Synthetic genetic elements for biomanufacturing

Abstract

The present invention describes recombinant constructs, cells, and devices for increasing the production of adeno-associated virus (AAV). The present invention also describes methods for producing recombinant AAV using the constructs and the cells.

Description

[0001] This application claims the benefit of the following U.S. Provisional Applications: U.S. Provisional Application 62 / 877,508, filed Jul. 23, 2019; U.S. Provisional Application 62 / 877,516, filed Jul. 23, 2019; U.S. Provisional Application 62 / 877,524, filed Jul. 23, 2019; U.S. Provisional Application 62 / 877,532, filed Jul. 23, 2019; U.S. Provisional Application 62 / 877,540, filed Jul. 23, 2019; U.S. Provisional Application 62 / 877,551, filed Jul. 23, 2019; U.S. Provisional Application 62 / 877,561, filed Jul. 23, 2019; and U.S. Provisional Application 62 / 877,577, filed Jul. 23, 2019, the entire contents of which are hereby incorporated by reference.

[0002] Electronically Submitted Sequence Listing

[0003] This application contains a Sequence Listing, which is submitted electronically via EFS-Web as an ASCII-formatted sequence listing, named “14620-192-228_SEQ_LISTING,” created on Jul. 16, 2020, and having a size of 152,403 bytes. The sequence listing submitted via EFS-Web is part of this specification and is hereby incorporated by reference in its entirety. Background of the Invention

[0004] Adeno-associated virus (AAV) has a linear single-stranded DNA (ssDNA) genome with two inverted terminal repeats (ITRs) at its termini. These ITRs flank two viral genes, rep (replication) and cap (capsid), which encode non-structural and structural proteins, respectively. The rep gene encodes four regulatory proteins, Rep78, Rep68, Rep52, and Rep40, by using two promoters and alternative splicing. More specifically, Rep78 and Rep68 are transcribed by the P5 promoter, and Rep40 and Rep52 are transcribed by the P19 promoter (which is embedded within the Rep78 and Rep68 reading frames). The P5 and P19 promoters are activated by the adenovirus E1A gene and are active in cells such as HEK293 cells transformed with the adenovirus E1 gene. These Rep proteins are involved in AAV genome replication. The cap gene gives rise to three capsid proteins, VP1 (virion protein 1), VP2, and VP3, by alternative splicing and translation initiation, and these three capsid proteins assemble into the nearly spherical protein shell of the virus. AAV viruses do not encode a polymerase and thus rely on cellular polymerases for genome replication.

[0005] If the AAV rep and cap genes can be stably integrated or maintained in mammalian cells and subsequently induced to produce AAV in high-density cultures, it would be possible to produce AAV on a large scale in mammalian cells. However, the expression of the Rep proteins can be cytotoxic or cytostatic to host cells, making it difficult to generate stable cell lines in hosts expressing the rep gene (such as those expressing the adenovirus E1 gene, such as HEK293 cells). Since AAV encodes four Rep proteins using overlapping reading frames and these four Rep proteins are generated by using two promoters and alternative splicing, it is not straightforward to control rep gene expression using an inducible promoter.

[0006] The cytotoxic or cytostatic nature of these four Rep proteins has precluded the generation of stable cell lines that can produce high-titer AAV using the native rep / cap promoters (Clark et al., (1995) Hum. Gene Ther. 6:1329-1341; Chadeuf et al., (2000) J. Gene med. 2:260-268). Several groups have attempted to recombinantly regulate Rep expression. Yang replaced the P5 promoter with the mouse metallothionein promoter. Although stable clones in HEK293 exhibited metal-inducible rep78 expression, only low levels of rep50 and rep42 expression (driven by the internal P19 promoter) were detected and the growth rate of the cells decreased significantly (Yang et al., (1994) J. Virol 68:4847-4856). Ogasawara replaced the P5 promoter with a ubiquitous promoter containing a filler fragment flanked by loxP, which can be activated by Cre recombinase. The rep52, rep40, and cap genes were not induced in stable clones infected with adenovirus-Cre, indicating that constitutive rep52 / rep40 expression is also harmful to cells (Ogasawara et al., (1999) J. Gen. Virol. 80:2477–2480).

[0007] Xiao and colleagues described another method for regulating rep expression (Qiao et al., (2002) J. Virol. 76:13015-13027; Yuan et al., (2011) Hum. Gene Ther. 22:613-624). Xiao inserted an artificial intron into the coding region shared by all four Rep proteins in the rep gene and inserted a loxP-flanked termination cassette containing a poly(A) sequence either alone or in combination with the puro puromycin resistance gene into this intron. Expression of all Rep proteins was inhibited, allowing the generation of stable cell lines in HEK293 cells. Delivery of Cre recombinase to the cells (by adenovirus infection) excised the termination cassette by recombination of the loxP sites, allowing transcription of the full-length precursor mRNA. The remaining intron sequence was then precisely removed by RNA splicing, restoring the coding sequences of all four Rep proteins and thus triggering AAV production from the integrated transgene flanked by ITRs. However, since the Cre recombinase recognizes two identical loxP sites, these loxP sites remain identical after recombination and thus may recombine again because Cre catalyzes both ligation and excision reactions.

[0008] The AAV rep gene is expressed only in cells that also express the adenovirus E1 (early region 1) gene. A number of stable rep / cap cell lines have been constructed in hosts that do not express the E1 gene, including HeLa (Clark et al., (1995) Hum. Gen. Therap. 6:1329-1341; Yang et al., (1994) J. Virol. 68:4847-4856; Gao et al., (1998) Hu, Gen. Ther. 9:2353-2362), A549 (Gao et al., (2002) Mol Ther. 5:644-659), and Vero (Beal et al., (2007), 10th Annual Meeting of American Society of Gene Therapy, Seattle, WA, May 30 - June 3, 2007). The biggest drawback of these cell lines is the requirement for E1-intact (and usually replication-competent) adenovirus to produce AAV, which may pose an increased safety risk in the form of contaminants in the AAV viral preparation. th Annual Meeting of American Society of Gene Therapy), Washington State Seattle (Seattle, WA), May 30 - June 3, 2007). The biggest drawback of these cell lines is the requirement for E1-intact (and usually replication-competent) adenovirus to produce AAV, which may pose an increased safety risk in the form of contaminants in the AAV viral preparation.

[0009] AAV production systems have been described that use several different viruses to provide helper functions and deliver recombinant transgenes and / or AAV genes to human cells, including herpesviruses (Thomas et al., (2009) Hum Gene Ther. 20:861–70; Clement et al., (2009) Hum Gene Ther. 20:796–806), vaccinia virus (Wang et al., (2017) Mol. Ther. Methods Clin Devel. 7:146-155), and adenovirus (Fisher et al., (1996) Hum gene Ther. 7:2079-2087; Gao et al., (1998) Hum Gene Ther. 2353-2362. Liu et al., (1999) GeneTher 6:293-299). These methods require the production of several different viruses (and in some cases recombinant host cell lines). Baculovirus has also been used to produce AAV in insect cells (Mietzsch et al., (2014) Hum Gene Ther. 25:212–22; Aslanidi et al., (2009) Proc Natl Acad Sci USA. 106:5059–5064; Cecchini et al., (2011) Hum Gene Ther. 22:1021–1030). Whether AAV produced in insect cells and human cells is functionally equivalent remains an open question.

[0010] There is a need to improve the production of AAV with recombinant constructs and cells. SUMMARY OF THE INVENTION

[0011] In one aspect, the present disclosure provides a non-naturally occurring nucleic acid molecule comprising a modified adeno-associated virus (AAV) rep gene. The modified AAV rep gene has an AAV rep gene encoding four Rep proteins, Rep78, Rep68, Rep52, and Rep40, and an artificial intron inserted into the coding sequence of the rep gene shared by these four Rep proteins. The artificial intron contains a termination cassette inserted downstream of the 5' splice site and upstream of the branch site of the artificial intron, and the termination cassette comprises, in the 5' to 3' order: (a) an attP site comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:7, preferably an attP site having the nucleotide sequence of SEQ ID NO:7; (b) a splice acceptor; (c) a terminator; and (d) an attB site comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:8 or SEQ ID NO:9, preferably an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9.

[0012] In one embodiment, the splice acceptor comprises the nucleotide sequence of SEQ ID NO:17.

[0013] In one embodiment, the terminator comprises a polyadenylation signal. In one embodiment, the terminator further comprises the nucleotide sequence of SEQ ID NO:19.

[0014] In one embodiment, the termination cassette contains a gene encoding a selectable marker, preferably a neomycin phosphotransferase expression cassette having the nucleotide sequence of SEQ ID NO:18.

[0015] In one embodiment, the artificial intron comprises, in the 5' to 3' order, the nucleotide sequence of SEQ ID NO:14, the termination cassette, and the nucleotide sequence of SEQ ID NO:15.

[0016] In one embodiment, the AAV rep gene comprises the rep gene of one of AAV1 to AAV8 or a hybrid thereof. In one embodiment, the AAV rep gene comprises the rep gene of human AAV2 with nucleotide numbers 190 to 2202 of the nucleotide sequence having GenBank accession number NC_001401.2. In one embodiment, an artificial intron is inserted between nucleotide numbers 996 and 1905 of the nucleotide sequence having GenBank accession number NC_001401.2. In one embodiment, the artificial intron is inserted immediately downstream of nucleotide number 1052, 1061, 1712, 1906, 1022, 1112, 1475, 1514, 1700, 1742, 1784 or 1340, preferably nucleotide number 1052, of the nucleotide sequence having GenBank accession number NC_001401.2.

[0017] In one aspect, provided herein is a non-naturally occurring nucleic acid molecule that comprises a modified AAV rep gene, the modified AAV rep gene comprising, in 5' to 3' order: (a) a 5' portion of the AAV rep gene having the nucleotide sequence of SEQ ID NO:55; (b) an artificial intron that comprises, in 5' to 3' order: (i) a 5' intron fragment having the nucleotide sequence of SEQ ID NO:14; (ii) a termination cassette that comprises, in 5' to 3' order: (1) an attP site having the nucleotide sequence of SEQ ID NO:7; (2) a splice acceptor having the nucleotide sequence of SEQ ID NO:17; (3) a neomycin phosphotransferase expression cassette having the nucleotide sequence of SEQ ID NO:17; (4) a terminator having the nucleotide sequence of SEQ ID NO:18; and (5) an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9; and (iii) a 3' intron fragment having the nucleotide sequence of SEQ ID NO:15; and (c) a 3' portion of the AAV rep gene having the nucleotide sequence of SEQ ID NO:56.

[0018] In one aspect, the present disclosure provides a non-naturally occurring nucleic acid molecule that includes a modified AAV rep gene. The modified AAV rep gene, in 5' to 3' order, includes: (a) a 5' portion of the AAV rep gene having the nucleotide sequence of SEQ ID NO: 73; (b) an artificial intron that, in 5' to 3' order, includes: (i) a 5' intron fragment having the nucleotide sequence of SEQ ID NO: 14; (ii) a termination cassette that, in 5' to 3' order, includes: (1) an attP site having the nucleotide sequence of SEQ ID NO: 7; (2) a splice acceptor having the nucleotide sequence of SEQ ID NO: 17; (3) a neomycin phosphotransferase expression cassette having the nucleotide sequence of SEQ ID NO: 17; (4) a terminator having the nucleotide sequence of SEQ ID NO: 18; and (5) an attB site having the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 9; and (iii) a 3' intron fragment having the nucleotide sequence of SEQ ID NO: 66; and (c) a 3' portion of the AAV rep gene having the nucleotide sequence of SEQ ID NO: 56. In one embodiment, the termination cassette includes the nucleotide sequence of SEQ ID NO: 16.

[0019] In one embodiment, the non-naturally occurring nucleic acid molecule further includes an AAV cap gene encoding three capsid proteins VP1, VP2, and VP3. In one embodiment, the AAV cap gene includes the cap gene of one of AAV1 to AAV9 and AAVDJ or a hybrid thereof. In one embodiment, the AAV cap gene includes the cap gene of human AAV9 having the nucleotide sequence of GenBank accession number AY530579.1. In one embodiment, the AAV cap gene further includes a polyadenylation signal, preferably the polyadenylation signal of AAV2 having the nucleotide numbers 4411 to 4466 of the nucleotide sequence of GenBank accession number NC_001401.2, and an enhancer, preferably the AAV2 rep P5 promoter having the nucleotide numbers 190 to 313 of the nucleotide sequence of GenBank accession number NC_001401.2, wherein both the polyadenylation signal and the enhancer are downstream of the coding sequence of the cap gene. In one embodiment, the non-naturally occurring nucleic acid molecule further includes a transgene flanked by a pair of AAV inverted terminal repeats (ITRs) downstream of the AAV cap gene.

[0020] In one embodiment, the non-naturally occurring nucleic acid molecule further comprises a first insulator upstream of the modified AAV rep gene and optionally a second insulator downstream of the transgene flanked by ITRs. Preferably, the first insulator and the second insulator are independently selected from: (a) the human insulator element 40 having the nucleotide sequence of SEQ ID NO:24; (b) the mouse insulator element 40 having the nucleotide sequence of SEQ ID NO:25; (c) the insulator element 04 having the nucleotide sequence of GenBank accession number AY190749.1; (d) the insulator element 06 having the nucleotide sequence of GenBank accession number AY190750.1; (e) the insulator element 07 having the nucleotide sequence of GenBank accession number AY190751.1; (f) the insulator element 12 having the nucleotide sequence of GenBank accession number AY190752.1; (g) the insulator element 13 having the nucleotide sequence of GenBank accession number AY190753.1; (h) the insulator element 35 having the nucleotide sequence of GenBank accession number AY190754.1; (i) the insulator element 36 having the nucleotide sequence of GenBank accession number AY190755.1; (j) the insulator element 52 having the nucleotide sequence of GenBank accession number AY190757.1; (k) the insulator element 53 having the nucleotide sequence of GenBank accession number AY190758.1; and (l) two or more copies of the chicken HS4 insulator having the nucleotide sequence of AY040835.1 from the globin locus. More preferably, the first insulator and the second insulator have the nucleotide sequences of SEQ ID NO:24 and SEQ ID NO:25, respectively. In one embodiment, the non-naturally occurring nucleic acid molecule comprises a first insulator upstream of the modified AAV rep gene and further comprises a first spacer sequence upstream of the transgene and a second spacer sequence downstream of the transgene, wherein the first spacer sequence and the second spacer sequence are independently selected from: (a) the nucleotide sequence of SEQ ID NO:67; and (b) the nucleotide sequence of SEQ ID NO:68. In one embodiment, the ITR has the nucleotide sequence of SEQ ID NO:20, the transgene comprises a promoter operably linked to a coding sequence, and the coding sequence is operably linked to a polyadenylation signal; preferably, the promoter has the nucleotide sequence of SEQ ID NO:21 and the polyadenylation signal has the nucleotide sequence of SEQ ID NO:23.

[0021] In one aspect, the present disclosure provides a non-naturally occurring nucleic acid molecule that, in the 5' to 3' order, comprises: (A) a first insulator, preferably the first insulator has the nucleotide sequence of SEQ ID NO: 24; (B) a modified AAV rep gene that, in the 5' to 3' order, comprises: (i) a 5' portion of the AAV rep gene, preferably the 5' portion of the AAV rep gene has the nucleotide sequence of SEQ ID NO: 55; (ii) an artificial intron that, in the 5' to 3' order, comprises: (a) a 5' intron fragment, preferably the 5' intron fragment has the nucleotide sequence of SEQ ID NO: 14; (b) a termination cassette that, in the 5' to 3' order, comprises: (1) an attP site having the nucleotide sequence of SEQ ID NO: 7; (2) a splice acceptor, preferably the splice acceptor has the nucleotide sequence of SEQ ID NO: 17; (3) a gene encoding a selectable marker, preferably a neomycin phosphotransferase expression cassette having the nucleotide sequence of SEQ ID NO: 18; (4) a terminator, preferably the terminator has the nucleotide sequence of SEQ ID NO: 19; and (5) an attB site having the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 9; and (c) a 3' intron fragment, preferably the 3' intron fragment has the nucleotide sequence of SEQ ID NO: 15; (iii) a 3' portion of the AAV rep gene, preferably the 3' portion of the AAV rep gene has the nucleotide sequence of SEQ ID NO: 56; (C) an AAV cap gene, preferably the AAV cap gene comprises the nucleotide sequence of SEQ ID NO: 57; (D) a transgene flanked by a pair of AAV ITRs, preferably the AAV ITR has the nucleotide sequence of SEQ ID NO: 20, and the transgene comprises a promoter operably linked to a coding sequence, and the coding sequence is operably linked to a polyadenylation signal; more preferably, the promoter has the nucleotide sequence of SEQ ID NO: 21 and the polyadenylation signal has the nucleotide sequence SEQ ID NO: 23; and (E) a second insulator, preferably the second insulator has the nucleotide sequence of SEQ ID NO: 25.

[0022] In one aspect, provided herein is a non-naturally occurring nucleic acid molecule that, in a 5' to 3' order, comprises: (A) a first insulator, preferably the first insulator has the nucleotide sequence of SEQ ID NO:24; (B) a modified AAV rep gene that, in a 5' to 3' order, comprises: (i) a 5' portion of the AAV rep gene, preferably the 5' portion of the AAV rep gene has the nucleotide sequence of SEQ ID NO:73; (ii) an artificial intron that, in a 5' to 3' order, comprises: (a) a 5' intron fragment, preferably the 5' intron fragment has the nucleotide sequence of SEQ ID NO:14; (b) a termination cassette that, in a 5' to 3' order, comprises: (1) an attP site having the nucleotide sequence of SEQ ID NO:7; (2) a splice acceptor, preferably the splice acceptor has the nucleotide sequence of SEQ ID NO:17; (3) a gene encoding a selectable marker, preferably a neomycin phosphotransferase expression cassette having the nucleotide sequence of SEQ ID NO:18; (4) a terminator, preferably the terminator has the nucleotide sequence of SEQ ID NO:19; and (5) an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9; and (c) a 3' intron fragment, preferably the 3' intron fragment has the nucleotide sequence of SEQ ID NO:66; (iii) a 3' portion of the AAV rep gene, preferably the 3' portion of the AAV rep gene has the nucleotide sequence of SEQ ID NO:56; (C) an AAV cap gene; (D) a transgene that is flanked by: (1) a pair of AAV ITRs, preferably the AAV ITRs have the nucleotide sequence of SEQ ID NO:20, and the transgene comprises a promoter operably linked to a coding sequence, and the coding sequence is operably linked to a polyadenylation signal; more preferably, the promoter has the nucleotide sequence of SEQ ID NO:21 and the polyadenylation signal has the nucleotide sequence SEQ ID NO:23; and (2) a pair of spacer sequences, preferably the spacer sequences have the nucleotide sequences of SEQ ID NO:67 and SEQ ID NO:68.

[0023] In one aspect, provided herein is a vector that comprises the above-described non-naturally occurring nucleic acid molecule; preferably, the vector is a plasmid; more preferably, the plasmid comprises the nucleotide sequence of SEQ ID NO:12.

[0024] In one aspect, the present disclosure provides a vector comprising the above-described non-naturally occurring nucleic acid molecule; preferably, the vector is a plasmid; more preferably, the plasmid comprises the nucleotide sequence of SEQ ID NO:70.

[0025] In one aspect, the present disclosure provides a method for preparing the above-described non-naturally occurring nucleic acid molecule. In a specific embodiment, the present disclosure provides a method for preparing a vector comprising the above-described non-naturally occurring nucleic acid molecule; preferably, the vector is a plasmid; more preferably, the plasmid comprises the nucleotide sequence of SEQ ID NO:12. In another embodiment, the present disclosure provides a method for preparing a vector comprising the above-described non-naturally occurring nucleic acid molecule; preferably, the vector is a plasmid; more preferably, the plasmid comprises the nucleotide sequence of SEQ ID NO:70.

