Synthetic genetic elements for biomanufacture

Abstract

To provide recombinant constructs, cells and means for improved production of Adeno-Associated Viruses (AAVs).SOLUTION: A non-naturally occurring nucleic acid molecule comprising a modified adeno-associated virus (AAV) rep gene having an AAV rep gene encoding four Rep proteins Rep78, Rep68, Rep52 and Rep40, and an artificial intron inserted into a coding sequence of the rep gene shared by the four Rep proteins is used.SELECTED DRAWING: Figure 8

Description

[Technical field]

[0001] (Reference to Related Application) This application is a continuation of U.S. Provisional Patent Application No. 62 / 877,508, filed on July 23, 2019. No. 62 / 877,516, filed July 23, 2019; U.S. Provisional Patent Application No. 62 / 877,524, filed July 23, 2019; U.S. Provisional Patent Application No. 62 / 877,532, filed July 23, 2019 U.S. Provisional Patent Application No. 62 / 877,540, filed on July 23, 2019 No. 62 / 877,551, filed July 23, 2019, and U.S. Provisional Patent Application No. Provisional Patent Application No. 62 / 877,561, and U.S. Patent No. filed on July 23, 2019 This application claims the benefit of Provisional Application No. 62 / 877,577, which is incorporated herein by reference. No. 6,399,433, filed on Oct. 23, 2003, and which are incorporated herein in their entireties.

[0002] (Reference to electronically submitted sequence listing) This application is filed under the file name "14620-192-228_SEQ_LISTING" and Created on July 16, 2020, with a size of 152,403 bytes in ASCII format. Sequence listings submitted electronically via EFS-Web as sequence listings for formulas. - The sequence listing submitted via the web is incorporated herein by reference in its entirety. INCORPORATED INTO THE SPECIFICATION. [Background technology]

[0003] Adeno-associated virus (AAV) contains two inverted terminal repeats at the ends of the A linear single-stranded DNA (single-stranded DNA) with inverted terminal repeats (ITRs) The ITR contains two viral genes, rep (replication) and cap (capsid) are adjacent, These encode nonstructural and structural proteins, respectively. It expresses four regulatory proteins through the use of two promoters and alternative splicing. It encodes Rep78, Rep68, Rep52, and Rep40. More specifically, Rep78 and Rep68 are transcribed from the P5 promoter, whereas Rep40 and Rep52 is transcribed from the P19 promoter (which is involved in the Rep78 and Rep68 readings). The P5 and P19 promoters are integrated into the adenovirus E1A HEK transformed with the adenovirus E1 gene activated by the gene These Rep proteins are active in cells such as 293. The cap gene is involved in three transcription factors through alternative splicing and translation initiation. The capsid protein, VP1 (virion protein 1, The protein 1, VP2, and VP3 assemble to form the spherical vesicle of the virus. The AAV virus does not encode a polymerase, so genome replication is It relies on cellular polymerases for synthesis.

[0004] The AAV rep and cap genes can be stably integrated into the cell or expressed intracellularly. mammalian, if capable of being maintained and later induced to produce AAV in high density culture. Large-scale production of AAV in cells may be possible. However, expression of Rep proteins may be cytotoxic or cytostatic to host cells, such as HEK293 cells. In a host in which the rep gene is expressed, such as one that expresses the adenovirus E1 gene This makes it difficult to develop stable cell lines. AAV contains two promoters and an alternating sequence. Four Rep proteins with overlapping reading frames resulting from the use of splicing Since it encodes the rep gene, the use of an inducible promoter to control rep gene expression is straightforward. isn't it.

[0005] The cytotoxic or cytostatic properties of the four Rep proteins are similar to those of the native rep / c Development of stable cell lines capable of producing high titers of AAV using the ap promoter (Clark et al. (1995) Hum. Gene Ther. 6 :1329-1341, Chadeuf et al. (2000) J. Gene me d.2:260-268). Several groups have attempted to recombinationally regulate Rep expression. Yang replaced the P5 promoter with the mouse metallothionein promoter. Stable clones in HEK293 showed metal-inducible rep78 expression, Rep50 and rep42 expression (driven by an internal P19 promoter) was expressed at low levels. The cells were only detected in the control group and the cell growth rate was substantially decreased (Yang et al. 994) J. Virol 68:4847-4856). Ogasawara is a The promoter contains loxP-flanked stuffers that can be activated by Cre recombinase. The promoters of the rep52, rep40, or cap genes were replaced with ubiquitous promoters. None of these were induced in stable clones infected with adenovirus-Cre. suggested that constitutive rep52 / rep40 expression was also detrimental to cells ( Ogasawara et al.(1999)J.Gen.Virol.80:247 7-2480).

[0006] Another approach for regulated rep expression was described by Xiao and coworkers. (Qiao et al. (2002) J. Virol. 76:1301 5-13027, Yuan et al. (2011) Hum.Gene Ther.2 2:613-624). Xiao is the coding region shared by all four Rep proteins. An artificial intron was inserted into the rep gene in the The loxP-flanked stop cassette was used alone or in combination with the puromycin resistance gene puromycin. The expression of all Rep proteins was inhibited in HEK293 cells. This allows the generation of stable cell lines in the presence of Cre recombinase. The adenovirus infection induces the stop cassette by recombining the loxP sites. The remaining end is then excised, allowing the full-length pre-mRNA to be transcribed. The Rep sequence is precisely removed by RNA splicing to produce all four Rep proteins. thus restoring the coding sequence of AAV from the integrated ITR-flanked transgene. However, Cre recombinase recognizes two identical loxP sites, Since the loxP sites recognize Additional recombinations may be possible to catalyze both the binding and excision reactions.

[0007] The AAV rep gene is expressed in cells that also express the adenovirus E1 (early region 1) gene. Several stable rep / cap cell lines are available, including HeLa (Clark et al(1995)Hum.Gen.Therap.6:1329-1341, Y ang et al. (1994) J. Virol. 68:4847-4856, Gao et.Al(1998)Hu,Gen.Ther.9:2353-2362), A54 9 (Gao et al. (2002) Mol Ther. 5:644-659), and Vero (Beal et al. (2007) 10 th Annual Meeting g of American Society of Gene Therapy,Se Attle, WA, May 30-June 3, 2007) expressing the E1 gene. The biggest drawback to these cell lines is that they lack E1 intact. A (usually replication-competent) adenovirus is required for AAV production, which is why AAV viruses As a contaminant of the drug preparation, it may pose an increased safety risk.

[0008] Provide helper functions and deliver recombinant transgenes and / or AAV genes to human cells To identify herpes (Thomas et al. (2009) Hum Gene The er.20:861-70, Clement et al. (2009) Hum Gen e 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 ge ne Ther.7:2079-2087, Gao et al. (1998) Hum Gene Ther.2353-2362.Liu et al.(1999)Gene Ther 6:293-299) using several different viruses, including AA V production systems have been described. These approaches have been used to In some cases, production of recombinant host cell lines is required. AAV is produced by baculovirus It has also been produced in insect cells using the method (Mietzsch et al. (2014) H um Gene Ther.25:212-22, Aslanidi et al.(2 009)Proc Natl Acad Sci USA.106:5059-5064 , Cecchini et al. (2011) Hum Gene Ther.22:1 021-1030). Are AAVs produced in insect cells versus human cells functionally equivalent? Whether this is the case remains an open question. Summary of the Invention [Problem to be solved by the invention]

[0009] There is a need for improved production of AAV using recombinant constructs and cells. [Means for solving the problem]

[0010] In one embodiment, four Rep proteins, Rep78, Rep68, Rep52, and Rep78. A modified adeno-associated virus (AAV) carrying the AAV rep gene encoding ep40 ) The rep gene and the coding sequence of the rep gene shared by the four Rep proteins and an artificial intron inserted into the sequence. The artificial intron is located downstream of the 5' splice site and upstream of the branch site of the artificial intron. and a stop cassette inserted therein, the stop cassette comprising, in 5' to 3' order, (a) SEQ ID NO: No. 7 and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, attP sites with 98%, 99% or 100% identical nucleotide sequences, preferably 7, and (b) an attP site having the nucleotide sequence of SEQ ID NO: 7; (c) a terminator; and (d) a sequence identical to SEQ ID NO:8 or SEQ ID NO:9, at least 90%, 1%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 % attB site with identical nucleotide sequence, preferably SEQ ID NO: 8 or SEQ ID NO: and an attB site having a nucleotide sequence of 314, 315, 316, 319.

[0011] In one embodiment, the splice acceptor comprises the nucleotide sequence of SEQ ID NO:17.

[0012] In one embodiment, the terminator comprises a polyadenylation signal. The terminator further comprises the nucleotide sequence of SEQ ID NO:19.

[0013] In one embodiment, the termination cassette is a gene encoding a selectable marker, preferably The neomycin phosphotransferase gene has the nucleotide sequence of SEQ ID NO:18. Includes current cassette.

[0014] In one embodiment, the artificial intron comprises, in the 5' to 3' order, the nucleotide sequence of SEQ ID NO: 14. The nucleic acid sequence includes a nucleotide sequence, a stop cassette, and the nucleotide sequence of SEQ ID NO:15.

[0015] In one embodiment, the AAV rep gene is one of the rep genes AAV1 to AAV8. In one embodiment, the AAV rep gene is The nucleotide sequence of the nucleotide bank accession number NC_001401.2 In one embodiment, the rep gene of human AAV2 having sequence numbers 190 to 2202 is The artificial intron was identified by the nucleotide sequence of GenBank accession number NC_001401.2. In one embodiment, the nucleotide sequence is inserted between nucleotide numbers 996 and 1905 of the nucleotide sequence. The artificial intron was identified by GenBank accession number NC_001401.2. Nucleotide numbers of the leucine sequence are 1052, 1061, 1712, 1906, 1022, 1112, 1475, 1514, 1700, 1742, 1784, or 1340, preferably The insertion is located immediately downstream of nucleotide number 1052.

[0016] In one aspect, a non-naturally occurring nucleic acid molecule comprising a modified AAV rep gene is provided herein. The modified AAV rep gene comprises, in 5' to 3' order: (a) the nucleotide sequence of SEQ ID NO:55 (b) the 5' portion of the AAV rep gene with a primer sequence; and (b) an artificial intron. and, in 5' to 3' order, (i) a 5' nucleotide sequence of SEQ ID NO: 14; (ii) a stop cassette comprising, in 5' to 3' order: (1) SEQ ID NO:7 (2) an attP site having the nucleotide sequence of SEQ ID NO:17 (3) a splice acceptor having the nucleotide sequence of SEQ ID NO:18 (4) a phosphotransferase expression cassette having the nucleotide sequence of SEQ ID NO:19; and (5) a terminator having the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 9. (iii) a stop cassette containing the nucleotide sequence of SEQ ID NO: 15. (c) a 3' intron fragment having the nucleotide sequence of SEQ ID NO: 56; and a 3' portion of the AAV rep gene having an octade sequence.

[0017] In one aspect, a non-naturally occurring nucleic acid molecule comprising a modified AAV rep gene is provided herein. The modified AAV rep gene comprises, in 5' to 3' order: (a) the nucleotide sequence of SEQ ID NO: 73 (b) the 5' portion of the AAV rep gene with a primer sequence; and (b) an artificial intron. and, in 5' to 3' order, (i) a 5' end having the nucleotide sequence of SEQ ID NO:14. (ii) a stop cassette comprising, in 5' to 3' order: (1) a cDNA fragment of SEQ ID NO:7; (2) an attP site having the nucleotide sequence of SEQ ID NO:17 (3) a splice acceptor having the nucleotide sequence of SEQ ID NO:18, (4) a phosphotransferase expression cassette having the nucleotide sequence of SEQ ID NO:19 and (5) a terminator having the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 9. tB site, and (iii) a stop cassette comprising the nucleotide sequence of SEQ ID NO: 66. (c) a 3' intron fragment having the nucleotide sequence of SEQ ID NO: 56; and a 3' portion of the AAV rep gene having a stop codon sequence. The set includes the nucleotide sequence of SEQ ID NO:16.

[0018] In one embodiment, the non-naturally occurring nucleic acid molecule comprises three capsid proteins: VP1, VP In one embodiment, the AAV further comprises an AAV cap gene encoding VP3, VP4, and VP5. The cap gene is one of the cap genes of AAV1 to AAV9 and AAVDJ, or and hybrids thereof. In one embodiment, the AAV cap gene is c of human AAV9 having the nucleotide sequence of accession number AY530579.1 In one embodiment, the AAV cap gene contains a polyadenylation signal. Preferably, the nucleotide sequence of GenBank accession number NC_001401.2 is The polyadenylation signal of AAV2, which has nucleotides 4411 to 4466 of the AAV2 polyadenylation sequence, and enhancers, preferably those listed in GenBank Accession No. NC_001401 AAV2 rep having nucleotide numbers 190 to 313 of the nucleotide sequence of .2 P5 promoter, and further comprising a polyadenylation signal and an enhancer, both of which are In one embodiment, the non-naturally occurring nucleic acid molecule is The transgene is flanked by a pair of AAV inverted terminal repeats (ITRs) downstream of the V cap gene. It further includes children.

[0019] In one embodiment, the non-naturally occurring nucleic acid molecule comprises a first An insulator and, optionally, a second insulator downstream of the transgene flanked by ITRs. Preferably, the first insulator and the second insulator are independently, (a) a human anti-repressor element 4 having the nucleotide sequence of SEQ ID NO:24 0, (b) mouse anti-repressor element 40 having the nucleotide sequence of SEQ ID NO: 25, (c ) having the nucleotide sequence of GenBank accession number AY190749.1 anti-repressor element 04, (d) GenBank accession number AY190750.1 (e) anti-repressor element 06 having the nucleotide sequence of GenBank accession no. (f) anti-repressor element 07 having the nucleotide sequence of sequence number AY190751.1; The antibody having the nucleotide sequence of GenBank accession number AY190752.1 (g) repressor element 12, GenBank accession number AY190753.1 Anti-repressor element 13 with nucleotide sequence, (h) GenBank accession Anti-repressor element 35 having the nucleotide sequence number AY190754.1, (i) G The anti-ribosome antibody having the nucleotide sequence of enBank accession number AY190755.1 (j) the nuclease repressor element 36, GenBank accession number AY190757.1 Anti-repressor element 52 with nucleotide sequence, (k) GenBank accession no. (l) an anti-repressor element having the nucleotide sequence of No. AY190758.1; and A group having the nucleotide sequence of AY040835.1 in two or more copies. The chicken HS4 insulator from the robin locus is preferably selected from the group consisting of Alternatively, the first insulator and the second insulator are, respectively, SEQ ID NO: 24 and and the nucleotide sequence of SEQ ID NO: 25. In one embodiment, the non-naturally occurring nucleic acid molecule The modified AAV rep gene contains a first insulator upstream of the gene and a second insulator upstream of the gene. and downstream of the first spacer sequence and a second spacer sequence, the second spacer sequence and the second spacer sequence are independently: (a) a nucleotide sequence of SEQ ID NO:67 and (b) the nucleotide sequence of SEQ ID NO: 68. The ITR has the nucleotide sequence of SEQ ID NO: 20, and the transgene is operatively linked to the coding sequence. The coding sequence includes a promoter operably linked to a polyadenylation signal. and preferably the promoter has the nucleotide sequence of SEQ ID NO: 21. The polyadenylation signal has the nucleotide sequence SEQ ID NO:23.

[0020] In one aspect, provided herein is a non-naturally occurring nucleic acid molecule, the non-naturally occurring nucleic acid molecule comprising: In order from 5' to 3', (A) a first insulator, preferably having the sequence represented by SEQ ID NO:2 (B) the first insulator, having the nucleotide sequence of pGFR-4; a nucleic acid sequence comprising, in 5' to 3' order: (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, comprising, in the order of 5' to 3', (a) a 5' intron fragment; a 5' intron fragment, preferably having the nucleotide sequence of SEQ ID NO: 14; (b) a stop cassette, comprising, in 5' to 3' order: (1) the nucleotide sequence of SEQ ID NO:7; (2) an attP site having a splice acceptor, preferably having the sequence (3) a splice acceptor having the nucleotide sequence of sequence number 17; A gene encoding a marker, preferably a neomycin having the nucleotide sequence of SEQ ID NO: 18. (4) a terminator, preferably (5) a terminator having the nucleotide sequence of SEQ ID NO: 8 or a stop cassette comprising an attB site having the nucleotide sequence of SEQ ID NO:9, and (c) a 3' intron fragment, preferably having the nucleotide sequence of SEQ ID NO: 15. (iii) an artificial intron, including a 3' intron fragment, of the AAV rep gene; 3' portion, preferably having the nucleotide sequence of SEQ ID NO:56. (C) a modified AAV rep gene, including the 3' portion of the AAV ep gene; and AAV cap gene, preferably comprising the nucleotide sequence of SEQ ID NO:57. and (D) a transgene flanked by a pair of AAV ITRs, preferably V ITR has the nucleotide sequence of SEQ ID NO: 20, and the transgene is inserted into the coding sequence. The coding sequence includes a promoter operably linked to a polyadenylation signal. More preferably, the promoter is operably linked to 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 comprising the nucleotide sequence of SEQ ID NO: 25. and a second insulator having a gate arrangement.

[0021] In one aspect, provided herein is a non-naturally occurring nucleic acid molecule, the non-naturally occurring nucleic acid molecule comprising: In order from 5' to 3', (A) a first insulator, preferably having the sequence represented by SEQ ID NO:2 (B) the first insulator, having the nucleotide sequence of pGFR-4; a nucleic acid sequence comprising, in 5' to 3' order: (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, comprising, in the order of 5' to 3', (a) a 5' intron fragment; a 5' intron fragment, preferably having the nucleotide sequence of SEQ ID NO: 14; (b) a stop cassette, comprising, in 5' to 3' order: (1) the nucleotide sequence of SEQ ID NO:7; (2) an attP site having a splice acceptor, preferably having the sequence (3) a splice acceptor having the nucleotide sequence of sequence number 17; A gene encoding a marker, preferably a neomycin having the nucleotide sequence of SEQ ID NO: 18. (4) a terminator, preferably (5) a terminator having the nucleotide sequence of SEQ ID NO: 8 or a stop cassette comprising an attB site having the nucleotide sequence of SEQ ID NO:9, and (c) a 3' intron fragment, preferably having the nucleotide sequence of SEQ ID NO: 66; (iii) an artificial intron, including a 3' intron fragment, of the AAV rep gene; 3' portion, preferably having the nucleotide sequence of SEQ ID NO:56. (C) a modified AAV rep gene, including the 3' portion of the AAV ep gene; and and (D)(1) a pair of AAV ITRs, preferably the AAV ITRs are The transgene has the nucleotide sequence of SEQ ID NO: 20 and is operably linked to a coding sequence. The coding sequence is operably linked to a polyadenylation signal. More preferably, the promoter has the nucleotide sequence of SEQ ID NO: 21 and A pair of AAV ITRs, the denylation signal having the nucleotide sequence SEQ ID NO:23 and (2) a pair of spacer sequences, preferably the spacer sequences are represented by SEQ ID NO: 67. and a pair of spacer sequences having the nucleotide sequence of SEQ ID NO:68, and

[0022] In one aspect, provided herein is a vector comprising the non-naturally occurring nucleic acid molecule described above, and is preferred. Alternatively, the vector is a plasmid, and more preferably the plasmid is SEQ ID NO: 12. The nucleotide sequence of

[0023] In one aspect, provided herein is a vector comprising the non-naturally occurring nucleic acid molecule described above, and is preferred. Alternatively, the vector is a plasmid, and more preferably the plasmid is SEQ ID NO: 70. The nucleotide sequence of

[0024] In one aspect, provided herein are methods for making the non-naturally occurring nucleic acid molecules described above. In certain embodiments, a method for making a vector comprising the non-naturally occurring nucleic acid molecule described herein is provided. Preferably, the vector is a plasmid, more preferably a plasmid. In another embodiment, the non-naturally occurring Provided herein is a method for making a vector comprising a nucleic acid molecule, preferably the vector comprises , a plasmid, more preferably the plasmid has the nucleotide sequence of SEQ ID NO: 70 Includes.

