Packaging and producer cell lines for recombinant adeno-associated virus, plasmids, and methods of making and using

Abstract

The present disclosure provides recombinant Adeno-associated packaging cells and recombinant Adeno-associated virus producer cells, as well as plasmids and methods of making and using the same. The stable recombinant Adeno-associated packaging cells and recombinant Adeno-associated virus producer cells include the assembly genes, replication genes, and helper genes, and optionally a transgene, integrating / inserting into genomic safe harbor sites.

Description

PACKAGING AND PRODUCER CELL LINES FOR RECOMBINANT ADENO-ASSOCIATEDVIRUS, PLASMIDS, AND METHODS OF MAKING AND USINGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and the benefit of, the U.S. Provisional Patent Application No. 63 / 662,862, filed 21 June 2024 and titled PACKAGING CELL LINES FOR RECOMBINANT ADENO-ASSOCIATED VIRUS, PRODUCER CELL LINES FOR RECOMBINANT ADENO-ASSOCIATED VIRUS, AND PLASMIDS AND METHODS OF MAKING AND USING THE SAME, which is incorporated by reference herein in its entirety for all purposes.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under 2100075 awarded by U.S. National Science Foundation. The government has certain rights in the inventionINCORPORATION BY REFERENCE

[0003] In compliance with 37 C.F.R. 1 52(e), the sequence information contained in electronic file named “UM10055PCT_Sequence_Listing.xml”, which was created on 9 June 2025 and 59,320 bytes in size, is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0004] The invention provides stable recombinant Adeno-associated virus packaging and producer cells lines, as well as reagents and methods for preparing the same.BACKGROUND

[0005] Adeno- Associated Virus (AAV)-mediated gene therapy is a quickly growing segment of the pharmaceutical market. The current transient transfection process to produce a recombinant AAV (rAAV), however, has many challenges, such as low productivity of rAAVfrom host cells, difficult scalability of the rAAV-producing bioprocess, and high levels of impurities (e.g., empty / partial capsid) materialized during production.

[0006] Developing stable cell lines that express AAV replication and packaging proteins will ensure consistent and efficient viral vector production. Numerous challenges, however, still need to be overcome before achieving this goal. A stable producer cell line requires the integration of not only the gene of interest (GOI), the Rep gene, and Cap gene that are important for genome replication and encapsidation, but also helper genes that initiate the rAAV replication. The main barrier to establishing a stable cell line is the cytotoxicity induced by the continuous expression of Rep and helper genes after integration. To meet the current and future demands of gene therapy products, developing an inducible stable cell line is a necessary and promising approach for rAAV production.

[0007] Multiple inducible promoters have been reported successfully controlling the expression of the most toxic protein Rep78 / 68 and adenovirus helper proteins E2A and E4. Recently, with the rapid development of synthetic biology, CEVEC Pharmaceuticals reported the feasibility of developing a producer cell line through stable, sequential transfection of Tet inducible rAAV production-related components using CEVEC’ s Amniocyte Production(CAP) cells and HEK293 cells as a host cell line while more details were not disclosed (Hein, K., et al., Generation of helper virus-free adeno-associated viral vector packaging / producer cell lines based on a human suspension cell line. 2018; Swiech, K., V. Pican^o-Castro, and D.T. Covas, Human cells: new platform for recombinant therapeutic protein production. Protein expression and purification, 2012. 84(1): p. 147-153). Lee et al. constructed the inducible stable cell line to produce AAV using synthetic biology combined with a transposon-mediated random integration strategy, wherein multiple inducible promoters were utilized to regulate the expression of different rAAV components (Lee, Z., et al., Construction of an rAAV Producer Cell Line through Synthetic Biology. ACS Synthetic Biology, 2022. 11(10): p. 3285-3295). Separate control over replication and packaging activities further allowed the manipulation and regulation of empty / full capsid ratio, another important product quality attribute. Those results demonstrate the feasibility of using inducible systems and applying synthetic biology technologies in generating stable rAAV packaging / producer cell lines.

[0008] Compared to the random integration strategy, site-specific integration allows the targeted integration of transgenes to pre-validated genome loci (Hamaker, N.K. and K.H. Lee,Site-specific integration ushers in a new era of precise CHO cell line engineering. Current opinion in chemical engineering, 2018. 22: p. 152-160). It can overcome fundamental problems in random integration, such as high clone variation, loss of transgene, and uncontrollable integration sites (Lee, J.S., et al., Mitigating clonal variation in recombinant mammalian cell lines. Trends in biotechnology, 2019. 37(9): p. 931-942). Furthermore, genomic safety harbors are regions of the human genome that allow stable expression of transgenes without affecting the host cells (Yu, J., et al., Human induced pluripotent stem cells free of vector and transgene sequences. Science, 2009. 324(5928): p. 797-801). The most extensively targeted genomic loci in human cells are AAVS1, CCR5, and ROSA26 (Pavani, G. and M. Amendola, Targeted gene delivery: where to land. Frontiers in genome editing, 2021 : p. 36). These aforementioned loci are acceptable for research purposes, but the clinical application needs to be further validated.

[0009] Synthetic inducible promoters were used herein to control the gene expression of refactored viral components and develop the baseline of the HEK293T stable cells via Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 mediated site-specific knock- in approach targeting human genomic safe harbor sites (ROSA26, AAVS1, and CCR5 locus). The refactored rAAV Rep, Cap, and helper genes under the control of inducible Tet-on promoters, however, resulted in low AAV productivity in the stable packaging cell lines. Additional optimization strategies (such as optimization of gene expression level and timing optimal for vector production, mitigation of toxic genes, and improvement of site-specific knock- in efficiency for large insertions) can be used to achieve high rAAV titer in the stable cell lines. The present disclosure represents a significant leap forward in the realm of establishing stable cell lines for AAV production.

[0010] Thus, an ongoing need exists in the art for stable recombinant Adeno-associated virus packaging and producer cells lines, as well as reagents and methods for preparing the same.SUMMARY

[0011] An aspect of the present disclosure provides a plasmid (e.g., an assembly plasmid or an assembly donor plasmid) comprising or consisting essentially of: (a) a left or 5’ homology arm sequence; (b) a first assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter,a CMV enhancer, or a combination thereof); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an inducible transactivator (e.g., a reverse tetracycline-controlled transactivator (rtTA)); (c) a second assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to the inducible transactivator (e.g., the inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., TRE3G or CMV promoter)); and (2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of an adeno-associated virus capsid gene (e.g., a sequence encoding an adeno-associated virus VP1, VP2, and VP3 proteins); an internal ribosome entry site (IRES) or a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A), preferably IRES; and a sequence encoding an adeno- associate virus Reg52 and Reg40 proteins; and (d) a right or 3’ homology arm sequence, wherein the left or 5’ homology arm sequence and the right or 3’ homology arm sequence facilitate homology directed repair (HDR) in a genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor).

[0012] In any aspect or embodiment described herein, the genomic safe harbor site comprises or is ROSA26, Adeno-Associated Virus Integration Site 1 (AAVS1), or C-C chemokine receptor type 5 (CCR5) locus, preferably ROSA26.

[0013] In any aspect or embodiment described herein, the second assembly expression cassette is optionally flanked by recombinase sequences (e.g., allB or attP, preferably allB).

[0014] In any aspect or embodiment described herein, at least one of (a) the plasmid further comprises or consists essentially of, 5’ of the left or 5’ homology arm sequence and 3’ of the right or 3’ homology arm sequence at least one of: an original of replication (ori); an ampicillin resistance expression cassette comprising, consisting essentially of, or consisting of a regulatory element or expression control element (e.g., a promoter, such as an ampicillin resistance promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an ampicillin resistance gene (e.g., AmpR); a aminoglycoside resistance expression cassette comprising, consisting essentially of, or consisting of a regulatory element or expression control element (e.g., a promoter, such as a ccdB promoter) and asynthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of neomycin resistance, kanamycin resistance, or a combination thereof (e.g., NeoR / KanR); an expression cassette comprising, consisting essentially of, or consisting of a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a catabolite activator protein (CAP) binding site, a lac promoter, a lac operator, a T3 promoter, a Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding a fluorescence protein (e.g., green fluorescent protein or enhanced green fluorescence protein); or a combination thereof; (b) the plasmid further comprises or consists essentially of one or more (e.g., 1, 2, 3, or more) antibiotic resistance expression cassette located 3’ of the left or 5’ homology arm sequence and 5’ of the right or 3’ homology arm sequence, wherein the antibiotic resistance expression cassette is at least one of: a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of a regulatory element or expression control element (e.g., a promoter, such as a SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR); a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e.g., BleoR); a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a T7 promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g., PuroR); or a combination thereof, preferably a blastocidin S resistance expression cassette; or (c) a combination thereof.

[0015] A further aspect of the present disclosure provides plasmid (e.g., an assembly plasmid, an assembly transposon plasmid, an assembly donor plasmid, or an assembly transposon donor plasmid) comprising or consisting essentially of, in order from 5’ to 3’: (a) a right or 3’ inverted transposase terminal repeat sequence; (b) an assembly transposon comprisingor consisting essentially of: (1) a first assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an inducible transactivator (e.g., a reverse tetracycline-controlled transactivator (rtTA)); (2) a second assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’: a regulatory element or expression control element that is responsive to the inducible transactivator (e.g., the inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., TRE3G or CMV promoter)); and a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: an adeno- associated virus capsid gene (e.g., a sequence encoding an adeno-associated virus VP1, VP2, and VP3 proteins); an internal ribosome entry site (IRES) or a sequence encoding a 2 A peptide (e.g., P2A, T2A, E2A, or F2A), preferably IRES; and a sequence encoding an adeno-associate virus Reg52 and Reg40 proteins; and (c) a left or 5’ inverted transposase terminal repeat sequence, wherein the left or 5’ inverted transposase terminal repeat sequence and the right or 3’ inverted transposase terminal repeat sequence facilitate excision of the transposon from the plasmid and insertion or integration of the assembly transposon in one or more (e g., about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5) 4-base insertion site in a genome (e.g., a mammalian genome or a human genome).

[0016] In any aspect or embodiment described herein, at least one of: the second assembly expression cassette is optionally flanked by recombinase sequences (e.g., c / / / B or attP, preferably c / Z / B); the 4-base insertion site is a TTAA site or a TTAT site, preferably a TTAA site; the left or 5’ inverted transposase terminal repeat sequence and the right or 3’ inverted transposase terminal repeat sequence are reverse complements; each of the left or 5’ inverted transposase terminal repeat sequence and the right or 3 ’ inverted transposase terminal repeat sequence is about 8 to about 1200 nucleotides (e.g., about 12 to about 200 nucleotides, about 28to about 200 nucleotides, about 28 to about 600 nucleotides, or about 200 to 1200 nucleotides); or a combination thereof.

[0017] In any aspect or embodiment described herein, at least one of: (a) the plasmid further comprises or consists essentially of, 5’ of the right or 3’ inverted transposase terminal repeat sequence and 3’ of the left or 5’ inverted transposase terminal repeat sequence, at least one of: an original of replication (ori); an ampicillin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as an ampicillin resistance promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an ampicillin resistance gene (e.g., AmpR); a aminoglycoside resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a ccdB promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of neomycin resistance, kanamycin resistance, or a combination thereof (e.g., NeoR / KanR); an expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e g., a promoter, such as a constitutively active promoter, such as a catabolite activator protein (CAP) binding site, a lac promoter, a lac operator, a T3 promoter, a Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding a fluorescence protein (e.g., green fluorescent protein or enhanced green fluorescence protein); or a combination thereof; (b) the plasmid further comprises or consists essentially of one or more (e.g., 1, 2, 3, or more) antibiotic resistance expression cassette located 3’ of the right or 3’ inverted transposase terminal repeat sequence and 5’ of the left or 5’ inverted transposase terminal repeat sequence, wherein the antibiotic resistance expression cassette is at least one of: a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of ,a regulatory element or expression control element (e.g., a promoter, such as a SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR); a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading framemolecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e.g., BleoR); a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a T7 promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g., PuroR); or a combination thereof, preferably a blastocidin S resistance expression cassette; or (c) a combination thereof.

[0018] In any aspect or embodiment described herein, the plasmid further comprises, consists essentially of, or consists of, at least one of: a kozak sequence (e.g., a classical Kozak sequence) that is located 3’ of the regulatory element or expression control element and 5’ of the synthetic open reading frame molecule of the antibiotic resistance expression cassette; a poly(A) sequence / tail / signal (e.g., bGH poly(A) signal or SV40 poly(A) signal, preferably SV40 poly(A) signal) that is located 3’ of the gene of the antibiotic resistance expression cassette; a kozak sequence (e.g., classical Kozak sequence) that is located 3’ of the regulatory element or expression control element and 5’ of at least one of the adeno-associated virus capsid gene, the sequence encoding the adeno-associated virus Reg 52 and Reg 40 proteins, the synthetic open reading frame molecule comprising or of an inducible transactivator, or a combination thereof; a poly(A) sequence / tail / signal (e.g., bGH poly(A) signal or SV40 poly(A) signal, preferably bGH poly(A) signal) that is located 3’ of at least one of the adeno-associated virus capsid gene, the sequence encoding the adeno-associated virus Reg 52 and Reg 40 proteins, the synthetic open reading frame molecule comprising or of an inducible transactivator, or a combination thereof; the first assembly expression cassette further comprises, consists essentially of, or consists of (e.g., 5’ of the inducible transactivator and / or 3’ of the regulatory element or expression control element of the first assembly expression cassette), a nuclear localization signal (e.g., SV40 nuclear localization signal); or a combination thereof.

[0019] Another aspect of the present disclosure provides a plasmid (e.g., a replication plasmid or a replication donor plasmid) comprising or consisting essentially of: (a) a left or 5’ homology arm sequence; (b) a first replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivatorthat binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter)); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an adeno-associate virus Reg78 and Reg68 proteins; (c) a second replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter)); (2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: a deoxyribonucleic acid (DNA)-binding protein (DBP) gene (e.g., Transcription Factor E2 -Alpha (E2a) gene); a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A) or an internal ribosome entry site (IRES)), preferably P2A or T2A; and an adenovirus E4 region open reading frame 6 (E4orf6) gene; and (d) a right or 3’ homology arm sequence, wherein the left or 5’ homology arm sequence and the right or 3’ homology arm sequence facilitate homology directed repair (HDR) in a genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor).

[0020] In any aspect or embodiment described herein, the genomic safe harbor site comprises or is ROSA26, Adeno-Associated Virus Integration Site 1 (AAVS1), or C-C chemokine receptor type 5 (CCR5) locus, preferably Adeno-Associated Virus Integration Site 1 (AAVS1).

[0021] In any aspect or embodiment described herein, (a) the plasmid further comprises or consists essentially of, 5’ of the left or 5’ homology arm sequence and 3’ of the right or 3’ homology arm sequence at least one of: an original of replication (ori); an ampicillin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as an ampicillin resistance promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an ampicillin resistance gene (e.g., AmpR); a aminoglycoside resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression controlelement (e.g., a promoter, such as a ccdB promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of neomycin resistance, kanamycin resistance, or a combination thereof (e.g., NeoR / KanR); an expression cassette comprising, consisting essentially of, or consisting of a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a catabolite activator protein (CAP) binding site, a lac promoter, a lac operator, a T3 promoter, a Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding a fluorescence protein (e.g., green fluorescent protein or enhanced green fluorescence protein); or a combination thereof; (b) the plasmid further comprises or consists essentially of one or more (e.g., 1, 2, 3, or more) antibiotic resistance expression cassette located 3’ of the left or 5’ homology arm sequence and 5’ of the right or 3’ homology arm sequence, wherein the antibiotic resistance expression cassette is at least one of: a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR); a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e.g., BleoR); a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a T7 promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g.,PuroR); or a combination thereof, preferably a glycopeptide antibiotic or bleomycin resistance expression cassette; or (c) a combination thereof.

[0022] A further aspect of the present disclosure a plasmid (e.g., a transfer / transgene plasmid or a transfer / transgene donor plasmid) comprising or consisting essentially of, in order from 5’ to 3’ : (a) a left or 5’ homology arm sequence; (b) a first inverted terminal repeat (ITR,such as an adeno-associated virus ITR); (c) a transgene or transfer expression cassette comprising, consisting essentially of, or consisting of: (1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a transgene or a gene of interest; (d) a second inverted terminal repeat (ITR, such as an adeno- associated virus ITR); (e) a virus-associated ribonucleic acids (VA RNAs) expression cassette comprising, consisting essentially of, or consisting of, a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an Adenovirus VA- RNAs; and (f) a right or 3’ homology arm sequence, wherein the left or 5’ homology arm sequence and the right or 3’ homology arm sequence facilitate homology directed repair (HDR) in a genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor).

[0023] In any aspect or embodiment described herein, the genomic safe harbor site comprises or is ROSA26, Adeno-Associated Virus Integration Site 1 (AAVS1), or C-C chemokine receptor type 5 (CCR5) locus, preferably Adeno-Associated Virus Integration Site 1 (AAVS1).

[0024] In any aspect or embodiment described herein, the transgene or transfer expression cassette is flanked by recombinase sequences (e.g., atlP or atlQ, preferably atlP).

[0025] In any aspect or embodiment described herein, (a) the plasmid further comprises or consists essentially of, 5’ of the left or 5’ homology arm sequence and 3’ of the right or 3’ homology arm sequence, at least one of: an original of replication (ori); an ampicillin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as an ampicillin resistance promoter) and a synthetic open reading frame molecule comprising or of an ampicillin resistance gene (e.g., AmpR); a aminoglycoside resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a ccdB promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one neomycin resistance, kanamycin resistance, or a combination thereof (e.g., NeoR / KanR); or a combination thereof; (b) the plasmid further comprises or consists essentially of one or more (e.g., 1, 2, 3, or more) antibiotic resistanceexpression cassette located 3’ of the left or 5’ homology arm sequence and 5’ of the right or 3’ homology arm sequence, wherein the antibiotic resistance expression cassette is at least one of: a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR); a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e.g., BleoR); a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a T7 promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g.,PuroR); or a combination thereof, preferably a puromycin resistance expression cassette; or (c) a combination there.

[0026] A further aspect of the present disclosure provides a plasmid (e.g., a transfer / transgene plasmid, a transfer / transgene transposon plasmid, a transfer / transgene donor plasmid, or a transfer / transgene transposon donor plasmid) comprising or consisting essentially of, in order from 5’ to 3’ : (a) a left or 3’ inverted transposase terminal repeat sequence; (b) a transfer transposon comprising or consisting essentially of: (1) a first inverted terminal repeat (ITR, such as an adeno-associated virus ITR); (2) a transgene or transfer expression cassette comprising, consisting essentially of, or consisting of: a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a transgene or a gene of interest; (3) a second inverted terminal repeat (ITR, such as an adeno-associated virus ITR); (4) a virus-associated ribonucleic acids (VA RNAs) expression cassette comprising, consisting essentially of, or consisting of, a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an Adenovirus VA-RNAs; and (c) a right or5’ inverted transposase terminal repeat sequence, wherein the left or 5’ inverted transposase terminal repeat sequence and the right or 3’ inverted transposase terminal repeat sequence facilitate excision of the transfer transposon from the plasmid and insertion or integration of the transfer transposon in one or more (e.g., about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5) 4- base insertion site in a genome (e.g., a mammalian genome or a human genome).

[0027] In any aspect or embodiment described herein, at least one of: the transgene or transfer expression cassette is flanked by recombinase sequences (e.g., cil / P or MB, preferably attP , the 4-base insertion site is a TTAA site or a TTAT site, preferably a TTAA site; the left or 5’ inverted transposase terminal repeat sequence and the right or 3’ inverted transposase terminal repeat sequence are reverse complements; each of the left or 5’ inverted transposase terminal repeat sequence and the right or 3’ inverted transposase terminal repeat sequence is about 8 to about 1200 nucleotides (e.g., about 12 to about 200 nucleotides, about 28 to about 200 nucleotides, about 28 to about 600 nucleotides, or about 200 to 1200 nucleotides); or a combination thereof.

[0028] In any aspect or embodiment described herein, at least one of: (a) the plasmid further comprises or consists essentially of, 5’ of the right or 3’ inverted transposase terminal repeat sequence and 3’ of the left or 5’ inverted transposase terminal repeat sequence, at least one of: an original of replication (ori); an ampicillin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e g., a promoter, such as an ampicillin resistance promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an ampicillin resistance gene (e.g., AmpR); a aminoglycoside resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a ccdB promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of neomycin resistance, kanamycin resistance, or a combination thereof (e.g., NeoR / KanR); or a combination thereof; (b) the plasmid further comprises or consists essentially of one or more (e.g., 1, 2, 3, or more) antibiotic resistance expression cassette located 3’ of the right or 3’ inverted transposase terminal repeat sequence and 5’ of the left or 5’ inverted transposase terminal repeat sequence, wherein the antibiotic resistance expression cassette is at least one of: a blastocidin S resistance expressioncassette comprising, consisting essentially of, or consisting of a regulatory element or expression control element (e.g., a promoter, such as a SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR); a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e.g., BleoR); and a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a T7 promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g.,PuroR); or a combination thereof, preferably a puromycin resistance expression cassette; or (c) a combination there.

[0029] In any aspect or embodiment described herein, the transfer / transgene plasmid further comprises, consists essentially of, or consists of, at least one of: a kozak sequence (e.g., a classical Kozak sequence) that is located 3’ of the regulatory element or expression control element and 5’ of the synthetic open reading frame molecule of the antibiotic resistance expression cassette; a poly(A) sequence / tail / signal (e.g., bGH poly(A) signal or SV40 poly(A) signal, preferably SV40 poly(A) signal) that is located 3’ of the gene of the antibiotic resistance expression cassette; a kozak sequence (e.g., classical Kozak sequence) that is located 3’ of the regulatory element or expression control element and 5’ of the transgene or the gene of interest; a poly(A) sequence / tail / signal (e.g., bGH poly(A) signal or SV40 poly(A) signal, preferably bGH poly(A) signal) that is located 3’ of the transgene or the gene of interest; or a combination thereof.

[0030] In any aspect or embodiment described herein, the transgene is a fusion protein comprising a gene of interest fused to: a sequence encoding a woodchuck hepatitis virus regulatory element (WPRE); a sequence encoding an endosome-disruptive peptide (e.g., a protein or peptide that facilitates endosomal escape, such as a protective antigen (PA)); or a combination of both.

[0031] An additional aspect of the present disclosure a method of making a packaging cell, the method comprising: (a) transfecting a cell (e.g., human embryonic kidney 293 cell (HEK293) or HEK293T) with an assembly donor plasmid of the present disclosure and a clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) nuclease (e.g., Cas9) plasmid for the assembly donor plasmid that facilitates the insertion of the sequences between the 5’ or left homology arm sequence and the right or 3’ homology arm sequence into a first genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette; and (b) transfecting the cell transfected with the assembly donor plasmid with a replication donor plasmid of the present disclosure and a clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) nuclease (e.g., Cas9) plasmid for the replication donor plasmid that facilitates the insertion of the sequences between the 5’ or left homology arm sequence and the right or 3’ homology arm sequence into a second genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette, wherein: the assembly donor plasmid and the replication donor plasmid include different antibiotic resistance genes (e.g., genes that confer resistance to different antibiotics); and the first genomic safe harbor site and the second genomic safe harbor site are different genomic safe harbor sites.

