.

[0091] As used herein, the terms “adeno-associated virus inverted terminal repeats” or “AAV ITRs” refer to regions flanking each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the virus. AAV ITRs, together with the AAV Rep coding region, can also provide for the efficient excision and integration of a polynucleotide sequence interposed between two flanking ITRs into a mammalian, avian, or insect genome. Typically a wild-type AAV ITR comprises 145 bases, but ITRs may comprise fewer or greater numbers of bases. As used herein, an “AAV ITR” does not necessarily comprise the wild-type polynucleotide sequence, which, in some embodiments, are altered, e.g., by the insertion, deletion or substitution of nucleotides. Additionally, the AAV ITRs are derived from any of several AAV serotypes selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV- PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV- HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ/8, AAV-Myo, AAV-NP40, AAV-NP59, AAV- NP22, AAV-NP66, AAV-HSC16, and any functional derivative thereof. Furthermore, 5’ and 3’ ITRs which flank a selected polynucleotide sequence in an 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 allow for the desired therapeutic or genome editing effect. [0092] As used herein, the term “engineered Kozak sequence” describes a reference Kozak sequence that has been modified (e.g., as compared to a reference or endogenous Kozak sequence) by one or more nucleotide changes to alter (e.g., increase or reduce) translation of a downstream gene product. A Kozak sequence as used herein is a sequence encoding a translation initiation site, and an engineered Kozak sequence changes translation relative to translation that occurs in the presence of a Kozak sequence that has not been engineered. A change in translation that occurs with an engineered Kozak sequence may be an increase or a decrease in a particular downstream gene product. For example, engineered Kozak sequences as provided herein are able to incorporate higher levels of VP1 into AAV capsids as compared to a reference Kozak sequence (e.g., prior to engineering).

[0093] As used herein, the term “functional derivative” of a biomolecule (e.g., a polynucleotide, e.g., polypeptide, e.g., viral particle (e.g., an AAV particle)) refers to a biomolecule that has been modified relative to a reference biomolecule, where the resulting biomolecule does not necessarily comprise 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. When the biomolecule is a polynucleotide or a 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 of the nucleic acid or amino acid sequence of the reference biomolecule (but not necessarily to a contiguous portion of a sequence). For example, a functional derivative of a polypeptide may be a polypeptide that contains one or more amino acid modifications relative to the reference biomolecule and retains certain functions, such as binding to a particular receptor, though binding could be stronger, equivalent, or weaker than that of the reference biomolecule as long as the binding of the functional derivative achieves at least a portion of the activity of the reference biomolecule. A functional derivative of a polynucleotide 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 as or similar to the 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. In addition, such a biomolecule or functional derivative thereof may be a “freestanding” functional unit or system (e.g., a modified or variant AAV) and/or part of a larger system, such as, for example, a vector (e.g., a gene cassette encoding an antibiotic resistance gene or functional portion thereof; e.g., a gene cassette encoding a particular Kozak sequence, etc.).

[0094] As used herein, the term “functional fragment” of a biomolecule, e.g., a polynucleotide or polypeptide, refers to a fragment of a reference biomolecule (i.e., shorter and/or smaller) with the same or similar functional activity of the reference biomolecule. It is contemplated that a similar functional activity could be greater, about equal, or less than the functional activity of the reference biomolecule, as long as the functional fragment achieves at least a portion of the activity of the reference biomolecule. When the reference biomolecule is a polypeptide, it is 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, when the reference biomolecule is a polypeptide, it is contemplated that the polypeptide fragment may be a polypeptide that has been cleaved or otherwise modified to be shorter and/or smaller than the reference polypeptide, but still retains functional activity of the reference polypeptide, such as binding to a particular receptor; when the reference biomolecule is a polynucleotide, it is contemplated that the polynucleotide fragment retains some of the same activity of the reference polynucleotide. For example, in the case of a polynucleotide encoding a protein, it is contemplated that the polynucleotide fragment encodes a protein having least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the activity of the protein encoded by the reference polynucleotide in a suitable assay. In the case of a polynucleotide that acts a regulatory element (e.g., a promoter or enhancer), it is contemplated that the polynucleotide fragment has at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the activity of the activity as reference polynucleotide in a suitable assay.

[0095] As used herein, the term “parvovirus” encompasses the family parvoviridae, comprising but not limited to autonomously replicating parvoviral genera and virusdependent genera. Autonomous parvoviruses comprise, for example, members of the genus Bocavirus (Bocavirus), the genus Dependovirus (dependovirus), the genus erythro (erythrovirus), the genus mink aleutis virus (Amdovirus), the genus Parvovirus (Parvovirus), the genus Densovirus (Densovirus), the genus repeat virus (iterovirus), the genus cottravirus (containvirus), the genus avarporvovirus, the genus Copiparvovirus, the genus Protoparvovirus (Protoparvovirus), the genus Tetraparvovirus (tetrapivorvirus), the genus ambisense Densovirus (Ambidensovirus), the genus brevicula (brevinnovovirus), the genus hepatodensovirus (hepdensovirus), the genus prawn Densovirus (pendensovirus). Exemplary autonomous parvoviruses comprise, 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, Hl parvovirus, muscovy duck parvovirus, snake parvovirus, and B19 virus.

[0096] As used herein, the terms “percent identity,” “% identity,” or “sequence identity” refer to the extent 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 % identity of the nucleotide sequence to SEQ ID NO: Y and is elaborated as X% of residues in the nucleotide sequence are identical to the corresponding residues of sequence disclosed in SEQ ID NO: Y. A sequence said to be X% identical to a reference sequence may contain more nucleotide or amino acid residues than specified in the reference sequence but must contain a sequence corresponding to the reference sequence. In most cases, the sequence in question will contain a sequence that corresponds to all of the specified reference sequences.

Generally, computer programs are employed for such calculations. Exemplary programs that compare and align pairs of sequences, comprise ALIGN, FASTA, gapped BLAST, BLASTP, BLASTN, or GCG.

[0097] As used herein, the term “plasmid” refers to an extrachromosomal element that carries genes that can replicate independently of the chromosomes of the cell. The plasmid can be in the form of a circular double-stranded DNA molecule. Such elements can include autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, as well as linear, circular or supercoiled, or single-or double-stranded DNA or RNA of any origin.

[0098] As used herein, the terms “polynucleotide,” and “nucleic acid,” are used interchangeably herein and refer to chains of nucleotides of any length, and comprise DNA and RNA. In some embodiments, the nucleotides are deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that is incorporated into a chain by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure is 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 is further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications comprise, for example, “caps,” substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates) and with charged linkages (e.g., phosphorothioates, phosphorodithioates), those containing pendant moi eties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L- lysine), those with intercalators (e.g., acridine, psoralen), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids), as well as unmodified forms of the polynucleotide(s). In some embodiments, any of the hydroxyl groups ordinarily present in the sugars are replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or are conjugated to solid supports. In some embodiments, the 5’ and 3’ terminal OH is phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. In some embodiments, polynucleotides also contain analogous forms of ribose or deoxyribose sugars, comprising, 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, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, 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 can include, but are not limited to, embodiments wherein 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”), in which each R or R’ is independently H or substituted or unsubstituted alkyl (1-20 C), optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, comprising RNA and DNA.

[0099] 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, be composed of different elements derived 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 inducer molecules. Promoters that cause a gene to be expressed in most cell types most of the time are commonly referred to as “constitutive promoters.” Promoters that cause the expression of genes in a particular cell and tissue type are commonly referred to as “cell-specific promoters” or “tissue-specific promoters,” respectively. Promoters that cause the expression of genes at specific stages of development or cell differentiation are commonly referred to as “development-specific promoters” or “cell differentiation-specific promoters.” Promoters that induce and result in the expression of genes after exposing or treating cells with agents, biomolecules, chemicals, ligands, light, etc. that induce the promoters are commonly referred to as “inducible promoters” or “regulatable promoters.” It is further recognized, in some embodiments, that since the exact boundaries of regulatory sequences have not been completely defined in most cases, DNA fragments of different lengths have the same promoter activity.

[0100] As used herein, “recombinant AAV vector (rAAV vector)” refers to a polynucleotide vector derived from an AAV and comprising one or more heterologous sequences (z.e., a nucleic acid sequence not of AAV origin) that are flanked by 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 that is expressing suitable helper functions) and that expresses AAV Rep and Cap gene products (i.e.. N Rep and Cap proteins). When a rAAV vector is incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection), then the rAAV vector are referred to as a “pro-vector” which are “rescued” by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions.

[0101] As used herein, the terms “gene-of-interest (GOI)” and “transgene” refer to a polynucleotide that is introduced into a cell and is capable of being transcribed into RNA by the cell and optionally, translated into a protein and/or expressed under appropriate conditions. The gene of interest or transgene may confer a desired property to a cell into which it was introduced or otherwise lead to a desired therapeutic or diagnostic outcome. [0102] As used herein, the term “prevent” means to stop or reduce something from occurring, at least for a period of time, such as, for example, stopping at least one symptom of a disease from manifesting, stopping production of a particular gene product, or reducing an amount of a gene product produced by a particular gene.

[0103] 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 tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0104] As used herein, the phrase “pharmaceutically acceptable carrier” as used herein refers to an agent (e.g, excipient, carrier, buffer, etc.) suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Standard pharmaceutical carriers may include, for example a phosphate buffered saline solution, water, emulsions (e.g, such as an oil/water or water/oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rd ed. 2020).

[0105] As used herein, “treat”, “treating”, and “treatment” refer to the treatment of a disease, disorder, or symptom or manifestation of such in a subject, e.g., in a human. This includes preventing a disease or disorder; inhibiting the disease, disorder, etc., i.e., slowing or arresting its progress or development; and relieving the disease, disorder, etc., i.e., causing regression of the disease state.

[0106] As used herein, “subject” and “patient” refer to an organism to be treated by compositions made in accordance with the present disclosure and/or methods as provided herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably includes humans.