[0025] In one embodiment, four Rep proteins, Rep78, Rep68, Rep52, and Rep78. A modified adeno-associated virus (AAV) carrying the AAV rep gene encoding ep40 ) The rep gene and the coding sequence of the rep gene shared by the four Rep proteins and an artificial intron inserted into the sequence. The artificial intron is provided downstream of the 5' splice site of the artificial intron and at the branched portion. The stop cassette includes a stop cassette inserted upstream of the stop cassette position, which is, in 5' to 3' order, ( a) at least 90%, 91%, 92%, 93%, 94%, 95%, 96% identical to SEQ ID NO: 7 , attP sites having 97%, 98%, 99%, or 100% identical nucleotide sequences , preferably an attP site having the nucleotide sequence of SEQ ID NO: 7; and (b) a splice (c) a terminator; and (d) SEQ ID NO: 8 or SEQ ID NO: 9 and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or an attB site having 100% identical nucleotide sequence, preferably SEQ ID NO: 8 or and an attB site having the nucleotide sequence of SEQ ID NO:9.

[0026] In one embodiment, the splice acceptor comprises the nucleotide sequence of SEQ ID NO:17.

[0027] In one embodiment, the terminator comprises a polyadenylation signal. The terminator further comprises the nucleotide sequence of SEQ ID NO:19.

[0028] In one embodiment, the termination cassette is a gene encoding a selectable marker, preferably The neomycin phosphotransferase gene has the nucleotide sequence of SEQ ID NO:18. Includes current cassette.

[0029] In one embodiment, the artificial intron comprises, in the 5' to 3' order, the nucleotide sequence of SEQ ID NO: 14. In another embodiment, the nucleic acid sequence comprises a nucleotide sequence, a stop cassette, and the nucleotide sequence of SEQ ID NO: 15. The artificial intron comprises, in the 5' to 3' order, the nucleotide sequence of SEQ ID NO: 14, The cassette comprises the nucleotide sequence of SEQ ID NO:66.

[0030] In one embodiment, the AAV rep gene is one of the rep genes AAV1 to AAV8. In one embodiment, the AAV rep gene is The nucleotide sequence of the nucleotide bank accession number NC_001401.2 In one embodiment, the rep gene of human AAV2 having sequence numbers 190 to 2202 is The artificial intron was identified by the nucleotide sequence of GenBank accession number NC_001401.2. In one embodiment, the nucleotide sequence is inserted between nucleotide numbers 996 and 1905 of the nucleotide sequence. The artificial intron was identified by GenBank accession number NC_001401.2. Nucleotide numbers of the leucine sequence are 1052, 1061, 1712, 1906, 1022, 1112, 1475, 1514, 1700, 1742, 1784, or 1340, preferably The insertion is located immediately downstream of nucleotide number 1052.

[0031] In one embodiment, a cell comprising a non-naturally occurring nucleic acid molecule comprising a modified AAV rep gene is described herein. As provided herein, the modified AAV rep gene comprises, in 5' to 3' order: (a) SEQ ID NO: (b) the 5' portion of the AAV rep gene having the nucleotide sequence of a nucleotide sequence comprising, in 5' to 3' order, (i) the nucleotide sequence of SEQ ID NO: 14; (ii) a stop cassette comprising, in 5' to 3' order: (1) (1) an attP site having the nucleotide sequence of SEQ ID NO:7; (3) a splice acceptor having the nucleotide sequence of SEQ ID NO: 18 (4) a neomycin phosphotransferase expression cassette, and (5) a terminator having the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 9. (iii) a stop cassette containing an attB site having the nucleotide sequence of SEQ ID NO: 15; (c) an artificial intron comprising a 3' intron fragment having an octide sequence; and and a 3' portion of the AAV rep gene having a nucleotide sequence of 6.

[0032] In one embodiment, a cell comprising a non-naturally occurring nucleic acid molecule comprising a modified AAV rep gene is described herein. As provided herein, the modified AAV rep gene comprises, in 5' to 3' order: (a) SEQ ID NO: (b) the 5' portion of the AAV rep gene having the nucleotide sequence of SEQ ID NO:73; and a nucleotide sequence comprising, in 5' to 3' order, (i) the nucleotide sequence of SEQ ID NO: 14; (ii) a stop cassette comprising, in 5' to 3' order: (1) (1) an attP site having the nucleotide sequence of SEQ ID NO:7; (3) a splice acceptor having the nucleotide sequence of SEQ ID NO: 18 (4) a neomycin phosphotransferase expression cassette, and (5) a terminator having the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 9. (iii) a stop cassette containing an attB site having the nucleotide sequence of SEQ ID NO: 66; (c) an artificial intron comprising a 3' intron fragment having an octide sequence; and and a 3' portion of the AAV rep gene having a nucleotide sequence of 6.

[0033] In one embodiment, the stop cassette comprises the nucleotide sequence of SEQ ID NO:16.

[0034] In one embodiment, the cell contains three capsid proteins VP1, VP2, and V In one embodiment, the AAV cap gene further comprises an AAV cap gene encoding P3. The gene is a cap gene of one of AAV1 to AAV9 and AAVDJ, or a high-level gene thereof. In one embodiment, the AAV cap gene is The cap gene of human AAV9 having the nucleotide sequence of subunit number AY530579.1 In one embodiment, the AAV cap gene comprises a hybrid cap gene of AAV9. Includes genes.

[0035] In one embodiment, the AAV cap gene contains a polyadenylation signal, preferably G The nucleotide sequence of enBank accession number NC_001401.2 The polyadenylation signal of AAV2 having sequence numbers 4411 to 4466 and the enhancer Preferably, the nucleotide sequence of GenBank accession number NC_001401.2 is AAV2 rep P5 promoter having nucleotide numbers 190 to 313 of the promoter sequence , and both the polyadenylation signal and the enhancer are located within the coding sequence of the cap gene. It's downstream in the line.

[0036] In one embodiment, the cell containing the cap gene comprises a pair of AAV cap genes downstream of the AAV cap gene. The transgene further comprises an AV inverted terminal repeat (ITR). The cells contain a first insulator upstream of the modified AAV rep gene, and, optionally, an I The TR further comprises a second insulator downstream of the flanking transgene, preferably a first The insulator and the second insulator are independently selected from the group consisting of (a) a nucleotide sequence of SEQ ID NO: 24 (b) the nucleotide sequence of SEQ ID NO: 25 of human anti-repressor element 40 having the nucleotide sequence (c) Mouse anti-repressor element 40 having GenBank accession number AY1 (d) GenBan anti-repressor element 04 having the nucleotide sequence 90749.1; The anti-repressor element having the nucleotide sequence of accession number AY190750.1 Element 06, (e) nucleotide of GenBank accession number AY190751.1 (f) Anti-repressor element 07 having the sequence, GenBank accession number AY19 (g) GenBank anti-repressor element 12 having the nucleotide sequence 0752.1 An anti-repressor element having the nucleotide sequence of accession number AY190753.1 13, (h) Nucleotide sequence of GenBank accession number AY190754.1 Anti-repressor element 35 having sequence (i) GenBank accession number AY190 Anti-repressor element 36, having the nucleotide sequence of 755.1, (j) GenBank Accession No. Anti-repressor element 5 having the nucleotide sequence of accession number AY190757.1 2. (k) Nucleotide sequence of GenBank accession number AY190758.1 and (l) an anti-repressor element 53 having AY04 in two or more copies. The chicken HS4 enzyme from the globin locus having the nucleotide sequence of 0835.1 More preferably, the first insulator and the second insulator are selected from the group consisting of The insulators have the nucleotide sequences of SEQ ID NO:24 and SEQ ID NO:25, respectively. In one embodiment, the cell contains a first insulator upstream of the modified AAV rep gene. A first spacer sequence and a second spacer sequence are located upstream and downstream of the transgene, respectively. and wherein the first spacer sequence and the second spacer sequence independently comprise: (a) sequence no. (b) the nucleotide sequence of SEQ ID NO: 67; and (b) the nucleotide sequence of SEQ ID NO: 68. It is selected.

[0037] In one embodiment, the ITRs have the nucleotide sequence of SEQ ID NO: 20 and the transgene is The coding sequence includes a promoter operably linked to the coding sequence, and the coding sequence includes a polyadenylation sequence. Preferably, the promoter is operably linked to a signal of the nucleotide sequence of SEQ ID NO: 21. The polyadenylation signal has the nucleotide sequence SEQ ID NO: 23. do.

[0038] In one aspect, provided herein is a cell comprising a non-naturally occurring nucleic acid molecule, The acid molecules are, in 5' to 3' order, (A) a first insulator, preferably (B) a first insulator having the nucleotide sequence of SEQ ID NO: 24; a rep gene, comprising, in 5' to 3' order: (i) a 5' portion of the AAV rep gene; Preferably, the AAV rep gene has the nucleotide sequence of SEQ ID NO: 55. (ii) an artificial intron, which is, in the order from 5' to 3', A 5' intron fragment, preferably having the nucleotide sequence of SEQ ID NO: 14. (b) a stop cassette, comprising, in 5' to 3' order: (1) SEQ ID NO:7; (2) a splice acceptor, preferably an attP site having a nucleotide sequence of or a splice acceptor having the nucleotide sequence of SEQ ID NO: 17; A gene encoding a possible marker, preferably having the nucleotide sequence of SEQ ID NO: 18. (4) a terminator, (5) a terminator having the nucleotide sequence of SEQ ID NO: 19; and A stop cassette comprising an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9. and (c) a 3' intron fragment, preferably comprising the nucleotide sequence of SEQ ID NO: 15. (iii) an artificial intron, including a 3' intron fragment having the sequence 3' part of the p gene, preferably having the nucleotide sequence of SEQ ID NO: 56; (C) a modified AAV rep gene, including a 3' portion of the AAV rep gene; and AAV cap gene, preferably comprising the nucleotide sequence of SEQ ID NO: 57. (D) a transgene flanked by a pair of AAV ITRs, In one embodiment, the AAV ITRs have the nucleotide sequence of SEQ ID NO: 20, and the transgene is The coding sequence comprises a promoter operably linked to a coding sequence, and the coding sequence comprises a polyadenylation signal transduction signal. More preferably, the promoter is operably linked to the nucleotide sequence of SEQ ID NO: 21. The polyadenylation signal has the nucleotide sequence SEQ ID NO: 23. (E) a second insulator, preferably having the sequence of SEQ ID NO: 25. and a second insulator having a nucleotide sequence.

[0039] In one aspect, provided herein is a cell comprising a non-naturally occurring nucleic acid molecule, The acid molecules are, in 5' to 3' order, (A) a first insulator, preferably (B) a first insulator having the nucleotide sequence of SEQ ID NO: 24; a rep gene, comprising, in 5' to 3' order: (i) a 5' portion of the AAV rep gene; Preferably, the AAV rep gene has the nucleotide sequence of SEQ ID NO: 73. (ii) an artificial intron, which is, in the order from 5' to 3', A 5' intron fragment, preferably having the nucleotide sequence of SEQ ID NO: 14. (b) a stop cassette, comprising, in 5' to 3' order: (1) SEQ ID NO:7; (2) a splice acceptor, preferably an attP site having a nucleotide sequence of or a splice acceptor having the nucleotide sequence of SEQ ID NO: 17; A gene encoding a possible marker, preferably having the nucleotide sequence of SEQ ID NO: 18. (4) a terminator, (5) a terminator having the nucleotide sequence of SEQ ID NO: 19; and A stop cassette comprising an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9. and (c) a 3' intron fragment, preferably comprising the nucleotide sequence of SEQ ID NO: 66. (iii) an artificial intron, including a 3' intron fragment having the sequence 3' part of the p gene, preferably having the nucleotide sequence of SEQ ID NO: 56; (C) a modified AAV rep gene, including a 3' portion of the AAV rep gene; and (D)(i) a pair of AAV ITRs, preferably The ITR has the nucleotide sequence of SEQ ID NO: 20, and the transgene is operably linked to the coding sequence. The coding sequence comprises a promoter 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, a pair of AA V ITR, and (ii) a pair of spacer sequences, preferably the spacer sequences being A pair of spacer sequences having the nucleotide sequences of SEQ ID NO:67 and SEQ ID NO:68 are adjacent to each other. and a transgene.

[0040] In one embodiment, the non-naturally occurring nucleic acid molecule has the nucleotide sequence of SEQ ID NO: 12, In another embodiment, the non-naturally occurring nucleic acid molecule is the nucleic acid of SEQ ID NO: 70. It has a nucleotide sequence and is episomal.

[0041] In one embodiment, the cell has a sequence identical to the amino acid sequence of SEQ ID NO:2, at least 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 7%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 9 The recombinase is encoded by coding a recombinase having an amino acid sequence identical to that of the target gene, which is 7%, 98%, 99%, or 100% of the target gene. The present invention further includes a nucleic acid molecule that encodes a nucleotide sequence similar to that of SEQ ID NO:3. At least 85%, at least 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% More preferably, the cell comprises the amino acid sequence of SEQ ID NO:2. A recombinant ΔE1 / ΔE3 adenovirus serotype 5 (Ad 5) Contains a virus.

[0042] In one embodiment, the cell further comprises the adenovirus E1A and E1B genes, preferably For example, the cells are 911 cells, pTG6559 cells, GH329 cells, N52.E6 cells, HeLa-E1 cells, UR cells, VLI-293 cells, HEK293 cells, or PER. These are C6 cells.

[0043] In one aspect, provided herein is a method for producing a recombinant AAV comprising a transgene, the method comprising: The method includes (A) obtaining a first host cell, the first host cell comprising: (i) a modified AAV a rep gene, comprising, in 5' to 3' order: (a) a 5' portion of the AAV rep gene; AAV rep gene, preferably having the nucleotide sequence of SEQ ID NO:55. (b) an artificial intron, which is arranged in the order of 5' to 3': (1) a 5' intron; A 5' intron fragment, preferably having the nucleotide sequence of SEQ ID NO: 14. (2) a stop cassette, comprising, in 5' to 3' order: (aa) SEQ ID NO: (bb) an attP site having a nucleotide sequence of: Preferably, the splice acceptor (cc) has the nucleotide sequence of SEQ ID NO: 17. ) a gene encoding a selectable marker, preferably the nucleotide sequence of SEQ ID NO: 18 Neomycin phosphotransferase expression cassette with sequence, (dd) terminator a terminator, preferably 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 comprising the nucleotide sequence of SEQ ID NO: 15. (c) an artificial intron comprising a 3' intron fragment having a nucleotide sequence; rep gene, preferably having the nucleotide sequence of SEQ ID NO:56. (ii) a modified AAV rep gene, comprising a 3' portion of the AAV rep gene; An AAV cap gene, preferably comprising the nucleotide sequence of SEQ ID NO: 57; (iii) a transgene flanked by a pair of AAV ITRs. Preferably, the ITR has the nucleotide sequence of SEQ ID NO: 20, and the transgene is , a promoter operably linked to a coding sequence, the coding sequence comprising a polyadenylation Preferably, the promoter is operably linked to the signal, more preferably the promoter is and a polyadenylation signal is represented by the nucleotide sequence SEQ ID NO: 23. (B) obtaining a first host cell comprising a transgene having the , at least 85%, 86%, 87%, 88%, 89%, or more relative to the amino acid sequence of SEQ ID NO:2 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 %, or 100% sequence identity The recombinant adenovirus is then infected with a second recombinant adenovirus containing a recombinase gene. and (C) under conditions in which a recombinant AAV containing the transgene is produced. (D) growing the second host cell; and, optionally, harvesting the recombinant AAV. Including,

[0044] In one aspect, provided herein is a method for producing a recombinant AAV comprising a transgene, the method comprising: The method includes (A) obtaining a first host cell, the first host cell comprising: (i) a modified AAV a rep gene, comprising, in 5' to 3' order: (a) a 5' portion of the AAV rep gene; AAV rep gene, preferably having the nucleotide sequence of SEQ ID NO: 73. (b) an artificial intron, which is arranged in the order of 5' to 3': (1) a 5' intron; A 5' intron fragment, preferably having the nucleotide sequence of SEQ ID NO: 14. (2) a stop cassette, comprising, in 5' to 3' order: (aa) SEQ ID NO: (bb) an attP site having a nucleotide sequence of: Preferably, the splice acceptor (cc) has the nucleotide sequence of SEQ ID NO: 17. ) a gene encoding a selectable marker, preferably the nucleotide sequence of SEQ ID NO: 18 Neomycin phosphotransferase expression cassette with sequence, (dd) terminator a terminator, preferably 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 comprising the nucleotide sequence of SEQ ID NO: 66. (c) an artificial intron comprising a 3' intron fragment having a nucleotide sequence; rep gene, preferably having the nucleotide sequence of SEQ ID NO:66. (ii) a modified AAV rep gene, comprising a 3' portion of the AAV rep gene; (iii)(a) a pair of AAV ITRs, Preferably, the ITRs have the nucleotide sequence of SEQ ID NO: 20 and the transgene is The coding sequence includes a promoter operably linked to a polyadenylation signal. More preferably, the promoter is operably linked to the nucleotide sequence of SEQ ID NO:21. a pair of nucleotide sequences having the polyadenylation signal having the nucleotide sequence SEQ ID NO: 23; and (b) a pair of spacer sequences, preferably a pair of spacer sequences having the nucleotide sequences of SEQ ID NO:67 and SEQ ID NO:68; (B) obtaining a first host cell comprising a transgene flanked by a first vector; and (C) detecting the first host cell. to the amino acid sequence of SEQ ID NO:2, 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9 Recombinase genes encoding recombinases with 9% or 100% sequence identity The recombinant adenovirus containing the gene is then infected to produce a second adenovirus containing a recombinase gene. (C) obtaining a host cell of the second type under conditions in which a recombinant AAV containing a transgene is produced; (D) optionally, harvesting the recombinant AAV. Includes and.

[0045] In one embodiment, the first host cell contains a first inci gene 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 independent. (a) a human anti-repressor element 40 having the nucleotide sequence of SEQ ID NO: 24; (b) (c) GenB Ank accession number AY190749.1 is a nucleotide sequence of the anti-repressor (d) nucleotide sequence of element 04, GenBank accession number AY190750.1 (e) Anti-repressor element 06 having a tid sequence, GenBank accession number AY (f) GenBasin 190751.1 anti-repressor element 07, having the nucleotide sequence An anti-repressor having the nucleotide sequence of nk accession number AY190752.1 Element 12, (g) the nucleotide sequence of GenBank accession number AY190753.1 (h) GenBank accession number AY1 Anti-repressor element 35 having the nucleotide sequence of 90754.1, (i) GenBan The anti-repressor element having the nucleotide sequence of accession number AY190755.1 Element 36, (j) nucleotide of GenBank accession number AY190757.1 Anti-repressor element 52 having the sequence, (k) GenBank accession number AY19 53, an anti-repressor element having the nucleotide sequence of 0758.1, and (l) two or three A globin gene having the nucleotide sequence of AY040835.1 in one or more copies chicken HS4 insulator from the roe locus, more preferably from the 1st The first insulator and the second insulator are respectively represented by SEQ ID NO: 24 and SEQ ID NO: It has a 25 nucleotide sequence.

[0046] In one embodiment, the first host cell contains a first inci gene upstream of the modified AAV rep gene. The gene includes a first spacer sequence and a second spacer sequence upstream and downstream of the gene, respectively. The method further comprises the steps of: (a) the nucleotide sequence of SEQ ID NO: 67 and (b) the nucleotide sequence of SEQ ID NO: 68 The compound is selected from the group consisting of:

[0047] In one embodiment, the first host cell contains a modified AAV rep gene, an AAV cap gene, A clone containing a transgene flanked by ITRs, a first insulator, and a second insulator. In one embodiment, the nucleic acid sequence is obtained by introducing into the cell one or more nucleic acid molecules, including In the first host cell, a first insulator, a modified AAV The rep gene, AAV cap gene, transgene flanked by ITRs, and the first insula a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO: 12, and a second insulator; The vector can be obtained by introducing a plasmid containing the vector sequence into a cell.

[0048] In one embodiment, the first host cell contains a modified AAV rep gene, an AAV cap gene, a transgene flanked by ITRs, a first insulator, a first spacer sequence, and The present invention relates to a method for producing a nucleic acid molecule comprising the steps of: In one embodiment, the first host cell expresses a modified AAV rep gene, an AAV ca p gene, a transgene flanked by ITRs, a first insulator, a first spacer sequence, and a second spacer sequence, It is obtained by introducing a plasmid containing the nucleotide sequence of number 70 into a cell.

[0049] In one embodiment, the recombinant adenovirus comprises a recombinant adenovirus comprising the nucleotide sequence of SEQ ID NO:3. The virus is a ΔE1 / ΔE3 adenovirus serotype 5 (Ad5) virus.