[0032] Another aspect of the present disclosure a method of making a packaging cell, the method comprising: (a) transfecting a cell (e.g., human embryonic kidney 293 cell (HEK293) or HEK293T) with a replication donor plasmid of the present disclosure and a clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) nuclease (e.g., Cas9) plasmid for the replication donor plasmid that facilitates the insertion of the sequences between the 5’ or left homology arm sequence and the right or 3’ homology arm sequence into a second genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette; and (b) transfecting the cell transfected with the replication donor plasmid with an assembly donor plasmid of the present disclosure and a clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) nuclease (e.g., Cas9) plasmid for the assembly donor plasmid that facilitates the insertion of the sequences between the 5’ or left homology armsequence and the right or 3’ homology arm sequence into a first genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette, wherein: the assembly donor plasmid and the replication donor plasmid include different antibiotic resistance genes (e.g., genes that confer resistance to different antibiotics); and the first genomic safe harbor site and the second genomic safe harbor site are different genomic safe harbor sites.

[0033] In any aspect or embodiment described herein, (a) the first genomic safe harbor site is ROSA26, (b) the second genomic safe harbor site is Adeno-Associated Virus Integration Site 1 (AAVS1), or (c) a combination thereof.

[0034] In any aspect or embodiment described herein, the Cas nuclease (e.g., Cas9) plasmid for the assembly donor plasmid, the Cas nuclease (e.g., Cas9) plasmid for the replication donor plasmid, or a combination thereof, comprises or consists essentially of, in order from 5’ to 3’: (a) a Cas nuclease expression cassette comprising or consisting essentially of: (1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a Cytomegalovirus (CMV) promoter and / or a CMV enhancer, U6 promoter, or a combination thereof); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a Cas nuclease gene (e.g., Cas9) and (b) a single guide RNA (sgRNA) expression cassette comprising or consisting essentially of: (1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a U6 promoter, a Cytomegalovirus (CMV) promoter and / or a CMV enhancer, or a combination thereof); and (2) a synthetic open reading frame molecule comprising or of a single guide RNA (sgRNA) comprising, consisting essentially of, or consisting of a trans-activating CRISPR RNA (tracrRNA) for the Cas nuclease (e.g., Cas9) and a CRISPR RNA (crRNA) comprising, consisting essentially of, or consisting of, a nucleotide sequence (e.g., 17 to 20 nucleotides) that is complementary to a genomic safe harbor (e.g., a mammalian genomic safe harbor or a human genomic safe harbor).

[0035] In any aspect or embodiment described herein, the Cas nuclease gene comprises or consists essentially of a sequence that encodes, in order from 5’ to 3’, a Cas nuclease (e.g., Cas9), a 2A peptide (e.g., P2A, T2A, E2A, or F2A, preferably T2A or P2A), and a fluorescenceprotein (e.g., mCherry, green fluorescent protein, or enhanced green fluorescence protein, preferably mCherry).

[0036] Another aspect of the present disclosure provides a packaging cell produced according to the method of producing a packaging cell of the present disclosure or comprising or consisting essentially of: (a) a first assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an inducible transactivator gene (e.g., a reverse tetracycline-controlled transactivator gene ( / ' / 7 1 )); (b) a second assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to the inducible transactivator (e.g., the inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., TRE3G or CMV promoter)); and (2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: an adeno-associated virus capsid gene (e.g., a sequence encoding an adeno-associated virus VP1, VP2, and VP3 proteins); an internal ribosome entry site (IRES) or a sequence encoding a 2 A peptide (e.g., P2A, T2A, E2A, or F2A), preferably IRES; and a sequence encoding an adeno-associate virus Reg52 and Reg40 proteins; (c) a first replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Teton promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter)); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an adeno-associate virus Reg78 and Reg68 proteins; and (d) a second replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to an inducibletransactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Teton promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter)); (2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of a deoxyribonucleic acid (DNA)-binding protein (DBP) gene (e.g., Transcription Factor E2-Alpha (E2a) gene); a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A) or an internal ribosome entry site (IRES)), preferably P2A or T2A; and an adenovirus E4 region open reading frame 6 (E4orf6) gene, wherein: the first assembly expression cassette and the second assembly expression cassette are integrated or inserted in a first genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor); and the first replication expression cassette and the second replication expression cassette are integrated or inserted in a second genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor).

[0037] In any aspect or embodiment described herein, the packaging further comprises or consists essentially of, at least one of an assembly antibiotic resistance expression cassette located between the first assembly expression cassette and the second assembly expression cassette; a replication antibiotic resistance expression cassette located between the first replication expression cassette and the second replication expression cassette; or a combination thereof, optionally, the assembly antibiotic resistance expression cassette, the replication antibiotic resistance expression cassette, or a combination thereof, is at least one of: a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR), preferably the assembly antibiotic resistance expression cassette is a blastocidin S resistance expression cassette; a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof(e g., BleoR), preferably the assembly antibiotic resistance expression cassette is glycopeptide antibiotic or bleomycin resistance; a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a T7 promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g.,PuroR).

[0038] A further aspect of the present disclosure provides a method of making a recombinant Adeno-associated virus producer cell, the method comprising: (a) providing a packaging cell of the present disclosure, or produced according to the method of making a recombinant Adeno-associated virus producer cell of the present disclosure; (b) transfecting the packaging cell with a transfer donor plasmid of the present disclosure and a clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) nuclease (e.g., Cas9) plasmid for the transfer donor plasmid that facilitates the insertion of the sequences between the 5’ or left homology arm sequence and the right or 3’ homology arm sequence into a third genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette, wherein: the assembly donor plasmid, the replication donor plasmid, and the transfer donor plasmid include different antibiotic resistance genes (e.g., genes that confer resistance to different antibiotics); and the first genomic safe harbor site, the second genomic safe harbor site, and the third genomic safe harbor site are different genomic safe harbor sites.

[0039] In any aspect or embodiment described herein, the third genomic safe harbor site is C-C chemokine receptor type 5 (CCR5) locus.

[0040] In any aspect or embodiment described herein, the Cas nuclease (e.g., Cas9) plasmid for the transfer donor plasmid comprises or consists essentially of, in order from 5’ to 3’: (a) a Cas nuclease expression cassette comprising or consisting essentially of: (1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a Cytomegalovirus (CMV) promoter and / or a CMV enhancer, U6 promoter, or a combination thereof); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a Cas nuclease gene (e.g., Cas9) and (b) a single guide RNA (sgRNA) expression cassette comprising or consisting essentially of (1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as aU6 promoter, a Cytomegalovirus (CMV) promoter and / or a CMV enhancer, or a combination thereof); and (2) a synthetic open reading frame molecule comprising or of a single guide RNA (sgRNA) comprising, consisting essentially of, or consisting of a trans-activating CRISPR RNA (tracrRNA) for the Cas nuclease (e.g., Cas9) and a CRISPR RNA (crRNA) comprising, consisting essentially of, or consisting of a nucleotide sequence (e.g., 17 to 20 nucleotides) that is complementary to a genomic safe harbor (e.g., a mammalian genomic safe harbor or a human genomic safe harbor).

[0041] In any aspect or embodiment described herein, the Cas nuclease gene comprises or consists essentially of a sequence that encodes, in order from 5’ to 3’, a Cas nuclease (e.g., Cas9), a 2A peptide (e.g., P2A, T2A, E2A, or F2A, preferably T2A or P2A), and a fluorescence protein (e.g., mCherry, green fluorescent protein, or enhanced green fluorescence protein, preferably mCherry).

[0042] Another aspect of the present disclosure relates to a method of making a recombinant Adeno-associated virus producer cell, the method comprising: (a) providing a packaging cell of the present disclosure, or produced according to the method of the present disclosure; and (b) transfecting the packaging cell with a transfer transposon donor plasmid of the present disclosure and a transposase plasmid for the transfer transposon donor plasmid that facilitates the insertion or integration of the transfer transposon in one or more (e.g., about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5) 4-base insertion site in a genome (e.g., a mammalian genome or a human genome), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette, wherein: the transposase plasmid comprises or consists essentially of a transposase expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’, a regulatory element or expression control element (e.g., a promoter), and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a transposase sequence; the assembly donor plasmid, the replication donor plasmid, and the transfer transposon donor plasmid include different antibiotic resistance genes (e.g., genes that confer resistance to different antibiotics); and the first genomic safe harbor site and the second genomic safe harbor site are different genomic safe harbor sites. In any aspect or embodiment described herein, the method of making a recombinant Adeno-associated virus producer cell further comprises: transfecting the packaging cell with an assembly transposondonor plasmid of the present disclosure and a transposase plasmid for the assembly transposon plasmid that facilitates the insertion or integration of the assembly transposon in one or more (e.g., about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5) 4-base insertion site in a genome (e.g., a mammalian genome or a human genome), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette, wherein: the transposase plasmid comprises or consists essentially of a transposase expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’, a regulatory element or expression control element (e.g., a promoter), and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a transposase sequence; optionally, the assembly donor plasmid, the replication donor plasmid, the transfer transposon donor plasmid, and the assembly transposon donor plasmid include different antibiotic resistance genes (e g., genes that confer resistance to different antibiotics); and the first genomic safe harbor site and the second genomic safe harbor site are different genomic safe harbor sites.

[0043] A further aspect of the present disclosure relates to a method of making a recombinant Adeno-associated virus producer cell, the method comprising: (a) providing a packaging cell of the present disclosure, or produced according to the method of the present disclosure (optionally, the packaging cell of the present disclosure was not transfected with an assembly donor plasmid of the present disclosure and / or does not include the first assembly expression cassette and / or the second assembly expression cassette); (b) transfecting the packaging cell with a transfer transposon donor plasmid of the present disclosure and a transposase plasmid for the transfer transposon donor plasmid that facilitates the insertion or integration of the transfer transposon in one or more (e.g., about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5) 4-base insertion site in a genome (e.g., a mammalian genome or a human genome), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette; and (c) transfecting the packaging cell with an assembly transposon donor plasmid of the present disclosure and a transposase plasmid for the assembly transposon donor plasmid that facilitates the insertion or integration of the assembly transposon in one or more (e.g., about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5) 4-base insertion site in a genome (e.g., amammalian genome or a human genome), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette, wherein: the transposase plasmid comprises or consists essentially of a transposase expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’, a regulatory element or expression control element (e.g., a promoter), and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a transposase sequence; the transposase plasmid comprises or consists essentially of a transposase expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’, a regulatory element or expression control element (e.g., a promoter), and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a transposase sequence; optionally, the optional assembly donor plasmid, the replication donor plasmid, the transfer transposon donor plasmid, and the assembly transposon donor plasmid include different antibiotic resistance genes (e.g., genes that confer resistance to different antibiotics); and the first genomic safe harbor site and the second genomic safe harbor site are different genomic safe harbor sites.

[0044] In any aspect or embodiment described herein, at least one of: (a) the transposase plasmid further comprises, consists essentially of, or consists of, at least one of: an original of replication (ori); an ampicillin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as an ampicillin resistance promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an ampicillin resistance gene (e.g., AmpR); a aminoglycoside resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a ccdB promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of neomycin resistance, kanamycin resistance, or a combination thereof (e.g., NeoR / KanR); an expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a catabolite activator protein (CAP) binding site, a lac promoter, a lac operator, a T3 promoter, a Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding a fluorescence protein (e.g., green fluorescent protein or enhanced green fluorescence protein); or a combinationthereof; (b) the transposase plasmid further comprises, consists essentially of, or consists of, one or more (e.g., 1, 2, 3, or more) antibiotic resistance expression cassette, wherein the antibiotic resistance expression cassette comprise, consists essentially of, or consists of, at least one of: a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e g., a promoter, such as a SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR); a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e g., BleoR); a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g., PuroR); or a combination thereof; or a combination thereof, preferably a glycopeptide or bleomycin resistance expression cassette (e.g., zeocin resistance); or (c) a combination thereof.

[0045] In any aspect or embodiment described herein, the transposase plasmid further comprises, consists essentially of, or consists of, at least one of: a kozak sequence (e.g., a classical Kozak sequence) that is located 3’ of the regulatory element or expression control element and 5’ of the synthetic open reading frame molecule of the antibiotic resistance expression cassette; a poly(A) sequence / tail / signal (e.g., bGH poly(A) signal or SV40 poly(A) signal, preferably SV40 poly(A) signal) that is located 3’ of the gene of the antibiotic resistance expression cassette; a kozak sequence (e.g., classical Kozak sequence) that is located 3’ of the regulatory element or expression control element and 5’ of the transposase sequence; a poly(A) sequence / tail / signal (e.g., bGH poly(A) signal or SV40 poly(A) signal, preferably bGH poly(A) signal) that is located 3’ of the transposase sequence; or a combination thereof.

[0046] In any aspect or embodiment described herein, performing antibiotic selection comprises administering an antibiotic for at least 2 weeks (e.g., 2 to 3 weeks or 2 weeks).

[0047] In any aspect or embodiment described herein, the antibiotic comprises or is blastocidin S, bleomycin, phleomycin, Zeocin, or puromycin.

[0048] In any aspect or embodiment described herein, the method of making a recombinant Adeno-associated virus producer cell further comprises, consists essentially of, or consists of: (a) after transfection and antibiotic selection, isolating a single clone comprising the inserted sequence; (b) after transfection and antibiotic selection, confirming site-specific integration of the inserted sequence (e.g., polymerase chain reaction (such as 573’ junction polymerase chain reaction and / or out-to-out polymerase chain reaction), copy number analysis (e.g., quantitative polymerase chain reaction), or a combination thereof); (c) after transfection and antibiotic selection, contacting the cell with an agent (e.g., tetracycline, doxycycline, or derivative thereof) the induces the inducible transactivator (e.g., a reverse tetracycline-controlled transactivator (rtTA)); (d) after transfection and antibiotic selection (e.g., after contacting the cell with the agent that induces the inducible transactivator), evaluating expression (e.g., reverse transcriptase polymerase chain reaction, reverse transcriptase quantitative polymerase chain reaction, western blot, or a combination thereof) of the inserted genes; or (e) a combination thereof.

[0049] In any aspect or embodiment described herein, transfecting comprises or consists essentially of: (a) inducing the cell to be in S / G2 phase, (b) contacting the cell with an inhibitor (e.g., a small molecule inhibitor) of the non-homologous end joining (NHEJ) pathway, (c) enriching the donor plasmid near the Cas9-induced double stranded break (DSB) in the DNA, (d) co-expressing one or more DNA repair protein involved in homology directed repair (HDR), (e) liposomal transfecting (e.g., transfecting with a cationic liposome based reagent), or (f) a combination thereof.

[0050] In any aspect or embodiment described herein, the method of making a recombinant Adeno-associated virus producer cell further comprises, consists essentially of, or consists of producing recombinant adeno-associated virus.

[0051] In any aspect or embodiment described herein, producing recombinant adeno- associate virus comprises contacting the recombinant Adeno-associated virus producer cell with an agent (e.g., tetracycline, doxycycline, or derivative thereof) the induces the inducible transactivator (e.g., a reverse tetracycline-controlled transactivator (rtTA)).

[0052] An additional aspect of the present disclosure provides a recombinant Adeno- associated virus producer cell produced according to the method of the present disclosure or comprising or consisting essentially of: (a) a first assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an inducible transactivator gene (e.g., a reverse tetracycline-controlled transactivator gene (r / 4)); (b) a second assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to the inducible transactivator (e.g., the inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., TRE3G or CMV promoter)); and (2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: an adeno-associated virus capsid gene (e.g., a sequence encoding an adeno-associated virus VP1, VP2, and VP3 proteins); an internal ribosome entry site (IRES) or a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A), preferably IRES; and a sequence encoding an adeno-associate virus Reg52 and Reg40 proteins; (c) a first replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Teton promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter)); and (2) a synthetic open reading frame molecule comprising a sequence encoding an adeno-associate virus Reg78 and Reg68 proteins; (d) a second replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’: (1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter)); (2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of or consisting of: a deoxyribonucleic acid (DNA)-binding protein (DBP) gene (e.g., Transcription Factor E2-Alpha (E2a) gene); a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A) or an internal ribosome entry site (IRES)), preferably P2A or T2A; and an adenovirus E4 region open reading frame 6 (E4orf6) gene; (e) a first inverted terminal repeat (ITR, such as an adeno-associated virus ITR); (f) a transgene or transfer expression cassette comprising, consisting essentially of, or consisting of: (1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a transgene or a gene of interest; (g) a second inverted terminal repeat (ITR, such as an adeno- associated virus ITR); and (h) a virus-associated ribonucleic acids (VA RNAs) expression cassette comprising, consisting essentially of, or consisting of, a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an Adenovirus VA-RNAs, wherein: the first assembly expression cassette and the second assembly expression cassette are integrated or inserted in a first genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor) of the recombinant Adeno- associated virus producer cell; the first replication expression cassette and the second replication expression cassette are integrated or inserted in a second genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor) of the recombinant Adeno- associated virus producer cell; and the first inverted terminal repeat, the transgene / transfer expression cassette, the second inverted terminal repeat, and the expression cassette for the Adenovirus virus-associated ribonucleic acids (VA RNAs) are integrated or inserted in a third genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor) of the recombinant Adeno-associated virus producer cell.

[0053] A further aspect of the present disclosure provides a recombinant Adeno- associated virus producer cell produced according to the method of the present disclosure or comprising or consisting essentially of: (a) a first assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such asCytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an inducible transactivator gene (e.g., a reverse tetracycline-controlled transactivator gene (r / 714)); (b) a second assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to the inducible transactivator (e.g., the inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., TRE3G or CMV promoter)); and (2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: an adeno-associated virus capsid gene (e.g., a sequence encoding an adeno-associated virus VP1, VP2, and VP3 proteins); an internal ribosome entry site (IRES) or a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A), preferably IRES; and a sequence encoding an adeno-associate virus Reg52 and Reg40 proteins; (c) a first replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Teton promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e g., CMV promoter)); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an adeno-associate virus Reg78 and Reg68 proteins; (d) a second replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Teton promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter)); and (2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: a deoxyribonucleic acid (DNA)-binding protein (DBP) gene (e.g., Transcription Factor E2-Alpha (E2a) gene); a sequence encoding a 2 A peptide (e.g., P2A, T2A, E2A, or F2A) or an internalribosome entry site (IRES)), preferably P2A or T2A; and an adenovirus E4 region open reading frame 6 (E4orf6) gene; (e) an assembly transposon comprising or consisting essentially of, in order from 5’ to 3’: (1) a first assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an inducible transactivator (e.g., a reverse tetracycline-controlled transactivator (rtTA)); (2) a second assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : a regulatory element or expression control element that is responsive to the inducible transactivator (e.g., the inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., TRE3G or CMV promoter)); and a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: an adeno- associated virus capsid gene (e.g., a sequence encoding an adeno-associated virus VP1, VP2, and VP3 proteins); an internal ribosome entry site (IRES) or a sequence encoding a 2 A peptide (e.g., P2A, T2A, E2A, or F2A), preferably IRES; and a sequence encoding an adeno-associate virus Reg52 and Reg40 proteins; (f) a transfer transposon comprising or consisting essentially of, in order from 5’ to 3’: (1) a first inverted terminal repeat (ITR, such as an adeno-associated virus ITR); (2) a transgene or transfer expression cassette comprising, consisting essentially of, or consisting of: a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a transgene or a gene of interest; (3) a second inverted terminal repeat (ITR, such as an adeno-associated virus ITR); (4) a virus-associated ribonucleic acids (VA RNAs) expression cassette comprising, consisting essentially of, or consisting of, a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an Adenovirus VA-RNAs, wherein: the first assembly expression cassette and the second assembly expression cassette are integrated or inserted in a first genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor) of the recombinantAdeno-associated virus producer cell; the first replication expression cassette and the second replication expression cassette are integrated or inserted in a second genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor) of the recombinant Adeno-associated virus producer cell; the assembly transposon is integrated or inserted into one or more (e.g., about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5) 4-base insertion site in a genome (e.g., a mammalian genome or a human genome) of the recombinant Adeno-associated virus producer cell; and the transfer transposon is integrated or inserted into one or more (e.g., about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5) 4-base insertion site in a genome (e.g., a mammalian genome or a human genome) of the recombinant Adeno-associated virus producer cell.

[0054] In any aspect or embodiment described herein, the packaging cell further comprises or consists essentially of: an assembly antibiotic resistance expression cassette located between the first assembly expression cassette and the second assembly expression cassette; a replication antibiotic resistance expression cassette located between the first replication expression cassette and the second replication expression cassette; a transfer / transgene antibiotic resistance expression cassette located between the transgene / transfer expression cassette and the expression cassette for an Adenovirus virus-associated ribonucleic acids; or a combination thereof, optionally, the assembly antibiotic resistance expression cassette, the replication antibiotic resistance expression cassette, the transfer / transgene antibiotic resistance expression cassette, or a combination thereof, is at least one of: a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR), preferably the assembly antibiotic resistance expression cassette is a blastocidin S resistance expression cassette; a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e.g.,BleoR), preferably the assembly antibiotic resistance expression cassette is glycopeptide antibiotic or bleomycin resistance; a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a T7 promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g.,PuroR), preferably the transfer / transgene antibiotic resistance expression cassette a puromycin resistance expression cassette; or a combination thereof.

[0055] A further aspect of the present disclosure provides a method of making a recombinant Adeno-associated virus, the method comprising: (a) providing a recombinant Adeno-associated virus producer cell of the present disclosure; and (b) producing recombinant Adeno-associated virus.

[0056] In any aspect or embodiment described herein, producing recombinant adeno- associate virus comprises contacting the recombinant Adeno-associated virus producer cell with an agent (e.g., tetracycline, doxycycline, or derivative thereof) the induces the inducible transactivator (e.g., a reverse tetracycline-controlled transactivator (rtTA)).BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The accompanying drawings, which are incorporated into and form a part of the specification, illustrate several embodiments of the present disclosure and, together with the description, serve to explain the principles of the disclosure. The drawings are only for the purpose of illustrating embodiments of the disclosure and are not to be construed as limiting the disclosure. Further objects, features and advantages of the disclosure will become apparent from the following detailed description taken in conjunction with the accompanying figures showing illustrative embodiments of the disclosure.

[0058] Figures 1A and IB. (1A) Workflow and (IB) vector design. Three viral cassettes, including assembly, replication, and transfer cassettes, were constructed and integrated into three different genomic safety harbor sites sequentially via site-specific integration methods. Several assessment criteria, such as junction polymerase chain reaction (PCR), genome copy number, and rAAV productivity, were employed to evaluate the established stable cell pools, andsingle clones for the 2ndand 3rdrounds were fully characterized as packaging cell lines and producer cell lines. More details can be found in the result sections.

[0059] Figures 2A, 2B, and 2C. The Donor plasmid maps. (2A) Assembly donor plasmid. (2B) Replication donor plasmid. (2C) Transfer donor plasmid.

[0060] Figures 3A, 3B, and 3C. (3 A) Junction PCR results for the stable cell pool after three rounds of integration. The sample names and expected sizes are shown in the right table. The green boxes in the gel image show the corresponding bands in the expected size. AAV production evaluation for each round of stable pool with 5 ug / mL doxycycline induction (+). Donor control (DC) represents the transient transfection process with three re-constructed donor plasmids. Data represent the mean and standard deviation of biological duplicates (n=2). (3B) Specific genome titer in vg / cell harvest at 68 hours after transfection (HPT). (3C) Specific capsid titer in cp / cell at 68 HPT.