[0107] As used herein, the term “vector” comprises a nucleic acid vector, e.g., a DNA vector, such as a plasmid, an RNA vector, or another suitable replicon (e.g., viral vector). A variety of vectors have been developed for the delivery of polynucleotides encoding exogenous polynucleotides or proteins into a prokaryotic or eukaryotic cell. Examples of such expression vectors are disclosed in, e.g., WO 1994/011026; incorporated herein by reference as it pertains to vectors suitable for the expression of a nucleic acid molecule of interest. Expression vectors suitable for use with the compositions and methods described herein contain a polynucleotide sequence as well as, e.g., additional sequence elements used for the expression of heterologous nucleic acid materials (e.g., a nucleic acid molecule) in a cell. Certain vectors that are used for expression of nucleic acid molecules provided herein can include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which can direct and/or otherwise impact gene transcription. In some embodiments, a promoter may be a compact bidirectional promoter, which, in some embodiments, does not contain an enhancer. Other useful vectors for expression of nucleic acid molecule agents disclosed herein contain polynucleotide sequences that enhance the rate of translation of these polynucleotides or improve the stability or nuclear export of the RNA that results from gene transcription. These sequence elements comprise, e.g., 5’ and 3’ untranslated regions, an internal ribosomal entry site (IRES), and a polyadenylation signal (poly A) in order to direct efficient transcription of the gene carried on the expression vector.

Producer Cell Systems

[0108] 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 to produce a stable and inducible producer cell line that can be used in manufacture of viral products. These strategies include combining, for the first time, three exogenous cell components that are stably integrated into a host cell and then infected with an inducing virus to produce recombinant viral products that can be used for commercial and/or clinically-relevant purposes.

[0109] In some embodiments, the present disclosure provides engineered viral producer cell lines that comprise inducible promoters, enhancer motifs, and engineered Kozak sequences. 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 needed to encode components needed for recombinant viral production, wherein the nucleic acids are stably-integrated and include, but are not limited to, a Rep, a 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). [0110] In some aspects, the present disclosure provides systems, engineered cell lines, and methods of manufacturing viral vectors and products. While use of any appropriate virus is contemplated, to give but one example adeno-associated viruses (AAV) have been frequently used in recombinant viral vector systems for therapeutic delivery. Wild-type AAV is a small, non-enveloped human Parvovirus that can infect, but is non-pathogenic to humans. The wild-type AAV genome comprises two open reading frames, Rep, and Cap flanked by two inverted terminal repeats (ITRs). These ITRs base pair to allow for synthesis of the complementary DNA strand. Rep and Cap are translated to produce multiple distinct proteins (Rep78, Rep68, Rep52, Rep40 - required for the AAV life cycle; VP1, VP2, VP3 - capsid proteins).

[0111] 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, a nucleic acid sequence may be provided using combinations of A, G, C, and/or T; and/or A, G, C, and/or U. That is, given context, in some embodiments, a polynucleotide provided herein may comprise one or more A, G, C, and/or T nucleobases. In some embodiments, a polynucleotide provided herein may comprise one or more A, G, C, and/or U nucleobases. As will be understood to those of skill in the art, given context, the indication of either a “T” or a “U” in a given polynucleotide will be considered interchangeable with one another under appropriate circumstances. For example, a polynucleotide having a sequence of CAGTTTATGGT can also understood to be and function as CAGUUUAUGGU given appropriate context and, as will be understood to those of skill in the art, can be used interchangeably under appropriate circumstances and/or contexts (e.g., DNA/cDNA vs. e.g., mRNA/RNA). It is to be understood that, throughout the description (e.g., as provided in the Sequence Listing, as 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 including one or more thymine nucleobases (“T”), another polynucleotide is also contemplated that comprises, consists essentially of, or consists of the same nucleotide sequence comprising a uracil nucleobase (“U”) in place of thymines (T); or, in each instance where a polynucleotide comprises, consists essentially of, or consists of a nucleotide sequence including one or more uracil nucleobases (“U”), another polynucleotide is also contemplated that comprises, consists essentially of, or consists of the same nucleotide sequence comprising a thymine nucleobase (“T”) in place of uracils (U). Thus, for any sequence provided herein, including as set forth in any of SEQ ID NOs: 1-191 or 210-216, any sequence comprising a “T” should also be understood to contemplate a polynucleotide comprising a “U” and be used in accordance with technologies provided herein as appropriate, given context and/or circumstances. [0112] Generation of a recombinant adeno-associated viral (rAAV) vector involves providing an AAV transfer plasmid where the transgene is placed between the two ITRs and Rep and Cap are supplied in trans. The transfer plasmid along with Rep, Cap, and additional helper plasmids are then provided to a cell (e.g., a HEK-293 cell) to produce a recombinant virus comprising the rAAV vector.

[0113] The present disclosure provides the surprising finding that stably integrating Rep, Cap, and ITR-GOI constructs into a host cell does not destabilize the host cell genome. In addition, the quality of the viral product is not reduced. This is achieved by using, among other things, engineered Kozak sequences and/or enhancer sequences. For example, it is contemplated that toxicity can be modulated in host cells by modulating Rep levels. Alternatively or additionally, in some embodiments, concatemerization strategies are used (e.g., generating concatemers of Rep or Cap constructs prior to stable integration into a cell genome). In some embodiments, a concatemer of the present disclosure comprises at least two monomers. In some embodiments, a concatemer comprises three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more monomers. Concatemers may be any size that comprises at least two monomers and is still small enough to be resolved and purified using standard techniques. For example, in some embodiments, such concatemers can be purified using standard gel purification methods known to those of skill in the art.

[0114] Technologies provided herein, such as methods or systems, achieve stable expression of Rep and Cap by providing engineered Kozak sequences, and/or enhancer sequences and/or optionally combined with concatemerization of constructs prior to contacting the cell in which the constructs are to be stably integrated. These approaches improve parameters such as stabilization and reproducibility, accelerate growth, improve efficiency of viral product production, etc.

[0115] In some embodiments, the engineered cells (e.g., virus-producing cells with stably integrated Rep, Cap, and ITR-GOI), are able to grow rapidly. In some embodiments, the cells are able to grow at least about 1 x 10 ⁷ 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). In some embodiments, the cells are able to grow at least about between 1-2 x 10 ⁷ 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). By way of non-limiting example, doubling times of Sf9 cells can be between about 18-30 hours in suspension cultures and up to greater than 72 hours during isolation of single cells. [0116] In some embodiments, the cells are able to grow to at least about 1 x 10 ⁹ ' 1 x 10 ¹⁵ 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). In some embodiments, the cells are able to grow to at least about 1 x 10 ¹⁵ cells/mL or greater after at least about 24 hours (e.g., at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 hours). In some embodiments, the cells double after 0, 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 hours or more. In some embodiments, doubling time for suspension cultures is between about 10 - 40 hours, about 15-35 hours, about 18-30 hours, about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours. In some embodiments, doubling time for clonal cells is longer than that for suspension cultures. In some such embodiments, doubling time can range from about 20 hours to about 84 hours; about 24 hours to about 72 hours, about 48 hours to about 72 hours, about 24, 48, or 72 hours. In some such embodiments, rapid growth from single cells facilitates or provides for ability to select cells to be free of rhabdovirus.

[0117] In some embodiments, cells with stably integrated Rep, Cap, and ITR-GOI constructs into a host cell does not destabilize the host cell genome. This can be confirmed by using sequencing analysis (e.g., next generation sequencing) to show that there is low to no percentage of reads/sequences that map to host- or vector-related nucleic acids. For example, in some embodiments, sequencing analyses show that there is low percentage of reads/sequences that map to Sf9 or baculovirus genomes, supporting minimal packaging of Sf9 and Baculovirus DNA in assembled capsids. Low quantity of baculovirus packaging confirms that the stable integration of three constructs is a reliable, reproducible, safe, and improved approach relative to cells that only integrate one or two components. The low quantity of Sf9 genome in particular is a surprising finding considering that the cells stably integrate Cap, Rep, and GOI-ITR constructs because those of skill in the art would expect that integrating an ITR-GOI into a cell with Cap and Rep would result in a greater amount of host cell components (e.g., genomic DNA) being packaged into the final viral product.

[0118] Without being bound by theory, it is contemplated that stable genomic integration facilitates induction of AAV production at high volumetric titers by active infection using a recombinant baculovirus devoid of AAV elements that mitigates BEV genome stability issues during scale-up, allowing for larger, more reproducible, and/or healthier numbers of cells, and quantities of viral product to be produced in accordance with the present disclosure. Systems disclosed herein are adaptable to multiple viruses. In some embodiments, the virus is AAV. In some embodiments, systems provided herein are adaptable to AAV capsid serotypes and effectively packages self-complimentary and single-stranded AAV genomes. [0119] Systems of the present disclosure can be used to produce engineered producer cell line populations. In some embodiments, such cell lines can be produced by rapidly screening image verified clonal producer cell populations. In some embodiments, the populations are identified as rhabdovirus free candidates. In some such embodiments, such populations are capable of exceeding E5 AAV gc/cell or E14 AAV gc/L. In some embodiments, systems disclosed herein produce cell lines that generate virus in a reproducible and consistent manner over many (e.g., 5, 10, 15, 20, 25, etc.) passages post-thaw. In some such embodiments, ability of the systems supports the linear scalability of the system, in contrast to non-linear scalability of many other previously described approaches. It is contemplated that these successes are achieved because technologies of the present disclosure provide and allow for Rep, Cap, and/or the GOI to be stably-integrated, preventing loss of potency of these genes, as well as modulating the expression of Rep, Cap, or both, and preventing toxicity of the cells. In contrast, previously described processes are often hindered by complications such as, for example, Rep toxicity or loss of one or more components needed for the cell to produce viral product. Importantly, virus produced using engineered cell lines as provided herein are at least as potent and effective as those derived using other system. Furthermore, in some embodiments, the engineered cells are insect cells and virus produced using them is at least as potent and effective as virus derived from mammalian-derived counterparts in vivo.

[0120] 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 genome of the host cell and an inducer virus that induces the host cell to start producing a 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 functional fragment thereof; a second nucleic acid comprising a second enhancer sequence and a Cap gene or 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 a host cell genome, when the host cell is contacted with an inducer virus, the inducer virus activates the integrated components resulting in production of a recombinant virus comprising the GOI. Engineered Cells

[0121] In some aspects, the present disclosure provides virus-producing cells. In some embodiments, the virus-producing cells have 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 functional fragment or functional derivative thereof; (b) a second nucleic acid comprising a second enhancer sequence and a Cap gene or functional fragment or functional derivative thereof; and (c) a third nucleic acid comprising a GOI.