[0050] In one embodiment, the host cell comprises the adenovirus E1A and E1B genes, preferably In general, the host cells are 911 cells, pTG6559 cells, GH329 cells, N52.E6 cells, etc. cells, HeLa-E1 cells, UR cells, VLI-293 cells, HEK293 cells, or PE R.C6 cells.

[0051] In one embodiment, the conditions for growing the second host cell include growing the second host cell with 2-aminopurine. In one embodiment, the 2-aminopurine concentration is about 1.25 mM. In one embodiment, the 2-aminopurine concentration is about 1 μM to about 1.25 mM. In one embodiment, the concentration of 2-aminopurine is about 10 μM to about 1.25 mM. In one embodiment, the 2-aminopurine concentration is about 100 μM to about 1.25 mM. In an embodiment, the 2-aminopurine concentration is about 1.25 mM.

[0052] In one embodiment, culturing the second cell with 2-aminopurine comprises culturing the recombinant adenovirus. The infection is initiated approximately 24 hours after infection of the first host cell with the vector.

[0053] In one embodiment, a cell comprising a nucleic acid molecule encoding a recombinase as described above and a 2-amino acid Provided herein is a composition comprising 2-aminopurine and 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 10 μM to about 1.25 mM. 1.25 mM. In one embodiment, the 2-aminopurine concentration is about 100 μM to about 1. In one embodiment, the 2-aminopurine concentration is about 1.25 mM.

[0054] In one embodiment, the amino acid sequence of SEQ ID NO:2 is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Serine recombiners having amino acid sequences with 98%, 99%, or 100% identity Provided herein is a non-naturally occurring nucleic acid molecule comprising a nucleotide sequence encoding an enzyme. In one embodiment, the non-naturally occurring nucleic acid molecule has a sequence similar to that of SEQ ID NO:3. At least 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% It includes nucleotide sequences that have identity.

[0055] In one embodiment, the amino acid sequence of SEQ ID NO:2 is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Serine recombiners having amino acid sequences with 98%, 99%, or 100% identity The present invention provides a vector comprising a non-naturally occurring nucleic acid molecule comprising a nucleotide sequence encoding an enzyme. Provided in the fine print.

[0056] In one embodiment, the amino acid sequence of SEQ ID NO:3 is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Serine recombiners having amino acid sequences with 98%, 99%, or 100% identity The present invention provides a vector comprising a non-naturally occurring nucleic acid molecule comprising a nucleotide sequence encoding an enzyme. Provided in the fine print.

[0057] In one embodiment, the vector contains a promoter, preferably a gene encoding a serine recombinase. operably linked to a nucleotide sequence encoding a cytomegalovirus s, CMV) promoter.

[0058] In one embodiment, the vector comprises a nucleotide sequence encoding a serine recombinase. operably linked, Simian Virus 40 (SV40) polyadenylation site; The nucleic acid further includes a polyadenylation signal, such as a polyadenylation signal.

[0059] In one embodiment, the vector is a DNA plasmid. , a recombinant adenoviral vector.

[0060] In one embodiment, the vector comprises the amino acid sequence of SEQ ID NO:2 under the control of a CMV promoter. A recombinant ΔE1 / E2 gene comprising a nucleotide sequence encoding a serine recombinase having a sequence ΔE3 adenovirus serotype 5 (Ad5) virus, the nucleotide sequence of which is identical to that of SV4 0 polyadenylation signal (NC_001669.1, nt 2550-2774) are operably linked.

[0061] In one embodiment, the amino acid sequence of SEQ ID NO:2 is at least 85%, e.g., at least Also 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% or 100% identity A non-naturally occurring nucleic acid comprising a nucleotide sequence encoding a serine recombinase having a sequence Provided herein is a cell comprising an acid molecule. In one embodiment, the cell comprises a nucleic acid molecule of SEQ ID NO:3. At least 85% to the nucleic acid sequence, e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, It includes nucleotide sequences having 98%, 99% or 100% identity.

[0062] In one embodiment, the amino acid sequence of SEQ ID NO:2 is at least 85%, e.g., at least Also 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% or 100% identity A non-naturally occurring nucleic acid comprising a nucleotide sequence encoding a serine recombinase having a sequence Provided herein is a cell comprising a vector comprising an acid molecule. In another embodiment, the vector comprises the sequence of SEQ ID NO:3. At least 85% for the amino acid sequence, e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Serine recombiners having amino acid sequences with 98%, 99%, or 100% identity and vectors comprising non-naturally occurring nucleic acid molecules comprising nucleotide sequences encoding the enzymes. Cells are provided herein.

[0063] In one embodiment, the cell is provided with a promoter, preferably a promoter encoding a serine recombinase. A cytomegalovirus (CMV) promoter operably linked to a nucleotide sequence Further includes:

[0064] In one embodiment, the cell is operably linked to a nucleotide sequence encoding a serine recombinase. operably linked to a polyadenylation signal, such as the Simian Virus 40 (SV40) polyadenylation signal; It further comprises a re-adenylation signal.

[0065] In one embodiment, the vector is a DNA plasmid. , a recombinant adenoviral vector.

[0066] In one embodiment, the recombinant adenoviral vector comprises a sequence under the control of a CMV promoter. The nucleotide sequence encoding the serine recombinase having the amino acid sequence of SEQ ID NO:2 is recombinant ΔE1 / ΔE3 adenovirus serotype 5 (Ad5) virus, The nucleotide sequence is the SV40 polyadenylation signal (NC_001669.1, nt 255 0 to 2774).

[0067] In one embodiment, the cell comprises the adenoviral E1A and E1B genes, preferably , cells are 911 cells, pTG6559 cells, GH329 cells, N52.E6 cells, He La-E1 cells, UR cells, VLI-293 cells, HEK293 cells, or PER.C6 It is a cell.

[0068] In one aspect, provided herein is a method for performing site-specific recombination in a cell, the method comprising: , (a) a sequence identical to SEQ ID NO: 7 and at least 90%, 91%, 92%, 93%, 94%, 95%, 9 attP having 6%, 97%, 98%, 99%, or 100% identical nucleotide sequence site, preferably an attP site having the nucleotide sequence of SEQ ID NO: 7, and 8 or SEQ ID NO: 9 and at least 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99%, or 100% identical nucleotide sequence to attB attB site having the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 9 (b) obtaining a cell comprising a nucleic acid molecule having a sequence identical to that of SEQ ID NO:2, and , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, or 100% identity to a serine recombinase (c) introducing into the cell a non-naturally occurring nucleic acid molecule encoding a serine recombinase; The enzyme is capable of catalyzing site-specific recombination between attP and attB sites. and growing the cells under conditions that result in

[0069] In one aspect, a product produced by a process that performs site-specific recombination in a cell. The present invention provides a method for detecting a nucleic acid sequence comprising: (a) detecting a nucleic acid sequence having at least 90%, 91%, or 9 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical An attP site having a nucleotide sequence, preferably the nucleotide sequence of SEQ ID NO:7 and attP site having at least 90%, 91%, 92% or more of SEQ ID NO:8 or SEQ ID NO:9. %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical An attB site having a nucleotide sequence, preferably the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 9. (b) obtaining a cell containing a nucleic acid molecule having an attB site with an attB sequence; At least 85%, 86%, 87%, 88%, 89%, 90%, 91% for column number 2 , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% A non-naturally occurring nucleic acid molecule encoding a serine recombinase having the same identity is introduced into the cell. (c) a serine recombinase is inserted into a site between the attP site and the attB site. and growing the cells under conditions that allow the cells to catalyze specific recombination.

[0070] In one aspect, provided herein is a process for obtaining a product from a cell, the process comprising: , (a) a sequence identical to SEQ ID NO: 7 and at least 90%, 91%, 92%, 93%, 94%, 95%, 9 attP having 6%, 97%, 98%, 99%, or 100% identical nucleotide sequence site, preferably an attP site having the nucleotide sequence of SEQ ID NO: 7, and 8 or SEQ ID NO: 9 and at least 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99%, or 100% identical nucleotide sequence to attB attB site having the nucleotide sequence of SEQ ID NO: 8 or SEQ ID NO: 9 (b) obtaining a cell comprising a nucleic acid molecule having a sequence identical to that of SEQ ID NO:2, and , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, or 100% identity to a serine recombinase (c) introducing into the cell a non-naturally occurring nucleic acid molecule encoding a serine recombinase; The enzyme is capable of catalyzing site-specific recombination between attP and attB sites. (d) growing the cells under conditions conferring a product thereon; and (d) producing and recovering the product from the cells. Includes and.

[0071] In one aspect, a non-naturally occurring system is provided herein, the non-naturally occurring system being an AAV-mediated transcription factor. The means for recombinant expression of the vector includes, optionally, a transgenic element. In one embodiment, a means for transferring a non-naturally occurring system is provided herein, the means comprising AAV-mediated transcriptional regulation. The recombinant vector includes a means for recombinantly transforming the recombinant vector, which optionally includes a transgenic element.

[0072] In one aspect, a non-naturally occurring system is provided herein, the non-naturally occurring system being an AAV-mediated transcription factor. recombination means for recombining the system, the means optionally comprising: The recombinant means includes a transgenic element, and the recombinant means includes at least one serine residue in the catalytic In one aspect, means for transferring non-naturally occurring systems are provided herein. The recombination means for recombining the system are provided, including means for AAV-mediated recombination. The recombinant means comprises a catalytic step, the means optionally comprising a transgenic element, The present invention includes the use of at least one serine residue in the

[0073] In one aspect, provided herein is a means for producing a molecule, comprising: The means may include and replicate any of the means described above.

[0074] In one aspect, provided herein is a process for AAV-mediated site-specific recombination, The process includes: (a) a step of obtaining a cell that contains the means for AAV-mediated recombination; (b) a method for producing a recombinant vector comprising the steps of: Under conditions that allow for site-specific recombination that uses at least one serine residue in catalysis and for performing the function of growing the cells. In one embodiment, the process , obtaining a product, optionally the product being a therapeutic product.

[0075] Further aspects, features, and advantages of the present invention are set forth in the detailed description of the invention, as well as in the preferred embodiments thereof. These and other aspects of the present invention will become apparent from the following disclosure, including the embodiments and appended claims. [Brief description of the drawings]

[0076] The above summary and the following detailed description of preferred embodiments of the present application should be taken in conjunction with the accompanying drawings. The present application will be better understood upon reading the accompanying drawings. It should be understood that there is no limitation to the precise embodiment. [Figure 1]FIG. 1 shows alignment statistics and sequence alignment of a putative serine recombinase identified in the genome of Bacillus safensis strain CCMA-560 (SEQ ID NO:2, Sbjct) with a length of 535 amino acids, sequence ID: WP_029708089.1, with the SPBetac2 integrase protein (SEQ ID NO:1, query). The two proteins have 64% sequence identity at the protein level ranging from amino acids 1 to 529. This putative serine recombinase is referred to herein as SR21 (serine recombinase 21). [Diagram 2] Identification of strains representing the pre-insertion locus: The whole-genome shotgun sequence of Bacillus safensis strain Fairview contig56_1 (Sbjct) with a length of 568093 nucleotides, nucleotides 464352 to 464839 of sequence ID: NZ_JFBY01000018.1, and the alignment statistics and sequence alignment with the CCMA-560 DNA sequence (query) are shown. [Diagram 3] The SR21 recombinase attP and attB sites are shown. The attP and attB sites are organized in dyad symmetry around a central dinucleotide recombination crossover site (underlined). Half sites are numbered. Spaces have been introduced into the attB sequence to show alignment of sequences predicted to be bound by the zinc ribbon domain (ZD) and recombinase domain (RD) extrapolating from previous studies (Rutherford et al. (2013) Nucleic Acids Res. 41:8341-8356). Residues that are identical in three or four of the ZD or RD domains are in bold. The attP (SEQ ID NO:7) alignment to two alternating attB sequences (SEQ ID NO:8) and (SEQ ID NO:9) is shown. [Figure 4]Illustrates recombinase activation of reporter genes. Plasmid P41 encodes two reporter gene transcripts. The first, 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 an SR21 recombinase attB site (SEQ ID NO: 9), followed by an inverted fusion protein coding region encoding mCherry and firefly luciferase linked by a P2A self-cleaving peptide linker, and an SR21 attP site. Neither luciferase nor mCherry is expressed because they are in opposite orientations relative to the promoter. When SR21 recombinase is expressed, the attB and attP sequences are recombined, which results in the inversion of the reporter gene and the expression of firefly luciferase and mCherry. [Diagram 5] 1 shows AAV capsid proteins in purified recombinant AAV samples produced according to one embodiment of the present application, purified from cells stably transfected with plasmid P439, grown and infected in Hyperflask vessels at 20 MOI (A) and 40 MOI (B), and subjected to PAGE and silver staining. [Figure 6] 1 shows a rep / cap expression cassette with an artificial intron with a stop cassette inserted therein, according to one embodiment of the present application. [Figure 7] 1 shows a vector (plasmid P439) according to one embodiment of the present application. [Figure 8]The location and sequence of RNA splice sites identified in P439 by RT-PCR are shown. The top figure represents the structure of the REP gene after excision of the stop cassette. The 5' and 3' halves of REP are separated by the upstream half of the beta-actin intron (SEQ ID NO:14), the SR21AttL element (SEQ ID NO:35), and the downstream half of the beta-actin intron (SEQ ID NO:15). Splicing between (2) the beta-actin splice donor (SEQ ID NO:71) and (3) the beta-actin splice acceptor (SEQ ID NO:72) is shown by a solid line. Splicing between (1) the upstream splice donor in the 5'REP sequence (SEQ ID NO:64) and (3) the 3' beta-actin acceptor (SEQ ID NO:72) is shown by a dotted line. The sequences of the splice donor and acceptor are shown below. The lower case sequences indicate intron sequences. [Figure 9] 1 shows a vector (plasmid P600) according to one embodiment of the present application. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0077] Various publications, articles and patents are cited or referenced in the Background and throughout this specification. Each of these references is incorporated herein by reference in its entirety. Any discussion of documents, operations, materials, devices, articles or the like which is included in the document is to be understood as providing a context for the invention. Such discussion is intended to provide an understanding that any or all of these matters may be disclosed or used in a manner that is not intended to be limiting. Not admitted to constitute prior art to any claimed invention. There is none.

[0078] Unless otherwise defined, all technical and scientific terms used herein refer to the It has the same meaning as commonly understood by one of ordinary skill in the art to which it pertains. Certain terms used herein have the meanings set forth herein. All patents, published patent applications and publications cited herein are hereby incorporated by reference. It is incorporated in its entirety as if set forth herein.

[0079] As used in this specification and the appended claims, the singular forms "a," "an," and "t" are used interchangeably. It is important to note that "he" includes multiple referents unless the context makes clear otherwise. be.

[0080] Unless otherwise noted, the term "at least" preceding a series of elements refers to every element in the series. It should be understood that the present invention refers to elements of the and recognize many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by this invention. It is illustrated.

[0081] Throughout this specification and the following claims, unless the context otherwise requires, the term "comprises" or "includes" will be used interchangeably. "comprises" and variations such as "comprises" and "comprising" are used herein as examples. Including the specified integer or step or group of integers or steps, but including any other integer or step It will be understood that the term "step" means "step" or "integer number" or "group of steps" is not meant to exclude any particular step or integer. As used herein, the term "comprising" includes the term "containing." "including" or "including" may be substituted or may be used interchangeably herein. When used herein, it may be substituted with the term "having."

[0082] As used herein, "consisting of" refers to a claim element. As used herein, the term "compound" refers to any element, step, or ingredient not specified in the specification. In this case, "consisting essentially of" is the basis of the claim. The present application does not exclude materials or steps that do not substantially affect the novel characteristics of the present invention. or when used herein in connection with the embodiments, to vary the scope of the disclosure, "comprising," "containing," "including," and " Any of the above terms "comprising" or "comprising" may be replaced with the terms "consisting of" or "consisting essentially of." It can be replaced.

[0083] As used herein, the conjunctive term "and / or" between multiple listed elements means Both individual and combined options are understood to be encompassing. For example, When elements are connected by "and / or," the first option may be used without the second option. The first element is applicable. The second option is that the second element can be applied without the first element. The third option refers to the first and second elements being applicable together. Any one of these options is within the meaning and therefore the As used in the specification, it is understood that the term "and / or" is used interchangeably. The simultaneous applicability of two or more of them is also included in the meaning, therefore the term "and / or" It is understood that the requirements are met.

[0084] Unless otherwise indicated, all numerical values, such as concentrations or concentration ranges, described herein are total values. In all cases, the above statements should be understood as being modified by the word "about." Thus, numerical values ​​typically include ±10% of the stated value. For example, 1 mg Concentrations of 1 mg / mL to 1.1 mg / mL are included. The concentration range of 0 mg / mL includes 0.9 mg / mL to 11 mg / mL. When using numerical ranges, unless the context clearly indicates otherwise, All possible subranges, including integers and fractions of values, and all individual numbers within the range are specified. Explicitly includes.

[0085] When used in reference to amino acid sequences, "percent (%) sequence identity" or "% identity" " or "% identical" refers to the number of amino acid residues that make up the entire length of the amino acid sequence. The number of identical amino acid matches in two or more aligned amino acid sequences ("Human Other terms using alignments of two or more sequences are provided. When the sequences are determined using sequence comparison algorithms known in the art, Compare and align for maximum agreement when aligned or manually and visually inspected. The percentage of amino acid residues that are the same when Over the course of 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, 99%, or 100% identity) can be determined. The resulting sequence may differ by amino acid substitutions, additions, or deletions. Suitable programs for aligning protein sequences are known to those skilled in the art. The percentage of identity can be calculated using, for example, CLUSTALW, Clustal Omega, F ASTA, or BLAST programs, for example, NCBI BLAST access The α-amino acid sequence can be determined using the α-amino acid sequence algorithm (Altschul SF, et al (1997) , Nucleic Acids Res. 25:3389-3402).

[0086] As used herein, a "non-naturally occurring" nucleic acid or polypeptide is one that does not occur in nature. "Non-naturally occurring" nucleic acids or polypeptides are those that have not been produced in the laboratory or and / or may be synthesized, processed, manufactured, and / or otherwise manipulated at the manufacturing site. For example, a non-naturally occurring nucleic acid or polypeptide may be a naturally occurring nucleic acid or polypeptide prior to treatment. Naturally occurring nucleic acids or polypeptides that have been treated, engineered, or manipulated to exhibit properties not present in As used herein, a "non-naturally occurring" nucleic acid or polypeptide may include The polypeptide may be a nucleic acid or polypeptide that is isolated or separated from the natural source in which it is found, It lacks covalent bonds to sequences associated with it in its natural source. The polypeptides may be produced recombinantly or through other methods, such as chemical synthesis.

[0087] As used herein, the term "hybrid" refers to any gene that is a member of the AAV cap gene. When used in combination, one serotype capsid in combination with a portion of a different serotype capsid is The term is also intended to mean the cap gene which contains a portion of the capsid. AAV ca, in which the raw AAV serotype sequence contains one or more non-naturally occurring mutations Contains p gene variants.

[0088] As used herein, the term "spacer sequence" refers to a sequence that serves to separate other genetic elements. In the present context, the term "coding sequence" is intended to mean a region of non-coding nucleotides that has no apparent function. .

[0089] As used herein, the term "operably linked" refers to a linkage or juxtaposition. and components so described are intended to function in their intended manner. For example, a promoter is a molecule that affects the transcription of a coding sequence. or a signal sequence operably linked to the amino acid sequence of interest is coupled to the membrane. If the target amino acid sequence can be secreted or transported, the coding sequence is operably linked to

[0090] To assist the reader of this application, the description is divided into various paragraphs or sections. These separations are not intended to be limiting or to the various embodiments of the present application. Separating a section or embodiment entity from another paragraph or section or embodiment entity On the contrary, those skilled in the art will appreciate that the disclosure herein is of broad application. The present invention includes all possible combinations of paragraphs, paragraphs, and sentences. It will be understood that the discussion of any embodiment is meant to be merely illustrative. It is to be understood that the scope of the present disclosure, including the claims, is not limited to these examples. For example, the uses described herein (e.g., in the manufacture of plasmids) are not intended to imply any An embodiment of a non-naturally occurring nucleic acid or recombinant vector (such as a recombinant DNA or viral vector) is Specific promoter sequences, enhancer or regulatory sequences, introns, arranged in a specific order Examples of the coding sequence include the AAV Rep and / or Cap coding sequence, and the polyadenylation signal sequence. Although specific components may be included, including but not limited to, those of skill in the art will appreciate the scope of the present disclosure. The concepts disclosed in other arrangements may be used in the nucleic acids or vectors of the present application. It will be understood that the present application is equally applicable to the components of the specific combinations. Whether or not explicitly stated, any of the following may be used in the nucleic acid or vector of the present application: Use of any of the applicable components in any combination with any sequence that can be used The intention is to

[0091] As used herein, a "vector" refers to a nucleic acid used to carry genetic material into a cell. It is an acid molecule that can be replicated and / or expressed therein. Any vector that is known to be useful in the present invention may be used. Examples of vectors include plasmids, viral vectors, and the like. vectors (bacteriophages, animal viruses, and plant viruses), cosmids, and artificial Preferably, the vector is a vector, including, but not limited to, a chromosome (e.g., a YAC). The vector is a DNA plasmid. Those of skill in the art, having regard to the present disclosure, will be able to make such vectors through standard recombinant techniques. The vector of the present application can be constructed in the above manner.