[0061] Figures 4A, 4B, and 4C. Gel electrophoresis images for 573’ junction PCR results in each round of single clones. Numerals represent the number of stable clones, and the junction PCR-positive clones are shown in red. M, 1-kb DNA ladder. The green boxes indicate the expected size of the amplicons. The knock-in efficiencies for integration were calculated by dividing the number of both junction PCR positive single clones by the number of total single clones, which were 5.5 % (3 out of 55) for ROSA26, 5.2 % (2 out of 38) for AAVS1, and 17.7 % (11 out of 62) for CCR5 locus.

[0062] Figure 5. Transcript level of viral genes fold change with 0, 0.5, and 5 pg / mL dox induction at 68 HPT for packaging single clone (SC4-7), normalized to housekeeping gene GAPDH. Key viral components include Cap, Rep52, Rep68, DBP, and E4orf6. Presented numbers for dox represent the actual concentration of inducers used in pg / mL. Data represent the mean and standard deviation of triplicate wells (n=3).

[0063] Figures 6A and 6B. Genome titer (6A) and capsid titer (6B) evaluation for packaging single clone SC4-7 with 0 and 5 pg / mL dox induction. Positive control (PC) represents traditional transfection with commercial triple plasmids. Donor control (DC) represents transfection with refactored donor plasmids with 5 pg / mL dox induction. LOD represents the lower limit of detection. Error bars represent the standard deviation of biological triplicates (n=3). Note: One data point for donor control and SC4-7 5 pg / mL dox was eliminated as an outlier for capsid titer measurement.

[0064] Figure 7. Out-to-out PCR for further confirmation of site-specific integration in the AAVS1 locus.

[0065] Figures 8A and 8B. The transposon plasmid maps. (8A) Transfer transposon plasmid. (8B) Assembly transposon plasmid.

[0066] Figures 9A, 9B, 9C, and 9D. (9A and 9B) Relative genome copy analysis for Assembly (Cap gene) and Transfer (GFP) cassettes. (9C) Genome titer in vg / L harvest at 68 hours after transfection (HPT). (9D) Capsid titer in cp / L at 68 HPT.

[0067] Figure 10. Single clone screening via relative genome copy assays.

[0068] Figure 11. Transcript level of viral genes fold change with 0 and 5 pg / mL dox induction at 68 HPT for producer single clone (SC4-9), normalized to housekeeping gene GAPDH. Key viral components include Cap, Rep52, Rep68, DBP, and E4orf6. Presented numbers for dox represent the actual concentration of inducers used in pg / mL. Data represent the mean and standard deviation of triplicate wells (n=3).

[0069] Figures 12A and 12B. Genome titer (12A) and capsid titer (12B) evaluation for producer single clone SC4-9 with 0 and 5 pg / mL dox induction. Error bars represent the standard deviation of biological triplicates (n=3).

[0070] Figures 13A and 13B. Cell growth performance (13A) and cell line stability (13B) evaluation for stable producer single clone SC4-9. In Figure 13A, the botom two curves represent viable cell density, while the top two curves depict cell viability. Error bars represent the standard deviation of biological triplicates (n=3).DETAILED DESCRIPTION

[0071] Presently described are recombinant Adeno-associated packaging cells and recombinant Adeno-associated virus producer cells, as well as plasmids and methods of making and using the same. It was surprising and expectedly discovered that stable recombinant Adeno- associated packaging cells and recombinant Adeno-associated virus producer cells could be produced by integrating / inserting the assembly genes, replication genes, and helper genes, and optionally a transgene, into genomic safe harbor sites.DEFINITIONS

[0072] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosurebelongs. The terminology used in the description is for describing particular embodiments only and is not intended to be limiting of the disclosure.

[0073] Where a range of values is provided, it is understood that each intervening value in the range, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise (such as in the case of a group containing a number of carbon atoms in which case each carbon atom number falling within the range is provided), between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either / or both of those included limits are also included in the disclosure.

[0074] The following terms are used to describe the present disclosure. In instances where a term is not specifically defined herein, that term is given an art-recognized meaning by those of ordinary skill applying that term in context to its use in describing the present disclosure.

[0075] The articles “a”, “an”, and “the” as used herein and in the appended claims are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article unless the context clearly indicates otherwise. By way of example, “an element” means one element or more than one element, unless otherwise indicated.

[0076] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended (that is, to mean including but not limited to). It is expressly contemplated that all embodiments, and claims reciting one of the open-ended transitional phrases can be written with any other transitional phrase, which may be more limiting (e.g., “consisting essentially of’ or “consisting of’), unless clearly precluded by the context or art. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0077] The term “about” as it is used herein, in association with numeric values or ranges, reflects the fact that there is a certain level of variation that is recognized and tolerated in the art due to practical and / or theoretical limitations. For example, minor variation is tolerated due to inherent variances in the manner in which certain devices operate and / or measurements are taken.In accordance with the above, the phrase “about” is normally used to encompass values within the standard deviation or standard error.

[0078] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0079] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

[0080] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a nonlimiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment,to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0081] The term “optional” or “optionally”, as used herein, means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0082] It should also be understood that, in certain methods or processes described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.

[0083] As used herein, the term “liposome” refers to a vesicular structure having lipid- containing membranes enclosing an aqueous interior. In cell biology, a vesicular structure is a hollow, lamellar, spherical structure, and provides a small and enclosed compartment, separated from the cytosol by at least one lipid bilayer. Liposomes can have one or more lipid membranes. Oligolamellar large vesicles and multilamellar vesicles have multiple, usually concentric, membrane layers and are typically larger than 100 nm. Liposomes with several nonconcentric membranes, i.e., several smaller vesicles contained within a larger vesicle, are termed multivesicular vesicles.

[0084] Liposomes can further comprise one or more additional lipids and / or other components such as sterols, e.g., cholesterol. Additional lipids can be included in the liposome compositions for a variety of purposes, such as to prevent lipid oxidation, to stabilize the bilayer, to reduce aggregation during formation or to attach ligands onto the liposome surface. Any of a number of additional lipids and / or other components can be present, including amphipathic, neutral, cationic, anionic lipids, and programmable fusion lipids. Such lipids and / or components can be used alone or in combination. One or more components of the liposome can comprise a ligand, e.g., a targeting ligand. Liposome compositions can be prepared by a variety of methods that are known in the art. Niosomes are non-phospholipid based synthetic vesicles that have properties and function like liposomes.

[0085] As used herein, the term “gene expression” includes both gene transcription, whereby DNA (or RNA in the case of some RNA-containing viruses) corresponding to a gene is transcribed to generate an RNA molecule and RNA translation, whereby an RNA molecule is translated to generate a protein encoded by the gene. As used herein, the term "protein expression" is used to refer both to gene expression comprising transcription of DNA (or RNA) to form an RNA molecule and subsequent processing and translation of the RNA molecule to form protein and to gene expression comprising translation of mRNA to form protein.

[0086] The term “encode” refers to any process whereby the information in one molecule is used to direct the production of a second molecule that has a different chemical nature from the first molecule. For example, a DNA molecule can encode an RNA molecule (e.g., by the process of transcription incorporating a DNA-dependent RNA polymerase enzyme). Also, an RNA molecule can encode a polypeptide, as in the process of translation. When used to describe the process of translation, the term "encode" also extends to the triplet codon that encodes an amino acid. In some aspects, an RNA molecule can encode a DNA molecule, e.g., by the process of reverse transcription incorporating an RNA-dependent DNA polymerase. In another aspect, a DNA molecule can encode a polypeptide, where it is understood that "encode" as used in that case incorporates both the processes of transcription and translation.

[0087] The term “contacting” as used herein, refers to bringing a disclosed agent and a cell, a target receptor, or other biological entity together in such a manner that the agent can affect the activity of the target (e.g., neuronal cell, axon etc.), either directly; i.e., by interacting with the target itself, or indirectly; i.e., by interacting with another molecule, co-factor, factor, or protein on which the activity of the target is dependent.

[0088] As used herein, the terms “protein”, “peptide”, and “polypeptide” are used interchangeably to designate a series of amino acid residues connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms “protein”, “peptide”, and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein”, “peptide”, and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof.

[0089] The term “small molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

[0090] “Nucleic acid sequence”, as used herein, refers to a polymer of nucleotides in which the 3' position of one nucleotide sugar is linked to the 5' position of the next by a phosphodiester bridge. In a linear nucleic acid strand, one end typically has a free 5' phosphate group, the other a free 3' hydroxyl group. Nucleic acid sequences may be used herein to refer to oligonucleotides, or polynucleotides, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin that may be single- or double-stranded and represent the sense or antisense strand. The polymer can be composed of deoxyribonucleotides, ribonucleotides, or modified nucleotides in a single- or double-stranded form and is intended to encompass nucleic acids bearing nucleotide analogs or modified backbone residues or linkages known in the art. For example, the term “nucleic acid” or “polynucleotide” includes single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine or pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide base.

[0091] Definitions of common terms in cell biology and molecular biology can be found in “The Merck Manual of Diagnosis and Therapy”, 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0- 632-02182-9); Immunology by Werner Luttmann, published by Elsevier, 2006. Definitions of common terms in molecular biology can also be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN- 10: 0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

[0092] Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Sambrook et al., Molecular Cloning: A Laboratory Manual (3 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2001);Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995); Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998) which are all incorporated by reference herein in their entireties.PLASMIDS

[0093] An aspect of the present disclosure provides a plasmid (e.g., an assembly plasmid or an assembly donor plasmid) comprising or consisting essentially of: (a) a left or 5’ homology arm sequence; (b) a first assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an inducible transactivator (e.g., a reverse tetracycline-controlled transactivator (rtTA)); (c) a second assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to the inducible transactivator (e.g., the inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., TRE3G or CMV promoter)); and (2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of an adeno-associated virus capsid gene (e.g., a sequence encoding an adeno-associated virus VP1, VP2, and VP3 proteins); an internal ribosome entry site (IRES) or a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A), preferably IRES; and a sequence encoding an adeno- associate virus Reg52 and Reg40 proteins; and (d) a right or 3’ homology arm sequence, wherein the left or 5’ homology arm sequence and the right or 3’ homology arm sequence facilitate homology directed repair (HDR) in a genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor).

[0094] In any aspect or embodiment described herein, the genomic safe harbor site comprises or is ROSA26, Adeno-Associated Virus Integration Site 1 (AAVS1), or C-C chemokine receptor type 5 (CCR5) locus, preferably ROSA26.

[0095] In any aspect or embodiment described herein, the second assembly expression cassette is optionally flanked by recombinase sequences (e.g., attB or attP, preferably attB).

[0096] In any aspect or embodiment described herein, at least one of (a) the plasmid further comprises or consists essentially of, 5’ of the left or 5’ homology arm sequence and 3’ of the right or 3’ homology arm sequence at least one of: an original of replication (ori); an ampicillin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as an ampicillin resistance promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an ampicillin resistance gene (e.g., AmpR); a aminoglycoside resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a ccdB promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of neomycin resistance, kanamycin resistance, or a combination thereof (e.g., NeoR / KanR); an expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a catabolite activator protein (CAP) binding site, a lac promoter, a lac operator, a T3 promoter, a Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding a fluorescence protein (e.g., green fluorescent protein or enhanced green fluorescence protein); or a combination thereof; (b) the plasmid further comprises or consists essentially of, one or more (e.g., 1, 2, 3, or more) antibiotic resistance expression cassette located 3’ of the left or 5’ homology arm sequence and 5’ of the right or 3’ homology arm sequence, wherein the antibiotic resistance expression cassette is at least one of: a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR); a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, aregulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e g., BleoR); a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a T7 promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g., PuroR); or a combination thereof, preferably a blastocidin S resistance expression cassette; or (c) a combination thereof.

[0097] A further aspect of the present disclosure provides plasmid (e.g., an assembly plasmid, an assembly transposon plasmid, an assembly donor plasmid, or an assembly transposon donor plasmid) comprising or consisting essentially of, in order from 5’ to 3’: (a) a right or 3’ inverted transposase terminal repeat sequence; (b) an assembly transposon comprising or consisting essentially of: (1) a first assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an inducible transactivator (e.g., a reverse tetracycline-controlled transactivator (rtTA)); (2) a second assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’: a regulatory element or expression control element that is responsive to the inducible transactivator (e.g., the inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., TRE3G or CMV promoter)); and a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: an adeno- associated virus capsid gene (e.g., a sequence encoding an adeno-associated virus VP1, VP2, and VP3 proteins); an internal ribosome entry site (IRES) or a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A), preferably IRES; and a sequence encoding an adeno-associate virus Reg52 and Reg40 proteins; and (c) a left or 5’ inverted transposase terminal repeat sequence,wherein the left or 5’ inverted transposase terminal repeat sequence and the right or 3’ inverted transposase terminal repeat sequence facilitate excision of the transposon from the plasmid and insertion or integration of the assembly transposon in one or more (e.g., about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5) 4-base insertion site in a genome (e g., a mammalian genome or a human genome).

[0098] In any aspect or embodiment described herein, at least one of: the second assembly expression cassette is optionally flanked by recombinase sequences (e.g., attB or attP, preferably c / / / B); the 4-base insertion site is a TTAA site or a TTAT site, preferably a TTAA site; the left or 5’ inverted transposase terminal repeat sequence and the right or 3’ inverted transposase terminal repeat sequence are reverse complements; each of the left or 5’ inverted transposase terminal repeat sequence and the right or 3’ inverted transposase terminal repeat sequence is about 8 to about 1200 nucleotides (e.g., about 12 to about 200 nucleotides, about 28 to about 200 nucleotides, about 28 to about 600 nucleotides, or about 200 to 1200 nucleotides); or a combination thereof.

[0099] In any aspect or embodiment described herein, at least one of: (a) the plasmid further comprises or consists essentially of, 5’ of the right or 3’ inverted transposase terminal repeat sequence and 3’ of the left or 5’ inverted transposase terminal repeat sequence, at least one of: an original of replication (ori); an ampicillin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as an ampicillin resistance promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an ampicillin resistance gene (e.g., AmpR); a aminoglycoside resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a ccdB promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of neomycin resistance, kanamycin resistance, or a combination thereof (e.g., NeoR / KanR); an expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a catabolite activator protein (CAP) binding site, a lac promoter, a lac operator, a T3 promoter, a Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof) and a synthetic open reading framemolecule comprising, consisting essentially of, or consisting of, a sequence encoding a fluorescence protein (e.g., green fluorescent protein or enhanced green fluorescence protein); or a combination thereof; (b) the plasmid further comprises or consists essentially of one or more (e.g., 1, 2, 3, or more) antibiotic resistance expression cassette located 3’ of the right or 3’ inverted transposase terminal repeat sequence and 5’ of the left or 5’ inverted transposase terminal repeat sequence, wherein the antibiotic resistance expression cassette is at least one of: a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of ,a regulatory element or expression control element (e.g., a promoter, such as a SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR); a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e.g., BleoR); a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a T7 promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g., PuroR); or a combination thereof, preferably a blastocidin S resistance expression cassette; or (c) a combination thereof.

[0100] In any aspect or embodiment described herein, the plasmid further comprises, consists essentially of, or consists of, at least one of: a kozak sequence (e.g., a classical Kozak sequence) that is located 3’ of the regulatory element or expression control element and 5’ of the synthetic open reading frame molecule of the antibiotic resistance expression cassette; a poly(A) sequence / tail / signal (e.g., bGH poly(A) signal or SV40 poly(A) signal, preferably SV40 poly(A) signal) that is located 3’ of the gene of the antibiotic resistance expression cassette; a kozak sequence (e.g., classical Kozak sequence) that is located 3’ of the regulatory element or expression control element and 5’ of at least one of the adeno-associated virus capsid gene, the sequence encoding the adeno-associated virus Reg 52 and Reg 40 proteins, the synthetic open reading frame molecule comprising or of an inducible transactivator, or a combination thereof; apoly(A) sequence / tail / signal (e.g., bGH poly(A) signal or SV40 poly(A) signal, preferably bGH poly(A) signal) that is located 3’ of at least one of the adeno-associated virus capsid gene, the sequence encoding the adeno-associated virus Reg 52 and Reg 40 proteins, the synthetic open reading frame molecule comprising or of an inducible transactivator, or a combination thereof; the first assembly expression cassette further comprises, consists essentially of, or consists of (e.g., 5’ of the inducible transactivator and / or 3’ of the regulatory element or expression control element of the first assembly expression cassette), a nuclear localization signal (e.g., SV40 nuclear localization signal); or a combination thereof.

[0101] Another aspect of the present disclosure provides a plasmid (e.g., a replication plasmid or a replication donor plasmid) comprising or consisting essentially of: (a) a left or 5’ homology arm sequence; (b) a first replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter)); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an adeno-associate virus Reg78 and Reg68 proteins; (c) a second replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter)); (2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: a deoxyribonucleic acid (DNA)-binding protein (DBP) gene (e.g., Transcription Factor E2 -Alpha (E2a) gene); a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A) or an internal ribosome entry site (IRES)), preferably P2A or T2A; and an adenovirus E4 region open reading frame 6 (E4orf6) gene; and (d) a right or 3’ homology arm sequence, wherein the left or 5’ homology arm sequence and the right or 3’ homology armsequence facilitate homology directed repair (HDR) in a genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor).

[0102] In any aspect or embodiment described herein, the genomic safe harbor site comprises or is ROSA26, Adeno-Associated Virus Integration Site 1 (AAVS1), or C-C chemokine receptor type 5 (CCR5) locus, preferably Adeno-Associated Virus Integration Site 1 (AAVS1).

[0103] In any aspect or embodiment described herein, (a) the plasmid further comprises or consists essentially of, 5’ of the left or 5’ homology arm sequence and 3’ of the right or 3’ homology arm sequence at least one of an original of replication (ori); an ampicillin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as an ampicillin resistance promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an ampicillin resistance gene (e.g., AmpR); a aminoglycoside resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a ccdB promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of neomycin resistance, kanamycin resistance, or a combination thereof (e.g., NeoR / KanR); an expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a catabolite activator protein (CAP) binding site, a lac promoter, a lac operator, a T3 promoter, a Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding a fluorescence protein (e.g., green fluorescent protein or enhanced green fluorescence protein); or a combination thereof; (b) the plasmid further comprises or consists essentially of, one or more (e.g., 1, 2, 3, or more) antibiotic resistance expression cassette located 3’ of the left or 5’ homology arm sequence and 5’ of the right or 3’ homology arm sequence, wherein the antibiotic resistance expression cassette is at least one of: a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR); a glycopeptide antibiotic or bleomycin resistance expressioncassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e.g., BleoR); a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a T7 promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g.,PuroR); or a combination thereof, preferably a glycopeptide antibiotic or bleomycin resistance expression cassette; or (c) a combination thereof.

[0104] A further aspect of the present disclosure a plasmid (e.g., a transfer / transgene plasmid or a transfer / transgene donor plasmid) comprising or consisting essentially of, in order from 5’ to 3’: (a) a left or 5’ homology arm sequence; (b) a first inverted terminal repeat (ITR, such as an adeno-associated virus ITR); (c) a transgene or transfer expression cassette comprising, consisting essentially of, or consisting of: (1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a transgene or a gene of interest; (d) a second inverted terminal repeat (ITR, such as an adeno-associated virus ITR);(e) a virus- associated ribonucleic acids (VA RNAs) expression cassette comprising, consisting essentially of, or consisting of, a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an Adenovirus VA-RNAs; and (f) a right or 3’ homology arm sequence, wherein the left or 5’ homology arm sequence and the right or 3’ homology arm sequence facilitate homology directed repair (HDR) in a genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor).

[0105] In any aspect or embodiment described herein, the genomic safe harbor site comprises or is ROSA26, Adeno-Associated Virus Integration Site 1 (AAVS1), or C-C chemokine receptor type 5 (CCR5) locus, preferably Adeno-Associated Virus Integration Site 1 (AAVS1).

[0106] In any aspect or embodiment described herein, the transgene or transfer expression cassette is flanked by recombinase sequences (e.g., attP or atfQ, preferably attP').

[0107] In any aspect or embodiment described herein, the transgene is a fusion protein comprising a gene of interest fused to: a sequence encoding a woodchuck hepatitis virus regulatory element (WPRE), a sequence encoding an endosome-disruptive peptide (e.g., a protein or peptide that facilitates endosomal escape, such as a protective antigen (PA)); or a combination of both.

[0108] In any aspect or embodiment described herein, (a) the plasmid further comprises or consists essentially of, 5’ of the left or 5’ homology arm sequence and 3’ of the right or 3’ homology arm sequence at least one of: an original of replication (ori); an ampicillin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e g., a promoter, such as an ampicillin resistance promoter) and a synthetic open reading frame molecule comprising or of an ampicillin resistance gene (e.g., AmpR); a aminoglycoside resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a ccdB promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of neomycin resistance, kanamycin resistance, or a combination thereof (e.g., NeoR / KanR); or a combination thereof; (b) the plasmid further comprises or consists essentially of one or more (1, 2, 3 or more) antibiotic resistance expression cassette located 3’ of the left or 5’ homology arm sequence and 5’ of the right or 3’ homology arm sequence, wherein the antibiotic resistance expression cassette is at least one of: a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of a regulatory element or expression control element (e.g., a promoter, such as a SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR); a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e.g., BleoR); a puromycin resistance expression cassette comprising, consisting essentially of, orconsisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a T7 promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g.,PuroR); or a combination thereof, preferably a puromycin resistance expression cassette; or (c) a combination there.

[0109] A further aspect of the present disclosure provides a plasmid (e.g., a transfer / transgene plasmid, a transfer / transgene transposon plasmid, a transfer / transgene donor plasmid, or a transfer / transgene transposon donor plasmid) comprising or consisting essentially of, in order from 5’ to 3’ : (a) a left or 3’ inverted transposase terminal repeat sequence; (b) a transfer transposon comprising or consisting essentially of: (1) a first inverted terminal repeat (ITR, such as an adeno-associated virus ITR); (2) a transgene or transfer expression cassette comprising, consisting essentially of, or consisting of: a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a transgene or a gene of interest; (3) a second inverted terminal repeat (ITR, such as an adeno-associated virus ITR); (4) a virus-associated ribonucleic acids (VA RNAs) expression cassette comprising, consisting essentially of, or consisting of, a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an Adenovirus VA-RNAs; and (c) a right or 5’ inverted transposase terminal repeat sequence, wherein the left or 5’ inverted transposase terminal repeat sequence and the right or 3’ inverted transposase terminal repeat sequence facilitate excision of the transfer transposon from the plasmid and insertion or integration of the transfer transposon in one or more (e.g., about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5) 4- base insertion site in a genome (e.g., a mammalian genome or a human genome).

[0110] In any aspect or embodiment described herein, at least one of: the transgene or transfer expression cassette is flanked by recombinase sequences (e.g., atfP or atfQ, preferably attVy, the 4-base insertion site is a TTAA site or a TTAT site, preferably a TTAA site; the left or 5’ inverted transposase terminal repeat sequence and the right or 3’ inverted transposase terminal repeat sequence are reverse complements; each of the left or 5’ inverted transposase terminal repeat sequence and the right or 3’ inverted transposase terminal repeat sequence is about 8 toabout 1200 nucleotides (e.g., about 12 to about 200 nucleotides, about 28 to about 200 nucleotides, about 28 to about 600 nucleotides, or about 200 to 1200 nucleotides); or a combination thereof.