[0122] The virus-producing cells can be any type of cell that can be used for recombinant virus production. In some embodiments, the cell is a eukaryotic cell, a mammalian cell, an immortalized cell, an insect cell, a yeast cell, a plant cell, a fungal cell, or a prokaryotic cell. In some embodiments, the cell is a eukaryotic cell (e.g., a plant cell, an animal cell, a protist cell, or a fungi cell), a mammalian cell (a Chinese hamster ovary (CHO) cell, a baby hamster kidney (BHK) cell, a human embryo kidney (HEK) cell, a mouse myeloma (NSO) cell, or a human retinal cell), an immortalized cell (e.g., a HeLa cell, a COS cell, a HEK-293T cell, a MDCK cell, a 3T3 cell, a PC 12 cell, a Huh7 cell, a HepG2 cell, a K562 cell, an N2a cell, or an 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 an Heliothis virescens cell), a yeast cell (e.g., a Saccharomyces cerevisiae cell, a Cryptococcus cell, or a Candida cell), a plant cell (e.g., a parenchyma cell, a collenchyma cell, or a sclerenchyma cell), a fungal cell (e.g., a Saccharomyces cerevisiae cell, a Cryptococcus cell, or a Candida cell), or a prokaryotic cell (e.g., an E. coll cell, a streptococcus bacterium cell, a streptomyces soil bacteria cell, or an archaea cell).

[0123] In some embodiments, the cell is from a cell line. In some embodiments, the cell is a primary cell.

[0124] In some embodiments, the cell is an A549 cell, a HEK -293 cell, a HEK-293T cell, a BHK cell, a CHO cell, a HeLa cell, an MRC5 cell, an Sf2 cell, an Sf9 cell, an Sf2 cell, a High Five™ cell, an Sf21 cell, a BTI-Tn-5Bl-4 cell, a Cos-1 cell, a Cos-7 cell, a Vero cell, a BSC 1 cell, a BSC 40 cell, a BMT 10 cell, a WI38 cell, a Saos cell, a C2C12 cell, an L cell, an HT1080 cell, a HepG2 cell, a Huh7 cell, a K562 cell, or any derivative thereof. In some embodiments, the cell is an Sf9 cell.

[0125] In some embodiments, the present disclosure provides improved engineered cells, the improvement comprising combining, into 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 functional fragment thereof; a second nucleic acid comprising a second enhancer sequence and a Cap gene or any functional derivative or functional fragment thereof; and a third nucleic acid comprising a GOI. In some such embodiments, any one of the nucleic acids further comprises an enhancer sequence and/or an a Kozak sequence, wherein, in some embodiments, the Kozak sequence is an engineered Kozak sequence.

[0126] In some embodiments, a cell may optionally further comprise a helper virus or functional derivative or fragment thereof and/or a helper plasmid, and/or 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 comprising Rep, and/or Cap, etc.). In some such embodiments, the helper is an AAV helper. [0127] 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. In some embodiments, stable integration of one or more components into the genome is sitespecific and/or directed to one or more particular locations in the host-cell genome.

[0128] Upon stable integration into a host cell genome, a host cell is then contacted with an inducer virus, which activates the integrated components resulting in production of a recombinant virus comprising the GOI. The engineered cell produces more virus, faster, and more reproducibly than previously described engineered cells.

Rep and Rep Constructs

[0129] Parvoviruses, including AAV, comprise a Rep gene that encodes four proteins that are required for viral genome replication and packaging. The four proteins are Rep78, Rep68, Rep52, and Rep40. Virus-producing cells having an engineered genome described herein, in some embodiments, comprise a first nucleic acid comprising a Rep gene or any functional derivative or functional fragment thereof. In some embodiments, the Rep gene encodes a protein selected from: Rep78, Rep68, Rep52, Rep40, and any functional derivative or functional fragment thereof.

[0130] In some embodiments, the Rep gene or functional derivative or functional fragment thereof is derived from an parvovirus. In some embodiments, the parvovirus is an AAV. In some embodiments, the Rep gene or functional derivative or functional fragment thereof is 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); that is, in some embodiments, the starting and/or engineered AAV is or is derived from a virus capable of infecting a mammalian or avian organism. In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV- rh74, AAV-rhM4-l, AAV-hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV- HSC9, AAV-HSC10, AAV-HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV- HSC15, AAV-TT, AAV-DJ, AAV-DJ/8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivative thereof.

[0131] In some embodiments, the first nucleic acid comprising the Rep gene or 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 combinations thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini promoter, or any functional fragment and/or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EFla, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, plO, pl 9, p40, Synapsin, CaMKII, GRK1, polH, EM7, OpIEl, and any functional fragment and/or functional derivative thereof.

[0132] In some embodiments, the first nucleic acid comprising the Rep gene 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 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.

[0133] In some embodiments, the first nucleic acid comprising the Rep gene or functional fragment thereof further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMBl, pBR322, ColEl, R6K, pl5A, pSClOl, ColE2, Fl, pUC, and any functional fragment and/or functional derivative thereof.

[0134] In some embodiments, the first nucleic acid comprising the Rep gene 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 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, for example, a portion of SEQ ID NO: 210 that encodes a Rep protein or functional portion thereof. In some embodiments, the first nucleic acid comprising the Rep gene 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 more, or 100%) sequence identity to a sequence encoding a rep gene or functional derivative or functional fragment thereof produced using the nucleic acid sequence of SEQ ID NO: 210.

Cap and Cap Constructs

[0135] The capsid proteins of AAV compose the exterior, 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 translated from the same mRNA. These three proteins are produced in different quantities and have different sizes: VP1 is about 87 kDa; VP2 is about 72 kDa; and VP3 is about 63 kDa. VP3 is produced in the highest quantity of the three products, VP1 and VP2 are produced in lower quantities (relative to the total quantity of capsid protein). An AAV capsid generally comprises its VP1, VP2, and VP3 proteins in a ratio of 1 : 1 : 10 of VP1 :VP2:VP3. As is known to those of skill in the art, there may be other proteins that, in some embodiments, are translated from Cap transcripts, depending on reading frame during translation (e.g., Assembly Activating Protein (AAP), e.g., membrane- associated accessory protein (MAAP)).

[0136] Virus-producing cells having an engineered genome described herein, in some embodiments, comprise a second nucleic acid comprising a Cap gene or any functional derivative or functional fragment thereof.

[0137] In some embodiments, the Cap gene or functional derivative or functional fragment thereof is derived from a parvovirus. In some embodiments, the parvovirus is an AAV. In some embodiments, the AAV is a mammalian (e.g., human, e.g., non-human primate) AAV or an AAAV; that is, in some embodiments, the starting and/or engineered AAV is or is derived from a virus capable of infecting a mammalian or avian organism. In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV- hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV- HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ/8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivative thereof.

[0138] 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 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 NOs: 192 or 193 encode the amino acid sequence of SEQ ID NO: 204. In some embodiments, the nucleic acid sequence of SEQ ID NOs: 194 or 195 encode the amino acid sequence of SEQ ID NO: 205. In some embodiments, the nucleic acid sequence of SEQ ID NOs: 196 or 197 encode the amino acid sequence of SEQ ID NO: 206. In some embodiments, the nucleic acid sequence of SEQ ID NOs: 198 or 199 encode the amino acid sequence of SEQ ID NO: 207. In some embodiments, the nucleic acid sequence of SEQ ID NOs: 200 or 201 encode the amino acid sequence of SEQ ID NO: 208. In some embodiments, the nucleic acid sequence of SEQ ID NOs: 202 or 203 encode the amino acid sequence of SEQ ID NO: 209.

[0139] In some embodiments, the second nucleic acid comprising the Cap gene or functional derivative or 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 combinations thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini promoter, or any functional fragment and/or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EFla, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, plO, pl9, p40, Synapsin, CaMKII, GRK1, polH, EM7, OpIEl, and any functional fragment and/or functional derivative thereof. As will be understood by those of ordinary skill in the art, given context, certain promoters may be classified as early promoters or late promoters and the skilled artisan is aware of how and when to use such promoters to achieve expression of components. [0140] In some embodiments, the second nucleic acid comprising the Cap gene or 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 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.

[0141] In some embodiments, the second nucleic acid comprising the Cap gene or functional fragment thereof further comprises an origin of replication. In some embodiments, the origin of replication is selected from: pMBl, pBR322, ColEl, R6K, pl5A, pSClOl, ColE2, Fl, pUC, and any functional fragment and/or functional derivative thereof.

[0142] 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 the Cap gene 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 more, or 100%) sequence identity to the nucleic acid sequence of any of SEQ ID NOs: 211-216 or any functional fragment and/or functional derivative thereof, such as, for example, a portion of any of SEQ ID NOs: 211-216 that encodes a Cap protein or functional portion thereof. In some embodiments, the second nucleic acid comprising the Cap gene 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 more, or 100%) sequence identity to a sequence encoding a Cap gene or functional derivative or functional fragment thereof produced using the nucleic acid sequence of any of SEQ ID NOs: 211-216.

Enhancer Sequences

[0143] Enhancers are small molecules used to enhance the viral transduction process and increase target gene expression. Provided herein, in some embodiments, are enhancer sequences for improving the viral transduction process. Virus-producing cells, in some embodiments, comprise a first nucleic acid comprising a first enhancer sequence and a second nucleic acid comprising a second enhancer sequence. [0144] In some embodiments, the first and the second enhancer sequences are the same. In some embodiments, the first and the second enhancer sequences are different. In some embodiments, the first enhancer sequence is a homologous region (hr) enhancer sequence. In some embodiments, the second enhancer sequence is an hr enhancer sequence. In some embodiments, an enhancer is or comprises a sequence from Autographa Californica Nucleopolyhedrovirus (NCBI Taxonomy ID 46015).

[0145] 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 the nucleic acid sequence of any of SEQ ID NOs: 1-10 or to a functional fragment or functional derivative thereof.

[0146] 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 the nucleic acid sequence of any of SEQ ID NOs: 1-10 or to a functional fragment or functional derivative thereof.

[0147] In some embodiments, the first enhancer sequence is derived from an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a Dengue virus, a lentivirus, a herpesvirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, or a retrovirus. In some embodiments, the first enhancer sequence is derived from a baculovirus. In some embodiments, the AAV is a mammalian (e.g., human, e.g., non-human primate) AAV or an avian AAV (AAAV); that is, in some embodiments, the starting and/or engineered AAV is or is derived from a virus capable of infecting a mammalian or avian organism. In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV- Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV- HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ/8, AAV- Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivative thereof. In some embodiments, the first enhancer sequence is derived from a baculovirus.