[0092] The vector of the present application may be an expression vector. The term "target" refers to any type of gene that contains a nucleic acid that encodes an RNA that is transcribable. The expression vector may be a DNA plasmid or a viral vector. Vectors for expressing recombinant proteins, and DNA plasmids or virus vectors Vectors for delivering nucleic acids into the body of a subject and expressing them in the tissues of the subject, such as vectors The expression vector design is based on the host cell to be transformed. It will be appreciated by those skilled in the art that the present invention may depend on factors such as the choice of cell, the level of expression of the desired protein, and the like. It will be understood that.

[0093] In some embodiments of the present application, the vector is a non-viral vector. Examples of vectors include DNA plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, and bacterial vectors. Preferably, the vector is a non-viral vector, but is not limited to a phage. The vector is a DNA plasmid. "DNA plasmid," which is used interchangeably with "plasmid DNA vector," is a It refers to a double-stranded and generally circular DNA sequence capable of autonomous replication in a suitable host cell. DNA plasmids used to express encoded polynucleotides are typically replicative. An origin, a multiple cloning site, and a selectable marker, which may be, for example, an antibiotic resistance gene. Examples of suitable DNA plasmids that can be used include those used in well-known expression systems. Commercially available expression vectors for the synthesis of ribozymes (including both prokaryotic and eukaryotic systems), e.g., E. It can be used for the production and / or expression of proteins in Scherichia coli. pSE420 (Invitrogen, San Diego, Calif.), yeast Used for production and / or expression in Saccharomyces cerevisiae strains pYES2 (Invitrogen, Thermo Fisher Scientific) may be used. ntific), MAXBAC®, which can be used for production and / or expression in insect cells; Target) Complete Baculovirus Expression System (Thermo Fisher Scientific ), which can be used for high-level constitutive protein expression in mammalian cells; ) or pcDNA3™ (Life Technologies, Thermo Fisher Scientific) isher Scientific), as well as most target proteins in mammalian cells. pVAX or pVAX-1 (Life Transfection) can be used for high-level transient expression of proteins. echnologies, Thermo Fisher Scientific The backbone of any commercially available DNA plasmid can be expressed in a host cell, but is not limited to the above. For example, routine techniques and readily available starting materials may be used to optimize protein expression. By using substances, it is possible to selectively modify specific elements (e.g., origin of replication and / or antibiotic resistance). The plasmid is then inserted with an endogenous promoter (e.g., the antisense cassette) to reverse the orientation of the The promoter in the biosensor cassette is replaced and / or the transcribed protein is A polynucleotide sequence encoding a protein (e.g., a coding sequence for an antibiotic resistance gene) (See, e.g., Sambrook et al., Mol ecular Cloning a Laboratory Manual,Secon d Ed. Cold Spring Harbor Press (1989) sea ​​bream).

[0094] Preferably, the DNA plasmid is an expression vector suitable for protein expression in a mammalian host cell. Suitable expression vectors for protein expression in mammalian host cells include: pUC, pcDNATM, pcDNA3TM, pVAX, pVAX-1, ADVAX, N TC8454, etc. For example, the vector may be pUC Based on pUC57 containing the replication origin of and the ampicillin resistance gene (SEQ ID NO: 30). It can be expressed by the herpes virus thymidine kinase gene promoter (SEQ ID NO: 26), puromycin N-acetyltransferase coding region (SEQ ID NO:27) , and a polyadenylation signal from the bovine growth hormone gene (SEQ ID NO:28) The vector may further comprise a mammalian puromycin resistance gene cassette. -Also, the Epstein Barr Virus (EBV) OriP origin of replication The fragment (SEQ ID NO: 29) can include the "bilateral symmetry" and "anti-antibody" regions of EBV. It represents a complex of "family of repeat sequences" regions.

[0095] The vector of the present application may also be a viral vector. Generally, a viral vector is , which has been rendered non-infectious but still contains the viral promoter and transgene; Thus, modified viruses that allow translation of transgenes through viral promoters. A viral vector is a genetically engineered virus that carries DNA or RNA. Because they often lack infectious sequences, they are often used as helper vectors for large-scale transfection. Examples of viral vectors that can be used include Adenovirus vectors, adeno-associated virus vectors, and poxvirus vectors -, enteric virus vector, Venezuelan equine encephalitis virus vector, Semliki Forest virus vectors, tobacco mosaic virus vectors, lentivirus vectors, etc. The vector can also be a non-viral vector, including, but not limited to, a viral vector.

[0096] Preferably, the viral vector is an adenoviral vector, e.g., a recombinant adenovirus. As used herein, a "recombinant adenovirus vector" is a viral vector. "combinant adenovirus vector" and "recombinant adenovirus vector" The terms "recombinant adenoviral vector" and "recombinant adenoviral particle" are used interchangeably. is used to insert a polynucleotide of interest into a eukaryotic cell, where the polynucleotide is then The term "virus of the present invention" refers to a genetically engineered adenovirus that is designed to express the Examples of adenoviruses that can be used as vectors include those of serotypes Ad2, Ad5, Adenovirus A, and Adenovirus B. d11, Ad12, Ad24, Ad26, Ad34, Ad35, Ad40, Ad48, A d49, Ad50, Ad52 (e.g., RhAd52), and Pan9 (AdC68) These vectors include those having or derived from the vectors known in the art. For example, humans, chimpanzees (e.g., ChAd1, ChAd3, ChAd7, ChAd8 , ChAd21, ChAd22, ChAd23, ChAd24, ChAd25, ChAd 26, ChAd27.1, ChAd28.1, ChAd29, ChAd30, ChAd3 1.1, ChAd32, ChAd33, ChAd34, ChAd35.1, ChAd36 , ChAd37.2, ChAd39, ChAd40.1, ChAd41.1, ChAd4 2.1, ChAd43, ChAd44, ChAd45, ChAd46, ChAd48, C hAd49, ChAd49, ChAd50, ChAd67, or SA7P), or Rhesus adenovirus (e.g., rhAd51, rhAd52, or rhAd53 For example, recombinant adenovirus vectors can be derived from human adenovirus (HA dV, or AdHu), or Simian adenovirus, e.g., chimpanzee or gorilla adenovirus (ChAd, AdCh, or SAdV), or rhesus macaque It may be derived from an adenovirus (rhAd).

[0097] Preferably, the adenoviral vector is a recombinant human adenoviral vector, e.g. , recombinant human adenovirus serotype 5, or recombinant human adenovirus serotype 26, 4, 35, 7, 48, etc. Recombinant viral vectors useful in the present application include It can be prepared using methods known in the art in light of the present disclosure. For example, Taking into account the degeneracy of the sequence, several nucleic acid sequences can be designed which encode the same polypeptide. A polynucleotide encoding a protein of interest is introduced into a host cell (e.g., a bacterial or mammalian host cell). Optionally, the gene may be codon-optimized to ensure proper expression in mammalian cells. Codon optimization is a technique that is widely applied in the art, and codon-optimized polypeptides are Methods for obtaining nucleotides will be known to those of skill in the art in light of the present disclosure.

[0098] The non-naturally occurring nucleic acid molecule or vector can contain one or more expression cassettes. An "expression cassette" is a nucleic acid that directs the cellular machinery to make RNA and proteins. An expression cassette is a molecule or part of a vector. 3' untranslated region (UTR), and optionally Contains a polyadenylation signal. Open reading frame An ORF (open reading frame) is a sequence that contains, from the start codon to the stop codon, the gene encoding the protein of interest (e.g., the gene encoding the protein of interest). Recombinase or recombination protein (Rep, Cap, recombinase, or recombination protein) coding sequence The regulatory elements of an expression cassette are the coding frame for the protein of interest. The polypeptide may be operably linked to a polynucleotide sequence comprising the polypeptide.

[0099] The non-naturally occurring nucleic acid molecules or vectors of the present application may contain a variety of regulatory sequences. As used herein, the term "regulatory sequence" refers to the administration of a nucleic acid or its derivative to a host cell or organism. Replication, duplication, transcription, splicing, translation, and stabilization of one of the conductors (i.e., mRNA) enable or contribute to the functional regulation of nucleic acid molecules, including their function, activity, and / or transport; Regulatory elements include promoters, enhancers, promoter sequences, and any other sequences that regulate the expression of a gene. Re-adenylation signal, translation stop codon, ribosome binding element, transcription terminator, selection These include, but are not limited to, markers, origins of replication, and the like.

[0100] The non-naturally occurring nucleic acid molecule or vector may be used to control the expression of a protein of interest. Preferably, the expression cassette may include a promoter sequence. The term "promoter" is used in its conventional sense to initiate transcription of an operably linked nucleotide sequence. A promoter is a nucleotide sequence located near the nucleotide sequence it transcribes. Promoters can be constitutive, inducible, or repressible. Promoters may be viral, bacterial, fungal, plant, insect, and The promoter may be derived from any source, including animals and mammals. a promoter derived from a different gene source) or a heterologous promoter (i.e., derived from a different vector or gene source). For example, when the vector used is a DNA plasmid, the promoter may be They may be endogenous to the plasmid (homologous) or may be derived from another source (heterologous). Alternatively, the promoter may be a promoter for a polynucleotide encoding a protein of interest within an expression cassette. It is located upstream of the Chid.

[0101] Exemplary promoters that can be used include Simian Virus 40 (SV40), Mouse Mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) Whole virus (human immunodeficiency virus, HIV) promoters, e.g., bovine immunodeficiency virus (BOV) promoters, The long terminal repeats of the whole virus (bovine immunodeficiency virus, BIV) inal repeat, LTR) promoter, Moloney virus promoter, Avian leukosis virus (avian leukosis virus, ALV) promoter, cytomegalovirus (CMV) promoter CMV immediate early promoter (CMV-IE), Epstein-Barr virus (EBV) promoter or Rous sarcoma virus (Rous sarcoma Promoters include, but are not limited to, promoters of the RSV virus. , human actin, human myosin, human hemoglobin, human muscle creatine, or human meta The promoter may be from a human gene such as rothionine. The promoter may be synthetic, tissue specific, such as a muscle or skin specific promoter. Alternatively, the promoter may be a cytomegalovirus (CMV) promoter (nt-672 to + 15), EF1-alpha promoter, herpes virus thymidine kinase gene promoter and strong eukaryotic promoters such as ribozyme T cell promoter (SEQ ID NO: 26).

[0102] The non-naturally occurring nucleic acid molecule or vector stabilizes the expressed transcript and inhibits the synthesis of the RNA transcript. Additional polynucleotides that enhance nuclear export and / or improve transcription-translation coupling Examples of such sequences include polyadenylation signals and enhancer sequences. Polyadenylation signals are typically included in the expression cassette of a vector. of the coding sequence for the protein of interest (e.g., Rep, Cap, recombinase) in Enhancer sequences are located downstream of the associated gene when bound by a transcription factor. Enhancer sequences are regulatory DNA sequences that enhance the transcription of a gene. Enhancer sequences are preferably located adjacent to the promoter sequence. It is downstream of the sequence and may be downstream or upstream of the coding sequence in the expression cassette of the vector.

[0103] Any polyadenylation signal known to those of skill in the art in light of the present disclosure may be used. For example, the polyadenylation signal may be the SV40 polyadenylation signal (e.g., SEQ ID NO:6). 0), AAV2 polyadenylation signal (bp 4411-4466, NC_001401 .2), the polyadenylation signal from the herpes simplex virus thymidine kinase gene ( SEQ ID NO: 23), LTR polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal adenylation signal, human growth hormone (hGH) polyadenylation signal, or human β-glucose Preferably, the polyadenylation signal is a robin polyadenylation signal. bGH polyadenylation signal (SEQ ID NO: 28), GenBank Accession No. Nucleotide number 441 of the nucleotide sequence of accession number NC_001401.2 AAV2 polyadenylation signal with 1-4466 or SV40 polyadenylation Signal (SEQ ID NO: 60).

[0104] Any enhancer sequence known to those of skill in the art in light of the present disclosure may be used. The Hansa sequence is derived from human actin, human myosin, human hemoglobin, human muscle creatine, or can be a viral enhancer, such as from CMV, HA, RSV, or EBV. Examples of specific enhancers include the Woodchuck HBV post-transcriptional regulatory element. HBV Post-transcriptional regulatory element (WPRE), human apolipoprotein Intron / exon sequences derived from the A1 precursor (apolipoprotein A1, ApoAI), Human T-cell leukemia virus type 1 The untranslated R-U5 domain of the long terminal repeat (LTR) of human HTLV-1 (human T-lymphotropic virus type 1) main, splicing enhancer, synthetic rabbit β-globin intron, or their Any combination may be used, but is not limited to these.

[0105] Preferably, the enhancer sequence comprises the P5 promoter of AAV. cis-acting Rep-dependent elements in the coding sequence of the rep gene CARE, when present in cis, is a part of the replication and capture CARE was also shown to enhance pups formation in several AAV-producing cell lines. It is important for amplification of the rep gene integrated into the chromosome, such as in AAV ITR (If there is no ). Without wishing to be bound by theory, The P5 promoter placed in the elution channel potentially increases Cap expression and therefore AAV yield. The P5 promoter acts as an enhancer for the expression of the chromosome 11. It is also contemplated that the gene may provide enhancer activity for amplifying the selected gene.

[0106] Non-naturally occurring nucleic acid molecules or vectors, such as DNA plasmids, can also be expressed in bacterial cells, e.g. For example, bacterial origins of replication and antibiotics for the selection and maintenance of plasmids in E. coli. The origin of replication (ORI) may contain a substance resistance expression cassette. This is the sequence at which replication begins and enables the plasmid to replicate and survive within the cell. Examples of ORIs suitable for use in the present application include ColE1, pMB1, pUC, pSC1 01, R6K, and 15A, preferably pUC. .

[0107] Vectors for selection and maintenance in bacterial cells typically contain antibiotic resistance genes. Preferably, the promoter sequence is operably linked to an antibiotic resistance gene. The operably linked promoter sequence regulates the length of the polynucleotide encoding the protein of interest. The antibiotic resistance gene is distinct from the promoter sequence operably linked to the codon The sequence composition of antibiotic resistance genes can be optimized, typically in bacteria, e.g., E. coli. Kanamycin resistance gene (Kan r), ampicillin resistance gene (Amp r ), and Tetra Tetracycline resistance gene (Tet r ), and chloramphenicol Resistance to cyclosporine, bleomycin, spectinomycin, carbenicillin, etc. These include, but are not limited to, genes that confer Any antibiotic resistance gene known in the art may be used.

[0108] Vectors for selection and maintenance in mammalian cells typically contain a selectable marker. The gene includes a promoter operably linked to a gene encoding a protein that confers Preferably, the gene further comprises a polyadenylation signal. The thromycin resistance gene cassette is driven by the herpes virus thymidine kinase gene promoter. (SEQ ID NO: 26), puromycin N-acetyltransferase coding region (sequence No. 27), and the polyadenylation signal from the bovine growth hormone gene (SEQ ID NO:28 ).

[0109] The production of recombinant AAV in human cells involves the synthesis of AAV replication (rep) and capsid (ca p) gene, adenoviral genes, and AAV inverted terminal repeats (ITRs) are flanked by The expression of an AAV-packageable transgene consisting of an expression cassette containing the Although all three components can be delivered to cells on separate plasmids for AAV production, Existing transfection methods are difficult to scale up to large-scale cultures. Incorporating some of these elements into a host cell line makes AAV production more efficient. However, some AAV and adenoviral genes are cytostatic. or cytotoxicity, limiting this approach.

[0110] This application relates to the AAV rep gene, the AAV cap gene, and packaging-competent The transgene may be maintained and / or integrated and propagated in a suitable host cell. As described above, a non-naturally occurring nucleic acid molecule, vector, is used to reversibly inactivate the AAV rep gene. The present invention describes a method for the production of a recombinant adenovirus expressing a recombinase. Infection of these cells with rep reactivates the rep gene, allowing AAV replication and packaging. The approach described by Xiao and coworkers (Qiao et al. l.(2002)J.Virol.76:13015-13027, Yuan et a l.(2011)Hum.GeneTher.22:613-624) (This is The tyrosine recombinase Cre, which recognizes the same loxP site in Unlike the conventional methods (which catalyze both the cleavage and excision reactions), the present invention is based on the inventors' approach A newly characterized serine recombinase, serine recombinase 21 (SR 21). Unlike Cre, SR21 uses attP and a Recognizes the ttB site. After the binding reaction catalyzed by SR21, attP and attB The sites are recombined and destroyed so that further recombination is not possible. The method of the present application may be more efficient than that catalyzed by Cre. The embodiment includes different artificial introns, enhancers, insulators, etc. These include additional features such as a stop cassette that allows for the The reversible inactivation / reactivation system of the present application improves the expression of AAV rep genes packaged in The effect of Rep on the function of the host cell is tightly regulated during cell development and thus the cellular response of Rep proteins to the host cell. This also allows the vector to avoid the cell growth inhibitory / cytotoxic effects. This results in strong induction of the AAV rep gene during production and high yields of AAV vector.

[0111] Serine recombinase Catalyzed by members of the large serine recombinase family (e.g., SR21) Site-specific recombination does not require the cellular machinery for homologous recombination. Amiviruses require a special recombinase to recognize the site and break and join the DNA. Based on amino acid sequence homology and mechanistic relatedness, most site-specific recombinases Two families: the tyrosine recombinase family and the serine recombinase family. Their name comes from the fact that they are used to attack DNA. from a conserved nucleophilic amino acid residue that binds to and becomes covalently bound to during strand exchange. do.

[0112] Serine recombinases bind to separate recombination recognition sites known as "attachment sites": attP a, "attached phage" and attB, "attached bacterial" chromosome, which binds and recombines with it. The ttP and attB sites are derived from dyad symmetry around a central dinucleotide recombination crossover site. The left and right halves of the attP and attB sites are zinc ribbons (ZDs). and the recombinase (RD) domain binds the recombinase monomer. (Rutherford et al. (2013) Nucleic Acid s Res.41:8341-8356).

[0113] As described in more detail in the Examples below, the term "serine recombinase" as used herein means a The serine recombinase referred to as "serine recombinase 21" or "SR21" is used in the present invention to A new SR2 gene was identified in the genome of Illus safensis strain CCMA-560. The attP and attB sites recognized by 1 were also characterized in the present invention. .

[0114] In one general aspect, the present application provides a method for identifying a nucleic acid sequence having at least 8 amino acids relative to the amino acid sequence of SEQ ID NO:2. 5%, e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% Nucleotide sequence encoding a serine recombinase having an amino acid sequence with identical identity Preferably, the non-naturally occurring nucleic acid molecule comprises the sequence The present invention relates to a method for the preparation of a serine recombinase having an amino acid sequence as set forth in claim 2. In one embodiment, the non-naturally occurring nucleic acid molecule has at least one amino acid sequence similar to the nucleotide sequence of SEQ ID NO:3. At least 85%, e.g., at least 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99%, or 100% identity.

[0115] In certain embodiments, the present application relates to vectors comprising non-naturally occurring nucleic acids. The present invention relates to a method for producing a serine recombinase in a cell of interest, such as a bacterial cell or a mammalian cell. In one embodiment, the vector is an expression vector that expresses the cytomegalovirus ( CMV) promoter or any other suitable promoter described herein or known in the art. The serine recombinase is expressed in mammalian cells under the control of a suitable promoter. In certain embodiments, the vector comprises a simian virus 40 (SV40) polyadenylation signal transduction vector. polyadenylation signals such as null or any of the polyadenylation signals described herein or known in the art. The gene may further include any other suitable polyadenylation signal.

[0116] In one embodiment, the vector is a plasmid P having the nucleotide sequence of SEQ ID NO:10. 175 and other DNA plasmids.