[0111] In any aspect or embodiment described herein, at least one of: (a) the plasmid further comprises or consists essentially of, 5’ of the right or 3’ inverted transposase terminal repeat sequence and 3’ of the left or 5’ inverted transposase terminal repeat sequence, at least one of an original of replication (ori); an ampicillin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as an ampicillin resistance promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an ampicillin resistance gene (e.g., AmpR); a aminoglycoside resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a ccdB promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of neomycin resistance, kanamycin resistance, or a combination thereof (e.g., NeoR / KanR); or a combination thereof; (b) the plasmid further comprises or consists essentially of one or more (e.g., 1, 2, 3, or more) antibiotic resistance expression cassette located 3’ of the right or 3’ inverted transposase terminal repeat sequence and 5’ of the left or 5’ inverted transposase terminal repeat sequence, wherein the antibiotic resistance expression cassette is at least one of: a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of a regulatory element or expression control element (e.g., a promoter, such as a promoter or SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR); a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e.g., BleoR); and a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a T7 promoter, a CMV enhance, or a combination thereof)and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g.,PuroR); or a combination thereof, preferably a puromycin resistance expression cassette; or (c) a combination there.

[0112] In any aspect or embodiment described herein, an expression cassette described herein comprises or consists essentially of a regulatory element or expression control element (e.g., a promoter, KOZAK sequences, enhancer, or a combination thereof), a termination, stop codon, a 3’ untranslated sequence, a poly(A) sequence (e.g., in expression cassettes expressed in eukaryotic cells, such as packaging cells and / or producer cells), of a combination thereof.

[0113] In any aspect or embodiment described herein, the plasmid further comprises, consists essentially of, or consists of, at least one of: a kozak sequence (e.g., a classical Kozak sequence) that is located 3’ of the regulatory element or expression control element and 5’ of the synthetic open reading frame molecule of the antibiotic resistance expression cassette; a poly(A) sequence / tail / signal (e.g., bGH poly(A) signal or SV40 poly(A) signal, preferably SV40 poly(A) signal) that is located 3’ of the gene of the antibiotic resistance expression cassette; a kozak sequence (e.g., classical Kozak sequence) that is located 3’ of the regulatory element or expression control element and 5’ of at least one of the adeno-associated virus capsid gene, the sequence encoding the adeno-associated virus Reg 52 and Reg 40 proteins, the synthetic open reading frame molecule comprising or of an inducible transactivator, or a combination thereof; a poly(A) sequence / tail / signal (e.g., bGH poly(A) signal or SV40 poly(A) signal, preferably bGH poly(A) signal) that is located 3’ of at least one of the adeno-associated virus capsid gene, the sequence encoding the adeno-associated virus Reg 52 and Reg 40 proteins, the synthetic open reading frame molecule comprising or of an inducible transactivator, or a combination thereof; the first assembly expression cassette further comprises, consists essentially of, or consists of (e.g., 5’ of the inducible transactivator and / or 3’ of the regulatory element or expression control element of the first assembly expression cassette), a nuclear localization signal (e.g., SV40 nuclear localization signal); or a combination thereof.

[0114] In any aspect or embodiment described herein, the transfer / transgene plasmid further comprises, consists essentially of, or consists of, at least one of: a kozak sequence (e.g., a classical Kozak sequence) that is located 3’ of the regulatory element or expression control element and 5’ of the synthetic open reading frame molecule of the antibiotic resistance expression cassette; a poly(A) sequence / tail / signal (e.g., bGH poly(A) signal or SV40 poly(A)signal, preferably SV40 poly(A) signal) that is located 3’ of the gene of the antibiotic resistance expression cassette; a kozak sequence (e.g., classical Kozak sequence) that is located 3’ of the regulatory element or expression control element and 5’ of the transgene or the gene of interest; a poly(A) sequence / tail / signal (e.g., bGH poly(A) signal or SV40 poly(A) signal, preferably bGH poly(A) signal) that is located 3’ of the transgene or the gene of interest; or a combination thereof.

[0115] In any aspect or embodiment described herein, the transposase plasmid further comprises, consists essentially of, or consists of, at least one of: a kozak sequence (e.g., a classical Kozak sequence) that is located 3’ of the regulatory element or expression control element and 5’ of the synthetic open reading frame molecule of the antibiotic resistance expression cassette; a poly(A) sequence / tail / signal (e g., bGH poly(A) signal or SV40 poly(A) signal, preferably SV40 poly(A) signal) that is located 3’ of the gene of the antibiotic resistance expression cassette; a kozak sequence (e.g., classical Kozak sequence) that is located 3’ of the regulatory element or expression control element and 5’ of the transposase sequence; a poly(A) sequence / tail / signal (e.g., bGH poly(A) signal or SV40 poly(A) signal, preferably bGH poly(A) signal) that is located 3’ of the transposase sequence; or a combination thereof.

[0116] In any aspect or embodiment described herein, the transgene is a fusion protein comprising a gene of interest fused to: a sequence encoding a woodchuck hepatitis virus regulatory element (WPRE); a sequence encoding an endosome-disruptive peptide (e.g., a protein or peptide that facilitates endosomal escape, such as a protective antigen (PA)); or a combination of both.

[0117] In any aspect or embodiment described herein, the regulatory element or expression control element as described herein comprises or consists essentially of a promoter, KOZAK sequences, enhancer, or a combination thereof.RECOMBINANT ADENO-ASSOCIATED PACKAGING CELLS AND METHODS OF MAKING THE SAME

[0118] An additional aspect of the present disclosure a method of making a packaging cell, the method comprising: (a) transfecting a cell (e.g., human embryonic kidney 293 cell (HEK293) or HEK293T) with an assembly donor plasmid of the present disclosure and a clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) nuclease (e.g., Cas9) plasmid for theassembly donor plasmid that facilitates the insertion of the sequences between the 5’ or left homology arm sequence and the right or 3’ homology arm sequence into a first genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette; and (b) transfecting the cell transfected with the assembly donor plasmid with a replication donor plasmid of the present disclosure and a clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) nuclease (e.g., Cas9) plasmid for the replication donor plasmid that facilitates the insertion of the sequences between the 5’ or left homology arm sequence and the right or 3’ homology arm sequence into a second genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette, wherein: the assembly donor plasmid and the replication donor plasmid include different antibiotic resistance genes (e.g., genes that confer resistance to different antibiotics); and the first genomic safe harbor site and the second genomic safe harbor site are different genomic safe harbor sites.

[0119] Another aspect of the present disclosure a method of making a packaging cell, the method comprising: (a) transfecting a cell (e.g., human embryonic kidney 293 cell (HEK293) or HEK293T) with a replication donor plasmid of the present disclosure and a clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) nuclease (e.g., Cas9) plasmid for the replication donor plasmid that facilitates the insertion of the sequences between the 5’ or left homology arm sequence and the right or 3’ homology arm sequence into a second genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette; and (b) transfecting the cell transfected with the replication donor plasmid with an assembly donor plasmid of the present disclosure and a clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) nuclease (e.g., Cas9) plasmid for the assembly donor plasmid that facilitates the insertion of the sequences between the 5’ or left homology arm sequence and the right or 3’ homology arm sequence into a first genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette, wherein: the assembly donor plasmid and the replication donor plasmid include differentantibiotic resistance genes (e.g., genes that confer resistance to different antibiotics); and the first genomic safe harbor site and the second genomic safe harbor site are different genomic safe harbor sites. In any aspect or embodiment described herein, the 5’ or left homology arm sequence comprises, consists essentially of, or consists of a 5’homology arm sequence (e.g., for ROSA26, AAVS1, or CCR5) as described herein.

[0120] In any aspect or embodiment described herein, the 3’ or right homology arm sequence comprises, consists essentially of, or consists of a 3’homology arm sequence (e.g., for ROSA26, AAVS1, or CCR5) as described herein.

[0121] In any aspect or embodiment described herein, the cell is a mammalian cell (e.g., a human cell).

[0122] In any aspect or embodiment described herein, (a) the first genomic safe harbor site is ROSA26, (b) the second genomic safe harbor site is Adeno-Associated Virus Integration Site 1 (AAVS1), or (c) a combination thereof.

[0123] In any aspect or embodiment described herein, the Cas nuclease (e.g., Cas9) plasmid for the assembly donor plasmid, the Cas nuclease (e.g., Cas9) plasmid for the replication donor plasmid, or a combination thereof, comprises or consists essentially of, in order from 5’ to 3’ : (a) a Cas nuclease expression cassette comprising or consisting essentially of: (1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a Cytomegalovirus (CMV) promoter and / or a CMV enhancer, U6 promoter, or a combination thereof); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a Cas nuclease gene (e.g., Cas9 and (b) a single guide RNA (sgRNA) expression cassette comprising or consisting essentially of: (1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a U6 promoter, a Cytomegalovirus (CMV) promoter and / or a CMV enhancer, or a combination thereof); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a single guide RNA (sgRNA) comprising, consisting essentially of, or consisting of, a trans-activating CRISPR RNA (tracrRNA) for the Cas nuclease (e.g., Cas9) and a CRISPR RNA (crRNA) comprising, consisting essentially of, or consisting of, a nucleotide sequence (e.g., 17 to 20 nucleotides) that is complementary to a genomic safe harbor (e.g., a mammalian genomic safe harbor or a human genomic safe harbor). For example, in any aspect or embodiment described herein, the crRNA comprising, consisting essentially of, or consistingof a crDNA sequence or a sgRNA target sequence (e.g., for ROSA26, AAVS1 , or CCR5) as described herein.

[0124] In any aspect or embodiment described herein, the Cas nuclease gene comprises or consists essentially of a sequence that encodes, in order from 5’ to 3’, a Cas nuclease (e.g., Cas9), a 2A peptide (e.g., P2A, T2A, E2A, or F2A, preferably T2A or P2A), and a fluorescence protein (e.g., mCherry, green fluorescent protein, or enhanced green fluorescence protein, preferably mCherry).

[0125] Another aspect of the present disclosure provides a packaging cell produced according to the method of producing a packaging cell of the present disclosure or comprising or consisting essentially of: (a) a first assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an inducible transactivator gene (e.g., a reverse tetracycline-controlled transactivator gene ( / V74)); (b) a second assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to the inducible transactivator (e.g., the inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., TRE3G or CMV promoter)); and (2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: an adeno-associated virus capsid gene (e.g., a sequence encoding an adeno-associated virus VP1, VP2, and VP3 proteins); an internal ribosome entry site (IRES) or a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A), preferably IRES; and a sequence encoding an adeno-associate virus Reg52 and Reg40 proteins; (c) a first replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Teton promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more) tet operator (TetO) and a promoter (e ., CMV promoter)); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an adeno-associate virus Reg78 and Reg68 proteins; and (d) a second replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Teton promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter)); (2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: a deoxyribonucleic acid (DNA)-binding protein (DBP) gene (e.g., Transcription Factor E2-Alpha (E2a) gene); a sequence encoding a 2 A peptide (e.g., P2A, T2A, E2A, or F2A) or an internal ribosome entry site (IRES)), preferably P2A or T2A; and an adenovirus E4 region open reading frame 6 (E4orf6) gene, wherein: the first assembly expression cassette and the second assembly expression cassette are integrated or inserted in a first genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor); and the first replication expression cassette and the second replication expression cassette are integrated or inserted in a second genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor).

[0126] In any aspect or embodiment described herein, the packaging further comprises or consists essentially of, at least one of: an assembly antibiotic resistance expression cassette located between the first assembly expression cassette and the second assembly expression cassette; a replication antibiotic resistance expression cassette located between the first replication expression cassette and the second replication expression cassette; or a combination thereof, optionally, the assembly antibiotic resistance expression cassette, the replication antibiotic resistance expression cassette, or a combination thereof, is at least one of: a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR), preferably the assembly antibiotic resistance expression cassette is a blastocidin S resistance expression cassette; a glycopeptide antibiotic orbleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e.g., BleoR), preferably the assembly antibiotic resistance expression cassette is glycopeptide antibiotic or bleomycin resistance; a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a T7 promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g., PuroR); or a combination thereof.RECOMBINANT ADENO-ASSOCIATED VIRUS PRODUCER CELLS AND METHODS OF MAKING THE SAME

[0127] A further aspect of the present disclosure provides a method of making a recombinant Adeno-associated virus producer cell, the method comprising: (a) providing a packaging cell of the present disclosure, or produced according to the method of making a recombinant Adeno- associated virus producer cell of the present disclosure; (b) transfecting the packaging cell with a transfer donor plasmid of the present disclosure and a clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) nuclease (e.g., Cas9) plasmid for the transfer donor plasmid that facilitates the insertion of the sequences between the 5’ or left homology arm sequence and the right or 3’ homology arm sequence into a third genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette, wherein: the assembly donor plasmid, the replication donor plasmid, and the transfer donor plasmid include different antibiotic resistance genes (e.g., genes that confer resistance to different antibiotics); and the first genomic safe harbor site, the second genomic safe harbor site, and the third genomic safe harbor site are different genomic safe harbor sites.

[0128] In any aspect or embodiment described herein, the 5’ or left homology arm sequence comprises, consists essentially of, or consists of a 5’homology arm sequence (e.g., for ROSA26, AAVS1, or CCR5) as described herein.

[0129] In any aspect or embodiment described herein, the 3’ or right homology arm sequence comprises, consists essentially of, or consists of a 3’homology arm sequence (e.g., for ROSA26, AAVS1, or CCR5) as described herein.

[0130] In any aspect or embodiment described herein, the cell is a mammalian cell (e.g., a human cell).

[0131] In any aspect or embodiment described herein, the third genomic safe harbor site is C-C chemokine receptor type 5 (CCR5) locus.

[0132] In any aspect or embodiment described herein, the Cas nuclease (e.g., Cas9) plasmid for the transfer donor plasmid comprises or consists essentially of, in order from 5’ to 3’: (a) a Cas nuclease expression cassette comprising or consisting essentially of: (1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a Cytomegalovirus (CMV) promoter and / or a CMV enhancer, U6 promoter, or a combination thereof); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a Cas nuclease gene (e.g., Cas9), and (b) a single guide RNA (sgRNA) expression cassette comprising or consisting essentially of (1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a U6 promoter, a Cytomegalovirus (CMV) promoter and / or a CMV enhancer, or a combination thereof); and (2) a synthetic open reading frame molecule comprising or of a single guide RNA (sgRNA) comprising, consisting essentially of, or consisting of a trans-activating CRISPR RNA (tracrRNA) for the Cas nuclease (e.g., Cas9) and a CRISPR RNA (crRNA) comprising, consisting essentially of, or consisting of a nucleotide sequence (e.g., 17 to 20 nucleotides) that is complementary to a genomic safe harbor (e.g., a mammalian genomic safe harbor or a human genomic safe harbor). For example, in any aspect or embodiment described herein, the crRNA comprising, consisting essentially of, or consisting of a crDNA sequence or a sgRNA target sequence (e.g., for ROSA26, AAVS1, or CCR5) as described herein.

[0133] In any aspect or embodiment described herein, the Cas nuclease gene comprises or consists essentially of a sequence that encodes, in order from 5’ to 3’, a Cas nuclease (e.g., Cas9), a 2 A peptide (e.g., P2A, T2A, E2A, or F2A, preferably T2A or P2A), and a fluorescenceprotein (e.g., mCherry, green fluorescent protein, or enhanced green fluorescence protein, preferably mCherry).

[0134] Another aspect of the present disclosure relates to a method of making a recombinant Adeno-associated virus producer cell, the method comprising: (a) providing a packaging cell of the present disclosure, or produced according to the method of the present disclosure; and (b) transfecting the packaging cell with a transfer transposon donor plasmid of the present disclosure and a transposase plasmid for the transfer transposon donor plasmid that facilitates the insertion or integration of the transfer transposon in one or more (e.g., about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5) 4-base insertion site in a genome (e.g., a mammalian genome or a human genome), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette, wherein: the transposase plasmid comprises or consists essentially of a transposase expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’, a regulatory element or expression control element (e.g., a promoter), and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a transposase sequence; the assembly donor plasmid, the replication donor plasmid, and the transfer transposon donor plasmid include different antibiotic resistance genes (e.g., genes that confer resistance to different antibiotics); and the first genomic safe harbor site and the second genomic safe harbor site are different genomic safe harbor sites. In any aspect or embodiment described herein, the method of making a recombinant Adeno-associated virus producer cell further comprises: transfecting the packaging cell with an assembly transposon donor plasmid of the present disclosure and a transposase plasmid for the assembly transposon donor plasmid that facilitates the insertion or integration of the assembly transposon in one or more (e.g., about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5) 4-base insertion site in a genome (e.g., a mammalian genome or a human genome), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette, wherein: the transposase plasmid comprises or consists essentially of a transposase expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’, a regulatory element or expression control element (e.g., a promoter), and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a transposase sequence; optionally, the assembly donor plasmid, the replicationdonor plasmid, the transfer transposon donor plasmid, and the assembly transposon donor plasmid include different antibiotic resistance genes (e.g., genes that confer resistance to different antibiotics); and the first genomic safe harbor site and the second genomic safe harbor site are different genomic safe harbor sites.

[0135] Thus, a further aspect of the present disclosure relates to a method of making a recombinant Adeno-associated virus producer cell, the method comprising: (a) providing a packaging cell of the present disclosure, or produced according to the method of the present disclosure (optionally, the packaging cell of the present disclosure was not transfected with an assembly donor plasmid of the present disclosure and / or does not include the first assembly expression cassette and / or the second assembly expression cassette); (b) transfecting the packaging cell with a transfer transposon donor plasmid of the present disclosure and a transposase plasmid for the transfer transposon donor plasmid that facilitates the insertion or integration of the transfer transposon in one or more (e g., about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5) 4-base insertion site in a genome (e.g., a mammalian genome or a human genome), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette; and (c) transfecting the packaging cell with an assembly transposon donor plasmid of the present disclosure and a transposase plasmid for the assembly transposon donor plasmid that facilitates the insertion or integration of the assembly transposon in one or more (e.g., about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5) 4-base insertion site in a genome (e.g., a mammalian genome or a human genome), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette, wherein: the transposase plasmid comprises or consists essentially of a transposase expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’, a regulatory element or expression control element (e.g., a promoter), and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a transposase sequence; the transposase plasmid comprises or consists essentially of a transposase expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’, a regulatory element or expression control element (e.g., a promoter), and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a transposase sequence; optionally, the optional assembly donor plasmid, thereplication donor plasmid, the transfer transposon donor plasmid, and the assembly transposon donor plasmid include different antibiotic resistance genes (e.g., genes that confer resistance to different antibiotics); and the first genomic safe harbor site and the second genomic safe harbor site are different genomic safe harbor sites.

[0136] In any aspect or embodiment described herein, at least one of: (a) the transposase plasmid further comprises, consists essentially of, or consists of, at least one of: an original of replication (ori); an ampicillin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as an ampicillin resistance promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an ampicillin resistance gene (e.g., AmpR); a aminoglycoside resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a ccdB promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of neomycin resistance, kanamycin resistance, or a combination thereof (e.g., NeoR / KanR); an expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a catabolite activator protein (CAP) binding site, a lac promoter, a lac operator, a T3 promoter, a Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding a fluorescence protein (e.g., green fluorescent protein or enhanced green fluorescence protein); or a combination thereof; (b) the transposase plasmid further comprises, consists essentially of, or consists of, one or more (e.g., 1, 2, 3, or more) antibiotic resistance expression cassette, wherein the antibiotic resistance expression cassette comprise, consists essentially of, or consists of, at least one of: a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a promoter or SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR); a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading framemolecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e.g., BleoR); a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g., PuroR); or a combination thereof; or a combination thereof, preferably a glycopeptide or bleomycin resistance expression cassette (e.g., zeocin resistance); or (c) a combination thereof.

[0137] In any aspect or embodiment described herein, the transposase plasmid further comprises, consists essentially of, or consists of, at least one of: a kozak sequence (e.g., a classical Kozak sequence) that is located 3’ of the regulatory element or expression control element and 5’ of the synthetic open reading frame molecule of the antibiotic resistance expression cassette; a poly(A) sequence / tail / signal (e.g., bGH poly(A) signal or SV40 poly(A) signal, preferably SV40 poly(A) signal) that is located 3’ of the gene of the antibiotic resistance expression cassette; a kozak sequence (e.g., classical Kozak sequence) that is located 3’ of the regulatory element or expression control element and 5’ of the transposase sequence; a poly(A) sequence / tail / signal (e.g., bGH poly(A) signal or SV40 poly(A) signal, preferably bGH poly(A) signal) that is located 3’ of the transposase sequence; or a combination thereof.

[0138] In any aspect or embodiment described herein, performing antibiotic selection comprises administering an antibiotic for at least 2 weeks (e.g., 2 to 3 weeks or 2 weeks).

[0139] In any aspect or embodiment described herein, the antibiotic comprises or is blastocidin S, bleomycin, phleomycin, Zeocin, or puromycin.

[0140] In any aspect or embodiment described herein, the method of making a recombinant Adeno-associated virus producer cell further comprises, consists essentially of, or consists of: (a) after transfection and antibiotic selection, isolating a single clone comprising the inserted sequence; (b) after transfection and antibiotic selection, confirming site-specific integration of the inserted sequence (e.g., polymerase chain reaction (such as 573’ junction polymerase chain reaction and / or out-to-out polymerase chain reaction), copy number analysis (e.g., quantitative polymerase chain reaction), or a combination thereof); (c) after transfection and antibiotic selection, contacting the cell with an agent (e.g., tetracycline, doxycycline, or derivative thereof)the induces the inducible transactivator (e.g., a reverse tetracycline-controlled transactivator (rtTA)); (d) after transfection and antibiotic selection (e.g., after contacting the cell with the agent that induces the inducible transactivator), evaluating expression (e.g., reverse transcriptase polymerase chain reaction, reverse transcriptase quantitative polymerase chain reaction, western blot, or a combination thereof) of the inserted genes; or (e) a combination thereof.

[0141] In any aspect or embodiment described herein, transfecting comprises or consists essentially of (a) inducing the cell to be in S / G2 phase, (b) contacting the cell with an inhibitor (e g., a small molecule inhibitor) of the non-homologous end joining (NHEJ) pathway, (c) enriching the donor plasmid near the Cas9-induced double stranded break (DSB) in the DNA, (d) co-expressing one or more DNA repair protein involved in homology directed repair (HDR), (e) liposomal transfecting (e.g., transfecting with a cationic liposome based reagent), or (f) a combination thereof.

[0142] In any aspect or embodiment described herein, the method of making a recombinant Adeno-associated virus producer cell further comprises, consists essentially of, or consists of producing recombinant adeno-associated virus.

[0143] In any aspect or embodiment described herein, producing recombinant adeno- associate virus comprises contacting the recombinant Adeno-associated virus producer cell with an agent (e.g., tetracycline, doxycycline, or derivative thereof) the induces the inducible transactivator (e.g., a reverse tetracycline-controlled transactivator (rtTA)).