[0148] In some embodiments, the second enhancer sequence is derived from an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a Dengue virus, a lentivirus, a herpesvirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, or a retrovirus. In some embodiments, the second enhancer sequence is derived from a baculovirus. In some embodiments, the AAV is a mammalian (e.g, human, e.g., non-human primate) AAV or an avian AAV (AAAV); that is, in some embodiments, the starting and/or engineered AAV is or is derived from a virus capable of infecting a mammalian or avian organism. In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV-hu37, AAV- Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV-HSC11, AAV- HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ/8, AAV- Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivative thereof. In some embodiments, the second enhancer sequence is derived from a baculovirus.

Kozak Sequences

[0149] The Kozak sequence is a nucleic acid motif that functions as the protein translation initiation site in most eukaryotic mRNA transcripts. Provided herein, in some embodiments, are engineered Kozak sequences that improve expression of viral proteins involved in recombinant viral production. Such sequences are may be chosen to facilitate preferential translation in insect cells relative to that in mammalian cells. Preferential translation refers to, for example, improved quantity and/or quality of downstream gene products in one cell type or condition as compared to another (e.g., in insect cells as compared to mammalian cells). Preferential translation does not mean that a given engineered Kozak sequence will not facilitate 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 an insect cell, whereas a different design that preferentially translations in a mammalian cell may work, but not as well, in an insect cell. Various engineered Kozak sequences are known in the art and may 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).

[0150] In some embodiments, a Kozak sequence is used in a nucleic acid comprising a rep coding sequence, and the Kozak sequence is compatible with both mammalian and insect cell systems. In some embodiments, the Kozak sequence preferentially facilitates translation in insect cells. In some embodiments, the Kozak sequence facilitates preferential translation in insect cells as compared to, e.g., mammalian cells.

[0151] 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 facilitates translation in insect cells. In some embodiments, the Kozak sequence facilitates preferential translation in insect cells as compared to, e.g., mammalian cells.

[0152] 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 facilitates translation in mammalian cells. In some embodiments, the Kozak sequence facilitates preferential translation in mammalian cells as compared to, e.g., insect cells. In some embodiments, the GOI is not translated in insect cells. In some embodiments, the GOI is translated in insect cells, but is removed during the manufacturing process, along with any other contaminants, such as from insect cells, etc.

[0153] In some embodiments, an engineered Kozak sequence is used to increase or improve expression of a particular protein (e.g., a Cap protein, e.g., VP1, etc.). In some embodiments, an engineered Kozak sequence is used to modulate (e.g., attenuate, reduce, change, 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 four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen. In some embodiments, a Kozak sequence comprises a ten nucleotide residue sequence having six of those nucleotide residues 5’ to a start codon sequence (e.g., ATG/AUG) and one nucleotide residue 3’ to the start sequence (e.g., G). In some embodiments, a Kozak sequence consists essentially of six nucleotide residues 5’ to a start codon sequence (e.g., ATG/AUG) and one nucleotide residue 3’ to the start sequence (e.g., G). In some embodiments, a Kozak sequence is selected from any of SEQ ID NOs: 11-191. In some embodiments, a Kozak sequence is selected based on serotype and sequence required to maintain an open reading frame of a given capsid (see, e.g., Table 1).

[0154] As will be understood to those in the art, when generating a comparator product using a different platform, such as mammalian cells (e.g., HEK293 cells), such engineered Kozak sequences optimized for insect cells do not necessarily need to be used. That is, in some embodiments, when generating a viral product (e.g., an rAAV comprising a gene-of- interest) in insect (e.g., Sf9) and mammalian (e.g., HEK293) systems, such as for comparison to one another, mammalian systems will use endogenous rep/cap sequences and insect systems will use sequences that comprise an engineered Kozak sequence (see, e.g., Table 1; see also SEQ ID NOs: 193, 195, 197, 199, 201, and 203, which each comprise an engineered Kozak sequence that preferentially translates a capsid in insect cells).

[0155] Engineered Kozak sequences preferred in accordance with the present disclosure are those that can result in higher levels of VP1 protein as compared to a non-engineered Kozak sequence. That is, in some embodiments, Kozak sequences that are compatible with preferential translation in a mammalian system may, when transferred into an insect system, result in lower quantities of certain proteins, such as, e.g., VP1. Lower quantities of such structural proteins result in reduced quality and quantity of viral capsids and, thus, overall reduced quality and quantity of the therapeutic.

[0156] Virus-producing cells, in some embodiments, comprise a first nucleic acid further comprising a first Kozak sequence.

[0157] In some embodiments, such virus-producing cells comprise a first engineered Kozak sequence. In some embodiments, the first engineered Kozak sequence has at least 80% (e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more or 100%) identity to the nucleic acid sequence of any one of SEQ ID NOs: 11-191, or to a functional fragment and/or functional derivative thereof.

[0158] Virus-producing cells, in some embodiments, comprise a second nucleic acid further comprising 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 and the second engineered Kozak sequences are the same. In some embodiments, the first and the second engineered Kozak sequences are different. In some embodiments, a first Kozak sequence is not engineered and a second Kozak sequence is engineered.

[0159] In some embodiments, a Kozak sequence comprises or consists essentially of a polynucleotide a sequence comprising five terminal residues set forth as AXGYY, wherein X = U or T and Y = A, G, C, or T/U. In some embodiments, a Kozak sequence may be a “leaky” Kozak sequence and comprise a formula of ACGYY, wherein the ACG region can result in “read-through” such that the ribosome does not bind as often and/or strongly as in the present of a Kozak sequence comprising the sequence ATGYY, where the ATG confers a Kozak sequence that is “stronger.” In some embodiments, a Kozak sequence comprises a polynucleotide having a sequence comprising five terminal residues set forth as AXGGC (SEQ ID NO: 217), wherein X = U or T. In some embodiments, a construct comprising, consisting essentially of, or consisting of a Kozak sequence comprising “AXGGC” may be desirable versus a Kozak sequence comprising AXGYY. In some embodiments, a Kozak sequence comprises or consists essentially of a polynucleotide a sequence comprising five terminal residues set forth as AXGYY, wherein X = U or T and Y = A, G, C, or T/U. In some embodiments, an engineered Kozak sequence has a formula comprising XXXXXXATGYY, wherein X is any nucleotide and Y is G or C. In some embodiments, an engineered Kozak sequence has a formula comprising XXXXXXATGXX, wherein X is A, G, C, or T/U. In some embodiments, a Kozak sequence is not a suboptimal sequence, such as one that comprises a “leaky” Kozak, such as ACG.

[0160] Without being bound by theory, in some such embodiments, such a sequence set forth at a terminal portion of a Kozak sequence in a construct encoding a polypeptide is capable of preserving the amino acid sequence of one or more polypeptides (e.g., a Rep, e.g., a Cap, e.g, an AAV Rep, e.g, an AAV Cap, etc.) . In some embodiments, a Kozak sequence is modified to encode a non-wild-type amino acid sequence, such as, a non-wild-type Rep and/or a non-wild-type Cap (e.g., by changing, for instance, the second amino acid encoded by the Kozak). In some embodiments, strategies provided by the present disclosure more faithfully recapitulate and/or preserve certain features of AAVs by, for example, not altering amino acids in the vicinity of a start codon of a given construct e.g., a Rep construct, e.g., a Cap construct, etc.).

[0161] In some embodiments, instead of or in addition to a Kozak sequence (that is optionally modified, if present), a construct may comprise one or more artificial introns. As is known to those of skill in the art (see, e.g., Mol Ther. 2008 May;16(5):924-30; US Pat. No. 8, 945,918), one or more artificial intronic sequences may be used to drive gene expression of one or more viral e.g., AAV) components, such as rep and cap genes, in insect cells. In such an approach, artificial introns are arranged in a nucleic acid sequence comprising, e.g., an artificial intron which also comprises an insect cell e.g., Sf9) promoter e.g., polH, e.g., plO, etc.). This arrangement can be organized such that the artificial intron facilitates translation of genes having overlapping open reading frames (such as rep and cap), such that different splice forms of the proteins get translated when the intron is present versus when it is spliced out (e.g., VP1 of cap translated when present, and VP2 and VP3 translated when spliced out). [0162] In some embodiments, the present disclosure contemplates that engineered Kozak sequences are preferred for modulation of Cap expression as compared to artificial introns. [0163] Certain engineered Kozak sequences may be preferred depending on serotype of the AAV used. Exemplary engineered Kozak sequences used in certain AAV serotypes are shown in Table 1.

Table 1. Exemplary engineered Kozak sequences and AAV serotypes

Gene-of-Interest

[0164] Virus-producing cells having an engineered genome described herein, in some embodiments, comprise a third nucleic 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 the second ITR sequences are wild-type ITR sequences. In some embodiments, the first and/or second ITR sequences are modified relative to wild-type sequences. In some embodiments, the first and/or second ITR sequences are derived from the same serotype. In some embodiments, the first and/or second ITR sequences are derived from different serotypes. In some embodiments, the first ITR sequence and the second ITR sequences are the same. In some embodiments, the first ITR sequence and the second ITR sequences are different. In some embodiments, the first ITR and second ITR sequences are asymmetric. For example, in some embodiments, a first ITR may be longer than a second ITR or vice- versa. In some embodiments, one or both ITRs may be “mutant” ITRs (e.g., modified relative to those that exist in nature, such as to delete certain portions, such as “trs” mutant ITRs, etc.).

[0165] 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 combinations thereof). In some embodiments, the promoter is selected from a constitutive promoter, an inducible promoter, a mini promoter, or a functional fragment and/or functional derivative thereof. In some embodiments, the promoter is selected from: CMV, CBA, EFla, CAG, PGK, TRE, U6, UAS, T7, Sp6, lac, araBad, trp, Ptac, p5, plO, pl9, p40, Synapsin, CaMKII, GRK1, polH, EM7, OpIEl, and any functional fragment and/or functional derivative thereof.