[0026] In one aspect, the present disclosure provides a cell comprising a non-naturally occurring nucleic acid molecule, the non-naturally occurring nucleic acid molecule comprising a modified adeno-associated virus (AAV) rep gene, the modified AAV rep gene having an AAV rep gene encoding four Rep proteins, Rep78, Rep68, Rep52, and Rep40, and an artificial intron inserted into the coding sequence of the rep gene shared by these four Rep proteins, wherein the artificial intron comprises a termination cassette inserted downstream of the 5' splice site and upstream of the branch site of the artificial intron, and the termination cassette comprises, in 5' to 3' order: (a) an attP site comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:7, preferably, an attP site having the nucleotide sequence of SEQ ID NO:7; (b) a splice acceptor; (c) a terminator; and (d) an attB site comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:8 or SEQ ID NO:9, preferably, an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9.

[0027] In one embodiment, the splice acceptor comprises the nucleotide sequence of SEQ ID NO:17.

[0028] In one embodiment, the terminator comprises a polyadenylation signal. In one embodiment, the terminator further comprises the nucleotide sequence of SEQ ID NO:19.

[0029] In one embodiment, the termination cassette contains a gene encoding a selectable marker, preferably a neomycin phosphotransferase expression cassette having the nucleotide sequence of SEQ ID NO: 18.

[0030] In one embodiment, the artificial intron contains, in 5' to 3' order, the nucleotide sequence of SEQ ID NO: 14, the termination cassette, and the nucleotide sequence of SEQ ID NO: 15. In another embodiment, the artificial intron contains, in 5' to 3' order, the nucleotide sequence of SEQ ID NO: 14, the termination cassette, and the nucleotide sequence of SEQ ID NO: 66.

[0031] In one embodiment, the AAV rep gene contains the rep gene of one of AAV1 to AAV8 or a hybrid thereof. In one embodiment, the AAV rep gene contains the rep gene of human AAV2 with nucleotide numbers 190 to 2202 having the nucleotide sequence of GenBank accession number NC_001401.2. In one embodiment, the artificial intron is inserted between nucleotide numbers 996 and 1905 of the nucleotide sequence of GenBank accession number NC_001401.2. In one embodiment, the artificial intron is inserted immediately downstream of nucleotide number 1052, 1061, 1712, 1906, 1022, 1112, 1475, 1514, 1700, 1742, 1784 or 1340, preferably nucleotide number 1052, of the nucleotide sequence of GenBank accession number NC_001401.2.

[0032] In one aspect, the present disclosure provides a cell comprising a non-naturally occurring nucleic acid molecule, the non-naturally occurring nucleic acid molecule comprising a modified AAV rep gene, the modified AAV rep gene comprising, in 5' to 3' order: (a) a 5' portion of the AAV rep gene having the nucleotide sequence of SEQ ID NO:55; (b) an artificial intron, the artificial intron comprising, in 5' to 3' order: (i) a 5' intron fragment having the nucleotide sequence of SEQ ID NO:14; (ii) a termination cassette, the termination cassette comprising, in 5' to 3' order: (1) an attP site having the nucleotide sequence of SEQ ID NO:7; (2) a splice acceptor having the nucleotide sequence of SEQ ID NO:17; (3) a neomycin phosphotransferase expression cassette having the nucleotide sequence of SEQ ID NO:18; (4) a terminator having the nucleotide sequence of SEQ ID NO:19; and (5) an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9; and (iii) a 3' intron fragment having the nucleotide sequence of SEQ ID NO:15; and (c) a 3' portion of the AAV rep gene having the nucleotide sequence of SEQ ID NO:56.

[0033] In one aspect, the present disclosure provides a cell comprising a non-naturally occurring nucleic acid molecule, the non-naturally occurring nucleic acid molecule comprising a modified AAV rep gene, the modified AAV rep gene comprising, in 5' to 3' order: (a) a 5' portion of the AAV rep gene having the nucleotide sequence of SEQ ID NO:73; (b) an artificial intron, the artificial intron comprising, in 5' to 3' order: (i) a 5' intron fragment having the nucleotide sequence of SEQ ID NO:14; (ii) a termination cassette, the termination cassette comprising, in 5' to 3' order: (1) an attP site having the nucleotide sequence of SEQ ID NO:7; (2) a splice acceptor having the nucleotide sequence of SEQ ID NO:17; (3) a neomycin phosphotransferase expression cassette having the nucleotide sequence of SEQ ID NO:18; (4) a terminator having the nucleotide sequence of SEQ ID NO:19; and (5) an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9; and (iii) a 3' intron fragment having the nucleotide sequence of SEQ ID NO:66; and (c) a 3' portion of the AAV rep gene having the nucleotide sequence of SEQ ID NO:56.

[0034] In one embodiment, the termination cassette comprises the nucleotide sequence of SEQ ID NO:16.

[0035] In one embodiment, the above-mentioned cells further comprise an AAV cap gene encoding three capsid proteins VP1, VP2 and VP3. In one embodiment, the AAV cap gene comprises the cap gene of one of AAV1 to AAV9 and AAVDJ or their hybrids. In one embodiment, the AAV cap gene comprises the cap gene of human AAV9 having the nucleotide sequence of GenBank accession number AY530579.1. In one embodiment, the AAV cap gene comprises the cap gene of a hybrid of AAV9.

[0036] In one embodiment, the AAV cap gene further comprises a polyadenylation signal, preferably the polyadenylation signal of AAV2 with nucleotide numbers 4411 to 4466 of the nucleotide sequence of GenBank accession number NC_001401.2, and an enhancer, preferably the AAV2 rep P5 promoter with nucleotide numbers 190 to 313 of the nucleotide sequence of GenBank accession number NC_001401.2, wherein both the polyadenylation signal and the enhancer are downstream of the coding sequence of the cap gene.

[0037] In one embodiment, a cell comprising a cap gene further comprises a transgene flanked by a pair of AAV inverted terminal repeats (ITRs) downstream of the AAV cap gene. In one embodiment, the cell further comprises a first insulator upstream of the modified AAV rep gene and optionally a second insulator downstream of the transgene flanked by ITRs. Preferably, the first insulator and the second insulator are independently selected from: (a) human insulator element 40 having the nucleotide sequence of SEQ ID NO:24; (b) mouse insulator element 40 having the nucleotide sequence of SEQ ID NO:25; (c) insulator element 04 having the nucleotide sequence of GenBank accession number AY190749.1; (d) insulator element 06 having the nucleotide sequence of GenBank accession number AY190750.1; (e) insulator element 07 having the nucleotide sequence of GenBank accession number AY190751.1; (f) insulator element 12 having the nucleotide sequence of GenBank accession number AY190752.1; (g) insulator element 13 having the nucleotide sequence of GenBank accession number AY190753.1; (h) insulator element 35 having the nucleotide sequence of GenBank accession number AY190754.1; (i) insulator element 36 having the nucleotide sequence of GenBank accession number AY190755.1; (j) insulator element 52 having the nucleotide sequence of GenBank accession number AY190757.1; (k) insulator element 53 having the nucleotide sequence of GenBank accession number AY190758.1; and (l) two or more copies of the chicken HS4 insulator having the nucleotide sequence of AY040835.1 from the globin locus. More preferably, the first insulator and the second insulator have the nucleotide sequences of SEQ ID NO:24 and SEQ ID NO:25, respectively. In one embodiment, the cell comprises a first insulator upstream of the modified AAV rep gene and further comprises a first spacer sequence upstream of the transgene and a second spacer sequence downstream of the transgene, wherein the first spacer sequence and the second spacer sequence are independently selected from: (a) the nucleotide sequence of SEQ ID NO:67; and (b) the nucleotide sequence of SEQ ID NO:68.

[0038] In one embodiment, the ITR has the nucleotide sequence of SEQ ID NO:20, the transgene comprises a promoter operably linked to a coding sequence, and the coding sequence is operably linked to a polyadenylation signal; preferably, the promoter has the nucleotide sequence of SEQ ID NO:21 and the polyadenylation signal has the nucleotide sequence SEQ ID NO:23.

[0039] In one aspect, the present disclosure provides a cell comprising a non-naturally occurring nucleic acid molecule that, in 5' to 3' order, comprises: (A) a first insulator, preferably the first insulator has the nucleotide sequence of SEQ ID NO: 24; (B) a modified AAV rep gene that, in 5' to 3' order, comprises: (i) a 5' portion of the AAV rep gene, preferably the 5' portion of the AAV rep gene has the nucleotide sequence of SEQ ID NO: 55; (ii) an artificial intron that, in 5' to 3' order, comprises: (a) a 5' intron fragment, preferably the 5' intron fragment has the nucleotide sequence of SEQ ID NO: 14; (b) a termination cassette that, in 5' to 3' order, comprises: (1) an attP site having the nucleotide sequence of SEQ ID NO: 7; (2) a splice acceptor, preferably the splice acceptor has the nucleotide sequence of SEQ ID NO: 17; (3) a gene encoding a selectable marker, preferably a neomycin phosphotransferase expression cassette having the nucleotide sequence of SEQ ID NO: 18; (4) a terminator, preferably the terminator has the nucleotide sequence of SEQ ID NO: 19; and (5) an attB site having the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 9; and (c) a 3' intron fragment, preferably the 3' intron fragment has the nucleotide sequence of SEQ ID NO: 15; (iii) a 3' portion of the AAV rep gene, preferably the 3' portion of the AAV rep gene has the nucleotide sequence of SEQ ID NO: 56; (C) an AAV cap gene, preferably the AAV cap gene comprises the nucleotide sequence of SEQ ID NO: 57; (D) a transgene flanked by a pair of AAV ITRs, preferably the AAV ITR has the nucleotide sequence of SEQ ID NO: 20, and the transgene comprises a promoter operably linked to a coding sequence, and the coding sequence is operably linked to a polyadenylation signal; more preferably, the promoter has the nucleotide sequence of SEQ ID NO: 21 and the polyadenylation signal has the nucleotide sequence of SEQ ID NO: 23; and (E) a second insulator, preferably the second insulator has the nucleotide sequence of SEQ ID NO: 25.

[0040] In one aspect, the present disclosure provides a cell comprising a non-naturally occurring nucleic acid molecule that, in a 5' to 3' order, comprises: (A) a first insulator, preferably the first insulator has the nucleotide sequence of SEQ ID NO: 24; (B) a modified AAV rep gene that, in a 5' to 3' order, comprises: (i) a 5' portion of the AAV rep gene, preferably the 5' portion of the AAV rep gene has the nucleotide sequence of SEQ ID NO: 73; (ii) an artificial intron that, in a 5' to 3' order, comprises: (a) a 5' intron fragment, preferably the 5' intron fragment has the nucleotide sequence of SEQ ID NO: 14; (b) a termination cassette that, in a 5' to 3' order, comprises: (1) an attP site having the nucleotide sequence of SEQ ID NO: 7; (2) a splice acceptor, preferably the splice acceptor has the nucleotide sequence of SEQ ID NO: 17; (3) a gene encoding a selectable marker, preferably a neomycin phosphotransferase expression cassette having the nucleotide sequence of SEQ ID NO: 18; (4) a terminator, preferably the terminator has the nucleotide sequence of SEQ ID NO: 19; and (5) an attB site having the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 9; and (c) a 3' intron fragment, preferably the 3' intron fragment has the nucleotide sequence of SEQ ID NO: 66; (iii) a 3' portion of the AAV rep gene, preferably the 3' portion of the AAV rep gene has the nucleotide sequence of SEQ ID NO: 56; (C) an AAV cap gene; and (D) a transgene flanked by a pair of AAV ITRs, preferably the AAV ITR has the nucleotide sequence of SEQ ID NO: 20, and the transgene comprises a promoter operably linked to a coding sequence, and the coding sequence is operably linked to a polyadenylation signal; more preferably, the promoter has the nucleotide sequence of SEQ ID NO: 21 and the polyadenylation signal has the nucleotide sequence SEQ ID NO: 23; and (ii) a pair of spacer sequences, preferably the spacer sequences have the nucleotide sequences of SEQ ID NO: 67 and SEQ ID NO: 68.

[0041] In one embodiment, the non-naturally occurring nucleic acid molecule is episomal and has the nucleotide sequence of SEQ ID NO: 12. In another embodiment, the non-naturally occurring nucleic acid molecule is episomal and has the nucleotide sequence of SEQ ID NO: 70.

[0042] In one embodiment, the cell further comprises a nucleic acid molecule encoding a recombinase having an amino acid sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 2; preferably, the nucleic acid comprises a nucleotide sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the nucleotide sequence of SEQ ID NO: 3; more preferably, the cell comprises a recombinant ΔE1 / ΔE3 adenovirus serotype 5 (Ad5) virus encoding the recombinase having the amino acid sequence of SEQ ID NO: 2.

[0043] In one embodiment, the cell further comprises the adenovirus E1A and E1B genes, and preferably the cell is a 911 cell, pTG6559 cell, GH329 cell, N52.E6 cell, HeLa-E1 cell, UR cell, VLI-293 cell, HEK293 cell or PER.C6 cell.

[0044] In one aspect, the present disclosure provides a method for generating recombinant AAVs containing a transgene, the method comprising: (A) obtaining a first host cell comprising: (i) a modified AAV rep gene, the modified AAV rep gene comprising, in 5' to 3' order: (a) a 5' portion of the AAV rep gene, preferably the AAV rep gene having the nucleotide sequence of SEQ ID NO:55; (b) an artificial intron, the artificial intron comprising, in 5' to 3' order: (1) a 5' intron fragment, preferably the 5' intron fragment having the nucleotide sequence of SEQ ID NO:14; (2) a termination cassette, the termination cassette comprising, in 5' to 3' order: (aa) an attP site having the nucleotide sequence of SEQ ID NO:7; (bb) a splice acceptor, preferably the splice acceptor having the nucleotide sequence of SEQ ID NO:17; (cc) a gene encoding a selectable marker, preferably a neomycin phosphotransferase expression cassette having the nucleotide sequence of SEQ ID NO:18; (dd) a terminator, preferably the terminator having the nucleotide sequence of SEQ ID NO:19; and (ee) an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9; and (3) a 3' intron fragment, preferably the 3' intron fragment having the nucleotide sequence of SEQ ID NO:15; (c) a 3' portion of the AAV rep gene, preferably the 3' portion of the AAV rep gene having the nucleotide sequence of SEQ ID NO:56; (ii) an AAV cap gene, preferably the AAV cap gene comprising the nucleotide sequence of SEQ ID NO:57; and (iii) a transgene flanked by a pair of AAV ITRs, preferably the ITR having the nucleotide sequence of SEQ ID NO:20, the transgene comprising a promoter operably linked to a coding sequence, and the coding sequence operably linked to a polyadenylation signal; more preferably, the promoter having the nucleotide sequence of SEQ ID NO:21 and the polyadenylation signal having the nucleotide sequence of SEQ ID NO:23; (B) infecting the first host cell with a recombinant adenovirus comprising a recombinase gene encoding a recombinase having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:2 to obtain a second host cell further comprising the recombinase gene; (C) growing the second host cell under conditions such that the recombinant AAV containing the transgene is produced; and (D) optionally collecting the recombinant AAV.

[0045] In one aspect, the present disclosure provides a method for generating recombinant AAVs containing a transgene, the method comprising: (A) obtaining a first host cell that contains: (i) a modified AAV rep gene that, in 5' to 3' order, comprises: (a) a 5' portion of the AAV rep gene, preferably the AAV rep gene has the nucleotide sequence of SEQ ID NO:73; (b) an artificial intron that, in 5' to 3' order, comprises: (1) a 5' intron fragment, preferably the 5' intron fragment has the nucleotide sequence of SEQ ID NO:14; (2) a termination cassette that, in 5' to 3' order, comprises: (aa) an attP site having the nucleotide sequence of SEQ ID NO:7; (bb) a splice acceptor, preferably the splice acceptor has the nucleotide sequence of SEQ ID NO:17; (cc) a gene encoding a selectable marker, preferably a neomycin phosphotransferase expression cassette having the nucleotide sequence of SEQ ID NO:18; (dd) a terminator, preferably the terminator has the nucleotide sequence of SEQ ID NO:19; and (ee) an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9; and (3) a 3' intron fragment, preferably the 3' intron fragment has the nucleotide sequence of SEQ ID NO:66; (c) a 3' portion of the AAV rep gene, preferably the 3' portion of the AAV rep gene has the nucleotide sequence of SEQ ID NO:66; (ii) an AAV cap gene; and (iii) a transgene that is flanked by: (a) a pair of AAV ITRs, preferably the ITRs have the nucleotide sequence of SEQ ID NO:20, the transgene comprises a promoter operably linked to a coding sequence, and the coding sequence is operably linked to a polyadenylation signal; more preferably, the promoter has the nucleotide sequence of SEQ ID NO:21 and the polyadenylation signal has the nucleotide sequence SEQ ID NO:23; and (b) a pair of spacer sequences, preferably the spacer sequences have the nucleotide sequences of SEQ ID NO:67 and SEQ ID NO:68; (B) infecting the first host cell with a recombinant adenovirus containing a recombinase gene encoding a recombinase that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:2 to obtain a second host cell that further contains the recombinase gene; (C) growing the second host cell under conditions such that the recombinant AAV containing the transgene is produced; and (D) optionally collecting the recombinant AAV.

[0046] In one embodiment, the first host cell further comprises a first insulator upstream of the modified AAV rep gene and optionally a second insulator downstream of the transgene flanked by ITRs. Preferably, the first insulator and the second insulator are independently selected from: (a) the human antirepressor element 40 having the nucleotide sequence of SEQ ID NO:24; (b) the mouse antirepressor element 40 having the nucleotide sequence of SEQ ID NO:25; (c) the antirepressor element 04 having the nucleotide sequence of GenBank accession number AY190749.1; (d) the antirepressor element 06 having the nucleotide sequence of GenBank accession number AY190750.1; (e) the antirepressor element 07 having the nucleotide sequence of GenBank accession number AY190751.1; (f) the antirepressor element 12 having the nucleotide sequence of GenBank accession number AY190752.1; (g) the antirepressor element 13 having the nucleotide sequence of GenBank accession number AY190753.1; (h) the antirepressor element 35 having the nucleotide sequence of GenBank accession number AY190754.1; (i) the antirepressor element 36 having the nucleotide sequence of GenBank accession number AY190755.1; (j) the antirepressor element 52 having the nucleotide sequence of GenBank accession number AY190757.1; (k) the antirepressor element 53 having the nucleotide sequence of GenBank accession number AY190758.1; and (l) two or more copies of the chicken HS4 insulator having the nucleotide sequence of AY040835.1 from the globin locus. More preferably, the first insulator and the second insulator have the nucleotide sequences of SEQ ID NO:24 and SEQ ID NO:25, respectively.

[0047] In one embodiment, the first host cell comprises a first insulator upstream of the modified AAV rep gene and further comprises a first spacer sequence and a second spacer sequence upstream and downstream of the transgene, respectively, wherein the first spacer sequence and the second spacer sequence are independently selected from: (a) the nucleotide sequence of SEQ ID NO:67; and (b) the nucleotide sequence of SEQ ID NO:68.

[0048] In one embodiment, a first host cell is obtained by introducing into a cell one or more nucleic acid molecules comprising a modified AAV rep gene, an AAV cap gene, a transgene flanked by ITRs, a first insulator, and a second insulator. In one embodiment, a first host cell is obtained by introducing into a cell a nucleic acid molecule comprising, in 5' to 3' order, a first insulator, a modified AAV rep gene, an AAV cap gene, a transgene flanked by ITRs, a first insulator, and a second insulator, preferably a plasmid comprising the nucleotide sequence of SEQ ID NO:12.

[0049] In one embodiment, a first host cell is obtained by introducing into a cell one or more nucleic acid molecules comprising a modified AAV rep gene, an AAV cap gene, a transgene flanked by ITRs, a first insulator, a first spacer sequence, and a second spacer sequence. In one embodiment, a first host cell is obtained by introducing into a cell one or more nucleic acid molecules comprising a modified AAV rep gene, an AAV cap gene, a transgene flanked by ITRs, a first insulator, a first spacer sequence, and a second spacer sequence, preferably a plasmid comprising the nucleotide sequence of SEQ ID NO:70.