[0117] In another embodiment, the vector is a viral vector, such as a recombinant adenoviral vector. -It is.

[0118] In one embodiment, the vector has a sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:2. Identity, e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% Recombinant ΔE1 containing a nucleotide sequence encoding a serine recombinase having the same identity / ΔE3 adenovirus serotype 5 (Ad5) virus, the coding sequence of which is The vector is under the control of a promoter functional in cells. Preferably, the promoter is a CMV promoter. More preferably, the recombinant Ad5 vector comprises, in 5' to 3' order, SEQ ID NO: operably linked to a nucleotide sequence encoding the amino acid sequence of It contains the SV40 polyadenylation signal (NC_001669.1, nt2 In one embodiment, the amino acid sequence of SEQ ID NO:2 is operably linked to the amino acid sequence of SEQ ID NO:550-2774. The nucleotide sequence encoding the amino acid sequence is the bacterial translation initiation codon "TTG" changed to "ATG". and three point mutations replace the restriction endonuclease recognition site in SEQ ID NO:3. This is the same as SEQ ID NO:3, except that these restriction ends were introduced to disrupt The nuclease recognition sites are Xba I site (TCTAGA), Sac I site (GAGC TC), and an EcoRI site (GAATTC).

[0119] The serine recombinant protein of the present application may be synthesized using any method known in the art in light of this disclosure. A vector can be made that encodes the binase.

[0120] As described in more detail in the Examples below, the att In a particular embodiment, the seryl nucleotide sequence of the present application is The recombinase is an attP site comprising the nucleotide sequence of SEQ ID NO:7 or a barrier site thereof. In certain embodiments, the serine recombinase of the present application comprises at least one of SEQ ID NO:7. At least 90%, for example, at least 91%, 92%, 93%, 94%, 95%, 96%, The attP sites containing 97%, 98%, 99%, or 100% identical nucleotide sequences were identified. Recognize.

[0121] In certain embodiments, the serine recombinase of the present application is SEQ ID NO: 8 or SEQ ID NO: 9, or a variant thereof. The serine recombinase of the present application has a sequence similar to SEQ ID NO: 8 or SEQ ID NO: 9 at least 90% , e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% %, 99%, or 100% identical nucleotide sequence to attB sites.

[0122] In one embodiment, the present application relates to a method for performing site-specific recombination in a cell. The law is 1) an attP site having a nucleotide sequence that is at least 90% identical to SEQ ID NO:7, and and an att sequence having at least 90% identity to SEQ ID NO:8 or SEQ ID NO:9 obtaining a cell containing a nucleic acid molecule having a B site; 2) at least 85% relative to SEQ ID NO:2, e.g., at least 85%, 86%, 87% %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% %, 98%, 99%, or 100% identity to introducing a non-naturally occurring nucleic acid molecule into a cell; 3) Serine recombinase mediates site-specific recombination between the attP and attB sites. and growing the cells under conditions that allow the cells to catalyze

[0123] In a preferred embodiment, the present application relates to a method for performing site-specific recombination in a cell. The method comprises: 1) an attP site having the nucleotide sequence of SEQ ID NO:7, and SEQ ID NO:8 or and obtaining a cell containing a nucleic acid molecule having an attB site having the nucleotide sequence of SEQ ID NO:9. and, 2) a non-naturally occurring nucleic acid encoding a serine recombinase having the amino acid sequence of SEQ ID NO:2 introducing the nucleic acid molecule of the present invention into a cell; 3) Serine recombinase mediates site-specific recombination between the attP and attB sites. and growing the cells under conditions that allow the cells to catalyze

[0124] Constructs, cells, and methods for the production of recombinant AAV As shown in the following examples, the newly identified serine recombinase of the present application can be used to can be used to improve the production of recombinant AAV.

[0125] Modified AAV rep gene constructs In one general aspect, the present application relates to a modified adeno-associated virus (AAV) rep gene. A modified adeno-associated virus (AAV) rep gene is , which encodes the four Rep proteins Rep78, Rep68, Rep52, and Rep40. The AAV rep gene, which encodes the rep gene, and the rep gene shared by the four Rep proteins. and an artificial intron inserted into the coding sequence of the gene. It contains a stop cassette inserted downstream of the 5' splice site of the thrombin and upstream of the branch site. The stop cassette comprises, in 5' to 3' order, (i) at least 90% identical to SEQ ID NO: 7, e.g. If possible, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, (ii) attP sites with 9% or 100% identical nucleotide sequence; (iii) a terminator; and (iv) SEQ ID NO: 8 or SEQ ID NO: 9 and at least one At least 90%, for example, at least 91%, 92%, 93%, 94%, 95%, 96%, attB sites with 97%, 98%, 99%, or 100% identical nucleotide sequences Preferably, the attP site has the nucleotide sequence of SEQ ID NO: 7, The B site has the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9.

[0126] As used herein, an "intron" refers to a sequence that can be removed by RNA splicing. "RNA splicing" is broadly defined as the sequence of nucleotides that make up the mature RNA. As used herein, the term refers to the excision of introns from pre-mRNA to form pre-mRNA. As used herein, an "artificial intron" refers to an intron that is not a naturally occurring intron of a gene, but is It refers to a sequence of nucleotides that is nevertheless removable by RNA splicing. For example, an "artificial intron" is a naturally occurring intron with an inserted stop cassette. It could be.

[0127] Introns, including artificial introns, are 5' splice sites or junctions, splice accesses, It contains the septa or branch point, and the 3' splice site or splice junction. The term "splice site" or "5' splice junction" refers to a junction located within a gene or nucleic acid. The 5' end of the RNA fragment is located between the 3' end of the fragment and the 5' end of the intron. The exon-intron sequence includes a consensus sequence at the 5' end of the intron required for intron-binding The term "splice acceptor" or "branch point" refers to the location of a splice junction. helps form the lariat structure during the first transesterification reaction during NA splicing, A nucleotide located approximately 20-50 bp from the 3' splice site, usually an adenosine The term "3' splice site" or "3' splice junction" refers to a junction that is Between the 5' end of the 3' fragment of a gene or nucleic acid fragment and the 3' end of an intron, The exon also contains a consensus sequence at the 3' end of the intron that is required for splicing. The term "consensus sequence" refers to the position of an intron-intron junction in an RNA sequence. Within either the 5' or 3' splice junction required for splicing and / or The term refers to the nucleotides adjacent to it, and these sequences are usually either invariant or highly variable. or stored in

[0128] Analysis of a large number of mRNAs has revealed that certain nucleotides are typical of introns and splices. For example, the nearly invariant bases in the introns are conserved at the junctions of the The 5' and 3' conserved regions are 5'-GU and 3'-AG. Bases are often found with unusual (non-random) frequency. Branch point adenosine, usually , and 20–50 bases from the 3' splice site are also conserved. Gao et al. (2008) Nuc See Figure 4 in Leic Acids Research 36:2257-2267. The entire contents of Gao et al. (2008) are hereby incorporated by reference. However, within the introns, which can range in length from 40 to 50,000 bases, The central region is generally not necessary for splicing to occur. Exons are removed from the RNA or pre-mRNA as lariat structures by the endosome. Pricing proceeds via two successive transesterification reactions.

[0129] The insertion of an intron into an expression sequence can be accomplished by any method known in the art. The context of the flanking exons, as well as the actual intron sequence used, will result in a new "intron" sequence. This plays a role in whether or not a "trantron" is efficiently spliced ​​out. Introns suitable for the invention are sequenced in silico and have online splice predictions. Use a program that gives a high enough score for efficient RNA splicing It can be tested by finding combinations of rep gene sequences and intron sequences. Introns, either in genomic or synthetic sequences, may be used in the constructs of the present invention in light of this disclosure. It can be tested and optimized for use.

[0130] All four rep open links, Rep78, Rep68, Rep52, and Rep40 To disrupt the expression of the reading frame, the artificial intron preferably consists of four R It is inserted into the coding sequence of the rep gene shared by the ep protein. Thus, in a particular embodiment, in order to disrupt all four ORFs, the artificial intron is Nucleotides 996 and up to 1905 (NC_001401.2) of AAV2 or another A The stop cassette is inserted after the corresponding position in the AV rep gene. In order to function when inserted into an intron, the intron must be inserted as far upstream as possible. It is preferably inserted into the rep gene.

[0131] In addition, the context of the exons immediately upstream and downstream of the intron insertion site may be a useful indicator of possible insertion sites. For example, the general codons of introns relative to exons discussed above. It is important to define the consensus sequence. (wherein ^ indicates where the insertion is made) is the rep gene in AAV2 as follows: The numbers indicate the last nucleotide of the AAV before the insertion: 105 2, 1061, 1712, and 1906. In another embodiment, the consensus sequence AAG^ G, AAV2 positions 1022 (used by Qiao), 1112, 1475, Occurs in 1514, 1700, 1742, and 1784. Other consensuses such as AAG^A A preferred insertion site is also provided herein. In addition, other AAV rep genes may be identified in light of this disclosure.

[0132] Artificial introns useful in the present invention can be derived from any source, such as a genomic library. Introns are polymerized from human DNA using primers as described below. This can be obtained by polymerase chain reaction (PCR). Any intron capable of RNA splicing may be used in the methods of the present invention. In the examples below, the intron is an intron of the human β-actin gene.

[0133] According to an embodiment of the present application, in addition to RNA splicing via an artificial intron, Expression of the Rep protein was also mediated by DNA sequencing via a stop cassette inserted into an artificial intron. The stop cassette is characterized by the present invention. The attP and attB sites specifically recognized by serine recombinase are adjacent to each other. In one embodiment, the terminator comprises one or more adjacent transcription terminators. In another embodiment, the terminator comprises a polyadenylation signal, preferably A polyadenylation sequence encoding a self-cleaving RNA motif having the nucleotide sequence of SEQ ID NO:19. For efficient transcription termination, such as sequences from the human β-globin gene downstream of the transcription signal Other ribozymes, such as hammerhead ribozymes, which cleave their own RNA, Other ribozymes that replace the beta globin element may also be used in the present invention. For the use of the system, see West (2008) Molecular Cell 29:60. For a description of ribozyme design, see Kharma (2016) Nucl. See, eic Acids Res. 44:e39, the contents of both of which are incorporated by reference. No. 6,399,433, which are incorporated herein in their entireties.

[0134] In one embodiment, the termination cassette further comprises a gene encoding a selectable marker. In one embodiment, the selectable marker gene is a mammalian promoter (e.g., a mouse promoter phosphoglycerate kinase 1) and a bacterial (e.g., Lac zya) promoter, followed by Neomycin, driven by a polyadenylation signal such as that from SV40 It contains a phosphotransferase expression cassette (neo) (SEQ ID NO: 18). This gene were shown to be effective against neomycin and kanamycin in mammalian and bacterial cells, respectively. Without wishing to be bound by theory, it is believed that the development of cell lines In addition to providing a selectable marker, the selectable marker gene is This further blocks transcription of the target gene, thereby improving the stability of host cells containing the modified rep gene. Other selectable marker genes that can be used in the present invention include The genes include antibiotic selection genes (puromycin, hygromycin, bleomycin, synthase), metabolic genes (e.g., glutamine synthase or hypoxanthine-guanine synthase), Phosphoribosyltransferase (hypoxanthine-guanine phosphoribosyltransferase) , HPRT), visual markers such as mCherry, and beta-glucosidase. enzyme, secreted alkaline phosphatase, or any other suitable marker gene. But not limited to these.

[0135] In another embodiment, the stop cassette is spliced ​​from the primary mRNA transcript. The splice acceptor is included to prevent the splice from being cut out by the splice. Any naturally occurring splice acceptor site, provided that the acceptor is not skipped According to an embodiment of the present application, the splice acceptor is The consensus (yTnAynn), where y is C or T and n is any nucleoside. a branch point sequence that matches the nucleotide sequence of ... " dinucleotide, and 20-80bp of eukaryotic gene exon sequence (or intron The sequence contains a synthetic sequence that acts like an exon when placed next to the sequence. With a confidence score of 0.4 or higher, the sequences were predicted using the NetGene2 splice 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 Molecule (or a similar splice) (using sequence prediction software) should be recognized as a splice acceptor site. , a score closer to 1.0 is better. In one embodiment, the splice acceptor , the nucleotide sequence of SEQ ID NO: 17 ((NC_000086.7, mouse HPRT gene Nucleotides 53001998 to 53002138 from the human agouti signal transduction A 29-nt region from the nuclease protein (NC_000020.11, nucleotide 34262 765~34262793).

[0136] According to an embodiment of the present application, the stop cassette is a 5' splice donor of an artificial intron. It is inserted downstream of the splice acceptor "branch point" and upstream of the stop cassette. may be inserted anywhere between the two sites, provided that the insertion does not impair the function of the sites. In one embodiment, the stop cassette is inserted in the middle of the two sites. In one exemplary embodiment described in the Examples, the stop cassette comprises a 5' intron fragment located at 14, and the 3' intron fragment has the nucleotide sequence of SEQ ID NO:15. It has been inserted into an intron of the human β-actin gene so as to have a tid sequence.

[0137] As provided herein, in some embodiments, the 3' intron fragment comprises a RE Spacer sequences that make the P / CAP gene too large to be packaged into AAV For example, the AAV packaging limit is about 5.0 kb. Spacer sequences that extend the REP / CAP gene beyond about 5.0 kb are provided herein. In some embodiments, the spacer sequence may be generated in accordance with the present disclosure. A 2 kb random spacer is inserted into the intron fragment. In an exemplary embodiment described herein, the stop cassette comprises a 5' intron fragment having the sequence of SEQ ID NO: 14 and the 3' intron fragment has the nucleotide sequence of SEQ ID NO:66. It has been inserted into an intron of the human β-actin gene, as shown in FIG. The spacer sequence need not be 2 kb, but may be greater than about 5.0 kb of the REP / CAP gene. It is understood that the gene may be of any length that results in a gene.

[0138] Any AAV rep gene may be included in the modified rep gene of the present invention. The AV rep gene is one of the rep genes of AAV1 to AAV8, or its hybrid. The sequence of the AAV rep gene can be found, for example, in GenBank The following GenBank accessions for various AAV genes are available: With numbers: AAV1, GenBank accession number NC_002077.1; AAV2, GenBank accession number NC_001401.2; AAV3, Ge nBank accession number NC_001729.1; AAV4, GenBank accession number Session number NC_001829.1; AAV5, GenBank accession number NC_006152.1; AAV6, GenBank accession number AF02870 4.1; AAV7, GenBank accession number NC_006260.1; and A AV8, GenBank accession number NC_006261.1.

[0139] In the following examples, the nucleic acid sequence of GenBank Accession No. NC_001401.2 is The rep gene of human AAV2, containing nucleotides 190 to 2202 of the nucleotide sequence is used.

[0140] In some embodiments, modification of a cryptic splice site in the rep gene is This can be done to eliminate splicing at a site where the DNA sequence is mutated. However, synonymous mutations in the DNA sequence can be made, where the mutation does not change the encoded amino acid. .

[0141] Constructs with modified AAV rep and cap genes In another general aspect, the present application provides a modified AAV rep gene of the present application, and an AAV The present invention relates to a non-naturally occurring nucleic acid molecule comprising the cap gene, or a hybrid thereof. In this example, the AAV cap gene is downstream of the modified AAV rep gene.

[0142] In one embodiment, the AAV cap gene is operably linked to the coding sequence of a gene. In an exemplary embodiment described in the Examples below, the A The AV2 polyadenylation signal (bp 4411 to 4466, NC_001401.2) , contained downstream of the AAV9 cap coding sequence.

[0143] In another embodiment, the AAV cap gene further comprises an enhancer. AAV2 rep P5 promoter (bp 190 to 313, NC_001401.2 ) is contained downstream of the AAV2 polyadenylation signal.

[0144] In certain embodiments, the AAV cap gene comprises the capsid proteins VP1, VP2 , and VP3.

[0145] In other embodiments, the AAV cap gene encodes fewer than three capsid proteins. For example, AAV serotypes 1 to 5 can be successfully packaged in cells without VP2. It has been reported that the .alpha.- 10-kDa gene can replicate intracellularly and transduce cells (Grieger et al. al., J Virol. 2005 Aug;79(15):9933-9944). death Thus, in one embodiment, the AAV cap gene is any of AAV1 to AAV5, or a hybrid thereof encoding VP1 and VP3 but not VP2.

[0146] Any AAV cap gene can be used in the present invention. For example, the AAV cap gene The cap gene of one of AAV1 to AAV8, AAV9, and AAVDJ, or In one embodiment, the cap gene can be an AAV9 variant. The sequence of the AAV cap gene is available, for example, from GenBank. For AAV1 to AAV8 genomes, see GenBank accession numbers above. The AAV9 genome is available under GenBank accession number AY530579.1 and AAVDJ has GenBank protein accession number 3J1Q_A. do.

[0147] In one embodiment, described in the Examples below, GenBank Accession No. AY5 Human AAV9 cap open reading frame with nucleotide sequence 30579.1 A frame is used.

[0148] Constructs with modified AAV rep genes, AAV cap genes, and transgenes In another general aspect, the present application provides a modified AAV rep gene, an AAV ca p gene and a transgene flanked by AAV inverted terminal repeats (ITRs), Concerning developmental nucleic acid molecules.

[0149] The ITRs are important cis-acting sequences in AAV biology. The key role of the ITRs is to In addition to their role in AAV replication, the ITRs also play a role in AAV DNA replication. AV genome packaging, transcription, negative regulation under non-permissive conditions, and site-specific integration is essential for

[0150] In one embodiment, the 130 bp ITR is derived from the 3' AAV2 ITR as set forth in SEQ ID NO:2. The nucleotide sequence of 0 (nucleotides 4535 to 4664, NC_001401.2) In another embodiment, shorter mutated ITRs are used to flank the transgene. For example, for shorter genes, the ITRs are mutated to become shorter. The gene folds into a double-stranded form, increasing expression and enhancing expression after infection. McCarty 2008 Mol Ther.2008;16 See (10):1648-56.

[0151] In another embodiment, the transgene is a promoter, preferably one that functions in mammalian cells. In the examples described below, the human EF1-alpha promoter ( The transgene (SEQ ID NO: 21) contains intron 1, intron 1, and part of exon 2. Included in.

[0152] In another embodiment, the transgene comprises a polyadenylation signal. In the example, the polyadenylation signal from the herpes simplex virus thymidine kinase gene ( SEQ ID NO: 23) is included in the transgene.

[0153] In yet another embodiment, the non-naturally occurring nucleic acid molecule is a modified AAV rep gene, an AAV It contains a cap gene and a pair of insulators flanking the transgene flanked by ITRs. In another embodiment, the non-naturally occurring nucleic acid molecule is a modified AAV rep gene, an AAV It contains a cap gene and a single insulator upstream of the transgene flanked by ITRs. In one embodiment, the insulator is a genomic insulator that blocks chromatin-associated repression of gene expression. elements (Kwaks et al. (2003) Nature Biotechnology Kwaks et al. (2003) Nature Biotechnology 21:822).

[0154] Any suitable insulator, such as those described herein, may be used in the present invention. In one embodiment, the insulator comprises a human antibody having the nucleotide sequence of SEQ ID NO:24. In another embodiment, the insulator is a nucleic acid sequence of SEQ ID NO: 25. The mouse anti-repressor element 40 has a nucleic acid sequence. In terms of morphology, the insulator is identified by GenBank accession number AY190749.1 In another embodiment, the anti-repressor element 04 has the nucleotide sequence The nucleotide sequence of GenBank accession number AY190750.1 was used. In another embodiment, the insulator is an anti-repressor 06 having the structure Anti-repressor 0 having the nucleotide sequence of accession number AY190751.1 7. In another embodiment, the insulator is GenBank Accession No. A The anti-repressor 12 has the nucleotide sequence of Y190752.1. In this study, the insulator was identified as In another embodiment, the insulator is an anti-repressor 13 having the nucleotide sequence , having the nucleotide sequence of GenBank accession number AY190754.1 In another embodiment, the insulator is an anti-repressor 35. The anti-repressor 36 has the nucleotide sequence of session number AY190755.1. In another embodiment, the insulator is selected from the group consisting of the sequence represented by GenBank Accession No. AY190. 757.1. The insulator was synthesized using the nucleotide sequence of GenBank accession number AY190758.1. In another embodiment, the insulator is an anti-repressor 53 having a bifurcated globin having the nucleotide sequence of AY040835.1 in three or more copies The chicken HS4 insulator from the locus.