[0166] The present disclosure contemplates that, in some embodiments, it may be preferable to reduce the amount of host DNA excised from a plasmid genome from which the ITR-GOI cassette is excised. As provided herein, the present disclosure contemplates that a way to reduce excess plasmid/prokaryotic DNA surrounding an ITR-GOI cassette is to use restriction enzyme digests to remove host DNA flanking an ITR-GOI-ITR cassette (see, e.g., FIG. 13). FIG. 13 depicts a simplified diagram of a cassette in which two ITRs (each of which may or may not be a mutant or modified ITR, and may be the same or different) flanks a GOI. When the plasmid comprising this construct is linearized into monomers, the enzyme is also used to digest away additional genomic sequence outside of the ITR-GOI-ITR cassette. In some embodiments, the total amount of plasmid/prokaryotic DNA on the outside segments of the ITRs (the side that does not comprise 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 of the GOI) does not have to be the same length - for example, in some embodiments, the DNA flanking one ITR may be longer or shorter than that flanking the other ITR. In some embodiments, flanking DNA on a 5’ end of a GOI cassette may be about 100, 200, 300, 400, 500, 600, or 700 nucleotides and/or flanking DNA on a 3’ end of a GOI cassette may be about 50, 60, 70, 80, 100, 200, 300, 400, 500, 750, 100, 1250, 1500, o 1750 nucleotides. Having less flanking DNA (e.g., from plasmid/prokaryotic sources) may be desirable for downstream purification processes and clinical manufacturing processes.

Inducer Viruses

[0167] Described herein, in some embodiments, are virus-producing cells, wherein the cells are infected with a virus to induce expression of the first, second, and/or third nucleic acid stably integrated into the host virus-producing cells. In some embodiments, the virus is selected from an alphavirus, a parvovirus, an adenovirus, an AAV, a baculovirus, a Dengue virus, a lentivirus, a herpesvirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, or a 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, the wild-type virus refers to a virus that is different than a virus found in nature, but is not a recombinant virus and/or does not comprise recombinant components such as a GOI or ITR, etc. In some embodiments, a wild-type virus is a virus that does not comprise any exogenous gene and/or an exogenous sequence (e.g., a GOI, e.g., an ITR). In some embodiments, a wild-type virus is a baculovirus that does comprise an exogenous gene (e.g., an antibiotic resistance gene, a detectable marker gene, etc.), but not a gene that is designed to be inserted into an engineered cell (e.g., a GOI, e.g., an ITR). In some embodiments, a wild-type virus is a baculovirus that has been engineered to remove certain endogenous functions such as enzymatic activity/protease function, e.g., by removing or rendering non-functional certain proteases such as ChlA or vCath. In some embodiments, a virus may be recombinant, but devoid of any heterologous or exogenous components designed to be inserted into a cell, such as, e.g., an AAV element for insertion into a host cell such as an insect cell. In some embodiments, the virus is not a recombinant virus.

[0168] 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, a parvovirus, an adenovirus, an AAV, a baculovirus, a Dengue virus, a lentivirus, a herpesvirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, and a retrovirus. In some embodiments, the AAV is a mammalian (e.g., human, e.g., non-human primate) AAV or an avian AAV (AAAV); that is, in some embodiments, the starting and/or engineered AAV is or is derived from a virus capable of infecting a mammalian or avian organism. In some embodiments, the AAV is selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-rh8, AAV-rhlO, AAV-rh20, AAV-rh39, AAV-rh74, AAV-rhM4-l, AAV- hu37, AAV-Anc80, AAV-Anc80L65, AAV-7m8, AAV-PHP.B, AAV-PHP.EB, AAV-2.5, AAV2tYF, AAV-3B, AAV-LK03, AAV-HSC1, AAV-HSC2, AAV-HSC3, AAV-HSC4, AAV-HSC5, AAV-HSC6, AAV-HSC7, AAV-HSC8, AAV-HSC9, AAV-HSC10, AAV- HSC11, AAV-HSC12, AAV-HSC13, AAV-HSC14, AAV-HSC15, AAV-TT, AAV-DJ, AAV-DJ/8, AAV-Myo, AAV-NP40, AAV-NP59, AAV-NP22, AAV-NP66, AAV-HSC16, and any functional derivative thereof.

[0169] Virus-producing cells described herein, in some embodiments, comprise an rAAV vector generated with improved qualities (e.g, reduced contaminants, improved potency, etc.). In some embodiments, the rAAV vector comprises less than 5%, 4%, 3%, 2%, or 1% contaminants from non- AAV components and a GOI. In some embodiments, the contaminants are from an alphavirus, a parvovirus, a baculovirus, a Dengue virus, a lentivirus, a poxvirus, an anellovirus, a bocavirus, a vaccinia virus, a herpesvirus, or a retrovirus. In some embodiments, the contaminants are from a baculovirus. In some embodiments, contaminants may be from host cell genome components (e.g, insect cell genomic contaminants).

Methods of Manufacturing

[0170] Processes for generating recombinant viruses comprising recombinant viral vectors, such as rAAV vectors used for gene therapies, face difficulty in large-scale manufacturing and production. Specifically, issues arise due to low vector concentration, oncogenic host cell DNA, and difficulty with purifying rAAV vectors from cells. As such, improved strategies are required 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 having a stably-integrated genome that includes the components necessary for rAAV production. The cells can also include components designed to operate in an insect cell such as hr enhancer elements and promoters (e.g., polH, plO, etc.). That is, as provided herein, the present disclosure provides, among other things, viral cells in which all components for making a recombinant gene therapy (e.g., rAAV) are added (via transformation) into the genome of a single cell e.g., an Sf9 cell) and, none of the components of the therapeutic (e.g., ITR-GOI) are introduced via, for example, a recombinant baculovirus. Rather, all components are present in a viral cell prior to infection such that baculovirus infection (e.g., with a wild-type baculovirus or other baculovirus not comprising any ITR or GOI components) is all that is needed to induce production of the recombinant virus (e.g., rAAV).

[0171] Further provided herein, in some embodiments, are methods for producing a recombinant virus comprising a rAAV vector using the virus-producing cells described herein that requires less steps (e.g., multiple transfections) and results in improved scalability, quality, and potency.

[0172] Also provided herein are methods for generating a rAAV vector, comprising: (a) providing a virus-producing cell having an engineered genome, wherein the cell comprises or consists essentially of: (i) a first nucleic acid comprising a first enhancer sequence and a Rep gene or functional fragment thereof; (ii) a second nucleic acid comprising a second enhancer sequence and a Cap gene or functional fragment thereof; and (iii) a third nucleic acid comprising a GOI; (b) contacting the cell of step (a) with a virus; and (c) after step (b), culturing the cells to produce a recombinant virus comprising the rAAV vector.

[0173] In some embodiments, the virus-producing cells comprise one or more of the first nucleic acid, the second nucleic acid, the third nucleic acid, or combinations thereof, stably integrated into the genome of the cell. In some embodiments, the virus-producing cells comprise the first nucleic acid, the second nucleic acid, and the third nucleic acid stably integrated into the genome of the cell. In some embodiments, the virus-producing cells are inducible.

[0174] In some embodiments, the virus-producing cells are selected for productivity by infecting each clonal population with a recombinant virus expression vector (e.g., BEV) void of any AAV elements. In some embodiments, clones were screened in antibiotic and/or serum-free conditions. Infection can initiate expression of genomically-integrated AAV Rep and AAV Cap genes. Expression of these genes can rescue integrated AAV genomes and packaging into assembled AAV capsids. Following the production phase, AAV can then be harvested from cell monolayers by freeze-thaw and nuclease treatment (or, in some embodiments, from suspension cultures by salt and/or detergent lysis), and the resulting AAV content produced by each clonal population is separate and, optionally, can be quantified by PCR. (See FIGS. 2A-2E for a schematic overview).

[0175] In some embodiments, the clones are then expanded to a production bioreactor to produce a recombinant viral vector. In some embodiments, the recombinant viral vector (e.g., rAAV) is subsequently harvested, any active virus inactivated (e.g., by heat) and/or removed e.g., with enzyme and/or physical separation), and the viral particles are purified. In some embodiments, recombinant viral vectors are purified and formulated. In some embodiments, contents of viral particles are released and further processed (e.g., processed, e.g., purified).

[0176] In some embodiments, suitable media is used for the production of recombinant vectors. These media comprise, without limitation, media appropriate for cell type (e.g., mammalian, insect, etc.), such as, for example, media produced by Hyclone Laboratories and JRH comprising Modified Eagle Medium (MEM), Roswell Park Memorial Institute (RPMI) 1640, Eagle’s Minimal Essential Medium (EMEM), Dulbecco’s Modified Eagle Medium (DMEM), ExpiSf-CD media (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 custom formulations, particularly with respect to custom media formulations for use in production of recombinant vectors.

[0177] In some embodiments, suitable production culture media of the present disclosure is supplemented with serum or serum-derived recombinant proteins at a level of 0.5-20 (v/v or w/v). In some embodiments, vectors are produced in serum-free conditions which are also referred to as media with no animal-derived products. In some embodiments, media may be chemically-defined. In some embodiments, commercial or custom media is designed to support production of vectors, comprising supplementation of without limitation glucose, vitamins, amino acids, and/or growth factors, in order to increase the titer and/or yield of vector in production cultures.

[0178] Vector production cultures comprise a variety of conditions (e.g., over a wide temperature range, for varying lengths of time, and the like) suitable to the particular host cell being utilized. Vector production cultures comprise attachment-dependent cultures which are cultured in suitable attachment-dependent vessels such as, for example, plates, flasks, cell stacks, roller bottles, hollow fiber filters, microcarriers, and packed-bed or fluidized-bed bioreactors. In some embodiments, vector production cultures comprise suspension-adapted host cells such as HeLa, HEK-293, and SF-9 cells which are cultured in a variety of ways comprising, for example, spinner flasks, stirred tank bioreactors, single use bioreactors such as Cytiva Xcellerex and Sartorius, and disposable systems such as the Wave bag system.

[0179] In some embodiments, viral particles of the disclosure are harvested from vector production cultures by lysis of the host cells of the production culture or by harvest of the spent media from the production culture, provided the cells are cultured under conditions to cause release of viral particles into the media from intact cells. Suitable methods of lysing cells comprise for example multiple freeze/thaw cycles, sonication, microfluidization, and treatment with chemicals, such as detergents and/or proteases.