[0050] In one embodiment, the recombinant adenovirus is a recombinant ΔE1 / ΔE3 adenovirus serotype 5 (Ad5) virus comprising the nucleotide sequence of SEQ ID NO:3.

[0051] In one embodiment, the host cell comprises adenovirus E1A and E1B genes, preferably the host cell is a 911 cell, a pTG6559 cell, a GH329 cell, an N52.E6 cell, a HeLa-E1 cell, a UR cell, a VLI-293 cell, a HEK293 cell, or a PER.C6 cell.

[0052] In one embodiment, the conditions for growing the second host cell include culturing the second cell with 2-aminopurine. In one embodiment, the 2-aminopurine concentration is less than about 1.25 mM. In one embodiment, the 2-aminopurine concentration is from about 1 μM to about 1.25 mM. In one embodiment, the 2-aminopurine concentration is from about 10 μM to about 1.25 mM. In one embodiment, the 2-aminopurine concentration is from about 100 μM to about 1.25 mM. In one embodiment, the 2-aminopurine concentration is about 1.25 mM.

[0053] In one embodiment, culturing the second cell with 2-aminopurine is started about 24 hours after infecting the first host cell with the recombinant adenovirus.

[0054] In one aspect, the present disclosure provides a composition comprising a cell having a nucleic acid molecule encoding a recombinase as described above and 2-aminopurine. In one embodiment, the concentration of 2-aminopurine is less than about 1.25 mM. In one embodiment, the concentration of 2-aminopurine is from about 1 μM to about 1.25 mM. In one embodiment, the concentration of 2-aminopurine is from about 10 μM to about 1.25 mM. In one embodiment, the concentration of 2-aminopurine is from about 100 μM to about 1.25 mM. In one embodiment, the concentration of 2-aminopurine is about 1.25 mM.

[0055] In one aspect, the present disclosure provides an unnatural nucleic acid molecule comprising a nucleotide sequence encoding a serine recombinase having an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:2. In one embodiment, the unnatural nucleic acid molecule comprises a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the nucleotide sequence of SEQ ID NO:3.

[0056] In one aspect, the present disclosure provides a vector comprising an unnatural nucleic acid molecule comprising a nucleotide sequence encoding a serine recombinase having an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:2.

[0057] In one aspect, the present disclosure provides a vector comprising an unnatural nucleic acid molecule comprising a nucleotide sequence encoding a serine recombinase having an amino acid sequence with at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:3.

[0058] In one embodiment, the vector further comprises a promoter operably linked to the nucleotide sequence encoding the serine recombinase, preferably the cytomegalovirus (CMV) promoter.

[0059] In one embodiment, the vector further comprises a polyadenylation signal operably linked to a nucleotide sequence encoding a serine recombinase, such as the Simian virus 40 (SV40) polyadenylation signal.

[0060] In one embodiment, the vector is a DNA plasmid. In one embodiment, the vector is a recombinant adenovirus vector.

[0061] In one embodiment, the vector is a recombinant ΔE1 / ΔE3 adenovirus serotype 5 (Ad5) virus that comprises a nucleotide sequence encoding a serine recombinase having the amino acid sequence of SEQ ID NO:2 under the control of a CMV promoter, wherein the nucleotide sequence is further operably linked to an SV40 polyadenylation signal (NC_001669.1, nt2550 to 2774).

[0062] In one aspect, provided herein is a cell that comprises a non-naturally occurring nucleic acid molecule that comprises a nucleotide sequence encoding a serine recombinase having an amino acid sequence that is at least 85% identical, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical, to the amino acid sequence of SEQ ID NO:2. In one embodiment, the cell comprises a nucleotide sequence that is at least 85% identical, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical, to the nucleotide sequence of SEQ ID NO:3.

[0063] In one aspect, provided herein is a cell that comprises a vector that comprises a non-naturally occurring nucleic acid molecule that comprises a nucleotide sequence encoding a serine recombinase having an amino acid sequence that is at least 85% identical, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical, to the amino acid sequence of SEQ ID NO:2. In another aspect, provided herein is a cell that comprises a vector that comprises a non-naturally occurring nucleic acid molecule that comprises a nucleotide sequence encoding a serine recombinase having an amino acid sequence that is at least 85% identical, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical, to the amino acid sequence of SEQ ID NO:3.

[0064] In one embodiment, the cell further comprises a promoter operably linked to a nucleotide sequence encoding a serine recombinase, preferably the cytomegalovirus (CMV) promoter.

[0065] In one embodiment, the cell further comprises a polyadenylation signal operably linked to the nucleotide sequence encoding a serine recombinase, such as the simian virus 40 (SV40) polyadenylation signal.

[0066] In one embodiment, the vector is a DNA plasmid. In one embodiment, the vector is a recombinant adenovirus vector.

[0067] In one embodiment, the recombinant adenovirus vector comprises a recombinant ΔE1 / ΔE3 adenovirus serotype 5 (Ad5) virus that contains a nucleotide sequence encoding a serine recombinase having the amino acid sequence of SEQ ID NO:2 under the control of the CMV promoter, wherein the nucleotide sequence is further operably linked to the SV40 polyadenylation signal (NC_001669.1, nt 2550 to 2774).

[0068] In one embodiment, the cell contains the adenovirus E1A and E1B genes. Preferably, the cell is a 911 cell, pTG6559 cell, GH329 cell, N52.E6 cell, HeLa-E1 cell, UR cell, VLI-293 cell, HEK293 cell or PER.C6 cell.

[0069] In one aspect, the present disclosure provides a method for site-specific recombination in a cell, the method comprising: (a) obtaining a cell comprising a nucleic acid molecule having: an attP site comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:7, preferably an attP site having the nucleotide sequence of SEQ ID NO:7, and an attB site comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:8 or SEQ ID NO:9, preferably an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9; (b) introducing into the cell a non-naturally occurring nucleic acid molecule encoding a serine recombinase having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:2; and (c) growing the cell under conditions that permit the serine recombinase to catalyze site-specific recombination between the attP and attB sites.

[0070] In one aspect, the present disclosure provides a product produced by a process of site-specific recombination in a cell, the process comprising: (a) obtaining a cell comprising a nucleic acid molecule having: an attP site comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:7, preferably an attP site having the nucleotide sequence of SEQ ID NO:7, and an attB site comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:8 or SEQ ID NO:9, preferably an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9; (b) introducing into the cell a non-naturally occurring nucleic acid molecule encoding a serine recombinase having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:2; and (c) growing the cell under conditions that permit the serine recombinase to catalyze site-specific recombination between the attP and attB sites.

[0071] In one aspect, provided herein is a process for obtaining a product from a cell, the process comprising: (a) obtaining a cell comprising a nucleic acid molecule having: an attP site comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:7, preferably an attP site having the nucleotide sequence of SEQ ID NO:7, and an attB site comprising a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:8 or SEQ ID NO:9, preferably an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9; (b) introducing into the cell a non-naturally occurring nucleic acid molecule encoding a serine recombinase having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:2; (c) growing the cell under conditions to allow the serine recombinase to catalyze site-specific recombination between the attP and attB sites; and (d) producing and recovering the product from the cell.

[0072] In one aspect, provided herein is a non-naturally occurring system comprising: means for AAV-mediated recombination, wherein the means optionally comprises a transgene element. In one aspect, provided herein is a means for transferring a non-naturally occurring system, the means comprising: means for AAV-mediated recombination, wherein the means optionally comprises a transgene element.

[0073] In one aspect, provided herein is a non-naturally occurring system comprising: a recombination means for recombining the system, the recombination means comprising: means for AAV-mediated recombination, wherein the means optionally comprises a transgene element, wherein the recombination means includes the use of at least one serine residue during catalysis. In one aspect, provided herein is a means for transferring a non-naturally occurring system, the means comprising: a recombination means for recombining the system, the recombination means comprising: means for AAV-mediated recombination, wherein the means optionally comprises a transgene element, wherein the recombination means includes the use of at least one serine residue during catalysis.

[0074] In one aspect, provided herein is a means for manufacturing a molecule, wherein the means for manufacturing a molecule comprises any of the above means and is capable of replication.

[0075] In one aspect, the present disclosure provides a process for AAV-mediated site-specific recombination, the process comprising: (a) performing the function of obtaining a cell that contains a device for AAV-mediated recombination, wherein the device optionally contains a transgene element; (b) performing the function of growing the cell under conditions that allow site-specific recombination using at least one serine residue during catalysis. In one embodiment, the process includes obtaining a product, wherein optionally the product is a therapeutic product.

[0076] Based on the following disclosure, including the detailed description of the present invention and its preferred embodiments as well as the appended claims, other aspects, features, and advantages of the present invention will be apparent. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] The foregoing summary of the invention and the following detailed description of the preferred embodiments of the present patent application can be better understood when read in conjunction with the accompanying drawings. However, it should be understood that the present patent application is not limited to the exact embodiments shown in the drawings.

[0078] Figure 1 Shown are the alignment statistics and sequence alignment of the SPBetac2 integrase protein (SEQ ID NO:1, sequence to be checked) with a putative serine recombinase (SEQ ID NO:2, target sequence) identified in the genome of Bacillus safensis strain CCMA-560 (sequence ID: WP_029708089.1, length 535 amino acids). These two proteins have 64% sequence identity at the protein level in the range of amino acids 1 to 529. This putative serine recombinase is named SR21 (serine recombinase 21) herein.

[0079] Figure 2 Shown are the alignment statistics and sequence alignment of the identification of the strain representing the pre-insertion locus: the CCMA-560 DNA sequence (sequence to be checked) with nucleotides 464352 to 464839 of the whole-genome shotgun sequence of Bacillus safensis strain Fairview contig 56_1 (target sequence) (sequence ID: NZ_JFBY01000018.1, length 568093 nucleotides).

[0080] Figure 3The attP and attB sites of the SR21 recombinase are shown. The attP and attB sites are composed of dyad symmetry surrounding the central dinucleotide recombination crossover site (underlined). One half of the sites are numbered. Gaps are introduced into the attB sequence to show the alignment of sequences predicted to be bound by zinc ribbon domains (ZD) and recombinase domains (RD) extrapolated from previous studies (Rutherford et al., (2013) Nucleic Acids Res. 41:8341-8356). Residues that are identical in three or four of these ZD or RD domains are bolded. The alignment of attP (SEQ ID NO:7) with two alternative attB sequences (SEQ ID NO:8) and (SEQ ID NO:9) is shown.

[0081] Figure 4 Recombinase activation of the reporter genes is shown. Plasmid P41 encodes two reporter gene transcripts. The first reporter gene transcript driven by the EF1α promoter is constitutively active and encodes a fusion protein between green fluorescent protein (GFP) and Renilla luciferase linked by a self-cleaving F2A peptide linker. The second transcript driven by CMV contains the SR21 recombinase attB site (SEQ ID NO:9), followed by the coding region for a reverse fusion protein encoding mCherry and firefly luciferase linked by a P2A self-cleaving peptide linker, and the SR21 attP site. Since luciferase and mCherry are in opposite orientations relative to the promoter, neither of them is expressed. When the SR21 recombinase is expressed, the attB and attP sequences recombine, which causes inversion of the reporter genes and expression of firefly luciferase and mCherry.

[0082] Figure 5 The AAV capsid proteins in a purified recombinant AAV sample produced according to an embodiment of the present application are shown. Samples were purified from cells stably transfected with plasmid P439, cultured and infected in a multilayer cell culture flask (Hyperflask) vessel at 20 MOI (A) and 40 MOI (B), and the samples were subjected to PAGE and silver staining.

[0083] Figure 6 The rep / cap expression cassette in which an artificial intron with a termination cassette is inserted according to an embodiment of the present application is shown.

[0084] Figure 7 The vector (plasmid P439) according to an embodiment of the present application is shown.

[0085] Figure 8Shows the positions and sequences of RNA splicing sites identified by RT-PCR in P439. The top figure represents the structure of the REP gene after termination cassette excision. The 5’ and 3’ halves of REP are separated by the upstream half of the β-actin intron (SEQ ID NO:14), the SR21 AttL element (SEQ ID 35), and the downstream half of the β-actin intron (SEQ ID NO:15). The splicing between (2) the β-actin splicing donor (SEQ ID NO:71) and (3) the β-actin splicing acceptor (SEQ ID NO:72) is represented by a solid line. The splicing between (1) the upstream splicing donor in the 5’ REP sequence (SEQ ID NO:64) and (3) the 3’ β-actin acceptor (SEQ ID NO:72) is shown by a dashed line. The sequences of the splicing donors and acceptors are shown below. The lowercase letter sequences represent intron sequences.

[0086] Figure 9 Shows a vector (plasmid P600) according to an embodiment of the present application. Detailed Description

[0087] Background and various publications, articles, and patents are cited or described throughout the specification; each of these references is hereby incorporated by reference in its entirety. The discussion of documents, acts, materials, devices, articles, etc. included in this specification is intended to provide context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed herein.

[0088] 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 invention belongs. Otherwise, certain terms used herein have the meanings set forth in this specification. All patents, published patent applications, and publications cited herein are hereby incorporated by reference as if fully set forth herein.

[0089] It should be noted that, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” as used herein and in the appended claims include plural referents.

[0090] Unless otherwise stated, the term “at least” before a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0091] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "include" and "contain" will be understood to imply the inclusion of the stated integers or steps or groups of integers or steps but not the exclusion of any other integers or steps or groups of integers or steps. When used herein, the term "comprise" may be replaced with the term "contains" or "includes", or sometimes when used herein, with the term "having".

[0092] When used herein, "consisting of excludes any element, step, or ingredient not specified in the claim elements. When used herein, "consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Whenever used herein in the context of one aspect or embodiment of the present application, any of the foregoing terms "comprising," "containing," "including," and "having" may be replaced with the terms "consisting of" or "consisting essentially of" to alter the scope of the present disclosure.

[0093] As used herein, the connection term "and / or" between multiple listed elements is understood to include both single options and combined options. For example, where two elements are connected by "and / or", the first option refers to the application of the first element without the second element. The second option refers to the application of the second element without the first element. The third option refers to the application of the first element and the second element together. Any of these options is understood to fall within the meaning and therefore meets the requirements of the term "and / or" as used herein. The parallel applicability of more than one option is also understood to fall within the meaning and therefore meets the requirements of the term "and / or".

[0094] Unless otherwise indicated, any numerical value, such as the concentration or concentration range described herein is understood to be modified by the term "about" in all cases. Therefore, numerical values ​​generally include the value ± 10%. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1 mg / mL to 10 mg / mL includes 0.9 mg / mL to 11 mg / mL. Unless otherwise clearly indicated by the context, as used herein, the numerical range used explicitly includes all possible subranges, all single values ​​within the range, including integers within such ranges and fractions within the range.

[0095] The phrase "percent sequence identity (%)" or "identity %" or "percent identity to ...", when used with reference to a reference amino acid sequence, describes the number of matching ("hits") of identical amino acids in those amino acid sequences compared to the number of amino acid residues in the total length of the amino acid sequences that make up two or more aligned amino acid sequences. In other words, when comparing and aligning two or more sequences to achieve maximum correspondence as measured by sequence comparison algorithms known in the art, or when manually aligning and visually inspecting, the alignment of these sequences can be used to determine the percentage of amino acid residues that are identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99% or 100% identity over the full length of the amino acid sequence). Thus, the sequences being compared for determining sequence identity can differ by amino acid substitutions, additions or deletions. Suitable programs for aligning protein sequences are known to those skilled in the art. For example, programs such as CLUSTALW, Clustal Omega, FASTA or BLAST can be used, e.g., using the NCBI BLAST algorithm to determine the percent sequence identity of protein sequences (Altschul SF et al., (1997), Nucleic Acids Res. 25:3389-3402).

[0096] As used herein, an "unnaturally occurring" nucleic acid or polypeptide refers to a nucleic acid or polypeptide that does not exist in nature. An "unnaturally occurring" nucleic acid or polypeptide can be synthesized, processed, manufactured and / or otherwise manipulated in a laboratory and / or manufacturing setting. In some cases, an unnaturally occurring nucleic acid or polypeptide can comprise a naturally occurring nucleic acid or polypeptide that has been processed, engineered or manipulated to exhibit a property that was not present in the naturally occurring nucleic acid or polypeptide prior to the processing. As used herein, an "unnaturally occurring" nucleic acid or polypeptide can be an isolated or separated nucleic acid or polypeptide from its natural source of discovery and that lacks covalent bonds to sequences that are associated with it in the natural source. An "unnaturally occurring" nucleic acid or polypeptide can be prepared recombinantly or via other methods such as chemical synthesis.

[0097] As used herein, the term "hybrid" when used with reference to an AAV cap gene is intended to mean a cap gene that comprises a combination of portions of one serotype capsid and portions of a different serotype capsid. The term also includes AAV cap gene variants in which a naturally occurring AAV serotype sequence contains one or more unnaturally occurring mutations.

[0098] As used herein, the term "spacer sequence" is intended to mean a region of non-coding nucleotides that has no apparent function other than to separate other genetic elements.

[0099] As used herein, the term "operably linked" refers to a connection or adjacency where the components so described are in a relationship that permits them to function in their intended manner. For example, if a promoter affects the transcription of a coding sequence, the promoter is operably linked to the coding sequence, or a signal sequence operably linked to an amino acid sequence of interest can enable the amino acid sequence of interest to be secreted or translocated across a membrane.

[0100] To assist the reader of this application, the specification has been divided into various paragraphs or sections, or relates to various embodiments of this application. These divisions should not be construed as separating the substance of a paragraph or section or embodiment from the substance of another paragraph or section or embodiment. On the contrary, those skilled in the art will understand that this specification has broad application and encompasses all combinations of the various sections, paragraphs, and sentences that can be envisioned. The discussion of any embodiment is intended to be exemplary only and is not intended to indicate that the scope of the disclosure (including the claims) is limited to these examples. For example, while embodiments of the non-naturally occurring nucleic acids or recombinant vectors (e.g., plasmid DNA or viral vectors) of the application described herein may include specific components, including but not limited to certain promoter sequences, enhancer or regulatory sequences, introns, coding sequences for AAV Rep and / or Cap, polyadenylation signal sequences, etc., arranged in a specific order, those of ordinary skill in the art should understand that the concepts disclosed herein are equally applicable to other components arranged in other orders that can be used in the nucleic acids or vectors of this application. This application contemplates the use of any applicable components in any combination with any sequence that can be used in the nucleic acids or vectors of this application, regardless of whether a specific combination is explicitly described.

[0101] As used herein, a "vector" is a nucleic acid molecule used to transport genetic material into a cell, which can replicate and / or express in that cell. Any vector known to those skilled in the art from this disclosure can be used. Examples of vectors include but are not limited to plasmids, viral vectors (phages, animal viruses, and plant viruses), cosmids, and artificial chromosomes (e.g., YACs). Preferably, the vector is a DNA plasmid. Those of ordinary skill in the art can construct the vectors of this application by standard recombinant techniques according to this disclosure.

[0102] The vectors of this application can be expression vectors. As used herein, the term "expression vector" refers to any type of gene construct that contains nucleic acid encoding an RNA that can be transcribed. Expression vectors include but are not limited to vectors for recombinant protein expression such as DNA plasmids or viral vectors, and vectors for delivering nucleic acids into a subject for expression in the tissues of the subject such as DNA plasmids or viral vectors. Those skilled in the art should understand that the design of an expression vector can depend on factors such as the choice of host cell to be transformed, the desired level of protein expression, etc.