[0144] An additional aspect of the present disclosure provides a recombinant Adeno- associated virus producer cell produced according to the method of the present disclosure or comprising or consisting essentially of: (a) a first assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an inducible transactivator gene (e.g., a reverse tetracycline-controlled transactivator gene (r / Z4)); (b) a second assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to the inducible transactivator (e.g., the inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, isinduced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., TRE3G or CMV promoter)); and (2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: an adeno-associated virus capsid gene (e.g., a sequence encoding an adeno-associated virus VP1, VP2, and VP3 proteins); an internal ribosome entry site (IRES) or a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A), preferably IRES; and a sequence encoding an adeno-associate virus Reg52 and Reg40 proteins; (c) a first replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Teton promoter or a tetracycline response unit, such as one or more (e g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter)); and (2) a synthetic open reading frame molecule comprising a sequence encoding an adeno-associate virus Reg78 and Reg68 proteins; (d) a second replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’: (1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter)); (2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: a deoxyribonucleic acid (DNA)-binding protein (DBP) gene (e.g., Transcription Factor E2-Alpha (E2a) gene); a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A) or an internal ribosome entry site (IRES)), preferably P2A or T2A; and an adenovirus E4 region open reading frame 6 (E4orf6) gene; (e) a first inverted terminal repeat (ITR, such as an adeno-associated virus ITR); (f) a transgene or transfer expression cassette comprising, consisting essentially of, or consisting of: (1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a transgene or a gene of interest; (g) a second inverted terminal repeat (ITR, such as an adeno-associated virus ITR); and (h) a virus-associated ribonucleic acids (VA RNAs) expression cassette comprising, consisting essentially of, or consisting of, a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an Adenovirus VA-RNAs, wherein: the first assembly expression cassette and the second assembly expression cassette are integrated or inserted in a first genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor) of the recombinant Adeno- associated virus producer cell; the first replication expression cassette and the second replication expression cassette are integrated or inserted in a second genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor) of the recombinant Adeno- associated virus producer cell; and the first inverted terminal repeat, the transgene / transfer expression cassette, the second inverted terminal repeat, and the expression cassette for the Adenovirus virus-associated ribonucleic acids (VA RNAs) are integrated or inserted in a third genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor) of the recombinant Adeno-associated virus producer cell.

[0145] A further aspect of the present disclosure provides a recombinant Adeno-associated virus producer cell produced according to the method of the present disclosure or comprising or consisting essentially of: (a) a first assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an inducible transactivator gene (e.g., a reverse tetracycline-controlled transactivator gene (r / 4)); (b) a second assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to the inducible transactivator (e.g., the inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., TRE3G or CMV promoter)); and (2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: an adeno-associated virus capsid gene (e.g., a sequence encoding an adeno-associated virus VP1, VP2, and VP3 proteins); an internal ribosome entry site (IRES)or a sequence encoding a 2 A peptide (e.g., P2A, T2A, E2A, or F2A), preferably IRES; and a sequence encoding an adeno-associate virus Reg52 and Reg40 proteins; (c) a first replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Teton promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter)); and (2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an adeno-associate virus Reg78 and Reg68 proteins; (d) a second replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : (1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Teton promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter)); and (2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: a deoxyribonucleic acid (DNA)-binding protein (DBP) gene (e.g., Transcription Factor E2-Alpha (E2a) gene); a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A) or an internal ribosome entry site (IRES)), preferably P2A or T2A; and an adenovirus E4 region open reading frame 6 (E4orf6) gene; (e) an assembly transposon comprising or consisting essentially of, in order from 5’ to 3’: (1) a first assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an inducible transactivator (e.g., a reverse tetracycline-controlled transactivator (rtTA)); (2) a second assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : a regulatory element or expression control element that is responsive to the inducible transactivator (e.g., the inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or atetracycline response unit, such as one or more (e g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., TRE3G or CMV promoter)); and a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: an adeno- associated virus capsid gene (e.g., a sequence encoding an adeno-associated virus VP1, VP2, and VP3 proteins); an internal ribosome entry site (IRES) or a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A), preferably IRES; and a sequence encoding an adeno-associate virus Reg52 and Reg40 proteins; (f) a transfer transposon comprising or consisting essentially of, in order from 5’ to 3’: (1) a first inverted terminal repeat (ITR, such as an adeno-associated virus ITR); (2) a transgene or transfer expression cassette comprising, consisting essentially of, or consisting of: a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a transgene or a gene of interest; (3) a second inverted terminal repeat (ITR, such as an adeno-associated virus ITR); (4) a virus-associated ribonucleic acids (VA RNAs) expression cassette comprising, consisting essentially of, or consisting of, a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an Adenovirus VA-RNAs, wherein: the first assembly expression cassette and the second assembly expression cassette are integrated or inserted in a first genomic safe harbor site (e g., a mammalian genomic safe harbor or a human genomic safe harbor) of the recombinant Adeno-associated virus producer cell; the first replication expression cassette and the second replication expression cassette are integrated or inserted in a second genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor) of the recombinant Adeno-associated virus producer cell; the assembly transposon is integrated or inserted into one or more (e.g., about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5) 4-base insertion site in a genome (e.g., a mammalian genome or a human genome) of the recombinant Adeno-associated virus producer cell; and the transfer transposon is integrated or inserted into one or more (e.g., about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5) 4-base insertion site in a genome (e.g., a mammalian genome or a human genome) of the recombinant Adeno-associated virus producer cell.

[0146] In any aspect or embodiment described herein, the packaging cell further comprises or consists essentially of: an assembly antibiotic resistance expression cassette located between the first assembly expression cassette and the second assembly expression cassette; a replication antibiotic resistance expression cassette located between the first replication expression cassette and the second replication expression cassette; a transfer / transgene antibiotic resistance expression cassette located between the transgene / transfer expression cassette and the expression cassette for an Adenovirus virus-associated ribonucleic acids; or a combination thereof, optionally, the assembly antibiotic resistance expression cassette, the replication antibiotic resistance expression cassette, the transfer / transgene antibiotic resistance expression cassette, or a combination thereof, is at least one of: a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR), preferably the assembly antibiotic resistance expression cassette is a blastocidin S resistance expression cassette; a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e.g., BleoR), preferably the assembly antibiotic resistance expression cassette is glycopeptide antibiotic or bleomycin resistance; a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a T7 promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g.,PuroR), preferably the transfer / transgene antibiotic resistance expression cassette a puromycin resistance expression cassette; or a combination thereof.

[0147] A further aspect of the present disclosure provides a method of making a recombinant Adeno-associated virus, the method comprising: (a) providing (e.g., producing) a recombinantAdeno-associated virus producer cell of the present disclosure; and (b) producing recombinantAdeno-associated virus.

[0148] In any aspect or embodiment described herein, producing recombinant adeno- associate virus comprises contacting the recombinant Adeno-associated virus producer cell with an agent (e.g., tetracycline, doxycycline, or derivative thereof) the induces the inducible transactivator (e.g., a reverse tetracycline-controlled transactivator (rtTA)).EXAMPLESExample 1. Production of Inducible, Stable Packaging Cell Line for rAAV Production via CRISPR-Cas9 Mediated Site-Specific Integration

[0149] Adeno-Associated Virus (AAV)-mediated gene therapy is a quickly growing segment of the pharmaceutical market. However, the current transient transfection process to produce a recombinant AAV (rAAV) has many challenges, such as low productivity of rAAV from host cells, difficult scalability of the rAAV-producing bioprocess, and high levels of impurities (e.g. empty / partial capsid) materialized during production. Stable cells are ideal for industrial large- scale continuous production processes, overcoming the drawbacks in the current transient transfection process and providing the streamlined rAAV manufacture. In this study, we proposed to use synthetic inducible promoters to control the viral component expression and develop the baseline of the human embryonic kidney 293 T (HEK293T) stable cells via CRISPR- Cas9 mediated site-specific integration targeting human genomic safe harbor sites (ROSA26, Adeno-Associated Virus Integration Site 1 (AAVS1), and C-C chemokine receptor type 5 (CCR5) locus). Stable cell pools were developed and evaluated after each round of the three rounds of integration. Single clones were characterized for each integration round. The 573’ junction polymerase chain reaction (PCR) results confirmed the site-specific integration to each locus of the stable pools. The genome copy result showed that AAV components, including Rep78 / 68, E2A, E4orf6, Cap, and Rep52, were successfully integrated into the host cell genome. Genome and capsid titer after induction confirmed rAAV production for stable cell pools in each round. The isolated single packaging cell line (after 2ndround integration) was able to produce rAAV, although the titer is ten-fold lower than the traditional triple plasmids transfection. The out-to-out PCR and quantitative PCR (qPCR) assay results further confirm the site-specific integration. Overall, the results described herein demonstrate the feasibility of developing theinducible stable cell line with the refactored viral vector components via a site-specific integration strategy.

[0150] Materials and Methods

[0151] Vector Design and Plasmids Construction

[0152] sgRNA-Cas9 Plasmid Construction. The single guide ribonucleic acid (sgRNA) sequences targeting the ROSA26, AAVS1 and CCR5 locus were designed following a prior publication (Shin, S., et al., Comprehensive analysis of genomic safe harbors as target sites for stable expression of the heterologous gene in HEK293 cells. ACS Synthetic Biology, 2020. 9(6): p. 1263-1269), annealed, and then integrated into the sgRNA-Cas9 backbone plasmid (pU6- (BbsI)_CBh-Cas9- T2A-mCherry) using golden gate assembly.

[0153] Donor Plasmid Construction. Homology arms were amplified via polymerase chain reaction (PCR) from donor backbone plasmids (AAVSl-eGFP donor, ROSA26-eGFP donor, and CCR5-eGFP donor, gift from KAIST) (Shin, S., et al., Comprehensive analysis of genomic safe harbors as target sites for stable expression of the heterologous gene in HEK293 cells. ACS Synthetic Biology, 2020. 9(6): p. 1263-1269; Hockemeyer, D., et al., Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nature biotechnology, 2009. 27(9): p. 851-857). The components of the AAV were generated either through synthesis by Twist Bioscience or PCR cloning from corresponding plasmids.Subsequently, the plasmids were assembled according to the vector design depicted in Figure IB using the golden gate assembly method. Then the homology arms sequences, selection genes, and well-prepared AAV components were assembled using NEBuilder HiFi DNA Assembly master mix (New England Biolabs, Ipswich, Massachusetts, United States) followed by transformation using Escherichia coli DH5a competent cells. Plasmids were verified by sequencing and prepared with the EndoFree plasmid maxi kit (Zymo Research Corporation, Irvine, California, United States), according to the manufacturer’s instructions. The information on the sgRNA sequences, donor sequences for AAVS1, ROSA26, and CCR5 loci, plasmids, and primers used in cloning is listed in Tables 1, 2, and 3. The sgRNA target sites, sequences, and 573’ homology arm sequences for AAVS1, ROSA26, and CCR5 locus were referred to in the previous literature (Fu, Q., etal., Critical challenges and advances in recombinant adeno- associated virus (rAAV) biomanufacturing. Biotechnology and Bioengineering, 2023).Table 1 . sgRNA Target Sites and 573’ Homology Arm Sequences* Fu, Q., et al., Design space determination to optimize DNA complexation and full capsid formation in transient rAAV manufacturing. Biotechnology and Bioengineering, 2023.** Wang, Y., et al., Transcriptomic features reveal molecular signatures associated with recombinant adeno-associated virus production in HEK293 cells. Biotechnology Progress, 2023 : p. e3346.*** Yang, Q., F. Chen, and J.P. Trempe, Characterization of cell lines that inducibly express the adeno-associated virus Rep proteins. Journal of virology, 1994. 68(8): p. 4847-4856.Table 2. Plasmid InformationTable 3. Primer Information

[0154] Cell Culture. Stable Transfection, and Single Clone Isolation. HEK293T / 17 (ATCC, CRL-11268; Manassas, Virginia, United States) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Waltham, Massachusetts, United States) supplemented with 10% fetal bovine serum (Gibco, Waltham, Massachusetts, United States). The cells were cultured in GenClone® T-flasks (Genesee Scientific, El Cajon, California, United States) with a working volume of 5 mL at 37 °C under 5% CO2 and passaged every 4 days. To establish stable cell pools, donor and sgRNA-Cas9 vectors targeting safety harbor sites at a 1 : 1 (w:w) were transfected using Lipofectamine™ 2000 (Invitrogen, Carlsbad, California, United States) followed by 2 weeks of selection with antibiotics: 5ug / mL blasticidin (InvivoGen, San Diego, California United States), 200ug / mL ZEOCIN® (InvivoGen, San Diego, California United States), and 2 pg / mL of puromycin (Sigma-Aldrich, Saint Louis, Missouri, United States). The viable cell density (VCD) and cell viability were assessed using a Cedex HiRes Analyzer (Roche LifeScience, Basel, Switzerland, United States) and the trypan blue assay. The single-cell clones were isolated from the stable pools using an MA900 cell sorter (Sony, Minato City, Tokyo, Japan) and seeded individually into 96-well plates.

[0155] Genomic DNA Extraction for Junction PCR and Copy Number Analysis. Sitespecific integration of the target gene was evaluated using 5' and 3' junction PCR, and out-to-out PCR was utilized for further confirmation. The cell pellets from stable pools were collected for genomic DNA extraction. Genomic DNA was extracted from stable cell pools using Quick- DNA™ Miniprep Plus Kit (Zymo Research Corporation, Irvine, California, United States) following the manufacturer’s instructions. Approximately 100 ng of extracted DNA was used as the template for junction PCR, as described below. For single-cell clones, DNA was isolated by adding 20 uL of QuickExtract DNA extraction solution when cells were confluent in 96-well plates. The mixture was then incubated at 65°C for 15 minutes, followed by 5 minute incubation at 98°C. Of this mixture, 2 uL were used as the template for junction PCR, as described below. The 573’ junction PCR was performed using PrimeSTAR HS Premix (Takara Bio, Kusatsu, Shiga, Japan) by touchdown PCR (98 °C for 3 minutes; 10X: 98 °C for 10 seconds, 65 °C-55 °C (-l°C / cycle) for 30 seconds, 72 °C for 2 minutes; 30X: 98 °C for 10 seconds, 55 °C for 30 seconds, 72 °C for 2 minutes; 72 °C for 10 minutes). The primer sequences used for the 573’ junction PCR are listed in Table 3.

[0156] For relative genome copy number analysis, qPCR was performed on genomic DNA samples using SYBR Green qPCR Master Mix (Thermo, Waltham, Massachusetts, United States) on the Bio-Rad system (Bio-Rad Laboratories, Hercules, California, United States). According to the manufacturer’s instructions, qPCR reaction mixtures contained 2X SYBR Green master mix, 400 nM of forward and reverse primers, 20 ng of genomic DNA, and up to 20 pL molecular biology water. Amplification was executed with the following conditions: 50 °C for 1 minutes; 95 °C for 10 minutes; 40 cycle: 95 °C for 15 seconds, 60 °C for 30 seconds, 72 °C for 1 :30 minutes. The primer sequences used for Cap, Rep52 / 40, Rep78 / 68, E2a, E4orf6, codon- optimized GFP, and GAPDH are listed in Table 3.

[0157] RNA Isolation for Gene Expression Evaluation. The gene expression assay, including RNA isolation, complementary DNA (cDNA) reverse transcription and gene expression by reverse transcription-polymerase chain reaction (RT-PCR), was previously described (Wang, Y., et al., Transcriptomic features reveal molecular signatures associated with recombinant adeno- associated virus production in HEK293 cells. Biotechnology Progress, 2023: p. e3346). The comparative cycle threshold (2’AACt) method was used to analyze the transcript level fold changes between different conditions, in this case the induction of different doxycycline concentrations.The primer sequences used for the Cap, Rep52 / 40, Rep78 / 68, E2a, E4orf6, codon-optimized GFP, and GAPDH assessment are listed in Tables 3.

[0158] rAAV Vector Production, rAAV Preparation and Analytical Methods (genome titer, capsid titer). Triple plasmid transfection was performed for rAAV production. Three AAV- related plasmids were used, sourced from Addgene: pAdDeltaF6, pAAV2 / 2, and AAV-CMV- GFP. Plasmid information is listed in Table 2. Cells in the logarithmic growth phase were seeded at a density of 5*1O5cells per well in a 6-well plate. After a 24-hour incubation, transfection for stable pool evaluation was performed following the PEIpro manufacturer's instructions (Polyplus-transfection®, Strasbourg, France, United States), and the optimal condition of transfection obtained from the literature was utilized for single clone evaluation (Grieger, J.C., S.M. Soltys, and R.J. Samulski, Production of recombinant adeno-associated virus vectors using suspension HEK293 cells and continuous harvest of vector from the culture media for GMP FIX andFLTl clinical vector. Molecular Therapy, 2016. 24(2): p. 287-297; Gu, B., et al., Establishment of a scalable manufacturing platform for in-silico-derived ancestral adeno- associated virus vectors. Cell & Gene Therapy Insights, 2018. 4(S1): p. 753-769; Zhao, H., et al., Creation of a high-yield AAV vector production platform in suspension cells using a design-of- experiment approach. Molecular Therapy -Methods & Clinical Development, 2020. 18: p. 312- 320). Specific transfection conditions can be found in Table 4 and Table 5.Table 4. Transfection condition for stable pool evaluationTable 5. Transfection condition for single clone evaluation

[0159] After 68 hours post-transfection, harvested cell cultures were aliquoted to ImL volume (cells and supernatant inclusive) in 1.5 mL centrifuge tubes either for immediate analysis or stored at -80°C for future analysis. The genome titer and capsid titer assays followed the previously published paper (Fu, Q., et al., Design space determination to optimize DNA complexation and full capsid formation in transient r AAV manufacturing. Biotechnology and Bioengineering, 2023). The specific productivity can be calculated as shown below.Titer2— Titer-.0 = .. . . p (2) IVCD

[0160] Statistical Analysis. GraphPad Prism 9 (Boston, Massachusetts, United States) was used for processing raw data and statistical analysis. The workflow diagram and vector design were made in Biorender.

[0161] RESULTS

[0162] Strategy and workflow. CRISPR-Cas9 technology was used to accomplish site specific integration of refactored viral components and develop a stable cell line that can be induced for rAAV production in HEK293T cells, serving as the baseline for HEK293 stable producers. Figure 1A shows the integration strategy and workflow. Three essential viral cassettes were first constructed, including assembly cassette, replication cassette, and transfer cassette. Detailed information about the components of each cassette can be found in the vector design section. Three cassettes were integrated into three different genomic safety harbors (ROSA26, AAVS1 and CCR5) respectively and sequentially. The first round of stable pool was generated after the stable integration of the assembly cassette and corresponding antibiotic selection. As intermediate checkpoints, this stable pool was evaluated by junction PCR for targeted integration at the ROSA26 locus, relative genome copy analysis of assembly components, and its capability to produce AAV with two other plasmids transfection. Once the results met those criteria, the stable cell pool was maintained for the single clonal screening and the next round of the integration to expedite the overall integration process. The same integration and assessment process was applied to the replication and transfer cassettes targeting the AAVS1 locus and the CCR5 locus. The isolated single clones with intact target integration units in the specific sites after the second round of integration were further characterized as packaging cell lines.Similarly, the single clones with correct integrations after the third round integration were then assessed as producer cell lines.

[0163] Vector design and construction. The AAV production required components, including Rep (Rep78 / 68, Rep52 / 40), Cap, Helper genes (E2A / DBP, E4orf6, VARNA), and the Gene of Interest (Gol) flanked by inverted terminal repeat (ITR), were refactored into three different cassettes based on the function of different viral components (Lee, Z., et al., Construction of anrAAV Producer Cell Line through Synthetic Biology. ACS Synthetic Biology, 2022. 11(10): p. 3285-3295). The detailed information for the assembly, replication, and transfer cassettes is shown in Figure IB and Figures 2A, 2B, and 2C. (FIG. 2A) Assembly cassette includes the Cap gene and Rep52 / 40 gene, both crucial for capsid assembly and packaging. These two genes are regulated under a tet-on inducible promoter and linked by an internal ribosome entry site (IRES). IRES can effectively mitigate the cytotoxic effects of Rep52 / 40 expression by reducing its transcript level. The cassette also contains attB recombinase sites that allow the convenient switch between serotypes. Tetracycline-controlled transactivator (rtTA) encodes proteins that regulate the expression of the gene of interest under the transcriptional control of the tetracycline-responsive promoter element (TRE). (FIG. 2B) Replication cassette comprises two tet-on inducible switches: one controls Rep68 / 78 expression, and another one controls E2a / DBP, and E4orf6. Minimum required viral helper components (DBP and E4orf6) are included in this cassette to reduce their cytotoxicity. (FIG. 2C) Transfer cassette contains ITR flanked green fluorescent protein (GFP), as an exemplary transgene, and another helper gene VA RNA. The inclusion of attB recombinase sites enables the easy transgene switch in the future.

[0164] Stable pool evaluation for each round. Assembly, replication, and transfer cassettes were sequentially integrated to their corresponding target genomic safety harbor sites. After 2 weeks of antibiotic selection, the stable pool generated in each round was carefully evaluated. Genomic DNA was extracted from the stable pool. The targeted integration of each cassette in a designated locus was verified by 573’ junction PCR. The relative genome copy number of integrated cassettes in the stable pool was further measured by qPCR assay. Lastly, the stable pool in each round was transiently transfected for rAAV production to assess the effect of integration on cassette expression and its functionality. To be more specific, transient transfection with three donor plasmids containing refactored viral components serves as donor control (DC). The first-round stable pool was transient transfected with the remaining two donor plasmids for rAAV production, and the second-round stable pool was transient transfected with transfer donor plasmid only. 5 ug / mL doxycycline was added for all the conditions to induce gene expression and vector production. Genome and capsid titer were used to evaluate the rAAV production.

[0165] After each round of integration, 573' junction PCR was conducted to confirm the target integration in the stable cell pool. The gel image confirmed the integration of the assemblycassette in the first-round stable pool and the integration of the replication cassette in the second- round stable pool (Data not shown). The gel electrophoresis image (Figure 3A) displayed the junction PCR results of the stable pool after the third round of integration, and the results affirmed the successful integration of all three cassettes at their intended target sites.

[0166] The relative gene copy numbers of viral components, including Cap, Rep52, Rep68, DBP, E4orf6, and transfer cassette (GFP), were assessed through qPCR assays after each round of integration, with GAPDH serving as the internal reference. As indicated in Table 6, copy numbers of housekeeping gene GAPDH remained consistent across all three stable cell pools and negative control (NC)Zparental cells. In comparison to the NC group, the Cq values for viral components were below 29 in the stable cell pool after each round of integration, indicating the detection and successful integration of at least one copy of each viral component into the host cell genome.Table 6. Relative genome copy analysis summary of viral components for stable cell pools after each round of integrationFootnote: The data here represent the mean and standard deviation of biological triplicates (n=3).NC represents negative control / parental cells. rtTA represents tet-on inducible promoter.

[0167] To further assess the stable integration process, transient transfections with unintegrated donor cassettes were performed as intermediate checkpoints. The specific genometiter (Figure 3B) and capsid titer (Figure 3C) after 68 hours of induction with 5 pg / mL doxycycline were utilized to evaluate rAAV productivity per cell. In general, the genome titer after the 1st (1.88E+2 viral genome / cell (vg / cell)) and 2nd (2.68E+2 vg / cell) round of integrations were comparable to that of the donor control (2.21E+2 vg / cell), but no genome titer was detected after the 3rd round. Similarly, the capsid titer after the 1st (2.46E+2 cp / cell) and 2nd (2.95E+2 capsid / cell (cp / cell)) integrations was comparable to that of the donor control (2.17E+2 cp / cell). Although the amount of capsids in the 3rd round of integration was detectable (74.47E+1 cp / cell), there was a significant decrease compared to the amount in donor control, 1stand 2ndround stable cell pools. In general, the genome titer and capsid titer confirmed the production of rAAV with refactored donor plasmids and the effective integration of viral components into the host genome. The detection of capsids after the 3rd round of integration confirmed capsid production. However, it was important to notice the absence of genome titer when utilizing the 3rdround stable pool to produce rAAV. This might result from inadequate viral replication due to the low transgene copy numbers, compared to those achieved in transient transfection. Together with limited viral proteins production, the relatively low packaging efficiency, an inherent barrier in rAAV production, further contributes to the undetectable genome titer. Such observation underscores the need to integrate multiple copies of transgene and cap-encoding plasmids to enhance overall productivity. Stable cell pools after each round of integration were then maintained for single clone screening.