[0180] In a further embodiment, the viral particles are purified. The term “purified” as used herein comprises a preparation of viral particles devoid of at least some of the other components that are present where the viral particles naturally occur or are initially prepared. Thus, for example, in some embodiments, isolated viral particles are prepared using a purification technique to enrich it from a source mixture, such as a culture lysate or production culture supernatant. In some embodiments, enrichment is measured in a variety of ways, such as, for example, by the proportion of DNase-resistant particles (DRPs) or genome copies (gc) present in a solution, or by infectivity, or measured in relation to a second, potentially interfering substance present in the source mixture, such as contaminants, comprising production culture contaminants or in-process contaminants, comprising helper virus, media components, and the like.

[0181] In some embodiments, the vector production culture harvest is clarified to remove host cell debris. In some embodiments, the production culture harvest is clarified by filtration through a series of depth filters comprising, for example, a grade DOHC Millipore Millistak+ HC Pod Filter, a grade A1HC Millipore Millistak+ HC Pod Filter, and a 0.2 pm 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 of 0.2 pm or greater pore size.

[0182] 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, the Benzonase® digestion is performed under standard conditions comprising, for example, a final concentration of at least 1-2.5 units/mL (and in some embodiments, up to 50 units/mL) of Benzonase® at a temperature ranging from ambient to 37 °C for a period of 30 minutes to several hours.

[0183] In some embodiments, viral particles are isolated or purified using one or more of exemplary purification steps: freeze-thaw; equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) for concentrating the 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 comprises all the steps and, optionally, in the order as described below.

[0184] In some embodiments, methods for generating a recombinant vector (e.g., rAAV) comprise providing stably-integrated virus-producing cells with a helper plasmid. In some embodiments, the cells are transfected with a helper plasmid that provides helper functions to the AAV. In some embodiments, the helper plasmid provides adenovirus functions including, but not limited to, El A, E1B, E4, and E2A. In some embodiments, the helper plasmid provides other virus functions including, but not limited to, VA RNA, Gag, Pol, Tat, Rev, Env, and VSV-G. The sequences of adenovirus gene providing these functions, in some embodiments, are obtained from any known adenovirus serotype, such as serotypes 2, 3, 4, 7, 12, and 40, and further comprising any of the presently identified human types. In some embodiments, the methods involve transfecting the cell with vectors expressing one or more genes necessary for AAV replication, AAV gene transcription, and/or AAV packaging.

[0185] The present disclosure also provides methods for generating a recombinant vector, wherein the methods comprise providing virus-producing cells comprising an engineered genome that is 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 cells are virus-producing cells comprising the selected components under the control of a constitutive promoter and other selected components under the control of one or more inducible promoters. For example, a virus-producing cell is generated which contains El helper functions under the control of a constitutive promoter, but which contains the Rep and/or Cap proteins under the control of one or more inducible promoters.

[0186] Products produced using platforms provided herein, such as in Sf9 cells, may also be compared to those produced using standard platforms such as mammalian cell systems (e.g. HEK293 cells). In such systems, appropriate rep and cap genes may be transfected into mammalian cells either on a single plasmid (e.g., a pRep/Cap) or using individual plasmids, each encoding a rep and a cap gene, where whether on a single or on more than one plasmid, the material transfected comprises sequences for producing a viral product (e.g., an rAAV) comprising a gene-of-interest. In general, the gene-of-interest can be introduced to the mammalian cell via a separate (e.g., in addition to one or more plasmids encoding rep and/or cap) plasmid comprising ITR-GOI components such that when the cell comprising the ITR- GOI, expresses the rep and cap components it generates an encapsidated viral gene product comprising the gene-of-interest. [0187] General approaches for transfecting, transforming and infecting mammalian cells to produce viral (e.g., AAV)-based gene therapies are known in the art. As will be evident to one of skill in the art, depending upon the circumstances, particular rep and cap genes for a serotype of interest and their regulatory sequences will be either the endogenous sequences for a particular serotype or those that correspond to particular capsids (e.g., AAV6TM, AAV7TM, AAV8TM, etc.). Such plasmids will be those that facilitate expression in mammalian systems and do not generally comprise engineered Kozaks such as those used in the insect cell systems disclosed herein. General principles of recombinant AAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial, and Immunol., 158:97-129. Various approaches are also described in Ratschin et al., Mol, Cell. Biol. 4:2072 (1984); Hermonat et al. , Proc. Natl. Acad. Sci. USA. 81 :6466 (1984); Tratschin et al. , Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al. , J. Virol., 62: 1963 (1988); and Lebkowski et al. 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol., 633822-3828): U.S. Pat. No. 5,173,414: WO 95/13365 and corresponding U.S. Pat. No. 5,658,776; WO 95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96.4423); WO 97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777): WO 97/06243 (PCT/FR96/01064); WO99/11764; Perrin et al. (1995) Vaccine 13: 1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark et al. (1996) Gene Therapy 3: 1124-1132; U.S. Pat. Nos. 5,786,211; 5,871,982; and 6,258,595. [0188] Methods disclosed herein result in improvements in recombinant viral vector manufacturing, including improved quantity of recombinant virus, more efficient and faster production time, and greater reproducibility and scalability without any decrease in efficacy of recombinant viral produce produced.

[0189] In some embodiments, the present disclosure provides an improvement in manufacturing recombinant viral products, the improvement comprising combining, into a single host cell, three stably integrated components, wherein the three stably integrated components comprise a first nucleic acid comprising a first enhancer sequence and Rep gene or fragment thereof; a second nucleic acid comprising a second enhancer sequence and a Cap gene 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 an engineered Kozak sequence. Upon stable integration into a host cell genome, a host cell is contacted with an inducer virus, which activates the integrated components resulting in production of a recombinant virus comprising the GOI. [0190] Other details for making and using recombinant viruses containing a gene of interest, including an AAV, can be found, for example, in PCT Publications Nos. WO 2010/114948 and WO 2017/181162.

[0191] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, and even if not explicitly recited as such therein, 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, unless specified as such, may occur in any order.

[0192] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present disclosure and does not pose a limitation 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 present disclosure.

[0193] Where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0194] Further, it should be understood that elements and/or features of a composition or a method provided herein can be combined in a variety of ways without departing from the spirit and scope of anything disclosed, whether explicit or implicit, herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions and/or in methods of the present disclosure, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present disclosure. For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of any invention(s) provided, described and/or depicted herein.

[0195] It should be understood that the order of steps or order for performing certain actions is immaterial so long as that which is disclosed and/or claimed remains operable regardless of order. Moreover, two or more steps or actions may be conducted simultaneously.

[0196] 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.

Pharmaceutical Compositions

[00191] Once produced, recombinant AAV particles as provided herein can be formulated into a pharmaceutical composition.

[00192] For therapeutic use, a composition comprising recombinant virus as provided herein is combined with a pharmaceutically acceptable carrier. Various carriers (e.g., diluents, excipients, etc.) used in formulating and preparing pharmaceutical compositions are known and/or readily accessible to those of skill in the art. Depending upon the circumstances, a carrier can include a liquid (e.g., a sterile liquid) or a solid. A carrier may be selected from or comprise water, aqueous solvents, non-aqueous solvents, dispersion media, surfactants, antioxidants, buffers, adjuvants, tonicity agents, stabilizers, bulking agents, lyoprotectants, metal ions, chelating agents, isotonic and absorption delaying agents and the like that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art. Typically a carrier is approved by 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 Pharmacopoeia. Suitable formulations for use in the present disclosure are found in see e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23 ^rd ed. 2020). For a brief review of methods for drug delivery, see, e.g., Langer (1990) SCIENCE 249: 1527-1533. The resulting pharmaceutical compositions are suitable for administration to a subject (e.g., an animal, e.g., a mammal, e.g., a human).

[00193] A pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, 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, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, polyethylene glycol (PEG), sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and/or pharmaceutical adjuvants (see e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23 ^rd ed. 2020)).

[00194] In certain embodiments, a pharmaceutical composition may contain a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled-delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Sustained-release preparations may include, e.g., 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 that can be prepared by any of several methods known in the art.

[00195] Depending upon the circumstances, a pharmaceutical composition may contain nanoparticles, or lipid droplets, e.g., polymeric nanoparticles, liposomes, or micelles (see Anselmo et al. (2016) BIOENG. TRANSL. MED. 1 : 10-29).

[00196] Pharmaceutical compositions containing rAAVs of the present disclosure may be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intraperitoneal, intradermal, inhalation, transdermal, intracerebroventricular (ICV), intraparenchymal, intra cisterna magna (ICM), intrathecal, intradural, etc.

[00197] Useful formulations can be prepared by methods known in the pharmaceutical art. For example, see e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23 ^rd ed. 2020). Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose. [00198] Suitable carriers will be known to those of skill in the art, for example, for intravenous administration, suitable carriers include physiological saline, bacteriostatic water, poly ethoxylated castor oil or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In the case of injection into the central nervous system (e.g., ICV, ICM, intraparenchymal, intrathecal, etc.) carriers will be adjusted appropriately. [00199] Pharmaceutical formulations preferably are sterile. Formulations or components thereof can be sterilized, for example, by methods appropriate to retain activity and stability of the GOI encoded therein. Sterilization can be accomplished by any suitable method, e.g., filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.

[00200] Depending upon the drug substance and formulation, the resulting dosage forms can be stable for extended periods of time, such as 1 month, 3 months, 6 months, 1 year, 2 years, 3 years, or more, when the dosage form is a liquid or solid. The formulations can be stable at room temperature or higher. It is contemplated that the dosage form is stable at ambient conditions in PBS. Alternatively the dosage form is frozen (e.g., a liquid or a lyophilizate) and stable under appropriate temperatures such as, e.g., -20°C, -80°C).

[00201] Depending upon the circumstances, the dosage forms can be formulated as a unit dose, which can include, for example a particular vg/L as provided herein.

[00202] The compositions described herein may be administered locally or systemically. It is contemplated that the compositions described herein can be administered by parenteral administration. In some embodiments, such administration is preferably 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 composition is administered subcutaneously or may be administered intravenously, e.g., via intravenous infusion. In certain embodiments, it is contemplated that the gene therapies disclosed herein can be administered by systemic administration.

[00203] The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient, the in vivo potency of the active component and any toxicity concerns, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood-level or tissue-level. Alternatively, the initial dosage can be smaller than the optimum, and the daily dosage may be progressively increased during the course of treatment. Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study. Dosing frequency can vary, depending on factors such as route of administration, dosage amount, and the disease, disorder, or condition being treated.

Exemplary dosing frequencies are once per day, once per week and once every two weeks.