[0103] In some embodiments of the present application, the vector is a non-viral vector. Examples of non-viral vectors include, but are not limited to, DNA plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, etc. Preferably, the non-viral vector is a DNA plasmid. "DNA plasmid", which can be used interchangeably with "DNA plasmid vector", "plasmid DNA", or "plasmid DNA vector", refers to a double-stranded and generally circular DNA sequence that can autonomously replicate in a suitable host cell. The DNA plasmid used for the expression of the polynucleotide to be encoded usually contains an origin of replication, a multiple cloning site, and a selectable marker, which can be, for example, an antibiotic resistance gene. Examples of suitable DNA plasmids that can be used include, but are not limited to, commercially available expression vectors for well-known expression systems (including prokaryotic systems and eukaryotic systems), such as pSE420 (Invitrogen, San Diego, Calif.), which can be used to produce and / or express proteins in Escherichia coli; pYES2 (Invitrogen, Thermo Fisher Scientific), which can be used to produce and / or express in Saccharomyces cerevisiae strains of yeast; the complete baculovirus expression system (Thermo Fisher Scientific), which can be used to produce and / or express in insect cells; TM pcDNA TM (Life Technologies, Thermo Fisher Scientific), which can be used for high-level constitutive protein expression in mammalian cells; and pVAX or pVAX-1 (Life Technologies, Thermo Fisher Scientific), which can be used for high-level transient expression of proteins of interest in most mammalian cells. The backbone of any commercially available DNA plasmid can be modified using conventional techniques and readily available starting materials to optimize protein expression in host cells, such as reversing the orientation of certain elements (e.g., the origin of replication and / or the antibiotic resistance cassette), replacing the promoter endogenous to the plasmid (e.g., the promoter in the antibiotic resistance cassette), and / or replacing the polynucleotide sequence encoding the transcribed protein (e.g., the coding sequence of the antibiotic resistance gene). (See, for example, Sambrook et al., Molecular Cloning a Laboratory Manual, Second Edition, Cold Spring Harbor Press (1989)).

[0104] Preferably, the DNA plasmid is an expression vector suitable for protein expression in mammalian host cells. Expression vectors suitable for protein expression in mammalian host cells include, but are not limited to, pUC, pcDNA™, pcDNA3™, pVAX, pVAX-1, ADVAX, NTC8454, etc. For example, the vector can be based on pUC57, which contains the pUC origin of replication and the ampicillin resistance gene (SEQ ID NO:30). It can also contain a mammalian puromycin resistance gene cassette constructed from the herpes simplex virus thymidine kinase gene promoter (SEQ ID NO:26), the puromycin N-acetyltransferase coding region (SEQ ID NO:27), and the polyadenylation signal from the bovine growth hormone gene (SEQ ID NO:28). The vector can also contain the Epstein-Barr virus (EBV) OriP origin of replication fragment (SEQ ID NO:29), which represents a complex of the "dyad symmetry" region and the "repeat family" region of EBV.

[0105] The vector of the present application can also be a viral vector. Generally speaking, a viral vector is a genetically engineered virus carrying modified viral DNA or RNA, which has been rendered non-infectious but still contains a viral promoter and a transgene, thus allowing the translation of the transgene through the viral promoter. Since viral vectors often lack infectious sequences, they require helper viruses or packaging lines for large-scale transfection. Examples of viral vectors that can be used include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, poxvirus vectors, enterovirus vectors, Venezuelan equine encephalitis virus vectors, Semliki Forest virus vectors, tobacco mosaic virus vectors, lentivirus vectors, etc. The vector can also be a non-viral vector.

[0106] Preferably, the viral vector is an adenoviral vector, such as a recombinant adenoviral vector. As used herein, the terms "recombinant adenovirus vector" and "recombinant adenoviral vector" and "recombinant adenovirus particle" are used interchangeably and refer to a genetically engineered adenovirus that is designed to insert a polynucleotide of interest into a eukaryotic cell such that the polynucleotide is subsequently expressed. Examples of adenoviruses that can be used as the viral vector of the present invention include those having or derived from serotypes Ad2, Ad5, Ad11, Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, Ad49, Ad50, Ad52 (e.g., RhAd52) and Pan9 (also known as AdC68); these vectors can be derived from, for example, humans, chimpanzees (e.g., ChAd1, ChAd3, ChAd7, ChAd8, ChAd21, ChAd22, ChAd23, ChAd24, ChAd25, ChAd26, ChAd27.1, ChAd28.1, ChAd29, ChAd30, ChAd31.1, ChAd32, ChAd33, ChAd34, ChAd35.1, ChAd36, ChAd37.2, ChAd39, ChAd40.1, ChAd41.1, ChAd42.1, ChAd43, ChAd44, ChAd45, ChAd46, ChAd48, ChAd49, ChAd49, ChAd50, ChAd67 or SA7P) or rhesus monkey adenoviruses (e.g., rhAd51, rhAd52 or rhAd53). The recombinant adenoviral vector can be derived, for example, from a human adenovirus (HAdV or AdHu) or a simian adenovirus such as a chimpanzee or gorilla adenovirus (ChAd, AdCh or SAdV) or a rhesus monkey adenovirus (rhAd).

[0107] Preferably, the adenoviral vector is a recombinant human adenoviral vector, such as recombinant human adenovirus serotype 5 or any one of recombinant human adenovirus serotypes 26, 4, 35, 7, 48, etc. The recombinant viral vector that can be used in the present application can be prepared by methods known in the art according to the present disclosure. For example, according to the degeneracy of the genetic code, several nucleic acid sequences encoding the same polypeptide can be designed. The polynucleotide encoding the protein of interest can optionally be codon-optimized to ensure proper expression in a host cell (e.g., a bacterial or mammalian cell). Codon optimization is a technique widely used in the art, and according to the present disclosure, the methods for obtaining codon-optimized polynucleotides will be well known to those skilled in the art.

[0108] Non-naturally occurring nucleic acid molecules or vectors can contain one or more expression cassettes. An "expression cassette" is a part of a nucleic acid molecule or vector that directs the cellular machinery to produce RNA and protein. An expression cassette can contain a promoter sequence, an open reading frame, a 3'-untranslated region (UTR), which optionally contains a polyadenylation signal. The open reading frame (ORF) is the reading frame that contains the coding sequence of the protein of interest (e.g., Rep, Cap, recombinase, or recombinant protein of interest) from the start codon to the stop codon. The regulatory elements of the expression cassette can be operably linked to the polynucleotide sequence encoding the protein of interest.

[0109] The non-naturally occurring nucleic acid molecules or vectors of the present application can contain a variety of regulatory sequences. As used herein, the term "regulatory sequence" refers to any sequence that permits, facilitates, or regulates the functional regulation of a nucleic acid molecule, which includes replication, multiplication, transcription, splicing, translation, stability, and / or transport into a host cell or organism of one of the nucleic acids or its derivatives (i.e., mRNA). Regulatory elements include, but are not limited to, promoters, enhancers, polyadenylation signals, translation termination codons, ribosome binding elements, transcription terminators, selectable markers, origins of replication, etc.

[0110] Non-naturally occurring nucleic acid molecules or vectors can contain a promoter sequence, preferably within the expression cassette, to control the expression of the protein of interest. The term "promoter" is used in its conventional sense and refers to a nucleotide sequence that initiates the transcription of an operably linked nucleotide sequence. The promoter is on the same strand and near the nucleotide sequence it transcribes. The promoter can be constitutive, inducible, or repressible. The promoter can be naturally occurring or synthetic. The promoter can be derived from sources including viruses, bacteria, fungi, plants, insects, and animals. The promoter can be a homologous promoter (i.e., derived from the same genetic source as the vector) or a heterologous promoter (i.e., derived from a different vector or genetic source). For example, if the vector to be used is a DNA plasmid, the promoter can be endogenous (homologous) to the plasmid or derived from other sources (heterologous). Preferably, the promoter is upstream of the polynucleotide encoding the protein of interest within the expression cassette.

[0111] Examples of promoters that can be used include, but are not limited to, the promoter from Simian virus 40 (SV40), the Mouse Mammary Tumor Virus (MMTV) promoter, the Human Immunodeficiency Virus (HIV) promoter such as the Bovine Immunodeficiency Virus (BIV) Long Terminal Repeat (LTR) promoter, the Moloney virus promoter, the Avian Leukosis Virus (ALV) promoter, the Cytomegalovirus (CMV) promoter such as the CMV immediate early promoter (CMV-IE), the Epstein-Barr virus (EBV) promoter, or the Rous Sarcoma Virus (RSV) promoter. The promoter can also be a promoter from a human gene such as human actin, human myosin, human hemoglobin, human muscle creatine, or human metallothionein. The promoter can also be a natural or synthetic tissue-specific promoter, such as a muscle or skin-specific promoter. Preferably, the promoter is a strong eukaryotic promoter, such as the Cytomegalovirus (CMV) promoter (nt –672 to +15), the EF1-α promoter, the Herpes simplex virus thymidine kinase gene promoter (SEQ ID NO:26), etc.

[0112] The non-naturally occurring nucleic acid molecule or vector can include additional polynucleotide sequences that stabilize the expressed transcript, enhance the nuclear export of the RNA transcript, and / or improve transcription-translation coupling. Examples of such sequences include polyadenylation signals and enhancer sequences. The polyadenylation signal is typically located downstream of the coding sequence of the protein of interest (e.g., Rep, Cap, recombinase) within the expression cassette of the vector. The enhancer sequence is a regulatory DNA sequence that enhances the transcription of the associated gene when bound by a transcription factor. The enhancer sequence is preferably downstream of the promoter sequence and can be downstream or upstream of the coding sequence within the expression cassette of the vector.

[0113] Any polyadenylation signal known to those skilled in the art based on the present disclosure can be used. For example, the polyadenylation signal can be the SV40 polyadenylation signal (e.g., SEQ ID NO:60), the AAV2 polyadenylation signal (bp 4411-4466, NC_001401.2), the polyadenylation signal from the Herpes simplex virus thymidine kinase gene (SEQ ID NO:23), the LTR polyadenylation signal, the Bovine Growth Hormone (bGH) polyadenylation signal, the Human Growth Hormone (hGH) polyadenylation signal, or the Human β-globin polyadenylation signal. Preferably, the polyadenylation signal is the Bovine Growth Hormone (bGH) polyadenylation signal (SEQ ID NO:28), the AAV2 polyadenylation signal of nucleotide numbers 4411 to 4466 of the nucleotide sequence with GenBank accession number NC_001401.2, or the SV40 polyadenylation signal (SEQ ID NO:60).

[0114] Any enhancer sequence known to those skilled in the art based on the present disclosure can be used. For example, the enhancer sequence can be a human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer, such as an enhancer from CMV, HA, RSV, or EBV. Examples of specific enhancers include, but are not limited to, the woodchuck hepatitis B virus post-transcriptional regulatory element (WPRE), the intron / exon sequence derived from human apolipoprotein A1 precursor (ApoAI), the untranslated R-U5 domain of the long terminal repeat (LTR) of human T-cell leukemia virus type 1 (HTLV-1), a splicing enhancer, a synthetic rabbit β-globin intron, or any combination thereof.

[0115] Preferably, the enhancer sequence comprises the P5 promoter of AAV. The P5 promoter is part of a cis-acting Rep-dependent element (CARE) within the coding sequence of the rep gene. The CARE has been shown to enhance replication and encapsidation when present in cis. As in some AAV producer cell lines, the CARE is also important for the amplification of the chromosomally integrated rep gene (if AAV ITRs are absent). Without wishing to be bound by theory, it is believed that the P5 promoter placed downstream of the cap coding sequence potentially acts as an enhancer to increase Cap expression, thus producing AAV, and it also provides enhancer activity for amplifying genes integrated into the chromosome.

[0116] Non-naturally occurring nucleic acid molecules or vectors such as DNA plasmids may also contain a bacterial origin of replication and an antibiotic resistance expression cassette for selection and maintenance in bacterial cells such as Escherichia coli (E. coli). The origin of replication (ORI) is the sequence that initiates replication, enabling the plasmid to replicate and survive within the cell. Examples of ORIs suitable for the present application include, but are not limited to, ColE1, pMB1, pUC, pSC101, R6K, and 15A, preferably pUC.

[0117] Vectors for selection and maintenance in bacterial cells typically contain a promoter sequence operably linked to an antibiotic resistance gene. Preferably, the promoter sequence operably linked to the antibiotic resistance gene is different from the promoter sequence operably linked to the polynucleotide sequence encoding the protein of interest. The antibiotic resistance gene can be codon-optimized, and generally the sequence composition of the antibiotic resistance gene is adjusted to accommodate the codon usage of bacteria such as Escherichia coli (E. coli). Any antibiotic resistance gene known to those skilled in the art based on the present disclosure can be used, including but not limited to the kanamycin resistance gene (Kan r ), the ampicillin resistance gene (Amp r ), and the tetracycline resistance gene (Tet r ), as well as genes conferring resistance to chloramphenicol, bleomycin, spectinomycin, carbenicillin, etc.

[0118] Vectors for selection and maintenance in mammalian cells typically contain a promoter sequence operably linked to a gene encoding a protein conferring a selectable marker. Preferably, the gene also contains a polyadenylation signal. For example, a mammalian puromycin resistance gene cassette can contain the herpes simplex virus thymidine kinase gene promoter (SEQ ID NO:26), the puromycin N-acetyltransferase coding region (SEQ ID NO:27), and the polyadenylation signal from the bovine growth hormone gene (SEQ ID NO:28).

[0119] Production of recombinant AAV in human cells requires expression of the AAV replication (rep) and capsid (cap) genes, adenovirus genes, and an AAV-packagable transgene consisting of an expression cassette flanked by AAV inverted terminal repeats (ITRs). All three components can be delivered to cells on separate plasmids for AAV production, but existing transfection methods are difficult to scale up to large-scale culture. Incorporating some of these elements into a host cell line can make AAV production more efficient, but some AAV and adenovirus genes are cytostatic or cytotoxic, thus limiting this approach.

[0120] This application describes non-naturally occurring nucleic acid molecules, vectors, cells, and methods for reversibly inactivating the AAV rep gene such that the AAV rep gene, AAV cap gene, and packagable transgene can be maintained and / or integrated into a suitable host cell and amplified. Infection of these cells with a recombinant adenovirus expressing a recombinase will reactivate the rep gene and induce AAV replication and packaging. Different from the method described by Xiao and colleagues (Qiao et al., (2002) J. Virol. 76:13015-13027; Yuan et al., (2011) Hum. Gene Ther. 22:613-624) which uses Cre, a tyrosine recombinase that recognizes two identical loxP sites and catalyzes both ligation and excision reactions, the present invention uses serine recombinase 21 (SR21), a serine recombinase newly characterized by the inventors of this application. Different from Cre, SR21 recognizes attP and attB sites with different sequences. After the ligation reaction catalyzed by SR21, the attP and attB sites recombine and are disrupted, thus making recombination impossible to occur again. Therefore, the method of this application can be more efficient than the method catalyzed by Cre. Certain embodiments of this application include additional features, such as different termination cassettes inserted in different artificial introns, enhancers, insulators, etc., which further improve the methods in the prior art. The reversible inactivation / reactivation system of this application allows strict control of the AAV rep gene during the growth of packaging cells, thus avoiding the cytostatic / cytotoxic effects of Rep proteins on host cells. It also provides strong induction of the AAV rep gene and high yields of these vectors during the production of AAV vectors.

[0121] Serine Recombinase

[0122] Site-specific recombination catalyzed by members of the large serine recombinase family, such as SR21, does not require the cellular machinery for homologous recombination. Generally, it requires specialized recombinases that recognize these sites, break, and ligate the DNA. Based on amino acid sequence homology and mechanistic relatedness, most site-specific recombinases are grouped into one of the following two families: the tyrosine recombinase family or the serine recombinase family. These names are derived from the conserved nucleophilic amino acid residues they use to attack DNA and covalently link to it during strand exchange.

[0123] Serine recombinases bind to and recombine separate recombination recognition sites, which are referred to as the following "attachment sites": attP "attachment phage" and attB "attachment bacterium" chromosome. The attP and attB sites consist of dyad symmetry around a central dinucleotide recombination crossover site. The left and right halves of the attP and attB sites are bound by recombinase monomers via zinc ribbon (ZD) and recombinase (RD) domains (Rutherford et al., (2013) Nucleic Acids Res. 41:8341-8356).

[0124] As described in more detail in the Examples below, a serine recombinase was newly identified in the genome of Bacillus safensis strain CCMA-560 in the present invention, which is referred to herein as "serine recombinase 21" or "SR21". The attP and attB sites recognized by SR21 were also characterized in the present invention.

[0125] In one general aspect, the present application relates to a non-naturally occurring nucleic acid molecule that comprises a nucleotide sequence encoding a serine recombinase having an amino acid sequence with at least 85% identity, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:2. Preferably, the non-naturally occurring nucleic acid molecule comprises a nucleotide sequence encoding a serine recombinase having the amino acid sequence of SEQ ID NO:2. In one embodiment, the non-naturally occurring nucleic acid molecule comprises a nucleotide sequence having at least 85% identity, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the nucleotide sequence of SEQ ID NO:3.

[0126] In certain embodiments, the present application relates to a vector that comprises the non-naturally occurring nucleic acid. The vector can be an expression vector that expresses the serine recombinase in a cell of interest, such as a bacterial cell or a mammalian cell. In one embodiment, the vector expresses the serine recombinase in a mammalian cell under the control of a cytomegalovirus (CMV) promoter or any other suitable promoter described herein or known in the art. In certain embodiments, the vector may further comprise a polyadenylation signal, such as a simian virus 40 (SV40) polyadenylation signal or any other suitable polyadenylation signal described herein or known in the art.

[0127] In one embodiment, the vector is a DNA plasmid, such as plasmid P175 having the nucleotide sequence of SEQ ID NO:10.

[0128] In another embodiment, the vector is a viral vector, such as a recombinant adenovirus vector.

[0129] In one embodiment, the vector is a recombinant ΔE1 / ΔE3 adenovirus serotype 5 (Ad5) virus that contains a nucleotide sequence encoding a serine recombinase having at least 85% identity, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to the amino acid sequence of SEQ ID NO:2, and the coding sequence is under the control of a promoter functional in mammalian cells. Preferably, the promoter is the CMV promoter. More preferably, the recombinant Ad5 vector contains, in 5' to 3' order, the CMV promoter operably linked to a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:2, which nucleotide sequence is operably linked to the SV40 polyadenylation signal (NC_001669.1, nt 2550 to 2774). In one embodiment, the nucleotide sequence encoding the amino acid sequence of SEQ ID NO:2 is the same as SEQ ID NO:3, except that the bacterial translation initiation codon "TTG" is replaced with "ATG", and three point mutations are introduced to disrupt the restriction endonuclease recognition sites within SEQ ID NO:3. These restriction endonuclease recognition sites are the Xba I site (TCTAGA); the Sac I site (GAGCTC); the EcoRI site (GAATTC).

[0130] According to the present disclosure, any method known in the art can be used to prepare the vector encoding the serine recombinase of the present application.

[0131] As described in more detail in the following examples, the attP and attB sites of the serine recombinase of the present application were identified in the present invention. In certain embodiments, the serine recombinase of the present application recognizes an attP site comprising the nucleotide sequence of SEQ ID NO:7 or a variant thereof. In certain embodiments, the serine recombinase of the present application recognizes an attP site comprising a nucleotide sequence having at least 90% identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to SEQ ID NO:7.

[0132] In certain embodiments, the serine recombinase of the present application recognizes an attB site comprising the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9 or a variant thereof. In certain embodiments, the serine recombinase of the present application recognizes an attB site comprising a nucleotide sequence having at least 90% identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to SEQ ID NO:8 or SEQ ID NO:9.

[0133] In one embodiment, the present application relates to a method for site-specific recombination in a cell. The method comprises:

[0134] 1) obtaining a cell comprising a nucleic acid molecule having: an attP site comprising a nucleotide sequence having at least 90% identity to SEQ ID NO:7, and an attB site comprising a nucleotide sequence having at least 90% identity to SEQ ID NO:8 or SEQ ID NO:9;

[0135] 2) introducing into the cell a non-naturally occurring nucleic acid molecule encoding a serine recombinase having at least 85% identity, such as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity, to SEQ ID NO:2; and

[0136] 3) growing the cell under conditions that allow the serine recombinase to catalyze site-specific recombination between the attP and attB sites.