[0155] A non-naturally occurring nucleic acid molecule containing a pair of insulators is inserted into a gene segment of interest. Adjacent pairs may have the same or different insulators. Different insulators are used in pairs flanking the gene segment of interest. In one exemplary embodiment, described in the Examples below, human anti-repressor element 40 (AY1 90756.1, SEQ ID NO: 24) and mouse anti-repressor element 40 (SEQ ID NO: 25), In another exemplary embodiment described in the Examples below, The anti-repressor element 40 (AY190756.1, SEQ ID NO: 24) is an insulator and It is used as such.

[0156] As provided herein, the constructs of the present disclosure may also be used to mispair other vector components. To reduce the risk of packaging, spacer sequences are inserted on both sides of the AAV transgene. In one embodiment, the non-naturally occurring nucleic acid molecule can be located upstream and downstream of the transgene. In certain embodiments, the spacer sequence is b spacer sequence. In certain embodiments, the non-naturally occurring nucleic acid molecule is a modified AAV r The first insulator is located upstream of the ep gene, and the second insulator is located upstream and downstream of the transgene. The first insulator and the second spacer arrangement further include a first spacer arrangement and a second spacer arrangement. The strings are independently: (a) the nucleotide sequence of SEQ ID NO:67; and (b) the nucleotide sequence of SEQ ID NO:68. The sequence is selected from the group consisting of:

[0157] Cells and methods for the production of recombinant AAV Expression of the Rep protein from the modified AAV rep gene of the present application is is under strict control by both the transcription and RNA splicing machinery and therefore Stable host cells containing the modified rep gene are generated in a bioreactor and cultured in large numbers. For AAV production, a modified AAV rep gene, AA Stable host cells containing the V cap gene and a transgene flanked by ITRs are First, they grow to large numbers, and then the attP and attB genes in the modified AAV rep gene are The host is infected with a replication-deficient adenovirus that expresses a serine recombinase that recognizes the site. Site-specific recombination between attP and attB sites catalyzed by serine recombinase The targeted recombination site splices out the stop cassette and is replaced by a functional intron. This results in the production of a pre-mRNA that contains both separate 5' and 3' coding sequences. Introns are excised by a ubiquitous cellular machinery (the spliceosome) and are split into four Re p protein, allowing production of AVV at high titers.

[0158] A transgene flanked by a modified AAV rep gene, an AAV cap gene, and ITRs A stable host cell containing a gene is a cell that is transformed with one or more nucleic acid molecules encoding the gene. In one embodiment, the stable host cell can be obtained by transducing modified A Transducing cells with a first nucleic acid molecule encoding an AV rep gene and an AAV cap gene. and introducing a first host cell containing a modified AAV rep gene and an AAV cap gene. and transfecting the first host cell with a second nucleic acid molecule encoding a transgene flanked by ITRs. In one embodiment, the modified AAV rep gene is further transduced into the cells. The vector and the AAV cap gene are stably integrated into the chromosome of the first host cell. In embodiments, the modified AAV rep gene and the AAV cap gene are expressed in a first host cell. Transgenes flanked by ITRs also remain stable in host cells. The vector may be integrated intracellularly or may remain episomal.

[0159] In another embodiment, the stable host cell contains a modified AAV rep gene, an AAV cap Transducing cells with a nucleic acid molecule encoding a gene and a transgene flanked by ITRs. The modified AAV rep gene, AAV cap gene, and ITRs are obtained by The flanking transgenes may be stably integrated into the host cell or may remain episomal. It could be.

[0160] Stable host cells were transfected with adenovirus expressing the serine recombinase prior to infection. , and can grow to high cell densities.

[0161] In light of the present disclosure, one or more of the serine glycoproteins of the present application may be synthesized using any method known in the art. A replication-deficient adenovirus expressing the recombinant adenovirus is introduced into a stable host cell. In an embodiment, the replication-deficient adenovirus has an amino acid sequence of SEQ ID NO:2 and at least 85 % identical, preferably 100% identical to SEQ ID NO:2. A recombinant ΔE1 / ΔE3 adenovirus serotype 5 (Ad5) virus containing the .DELTA.E1 / .DELTA.E3 sequence. For example, the adenovirus has a sequence at least 85% identical to SEQ ID NO: 3, preferably SEQ ID NO: The nucleic acid sequence may comprise a nucleotide sequence which is at least 95% identical to SEQ ID NO:3.

[0162] As disclosed herein, the present disclosure also provides a method for treating a cell as described herein with 2 aminopurine. Methods and compositions for increasing AAV production by contact with (2-AP) In the later stages of the adenoviral life cycle, the virus binds to the host protein This is due in part to the inhibition of cap initiation complex eIF4F to Mnk1 kinase. , leading to dephosphorylation of eIF4E and inhibition of cap-dependent mRNA translation. It is caused by the action of late adenovirus 100-kilodalton (kDa) proteins (e.g. See, for example, Cuesta (2004), J. Virology 78:7707-7716. (See references below.) Adenovirus late gene transcripts are expressed in a manner known as ribosome shunting. They contain tripartite leader sequences at their 5' ends that facilitate translation by a specific mechanism (e.g., See, for example, Yueh (2000) Genes Dev 14:414-421. In the context of AAV-producing cell lines, inhibition of cap-dependent translation is mediated by AAV REP and CAP. Early adenoviral proteins required for gene expression, and AAV replication and packaging Thus, in some embodiments, the cells are To increase the efficiency of AAV-producing cell lines using virus inducers, host proteins They are incubated with a chemical that blocks translation arrest.

[0163] In certain embodiments, the chemical that blocks host protein translation termination is 2-aminopurine. 2-AP is a host protein induced by adenovirus. It has been shown that the cessation of synthesis of ribozymes is blocked by ribozymes (e.g., Zhang and Schnei der(1994) J. Virology 68:2544-2555, Huang a See Schneider (1990) PNAS 87:7115-7119. Treatment of AAV-producing cells with 2-AP inhibits the production of 2-AP, which is normally degraded by late infection. It is possible to reduce the cytopathic effects of infection, including restoration of the cytokeratin network. (Zhang and Schneider (1994) J. Virology 68:2544-2555). 2-AP inhibits the RNA-dependent protein kinase PKR ( karyotic translation initiation factor 2 alpha kinase 2 (eukaryotic translati on initiation factor 2 alpha kinase 2), E It inhibits a number of kinases in vitro, including IF2AK2 (also known as DeB enedetti(1983)J Biol Che,258:14556-14562 ) and phosphorylation of eIF-2α following intracellular PKR activation and adenovirus infection. It was not possible to block it (Huang and Schneider (1990) P NAS 87:7115-7119). 2-AP did not increase mRNA levels. Early adenovirus DNA-binding protein n, DBP) levels increased 10-20 fold (Huang and Schneider (1990)PNAS 87:7115-7119), and its effect on cap-dependent translation It matches.

[0164] Thus, in some embodiments, a method for producing a recombinant AAV comprising a transgene is provided. In some embodiments, the method comprises culturing a cell 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 about 10 mM to about 1.25 mM. In some embodiments, the 2-aminopurine concentration is about 1 μM to about 1.25 mM. In some embodiments, the 2-aminopurine concentration is It is about 1.25 mM.

[0165] In certain embodiments, the cells of the present disclosure are cultured within about 24 hours of infection with a recombinant adenovirus. In some embodiments, the cells of the present disclosure are subsequently contacted with 2-aminopurine. Approximately 20 hours after infection with the adenovirus, the subject is exposed to 2-aminopurine. In one embodiment, the cells of the present disclosure express 2-aminobutyric acid at approximately 12 hours after infection with the recombinant adenovirus. In some embodiments, the cells of the present disclosure are contacted with a recombinant adenovirus. In some embodiments, the contact with 2-aminopurine is about 30 hours after infection with the The disclosed cells were challenged with 2-aminopurine approximately 36 hours after infection with the recombinant adenovirus. In some embodiments, the cells of the present disclosure are infected with a recombinant adenovirus. Contact with 2-aminopurine approximately 48 hours later. EXAMPLES

[0166] The following examples of the present application are intended to further illustrate the principles of the present application. It will be appreciated that changes may be made to the embodiments described above without departing from the broad inventive concept. It will be understood, therefore, that the invention is not limited to the particular embodiments disclosed, but rather that the invention may be modified in a manner consistent with the teachings of this description. It is intended to cover modifications within the spirit and scope of the invention as defined by It is understood that.

[0167] material Cells: HEK293 cells (American Type Culture Colle ction (ATCC), Manassas, VA, catalog no. CRL-1573); PEAK-rapid (ATCC, Manassas, VA, catalog no. CRL282 8).

[0168] Tissue culture media and reagents: OptiMEM medium (Thermo-fisher, Walt Ham, MA; Catalog No. 31985-062; DMEM, high glucose (Ther mo-fisher, Catalog No. 10569-010); DMEM, Phenol Red None (Thermo-fisher; Catalog No. A14430-01); Hyclon eDialyzed fetal bovine serum (Thermo fisher; catalogue no. SH30079.03 ); 96-well TC plate (Corning, Corning NY; Catalog no. 3 596);6-well tissue culture plate, clear (Corning catalog no. 3516); Culture Plate 96, milky white (PerkinElmer, Waltham, MA; catalog No. 6005680); TrypLE Select Cell Dissociation Reagent (Thermo-fish r, Catalog No. 12563-011); Dulbecco's Phosphate Buffered Saline, Calcium None, no magnesium, D-PBS (Thermo-fisher, Cat. No. 14 190-144);Geneticin(G418)50mg / mL(Thermo-f isher, Catalog No. 10131-027);Streptomyces albo Puromycin dihydrochloride from niger (Sigma Aldrich P9620 ); T150 tissue culture flask 150 mm2 (Corning, Catalog No. CLS43 0825); GlutaMax 100x (Thermo-Fisher, catalog no. 35050-061); non-tissue culture treated 6-well culture plates (Corning, Catalog Hyperflask M vessel (Corning, Cat. No. 351146) 10030); 2.5% ClonaCell methylcellulose (L-glucose) in DMEM (containing glucose, sodium pyruvate, and sodium bicarbonate, without tamin) (StemCell Technologies, Vancouver, Britain Columbia, Canada, catalog number 03899-DI).

[0169] Transfection reagent: Fugene-HD transfection reagent (Promega) ega, Madison WI, Catalog No. E2311); Lipofectamin e 3000 transfection reagent (Thermo-fisher catalog number L3 000008); deoxynucleotides (Millipore-Sigma, St.Lo uis, MO, Catalog No. D7295-2ML).

[0170] Tubes: 15mL conical tubes (Corning, catalog number 430053) 1.5mL screw cap tube (Sarstedt AG & Co., KG ,Germany, catalog number 72.692.005).

[0171] Purification kit and assay reagents: Plasmid Spin Miniprep Kit ( Qiagen, Hilden, Germany, Catalog No. 27106);CHROM A SPIN™ + TE-1000 column (Takara Bio USA, Mou Intainview CA, Catalog No. 636079);Dual-Glo Luci Ferase Assay System (Promega, Madison WI, USA) Catalog number E2940; Silver Staining Kit (Thermo-fisher Catalog number 24 600);Trizol Plus RNA Purification with Phase-maker Tubes Kit (Thermo-fisher Catalog No. A33254); DNA-Free Kit (Thermo-Fisher Catalog No. AM1906); Nucleospi n Gel and PCR Cleanup Kit(Takara Bio USA , catalog number 740609.5).

[0172] Enzyme: Spe I-HF (New England Biolabs, Ipswich) ,MA, Catalog No. (R3133S); DNAse I Grade II from bovine pancreas ( Sigma-Aldrich, Catalog No. 10104159001);NEXT Ul tra II Q5 Master Mix (New England Biolabs , Catalog Number M05445S).

[0173] Buffers and Chemicals: CutSmart® Buffer (1x Buffer Components: 5 0 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 10 0 μg / mL BSA, pH 7.9, at 25°C) (New England Bio bs, Catalog No. B7204S); Benzonase Nuclease (Sigma a-Aldrich, Cat. No. E1014-25K); containing 1.5 mM MgCl2 10x GeneAmp PCR Buffer I (Thermo-fisher Catalog number N8080006; sodium deoxycholate (Sigma-Aldrich ich, Catalog No. D6750-25g; 1M TRIS-HCL PH8.5(T hermo-fisher, Catalog No. T1085); 10x GeneAmp PCR Buffer I (Thermo-Fisher catalog number N8080006) [1 00 mM Tris-HCl, pH 8.3 (at 25 °C); 500 mM KCl; 15 mM MgCl2; 0.01% gelatin in autoclaved, deionized, ultrafiltered water 10% Pluronic F-68 (Thermo-Fisher catalogue no. 24040-032); sheared salmon sperm DNA (10mg / mL) (Thermo-Fis her Catalog No. AM9680; Virus Dilution Buffer n Buffer, VDB) [1× GeneAmp PCR Buffer I, 2 μg / mL sheared salmon sperm DNA, and 0.05% Pluronic F-68); β-methyl amphotericin A (Sigma-Aldrich, Cat. No. M3148); Virus formulation buffer (10 mM Tris (pH 7.4), 1 mM MgCl, 75 mM NaCl, 5% sucrose, 0.02% polysorbate 80, 0.1 mM EDTA, 10mM histidine, 0.5% EtOH); 2-aminopurine, DMEM + 2% F Nitrate (Sigma-Aldrich, Cat. No. A2 380).

[0174] RT-PCR Reagent: SuperScript III First-Strand S synthesis system (Thermo-Fisher catalog number 18808 0-051);Q5 Hot Start High-Fidelity 2×Mast er Mix (New England Biolabs, catalog number M0494S) ; 1% TAE Mini ReadyAgarose Gel with Ethidium Bromide ( Bio-RAD, Catalog No. 1613016);Dark Reader Blue Light Transilluminator (Clare Chemicals, D olores, CO, Catalog Number DR46B)

[0175] Digital Droplet PCR: 2× SuperMix for Probes (Bio-Rad Catalog Number 186-3026;DG32 AutoDG Cartridges(B io-Rad Catalog No. 1864108);Auto Droplet Generator Tor Oil in PBS (Bio-Rad Catalog No. 1864110); -da oil (Bio-Rad catalog number 1863004); Eppendorf tw in.tec 96-Well PCR Plates (Cat. No. 95102034 6); Automated Droplet Generator (Bio-Rad Catalog) Log number 186-4101; QX200 Droplet Reader (Bio-R ad Catalog Number 186-4003);C1000Touch Thermal Cyc ler with Deep Well Reaction Module(Bio-R ad catalog number 185-1197).

[0176] PrimeTime qPCR Assays: 20x stocks of these assays are available Forward and reverse PCR primers (18 μM) and the fluorescence quenchers ZEN and Black Ho le Quencher 1 (3IABkFQ) and either FAM or HEX and a 5' nuclease probe (5 μM) containing a photoreporter dye. , Integrated DNA Technologies, Inc., Coralv Synthesized by ille IA. Primers and probes for qPCR assays The sequence is as follows: mCherry: primer 1 (SEQ ID NO: 36, 5'-CTGTTCCACGATGG TGTAGTC-3'); primer 2 (SEQ ID NO: 37, 5'-TGAGGTCAAGA CCACCTACA-3'; probe (SEQ ID NO: 38, 5'-FAM-TTGGACA TC-ZEN-ACCTCCCACAACGAG-3IABkFQ-3'); Adenovirus exon 2 (Ad5E2): Primer 1 (SEQ ID NO: 39, 5'-GG GTGATGCAGTAGAAGGTAAG-3'); primer 2 (SEQ ID NO: 40, 5 '-ATGAAGTTCGGCGGAGATG-3'; probe (SEQ ID NO: 41, 5' -HEX-TC TTGTTCC-Zen-CAGCGGTCCCATC-3IABkF Q-3'); P5 (P5 promoter region of AAV): Primer 1 (SEQ ID NO: 42, 5'-GTGG TCACGCTGGGTATTTA-3'); primer 2 (SEQ ID NO: 43, 5'-GG GACCTTAATCACAATCTCGT-3'; probe (SEQ ID NO: 44, 5'- FAM-TTTGAAGCG-ZEN-GGAGGTTTGAACGC-31ABkFQ -3'); AAV REP gene: primer 1 (SEQ ID NO: 45, 5'-GTCCGTGAGTG AAGCAGATATT-3'); primer 2 (SEQ ID NO: 46, 5'-TTCGATC AACTACGCAGACAG-3'; probe (SEQ ID NO: 47, 5'-FAM-TC TGATGCT-ZEN-GTTTCCCTGCAGACA-3IABkFQ-3'); AAV9 CAP gene: primer 1 (SEQ ID NO: 48, 5'-CCGGGTCCAA GGTATTTGTAA-3'); primer 2 (SEQ ID NO: 49, 5'-CTCAACC CAAGGCAAATCAAC-3'); probe (SEQ ID NO: 50, 5'-FAM-AC ATCAAGA-ZEN-CAACGCTCGAGGTCT-3IABkFQ-3'); and Beta-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').

[0177] PAGE electrophoresis: 4× NuPAGE LDS sample buffer (ThermoFisher r, Catalog No. NP0007); 4-12% Bis-Tris in 1x MOPS running buffer PAGE gel (Thermo-Fisher, Cat. No. NP0322PK2); 20 ×NuPAGE MOPS SDS Running Buffer(Thermo-F isher, catalog number NP0001).

[0178] AAV purification buffers and supplies: 0.2 μm PES membrane filters (ThermoFi sher catalog number 567-0020); POROS CaptureSelect 0.5 x 5 cm POROS GoPure chromatograph pre-packed with AAVX resin Ficollum (Thermo-fisher catalog number A36652); Amicon 15 100kDa MWCO Filter (Millipore-Sigma Catalog Product number UFC910024; CIM QA Disk 0.34mL volume (BIA S Eppendorf, Slovenia); Buffer A (20 mM Tris, pH 7.5 , 400 mM NaCl); buffer B (25 mM Tris, pH 7.5, 40 mM Na Cl, and 1.5 mM MgCl; buffer C (20 mM sodium citrate, pH 2.5, 400 mM NaCl); Buffer D (100 mM sodium citrate, 10 mM Buffer E (20 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-Tri Spropane pH 10.0, 0.001% Pluronic F68, 400mM Na Cl); Bis-tris propane (BTP) (Mil lipore Sigma catalog number B4679).

[0179] Other equipment: AKTA Explorer FPLC system (GE Healthcare re Life Sciences, Marlborough, MA);AKTA Pu rifier system (GE Healthcare Life Sciences); Envision Multi-Label Reader Model 2104 (PerkinElmer, Waltham, MA).

[0180] Identification and recombinant expression of SR21 recombinase As a query, the SPBeta c2 integrase protein (query, SEQ ID NO: 1) Using a BLAST search of the non-redundant protein database at NCBI with Serine recombinase (Sbjct, SEQ ID NO: 2) was identified by 64% sequence identity at the protein level. In the genome of Bacillus safensis strain CCMA-560, which has the same The putative serine recombinase or integrase was identified as a putative pro- This recombinase is named SR21 (serine recombinase 21). The DNA sequence encoding SR21 is shown in SEQ ID NO:3.

[0181] A bacterial strain closely related to CCMA-560 that does not contain a prophage insertion ("Fair The “view” strain was used as a query (SEQ ID NO: 58), which was the 3′ end of the recombinase coding region. Sequence data from NCBI using CCMA-560 DNA sequences from the end and beyond The putative promoter in CCMA-560 was identified by a BLAST search of the base (Figure 2). The DNA sequence of the Fairview strain corresponding to the upstream and downstream sequences of the phage insertion site is This sequence, referred to herein as the "pre-insertion sequence," is shown in SEQ ID NO: 4. This sequence (SEQ ID NO: 4) as a query to BLAST the genome sequence of strain CCMA-560. identified another prophage-host DNA junction 94 kb upstream. Right and left prophage-host DNA of Illus safensis strain CCMA-560 The sequences of the A junctions are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0182] The sequence upstream of the central identity region ("ACTGACAAAGCGGT") (SEQ ID NO:54) The att site boundaries, which were exchanged and maximized for the central dinucleotide and dyad symmetry, were By selecting attB-CCMA-560 (sequence number 8), SR21 The recombinase attP and attB sequences were inserted at the host DNA junctions (SEQ ID NO:5), respectively. ) and (SEQ ID NO: 6). Fair The attB sequence (SEQ ID NO:9) derived from the host DNA junction of the view strain (SEQ ID NO:4) , two mitogens were added to the reconstructed attB sequence from strain CCMA-560 (SEQ ID NO: 8). FIG. 3 shows the alignment of attP with these two alternating attB sequences. This shows the symmetry of the two rotations and emphasizes the position of the two rotations.