Methods Of Use And Treatment

[00204] The rAAVs provided herein can be used in a variety of different approaches. For example, the rAAVs can be used in a method of treating a disease, disorder, or condition, associated with a dysfunctional gene-of-interest. The method comprises contacting a cell that in a subject in need thereof, wherein the composition comprises a nucleic acid encoding a gene-of-interest, which, when expressed, will treat the disease, disorder, or condition associated with the dysfunctional GOI.

[00205] In some embodiments, the disease, disorder, or condition is associated with a dysfunctional gene that is expressed or impacts one or more cells of the central nervous system and/or the peripheral nervous system.

[00206] It is contemplated that therapy can be accomplished using an rAAV alone, as a monotherapy, or as part of a combination therapy. The combination therapy may include one or more additional agents or therapeutic approaches known to those of skill in the art for treating inflammatory and/or autoimmune diseases, and may have been previously used, be already ongoing, or added to a treatment for a subject in need thereof. [00207] A subject may be evaluated, e.g., by a healthcare provider, before, during, and/or after treatment with a composition provided herein. Depending on the outcome of the evaluation, a treatment may be continued or ceased, treatment frequency or dosage may change, or the patient may be treated with a different gene therapy. Subjects may be administered a composition comprising a gene therapy as provided herein for a discrete period of time according to dosage paradigms described herein, including, optionally, until the disease, disorder, or condition is treated.

INCORPORATION BY REFERENCE

[00208] All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety for all purposes. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

EQUIVALENTS

[00209] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

EXAMPLES

[0208] Below are examples of specific embodiments for carrying out the present disclosure. The examples are offered for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way.

EXAMPLE 1. Production of AAV using an engineered cell line with stably integrated exogenous viral components

[0209] This example generally describes production of AAV using the engineered cell lines described herein. An engineered cell line was produced to stably-integrate AAV components into Sf9 cells. The engineered cell comprised AAV Cap components, AAV Rep components, and GOIs, which were stably integrated into the genome of the cell. The Cap, Rep, and GOI components were integrated into the genome using engineered constructs. Various AAV serotypes were tested.

[0210] Kozak-optimized AAV capsid sequences and AAV Rep sequences and GOIs were stably-integrated into Sf9 cells by random integration and antibiotic selection to generate a heterogeneous pool of stably-transformed Sf9 producer cells. This is further described in Example 2.

[0211] For each serotype, Kozak sequences were first screened by SDS-PAGE and Western blot resulting in a range of capsid stoichiometries. Leading candidate sequences that enhance VP1 incorporation into AAV capsids were cloned into cell line integrating vectors alongside enhancer elements.

[0212] Clonal Sf9 cell lines were derived from heterogeneous pools by a process of single cell seeding and whole-well imaging. Rapidly growing clones that exhibit favorable growth kinetics were scaled up for productivity screening. This is further described in Example 3.

[0213] Clones were selected for productivity by infecting with a baculovirus and harvesting AAV produced by each clone. Clones that produced the highest amounts of AAV were further scaled up to analyze product attributes and improve production parameters.

[0214] Infection parameters were determined using small scale stirred tank bioreactors. This scale of production was sufficient to examine process and product critical quality attributes for AAVs derived from each clonal cell line.

[0215] AAVs manufactured using the engineered Sf9 producer cell line platform and mammalian HEK293 triple transfection platform were purified and evaluated head-to-head in rodent models. This is further described in Example 4. EXAMPLE 2: Isolation and Characterization of Sf9 Producer Cell Lines

[0216] Sf9 producer cell lines, as described in Example 1, were designed to increase productivity and potency. The producer cells were engineered to produce AAV using homologous region (hr) enhancer elements and engineered Kozak sequences. Exemplary construct designs are shown in FIG. 1A. Hr elements used in this Example were native to the baculovirus genome, where they act as transcriptional enhancers for adjacent transgenes. This mechanism was preserved and incorporated into the Sf9 cell genome in the form of DNA concatemers encoding AAV Rep and AAV Cap proteins. Engineered Kozak sequences were screened and compared to determine ability to incorporate higher levels of VP1 into AAV capsids. These higher levels of VP1 may facilitate higher probability of endosomal escape and increased capsid potency. This engineering approach functions across AAV serotypes as evidenced in FIG. IB (Western blot showing AAV1 and AAV5 capsids) and FIG. 1C (affinity purified AAV7, AAV8, and AAV9 capsids).

[0217] Clonal populations of cells were produced by single cell seeding and whole-well imaging. Random integration of DNA concatemers and linearized ITR-flanked gene of interest constructs were performed by transfection of DNA into Sf9 cells and antibiotic selection of stable transformants. A heterogeneous pool of stably transformed Sf9 AAV producer cell lines was scaled up to facilitate initial testing of ability to produce and purify AAV products using these engineered cells. Clonal populations of these Sf9 AAV producer cell lines were isolated by single cell seeding and whole-well imaging. Verified clonal populations were monitored over a period of 3-4 weeks before transferring to 96-well plates for head-to-head screening.

[0218] Producer cell lines produced as described herein were selected for productivity by infecting each clonal population with a recombinant baculovirus expression vector (BEV) void of any AAV elements. As shown in FIGS. 2A-2E, clones were seeded in a well-plate (FIG. 2A) and grown to a confluent monolayer (FIG. 2B and 2C). Plates were imaged prior to infection to determine confluency. The BEV contained a visualizable reporter to facilitate detection and was also devoid of naturally-occurring proteases (e.g., VCath, ChlA). Visualization allowed purification (prior to infection) as well as monitorization of rate of infection (e.g., monitoring how many cells show the detectable label). In most instances 90- 95% of the cells were fluorescent. Clones were screened in antibiotic and serum-free conditions, mirroring conditions of a mature manufacturing process.

[0219] Following baseline measurement of confluency to estimate the number of cells, each monolayer consisting of cells from a single clone were infected with a “WT” BEV (meaning it was devoid of any AAV elements such as ITRs and/or a GOI (FIG. 2C). Infection initiated expression of genomically-integrated AAV Rep and AAV Cap genes and production of AAV was induced (FIG. 2C).

[0220] Expression of these genes resulted in rescue of integrated AAV genomes and packaging of the gene of interest (GOI) into assembled AAV capsids.

[0221] Following the production phase AAV was harvested from cell monolayers using freeze thaw and nuclease treatment. Briefly the media was discarded (including any BEV and any unpackaged components) (FIG. 2D) and AAV was isolated from cell monolayer (by freeze/thaw cycles and nuclease treatment) and subsequent separation/clarification of cellular debris from AAV (FIG. 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 of FIG. 2E, right panel.

[0222] To identify high-producing clones, clonal Sf9 AAV producer candidates were selected based on the amount of AAV they produce. Adherent clone screening assays were refined to select producer clones that yield >E14vg/L in more mature suspension-based manufacturing processes prior to optimization. That is, although screening was performed in adherent cultures, clones producing in the E10 vg/mL range during the screening assay typically yield in the high El 3 - low E14 vg/L range in a more mature suspension-based manufacturing process prior to optimization. Top producers identified (see FIG. 3) were scaled-up and analyzed further for product analysis and production optimization.

[0223] A scale-up process was developed to reduce number of passages required to transition cells from 96-well adherent cultures to suspension. This greatly accelerated growth and reduced the time to establish a suspension-ready research cell bank as can be seen in FIG. 4. Growth acceleration using the scale up process resulted in twice the number of cells in half the time (i.e., a 4x increase). The scale up process from transfection, clone selection, AAV product analysis, and banking of RCBs was approximately 3 months.

EXAMPLE 3: Sf9 Quality Analysis

[0224] This Example describes quality of Sf9 cells produced in accordance with systems, methods, and compositions (e.g., engineered cells) provided herein.

[0225] Purification of Sf9-derived AAVs by affinity capture demonstrated preservation of capsid stoichiometries and enhanced levels of VP1 incorporation into purified capsids. Anion exchange methods were developed to enrich for genome containing capsids with enrichment currently 70%-80% depending on serotype. Downstream purification methods removed host cell protein in addition to host cell and baculovirus DNA impurities in the final AAV preparation.

[0226] FIGS. 5A-5D show measurements of amount of host cell protein (HCP) and activating virus present over the course of production and pre- and post-purification, demonstrating that both host cell proteins and activating virus impurities were reduced over the course of purification and enhanced stoichiometry did not impact ability to purify recombinant viruses from host cells or other potential contaminants from viruses involved in manufacturing (e.g., activating baculovirus). FIGS. 5A and 5B are gels showing reduction in impurities over the course of purification. FIG. 5C is a bar graph showing HCP in ng/mL detected in fractions from various purification steps. FIG. 5D is a bar graph showing DNA concentration of Sf9 and BEV DNA detected in eluates from AAVX and AEX column purifications; left bars on each of AAVX and AEX are from Sf9 DNA measurements and right bars are from BEV DNA measurements. These data confirm that the systems, methods, and cells provided herein provide end products with biophysical and biochemical properties that are at least identical to those that have traditionally been obtained using other (e.g., HEK293)-based cell systems and that already existing equipment (e.g., AEX columns, affinity purification columns, etc.) can be successfully used to purify virus produced using the methods provided herein. As Sf9 cells are a heterogeneous population of rhabdovirus- positive and rhabdovirus-negative cells, producer lines were screened for presence of rhabdovirus. During single cell isolation and clone selection, rhabdovirus-free Sf9 AAV producer cell lines were identified, eliminating the presence of rhabdovirus in the manufacturing process as seen in Table 2.

Table 2. Rhabdovirus status of AAV Producer Cell Lines [0227] Sf9 AAV producer cell lines exhibit robust manufacturing capabilities. A stability study was performed by thawing exemplary producer clones. In this example, the exemplary producer clones were independent research cell banks of Sf9 AAV5 producer clones.

Thawed cultures underwent an additional 20 passages with a subset of the culture infected at passages 5, 10, 15, and 20. AAV was harvested from each clone at each of the infection timepoints and quantified by droplet Digital PCR (ddPCR). As shown in FIG. 6, no drift in volumetric (vg/L) or unit (vg/cell) titer was observed over 20 passages, indicating stable integration of AAV-producing components.

[0228] Sf9 AAV producer cell lines were selected based on growth and infection kinetics analogous to traditional IC-BEV manufacturing methods. Cell density (cells/mL) and percent viability were measured over time in growth and infection phases. Stable integration of genetic components did not affect the ability of Sf9 cells to double (24-30 hours), grow to high densities (>1 x 10 ⁷ cells/mL), or alter the infection kinetics in response to baculovirus infection as shown in FIG. 7.