[0137] In a preferred embodiment, the present application relates to a method for site-specific recombination in a cell. The method comprises:

[0138] 1) obtaining a cell comprising a nucleic acid molecule having: an attP site having the nucleotide sequence of SEQ ID NO:7, and an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9;

[0139] 2) introducing into the cell a non-naturally occurring nucleic acid molecule encoding a serine recombinase having the amino acid sequence of SEQ ID NO:2; and

[0140] 3) growing the cell under conditions that allow the serine recombinase to catalyze site-specific recombination between the attP and attB sites.

[0141] Constructs, Cells, and Methods for Producing Recombinant AAV

[0142] As shown in the following examples, the newly identified serine recombinase of the present application can be used to increase the production of recombinant AAV.

[0143] Modified AAV rep Gene Construct

[0144] In one general aspect, the present application relates to a non-naturally occurring nucleic acid molecule that comprises a modified adeno-associated virus (AAV) rep gene, the modified AAV rep gene having an AAV rep gene encoding four Rep proteins, Rep78, Rep68, Rep52, and Rep40, and an artificial intron inserted into the coding sequence of the rep gene shared by these four Rep proteins. The artificial intron comprises a termination cassette inserted downstream of the 5' splice site and upstream of the branch site of the artificial intron, and the termination cassette comprises, in 5' to 3' order: (i) an attP site comprising a nucleotide sequence having at least 90% identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, with SEQ ID NO:7; (ii) a splice acceptor; (iii) a terminator; and (iv) an attB site comprising a nucleotide sequence having at least 90% identity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, with SEQ ID NO:8 or SEQ ID NO:9. Preferably, the attP site has the nucleotide sequence of SEQ ID NO:7 and the attB site has the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9.

[0145] As used herein, "intron" is broadly defined as a nucleotide sequence that can be removed by RNA splicing. "RNA splicing" means excising introns from precursor mRNA to form mature mRNA. As used herein, "artificial intron" refers to a nucleotide sequence that is not a naturally occurring intron of a gene but can still be removed by RNA splicing. For example, an "artificial intron" can be a naturally occurring intron into which a termination cassette has been inserted.

[0146] Introns (including artificial introns) contain a 5′ splice site or splice junction, a splice acceptor or branch point, and a 3′ splice site or splice junction. The term “5′ splice site” or “5′ splice junction” means the position of the exon-intron junction where the junction is between the 3′ end of the 5′ segment of a gene or nucleic acid fragment and the 5′ end of the intron, and contains a consensus sequence required for RNA splicing at the 5′ end of the intron. The term “splice acceptor” or “branch point” refers to a nucleotide (usually adenosine) located approximately 20 to 50 bp from the 3′ splice site, which aids in the formation of a lariat structure during the first transesterification reaction in RNA splicing. The term “3′ splice site” or “3′ splice junction” means the position of the exon-intron junction where the junction is between the 5′ end of the 3′ segment of a gene or nucleic acid fragment and the 3′ end of the intron, and also contains a consensus sequence required for RNA splicing at the 3′ end of the intron. The term “consensus sequence” means the nucleotides required for RNA splicing in or near the 5′ or 3′ splice junction; these sequences are usually invariant or highly conserved.

[0147] Analysis of a large number of mRNAs has revealed that certain nucleotides are conserved in typical introns and splice junctions. For example, the almost invariant bases of an intron are 5′-GU and 3′-AG. Certain bases flanking these 5′ and 3′ conserved regions are usually present at abnormal (non-random) frequencies. Also conserved is the branch point adenosine, usually 20 to 50 bases from the 3′ splice site. See, for example, Gao et al. (2008) Nucleic Acids Research 36:2257-2267, Figure 4 which shows the general consensus regions of an intron in the context of an exon, the entire content of Gao et al. (2008) being incorporated herein by reference. However, the central region of an intron, which can range in length from 40 to 50,000 bases, is generally not necessary for splicing. The intron is removed from the RNA or pre-mRNA in the form of a lariat structure by the spliceosome. The exons are spliced together via two consecutive transesterification reactions.

[0148] Insertion of an intron into an expressed sequence can be accomplished by any method known in the art. The flanking exon context as well as the actual intron sequence to be used play a role in whether the new “intron” will be effectively spliced out. Introns suitable for the present invention can be tested by preparing composite sequences in silico and using an online splicing prediction program to find combinations of rep gene sequences and intron sequences that give a high enough score for effective RNA splicing. According to the present disclosure, any intron in a genomic or synthetic sequence can be tested and optimized for use in the constructs of the present invention.

[0149] To disrupt the expression of all four rep open reading frames of Rep78, Rep68, Rep52, and Rep40, an artificial intron is preferably inserted into the coding sequence of the rep gene shared by these four Rep proteins. Thus, in certain embodiments, to disrupt all four ORFs, the artificial intron is inserted at nucleotide 996 of AAV2 and extending to 1905 (NC_001401.2) or at the corresponding position in another AAV rep gene. However, to allow the termination cassette to function when inserted into the artificial intron, it is preferred that the intron be inserted as upstream as possible in the rep gene.

[0150] In addition, the exon context immediately upstream and downstream of the intron insertion site is important for defining what will serve as a possible insertion site, such as the consensus common region of the intron in the exon context discussed above. In one embodiment, the consensus sequence CAG^G (where ^ marks where the insertion will occur) occurs in the relevant region of the rep gene in AAV2 as follows, where the number indicates the last nucleotide of AAV before the insertion: 1052, 1061, 1712, and 1906. In another embodiment, the consensus sequence AAG^G occurs at positions 1022 (as used by Qiao), 1112, 1475, 1514, 1700, 1742, and 1784 in AAV2. Other consensus sites such as AAG^A occur, for example, at nucleotide 1340 of AAV2. Preferred insertion sites have also been identified in the rep genes of other AAVs according to the present disclosure.

[0151] The artificial introns useful in the present invention can be derived from any source, such as from a genomic library. As described below, the introns can be obtained from human DNA using primers by polymerase chain reaction (PCR). Any intron capable of performing RNA splicing in a cell can be used in the method of the present invention. In the following examples, the intron is the intron of the human β-actin gene.

[0152] According to an embodiment of the present application, in addition to RNA splicing via an artificial intron, the expression of the Rep protein is also regulated by DNA splicing via a termination cassette inserted into the artificial intron. The termination cassette contains a transcription terminator flanked by attP and attB sites specifically recognized by a serine recombinase (such as the serine recombinase characterized in the present invention). In one embodiment, the terminator contains one or more polyadenylation signals. In another embodiment, the terminator contains another sequence for efficient transcriptional termination, such as a sequence from the human β-globin gene, downstream of the polyadenylation signal and encoding a self-cleaving RNA motif, which preferably has the nucleotide sequence of SEQ ID NO:19. Other terminators can also be used in the present invention, such as a hammerhead ribozyme that cleaves its own RNA. See West (2008) Molecular Cell 29:600–610 for replacing the β-globin element with other ribozymes, and see Kharma (2016) Nucleic Acids Res. 44:e39 for a description of designing ribozymes, the contents of both of which are incorporated herein by reference in their entirety.

[0153] In one embodiment, the termination cassette further contains a gene encoding a selectable marker. In one embodiment, the selectable marker gene contains a neomycin phosphotransferase expression cassette (neo) (SEQ ID NO:18), which is driven by a mammalian promoter (e.g., mouse phosphoglycerate kinase 1) and a bacterial (e.g., Lac zya) promoter and followed by a polyadenylation signal (such as the polyadenylation signal from SV40). This gene confers resistance to neomycin and kanamycin in mammalian cells and bacterial cells, respectively. Without wishing to be bound by theory, it is believed that in addition to using the selectable marker for cell line formation, the selectable marker gene can also block the transcription of the rep gene, thereby increasing the stability of host cells containing the modified rep gene. Other selectable marker genes that can be used in the present invention include, but are not limited to, antibiotic selection genes (puromycin, hygromycin, bleomycin), metabolic genes (e.g., glutamine synthetase or hypoxanthine-guanine phosphoribosyltransferase (HPRT)), visual markers such as mCherry, enzymes such as β-galactosidase, secreted alkaline phosphatase, or any other suitable marker gene.

[0154] In another embodiment, the termination cassette comprises a splice acceptor to prevent splicing out of the termination cassette from the primary mRNA transcript. Any naturally occurring splice acceptor site or synthetic sequence may be used, so long as the splice acceptor is not skipped. According to an embodiment of the present application, the splice acceptor comprises a branch point sequence conforming to the consensus region (yTnAynn), where y is C or T and n is any nucleotide, polypyrimidine tract (4 to 24 nt), "AG" dinucleotide, and a 20 to 80 bp eukaryotic gene exon sequence (or a synthetic sequence that behaves like an exon when placed next to an intron sequence). This sequence should be recognized as a splice acceptor site by the NetGene2 splicing prediction software (www.cbs.dtu.dk / services / NetGene2 / ; Brunak, S., Engelbrecht, J., and Knudsen, S.: Prediction of Human mRNA Donor and Acceptor Sites from the DNA Sequence, Journal of Molecular Biology, 1991, 220, 49-65), with a confidence score of 0.4 or better (or using similar splicing prediction software); the closer the score is to 1.0, the better. In one embodiment, the splice acceptor comprises the nucleotide sequence of SEQ ID NO: 17 (NC_000086.7, nucleotides 53001998 to 53002138 from the mouse HPRT gene, plus a 29 nt region from the human agouti signaling protein (NC_000020.11, nucleotides 34262765 to 34262793).

[0155] According to an embodiment of the present application, the termination cassette is inserted downstream of the 5' splice donor site of an artificial intron and upstream of the splice acceptor "branch point". The termination cassette may be inserted at any position between these two sites, so long as the insertion does not damage the function of these sites. In one embodiment, the termination cassette is inserted in the middle of these two sites. In one exemplary embodiment described in the following examples, the termination cassette is inserted into the intron of the human β-actin gene such that the 5' intron fragment has the nucleotide sequence of SEQ ID NO: 14 and the 3' intron fragment has the nucleotide sequence of SEQ ID NO: 15.

[0156] As provided herein, in some embodiments, the 3' intron fragment may contain a spacer sequence such that the REP / CAP gene is too large to be packaged in AAV. For example, the AAV packaging limit is approximately 5.0 kb. Thus, according to the present disclosure provided herein, a spacer sequence can be generated that makes the REP / CAP gene larger than approximately 5.0 kb. In some embodiments, the spacer sequence is a 2 kb random spacer region inserted into the 3' intron fragment. Thus, in the exemplary embodiments described in the following examples, a termination cassette was inserted into the intron of the human β-actin gene such that the 5' intron fragment has the nucleotide sequence of SEQ ID NO:14 and the 3' intron fragment has the nucleotide sequence of SEQ ID NO:66. However, it should be understood that the spacer sequence need not be 2 kb and can be any length that makes the REP / CAP gene larger than approximately 5.0 kb.

[0157] Any AAV rep gene can be included in the modified rep gene of the present invention. For example, the AAV rep gene can include the rep gene of one of AAV1 to AAV8 or a hybrid thereof. The sequences of the AAV rep genes are available from, for example, GenBank, and the various AAV genomes have the following GenBank accession numbers: AAV1, GenBank accession number NC_002077.1; AAV2, GenBank accession number NC_001401.2; AAV3, GenBank accession number NC_001729.1; AAV4, GenBank accession number NC_001829.1; AAV5, GenBank accession number NC_006152.1; AAV6, GenBank accession number AF028704.1; AAV7, GenBank accession number NC_006260.1; and AAV8, GenBank accession number NC_006261.1.

[0158] In the following examples, the rep gene of human AAV2 with nucleotide numbers 190 to 2202 having the nucleotide sequence of GenBank accession number NC_001401.2 was used.

[0159] In some embodiments, cryptic splice sites in the rep gene can be modified to eliminate splicing at that site. For example, synonymous mutations can be made to the DNA sequence, where the DNA sequence is mutated but the mutation does not change the encoded amino acid.

[0160] Constructs with Modified AAV rep Gene and AAV cap Gene

[0161] In another general aspect, the present application relates to a non-naturally occurring nucleic acid molecule that comprises a modified AAV rep gene and an AAV cap gene of the present application or a hybrid thereof. Preferably, the AAV cap gene is downstream of the modified AAV rep gene.

[0162] In one embodiment, the AAV cap gene further comprises a polyadenylation signal operably linked to the coding sequence of the gene. In the exemplary embodiment described in the following examples, the AAV2 polyadenylation signal (bp 4411-4466, NC_001401.2) is included downstream of the AAV9 cap coding sequence.

[0163] In another embodiment, the AAV cap gene further comprises an enhancer. In the following examples, the AAV2 rep P5 promoter (bp 190-313, NC_001401.2) is included downstream of the AAV2 polyadenylation signal.

[0164] In certain embodiments, the AAV cap gene encodes all three capsid proteins VP1, VP2, and VP3.

[0165] In other embodiments, the AAV cap gene encodes fewer than three of these capsid proteins. For example, AAV serotypes 1 to 5 have been reported to be able to be successfully packaged, replicated, and transduced in cells in the absence of VP2 (Grieger et al., J Virol. August 2005; 79(15):9933–9944). Thus, in one embodiment, the AAV cap gene encodes VP1 and VP3 of any one of AAV 1 to AAV5 or a hybrid thereof, and does not encode VP2.

[0166] Any AAV cap gene can be used in the present invention. For example, the AAV cap gene can be the cap gene of one of AAV1 to AAV8, AAV9, AAVDJ, or a hybrid thereof. In one embodiment, the cap gene is an AAV9 variant. The sequences of the AAV cap genes are available from, for example, GenBank. See the above GenBank accession numbers for the AAV1 to AAV8 genomes. The AAV9 genome has GenBank accession number AY530579.1, and AAVDJ has GenBank protein accession number 3J1Q_A.

[0167] In one embodiment described in the following examples, the cap open reading frame of human AAV9 having the nucleotide sequence of GenBank accession number AY530579.1 is used.

[0168] Constructs with Modified AAV rep Gene, AAV cap Gene, and Transgene

[0169] In another general aspect, the present application relates to a non-naturally occurring nucleic acid molecule that comprises a modified AAV rep gene, an AAV cap gene, and a transgene flanked by AAV inverted terminal repeats (ITRs) of the present application.

[0170] ITRs are important cis-acting sequences in AAV biology. ITRs play a key role in AAV DNA replication. In addition to its role in AAV replication, ITRs are also required for AAV genome packaging, transcription, negative regulation under non-permissive conditions, and site-specific integration.

[0171] In one embodiment, the 130 bp ITR comprises the nucleotide sequence of SEQ ID NO:20 (nucleotides 4535 to 4664, NC_001401.2) derived from the 3' AAV2 ITR, which is used to flank the transgene. In another embodiment, a shorter mutant ITR is used. For example, for shorter genes, the ITR is mutated to be shorter and the gene can fold into a double-stranded form to increase expression and accelerate expression after infection. See McCarty 2008 Mol Ther. 2008;16(10):1648–56.

[0172] In another embodiment, the transgene comprises a promoter, preferably a promoter that is functional in mammalian cells. In the following examples, the human EF1-α promoter (comprising exon 1, intron 1, and a portion of exon 2) (SEQ ID NO:21) is included in the transgene.

[0173] In another embodiment, the transgene comprises a polyadenylation signal. In the following examples, the polyadenylation signal from the herpes simplex virus thymidine kinase gene (SEQ ID NO:23) is included in the transgene.

[0174] In yet another embodiment, the non-naturally occurring nucleic acid molecule comprises a pair of insulators flanking the modified AAV rep gene, an AAV cap gene, and a transgene flanked by ITRs. In another embodiment, the non-naturally occurring nucleic acid molecule comprises a single insulator upstream of the modified AAV rep gene, an AAV cap gene, and a transgene flanked by ITRs. In one embodiment, the insulator comprises a genomic element that blocks chromatin-associated repression of gene expression (Kwaks et al., (2003) Nature Biotechnology 21:554-558; Kwaks et al., (2003) Nature Biotechnology 21:822).

[0175] Any suitable insulator (such as those described herein) can be used in the present invention. In one embodiment, the insulator is a human insulator element 40 having the nucleotide sequence of SEQ ID NO:24. In another embodiment, the insulator is a mouse insulator element 40 having the nucleotide sequence of SEQ ID NO:25. In another embodiment, in another embodiment, the insulator is an insulator element 04 having the nucleotide sequence of GenBank accession number AY190749.1. In another embodiment, the insulator is an insulator element 06 having the nucleotide sequence of GenBank accession number AY190750.1. In another embodiment, the insulator is an insulator element 07 having the nucleotide sequence of GenBank accession number AY190751.1. In another embodiment, the insulator is an insulator element 12 having the nucleotide sequence of GenBank accession number AY190752.1. In another embodiment, the insulator is an insulator element 13 having the nucleotide sequence of GenBank accession number AY190753.1. In another embodiment, the insulator is an insulator element 35 having the nucleotide sequence of GenBank accession number AY190754.1. In another embodiment, the insulator is an insulator element 36 having the nucleotide sequence of GenBank accession number AY190755.1. In another embodiment, the insulator is an insulator element 52 having the nucleotide sequence of GenBank accession number AY190757.1. In another embodiment, the insulator is an insulator element 53 having the nucleotide sequence of GenBank accession number AY190758.1. In another embodiment, the insulator is the chicken HS4 insulator having two or more copies of the nucleotide sequence of AY040835.1 from the globin locus.

[0176] The non-naturally occurring nucleic acid molecule comprising a pair of insulators can have the same or different insulators as a pair flanking the gene segment of interest. Preferably, different insulators are used as a pair to flank the gene segment of interest. In one exemplary embodiment described in the following examples, the human insulator element 40 (AY190756.1, SEQ ID NO:24) and the mouse insulator element 40 (SEQ ID NO:25) are used as insulators. In another exemplary embodiment described in the following examples, the human insulator element 40 (AY190756.1, SEQ ID NO:24) is used as an insulator.

[0177] As provided herein, the constructs of the disclosure may also include spacer sequences flanking the AAV transgene to reduce the risk of mis-packaging other vector components. In one embodiment, the non-naturally occurring nucleic acid molecule includes a first spacer sequence and a second spacer sequence upstream and downstream of the transgene, respectively. In certain embodiments, these spacer sequences are 2 kb spacer sequences. In a specific embodiment, the non-naturally occurring nucleic acid molecule includes a first insulator upstream of the modified AAV rep gene and also includes a first spacer sequence and a second spacer sequence upstream and downstream of the transgene, respectively, wherein the first insulator and the second spacer sequence are independently selected from: (a) the nucleotide sequence of SEQ ID NO: 67; and (b) the nucleotide sequence of SEQ ID NO: 68.

[0178] Cells and Methods for Producing Recombinant AAV

[0179] Expression of the Rep protein from the modified AAV rep gene of the present application is tightly controlled by DNA splicing and RNA splicing mechanisms, thus allowing the generation of stable host cells containing the modified rep gene in a bioreactor and growing these cells to a very high number. For AAV production, first, stable host cells containing the modified AAV rep gene, the AAV cap gene, and a transgene flanked by ITRs are grown to a very high number and then infected with a replication-defective adenovirus expressing a serine recombinase that recognizes the attP and attB sites in the modified AAV rep gene. Site-specific recombination between the attP and attB sites catalyzed by the serine recombinase excises the termination cassette, thereby generating a precursor mRNA containing 5′ and 3′ rep coding sequences separated by a functional intron. Then, the ubiquitous cellular machinery (spliceosome) excises the intron, resulting in an mRNA encoding the four Rep proteins, thus allowing the production of AAV at high titers.