[0183] Measurement of recombinase activity in mammalian cells In mammalian cells, under the control of the CMV promoter followed by the SV40 polyadenylation signal To express the SR21 recombinase in ) was constructed by gene synthesis (GENEWIZ, Plainfield, NJ). The R21 recombinase open reading frame begins with the bacterial translation initiation codon "TTG". is replaced by "ATG" and three point mutations create restriction endonuclease recognition sites. Same as SEQ ID NO:3, except that it was introduced to disrupt the open reading frame. These changes in the coding frame are does not result in any change in

[0184] The recombinase reporter plasmid (P41) was also synthesized by Gene Synthesis (GENEWIZ, Plainfield, NJ) (SEQ ID NO:11; FIG. 4). Constitutively expressed green fluorescent protein (GFP) driven by the 1α promoter - Self-cleaving F2A-Renilla luciferase (rLUC) fusion protein It also encodes the SR21 gene in antisense orientation to the CMV promoter. The recombinase is flanked by attP (SEQ ID NO: 7) and attB (SEQ ID NO: 9) signals. P2A-activated mCherry-self-cleaving P2A-firefly luciferase rase, fLUC) reporter gene. SR21 recombinase encodes attP and Upon recombination of the attB signal, the coding region is inverted into the sense orientation, expressing mCherry -P2A-fLUC protein is expressed (see FIG. 4).

[0185] To measure SR21 recombinase activity in human cells, 75,000 HEK 293 cells were plated in 96-well plates in 100 μL of high glucose DMEM + 10% fetal bovine serum. The recombinase reporter plasmid (P041 ) ± SR21 recombinase expression plasmid (P175) + deoxynucleotides (DN A) were added to OptiMEM medium containing Fugene- Triplicate cells were seeded and complexed with HD transfection reagent for 15 min at room temperature. Plates were incubated at 37°C for 48 hours.

[0186] [Table 1]

[0187] Firefly luciferase (fLUC) and Renilla luciferase (rLUC) were Using the Dual Glo Assay Kit from Mega, transfected wells were The medium was then transferred to the transfected wells of the tissue culture plate. and 100 μL of DMEM medium (without phenol red) and Dual Glo A 1:1 mixture of luciferase + fLUC substrate was added. The plate was incubated at room temperature for 10 min. Lysates were transferred to opaque 96-well plates. fLUC activity was assayed using E Measured using an nvision multi-label reader. Then, 50 μL per well Add 100 ml of Stop-and-Glo buffer + Renilla substrate and gently shake the plate. The Renilla luciferase signal was measured using the same Envisi Read with on reader.

[0188] Results: The recombinase reporter was cotransfected with the recombinase expression plasmid. When co-transfected with deoxynucleotides, The renilla luciferase produced 1,535 times more firefly luciferase than the renilla luciferase (Table 2). The reporter enzyme (rLUC) activity was 5-fold higher in transfections of the reporter alone. Therefore, this data cannot be explained by different transfection efficiencies. 1 recombinase is highly active in human cells, and this result was confirmed in three independent experiments This demonstrates that

[0189] [Table 2]

[0190] Construction of REP / CAP+ transgene plasmid The AAV replication (REP) and capsid (CAP) genes are stably integrated into cells. in mammalian cells, where they can be subsequently induced to produce AAV in high-density cultures. Large-scale production of AAV may be possible. However, expression of the REP protein may result in toxicity. and may be a RE such as one expressing the adenovirus E1 gene, such as HEK293 cells. This makes it difficult to develop stable cell lines in hosts in which the P gene is expressed. AAV types contain overlapping genomes that arise from the use of two promoters and alternative splicing. The transcription factor α encodes four REP proteins in a transcriptional codon. The use of inducible promoters to control the expression of genes is not straightforward. Previous studies have used artificial introns. A "stop cassette" was inserted into the REP coding region in the HEK293 stable cell line. They demonstrated that it is possible to produce β-lactam esters in cells (Qiao et al. (200 2) J. Virol.76:13015, Yuan et al. (2011) Hum Gene Therapy. 22:613-624). Delivered by adenovirus infection. Excision of the stop cassette using Cre recombinase restores REP expression and inhibits the IT In this example, the ITRs initiated AAV replication of the transgene flanked by Rs. In association with the REP / CAP expression cassette in the plasmid containing the gene, the recombinase An improved version of the activated REP gene was constructed.

[0191] AAV2 REP gene (bp 190 to 2202 of human AAV2, NC_001401 .2), followed by the AAV9 CAP open reading frame (AY530579.1 ), AAV2 polyadenylation signal (bp 4411–4466, NC_001401. 2), and a second copy of the AAV2 REP P5 promoter (bp 190 to 313, N C_001401.2) was used to transcribe the AAV REP / CAP9 expression cassette (SEQ ID NO: 13) was constructed.

[0192] Splice site prediction software (www.cbs.dtu.dk / services NetGene2 in / NetGene2 / ; Brunak, S., Engelbrech t, J., and Knudsen, S.: Prediction of Human. mRNA Donor and Acceptor Sites from the D NA Sequence,Journal of Molecular Biology , 1991, 220, 49-65) was used to obtain an introductory fragment from the human β-actin gene. A suitable position was selected for inserting the gene into the REP coding region. In the common region, nucleotide number 105 of AAV2 (NC_001401.2) An intron was inserted downstream of 2. Both the intron and the insertion position were as described in Qiao et al. (2002) J. Virol. 76:13015) Then, the upstream and downstream halves of this β-actin intron (SEQ ID NO: 1, respectively) are A stop cassette (below) was inserted between numbers 14 and 15).

[0193] Stop Cassette The transcription termination cassette (SEQ ID NO: 16) was composed of the following elements: SR21 attP (SEQ ID NO: 7) Strong splice acceptor (SEQ ID NO: 17) (NC_000086.7, mouse HP Nucleotides 53001998 to 53002138 from the RT gene, and human agouti A 29-nt region from a signaling protein (NC_000020.11, nucleotide This is because the stop cassette is not part of the primary mRNA transcript. The nucleotide sequence was included to prevent excision by splicing. The neomycin phosphotransferase expression cassette (SEQ ID NO: 18) is Promoter (mouse phosphoglycerate kinase 1) and bacterial (Lac zya) promoter The gene was driven by the nucleotide sequence followed by a polyadenylation signal from SV40. , and the anti-neomycin and anti-kanamycin activities in mammalian and bacterial cells, respectively. It confers resistance to Polyadenylation encodes a self-cleaving RNA motif that is important for efficient transcription termination Sequences from the human β-globin gene downstream of the signal (Teixeira et al. (2004) Nature 432:526-30; sequence number 19). SR21 attB (SEQ ID NO: 8).

[0194] AAV transgene The transgene is flanked by AAV inverted terminal repeats (ITRs), The 130 bp ITR (SEQ ID NO: 12) was encoded in the P439 vector downstream of the region. Column number 20) indicates the 3' AAV2 ITR (nucleotides 4535 to 4664, NC_00 1401.2) and upstream and downstream of the HPRT-E2A-mCherry transgene. It was inserted in the reverse 3' orientation of the gene.

[0195] The transgene was driven by the human EF1-alpha promoter (exon 1, intron 1, and endon 2). (SEQ ID NO: 21), mCherry-self-splicing E2A Linker-human HPRT fusion gene coding sequence (SEQ ID NO: 22), and herpes simplex from the polyadenylation signal from the human thymidine kinase gene (SEQ ID NO: 23) It became.

[0196] Insulator REP / CAP and ITR-transgene elements mediate chromatin-associated repression of gene expression. Blocking genomic elements (Kwaks et al. (2003) Nature Biol. chnology 21:554-558, Kwaks et al. (2003)Na ture Biotechnology 21:822): Human anti-repressor element 40 ( AY190756.1, SEQ ID NO: 24) and mouse anti-repressor element 40 (SEQ ID NO: 25 ) was adjacent.

[0197] Plasmid backbone The plasmid backbone contains the following elements: Herpes virus thymidine kinase gene promoter (SEQ ID NO: 26), Puroma isin N-acetyltransferase coding region (SEQ ID NO:27), and bovine growth hormone A mammalian p53 construct was constructed from the polyadenylation signal from the mon gene (SEQ ID NO:28). Euromycin resistance gene cassette. Epstein Barr Virus (EBV) OriP replication origin fragment ( SEQ ID NO: 29) (which corresponds to the "dyad symmetry" and "family of repeats" regions of EBV) (representing a complex of regions). pUC57 vector sequence encoding the plasmid replication origin and ampicillin resistance gene String (SEQ ID NO:30).

[0198] The sequence of the complete plasmid P439 is shown in SEQ ID NO:12.

[0199] Testing efficiency of removal of stop cassettes by SR21 recombinase Whether the stop cassette can be precisely removed by SR21 recombinase in human cells To test whether the vector P439 (SEQ ID NO: 12) and SR21 recombinase The expression vector P175 (SEQ ID NO: 10) was transformed with Lipofectamine 3000. and co-transfected into PEAK-Rapid cells according to the manufacturer's instructions. in medium containing DMEM and 10% FBS for 3 days at 37°C in 5% CO The medium was removed, and the cells were washed once with D-PBS and then incubated at 37°C for 5 min. The cells were then transferred to a sterile microfuge tube and centrifuged. The bacteria were pelleted with PBS, washed once with 1 mL of D-PBS, and pelleted again. Qiagen Spin Miniprep Kit designed for isolating plasmids from Episomal plasmids were recovered by alkaline lysis using a PBS.

[0200] Aliquots of the recovered DNA were then used to destroy unmodified plasmid DNA. The cut was digested with the enzyme Spe I-HF in 1× CutSmart Buffer at 37°C. The DNA was digested at 37 °C for 1 hour and at 80 °C for 20 minutes. Using the conditions, NEXT Ultra II Q5 Master Mix was used to PC using primers P349F3 (SEQ ID NO: 32) and P349R9 (SEQ ID NO: 33) R was subjected to amplification: 98°C 1 min; 35x (98°C 10 sec, 72°C 10 sec); 5 min 72°C. When subjected to electrophoresis on a 100% agarose gel, a single PCR product of the predicted size was observed. Size-exclusion chromatography was performed using a CHROMA SPIN™ TE-1000 column. The PCR products were purified by chromatography. The same primers were used to determine the sequence (GeneWiz). The resulting sequence (SEQ ID NO: 34) ) is a stop cassette comprising the attP (SEQ ID NO: 7) and attB (SEQ ID NO: 8) sequences. SR21 recombination was performed by recombining the SR21 recombination sequence to produce the attL recombination sequence (SEQ ID NO: 35). It was demonstrated that the ribosome-specific cleavage site was accurately removed from plasmid P439 by the enzyme.

[0201] Construction of recombinant adenovirus serotype 5 expressing SR21 recombinase Recombinant ΔE1 / ΔE3 adenovirus serotype 5 (Ad5) viruses were generated using a recombinant vector lacking the E3 region. The modified cosmid (pWE / Ad5.AflII-rITRsp.ΔE3, published in U.S. Patent Application Regarding the production of E1 deleted vectors, except that the E1 deleted vector (No. 6340595(B1)) was used As previously described (Havenga et al. (2001) J. Vir ol 75:3335-3342), homologous recombination procedure in PER.C6 cells (Fal laux et al.(1998)Hum Gene Ther.9:1909-19 17) by Batavia Biosciences (Leiden, the Netherlands). This cosmid, as well as Ad5 sequences 1 to 454 (left) ITR and packaging signal), cytomegalovirus (CMV) promoter A cassette for transgene expression (nt -672 to +15), containing the SR21 recombinase nucleotide sequence, simian virus 40 (SV40) polyadenylation signal (NC_0 01669.1, nt2550-2774), and nt3511-6095 PER.C6 cells were transfected with plasmid P321 (SEQ ID NO: 31), which contains two Ad5 sequences. Co-expression of P321 Ad5 sequence (nt 3511-6095) in PER.C6 cells Homologous recombination between the cosmid pWE / Ad5.AflII-rITRsp.ΔE3 Producing recombinant adenovirus. Purified virus stocks were purified using a two-step CsCl gradient banding procedure. The isolated virus stock obtained by the above procedure was diluted with 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 The mixture was dialyzed against 100% EtOH.

[0202] Generation of stable cell lines Plasmid P439 (SEQ ID NO: 12) was transfected using Lipofectamine 3000. Transfect adherent PEAK-RAPID cells using the following kit according to the manufacturer's instructions: , 37℃, DMEM + 10% FBS + 0.05 mg / mL Genetic After 24 hours, the cells were treated with TrypLE and diluted with DMEM. +10% FBS + 0.05mg / mL Geneticin + 0.5μg / mL Purified water The cells were then transferred to a T75 flask containing 1:1 broth and 1:2 broth per flask containing 1:1 broth per flask. The cells were then re-cultured in the same medium every week for two consecutive weeks. Three weeks after transfection, the cells were resuspended in DMEM + 10% FB S + 0.05 mg / mL Geneticin + 5.0 μg / mL Puromycin The cells were split 1:10 weekly for 3 weeks into culture medium containing 100 mM MgCl2.

[0203] DMEM + 30% FBS + 1x GlutaMax + 5μg / mL puromycin + 1% ClonaCell Methyl Cellulose in 0.05mg / mL Geneticin Dilute the cells into a medium containing 100% ethanol, seed them into non-tissue culture treated 6-well plates and culture them for 3 weeks at 37 °C. Single cell clones were generated by mixing the clones with methylcellulose using a pipettor. From the monocellulose plate, DMEM + 10% FBS + 0.05 mg / mL Gene ticin + 5.0 μg / mL puromycin in a 96-well TC-treated plate Clones were grown in the same medium by standard methods.

[0204] Screening of clones To screen clones for AAV production, cells were cultured in 100 µL of DM Duplicate plates were seeded into 96-well plates in EM + 10% FBS and incubated overnight at 37°C. SR21 adenovirus was incubated in serum-free DMEM at 1E8 virus gel per mL. The medium from the plated cells was replaced with 100 μL of diluted adenovirus. The plates were incubated at 37°C for 4 days. 10 μL of the following mixture was added: Cells were lysed by: 5% deoxycholic acid in PBS + 10 units of 5% Benzonase. The plates were incubated at 37°C for 2 hours. The cells were centrifuged at 4 °C for 5 min at 200 rpm to pellet the cell debris, and the AAV virus in the supernatant was digitized. The results were quantified by digital droplet PCR (ddPCR).

[0205] Digital Droplet PCR (ddPCR) ddPCR quantification was performed according to Lock et al. (2014) Human Gene T Based on the method described by Herapy Methods 23:115-125 2 μL of the lysate was incubated in a 96-well plate in a thermal cycler for 1 h at 37°C. 2x PCR buffer + 20 mM Tris pH 8.5 + 8 units of DNAse I DNAse digestion was performed in 100 μL of reaction. 2 μL of DNAse digested sample was added to 98 μL of virus. Dilute this dilution in 1x PCR SuperM buffer (VDB) and add 2 µL of this dilution to the 1x PCR SuperM ix + 1 × PCR primer / probe for mCherry transgene (Materials section) The ddPCR droplets were added to a ddPCR reaction containing 100 μl of 10 ... The PCR cycle was as follows: There were: 95°C 10 min; 42x (94°C 30 sec, 60°C 1 min, 72°C 15 sec, all three Cycle time: 2°C per second; 98°C 10 min; 4°C hold. FAM fluorescence was measured using Bio-R ad droplet reader and detected according to the manufacturer's instructions. FAM-detected as fluorescent positive droplets. The clones that produced the highest DNAse-resistant particles as determined were expanded and further screened. It was served for ning.

[0206] Screening of clones – second assay 1.25E6 cells of the clone to be screened were cultured in 3 mL of DMEM + 10% F Cells were seeded into a single well of a 6-well plate in BS and incubated at 37° C. for 2 days. The growth medium was 3 mL of DMEM+10 containing 5E8 Ad5-SR21 viral particles. The plates were returned to 37°C and incubated for 3 days. Transfer the culture medium to a 15 mL tube and freeze-thaw it for three times (dry ice / 37°C). Incubation was then performed, followed by centrifugation at 3000 rpm for 5 min to pellet the cell debris. 2 μL of each sample was subjected to DNAse digestion using the mCherry assay described above. and ddPCR quantification. Most AAV was produced upon infection and was selected for further characterization (Table 3).

[0207] [Table 3]

[0208] Time lapse experiment To determine the kinetics of AAV production and secretion in culture medium at two different growth temperatures, A new experiment was performed. 2 mL of the non-enzymatic dissociation solution was added to P439-C in a T150 flask. 4 was added to the PBS-washed monolayer of cells and the flask was incubated at 37°C for 5 min. Wash the tube with 8 mL of DMEM + 10% FBS and transfer the cells to a 50 mL centrifuge tube. The cells were centrifuged at 1500 rpm for 5 min and the pellet was resuspended in DMEM + 2% FBS. The cells were then resuspended in 1 mL of the same medium. The cells were then diluted to 1.25E6 cells per mL. Cells were seeded into each well of four 6-well plates. 1 mL of DMEM + 2% FBS (2E8vp) of Ad5-CMV-SR21 adenovirus was added to the wells. Plates were incubated at 37°C and two plates were incubated at 32°C in 5% CO2. After each of the 8 days, cells and medium were harvested using a cell scraper. The adherent cells were removed by stirring and the sample was transferred to a 15 mL conical tube. Spin at 100 rpm for 5 min and aliquot into 1.5 mL screw-cap tubes. The plates were transferred and frozen at -20°C until ddPCR assay.

[0209] Samples were DNAse treated in duplicate as above and VDB was added to each DNAse treated sample. The samples were diluted in 1x PCR Master Mix + 1x mChe rry-FAM assay + 1x Ad5E2-HEX assay (see Materials section) The PCR was performed as described above. .

[0210] result: Adenovirus and AAV in cell culture medium increased over an 8-day time course (Table 4). Adenovirus replication was slower at 32°C, probably due to delayed adenovirus Cytopathic effects resulted in higher AAV production. AAV production at 32°C was , exceeding 14,000 genome copies per cell.

[0211] [Table 4]

[0212] Hyperflask cultures 8.3E07 P439C4 cells were cultured in 550 mL of DMEM + 10% FBS + 0.5 2 Hyper in 50.0 μg / mL puromycin + 50.0 μg / mL G418 The seeds were inoculated into flask M vessels and incubated at 37°C for 3 days. The cell density was estimated to be 3.6E8 cells per flask. 550 mL of DMEM+10 Dilute the virus in 100% FBS and replace the medium in the HyperFlask with the diluted virus. The flasks were then incubated at 40 MOI (1.4E10 vp) or 20 MOI (7 The cells were then infected with .2E09 vp. The cells were incubated at 32°C in 5% CO2 for 7 days. Supernatants were harvested 7 days post-infection and passed through a 0.2 μm PES membrane filter. It was purified by

[0213] AAVX purification 1mL total volume, mounted on an AKTA Explorer FPLC system Pre-filled with POROS CaptureSelect AAVX resin, 0.5× A 5 cm POROS GoPure chromatography column was run at a flow rate of 3 mL / min for 10 min. Column volume (CV) Buffer A (20 mM Tris, pH 7 The virus suspension was fed at 4.5 mL / min. The column was loaded at flow rate 10 mL, followed by 10 mL of buffer A to wash out unbound sample. low salt benzonase buffer, buffer B (25 mM Tris, pH 7.5, 40 mM Na The column was equilibrated with 250 units / mL of 1.5 mM NaCl, and 1.5 mM MgCl2. The column was packed with 15 mL of buffer B containing Benzonase. The column was then incubated at room temperature for 30 minutes and then incubated for 30 minutes. The virus was then washed for 15 CV with buffer A. The virus was then washed for 15 CV with buffer A (20 mM citric acid). The column was eluted in 0.5 mL fractions with 0.01% sodium chloride, pH 2.5, 400 mM NaCl. The single particles were immediately neutralized with 25 μL of 500 mM Bis-Tris propane, pH 10.0. All fractions under the curve were pooled and subjected to three rounds of buffer addition / centrifugation. Amicon 15 100kDa MWCO (catalog number UFC91 The mixture was concentrated using buffer D (100 mM sodium citrate, The buffer was exchanged into 10 mM Tris, pH 8.0. The affinity chromatography product was subjected to anion exchange chromatography to obtain the empty capsid. The AAV was further purified from the culture.