[0168] Knockin Efficiency Results. Approximately 30-70 single clones were isolated and screened from each round of stable cell pools. Two criteria were defined: site-specific integration with positive 573’ junction PCR results; and detection of all the viral components in the host cell genome via relative genome copy assay. Any clones in the 2ndand 3rdrounds pass that two criteria were further explored and fully characterized for rAAV production.

[0169] Table 7 shows the summary of junction PCR screening results for targeted integration, and the gel electrophoresis image of junction PCR screening is shown in Figures 4A, 4B, and 4C. Out of 55 clones picked from 1stround stable pool, 3 clones were 573’ junction (ROSA26) positive (Figure 4A). Out of 38 clones from 2ndround stable pool, 2 clones were 573’ junction (AAVS1) positive (Figure 4B). Out of 62 clones from 3rdround stable pool, 12 clones were 573’ junction (CCR5) positive (Figure 4C). It was observed that with the larger insertionsize, CRTSPR-Cas9 mediated knock-in efficiency tends to be lower. Further explanation can be found in the discussion.Table 7. Knock-in efficiency summary for three rounds of integrations

[0170] Clones with targeted integration were further assessed for their genome copy of viral components. Table 8 shows the relative genome copy of all the required viral components for the 1stround single clones (Cap and Rep52), and the 2ndand 3rdsingle clones (Cap, Rep52, Rep68, DBP, and E4orf6). Genome copy results for stable cell pool were listed again at the end of each table here for comparison. According to the Cq value of viral components in the NC group (Cq >30), any viral components in single clones with genome copy Cq value greater than 29 were defined as missing. Overall, 2 clones out of 3 had Cap and Rep52 successfully integrated in the first round; 2 clones out of 2 had all viral components integrated for the second round; no clones had all the components detected after 3rdround integration, either missing Rep or Cap genes. Single clones picked up after 2ndround of integration were evaluated and fully characterized as the packaging cell lines in the next section.Table 8. Genome copy analysis summary for isolated single clones from each stable cell poolFootnote: Italicized represents the missing of corresponding viral genes. Bold and italicized highlight means the isolated single clones have all the components integrated and can be confirmed by genome copy analysis.

[0171] Single Clone Evaluation (Packaging Cell Line). Single clone 4-7 in the 2ndround was selected and fully characterized as the packaging cell line. The relative transcript level of viral genes in the packaging cell 4-7 after induction was measured by qPCR assay. Different doxycycline concentrations, 0 pg / mL, 0.5 pg / mL and 5 pg / mL were added to induce the gene expression (Figure 5 / Table 9). Detected signals of viral components at 0 pg / mL induction shown in Table 9 indicated the leakage of the designed inducible promoter. The extent of leakiness varied among the genes. Despite being constructed within the same vector, DBP exhibited the lowest degree of expression leakage compared to Rep68 and E4orf6. The variations in transcript level and the extent of leakage could potentially relate to different primer amplification efficiencies and promoter interference. Addition of 0.5 and 5 pg / mL doxycycline were both able to induce the viral gene expressions. The fold change of transcript level in 0.5 and 5 pg / mL doxycycline relative to that 0 ug / mL dox was shown in Figure 5. The trend further confirmed that 5 pg / mL doxycycline can be used to maximize gene expression for later rAAV production. The transcript levels (Cq values) for Rep68, DBP, and E4orf6 following 5 pg / mL doxycycline induction were comparable, suggesting the consistent expression from the same cassette. Compared with the Cap gene, the higher transcript level of Rep52 at 0 and 5ug / mL dox might result from the potential leakage expression of Rep68, although the pl9 promoter was mutated to prevent any Rep52 synthesis from the replication cassette. Transcript levels of Rep and helper genes were elevated to a great extent as expected, after the addition of doxycycline. Thetranscript level of the Cap gene showed a roughly two-fold increase after induction, but this increase was comparatively lower than the changes in expression levels observed in other viral genes at 0 and 5 ug / mL dox induction. This suggests the need for further optimization of the inducible system.Table 9. Transcript Cq value summary of all the viral components in the single clone packaging cell line SC4-7 with 0, 0.5, 5 ug / mL doxycycline induction via RT-PCR assay.

[0172] Packaging cell line 4-7 was transfected with transfer donor plasmid for rAAV production. Cell cultures induced with 5 pg / mL doxycycline and uninduced (0 ug / mL dox) cell culture were compared in parallel to understand the extent of promoter leakage. Positive control, traditional triple transient transfection with commercial plasmids (Addgene), and donor control, transient transfection with refactored donor plasmids, were used as the reference for later optimization of rAAV productivity in inducible stable cell lines. Figures 6A and 6B demonstrated that compared to positive control, donor control resulted in approximately 10-fold lower genome titer (Figure 6A) and 100-fold lower capsid titer (Figure 6B), indicating room to improve productivity based upon the optimization of inducible system and vector design.Packaging cell line SC4-7 was expected to have no titer at 0 ug / mL dox induction. Sensitivity of qPCR titer assay and leakage of the designed inducible system can be potential reasons for the SC4-7 0 dox genome titer result shown in Figure 6B. With the addition of 5 pg / mL doxycycline, genome titer for SC4-7 (8.46E+11 vg / L) was slightly lower than that for donor control (1.15E+12 vg / L); whereas capsid titer (8.79E+10 cp / L) was 3-fold lower than that for donorcontrol (2.58E+11 cp / L). The big variation between genome and capsid titer might result from the sensitivity and limitations of each assay in the low titer profde.

[0173] The genome titer variation at 0 and 5 pg / mL dox induction was overall consistent with the transcript level changes of viral components. Low genome titer at 0 dox induction suggested that the leaky expression of viral components was not sufficient to support rAAV replication. After the addition of 5 pg / mL doxycycline, there was a noticeable increase in genome titer together with the increase in transcript levels. However, there was a slight discrepancy observed in capsid titers. With only 2-fold difference in Cap gene transcript levels (Figure 5), the capsid titer was undetectable at 0 pg / mL dox induction, but became detectable at 5 pg / mL dox induction. The Cap expression in 0 pg / mL dox condition was largely due to the leakiness of the designed promoter, but incorrect VP stoichiometric ratio and improper assembly process potentially resulted from insufficient expression of other rAAV viral components might lead to the capsid titer lower than the detection limit. Overall, the performance in the transcript level, genome titer, and capsid titer confirmed that this cell line was able to produce rAAV, although the productivity was significantly lower than that in traditional triple transfection. Potential optimization strategies are discussed below.

[0174] Site-specific integration was further confirmed by out-to-out PCR (Figure 7). Due to the large insertion size, two pairs of primers were designed to separately amplify left (5’) and right (3’) insert fragments with 376 bp overlap. The primer design strategy is shown in Figure 7. PCR products were further purified and assessed for their relative genome copies of viral components via qPCR assay. This validated that at least one copy of Rep68, DBP, and E4orf6 was precisely integrated into the target locus AAVS1.

[0175] Discussion. An inducible stable cell line for rAAV production with site-specific integration was produced herein. High expression levels of viral components, such as Rep proteins and helper genes, and cytotoxicity associated with these components are challenges to constructing stable producers for viral vectors. With the design of inducible circuit, it can effectively regulate the level of gene expression. Additionally, site-specific integration allows the targeted integration of transgenes to pre-validated genome loci. In contrast to the widely used random integration method for generating stable monoclonal antibody production in Chinese hamster ovary (CHO) cells, this approach necessitates less time-consuming isolation and screening of clones to obtain stable high producers.

[0176] Several HEK293T clones were isolated as packaging cell lines and the established stable cell lines fully characterized. In the selected single clone 4-7, at least one copy of each viral component was detected. With the addition of the inducing reagent, the transcript levels of the integrated viral components were significantly boosted. It can result in genome titer 1.24E+3 vg / cell and capsid titer 1.38E+2 cp / cell. The site-specific integration was confirmed by 573’ junction PCR and out-to-out PCR for replication cassette targeting the AAVS1 locus.

[0177] The proof of concept for developing a stable AAV cell line was demonstrated herein by the characterized packaging cell line, involving the redesign of viral components under the regulation of inducible promoters through site-specific integration methods. However, there are several inherent limitations expected for this study: overall low knock-in efficiency for CRISPR- Cas9 mediated large fragment insertion; low genome titer resulting from refactoring of viral components; and promoter leakage and inducibility.

[0178] Low knock-in efficiency and potential strategies to improve. Approximately 400-500 single clones were isolated from stable cell pools in each round initially. Only 30-60 clones were able to survive and grow. Out of these viable clones, only 5% of clones had inserts successfully integrated into the target loci. This observation is in agreement with the reported challenges and hurdles in existing CRISPR-Cas9 technology: overall low knock-in efficiency and worse with larger inserts (more than 3000 bp) in mammalian cell lines. Double strand break (DSB) induced by active Cas9 can be repaired by two major pathways: non-homologous end joining (NHEI) and homology directed repair (HDR). Targeted integration harnesses CRISPR / Cas9 and HDR to incorporate the inserts containing homology arms (HA) precisely to the locus. NHEJ is one of the fastest and an active repair pathway existing in almost all the cell cycle phases, whereas HDR is primarily active in G2 / S phase. Low HDR efficiency becomes the major reason for relatively low knock-in efficiency. This might also explain why important viral components integrated earlier were lost for the majority of isolated producer clones, after three rounds of integration and selection process. It is also worthwhile to mention here that initially both HEK293 and HEK293T were attempted for stable cell line development. After the integration of the replication cassette in the second round, the loss of integrated assembly components was observed during the antibiotic selection process for the HEK293 stable cell pool (data not shown). Thus, to develop stable producers in the future, it is essential to increase HDR for sitespecific integration. Several strategies have been attempted and developed in different models:such as adding small molecules to inhibit NHEJ pathways; controlling cell cycles to keep cells in HDR rich S / G2 phase; enriching the donor near Cas9-induced DSB; and co-expressing DNA repair protein involved in HDR. These strategies can be applied and explored in future studies.

[0179] Low genome titer and future directions to optimize. Here, rAAV Rep, Cap, and helper genes were refactored and regulated under three inducible Tet-on promoters. The genome titer obtained by transient transfection with refactored donor plasmids was lower than that by traditional transient transfection with commercially available plasmids. With the same viral gene sequences, the low titer in donor control indicates the potential for optimization and improvement in inducible systems. Tet-on inducible promoter was used here with a total number of three to separately control the different viral components. The use of one type inducible promoter simplifies the production process by minimizing the addition of various inducible reagents. However, the modulation of gene expression levels and timing for diverse viral components to achieve optimal production demands becomes challenging. In order to better regulate the gene expression level and timing, the inducible promoters can be tuned by the number and the spacing between of tetO sites. Protein expression control can be achieved by screening Kozak sequence variants and modulating the translational efficiency. Various factors in circuit designs, such as inducibility, leakage, control of gene expression, and expression timing, can be screened and evaluated during transient transfection. The inducible system can be adjusted to achieve maximum productivity and optimal quality before progressing to stable integration.

[0180] The cytotoxicity associated with viral components remains as a challenge in stable packaging / producer cell line development. It was observed that cells in stable pools after the second round of integration and packaging cell line SC4-7 grew much slower than parental cells (data not shown). Moreover, the transcript data revealed detectable levels of all viral components in the packaging cells at 0 ug / mL dox induction, indicating the leakage of the designed inducible circuit. The low cell growth rate in the stable pools and packaging cell line is being attributed to the leaky expression of the toxic genes, particularly the Rep78. Rep78 has been shown to activate caspase-3 and induce cell apoptosis, decrease Cdc25A activity, and block the cell cycle in the S phase. Therefore, it is hypothesized that the leakage of these toxic genes could significantly impact cell physiology, thereby potentially impeding AAV production. In future studies, additional regulations, such as the use of a second inducible circuit, can be proposed totightly control the expression of those toxic genes. In addition, the literature reported that a conditional degron tag can be used to modulate protein stability and regulate protein expression at the post-translational level. That is to say, the dynamics of protein expression can be regulated by controlling its degradation rate after translation. Therefore, the degron tag can be used in the future to tune Rep78 / 68 lifetime, regulate its degradation, and eliminate the cytotoxicity associated with it. Furthermore, the mechanistic study of the cytotoxicity associated with Rep genes and helper genes from the host cell perspective will be also necessary and useful for rational modifications to enhance the tolerance of the host cells. Omics study and Crispr-Cas9 genome-wide screening can be conducted to investigate the interactions between Rep / helper viral proteins and host cell proteins by using the cell lines that express rep protein or helper gene under the control of inducible promoters. It might offer valuable insights about potential strategies to allow the host cells to be more tolerant to cytotoxicity.

[0181] Conclusion. An inducible stable packaging cell line for AAV production was successfully established here through a site-specific integration strategy. The introduction of the inducing reagent resulted in a significant increase in transcript levels of integrated viral components, yielding a genome titer of 1.24E+3 vg / cell and a capsid titer of 1.38E+2 cp / cell. Site-specific integration was confirmed through junction PCR and out-to-out PCR. The characterization of the isolated inducible packaging cell line underscores its potential for developing optimized stable cell lines in the future. Efforts can be concentrated on addressing challenges such as low knock-in efficiency, optimizing gene expression levels and timing for various viral components, and minimizing the leakage of toxic genes.Example 2. Development of an Inducible, Stable Producer Cell Line for rAAV Production

[0182] The traditional rAAV production process is through triple plasmid transient transfection in HEK293 cells. However, there are several drawbacks associated with transient production process, including complicated and expensive process consuming considerable amounts of transfection reagents and plasmid, the lot-to-lot variation of the products, and difficulty in scaling up. To achieve consistent and scalable production, it is necessary to establish a stable cell line for rAAV production.

[0183] The major challenge in developing a stable producer is the cytotoxicity induced by continuous expression of rep and helper genes after stable integration. A stable packaging cell line with site-specific integration of refactored and inducible Tet-on promoters controlled viralcomponents was successfully produced in Example 1 . To produce rAAV with this packaging cell line, transient transfection with transgene plasmid was required before doxycycline induction. A comprehensive characterization from genomic, transcript, protein level, and ability to produce rAAV was conducted for this cell line before and after doxycycline addition. It was observed that the genome titer was ten-fold lower than that of rAAV products done with triple plasmid transfection.

[0184] Same integration strategy has been applied to establish a stable producer cell line, with all viral components integrated sequentially. Relative genome copy analysis of all viral components, including Rep68, Rep52, Cap, DBP, E4orf6, and GFP, in the stable cell pool indicated successful integration; however, no genome titer and only a very faint capsid titer signal were detected. Limited copy number of transgene cassette with site-specific integration and low packaging efficiency could potentially contribute to this result. Genome replication is one limitation: site specific integration offers control over the number of integrated transgene copies, which must be significantly lower than the high copy numbers typically achieved during transient plasmid transfection. Genome encapsidation could be another bottleneck, with one study reporting only 0.3%-3% of total genomes were encapsidated. Thus, it is hypothesis that increasing the copy number of transgene and Cap gene can increase the likelihood of detecting genome and capsid titer signals.

[0185] DNA transposon-based integration is an effective method to enhance transgene integration levels and target transcriptionally active regions of host cell genome with greater affinity. Additionally, transposon allows the development of stable cell factories with higher homogeneity compared to cell line generated by random integration. Transposon system comprises a donor vector expressing gene of interest flanked by the ITRs, along with a helper plasmid expressing the transposases, which facilitates the transgene’s transposition from donor plasmid into host cell genome. Several commercially available transposase systems, including Sleeping Beauty Transposon™ System, piggyBac®, and Leap-In Transposase®, are widely used in biopharmaceutical research. Transposition efficiency and vector copy number vary greatly in different cell types, so it is worthwhile optimizing the co-transfection ratio of transposase to transposon. With a hyperactive transposase, optimized transposon, and optimal co-transfection ratio, 50-100 copies of the insert are expected to be stably integrated into the host genome. Thegenome titer is anticipated to increase as a result of the elevated copy number of gene of interest (Gol).

[0186] In Example 2, a stable producer was developed by conducting transposon mediated integration of transfer (containing green fluorescent protein (GFP)) and assembly (containing Cap) cassettes in inducible stable packaging cell line using transposon-mediated integration packaging cell line. Genome copy analysis of all viral components and rAAV production were initially evaluated in the stable cell pool. After confirming the genome titer signal detection in the stable pool, single clones were isolated. These clones were screened for their copy number of GFP and Cap. Clones with genome copy numbers comparable to or higher than those in stable pools were selected and tested for the genome copy of all viral components. The top-performing clone was identified and fully characterized after all viral component genome copy evaluations. Following doxycycline induction, genome titer (3.73E+11 vg / L) and capsid titer (2.01E+11 cp / L) were detected, confirming the successful establishment of the inducible stable producer cell line.

[0187] Thus, this examples demonstrates the ability to develop inducible stable cell lines for rAAV production through refactored viral vector components and a combination of site-specific and transposon-mediated integration approaches.

[0188] Materials and Methods

[0189] Vector design and plasmids construction, piggy Bac® transposon plasmid construction: piggyBac® transposon backbone with Long Terminal Repeats (LTR) were amplified via PCR from plasmid pTS1019_Tier3(PB), which was a gift from Martin Fussenegger (Addgene plasmid # 169649). The components of the AAV were generated either through synthesis by Twist Bioscience or PCR cloning from corresponding plasmids. Subsequently, they were assembled according to the vector design using the golden gate assembly method as shown in Example 1. Then the piggyBac® transposon sequences, selection genes, and well-prepared AAV components were assembled using NEBuilder® HiFi DNA Assembly Master Mix (New England Biolabs®, Ipswich, Massachusetts, United States) followed by transformation using E. coli DH5a competent cells. Plasmids were verified by sequencing and prepared with the EndoFree plasmid maxi kit (Zymo Research Corporation, Irvine, California, United States), according to the manufacturer’s instructions. The information on the plasmids and primers used in cloning is listed in Table 10 and Table 11.Table 10. Plasmid informationTable 11. Primer information

[0190] Cell culture, stable transfection, and single clone isolation. Packaging cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Waltham, Massachusetts, United States) supplemented with 10% fetal bovine serum (Gibco, Waltham, Massachusetts, United States), 5ug / mL blasticidin (InvivoGen, San Diego, California, United States) and 200ug / mL zeocin (InvivoGen, San Diego, California, United States). The cells were cultured in GenClone® T-flasks (Genesee Scientific, El Cajon, California, United States) with a working volume of 5 mb at 37 °C under 5% CO2 and passaged every 4 days. To establish stable cell pools, we transfected transposon plasmid and Hyperactive PiggyBac transposase plasmid (VectorBuilder, Chicago, Illinois, United States) at a 1 :1 (w:w) using Lipofectamine™ 2000 (Invitrogen, Carlsbad, California, United States) followed by 2 weeks of selection with antibiotics: 200ug / mL zeocin(InvivoGen, San Diego, California, United States), 15ug / mL blasticidin (InvivoGen, San Diego, California, United States) and 2 pg / mL of puromycin (Sigma- Aldrich, Saint Louis, Missouri, United States). The viable cell density (VCD) and cellviability were assessed using a BioProfile® FLEX2 Analyzer (Nova Biomedical, Waltham, Massachusetts, United States). The single-cell clones were isolated from the stable pools using limited dilution and seeded with 0.8 cells per well into 96-well plates.

[0191] Genome copy number analysis. The cell pellets from stable pools and single clones were collected for genomic DNA extraction. Genomic DNA was extracted using Quick-DNA™ Miniprep Plus Kit (Zymo Research Corporation, Irvine, California, United States) following the manufacturer’s instructions. qPCR was performed on genomic DNA samples using SYBR Green qPCR Master Mix (Thermo, Waltham, Massachusetts, United States) on the Bio-Rad system (Bio-Rad Laboratories, Hercules, California, United States). According to the manufacturer’s instructions, qPCR reaction mixtures contained 2X SYBR Green master mix, 400 nM of forward and reverse primers, 20 ng of genomic DNA, and up to 20 pL molecular biology water. Amplification was executed with the following conditions: 50 °C for 1 minute; 95 °C for 10 minutes; 40 cycles of: 95 °C for 15 seconds, 60 °C for 30 seconds, and 72 °C for 1:30 minutes. The primer sequences used for Cap, Rep52 / 40, Rep78 / 68, E2a, E4orf6, codon-optimized green fluorescent protein (GFP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are listed in Table 11.

[0192] RNA isolation for gene expression evaluation. The gene expression assay, including RNA isolation, cDNA reverse transcription and gene expression by RT-PCR, was previously described (Wang, Y., et al., Transcriptomic features reveal molecular signatures associated with recombinant adeno-associated virus production in HEK293 cells. Biotechnology Progress, 2023: p. e3346). The comparative cycle threshold (2-AACt) method was used to analyze the transcript level fold changes between different conditions, in this case the induction of different doxycycline concentrations. The primer sequences used for Cap, Rep52 / 40, Rep78 / 68, E2a, E4orf6, codon-optimized green fluorescent protein (GFP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are listed in Table 11.

[0193] rAAV Vector Production, rAAV Preparation and Analytical methods (genome titer, capsid titer). Cells in the logarithmic growth phase were seeded at a density of 5E+5 cells per well in a 6-well plate. After a 24-hour incubation, induction was performed for rAAV production with the addition of 5ug / mL doxycycline. After 68 hours post-induction, harvested cell cultures were aliquoted to 1 m volume (cells and supernatant inclusive) in 1.5 mL centrifuge tubes either for immediate analysis or stored at -80°C for future analysis. The genome titer and capsidtiter assays followed the previously published paper (Fu, Q., et al., Design space determination to optimize DNA complexation and full capsid formation in transient rAA V manufacturing.Biotechnology and Bioengineering, 2023). The specific productivity can be calculated as shown below.

[0194] Statistical Analysis. GraphPad Prism 9 was used for processing raw data and statistical analysis.

[0195] Results

[0196] Strategy and workflow. A stable producer cell line was established by performing transposon-mediated integration of the transfer cassette (containing GFP / transgene) and the assembly cassette (containing Cap) into the isolated packaging cell line, derived from the packaging cell line developed in Example 1. To achieve this goal, two strategies were used for stable integration, one was to integrate the assembly and transfer transposon donor vector sequentially (3rdSC4-7 seq pool), and another was to integrate the assembly and transfer transposon donor vector at one integration experiment (3rdSC4-7 double pool). The stable cell pools were generated after stable transfection of transposase and transposon donor vectors and antibiotic selection. Genome copy number of all viral components and rAAV productivity were first evaluated for stable cell pools. After confirming the detection of viral genome and capsid titer signal, single clone isolation was further conducted.

[0197] The clones were screened for their GFP and Cap copy numbers. Clones with genome copy numbers comparable or higher than those in the stable pools were selected and assessed for the genome copies of all viral components. The top-performing clone was identified and thoroughly characterized, including genome copy analysis, genome / capsid titer, cytotoxicity, and cell line stability.

[0198] Plasmid construction. Transposon donor vectors containing transgene and assembly cassette were constructed.

[0199] Bacteria clones were banked and subjected to plasmid extraction following Gibson assembly and transformation. Gel electrophoresis was utilized to verify and screen the size of the extracted plasmids from the clones.

[0200] Clones displaying the correct plasmids size were sent out for the whole plasmid sequencing. The corresponding plasmid maps are shown in Figure 8. Clones with confirmed sequences were banked, extracted, and filtered for transposon-mediated integration.