[0180] Stable host cells containing a modified AAV rep gene, an AAV cap gene, and a transgene flanked by ITRs can be obtained by transducing cells with one or more nucleic acid molecules encoding these genes. In one embodiment, stable host cells are obtained by transducing cells with a first nucleic acid molecule encoding a modified AAV rep gene and an AAV cap gene to obtain a first host cell containing the modified AAV rep gene and the AAV cap gene, and further transducing the first host cell with a second nucleic acid molecule encoding a transgene flanked by ITRs. In one embodiment, the modified AAV rep gene and the AAV cap gene are stably integrated into the chromosome of the first host cell. In another embodiment, the modified AAV rep gene and the AAV cap gene remain episomal in the first host cell. The transgene flanked by ITRs can also be stably integrated into the host cell or remain episomal.

[0181] In another embodiment, stable host cells are obtained by transducing cells with a nucleic acid molecule encoding a modified AAV rep gene, an AAV cap gene, and a transgene flanked by ITRs. The modified AAV rep gene, the AAV cap gene, and the transgene flanked by ITRs can be stably integrated into the host cell or remain episomal.

[0182] The stable host cells can be grown to a high cell density prior to infection with an adenovirus expressing a serine recombinase.

[0183] According to the present disclosure, any method known in the art can be used to introduce a replication-deficient adenovirus expressing the serine recombinase of the present application into the stable host cells. In one embodiment, the replication-deficient adenovirus is a recombinant ΔE1 / ΔE3 adenovirus serotype 5 (Ad5) virus that contains a nucleotide sequence encoding an amino acid sequence having at least 85% identity, preferably 100% identity, to the amino acid sequence of SEQ ID NO: 2. For example, the adenovirus can contain a nucleotide sequence having at least 85% identity, preferably at least 95% identity, to SEQ ID NO: 3.

[0184] As disclosed herein, the present disclosure also includes methods and compositions for increasing AAV production by contacting the cells described herein with 2-aminopurine (2-AP). In the late stage of the adenovirus life cycle, the virus inhibits host protein synthesis. This is due in part to the action of the late adenovirus 100 kilodalton (kDa) protein, which displaces the Mnk1 kinase from the cap initiation complex eIF4F, resulting in dephosphorylation of eIF4E and inhibition of cap-dependent mRNA translation (see, e.g., Cuesta (2004), J. Virology 78:7707-7716). Adenovirus late gene transcripts contain a tripartite leader sequence at their 5' end that promotes translation by a mechanism called ribosome shunting (see, e.g., Yueh (2000) Genes Dev 14:414-421). In the context of an AAV producer cell line, inhibition of cap-dependent translation is predicted to block the expression of the AAV REP and CAP genes as well as early adenovirus proteins required for AAV replication and packaging. Thus, in some embodiments, these cells are cultured with a chemical that blocks the shutoff of host protein translation to increase the efficiency of the AAV producer cell line using an adenovirus inducer.

[0185] In certain embodiments, the chemical that blocks the shutoff of host protein translation is 2-aminopurine (2-AP). 2-AP has been shown to block the shutoff of host protein synthesis induced by adenovirus (see, e.g., Zhang and Schneider (1994) J. Virology 68:2544-2555; Huang and Schneider (1990) PNAS 87:7115-7119). Treatment of AAV-producing cells with 2-AP can reduce the cytopathic effect of infection, including the restoration of the cytokeratin network that is normally degraded by late infection (Zhang and Schneider (1994) J. Virology 68:2544-2555). 2-AP inhibits a variety of kinases in vitro, including the RNA-dependent protein kinase PKR (also known as eukaryotic translation initiation factor 2α kinase 2, EIF2AK2) (DeBenedetti (1983) J Biol Che, 258:14556-14562), but is unable to block PKR activation in cells and the phosphorylation of eIF-2α that occurs after adenovirus infection (Huang and Schneider (1990) PNAS 87:7115-7119). 2-AP increases the level of the early adenovirus DNA-binding protein (DBP) by 10 to 20-fold but does not increase the mRNA level (Huang and Schneider (1990) PNAS 87:7115-7119), which is consistent with its effect on cap-dependent translation.

[0186] Thus, in some embodiments, methods of producing recombinant AAVs comprising transgenes include culturing the cells of the present disclosure with 2-aminopurine. In some embodiments, the 2-aminopurine concentration is less than about 10 mM. In some embodiments, the 2-aminopurine concentration is less than about 5 mM. In some embodiments, the 2-aminopurine concentration is less than about 2.25 mM. In some embodiments, the 2-aminopurine concentration is less than about 1.25 mM. In some embodiments, the 2-aminopurine concentration is from about 1 μM to about 1.25 mM. In some embodiments, the 2-aminopurine concentration is from about 10 μM to about 1.25 mM. In some embodiments, the 2-aminopurine concentration is from about 100 μM to about 1.25 mM. In some embodiments, the 2-aminopurine concentration is about 1.25 mM.

[0187] In certain embodiments, the cells of the present disclosure are contacted with 2-aminopurine about 24 hours after infection with recombinant adenovirus. In some embodiments, the cells of the present disclosure are contacted with 2-aminopurine about 20 hours after infection with recombinant adenovirus. In some embodiments, the cells of the present disclosure are contacted with 2-aminopurine about 12 hours after infection with recombinant adenovirus. In some embodiments, the cells of the present disclosure are contacted with 2-aminopurine about 30 hours after infection with recombinant adenovirus. In some embodiments, the cells of the present disclosure are contacted with 2-aminopurine about 36 hours after infection with recombinant adenovirus. In some embodiments, the cells of the present disclosure are contacted with 2-aminopurine about 48 hours after infection with recombinant adenovirus.

[0188] Examples

[0189] The following examples of this patent application will further illustrate the nature of this patent application. Those skilled in the art should understand that the above embodiments can be modified without departing from the broad inventive concept of the present invention. Therefore, it should be understood that the present invention is not limited to the specific embodiments disclosed, but the present invention is intended to cover modifications within the spirit and scope of the present invention as defined in this specification.

[0190] Materials

[0191] Cells: HEK293 cells (American Type Culture Collection (ATCC), Manassas, VA, catalog number CRL-1573); PEAK-rapid (ATCC, Manassas, VA, catalog number CRL2828).

[0192] Tissue culture media and reagents: OptiMEM medium (Thermo-fisher, Waltham, MA; catalog number 31985-062); DMEM, high glucose (Thermo-fisher, catalog number 10569-010); DMEM, phenol red-free (Thermo-fisher; catalog number A14430-01); Hyclone dialyzed fetal bovine serum (Thermo-fisher; catalog number SH30079.03); 96-well TC plate (Corning, Corning, NY; catalog number 3596); 6-well tissue culture plate, clear (Corning catalog number 3516); 96-well culture plate, opaque white (PerkinElmer, Waltham, MA; catalog number 6005680); TrypLE Select cell dissociation reagent (Thermo-fisher, catalog number 12563-011); Dulbecco's phosphate buffered saline, calcium-free, magnesium-free, D-PBS (Thermo-fisher, catalog number 14190-144); Geneticin (G418) 50 mg / ml (Thermo-fisher, catalog number 10131-027); puromycin dihydrochloride from Streptomyces alboniger (Sigma Aldrich P9620); T150 cell culture flask 150 mm2 (Corning, catalog number CLS430825); GlutaMax 100x (Thermo-fisher, catalog number 35050-061); 6-well culture plate, not tissue culture treated (Corning, catalog number 351146); multi-layer cell culture flask M container (Corning, catalog number 10030); 2.5% ClonaCell methylcellulose in DMEM (without L-glutamine and containing glucose, sodium pyruvate and sodium bicarbonate) (StemCell Technologies, Vancouver, British Columbia, Canada, catalog number 03899-DI).

[0193] Transfection reagents: Fugene-HD transfection reagent (Promega, Madison, WI, catalog number E2311); Lipofectamine 3000 transfection reagent (Thermo-fisher catalog number L3000008); deoxynucleotides (Millipore-Sigma, St. Louis, MO, catalog number D7295-2ML).

[0194] Tubes: 15 ml conical tubes (Corning, catalog number 430053); 1.5 ml screw-cap tubes (Sarstedt AG & Co. KG, Germany, catalog number 72.692.005).

[0195] Purification kits and assay reagents: Plasmid Mini Kit (Qiagen, Hilden, Germany, catalog number 27106); CHROMA SPIN TM +TE-1000 column (Takara Bio USA, Mountainview, CA, catalog number 636079); Dual-Glo Luciferase Assay System (Promega, Madison, WI, catalog number E2940); Silver Staining Kit (Thermo-fisher catalog number 24600); Trizol Plus RNA Purification Kit with Phase-maker tubes (Thermo-fisher catalog number A33254); DNA Removal Kit (Thermo-fisher catalog number AM1906); Nucleospin Gel and PCR Clean-up Kit (Takara Bio USA, catalog number 740609.5).

[0196] Enzymes: Spe I-HF (New England Biolabs, Ipswich, MA, catalog number R3133S); DNase I, Grade II from bovine pancreas (Sigma-Aldrich, catalog number 10104159001); NEXT UltraII Q5 Premix (New England Biolabs, catalog number M05445S).

[0197] Buffers and chemicals: Buffer (1X buffer components: 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 100 μg / ml BSA, pH 7.9 at 25°C) (New England Biolabs, catalog number B7204S); Benzonase Nuclease (Sigma-Aldrich, catalog number E1014-25K); 10x GeneAmp PCR Buffer I containing 1.5 mM MgCl2 (Thermo-fisher catalog number N8080006); Sodium deoxycholate (Sigma-Aldrich, catalog number D6750-25g); 1 M TRIS-HCL PH8.5 (Thermo-fisher, catalog number T1085); 10x GeneAmp PCR Buffer I (Thermo-Fisher catalog number N8080006) [100 mM Tris-HCl, pH 8.3 (at 25°C); 500 mM KCl; 15 mM MgCl2; 0.01% gelatin, dissolved in autoclaved, deionized, ultrafiltered water]; 10% Pluronic F-68 (Thermo-Fisher catalog number 24040-032); Sheared salmon sperm DNA (10 mg / ml) (Thermo-Fisher catalog number AM9680); Virus Dilution Buffer (VDB) [1x GeneAmp PCR Buffer I, 2 μg / ml sheared salmon sperm DNA and 0.05% Pluronic F-68]; β-Mercaptoethanol (Sigma-Aldrich, catalog number M3148); Adenovirus Preparation Buffer (10 mM Tris (pH 7.4), 1 mM MgCl2, 75 mM NaCl, 5% sucrose, 0.02% polysorbate 80, 0.1 mM EDTA, 10 mM histidine, 0.5% EtOH); 2-Aminopurine, nitrate (Sigma-Aldrich, catalog number A2380), dissolved to 100 mM in DMEM + 2% FBS.

[0198] RT-PCR reagents: SuperScript III First-Strand Synthesis System (Thermo-fisher catalog number 188080-051); Q5 Hot Start High-Fidelity 2X Premix (New England Biolabs, catalog number M0494S); 1% TAE Mini-Prep Agarose Gel containing ethidium bromide (Bio-RAD, catalog number 1613016); Dark Reader Blue Light Transilluminator (ClareChemicals, Dolores, CO, catalog number DR46B).

[0199] Digital droplet PCR: 2x Probe Supermix (Bio-Rad catalog number 186-3026); DG32AutoDG columns (Bio-Rad catalog number 1864108); Auto Droplet Generation Oil in PBS (Bio-Rad catalog number 1864110); Droplet Reader Oil (Bio-Rad catalog number 1863004); Eppendorf twin.tec 96-well PCR plates (catalog number 951020346); Automated Droplet Generator (Bio-Rad catalog number 186-4101); QX200 Droplet Reader (Bio-Rad catalog number 186-4003); C1000 Touch Thermal Cycler with Deep Well Reaction Module (Bio-Rad catalog number 185-1197).

[0200] PrimeTime qPCR assays: 20x stock solutions of these assays consisted of forward and reverse PCR primers (18 μM) and 5′ nuclease probes (5 μM) containing the fluorescent quencher ZEN and Black Hole Quencher 1 (3IABkFQ) and FAM or HEX fluorescent reporter dyes. Assay samples were synthesized by Integrated DNA Technologies, Inc., Coralville, IA. Primer and probe sequences for qPCR assays were as follows:

[0201] mCherry: Primer 1 (SEQ ID NO:36, 5'-CTGTTCCACGATGGTGTAGTC-3'); Primer 2 (SEQ ID NO:37, 5'-TGAGGTCAAGACCACCTACA-3'); Probe (SEQ ID NO:38, 5’-FAM-TTGGACATC-ZEN-ACCTCCCACAACGAG-3IABkFQ-3’);

[0202] Adenovirus exon 2 (Ad5E2): Primer 1 (SEQ ID NO:39, 5'-GGGTGATGCAGTAGAAGGTAAG-3'); Primer 2 (SEQ ID NO:40, 5'-ATGAAGTTCGGCGGAGATG-3'); Probe (SEQ ID NO:41, 5’-HEX-TCTTGTTCC-Zen-CAGCGGTCCCATC-3IABkFQ-3’);

[0203] P5 (P5 promoter region of AAV): Primer 1 (SEQ ID NO:42, 5'-GTGGTCACGCTGGGTATTTA-3'); Primer 2 (SEQ ID NO:43, 5'-GGGACCTTAATCACAATCTCGT-3'); Probe (SEQ ID NO:44, 5’-FAM-TTTGAAGCG-ZEN-GGAGGTTTGAACGC-31ABkFQ-3’);

[0204] AAV REP gene: Primer 1 (SEQ ID NO:45, 5'-GTCCGTGAGTGAAGCAGATATT-3'); Primer 2 (SEQ ID NO:46, 5'-TTCGATCAACTACGCAGACAG-3’); Probe (SEQ ID NO:47, 5’-FAM-TCTGATGCT-ZEN-GTTTCCCTGCAGACA-3IABkFQ-3’);

[0205] AAV9 CAP gene: Primer 1 (SEQ ID NO:48, 5'-CCGGGTCCAAGGTATTTGTAA-3'); Primer 2 (SEQ ID NO:49, 5’-CTCAACCCAAGGCAAATCAAC-3'); Probe (SEQ ID NO:50, 5’-FAM-ACATCAAGA-ZEN-CAACGCTCGAGGTCT-3IABkFQ-3’); and

[0206] β-lactamase (ampicillin resistance) gene: Primer 1 (SEQ ID NO:51, 5'-CCAGAAACGCTGGTGAAAGTA-3’); Primer 2 (SEQ ID NO:52, 5'-CTCAAGGATCTTACCGCTGTTG-3’); Probe (SEQ ID NO:53, 5’-FAM-TGCACGAGT-ZEN-GGGTTACATCGAACT-3IABkFQ-3’).

[0207] PAGE electrophoresis: 4x NuPAGE LDS sample buffer (Thermo-Fisher, catalog number NP0007); 4% to 12% Bis-Tris PAGE gel in 1x MOPS running buffer (Thermo-Fisher, catalog number NP0322PK2); 20x NuPAGE MOPS SDS running buffer (Thermo-Fisher, catalog number NP0001).

[0208] AAV Purification Buffers and Supplies: 0.2 μm PES membrane filter (Thermo-Fisher catalog number 567-0020); 0.5 × 5 cm POROS GoPure chromatography column pre-packed with POROS CaptureSelect AAVX resin (Thermo-fisher catalog number A36652); Amicon 15 100 kDa MWCO filter (Millipore-Sigma catalog number UFC910024); CIM QA Disk 0.34 ml volume (BIA Separations, Slovenia); Buffer A (20 mM Tris pH 7.5, 400 mM NaCl); Buffer B (25 mM Tris pH 7.5, 40 mM NaCl and 1.5 mM MgCl2); Buffer C (20 mM sodium citrate pH 2.5, 400 mM NaCl); Buffer D (100 mM sodium citrate, 10 mM Tris, pH 8.0); Buffer E (20 mM BTP pH 10.0, 0.001% Pluronic F68, 10 mM NaCl); Buffer F (20 mM Bis-TRIS propane pH 10.0, 0.001% Pluronic F68, 400 mM NaCl); Bis-TRIS propane (BTP) (Millipore Sigma catalog number B4679).

[0209] Other Equipment: AKTA Explorer FPLC system (GE Healthcare Life Sciences, Marlborough, MA); AKTA purifier system (GE Healthcare Life Sciences); Envision multimode plate reader model 2104 (PerkinElmer, Waltham, MA).

[0210] Identification and Recombinant Expression of SR21 Recombinase

[0211] Using a BLAST search of the non-redundant protein database at NCBI and the SPBeta c2 integrase protein (query sequence, SEQ ID NO: 1) as the query sequence, a putative serine recombinase (target sequence, SEQ ID NO: 2) was identified in the genome of Bacillus safensis strain CCMA-560, which has 64% sequence identity at the protein level ( Figure 1)。The putative serine recombinase or integrase is part of a putative prophage insertion fragment. The recombinase is named SR21 (serine recombinase 21). The DNA sequence encoding SR21 is shown in SEQ ID No:3.

[0212] The bacterial strain (“Fairview” strain) closely related to CCMA-560 without the prophage insertion fragment was identified by performing a BLAST search of the sequence database at NCBI using the CCMA-560 DNA sequence from the 3’ end and beyond of the recombinase coding region as the query sequence (SEQ ID NO:58)( Figure 2 )。The DNA sequence of the Fairview strain corresponding to the upstream and downstream sequences of the putative prophage insertion site in CCMA-560 is referred to herein as the “pre-insertion sequence” and is shown in SEQ ID NO:4. Using this sequence (SEQ ID NO:4) as the query sequence for BLAST analysis of the genomic sequence of the CCMA-560 strain, other prophage-host DNA junctions at 94 kb upstream were identified. The sequences of the right and left prophage-host DNA junctions of Bacillus safensis strain CCMA-560 are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0213] The attP and attB sequences of the SR21 recombinase were reconstructed from these host DNA junctions (SEQ ID NO:5) and (SEQ ID NO:6), respectively, by swapping the sequences upstream of the central identical region (“ACTGACAAAGCGGT”) (SEQ ID NO:54) and selecting the central dinucleotide and the att site boundaries that maximize dyad symmetry: attP (SEQ ID NO:7); attB-CCMA-560 (SEQ ID NO:8). The attB sequence (SEQ ID NO:9) of the host DNA junction (SEQ ID NO:4) from the Fairview strain of Bacillus safensis contains two mismatches relative to the reconstructed attB sequence (SEQ ID NO:8) from strain CCMA-560. Figure 3 An alignment of attP with these two alternative attB sequences is shown, highlighting the positions of dyad symmetry.

[0214] Measuring Recombinase Activity in Mammalian Cells

[0215] The vector (P175) (SEQ ID NO:10) was constructed by gene synthesis (GENEWIZ, Plainfield, NJ) to express the SR21 recombinase in mammalian cells under the control of the CMV promoter and followed by the SV40 polyadenylation signal. The SR21 recombinase open reading frame is identical to SEQ ID NO:3, except that the bacterial translation start codon "TTG" was replaced with "ATG", and three point mutations were introduced to disrupt restriction endonuclease recognition sites. These changes in the open reading frame do not cause any changes in the amino acid sequence of the encoded SR21 recombinase.

[0216] The recombinase reporter plasmid (P41) (SEQ ID NO:11; Figure 4 ) was also constructed by gene synthesis (GENEWIZ, Plainfield, NJ). It encodes a constitutively expressed green fluorescent protein (GFP)-self-cleaving F2A-renilla luciferase (rLUC) fusion protein driven by the EF1α promoter. It also encodes a recombinase-activated mCherry-self-cleaving P2A-firefly luciferase (fLUC) reporter gene flanked by SR21 attP (SEQ ID NO:7) and attB (SEQ ID NO:9) signals in the antisense orientation relative to the CMV promoter. When the SR21 recombinase recombines the attP and attB signals, the coding region is inverted into the sense orientation and the mCherry-P2A-fLUC protein is expressed (see Figure 4 ).