[0229] AAV capsids manufactured in both Sf9 and HEK293 platforms (as further described in Example 4) were analyzed by LCMS for post-translation modifications (see Table 3) As previously reported, alternate patterns of post-translational modifications were observed between manufacturing methods. Next generation Illumina sequencing of purified Sf9 AAVs was conducted to quantify any contaminants from Sf9 or baculovirus genomes. As illustrated in FIG. 8 and shown in Table 3, there was a very low percentage of reads/ sequences that mapped to Sf9 or baculovirus genomes, supporting minimal packaging of Sf9 and Baculovirus DNA in assembled capsids. Low quantity of baculovirus packaging confirms that the stable integration of three constructs is a reliable, reproducible, safe, and improved approach relative to cells that only integrate one or two components. The low quantity of Sf9 genome, in particular, is a surprising finding considering that the cells stably integrate Cap, Rep, and GOLITR constructs because those of skill in the art would expect that integrating an ITR-GOI into a cell with Cap and Rep would result in a greater amount of host cell genome packaging.

Table 3. LCMS analysis of post-translational modifications

- = no modification detected

EXAMPLE 4. Virus-producing Sf9 Cells with Engineered Genomes

[0230] This Example generally describes the process for making virus-producing cells with engineered genomes.

[0231] First, the plasmids comprising 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 functional derivative thereof), engineered Kozak sequence (which may be optionally engineered and selected from any of SEQ ID NOs: 1-191 or any functional fragment or functional derivative thereof in the construct comprising the Cap sequence), promoter (e.g., inducible promoter), and a Rep sequence (encoded by a plasmid such as in SEQ ID NO: 210) or Cap sequence such as a nucleic acid (SEQ ID NOs: 192-203) or plasmid (SEQ ID NOs: 211-261) that encodes a protein of any of amino acid (SEQ ID NOs: 204- 209), can be linearized at a unique Type IIS restriction enzyme site to generate monomers of DNA.

[0232] As exemplified herein, for Sf9 produced viral gene therapy products, plasmids for each of the rep and cap sequences included an enhancer sequence of SEQ ID NO: 1, a promoter (polH or plO), and an antibiotic selection cassette encoding blasticidin. The rep gene included the rep sequence in the plasmid of SEQ ID NO: 201 and the cap proteins were each selected by serotype (AAV1 : SEQ ID NO: 192 (nucleic acid without engineered Kozak), SEQ ID NO: 193 (nucleic acid with engineered Kozak used in insect cell platforms), SEQ ID NO: 204 (amino acid sequence of 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 insect cell platforms), SEQ ID NO: 205 (amino acid sequence of 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 insect cell platforms), SEQ ID NO: 206 (amino acid sequence of capsid protein); and SEQ ID NO: 213 (plasmid); AAV7TM: SEQ ID NO: 198 (nucleic acid without engineered Kozak), SEQ ID NO: 199 (nucleic acid with engineered Kozak used in insect cell platforms), SEQ ID NO: 207 (amino acid sequence of capsid protein); and SEQ ID NO: 214 (plasmid); AAV8TM: SEQ ID NO: 200 (nucleic acid without engineered Kozak), SEQ ID NO: 201 (nucleic acid with engineered Kozak used in insect cell platforms), SEQ ID NO: 208 (amino acid sequence of 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 insect cell platforms), SEQ ID NO: 209 (amino acid sequence of capsid protein); and SEQ ID NO: 216 (plasmid)) and according to the sequences set forth in any of SEQ ID NOs: 192-216. Cap genes transformed into Sf9 genomes also further comprised an engineered Kozak sequence selected from SEQ ID NO: 11 or 13 (which are also represented in the capsid sequences of SEQ ID NOs: 193 (AAV1), 195 (AAV5), 197 (AAV7), 199 (AAV7TM), 201 (AAV8TM), and 203 (AAV9)).

[0233] For the insect cell platform provided herein DNA for each nucleic acid (rep and cap) was purified and then assembled into monomers together into a very high molecular weight concatemers in head-to-tail orientation by the process of ligation. These high molecular weight concatemers were then purified further and quantified for transfection. [0234] The GOI was linearized into monomers using restriction enzymes (FIG. 9). In brief, restriction digest was performed on plasmid DNA that included restriction endonuclease sites and engineered inverted terminal repeats flanking a nucleotide sequence encoding a gene-of-interest. This restriction digestion process removes undesirable sequences, such as the antibiotic resistance marker that is used for propagation in bacteria. These GOI monomers (comprising the GOI flanked by ITRs) were purified and ready for transfection to integrate them into the Sf9 genome. The restriction digest removed a substantial amount of plasmid DNA elements/prokaryotic DNA sequences such that the total length of flanking genomic DNA was about 1700 base pairs.

[0235] Prior to transfection into Sf9 cells, the DNA was analyzed to calculate the volume of DNA of each of the components to transfect into the Sf9 cells. For transfection, Sf9 cells were first seeded at about 80% confluence. Sf9 cells were transfected according to manufacturing protocol and subject to antibiotic resistance 48 hours following transfection. Sf9 cells were either transfected with the DNA or with sham. Cells were then assayed for viability 48 hours post-transfection. Cells were viable and able to produce AAVs that were purified and administered to murine test subjects as shown in Example 5

[0236] For comparison to gene therapy products produced in Sf9 cells, AAV particles were also produced using HEK293 systems. Virus-producing HEK293 cells were generated using standard transfection methods known to those in the art using: (a) (i) endogenous rep/cap genes for each serotype of interest (e.g., AAV1, 5, 7, 8, and 9) or (ii) appropriately modified sequences different from endogenous AAV sequences for variant capsid proteins (e.g., for AAV7TM, e.g., AAV8TM), where the rep/cap genes were expressed on the same plasmid; and (b) an ITR-GOI nucleic acid such as that exemplified in Example 5. Recombinant viral production (of encapsidated GOI) was induced via infection of HEK293 cells.

EXAMPLE 5. In Vivo Head-To-Head Comparison of Sf9-Derived AAVs versus HEK293-Derived AAVs

[0237] This Example compares AAVs derived from Sf9 and HEK293 host cells. AAVs produced using Sf9 systems made in accordance with the present disclosure exhibited a similar therapeutic effect as compared to AAVs make using HEK293 cells.

[0238] AAV5 (SEQ ID NO: 194 for mammalian (HEK) cells or SEQ ID NO: 195 for insect (Sf9) cells) encoding a single-stranded GOI encoding an exemplary enzyme was generated using the Sf9 platform in accordance with the present disclosure or using a standard HEK293 platform as described in Example 4, and administered via intra-cistema magna (ICM) injection. Enzyme levels were measured in cerebellum of wild-type, knockout, and Sf9 AAV- or HEK293 AAV-treated mice. As shown in FIG. 10, a head-to-head comparison between AAVs produced by Sf9 cells made as provided herein and HEK293 cells revealed no significant difference in the level of resulting enzyme activity measured in the cerebella of animals treated with capsids manufactured using different platforms (HEK293 cells versus Sf9 cells made in accordance with the present disclosure), and enzyme levels in each AAV-treated group were higher than wild-type and knock-out animals in the exemplary mouse model.

[0239] A self-complementary AAV, a variant of AAV7 (SEQ ID NO: 198 for mammalian (HEK) cells or SEQ ID NO: 199 for insect (Sf9) cells), encoding an exemplary GOI was manufactured using Sf9 producer cell lines of the present disclosure and compared to HEK293 -produced AAV described in Example 4 in an exemplary mouse model of disease. Head-to-head comparison of the Sf9-produced AAV (containing the GOI) with that of the AAV (containing the GOI) using the HEK293 platform showed equal biodistribution of vector genomes in target tissue (FIG. 11 A) and efficacy in extending survival of the disease model (FIG. 11B).

[0240] A variant of AAV8 (SEQ ID NO: 200 for mammalian (HEK) cells or SEQ ID NO: 201 for insect (Sf9) cells) encoding an exemplary therapeutic GOI sequence was manufactured using both Sf9 producer cell lines made in accordance with the present disclosure and those made using a standard HEK293 platform, as described in Example 4. AAV-gene therapy products manufactured in each platform were administered to neonatal mice at equal doses. Ten weeks post-administration, tissues were harvested and analyzed for resulting enzyme activity. No differences were observed in enzyme levels as a result of treatment with vectors made with different manufacturing methods (Sf9 or HEK) as demonstrated by levels of enzyme measured in forebrains (FIG. 12A), hindbrains (FIG. 12B) and cerebella (FIG. 12C), or in resulting biodistribution and expression of the therapeutic transgene (not shown).

[0241] Importantly, these data also demonstrate that the Sf9 producer lines are appropriate for both self-complementary (see, e.g., FIGS. HA and 11B) and single stranded AAV (see, e.g., FIGS. 12A and 12B).

[0242] An AAV9 (SEQ ID NO: 202 for mammalian (HEK) cells or SEQ ID NO: 203 for insect (Sf9) cells) encoding a GFP reporter construct was manufactured in both Sf9 insect cell and HEK293 mammalian platforms as described in Example 4. AAVs derived from three independent Sf9 clones were compared to material derived from triple-transfection of HEK293 cells. Following intra-cistema magna (ICM) administration, samples were blinded and analyzed by a neurohistologist. No appreciable difference was noted between the biodistribution profiles of AAV vectors derived from either the Sf9 platform or mammalian HEK293 platform (see FIG. 13, arrows, and Table 4).

[0243] In each serotype and with each GOI, AAV-gene therapy products produced using Sf9 insect cells as provided herein did not show any difference from those produced using an HEK293-based mammalian cell platform. Moreover, the insect cell-based therapeutics were scalable and reproducible, and can therefore provide commercially useful amounts of AAV particles suitable for gene therapy.

[0244] These results provide various examples that demonstrate that such insect-cell- based approaches can be used with various serotypes and GOIs. Thus, such insect-cell approaches are able to produce viral-based gene therapies at least as well as mammalian cell systems, while also providing several advantages over such HEK293/mammalian cell platforms that can be used to improve production of recombinant gene therapy products.

Table 4. Sf9 versus HEK293 Biodistribution

SEQUENCE LISTING