[0201] Stable pool evaluation. Reconstructed transfer and assembly donor cassettes were transfected along with hyperactive piggyBac® transposase plasmid in packaging cell line (2ndSC4-7). Antibiotic concentration, blasticidin, was elevated to 15 pg / mL to select cells with higher copy number of assembly cassettes. Puromycin (2 pg / mL) was used to select for cells containing transfer cassettes. After two weeks of antibiotic selection, the stable pool was established and evaluated.

[0202] Genome copy analysis of all viral components for stable pool was conducted. The absolute Cq value for all viral genes were shown in Table 12. The relative fold change before and after transposon integration were demonstrated in Figure 9A and Figure 9B. The viral components integrated from the replication cassettes had comparable genome copies in the stable cell pool as observed in the HEK293T packaging cell line (2ndSC4-7). This suggests that no replication genes were lost following transposon integration. Additionally, the copy number of Cap gene in stable pool increased up to 3.1-fold, compared to prior packaging cell line; and the copy number of GFP in stable pool increased up to 38-fold, compared to producer cell pool after 3 rounds of site-specific integration.Table 12. Relative genome copy analysis summary of viral components for stable cell poolsFootnote: 2ndSC 4-7: single clone, packaging cell line; 3rdSC 4-7 seq and double pools: the transposon mediated producer cell pools; SSI 3rd pool: site- specific integration mediated producer cell pool.

[0203] rAAV production was then evaluated for the stable pool. After the addition of 5 pg / mL doxycycline, the stable cell pool achieved 1.4E+10 vg / L genome titer and 1.3E+11 cp / L capsid titer (Figure 9C and Figure 9D). The genome titer generated by the stable cell pool was much lower than that achieved by the packaging cell line after induction. The high genome titer in the packaging cells might result from the incomplete digestion of the replicated genome and transgene plasmids during the sample treatment. Additionally, capsid titer in the stable pool was consistent with the results shown in the genome copy analysis, comparable and slightly higher than that in the packaging cell line.

[0204] Single clone isolation and genome copy screening. Approximately 500 wells were plated for single clone isolation. Out of 500 clones, 37 clones were expanded and screened for their genome copies of GFP and Cap. With transposon-mediated integration, higher homogeneity was expected as compared to cell line generated by random integration. Figure 10 shows the relative genome copies of GFP and Cap for all 37 clones, compared to these for the stable pool. The result suggests that only 3 clones out of 37 clones achieved comparable or higher genome copy of GFP and Cap than that in stable pool. Two clones (SC4-9, SC 1-8) along with other 2 randomly selected clones (SC2-2, SC2-3) were evaluated for their genome copies of all viral components, details shown in Table 13. It further confirmed that viral components (e.g. Rep68, DBP and E4orf6 from replication cassette) were retained from the packaging cell line and viral components (e.g. Cap and GFP) were comparable / higher than stable pool.Table 13. Relative genome copy analysis summary of viral components for selected single clones

[0205] Single clone 4-9 characterization and rAAV production. SC4-9 was selected as the top clone and fully characterized for rAAV production. The addition of 5 pg / mL doxycycline inducible reagent activated the viral gene expression and initiated the rAAV production, following the prior packaging cell line development study. The relative transcript level of these viral components was measured by RT-qPCR before and after induction. The transcript level of all viral components, controlled by inducible promoters, achieved 20-80-fold increase after doxycycline induction, as shown in Figure 11.

[0206] The absolute Cq value for SC4-9 before induction shown in Table 14 demonstrated the detection of viral gene transcription without the addition of doxycycline and indicated the leakage of the designed inducible circuit. The vector production capability of this stable cell line was further evaluated by measuring genome and capsid titers. Genome titer reached 3.73E+11 vg / L and capsid titer reached 2.01E+11 cp / L after the addition of doxycycline, achieving 27-fold and 1.4-fold increase respectively compared to non-induction condition (Figure 12 A and Figure 12B). Limited increase in Cap expression before and after induction indicated the leakage of circuit design. This result aligned with the transcript level changes described above, with the Cap gene showing the smallest increase. Additional translation and viral protein assembly steps contributed to the nonlinear relationship between fold changes in transcript level and in capsid titer. Limited amount of assembled capsids remained as a bottleneck for the low productivity of the established stable producer cell line. It was also noted that genome titer (3.73E+11 vg / L) was higher than capsid titer (2.01E+1 1 cp / L). This result might be due to the sensitivity and limitations of each assay in the low titer profile.Table 14. Transcript Cq value summary of all the viral components in the single clone producer cell line SC4-9 with 0 and 5 ug / mL doxycycline induction via RT-PCR assay

[0207] The cell growth performance and the cell line stability of the established stable producer cell line were further assessed, with results presented in Figures 13A and 13B. For growth performance (Figure 13 A), both the parental and producer cell line were seeded at the same initial density (5E4 cells per well) in 12-well plates. Viable cell density (VCD) and cell viability were monitored daily over 7 days. SC4-9 exhibited much slower growth rate compared to the 293T parental cells, indicating potential leakiness in viral component expression that may contribute to cytotoxicity. Regarding cell line stability (Figure 13B), SC4-9 was maintained for over 10 passages, with genome titers evaluated every five passages. The results demonstrated comparable titers, remaining within a similar order of magnitude throughout the 10 passages.

[0208] Discussion

[0209] A stable producer cell line for rAAV production was established in Example 2. In Example 1, a stable packaging cell line was developed with the design of an inducible circuit. The expression of viral components, such as Rep and helper genes, were controlled under an inducible circuit, to minimize the cytotoxicity. Viral components required for rAAV production were refactored to three cassettes based upon their functionality (replication, assembly, and transgene cassettes) and then integrated sequentially to pre-validated genome loci via sitespecific integration. The packaging cell line was derived after site-specific integration of replication and assembly cassettes. It requires additional transfection of transgene cassette for rAAV production and thus allows the easy switch between different transgenes. Due to the large insertion size, only 2 clones out of total 38 clones got further confirmed with targeted integration. The introduction of the doxycycline resulted in a significant increase in transcript levels of integrated viral components, yielding a genome titer of 8.46E+11 vg / L and a capsid titer of 8.79E+10 cp / L. However, after 3rd round of integration, it was observed that only limited amounts of capsids was produced, and no genome titer signal was detected at all. This might result from inadequate viral replication due to the low transgene copy numbers.

[0210] Genome replication is the one key limitation, as site-specific integration produces significantly fewer genome copies compared to the high numbers achieved through transient plasmid transfection . Genome encapsidation may be another bottleneck, with one study reporting only 0.3%-3% of total genomes were encapsidated. The detection of capsid confirmed the capsid production, but the relatively low amount of assembled capsids along with limited copy number of transgenes might be the reason for no genome titer in the stable producer derivedby multiple rounds of site-specific integration. The relatively low packaging efficiency, an inherent barrier in rAAV production, further contributes to the undetectable genome titer.

[0211] Thus here, in Example 2 transposon-mediated integration in the previous derived packaging cell line was performed, integrating additional cap gene and transgene.

[0212] Although random integration emerges as the gold standard for generating stable cell lines, it is demonstrated that this approach introduces significant challenges to cell line development process, such as cell-to-cell variability, transgene rearrangement, loss of copy number, and lack of molecular mechanisms. Increased risk of inducing cell line instability through random integration indicates the necessity of developing and utilizing alternative technologies. Transposase-mediated genomic integration is such a method that can allow increased level of transgene integration and better stability. By utilizing a hyperactive transposase, an optimized transposon, and optimal co-transfection ratio, it is expected that 50- 100 copies of the insert will stably integrate into the host genome. This increase in genome copy of gene of interest is anticipated to boost the genome and capsid titer accordingly.

[0213] Overall, transposon mediated integration of assembly and transgene cassettes in the packaging cell line increased the copy number of Cap gene up to 3 -fold and GFP up to 38-fold. The antibiotic concentration was increased to further select for cells with a higher number of integrated assembly cassettes. The limited increase in Cap gene expression could be attributed to the presence of pre-existing site-specific integrations, hindering the effective selection of cells containing high copy numbers. The increase in genome copy of GFP transgene was as expected. One outstanding single clone was isolated and characterized for rAAV production. The isolated stable producer cell line was able to result in 3.73E+1 Ivg / L genome titer and 2.01E+11 cp / L capsid titer, leaving room for improvement in the future study. This was only a proof-of-concept study aimed at stablishing the baseline producer cell.

[0214] Similar synthetic biology approach has been applied before for rAAV production: multiple inducible promoters were used to control the expression of viral components; three separate modules were integrated into HEK293s cells by Leap-in transposase mRNA mediated transfection. The baseline they isolated was about two orders magnitude lower than traditional triple-based production (Lee, Z., et al., Construction of an rAAV producer cell line through synthetic biology. ACS Synthetic Biology, 2022. 11(10): p. 3285-3295). Independent control over replication and packaging activity by varying the inducer levels and altering the viralcomponents led to higher capsid titer. To further understand the bottlenecks that limit the inducible cell line productivity, they conducted a comprehensive analysis of viral production kinetics using proteomics and other physical assays of viral components. They show that reducing the excessive expression of transgene resulted in higher genome and capsid titer. With additional optimization of induction profile and the addition of proteasome inhibitor, the rAAV produced reached the similar level as that in transient transfection (Lu, M., et al., Enhancing the production of recombinant adeno-associated virus in synthetic cell lines through systematic characterization. Biotechnology and bioengineering, 2024. 121(1): p. 341-354). Finally, multi- omics analysis further demonstrated that the balance of viral genome and capsid protein was key to productivity and quality. Thus, sequential integration of multiple copies of cap gene allowed the cap gene expression, further boosting rAAV productivity, leading to the final inducible rAAV high producing cell line (Lu, M., et al., Tuning capsid formation dynamics in recombinant adeno-associated virus producing synthetic cell lines to enhance full particle productivity. Biotechnology Journal, 2024. 19(3): p. 2400051).

[0215] Here, the titer achieved in the baseline producer cell was significantly lower than obtained by traditional transient transfection with commercially available plasmids, indicating the potential for optimization and improvement in inducible system. With the use of a single type of Tet-on promoter, it can reduce the need for multiple inducible reagents and simplify the process, but fine-tuning gene expression levels and timing to meet optimal production requirements becomes more complex. To better regulate the gene expression level and timing, the inducible promoters can be tuned by the number and the spacing between of tetO sites. Protein expression control can be achieved by screening Kozak sequence variants and modulating the translational efficiency. Additionally, a systematic characterization of rAAV production in inducible cell line can be conducted to identify potential factors that restrain the productivity. Corresponding tuning strategies can then be applied to increase the productivity of these synthetic cell lines.

[0216] Conclusion

[0217] An inducible stable producer cell line for AAV production was successfully produced using a combination of site-specific and transposon-mediated integration approaches.. Upon the addition of the inducing reagent, transcript levels of integrated viral components significantly increased, resulting in a genome titer of 3.73E+11 vg / L and a capsid titer of 2.01E+11 cp / L.However, cell growth was notably slower compared to the parental cells, likely due to leakage of toxic genes. Stability testing confirmed that the cell line remained stable for at least 10 passages. Future research will focus on optimizing gene expression levels and timing for various viral components, as well as reducing toxic gene leakage, to further enhance the productivity and efficiency of the inducible rAAV-producing stable cell line.

[0218] The preceding general areas of utility are given by way of example only and are not intended to be limiting on the scope of the present disclosure and appended claims. Additional objects and advantages associated with the compositions, methods, and processes of the present disclosure will be appreciated by one of ordinary skill in the art in light of the instant claims, description, and examples. For example, the various aspects and embodiments of the disclosure may be utilized in numerous combinations, all of which are expressly contemplated by the present description. These additional aspects and embodiments are expressly included within the scope of the present disclosure. The publications and other materials used herein to illuminate the background of the disclosure, and in particular cases, to provide additional details respecting the practice, are incorporated by reference.

[0219] Thus, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims. It is understood that the detailed examples and embodiments described herein are given by way of example for illustrative purposes only, and are in no way considered to be limiting to the disclosure. Various modifications or changes in light thereof will be suggested to persons skilled in the art and are included within the spirit and purview of this application and are considered within the scope of the appended claims. For example, the relative quantities of the ingredients may be varied to optimize the desired effects, additional ingredients may be added, and / or similar ingredients may be substituted for one or more of the ingredients described. Additional advantageous features and functionalities associated with the systems, methods, and processes of the present disclosure will be apparent from the appended claims. Moreover, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMSWhat Is Claimed Is:

1. A plasmid (e.g., an assembly plasmid or an assembly donor plasmid) comprising or consisting essentially of:(a) a left or 5’ homology arm sequence;(b) a first assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ :(1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and(2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an inducible transactivator (e.g., a reverse tetracycline-controlled transactivator (rtTA));(c) a second assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ :(1) a regulatory element or expression control element that is responsive to the inducible transactivator (e.g., the inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., TRE3G or CMV promoter)); and(2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: an adeno-associated virus capsid gene (e.g., a sequence encoding an adeno- associated virus VP1, VP2, and VP3 proteins); an internal ribosome entry site (IRES) or a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A), preferably IRES; and a sequence encoding an adeno-associate virus Reg52 and Reg40 proteins; and(d) a right or 3’ homology arm sequence,wherein the left or 5’ homology arm sequence and the right or 3’ homology arm sequence facilitate homology directed repair (HDR) in a genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor).

2. The plasmid of claim 1, wherein at least one of: the genomic safe harbor site comprises or is ROSA26, Adeno- Associated Virus Integration Site 1 (AAVS1), or C-C chemokine receptor type 5 (CCR5) locus, preferably ROSA26; the second assembly expression cassette is optionally flanked by recombinase sequences (e.g., attB or attP, preferably c / / / B); or a combination thereof.

3. The plasmid of claim 1 or 2, wherein at least one of:(a) the plasmid further comprises or consists essentially of, 5’ of the left or 5’ homology arm sequence and 3’ of the right or 3’ homology arm sequence, at least one of: an original of replication (ori); an ampicillin resistance expression cassette comprising, consisting essentially of, or consisting of a regulatory element or expression control element (e.g., a promoter, such as an ampicillin resistance promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of an ampicillin resistance gene (e.g., AmpR); a aminoglycoside resistance expression cassette comprising, consisting essentially of, or consisting of a regulatory element or expression control element (e.g., a promoter, such as a ccdB promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of a gene that provides at least one of neomycin resistance, kanamycin resistance, or a combination thereof (e.g., NeoR / KanR); an expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a catabolite activator protein (CAP) binding site, a lac promoter, a lac operator, a T3 promoter, a Cytomegalovirus(CMV) promoter, a CMV enhancer, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding a fluorescence protein (e.g., green fluorescent protein or enhanced green fluorescence protein); or a combination thereof;(b) the plasmid further comprises or consists essentially of one or more (e.g., 1, 2, 3, or more) antibiotic resistance expression cassette located 3’ of the left or 5’ homology arm sequence and 5’ of the right or 3’ homology arm sequence, wherein the antibiotic resistance expression cassette is at least one of a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting, of a blastocidin S resistance gene (e.g., BlastR); a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e.g., BleoR); a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a T7 promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting, of a puromycin resistance gene (e.g., PuroR); or a combination thereof, preferably a blastocidin S resistance expression cassette; or(c) a combination thereof.

4. A plasmid (e.g., an assembly plasmid, an assembly transposon plasmid, an assembly donor plasmid, or an assembly transposon donor plasmid) comprising or consisting essentially of, in order from 5’ to 3’ :(a) a right or 3’ inverted transposase terminal repeat sequence;(b) an assembly transposon comprising or consisting essentially of:(1) a first assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an inducible transactivator (e.g., a reverse tetracycline- controlled transactivator (rtTA));(2) a second assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : a regulatory element or expression control element that is responsive to the inducible transactivator (e.g., the inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., TRE3G or CMV promoter)); and a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: an adeno-associated virus capsid gene (e.g., a sequence encoding an adeno-associated virus VP1, VP2, and VP3 proteins); an internal ribosome entry site (IRES) or a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A), preferably IRES; and a sequence encoding an adeno-associate virus Reg52 and Reg40 proteins; and(c) a left or 5’ inverted transposase terminal repeat sequence,wherein the left or 5’ inverted transposase terminal repeat sequence and the right or 3’ inverted transposase terminal repeat sequence facilitate excision of the transposon from the plasmid and insertion or integration of the assembly transposon in one or more (e.g., about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5) 4-base insertion site in a genome (e.g., a mammalian genome or a human genome).

5. The plasmid of claim 4, wherein at least one of: the second assembly expression cassette is optionally flanked by recombinase sequences (e.g., attQ or attP preferably c / / / B); the 4-base insertion site is a TTAA site or a TTAT site, preferably a TTAA site; the left or 5’ inverted transposase terminal repeat sequence and the right or 3’ inverted transposase terminal repeat sequence are reverse complements; each of the left or 5’ inverted transposase terminal repeat sequence and the right or 3’ inverted transposase terminal repeat sequence is about 8 to about 1200 nucleotides (e.g., about 12 to about 200 nucleotides, about 28 to about 200 nucleotides, about 28 to about 600 nucleotides, or about 200 to 1200 nucleotides); or a combination thereof.

6. The plasmid of claim 4 or 5, wherein at least one of:(a) the plasmid further comprises or consists essentially of, 5’ of the right or 3’ inverted transposase terminal repeat sequence and 3’ of the left or 5’ inverted transposase terminal repeat sequence, at least one of: an original of replication (ori); an ampicillin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as an ampicillin resistance promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an ampicillin resistance gene (e.g., AmpR); a aminoglycoside resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., aI l lpromoter, such as a ccdB promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of neomycin resistance, kanamycin resistance, or a combination thereof (e.g., NeoR / KanR); an expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a catabolite activator protein (CAP) binding site, a lac promoter, a lac operator, a T3 promoter, a Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding a fluorescence protein (e.g., green fluorescent protein or enhanced green fluorescence protein); or a combination thereof;(b) the plasmid further comprises or consists essentially of one or more (e.g., 1, 2, 3, or more) antibiotic resistance expression cassette located 3’ of the right or 3’ inverted transposase terminal repeat sequence and 5’ of the left or 5’ inverted transposase terminal repeat sequence, wherein the antibiotic resistance expression cassette is at least one of: a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of ,a regulatory element or expression control element (e.g., a promoter, such as a SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR); a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e.g., BleoR); and a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., apromoter, such as a cytomegalovirus (CMV) promoter, a T7 promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g., PuroR); or a combination thereof, preferably a blastocidin S resistance expression cassette; or(c) a combination thereof.

7. A plasmid (e.g., a replication plasmid or a replication donor plasmid) comprising or consisting essentially of(a) a left or 5’ homology arm sequence;(b) a first replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ :(1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter)); and(2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an adeno-associate virus Reg78 and Reg68 proteins;(c) a second replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ :(1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter));(2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: a deoxyribonucleic acid (DNA)-binding protein (DBP) gene (e.g., Transcription Factor E2-Alpha (E2a) gene); a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A) or an internal ribosome entry site (IRES)), preferably P2A or T2A; and an adenovirus E4 region open reading frame 6 (E4orf6) gene; and(d) a right or 3’ homology arm sequence, wherein the left or 5’ homology arm sequence and the right or 3’ homology arm sequence facilitate homology directed repair (HDR) in a genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor).

8. The plasmid of claim 7, wherein the genomic safe harbor site comprises or is ROSA26, Adeno- Associated Virus Integration Site 1 (AAVS1), or C-C chemokine receptor type 5 (CCR5) locus, preferably Adeno- Associated Virus Integration Site 1 (AAVS1).

9. The plasmid of claim 7 or 8, wherein:(a) the plasmid further comprises or consists essentially of, 5’ of the left or 5’ homology arm sequence and 3’ of the right or 3’ homology arm sequence, at least one of: an original of replication (ori); an ampicillin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as an ampicillin resistance promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an ampicillin resistance gene (e.g., AmpR); a aminoglycoside resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a ccdB promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of neomycin resistance, kanamycin resistance, or a combination thereof (e.g., NeoR / KanR);an expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a catabolite activator protein (CAP) binding site, a lac promoter, a lac operator, a T3 promoter, a Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding a fluorescence protein (e.g., green fluorescent protein or enhanced green fluorescence protein); or a combination thereof;(b) the plasmid further comprises or consists essentially of one or more (e.g., 1, 2, 3, or more) antibiotic resistance expression cassette located 3’ of the left or 5’ homology arm sequence and 5’ of the right or 3’ homology arm sequence, wherein the antibiotic resistance expression cassette is at least one of: a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a SV40 promoter) and a synthetic open reading frame molecule comprising or of a blastocidin S resistance gene (e.g., BlastR); a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e.g., BleoR); a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a T7 promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g.,PuroR); or a combination thereof,preferably a glycopeptide antibiotic or bleomycin resistance expression cassette; or(c) a combination thereof.

10. A plasmid (e.g., a transfer / transgene plasmid or a transfer / transgene donor plasmid) comprising or consisting essentially of, in order from 5’ to 3’ :(a) a left or 5’ homology arm sequence;(b) a first inverted terminal repeat (ITR, such as an adeno-associated virus ITR);(c) a transgene or transfer expression cassette comprising, consisting essentially of, or consisting of:(1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and(2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a transgene or a gene of interest;(d) a second inverted terminal repeat (ITR, such as an adeno-associated virus ITR);(e) a virus-associated ribonucleic acids (VA RNAs) expression cassette comprising, consisting essentially of, or consisting of, a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an Adenovirus VA-RNAs; and(f) a right or 3 ’ homology arm sequence, wherein the left or 5’ homology arm sequence and the right or 3’ homology arm sequence facilitate homology directed repair (HDR) in a genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor).

11. The plasmid of claim 10, wherein at least one of: the genomic safe harbor site comprises or is ROSA26, Adeno- Associated Virus Integration Site 1 (AAVS1), or C-C chemokine receptor type 5 (CCR5) locus, preferably Adeno- Associated Virus Integration Site 1 (AAVS1); the transgene or transfer expression cassette is flanked by recombinase sequences (e.g., attP or attB, preferably attP) or a combination thereof.

12. The plasmid of claim 10 or 1 1, wherein:(a) the plasmid further comprises or consists essentially of, 5’ of the left or 5’ homology arm sequence and 3’ of the right or 3’ homology arm sequence, at least one of: an original of replication (ori); an ampicillin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as an ampicillin resistance promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an ampicillin resistance gene (e.g., AmpR); a aminoglycoside resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a ccdB promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of neomycin resistance, kanamycin resistance, or a combination thereof (e.g., NeoR / KanR); or a combination thereof(b) the plasmid further comprises or consists essentially of one or more (e.g., 1, 2, 3, or more) antibiotic resistance expression cassette located 3’ of the left or 5’ homology arm sequence and 5’ of the right or 3’ homology arm sequence, wherein the antibiotic resistance expression cassette is at least one of: a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR); a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene thatprovides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e.g., BleoR); a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a T7 promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g.,PuroR); or a combination thereof, preferably a puromycin resistance expression cassette; or(c)a combination there.