[0217] To measure SR21 recombinase activity in human cells, 75,000 HEK293 cells were seeded into 100 μl of high glucose DMEM + 10% fetal bovine serum in each well of a 96-well tissue culture plate. The recombinase reporter plasmid (P041) ± the SR21 recombinase expression plasmid (P175) + deoxynucleotides (to normalize the DNA amount) were complexed with Fugene-HD transfection reagent in OptiMEM medium for 15 minutes at room temperature (as shown in Table 1) and transfected into three replicate wells of the seeded cells. The plate was incubated at 37 °C for 48 hours.

[0218] Table 1. Transfection Conditions

[0219]

[0220] The Dual Glo assay kit from Promega was used to sequentially measure firefly luciferase (fLUC) and Renilla luciferase (rLUC) in the transfected wells. The medium was removed from the transfected wells of the cell culture plate and 100 μl of DMEM medium (without phenol red) with a 1:1 mixture of Dual Glo luciferase + fLUC substrate was added. The plate was incubated at room temperature for 10 minutes. The lysate was transferred to an opaque white 96-well plate. The fLUC activity was measured using an Envision multimode plate reader. Next, 50 μl of Stop-and-Glo buffer + Renilla substrate was added per well and the plate was incubated for 10 minutes with gentle shaking. The Renilla luciferase signal was read on the same Envision plate reader.

[0221] Results: The recombinase reporter produced 1535-fold more firefly luciferase when co-transfected with the recombinase expression plasmid compared to when co-transfected with deoxynucleotides (Table 2). This difference could not be explained by different transfection efficiencies as the Renilla luciferase (rLUC) activity was 5-fold higher in the single reporter transfection. This data indicates that the SR21 recombinase has high activity in human cells and the results represent three independent experiments.

[0222] Table 2. Recombinase Activity in HEK293 Cells

[0223] Sample Description fLUC rLUC Fold Increase in fLUC Activity 1 Reporter Alone 4.3E03±1.2E03 1.4E07±2.6E06 2 Reporter + Recombinase 6.6E06±3.9E05 3.4E06±1.6E05 1535

[0224] Construct REP / CAP + Transgene Plasmid

[0225] If the AAV replication (REP) and capsid (CAP) genes can be stably integrated and subsequently induced to produce AAV in high-density cultures, it is possible to produce AAV on a large scale in mammalian cells. However, the expression of the Rep proteins is toxic, making it difficult to generate stable cell lines in hosts that express the REP gene (such as those that express the adenovirus E1 gene, such as HEK293 cells). Wild-type AAV encodes four Rep proteins using overlapping reading frames, which are generated by the use of two promoters and alternative splicing. Thus, using an inducible promoter to control REP expression is not straightforward. Previous work has demonstrated that the insertion of a "stop cassette" into the REP coding region within an artificial intron allows the generation of stable cell lines in HEK293 cells (Qiao et al., (2002) J. Virol. 76:13015; Yuan et al., (2011) Hum Gene Therap. 22:613-624). The excision of the stop cassette using Cre recombinase delivered by adenovirus infection restored REP expression and initiated AAV replication of the transgene flanked by ITRs. In this example, a modified form of the recombinase-activated REP gene was constructed in the context of a REP / CAP expression cassette in a plasmid containing a transgene flanked by ITRs.

[0226] The AAV REP / CAP9 expression cassette (SEQ ID NO:13) was constructed using the AAV2 REP gene (bp 190-2202 of human AAV2, NC_001401.2), followed by the AAV9 CAP open reading frame (AY530579.1), the AAV2 polyadenylation signal (bp4411-4466, NC_001401.2), and a second copy of the AAV2 REP P5 promoter (bp 190-313, NC_001401.2).

[0227] The appropriate positions were selected using a splicing site prediction software (NetGene2 at www.cbs.dtu.dk / services / NetGene2 / ; Brunak, S., Engelbrecht, J. and Knudsen, S.: Prediction of Human mRNA Donor and Acceptor Sites from the DNA Sequence, Journal of Molecular Biology, 1991, 220, 49 - 65) to insert an intron from the human β-actin gene into the REP coding region. The intron was inserted downstream of nucleotide number 1052 of AAV2 (NC_001401.2) and in a region common to all four REP transcripts. The intron and the insertion position were different from those used by Qiao et al., (2002) J. Virol. 76:13015. Subsequently, the termination cassette (described below) was inserted between the upstream and downstream halves of this β-actin intron (SEQ ID 14 and 15, respectively).

[0228] Termination Cassette

[0229] The transcription termination cassette (SEQ ID NO:16) consists of the following elements:

[0230] · SR21 attP (SEQ ID NO:7)

[0231] · A strong splice acceptor (SEQ ID NO:17) (NC_000086.7, nucleotides 53001998 to 53002138 from the mouse HPRT gene, plus a 29 nt region from the human agouti signaling protein (NC_000020.11, nucleotides 34262765 to 34262793). This element was included to prevent the termination cassette from being spliced out of the primary mRNA transcript.

[0232] · The neomycin phosphotransferase expression cassette (SEQ ID NO:18) is driven by a mammalian promoter (mouse phosphoglycerate kinase 1) and a bacterial (Lac zya) promoter and is followed by a polyadenylation signal from SV40. This gene confers resistance to neomycin and kanamycin in mammalian and bacterial cells, respectively.

[0233] · A sequence from the human β-globin gene encoding a self-cleaving RNA motif (Teixeira et al., (2004) Nature 432:526 - 30; SEQ ID No:19) that is important for efficient transcriptional termination, downstream of the polyadenylation signal.

[0234] · SR21 attB (SEQ ID NO:8).

[0235] AAV Transgene

[0236] A transgene with AAV inverted terminal repeats (ITRs) is encoded downstream of the AAV REP / CAP region in the P439 vector (SEQ ID NO:12). The 130 bp ITR (SEQ ID NO:20) is derived from the 3’ AAV2 ITR (nucleotides 4535 to 4664, NC_001401.2) and is inserted upstream of the HPRT-E2A-mCherry transgene and in the 3’ reverse direction of this transgene.

[0237] The transgene consists of a human EF1-α promoter (including exon 1, intron 1, and part of exon 2) (SEQ ID NO:21), a sequence encoding the mCherry-self-cleaving E2A linker–human HPRT fusion gene (SEQ ID NO:22), and a polyadenylation signal from the herpes simplex virus thymidine kinase gene (SEQ ID NO:23).

[0238] Insulator

[0239] The REP / CAP and ITR-transgene elements are flanked by genomic elements that block chromatin-associated repression of gene expression (Kwaks et al., (2003) Nature Biotechnology 21:554-558; Kwaks et al., (2003) Nature Biotechnology 21:822): the human antirepressor element 40 (AY190756.1, SEQ ID NO:24) and the mouse antirepressor element 40 (SEQ ID NO:25).

[0240] Plasmid Backbone

[0241] The plasmid backbone contains the following elements:

[0242] · A mammalian puromycin resistance gene cassette constructed from the herpes simplex virus thymidine kinase gene promoter (SEQ ID NO:26), the puromycin N-acetyltransferase coding region (SEQ ID NO:27), and a polyadenylation signal from the bovine growth hormone gene (SEQ ID NO:28).

[0243] · An Epstein-Barr virus (EBV) OriP origin of replication fragment (SEQ ID NO:29), which represents a complex of the “dyad symmetry” region and the “repeat family” region of EBV

[0244] · The pUC57 vector sequence (SEQ ID NO:30) encoding the plasmid replication origin and ampicillin resistance gene.

[0245] The sequence of the complete plasmid P439 is given in SEQ ID NO:12.

[0246] Test Efficiency of SR21 Recombinase for Removal of Termination Cassette

[0247] To test whether the SR21 recombinase can precisely remove the termination cassette in human cells, the vector P439 (SEQ ID NO:12) and the SR21 recombinase expression vector P175 (SEQ ID NO:10) were co-transfected into PEAK-Rapid cells using Lipofectamine 3000 according to the manufacturer's instructions and cultured in a medium containing DMEM and 10% FBS at 5% CO2 and 37 °C for three days. The medium was removed, the cells were washed once with D-PBS, and then the cells were incubated with TrypLE at 37 °C for 5 minutes. The cells were transferred to a sterile microcentrifuge tube, pelleted by centrifugation, washed once with 1 ml D-PBS, and pelleted again. The episomal plasmid was recovered by alkaline lysis using the Qiagen Spin Miniprep Kit designed for plasmid isolation from bacteria.

[0248] To destroy the unrecombined plasmid DNA, an aliquot of the recovered DNA was digested with the enzyme Spe I-HF in 1x CutSmart buffer at 37 °C for 1 hour and at 80 °C for 20 minutes. The recovered DNA was PCR amplified using primers P349F3 (SEQ ID NO:32) and P349R9 (SEQ ID NO:33) with NEXT Ultra II Q5 premix under the following cycling conditions: 98 °C 1 min; 35x (98 °C 10 s, 72 °C 10 s); 72 °C 5 min. A single PCR product of the predicted size was observed when electrophoresed on a 1% agarose gel. The PCR product was purified by size exclusion chromatography using a CHROMA SPIN TM +TE-1000 column. The PCR product was sequenced (GeneWiz) using the same primers used for PCR. The resulting sequence (SEQ ID NO:34) indicated that the SR21 recombinase had precisely removed the termination cassette from plasmid P439 by recombining the attP (SEQ ID NO:7) and attB (SEQ ID NO:8) sequences, generating the attL recombinant sequence (SEQ ID NO:35).

[0249] Construction of Recombinant Adenovirus Serotype 5 Expressing SR21 Recombinase

[0250] Recombinant ΔE1 / ΔE3 adenovirus serotype 5 (Ad5) virus was generated by homologous recombination protocols in PER.C6 cells at Batavia Biosciences (Leiden, the Netherlands) (Fallaux et al., (1998) Hum Gene Ther. 9:1909-1917), as previously described for the generation of E1-deleted vectors (Havenga et al., (2001) J. Virol 75:3335-3342), except that a modified cosmid (pWE / Ad5.AflII-rITRsp.ΔE3, patent US6340595B1) lacking the E3 region was used. The cosmid and plasmid P321 (SEQ ID NO:31) were co-expressed in PER.C6 cells, and the plasmid contained: Ad5 sequences from 1 to 454 (left ITR and packaging signal), a transgene expression cassette containing a cytomegalovirus (CMV) promoter (nt -672 to +15), the SR21 recombinase coding region, a simian virus 40 (SV40) polyadenylation signal (NC_001669.1, nt 2550 to 2774), and a second Ad5 sequence in the range of nt 3511 to 6095. Homologous recombination between the P321 Ad5 sequence (nt 3511-6095) and the cosmid pWE / Ad5.AflII-rITRsp.ΔE3 in PER.C6 cells generated recombinant adenovirus. The purified virus stock was obtained by a two-step CsCl gradient banding protocol, and the isolated virus stock was dialyzed into adenovirus formulation buffer (10 mM Tris (pH 7.4), 1 mM MgCl2, 75 mM NaCl, 5% sucrose, 0.02% polysorbate 80, 0.1 mM EDTA, 10 mM histidine, 0.5% EtOH).

[0251] Stable Cell Line Generation

[0252] Plasmid P439 (SEQ ID NO:12) was transfected into adherent PEAK-RAPID cells using Lipofectamine 3000 according to the manufacturer's instructions and cultured at 37 °C in DMEM + 10% FBS + 0.05 mg / ml geneticin in a T25 flask. After 24 hours, the cells were treated with TrypLE and transferred to a T75 flask containing DMEM + 10% FBS + 0.05 mg / ml geneticin + 0.5 μg / ml puromycin. The cells were split 1:10 into the same medium weekly for two consecutive weeks. In the third week after transfection, the cells were split 1:10 weekly into a medium containing DMEM + 10% FBS + 0.05 mg / ml geneticin + 5.0 μg / ml puromycin for three weeks.

[0253] Single cell clones were prepared by diluting the cells to 1% ClonaCell methylcellulose in DMEM + 30% FBS + 1x GlutaMax + 5 μg / ml puromycin + 0.05 mg / ml geneticin, inoculating into a 6-well plate untreated for tissue culture, and culturing for three weeks at 37 °C. Using a pipette, the clones were transferred from the methylcellulose plate to a TC-treated 96-well plate containing DMEM + 10% FBS + 0.05 mg / ml geneticin + 5.0 μg / ml puromycin. The clones were expanded in the same medium by standard methods.

[0254] Screening Clones

[0255] To screen clones for AAV production, the cells were seeded in duplicate into 100 μl of DMEM + 10% FBS in a 96-well plate and incubated overnight at 37 °C. SR21 adenovirus was diluted to 1E8 viral genomes per ml in serum-free DMEM. The medium from the seeded cells was replaced with 100 μl of the diluted adenovirus and the plate was incubated at 37 °C for four days. The cells were lysed by adding 10 μl of the following mixture: 5% deoxycholate in PBS + 10 units of benzonase nuclease. The plate was incubated at 37 °C for 2 hours. The plate was centrifuged at 3000 rpm for 5 minutes to pellet cell debris, and the AAV virus in the supernatant was quantified by digital droplet PCR (ddPCR).

[0256] Digital Droplet PCR (ddPCR)

[0257] ddPCR quantification is based on the method described by Lock et al., (2014) Human Gene Therapy methods 23:115-125. 2 μl of lysate was subjected to DNase digestion at 37 °C for 1 hour in 20 μl of reaction solution containing 1x PCR buffer + 20 mM Tris pH 8.5 + 8 units of DNase I in a 96-well plate in a thermal cycler. 2 μl of the DNase-digested sample was diluted with 98 μl of viral dilution buffer (VDB), and 2 μl of this dilution was added to the ddPCR reaction solution containing 1x PCR Supermix + 1x PCR primer / probe for the mCherry transgene (see Materials section). ddPCR droplets were formed using a Bio-Rad automated droplet generator. The PCR cycle was as follows: 95 °C for 10 min; 42x (94 °C for 30 s, 60 °C for 1 min, 72 °C for 15 s, all three with a cycling time of 2 °C per second); 98 °C for 10 min; hold at 4 °C. FAM fluorescence was detected on a Bio-Rad droplet reader according to the manufacturer's instructions. Clones of the highest DNase-resistant particles that produced FAM-fluorescent positive droplets were amplified and further screened.

[0258] Screening Clones - Second Assay

[0259] 1.25E6 cells of the clone to be screened were seeded into 3 ml of DMEM + 10% FBS in a single well of a 6-well plate and incubated at 37 °C for 2 days. The growth medium was replaced with 3 ml of DMEM + 10% FBS containing 5E8 Ad5-SR21 virus particles. The plate was returned to 37 °C to incubate for 3 days. The cells and medium were transferred to a 15-ml tube and subjected to 3 freeze-thaw cycles (dry ice / 37 °C incubation), followed by centrifugation at 3000 rpm for 5 minutes to pellet cell debris. DNase digestion and ddPCR quantification were performed on 2 μl of each sample using the mCherry assay described above. P439C4 cells produced the most AAV when infected with Ad5-SR21 virus and these cells were selected for further characterization (Table 3).

[0260] Table 3. AAV Yields in Screening Assays

[0261] Clone Number Total AAV (DNase-Resistant Particles) Clone 1 4.4E+08±7.6E+07 Clone 3 2.5E+09±3.4E+08 Clone 4 4.4E+09±5.6E+07 Clone 5 1.8E+09±1.9E+08 Clone 12 7.5E+08±9.8E+07 Clone 18 1.6E+08±2.6E+07 Clone 20 9.8E+07±1.4E+07 Clone 25 1.2E+09±6.4E+07 Clone 28 1.3E+08±1.2E+07 Clone 32 1.5E+09±1.3E+08 Clone 36 2.8E+08±3.0E+07 Clone 41 1.0E+09±8.9E+07

[0262] Time-Course Experiment

[0263] New experiments were conducted to determine the kinetics of AAV production and secretion in culture medium at two different growth temperatures. 2 ml of non-enzymatic dissociation solution was added to the PBS-washed P439-C4 cell monolayer in a T150 flask, and the flask was incubated at 37 °C for 5 minutes. The flask was washed with 8 ml of DMEM + 10% FBS and the cells were transferred to a 50 ml centrifuge tube. The cells were centrifuged at 1500 rpm for 5 minutes and the pellet was resuspended in DMEM + 2% FBS. The cells were diluted in the same medium to 1.25E6 cells per ml. 4 ml of cells were seeded into each well of four 6-well plates. 1 ml (2E8 viral particles) of Ad5-CMV-SR21 adenovirus in DMEM + 2% FBS was added to the wells. Two plates were incubated at 37 °C and two plates were incubated at 5% CO2 and 32 °C. Attached cells were scraped off daily (for a total of 8 days) using a cell scraper to recover the cells and medium, and the samples were transferred to 15 ml conical tubes. The tubes were centrifuged at 3000 rpm for 5 minutes, and aliquots were transferred to 1.5 ml screw-cap tubes and frozen at -20 °C until ddPCR assay.

[0264] The samples were treated with DNase in duplicate as described above, and each DNase-treated sample was serially diluted three times in VDB. The samples were quantified in a ddPCR reaction mixture containing 1x PCR premix + 1x mCherry-FAM assay + 1x Ad5E2-HEX assay (see Materials section). ddPCR was performed as described above.

[0265] Results :

[0266] Adenovirus and AAV in the cell culture medium increased over the 8-day time course (Table 4). Adenovirus replication was slower at 32 °C, leading to higher AAV yields, which may be due to the delayed adenovirus cytopathic effect. The AAV yield at 32 °C exceeded 14,000 genome copies / cell.

[0267] Table 4. AAV and Adenovirus Yields during 8-Day Time Course

[0268]

[0269] Multi-Layer Cell Culture Flask Culture

[0270] 8.3E07 P439C4 cells were seeded into 550 ml of DMEM + 10% FBS + 0.5 μg / mL puromycin, +50.0 μg / mL G418 in two multi-layer cell culture flask M containers and incubated at 37 °C for 3 days. The density after 3 days of growth was estimated to be 3.6E8 cells per flask. The flasks were infected at 40 MOI (1.4E10 viral particles) or 20 MOI (7.2E09 viral particles) by the following method: The virus was diluted in 550 ml of DMEM + 10% FBS and the medium in the multi-layer cell culture flask was replaced with the diluted virus. The cells were incubated at 5% CO2 and 32 °C for 7 days. After 7 days, the supernatant was collected from the infection and the supernatant was clarified by passing through a 0.2 μm PES membrane filter.

[0271] AAVX Purification

[0272] A 0.5 × 5 cm POROS GoPure chromatography column (pre-packed with POROS CaptureSelect AAVX resin to a 1 mL bed volume and attached to an AKTA Explorer FPLC system) was equilibrated with 10 column volumes (CV) of buffer A (20 mM Tris pH 7.5, 400 mM NaCl) at a flow rate of 3 ml / min. The virus suspension was loaded at a flow rate of 4.5 mL / min, followed by 10 mL of buffer A to wash away unbound sample. On-column DNA digestion was performed by the following method: The column was equilibrated with 5 ml of low salt universal nuclease buffer, buffer B (25 mM Tris pH 7.5, 40 mM NaCl and 1.5 mM MgCl2), and then 15 ml of buffer B containing 250 units / ml of universal nuclease was loaded onto the column. The column was then incubated at room temperature for 30 minutes, after which it was washed with 15 column volumes of buffer A. The virus was eluted with 15 column volumes of buffer C (20 mM sodium citrate pH 2.5, 400 mM NaCl) to give 0.5 mL fractions, which were immediately neutralized with 25 μL of 500 mM Bis-TRIS propane pH 10.0. A single peak elution was observed. All fractions under the curve were pooled, concentrated using an Amicon 15 100 kDa MWCO (catalog number UFC910024, Fisher), and the buffer was changed to buffer D (100 mM sodium citrate, 10 mM Tris, pH 8.0) by three rounds of buffer addition / centrifugation. The buffer-changed and concentrated affinity chromatography product was subjected to anion exchange chromatography to further purify AAV from empty capsids.

[0273] Ion Exchange Chromatography

[0274] Dilute the affinity chromatography product (virus suspension) to 45 mL in buffer E (20 mM BTP pH 10.0, 0.001% Pluronic F68, 10 mM NaCl) and load it onto a CIM QA Disk (BIA Separations, 0.34 mL volume) on an AKTA purifier system (GE Healthcare Life Sciences) at a flow rate of 2 ml / min. Wash the column with 10 column volumes of sterile filtered buffer E (20 mM BTP pH 10.0, 0.001% Pluronic F68, 10 mM NaCl). Elute the virus with 60 column volumes of a gradient from 100% buffer E to 100% buffer F (20 mM Bis-TRIS propane pH 10.0, 0.001% Pluronic F68, 400 mM NaCl), collecting 0.5 mL fractions. Pool all the fractions under the curve and concentrate them by centrifugation at 2000×g for 5 min using an Amicon 15 100 kDa MWCO (Catalog No.: UFC910024, Fisher), and change the buffer to buffer D (100 mM sodium citrate, 10 mM Tris, pH 8.0).

[0275] Protein Chromogenic Reaction

[0276] Heat-denature 2 μL of the concentrated eluate in NuPage LDS sample buffer (4x) supplemented with 5% β-mercaptoethanol (95 °C for 10 min) and electrophorese it on a 4% to 12% Bis-Tris PAGE gel in 1x MOPS running buffer. Silver stain the gel according to the manufacturer's instructions.

[0277] ddPCR

[0278] Measure the virus concentration by digital droplet PCR using the mCherry assay as described above.

[0279] Results

[0280] Infection and growth of P439C4 cells in a multi-layer cell culture flask container produced 1.9E13 and 7.0E13 genomic copies (GC) at infections of 20 and 40 MOI, respectively. For 20 and 40 MOI infections, these corresponded to 5.2E4 and 1.9E5 GC / cell, respectively. Examine the purity of the virus samples by PAGE electrophoresis and silver staining. Only three bands corresponding to the expected sizes of the three AAV9 capsid isotypes (VP1, VP2, and VP3) were visible ( Figure 5)。The capsid proteins (VP1 (87 kDa), VP2 (72 kDa), and VP3 (62 kDa)) are present in an approximate stoichiometry of 1:1:10, as previously reported for other recombinant AAV vectors (Daya and Berns (2008) Clin Microbiol Rev. 21:583–593).

[0281] Measuring the Level of Mis-Packaged DNA

[0282] Sequences encoding the AAV REP or CAP genes, as well as prokaryotic sequences derived from plasmid vectors used during production, can be non-specifically packaged into AAV particles and represent a potential safety risk when used for gene therapy (see, e.g., Schnodt and Buning, Hum Gene Ther Methods., 2017; 28(3):101-108). Risks include the generation of replication-competent AAV through homologous recombination, cytotoxic T lymphocyte responses triggered by capsid gene expression, and inflammatory responses and / or gene silencing due to the recognition of prokaryotic sequences by the immune system. Encapsidated rep, cap, and prokaryotic sequences of 2%, 0.4% to 1.0%, and 1.3% to 6.3% have been reported, respectively, in purified recombinant AAV preparations produced by triple transfection or in the cell lines generated (Nony et al., (2003) J. Virology 77:776-781; Gao et al., (2008) Molecular Therapy 16:S105; Chaudeuf et al., (2005) Molecular therapy 12:744-753).

[0283] To determine the level of mispackaging associated with the producer systems described above, the abundance of four sequences in the transfected vector (outside the transgene flanked by ITRs) was determined by ddPCR: a) the P5 promoter; b) the AAV REP gene; c) the AAV9 CAP gene; and d) the β-lactamase (ampicillin resistance) gene. Purified viral preparations from the previously described 20 and 40 MOI multi-layer cell culture flask cultures were digested with DNase in triplicate, serially diluted in VDB, and then subjected to ddPCR. The concentration of viral particles containing these sequences was expressed as a percentage of AAV particles containing the mCherry transgene (Table 5). The P5 promoter in preparation A of virus (produced with 20 MOI infectious recombinant adenovirus) had the highest encapsidation rate of 0.04%. However, the P5 encapsidation rate in preparation B of virus (40 MOI), which had a much higher AAV yield, was only 0.007%. The REP, CAP, and ampicillin gene sequences were the same or lower. The CAP level was 0.007% to 0.009%, which was lower than the 0.016% to 0.021% cap encapsidation rates reported previously for four clinical batches of recombinant AAV2 produced for hemophilia B gene therapy trials (Hauck et al., (2009) Molecular Therapy 17:144-152). Thus, the method for producing and purifying recombinant AAV described herein results in a very low rate of mispackaged DNA that meets the requirements of clinical gene therapy programs.

[0284] Table 5. Abundance of Non-Transgene Sequences Packaged in Purified Viruses

[0285]

[0286] The mean percentage ± standard deviation of DNase-resistant particles for the four probes relative to particles containing the mCherry transgene is shown for the analysis of two AAV vector preparations.

[0287] RT-PCR Analysis of RNA Splicing of REP Gene after Termination Cassette Excision

[0288] To determine whether the intron in the REP gene inserted into construct P439 was accurately spliced during excision of the termination cassette, RT-PCR experiments were performed.

[0289] Ten million cells from the P439 stable pool in PEAK-RAPID cells were pelleted by centrifugation and resuspended in 15 ml of DMEM + 2% FBS + 1E9 Ad5-CMV-SR21 virus particles. The cells were seeded into a T75 flask and incubated at 37 °C for forty-eight hours. The cells were detached using a cell scraper. The medium and cells were transferred to a 15 ml centrifuge tube and centrifuged at 1500 rpm for 10 minutes to pellet the cells. RNA was purified from the cell pellet using the Trizol Plus RNA Purification Kit with Phase-maker tubes.

[0290] To remove any contaminating DNA, 31 μg of RNA was treated with 1 μl of DNase from a DNA removal kit in 1x digestion buffer at 37 °C for 30 minutes. 5 μl of DNase inactivation slurry was added and the sample was inverted several times during a 2-minute incubation. The RNA sample was centrifuged at 10,000 × g for 5 minutes and the RNA was transferred to a new sterile tube.

[0291] The RNA was reverse transcribed using the SuperScript III First-Strand Synthesis System. 80 μg of RNA, 1 μl of 50 μM Oligo-DT, and 1 μl of 10 mM dNTPs were mixed in a sterile tube and incubated at 65 °C for 5 minutes and on ice for 2 minutes. 10 μl of 2x mix (2x RT buffer, 10 mM MgCl2, 20 mM dithiothreitol, 0.5 μl of RNase inhibitor (RNAse-out), and 0.5 μl of reverse transcriptase) was added. A mock RT reaction mix was identical except that the reverse transcriptase was replaced with water. The reaction mix was incubated at 50 °C for 50 minutes and on ice for 2 minutes. 1 μl of RNase H was added and the sample was incubated at 37 °C for 20 minutes.

[0292] A 50 μl PCR reaction mix contained 1 μl of reverse-transcribed RNA, 25 μl of Q5 Hot Start High-Fidelity 2X Premix, and 0.5 μM of two primers. The reaction mix was thermocycled as follows:

[0293] 98 °C for 1 min; 35 cycles (98 °C for 10 s, 69 °C for 10 s, 72 °C for 36 s); 72 °C for 5 min.

[0294] The 5 μl reaction was resolved on a 1% agarose gel in 1x TAE buffer and ethidium bromide. The bands were visualized under blue light illumination of a Dark Reader transilluminator. DNA was recovered from the excised bands using a Nucleospin Gel and PCR Clean-up Kit. The DNA was sequenced using PCR primers and dye terminator chemistry at GeneWiz (South Plainfield, NJ).

[0295] Results :

[0296] The PCR reaction from the mock-RT template did not produce a detectable product, indicating that genomic DNA had been removed from the RNA sample. PCR using primers AAVRT-F1 (SEQ ID NO:62) and P349R9 (SEQ ID NO:63) produced two PCR products of similar fluorescence intensity, which originated from transcripts spliced after excision of the stop cassette from P439. One product was generated by splicing at the engineered β-actin splice donor and acceptor sites (SEQ ID NO:14 and SEQ ID NO:15, respectively; Figure 8 ). The second product was generated by splicing between the donor site in the 5’ REP gene (SEQ ID NO:64) and the downstream β-actin acceptor ( Figure 8 ). This splicing event was predicted to remove 64 bp of the REP coding sequence relative to wild-type AAV2, creating a frameshift and producing a truncated REP protein. This suggests that mutating this upstream splice donor site could increase the abundance of the active REP protein and make AAV production more efficient.

[0297] Updated AAV Construct: P600 (SEQ ID NO:70)

[0298] Several changes were made to plasmid P439 (SEQ ID NO:12) to yield construct P600 (SEQ ID NO:70). First, the splice donor site of the REP gene upstream of the stop cassette was mutated. Briefly, the nucleotides GT of the splice donor site (SEQ ID NO:64) identified in the 5’ REP sequence were mutated to AT (SEQ ID NO:65). This mutation was predicted to eliminate splicing at this site without altering the REP protein sequence.

[0299] To reduce the likelihood that the REP / CAP genes can be packaged into the AAV capsid after termination cassette excision, a 2 kb random sequence (SEQ ID NO:66) was designed and inserted downstream of the attB sequence and upstream of the actin splice acceptor to increase the size of the engineered intron. Potential splice sites were identified using NetGene 2 software (cited above) and these potential splice sites were removed. This insertion increased the size of the REP / CAP genes from 4.3 kb to 6.4 kb, which is well above the 5.0 kb AAV packaging limit.

[0300] Based on the hypothesis that sequences adjacent to the AAV ITRs can also be amplified from the genome during transgene rescue and potentially be mispackaged into the AAV capsid (see, for example, Schnodt and Buning, Hum Gene Ther Methods., 2017;28(3):101 - 108), two random 2 kb non - coding spacer elements were designed to flank the transgene, thereby reducing the potential impact of mispackaged DNA. One element (SEQ ID NO:67) was inserted upstream of the left AAV ITR and a second element (SEQ ID NO:68) replaced the murine repressor element 40 (SEQ ID NO:25) downstream of the right AAV ITR.

[0301] In addition, the cap gene is an AAV9 variant (see, for example, Hinderer et al., Hum Gene Ther. 2018;29(3):285 - 298).

[0302] Finally, the coding sequence of the transgene flanked by ITRs in P439 was replaced with SEQ ID NO:69 encoding the mCherry - IRES - SEAP (secreted alkaline phosphatase) protein.

[0303] The complete sequence of the resulting construct P600 is disclosed in SEQ ID NO:70 and a schematic of the plasmid is shown in Figure 9 as shown.

[0304] Production of AAV from P600 in Stable Pool

[0305] Substantially as described above for P439 cells, the construct P600 was transfected into Peak - RAPID cells and a stable pool was generated by selection with 0.5 μg / ml puromycin. The cells were passaged 1:10 for 6 weeks before measuring AAV production.

[0306] 2.5 E6 P600-PEAK-Rapid p6 cells were seeded into 5 ml of DMEM + 10% FBS in three T25 flasks and incubated at 37 °C for three days. The cell density on the day of infection was determined to be 6.6 E6 live cells in one of these flasks. The cells were harvested using TrypLE in this flask and stained with trypan blue exclusion. The medium in the remaining two flasks was replaced with 11 ml of DMEM + 2% FBS containing 2.6 E8 Ad5-CMV-SR21 viral particles. These flasks were incubated at 32 °C for 24 hours. 1 ml of 1.25 mM 2-aminopurine in DMEM + 2% FBS was added to one flask. 1 ml of DMEM + 2% FBS was added to the other flask, and these two flasks were incubated at 32 °C for an additional 7 days. The medium was harvested from these flasks and centrifuged at 3,000 rpm for 5 minutes to pellet cells and debris. DNase digestion and ddPCR quantification were performed on 2 μl of each sample supernatant using the mCherry assay described above.

[0307] AAV production levels are shown in Table 6. The P600 stable pool actively produces AAV upon infection with Ad5-CMV-SR21. AAV viral production increases 2.5-fold in the presence of 2-aminopurine, a drug reported to block adenovirus-induced inhibition of cap-dependent mRNA translation (see, e.g., Zhang and Schneider (1994) J. Virology 68:2544-2555; and Huang and Schneider (1990) PNAS 87:7115-7119). While treatment with 10 mM 2-AP 1 to 2 hours post-infection has been reported to block the cytopathic effect of adenovirus infection and is non-toxic for at least three days (see, e.g., Zhang and Schneider (1994) J. Virology 68:2544-2555; and Huang and Schneider (1990) PNAS 87:7115-7119), we found that concentrations above 1.25 mM and addition earlier than 24 hours were inhibitory for AAV production in our AAV producer cell system. These data suggest that inhibiting the late adenovirus gene program, particularly the shut-off of cap-dependent mRNA translation, is a useful strategy for increasing AAV production in producer cell lines.

[0308] Table 6.

[0309]

[0310] Production of recombinant adeno-associated virus (AAV) in human cells requires expression of the AAV replication (REP) and capsid (CAP) genes, adenovirus genes, and an AAV-packagable transgene consisting of an expression cassette flanked by AAV inverted terminal repeats (ITRs). All three components can be delivered into cells on separate plasmids for AAV production, but existing transfection methods are difficult to scale up to large-scale culture. Incorporating some of these elements into a host cell line can make AAV production more efficient, but some AAV and adenovirus genes are cytostatic or cytotoxic, thus limiting this approach. The present invention describes a way to reversibly inactivate the AAV REP gene such that AAV REP, CAP, and the packagable transgene can be integrated into a suitable host cell and amplified. Infection of these cells with a replication-deficient recombinant adenovirus expressing a recombinase (e.g., ΔE1 / ΔE3) will reactivate the REP gene and induce AAV replication and packaging.

Claims

1. A mammalian cell, the cell comprising (i) a non-naturally occurring nucleic acid molecule, wherein the sequence of the nucleic acid molecule is a nucleotide sequence encoding a serine recombinase as shown in the amino acid sequence of SEQ ID NO:2; (ii) a nucleic acid sequence comprising an attP site, wherein the attP site is the nucleotide sequence of SEQ ID NO:7; and (iii) a nucleic acid sequence comprising an attB site, wherein the attB site is the nucleotide sequence of SEQ ID NO:8 or 9.

2. The cell according to claim 1, wherein the nucleotide sequence encoding the serine recombinase is codon-optimized for expression in mammalian cells.

3. The cell according to claim 1, wherein the nucleotide sequence encoding the serine recombinase comprises the nucleotide sequence of SEQ ID NO:

3.

4. The cell according to claim 1, the cell further comprising a promoter operably linked to the nucleic acid molecule.

5. The cell according to claim 4, wherein the promoter is a cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter.

6. The cell according to claim 4, wherein the promoter is a tissue-specific promoter.

7. The cell according to claim 2, the cell further comprising a promoter operably linked to the nucleic acid molecule.

8. The cell according to claim 7, wherein the promoter is a cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter.

9. The cell according to claim 7, wherein the promoter is a tissue-specific promoter.

10. The cell according to claim 3, the cell further comprising a promoter operably linked to the nucleic acid molecule.

11. The cell according to claim 10, wherein the promoter is a cytomegalovirus (CMV) promoter, a simian virus 40 (SV40) promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, an Epstein-Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter.

12. The cell according to claim 10, wherein the promoter is a tissue-specific promoter.

13. The cell according to any one of claims 1 to 12, the cell further comprising a polyadenylation signal operably linked to the nucleic acid molecule.

14. The cell according to claim 13, wherein the polyadenylation signal is a simian virus 40 (SV40) polyadenylation signal, a bovine growth hormone (bGH) polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal.

15. The cell according to any one of claims 1 to 12, wherein the nucleic acid molecule is in a vector.

16. The cell according to claim 15, wherein the vector is a DNA plasmid or a viral vector.

17. The cell according to claim 16, wherein the viral vector is an adenovirus vector, an adeno-associated virus vector, a poxvirus vector, an enterovirus vector, a Venezuelan equine encephalitis virus vector, a Semliki Forest virus vector, a tobacco mosaic virus vector, or a lentivirus vector.

18. The cell according to claim 16, wherein the viral vector is a recombinant adenovirus vector.

19. The cell according to claim 18, wherein the cell further comprises adenovirus E1A and E1B genes.

20. The cell according to claim 17, wherein the cell is selected from the group consisting of 911 cells, pTG6559 cells, GH329 cells, N52.E6 cells, HeLa-E1 cells, UR cells, VLI-293 cells, HEK293 cells, and PER.C6 cells.

21. The cell according to claim 19, wherein the cell is selected from the group consisting of 911 cells, pTG6559 cells, GH329 cells, N52.E6 cells, HeLa-E1 cells, UR cells, VLI-293 cells, HEK293 cells, and PER.C6 cells.

22. The cell according to claim 13, wherein the nucleic acid molecule is in a vector.

23. The cell according to claim 22, wherein the vector is a DNA plasmid or a viral vector.

24. The cell according to claim 23, wherein the viral vector is an adenovirus vector, an adeno-associated virus vector, a poxvirus vector, an enterovirus vector, a Venezuelan equine encephalitis virus vector, a Semliki Forest virus vector, a tobacco mosaic virus vector, or a lentivirus vector.

25. The cell according to claim 23, wherein the viral vector is a recombinant adenovirus vector.

26. The cell according to claim 25, wherein the cell further comprises adenovirus E1A and E1B genes.

27. The cell according to claim 23, wherein the cell is selected from the group consisting of 911 cells, pTG6559 cells, GH329 cells, N52.E6 cells, HeLa-E1 cells, UR cells, VLI-293 cells, HEK293 cells, and PER.C6 cells.

28. The cell according to claim 26, wherein the cell is selected from the group consisting of 911 cells, pTG6559 cells, GH329 cells, N52.E6 cells, HeLa-E1 cells, UR cells, VLI-293 cells, HEK293 cells, and PER.C6 cells.

29. The cell according to claim 14, wherein the nucleic acid molecule is in a vector.

30. The cell according to claim 29, wherein the vector is a DNA plasmid or a viral vector.

31. The cell according to claim 30, wherein the viral vector is an adenovirus vector, an adeno-associated virus vector, a poxvirus vector, an enterovirus vector, a Venezuelan equine encephalitis virus vector, a Semliki Forest virus vector, a tobacco mosaic virus vector, or a lentivirus vector.

32. The cell according to claim 30, wherein the viral vector is a recombinant adenovirus vector.

33. The cell according to claim 32, wherein the cell further comprises adenovirus E1A and E1B genes.

34. The cell according to claim 31, wherein the cell is selected from 911 cells, pTG6559 cells, GH329 cells, N52.E6 cells, HeLa-E1 cells, UR cells, VLI-293 cells, HEK293 cells, and PER.C6 cells.

35. The cell according to claim 33, wherein the cell is selected from 911 cells, pTG6559 cells, GH329 cells, N52.E6 cells, HeLa-E1 cells, UR cells, VLI-293 cells, HEK293 cells, and PER.C6 cells.

36. The cell according to any one of claims 1 to 12, wherein the cell is selected from 911 cells, pTG6559 cells, GH329 cells, N52.E6 cells, HeLa-E1 cells, UR cells, VLI-293 cells, HEK293 cells, and PER.C6 cells.

37. A method for site-specific recombination in mammalian cells, the method comprising: (a) obtaining a mammalian cell comprising a nucleic acid molecule comprising an attP site and an attB site, wherein the attP site is the nucleotide sequence of SEQ ID NO:7, and the attB site is the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9; (b) introducing into the mammalian cell a non-naturally occurring nucleic acid molecule, the sequence of which is the nucleotide sequence encoding a serine recombinase as shown in the amino acid sequence of SEQ ID NO:2; and (c) growing the mammalian cell under conditions that allow the serine recombinase to catalyze the site-specific recombination between the attP site and the attB site.

38. The cell according to claim 37, wherein the nucleotide sequence encoding the serine recombinase is codon-optimized for expression in mammalian cells.

39. The cell according to claim 37, wherein the nucleotide sequence encoding the serine recombinase comprises the nucleotide sequence of SEQID NO:3.

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