13. A plasmid (e.g., a transfer / transgene plasmid, a transfer / transgene transposon plasmid, a transfer / transgene donor plasmid, or a transfer / transgene transposon donor plasmid) comprising or consisting essentially of, in order from 5’ to 3’:(a) a left or 3’ inverted transposase terminal repeat sequence;(b) a transfer transposon comprising or consisting essentially of:(1) a first inverted terminal repeat (ITR, such as an adeno-associated virus ITR);(2) a transgene or transfer expression cassette comprising, consisting essentially of, or consisting of: a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a transgene or a gene of interest;(3) a second inverted terminal repeat (ITR, such as an adeno-associated virus ITR);(4) a virus-associated ribonucleic acids (VA RNAs) expression cassette comprising, consisting essentially of, or consisting of, a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an Adenovirus VA-RNAs; and(c) a right or 5’ inverted transposase terminal repeat sequence, wherein the left or 5’ inverted transposase terminal repeat sequence and the right or 3’ inverted transposase terminal repeat sequence facilitate excision of the transfer transposon from the plasmid and insertion or integration of the transfer transposon in one or more (e.g., about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5) 4-base insertion site in a genome (e.g., a mammalian genome or a human genome).

14. The plasmid of claim 13, wherein at least one of: the transgene or transfer expression cassette is flanked by recombinase sequences (e.g., atfP or altB, preferably a / tPy the 4-base insertion site is a TTAA site or a TTAT site, preferably a TTAA site; the left or 5’ inverted transposase terminal repeat sequence and the right or 3’ inverted transposase terminal repeat sequence are reverse complements; each of the left or 5’ inverted transposase terminal repeat sequence and the right or 3’ inverted transposase terminal repeat sequence is about 8 to about 1200 nucleotides (e.g., about 12 to about 200 nucleotides, about 28 to about 200 nucleotides, about 28 to about 600 nucleotides, or about 200 to 1200 nucleotides); or a combination thereof.

15. The plasmid of claim 13 or 14, wherein at least one of:(a) the plasmid further comprises or consists essentially of, 5’ of the right or 3’ inverted transposase terminal repeat sequence and 3’ of the left or 5’ inverted transposase terminal repeat sequence, at least one of: an original of replication (ori); an ampicillin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as an ampicillin resistance promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an ampicillin resistance gene (e.g., AmpR);a aminoglycoside resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a ccdB promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of neomycin resistance, kanamycin resistance, or a combination thereof (e.g., NeoR / KanR); or a combination thereof(b) the plasmid further comprises or consists essentially of one or more (e.g., 1, 2, 3, or more) antibiotic resistance expression cassette located 3’ of the right or 3’ inverted transposase terminal repeat sequence and 5’ of the left or 5’ inverted transposase terminal repeat sequence, wherein the antibiotic resistance expression cassette is at least one of: a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of a regulatory element or expression control element (e.g., a promoter, such as a SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR); a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e.g., BleoR); a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a T7 promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g.,PuroR); or a combination thereof, preferably a puromycin resistance expression cassette; or(c) a combination there.

16. The plasmid of any one of claims 10-15, wherein the transgene is a fusion protein comprising a gene of interest fused to: a sequence encoding a woodchuck hepatitis virus regulatory element (WPRE); a sequence encoding an endosome-disruptive peptide (e.g., a protein or peptide that facilitates endosomal escape, such as a protective antigen (PA)); or a combination of both.

17. A method of making a packaging cell, the method comprising:(a) transfecting a cell (e g., human embryonic kidney 293 cell (HEK293) or HEK293T) with an assembly donor plasmid of any one of claims 1-3 and a clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) nuclease (e.g., Cas9) plasmid for the assembly donor plasmid that facilitates the insertion of the sequences between the 5’ or left homology arm sequence and the right or 3’ homology arm sequence into a first genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette; and(b) transfecting the cell transfected with the assembly donor plasmid with a replication donor plasmid of any one of claims 7-9 and a clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) nuclease (e.g., Cas9) plasmid for the replication donor plasmid that facilitates the insertion of the sequences between the 5’ or left homology arm sequence and the right or 3’ homology arm sequence into a second genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette, wherein: the assembly donor plasmid and the replication donor plasmid include different antibiotic resistance genes (e.g., genes that confer resistance to different antibiotics); and the first genomic safe harbor site and the second genomic safe harbor site are different genomic safe harbor sites.

18. A method of making a packaging cell, the method comprising:(a) transfecting a cell (e.g., human embryonic kidney 293 cell (HEK293) or HEK293T) with a replication donor plasmid of any one of claims 7-9 and a clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) nuclease (e.g., Cas9) plasmid for the replication donor plasmid that facilitates the insertion of the sequences between the 5’ or left homology arm sequence and the right or 3’ homology arm sequence into a second genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette; and(b) transfecting the cell transfected with the replication donor plasmid with an assembly donor plasmid of any one of claims 1-3 and a clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) nuclease (e.g., Cas9) plasmid for the assembly donor plasmid that facilitates the insertion of the sequences between the 5’ or left homology arm sequence and the right or 3’ homology arm sequence into a first genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette, wherein: the assembly donor plasmid and the replication donor plasmid include different antibiotic resistance genes (e.g., genes that confer resistance to different antibiotics); and the first genomic safe harbor site and the second genomic safe harbor site are different genomic safe harbor sites.

19. The method of claim 17 or 18, wherein (a) the first genomic safe harbor site is ROSA26, (b) the second genomic safe harbor site is Adeno-Associated Virus Integration Site 1 (AAVS1), or (c) a combination thereof.

20. The method of any one of claims 17-19, wherein the Cas nuclease (e.g., Cas9) plasmid for the assembly donor plasmid, the Cas nuclease (e.g., Cas9) plasmid for the replication donor plasmid, or a combination thereof, comprises or consists essentially of, in order from 5’ to 3’:(a) a Cas nuclease expression cassette comprising or consisting essentially of:(1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a Cytomegalovirus (CMV) promoter and / or a CMV enhancer, U6 promoter, or a combination thereof); and(2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a Cas nuclease gene (e.g., Cas9 and(b) a single guide RNA (sgRNA) expression cassette comprising or consisting essentially of:(1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a U6 promoter, a Cytomegalovirus (CMV) promoter and / or a CMV enhancer, or a combination thereof); and(2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a single guide RNA (sgRNA) comprising, consisting essentially of, or consisting of, a trans-activating CRISPR RNA (tracrRNA) for the Cas nuclease (e.g., Cas9) and a CRISPR RNA (crRNA) comprising, consisting essentially of, or consisting of, a nucleotide sequence (e.g., 17 to 20 nucleotides) that is complementary to a genomic safe harbor (e.g., a mammalian genomic safe harbor or a human genomic safe harbor).

21. The method of claim 20, wherein the Cas nuclease gene comprises or consists essentially of a sequence that encodes, in order from 5’ to 3’, a Cas nuclease (e.g., Cas9), a 2A peptide (e.g., P2A, T2A, E2A, or F2A, preferably T2A or P2A), and a fluorescence protein (e.g., mCherry, green fluorescent protein, or enhanced green fluorescence protein, preferably mCherry).

22. A packaging cell line produced according to the method of anyone of claims 17- 21 or comprising or consisting essentially of:(a) a first assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ :(1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and(2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an inducible transactivator gene (e.g., a reverse tetracycline- controlled transactivator gene ( / 7 )),(b) a second assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ :(1) a regulatory element or expression control element that is responsive to the inducible transactivator (e.g., the inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., TRE3G or CMV promoter)); and(2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: an adeno-associated virus capsid gene (e.g., a sequence encoding an adeno- associated virus VP1, VP2, and VP3 proteins); an internal ribosome entry site (IRES) or a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A), preferably IRES; and a sequence encoding an adeno-associate virus Reg52 and Reg40 proteins;(c) a first replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ :(1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter)); and(2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an adeno-associate virus Reg78 and Reg68 proteins; and(d) a second replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ :(1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter)); and(2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: a deoxyribonucleic acid (DNA)-binding protein (DBP) gene (e.g., Transcription Factor E2-Alpha (E2a) gene); a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A) or an internal ribosome entry site (IRES)), preferably P2A or T2A; and an adenovirus E4 region open reading frame 6 (E4orf6) gene; wherein: the first assembly expression cassette and the second assembly expression cassette are integrated or inserted in a first genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor); and the first replication expression cassette and the second replication expression cassette are integrated or inserted in a second genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor).

23. The packaging cell line of claim 22, further comprising or consisting essentially of, at least one of: an assembly antibiotic resistance expression cassette located between the first assembly expression cassette and the second assembly expression cassette; a replication antibiotic resistance expression cassette located between the first replication expression cassette and the second replication expression cassette; or a combination thereof, optionally, the assembly antibiotic resistance expression cassette, the replication antibiotic resistance expression cassette, or a combination thereof, is at least one of:a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR), preferably the assembly antibiotic resistance expression cassette is a blastocidin S resistance expression cassette; a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising or of a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e.g., BleoR), preferably the assembly antibiotic resistance expression cassette is glycopeptide antibiotic or bleomycin resistance; and a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a T7 promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising or of a puromycin resistance gene (e.g.,PuroR).

24. A method of making a recombinant Adeno-associated virus producer cell, the method comprising:(a) providing a packaging cell of claim 22 or 23, or produced according to the method of any one of claims 17-21; and(b) transfecting the packaging cell with a transfer donor plasmid of any one of claims 10- 12, 15, or 16 and a clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) nuclease (e.g., Cas9) plasmid for the transfer donor plasmid that facilitates the insertion of the sequences between the 5’ or left homology arm sequence and the right or 3’ homology arm sequence into a third genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette,wherein: the assembly donor plasmid, the replication donor plasmid, and the transfer donor plasmid include different antibiotic resistance genes (e.g., genes that confer resistance to different antibiotics); and the first genomic safe harbor site, the second genomic safe harbor site, and the third genomic safe harbor site are different genomic safe harbor sites.

25. The method of claim 24, wherein at least one of: the third genomic safe harbor site is C-C chemokine receptor type 5 (CCR5) locus; the Cas nuclease (e.g., Cas9) plasmid for the transfer donor plasmid comprises or consists essentially of, in order from 5’ to 3’ :(a) a Cas nuclease expression cassette comprising or consisting essentially of:(1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a Cytomegalovirus (CMV) promoter and / or a CMV enhancer, U6 promoter, or a combination thereof); and(2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a Cas nuclease gene (e.g., Cas9), and(b) a single guide RNA (sgRNA) expression cassette comprising or consisting essentially of:(1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a U6 promoter, a Cytomegalovirus (CMV) promoter and / or a CMV enhancer, or a combination thereof); and(2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a single guide RNA (sgRNA) comprising, consisting essentially of, or consisting of a trans-activating CRISPR RNA (tracrRNA) for the Cas nuclease (e.g., Cas9) and a CRISPR RNA (crRNA) comprising, consisting essentially of, or consisting of, a nucleotide sequence (e.g., 17 to 20 nucleotides) that is complementary to a genomic safe harbor (e.g., a mammalian genomic safe harbor or a human genomic safe harbor); or a combination thereof.

26. The method of claim 25, wherein the Cas nuclease gene comprises or consists essentially of a sequence that encodes, in order from 5’ to 3’, a Cas nuclease (e.g., Cas9), a 2A peptide (e.g., P2A, T2A, E2A, or F2A, preferably T2A or P2A), and a fluorescence protein (e.g., mCherry, green fluorescent protein, or enhanced green fluorescence protein, preferably mCherry).

27. A method of making a recombinant Adeno-associated virus producer cell, the method comprising:(a) providing a packaging cell of claim 22 or 23, or produced according to the method of any one of claims 17-21; and(b) transfecting the packaging cell with a transfer transposon donor plasmid of any one of claims 13-16 and a transposase plasmid for the transfer transposon donor plasmid that facilitates the insertion or integration of the transfer transposon in one or more (e.g., about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5) 4-base insertion site in a genome (e.g., a mammalian genome or a human genome), and performing antibiotic selection for the antibiotic resistance gene in the antibiotic resistance expression cassette, wherein: the transposase plasmid comprises or consists essentially of a transposase expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’, a regulatory element or expression control element (e.g., a promoter), and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a transposase sequence; the assembly donor plasmid, the replication donor plasmid, and the transfer transposon donor plasmid include different antibiotic resistance genes (e.g., genes that confer resistance to different antibiotics); and the first genomic safe harbor site and the second genomic safe harbor site are different genomic safe harbor sites.

28. The method of claim 27, wherein at least one of:(a) the transposase plasmid further comprises, consists essentially of, or consists of, at least one of: an original of replication (ori); an ampicillin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as an ampicillin resistance promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an ampicillin resistance gene (e.g., AmpR); a aminoglycoside resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a ccdB promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of neomycin resistance, kanamycin resistance, or a combination thereof (e.g., NeoR / KanR); an expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as a catabolite activator protein (CAP) binding site, a lac promoter, a lac operator, a T3 promoter, a Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding a fluorescence protein (e.g., green fluorescent protein or enhanced green fluorescence protein); or a combination thereof;(b) the transposase plasmid further comprises, consists essentially of, or consists of, one or more (e.g., 1, 2, 3, or more) antibiotic resistance expression cassette, wherein the antibiotic resistance expression cassette comprise, consists essentially of, or consists of, at least one of: a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a SV40 promoter) and a synthetic open reading frame moleculecomprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR); a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e.g., BleoR); a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g., PuroR); or a combination thereof; or a combination thereof, preferably a glycopeptide or bleomycin resistance expression cassette (e.g., zeocin resistance);(c) a combination thereof.

29. The method of any one of claim 17-21 or 24-28, wherein performing antibiotic selection comprises administering an antibiotic for at least 2 weeks (e.g., 2 to 3 weeks or 2 weeks).

30. The method of any one of claim 17-21 or 24-29, wherein the antibiotic comprises or is blastocidin S, bleomycin, phleomycin, Zeocin, or puromycin.

31. The method of any one of claim 17-21 or 24-30, further comprising, consisting essentially of, or consisting of: after transfection and antibiotic selection, isolating a single clone comprising the inserted sequence;after transfection and antibiotic selection, confirming site-specific integration of the inserted sequence (e.g., polymerase chain reaction (such as 573’ junction polymerase chain reaction and / or out-to-out polymerase chain reaction), copy number analysis (e.g., quantitative polymerase chain reaction), or a combination thereof); after transfection and antibiotic selection, contacting the cell with an agent (e.g., tetracycline, doxycycline, or derivative thereof) the induces the inducible transactivator (e.g., a reverse tetracycline-controlled transactivator (rtTA)); after transfection and antibiotic selection (e.g., after contacting the cell with the agent that induces the inducible transactivator), evaluating expression (e.g., reverse transcriptase polymerase chain reaction, reverse transcriptase quantitative polymerase chain reaction, western blot, or a combination thereof) of the inserted genes; or a combination thereof.

32. The method of any one of claims 17-21 or 24-31, wherein transfecting comprises or consists essentially of: (a) inducing the cell to be in S / G2 phase, (b) contacting the cell with an inhibitor (e.g., a small molecule inhibitor) of the non-homologous end joining (NHEJ) pathway,(c) enriching the donor plasmid near the Cas9-induced double stranded break (DSB) in the DNA,(d) co-expressing one or more DNA repair protein involved in homology directed repair (HDR),(e) liposomal transfecting (e.g., transfecting with a cationic liposome based reagent), or (f) a combination thereof.

33. The method of claim 24-32, further comprising or consisting essentially of producing recombinant adeno-associated virus.

34. The method of claim 33, wherein producing recombinant adeno-associate virus comprises contacting the recombinant Adeno-associated virus producer cell with an agent (e.g., tetracycline, doxycycline, or derivative thereof) the induces the inducible transactivator (e.g., a reverse tetracycline-controlled transactivator (rtTA)).

35. A recombinant Adeno-associated virus producer cell produced according to the method of anyone of claims 24-26 or comprising or consisting essentially of:(a) a first assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ :(1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and(2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an inducible transactivator gene (e.g., a reverse tetracycline- controlled transactivator gene (r / 774));(b) a second assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ :(1) a regulatory element or expression control element that is responsive to the inducible transactivator (e g., the inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., TRE3G or CMV promoter)); and(2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: an adeno-associated virus capsid gene (e.g., a sequence encoding an adeno- associated virus VP1, VP2, and VP3 proteins); an internal ribosome entry site (IRES) or a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A), preferably IRES; and a sequence encoding an adeno-associate virus Reg52 and Reg40 proteins;(c) a first replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ :(1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter)); and(2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an adeno-associate virus Reg78 and Reg68 proteins;(d) a second replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ :(1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter)); and(2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: a deoxyribonucleic acid (DNA)-binding protein (DBP) gene (e.g., Transcription Factor E2-Alpha (E2a) gene); a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A) or an internal ribosome entry site (IRES)), preferably P2A or T2A; and an adenovirus E4 region open reading frame 6 (E4orf6) gene;(e) a first inverted terminal repeat (ITR, such as an adeno-associated virus ITR);(f) a transgene or transfer expression cassette comprising, consisting essentially of, or consisting of:(1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and(2) a synthetic open reading frame molecule comprising or of a transgene or a gene of interest;(g) a second inverted terminal repeat (ITR, such as an adeno-associated virus ITR); and(h) a virus-associated ribonucleic acids (VA RNAs) expression cassette comprising, consisting essentially of, or consisting of, a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an Adenovirus VA-RNAs,wherein: the first assembly expression cassette and the second assembly expression cassette are integrated or inserted in a first genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor) of the recombinant Adeno-associated virus producer cell; the first replication expression cassette and the second replication expression cassette are integrated or inserted in a second genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor) of the recombinant Adeno- associated virus producer cell; and the first inverted terminal repeat, the transgene / transfer expression cassette, the second inverted terminal repeat, and the expression cassette for the Adenovirus virus- associated ribonucleic acids (VA RNAs) are integrated or inserted in a third genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor) of the recombinant Adeno-associated virus producer cell.

36. A recombinant Adeno-associated virus producer cell produced according to the method of anyone of claims 27-33 or comprising or consisting essentially of:(a) a first assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ :(1) a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and(2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an inducible transactivator gene (e.g., a reverse tetracycline- controlled transactivator gene (rlTA) ,(b) a second assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ :(1) a regulatory element or expression control element that is responsive to the inducible transactivator (e.g., the inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit,such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., TRE3G or CMV promoter)); and(2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of an adeno-associated virus capsid gene (e.g., a sequence encoding an adeno- associated virus VP1, VP2, and VP3 proteins); an internal ribosome entry site (IRES) or a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A), preferably IRES; and a sequence encoding an adeno-associate virus Reg52 and Reg40 proteins;(c) a first replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ :(1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter)); and(2) a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an adeno-associate virus Reg78 and Reg68 proteins;(d) a second replication expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ :(1) a regulatory element or expression control element that is responsive to an inducible transactivator (e.g., an inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., CMV promoter)); and(2) a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting ofa deoxyribonucleic acid (DNA)-binding protein (DBP) gene (e.g., Transcription Factor E2-Alpha (E2a) gene); a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A) or an internal ribosome entry site (IRES)), preferably P2A or T2A; and an adenovirus E4 region open reading frame 6 (E4orf6) gene;(e) an assembly transposon comprising or consisting essentially of, in order from 5’ to 3’ :(1) a first assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, an inducible transactivator (e.g., a reverse tetracycline- controlled transactivator (rtTA));(2) a second assembly expression cassette comprising, consisting essentially of, or consisting of, in order from 5’ to 3’ : a regulatory element or expression control element that is responsive to the inducible transactivator (e.g., the inducible transactivator that binds and induces expression when the regulatory element or expression control element, such as a promoter, is induced, such as a Tet-on promoter or a tetracycline response unit, such as one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tet operator (TetO) and a promoter (e.g., TRE3G or CMV promoter)); and a polycistronic synthetic open reading frame molecule comprising, consisting essentially of, or consisting of: an adeno-associated virus capsid gene (e.g., a sequence encoding an adeno-associated virus VP1, VP2, and VP3 proteins); an internal ribosome entry site (IRES) or a sequence encoding a 2A peptide (e.g., P2A, T2A, E2A, or F2A), preferably IRES; and a sequence encoding an adeno-associate virus Reg52 and Reg40 proteins;(f) a transfer transposon comprising or consisting essentially of, in order from 5’ to 3’:(1) a first inverted terminal repeat (ITR, such as an adeno-associated virus ITR);(2) a transgene or transfer expression cassette comprising, consisting essentially of, or consisting of a regulatory element or expression control element (e.g., a promoter, such as a constitutively active promoter, such as Cytomegalovirus (CMV) promoter, a CMV enhancer, or a combination thereof); and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a transgene or a gene of interest;(3) a second inverted terminal repeat (ITR, such as an adeno-associated virus ITR);(4) a virus-associated ribonucleic acids (VA RNAs) expression cassette comprising, consisting essentially of, or consisting of, a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a sequence encoding an Adenovirus VA-RNAs, wherein: the first assembly expression cassette and the second assembly expression cassette are integrated or inserted in a first genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor) of the recombinant Adeno-associated virus producer cell; the first replication expression cassette and the second replication expression cassette are integrated or inserted in a second genomic safe harbor site (e.g., a mammalian genomic safe harbor or a human genomic safe harbor) of the recombinant Adeno- associated virus producer cell; the assembly transposon is integrated or inserted into one or more (e.g., about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5) 4-base insertion site in a genome (e.g., a mammalian genome or a human genome) of the recombinant Adeno-associated virus producer cell; and the transfer transposon is integrated or inserted into one or more (e.g., about 1 to about 60, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20,about 1 to about 10, or about 1 to about 5) 4-base insertion site in a genome (e g., a mammalian genome or a human genome) of the recombinant Adeno-associated virus producer cell.

37. The producer cell of claim 35 or 36, further comprising or consisting essentially of at least one of: an assembly antibiotic resistance expression cassette located between the first assembly expression cassette and the second assembly expression cassette; a replication antibiotic resistance expression cassette located between the first replication expression cassette and the second replication expression cassette; a transfer / transgene antibiotic resistance expression cassette located between the transgene / transfer expression cassette and the expression cassette for an Adenovirus virus- associated ribonucleic acids; or a combination thereof, optionally, the assembly antibiotic resistance expression cassette, the replication antibiotic resistance expression cassette, the transfer / transgene antibiotic resistance expression cassette, or a combination thereof, is at least one of: a blastocidin S resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a SV40 promoter) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a blastocidin S resistance gene (e.g., BlastR), preferably the assembly antibiotic resistance expression cassette is a blastocidin S resistance expression cassette; a glycopeptide antibiotic or bleomycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a gene that provides at least one of bleomycin resistance, phleomycin resistance, Zeocin resistance, or a combination thereof (e.g., BleoR), preferably the assemblyantibiotic resistance expression cassette is glycopeptide antibiotic or bleomycin resistance; a puromycin resistance expression cassette comprising, consisting essentially of, or consisting of, a regulatory element or expression control element (e.g., a promoter, such as a cytomegalovirus (CMV) promoter, a T7 promoter, a CMV enhance, or a combination thereof) and a synthetic open reading frame molecule comprising, consisting essentially of, or consisting of, a puromycin resistance gene (e.g.,PuroR), preferably the transfer / transgene antibiotic resistance expression cassette a puromycin resistance expression cassette; or a combination thereof.

38. A method of making a recombinant Adeno-associated virus, the method comprising:(a) providing a recombinant Adeno-associated virus producer cell of any one of claims 35-37; and(b) producing recombinant Adeno-associated virus.

39. The method of claim 38, wherein producing recombinant adeno-associate virus comprises contacting the recombinant Adeno-associated virus producer cell with an agent (e.g., tetracycline, doxycycline, or derivative thereof) the induces the inducible transactivator (e.g., a reverse tetracycline-controlled transactivator (rtTA)).

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