[0214] Anion Exchange Chromatography The affinity chromatography product (virus suspension) was diluted with Buffer E (20 mM BTP, pH 10.0, 0.001% Pluronic F68, 10 mM NaCl) Dilute to 5 mL and purify using the AKTA Purifier system (GE Healthcare Life Sciences) at a flow rate of 2 mL / min. The column was packed with 10 CV of sterile filtered Buffer E ( 20 mM BTP, pH 10.0, 0.001% Pluronic F68, 10 mM The virus was washed with 100% buffer E to 100% buffer F (20 mM NaCl). M Bis-Trispropane, pH 10.0, 0.001% Pluronic F68; The column was eluted with a 60 CV gradient of 400 mM NaCl and 0.5 mL fractions were collected. All fractions were pooled and purified by centrifugation at 2000 x g for 5 min using Amicon 1 5 100kDa MWCO (catalog number UFC910024, Fisher) was used. The mixture was concentrated and resuspended in buffer D (100 mM sodium citrate, 10 mM Tris, pH 8. The buffer was exchanged to 0).

[0215] Protein visualization 2 μL of concentrated eluate was transferred to NuPage L supplemented with 5% β-mercaptoethanol. Heat denaturation (10 min at 95°C) in DS sample buffer (4x) and denaturation in 1x MOPS running buffer The gels were electrophoresed on a 4-12% Bis-Tris PAGE gel according to the manufacturer's instructions. Therefore, it was subjected to silver staining.

[0216] ddPCR Virus concentrations were determined by digital droplet PCR using the mCherry assay described above. and measured.

[0217] result Infection and growth of P439C4 cells in Hyperflask vessels was performed at 20 and 40 M When infected with OI, the number of genome copies was 1.9E13 and 7.0E13, respectively. GC) for 20 and 40 MOI infections, respectively. The purity of the virus samples was confirmed by PAGE electrophoresis. The three AAV9 capsid isoforms (VP1, Only three bands corresponding to the expected sizes for VP1, VP2, and VP3 were visible. The capsid proteins (VP1 (87 kDa), VP2 (72 kDa), and and VP3 (62 kDa) were expressed as previously reported for other recombinant AAV vectors. They exist in an expected stoichiometry of about 1:1:10 (Daya and Berns (2008)Clin Microbiol Rev.21:583-593).

[0218] Measurement of levels of mispackaged DNA The sequences encoding the AAV REP or CAP genes and the plasmids used during production Prokaryotic sequences derived from the vector are non-specifically packaged into AAV particles and May pose potential safety risks when used in gene therapy (e.g., Sch nodt and Buning,Hum Gene Ther Methods.,2 017;28(3):101-108). The risk is This includes the generation of replication-competent AAV, capsid gene expression, and the induction of cytotoxic T lymphocyte responses. Triggering immune system recognition of prokaryotic sequences resulting in an inflammatory response and / or gene silencing The encapsidation rates were 2%, 0.4%-1.0%, and 1.3%-6.3%, respectively. The rep, cap and prokaryotic sequences are transfected by triple transfection or Purified recombinant AAV preparations produced from producer cell lines have been reported (Nony et al. (2003) J. Virology 77:776-781, Gao et al. (2008) Molecular Therapy 16:S105, Chaudeu f et al. (2005)Molecular therapy 12:744-7 53).

[0219] To determine the level of mispackaging associated with the above production systems, The presence of four sequences (outside the transgene flanked by ITRs) in the injected vector The amounts were determined by ddPCR: a) P5 promoter, b) AAV REP gene, c ) the AAV9 CAP gene, and d) the beta-lactamase (ampicillin resistance) gene. Purified virus preparations from the 20 and 40 MOI Hyperflask cultures described above were incubated for 3 h at 4 °C for 3 min. The clones were digested with DNAse in parallel, serially diluted in VDB, and subjected to ddPCR. The concentration of viral particles containing the mCherry transgene was calculated as the percentage of AAV particles containing the mCherry transgene. The highest encapsidation rate of 0.04% was observed in the A virus preparation. The P5 promoter in the preparation was from the P5 promoter in the recombinant adenovirus that was infected (20 M However, the AAV yield was much higher in preparation B (40 M The P5 encapsidation rate in the OI was only 0.007%. , and ampicillin gene sequences were equal to or less than 0.00. 7% to 0.009%, which is the percentage of recombinant hemophilia B gene therapy trials Previously reported results for four clinical lots of AAV2 ranged from 0.016% to 0.021%. capsid formation rate was lower than that of capsid formation rate of capsid 1 (Hauck et al. (2009) Mol Therefore, recombinant AAV The methods described herein for producing and purifying ribozymes are needed for clinical gene therapy programs. This results in a very low percentage of mispackaged DNA, consistent with what can be achieved.

[0220] [Table 5]

[0221] DNAse for the four probes compared to particles containing the mCherry transgene The mean percentage of resistant particles ± standard deviation for the analysis of two AAV vector preparations. Shown below.

[0222] RT-PCR analysis of RNA cysting of the REP gene after STOP cassette excision When the stop cassette was excised, the insert in the REP gene of construct P439 To determine whether the genes are correctly spliced, we performed RT-PCR experiments. Ta.

[0223] 10 million cells from a stable pool of P439 in PEAK-RAPID cells Pellet by centrifugation and resuspend in 15 mL of DMEM + 2% FBS + 1E9 Ad5- The cells were resuspended in CMV-SR21 viral particles. The cells were seeded into T75 flasks and incubated at 37°C. The cells were then incubated at 4°C for 48 hours. The cells were detached using a cell scraper. Transfer the cells to a 15 mL centrifuge tube and centrifuge at 1500 rpm for 10 minutes to separate the cells. Pelleted. Trizol Plus RN with Phase-maker tubes RNA was purified from cell pellets using a Purification Kit.

[0224] To remove any contaminating DNA, 31 μg of RNA was diluted with DNA in 1× digestion buffer. Treat with 1 μL of DNAse from the A-Free kit for 30 min at 37°C. 1 μL of DNAse inactivation slurry was added and the samples were incubated for 2 min. The RNA samples were centrifuged at 10,000 x g for 5 minutes and the RNA was transferred to fresh sterile Transferred to a tube.

[0225] RNA was synthesized using SuperScript III First Strand Synthesis The reverse transcription was performed using the esis system. 80 μg of RNA, 1 μL of 50 μM oligo-D Mix 1 μL of 10 mM dNTPS and 1 μL of 10 mM dNTPS in a sterile tube and incubate at 65° C. for 5 min. Incubate on ice for 2 minutes. Add 10 μL of 2× mixture (2× RT buffer, 10 mM MgCl2, 20 mM dithiothreitol, 0.5 μL RNAse-out, and 0 A mock PCR reaction was performed except that the reverse transcriptase was replaced with water. The reactions were identical. Reactions were incubated at 50°C for 50 min and on ice for 2 min. 1 μL of RNAse H was added and the samples were incubated at 37° C. for 20 minutes.

[0226] A 50 μL PCR reaction consisted of 1 μL reverse transcribed RNA, 25 μL Q5 Hot Star t High-Fidelity 2x Master Mix and 0.5μM The reactions were thermocycled as follows: 98°C 1 min; 35 cycles of (98°C 10 s, 69°C 10 s, 72°C 36 s); 7 cycles of 5 min 2°C.

[0227] 5 μL of the reaction was run on a 1% agarose gel in 1× TAE buffer and ethidium bromide. Bands were visualized under blue light illumination on a Dark Reader transluminator. The excised fragments were purified using Nucleospin gel and PCR clean-up kits. DNA was recovered from the band that was detected by cycle sequencing and dye-terminator amplification. Using the gene expression system, PCR primers were used to generate the The sequence was determined by the National Institute of Genomics (NIG) at 14:45–47.

[0228] result: PCR reactions from mock RT templates did not produce detectable products, indicating that genomic DNA A was eliminated from the RNA sample. Primer AAVRT-F1 (SEQ ID NO: PCR using P349R9 (SEQ ID NO: 62) and P349R9 (SEQ ID NO: 63) revealed that the stop cassette was P43 Two transcripts with similar fluorescence intensities were derived from the spliced ​​transcripts after excision from 9. PCR products were generated. One product contained an engineered beta-actin splice donor. Splices at the acceptor and acceptor sites (SEQ ID NO: 14 and SEQ ID NO: 15, respectively; FIG. 8). The second product was generated from lysis of the donor in the 5'REP gene (SEQ ID NO: 64). It results from splicing between this site and the downstream beta-actin acceptor (Figure 8). This splicing event removes 64 bp of the REP coding sequence relative to wild-type AAV2. This is predicted to create a frameshift and produce a truncated REP protein. By mutating the splice donor site upstream of These results suggest that AAV production can be made more efficient by increasing the expression of ribosomal protein.

[0229] Updated AAV construct: P600 (SEQ ID NO: 70) Several modifications were made to plasmid P439 (SEQ ID NO: 12) to create construct P600 (SEQ ID NO: 13). First, the splice donor site of the REP gene upstream of the stop cassette was obtained. Briefly, the splice identified in the 5'REP sequence (SEQ ID NO:64) were mutated. The nucleotides GT at the donor site were mutated to AT (SEQ ID NO: 65). To eliminate splicing at this site without altering the REP protein sequence, It is predicted that this will happen.

[0230] The REP / CAP gene is packaged into the AAV capsid after excision of the stop cassette. In order to reduce the possibility of this happening, a 2 kb random sequence (SEQ ID NO: 66) was designed and The engineered insert was inserted downstream of the tB sequence and upstream of the actin splice acceptor. The size of the gene was increased. Potential splice sites were identified using the NetGene 2 software. The insertion was identified and removed using the method described above. The size of the REP / CAP gene was increased, which resulted in a 5.0 kb AAV packaging region. Well above the limit.

[0231] Sequences flanking the AAV ITRs can also be amplified during transgene rescue from the genome. Based on the hypothesis that , , can be mispackaged into the AAV capsid (e.g., Sc hnodt and Buning,Hum Gene Ther Methods. 2017;28(3):101-108), two random 2kb non-codons The spacer elements flank the transgene to prevent potential mispackaged DNA. One element (SEQ ID NO: 67) was inserted into the left AAV I A second (SEQ ID NO: 68) replaced mouse anti-repressor element 4 was inserted upstream of TR. 0 (SEQ ID NO:25) was inserted downstream of the right AAV ITR.

[0232] In addition, the cap gene was an AAV9 variant (e.g., Hinderer et al.,Hum Gene Ther.2018;29(3):285-298 Please refer to.

[0233] Finally, the coding sequence of the transgene flanked by the ITRs of P439 was transfected with mCherry-I SEQ ID NO:69, which encodes the RES-SEAP (secreted alkaline phosphatase) protein was replaced by.

[0234] The complete sequence of the resulting construct, P600, is disclosed in SEQ ID NO: 70 and an exemplary plasmid is shown in FIG. Shown in Figure 9.

[0235] AAV production from P600 in a stable pool Construct P600 was transfected into Peak-RAPID cells essentially as described above. As described for P439 cells, 0.5 μg / mL puromycin was used. Through selection, stable pools were generated. Cells were cultured for 6 weeks before assaying for AAV production. The cells were passaged at a ratio of 1:10.

[0236] 2.5E6 P600-PEAK-Rapid p6 cells were cultured in 5 mL of DMEM+10 Three T25 flasks were seeded in 10% FBS and incubated at 37° C. for 3 days. The cell density on the day of staining was determined by harvesting the cells with TrypLE and counting using trypan blue exclusion. In one of the flasks, 6.6E6 viable cells were determined. The medium in the other flask was then replated with 2.6E8 Ad5-CMV-SR21 viral particles containing The flasks were then incubated at 32°C for 24 hours. 1 mL of 1.25 mM 2-aminopurine in DMEM + 2% FBS was added to 1 mL of DMEM + 2% FBS was added to one flask. 1 mL of DMEM + 2% FBS was added to the other flask. Both flasks were incubated for an additional 7 days at 32° C. The medium was harvested from the flasks and Cells and debris were pelleted by centrifugation at 3,000 rpm for 5 minutes. The supernatant was subjected to DNAse digestion and ddPCR quantification using the mCherry assay described above. It was used for transformation.

[0237] AAV production levels are shown in Table 6. The stable pool of P600 was Ad5-CMV-SR 21 is active in producing AAV upon infection. AAV virus production is CAP-dependent. 2-aminobutyric acid, a drug reported to block adenovirus-induced inhibition of dependent mRNA translation It increased 2.5-fold in the presence of aminopurine (e.g., Zhang and Schnei der (1994) J. Virology 68:2544-2555, and Huang See Schneider and Schneider (1990) PNAS 87:7115-7119. Treatment with 10 mM 2-AP 1-2 hours after infection significantly improved the survival of cells infected with adenovirus. It has been reported that it blocks the cell degenerative effect and is non-toxic for at least 3 days (e.g. Zhang and Schneider (1994) J. Virology 68 :2544-2555, and Huang and Schneider (1990) PN AS 87:7115-7119), and in our AAV-producing cell line, Because it is inhibitory to AV production, it is These data suggest that the late adenovirus gene program, especially ca Inhibiting p-dependent mRNA translation arrest increases AAV production in producer cell lines These results suggest that this may be a useful strategy to

[0238] [Table 6]

[0239] Production of recombinant adeno-associated virus (AAV) in human cells is based on AAV replication (REP) ) and capsid (CAP) genes, adenovirus genes, and AAV inverted terminal repeats AAV-packageable transgene consisting of an expression cassette flanked by sequences (ITRs) All three components are required for expression on separate plasmids for AAV production. Although it is possible to deliver the nucleotides to cells, existing transfection methods are not feasible for scaling up to large-scale cultures. It is difficult to integrate some of these elements into a host cell line. Although production can be made more efficient, some of the AAV and adenovirus genes Some of these are cytostatic or cytotoxic, limiting this approach. The AAV REP, CAP, and packageable transgene are then incorporated into a suitable host cell. A method to reversibly inactivate the AAV REP gene so that the vector can be transfected and propagated. The formula is as follows: A replication-deficient recombinant adenovirus expressing a recombinase (e.g., Δ Infection of these cells with ΔE1 / ΔE3 reactivates the REP gene, leading to AAV replication and and induces packaging. The inventions described in the original claims of this application are listed below. [Invention 1] 1. A non-naturally occurring nucleic acid molecule comprising a modified adeno-associated virus (AAV) rep gene, the 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 the four Rep proteins, the artificial intron comprising a stop cassette inserted downstream of the 5' splice site and upstream of the branch site of the artificial intron, the stop cassette comprising, in 5' to 3' order: (a) an attP site having a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical 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) a non-naturally occurring nucleic acid molecule comprising an attB site having a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical 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. [Invention 2] The non-naturally occurring nucleic acid molecule according to claim 1, wherein the splice acceptor comprises the nucleotide sequence of SEQ ID NO:17. [Invention 3] 3. The non-naturally occurring nucleic acid molecule according to claim 1 or 2, wherein the terminator comprises a polyadenylation signal. [Invention 4] 4. The non-naturally occurring nucleic acid molecule according to claim 3, wherein the terminator further comprises the nucleotide sequence of SEQ ID NO:19. [Invention 5] 5. The non-naturally occurring nucleic acid molecule according to any one of claims 1 to 4, wherein said termination cassette comprises a gene encoding a selectable marker, preferably a neomycin phosphotransferase expression cassette having the nucleotide sequence of SEQ ID NO: 18. [Invention 6] 6. The non-naturally occurring nucleic acid molecule according to any one of claims 1 to 5, wherein the artificial intron comprises, in 5' to 3' order, the nucleotide sequence of SEQ ID NO: 14, the stop cassette, and the nucleotide sequence of SEQ ID NO: 15. [Invention 7] 7. The non-naturally occurring nucleic acid molecule according to any one of claims 1 to 6, wherein the AAV rep gene comprises one of the rep genes of AAV1 to AAV8, or a hybrid thereof. [Invention 8] 8. The non-naturally occurring nucleic acid molecule according to claim 7, wherein the AAV rep gene comprises the human AAV2 rep gene having nucleotide numbers 190 to 2202 of the nucleotide sequence of GenBank Accession No. NC_001401.2. [Invention 9] 9. The non-naturally occurring nucleic acid molecule according to claim 8, wherein the artificial intron is inserted between nucleotide numbers 996 and 1905 of the nucleotide sequence of GenBank Accession No. NC_001401.2. [Invention 10] 10. The non-naturally occurring nucleic acid molecule according to claim 9, wherein the artificial intron is inserted immediately downstream of nucleotide number 1052, 1061, 1712, 1906, 1022, 1112, 1475, 1514, 1700, 1742, 1784, or 1340, preferably immediately downstream of nucleotide number 1052, of the nucleotide sequence of GenBank Accession No. NC_001401.2. [Invention 11] 1. A non-naturally occurring nucleic acid molecule comprising a modified AAV rep gene, said 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, in the 5' to 3' order: (i) a 5' intron fragment having the nucleotide sequence of SEQ ID NO: 14; (ii) a stop 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) a stop cassette comprising an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9; and (iii) an artificial intron comprising a 3' intron fragment having the nucleotide sequence of SEQ ID NO: 15; (c) a 3' portion of the AAV rep gene having the nucleotide sequence of SEQ ID NO:56. [Invention 12] 1. A non-naturally occurring nucleic acid molecule comprising a modified AAV rep gene, said 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, in the 5' to 3' order: (i) a 5' intron fragment having the nucleotide sequence of SEQ ID NO: 14; (ii) a stop 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) a stop cassette comprising 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. [Invention 13] 13. The non-naturally occurring nucleic acid molecule according to claim 11 or 12, wherein the termination cassette comprises the nucleotide sequence of SEQ ID NO:16. [Invention 14] 14. The non-naturally occurring nucleic acid molecule according to any one of claims 1 to 13, further comprising an AAV cap gene encoding the three capsid proteins VP1, VP2, and VP3. [Invention 15] 15. The non-naturally occurring nucleic acid molecule according to claim 14, wherein the AAV cap gene comprises one of the cap genes of AAV1 to AAV9 and AAVDJ, or a hybrid thereof. [Invention 16] 16. The non-naturally occurring nucleic acid molecule of claim 15, wherein the AAV cap gene comprises the human AAV9 cap gene having the nucleotide sequence of GenBank Accession No. AY530579.1. [Invention 17] 17. The non-naturally occurring nucleic acid molecule according to any one of inventions 14 to 16, wherein the AAV cap gene further comprises a polyadenylation signal, preferably an AAV2 polyadenylation signal having nucleotide numbers 4411 to 4466 of the nucleotide sequence of GenBank Accession No. NC_001401.2, and an enhancer, preferably an AAV2 rep P5 promoter having nucleotide numbers 190 to 313 of the nucleotide sequence of GenBank Accession No. NC_001401.2, wherein said polyadenylation signal and said enhancer are both downstream of the coding sequence of the cap gene. [Invention 18] 18. The non-naturally occurring nucleic acid molecule according to any one of Inventions 14 to 17, further comprising a transgene flanked by a pair of AAV inverted terminal repeats (ITRs) downstream of the AAV cap gene. [Invention 19] a first insulator upstream of the modified AAV rep gene, and optionally a second insulator downstream of the transgene flanked by the ITRs, preferably wherein the first insulator and the second insulator are independently: (a) a human anti-repressor element 40 having the nucleotide sequence of SEQ ID NO: 24; (b) a mouse anti-repressor element 40 having the nucleotide sequence of SEQ ID NO: 25; (c) anti-repressor element 04 having the nucleotide sequence of GenBank accession number AY190749.1; (d) anti-repressor element 06 having the nucleotide sequence of GenBank accession number AY190750.1; (e) anti-repressor element 07 having the nucleotide sequence of GenBank accession number AY190751.1; (f) anti-repressor element 12 having the nucleotide sequence of GenBank accession number AY190752.1; (g) anti-repressor element 13 having the nucleotide sequence of GenBank accession number AY190753.1; (h) anti-repressor element 35 having the nucleotide sequence of GenBank accession number AY190754.1; (i) anti-repressor element 36 having the nucleotide sequence of GenBank accession number AY190755.1; (j) anti-repressor element 52 having the nucleotide sequence of GenBank accession number AY190757.1; (k) anti-repressor element 53 having the nucleotide sequence of GenBank accession number AY190758.1, and (l) a chicken HS4 insulator from the globin locus having the nucleotide sequence of AY040835.1 in two or more copies; More preferably, the non-naturally occurring nucleic acid molecule according to invention 18, wherein said first insulator and said second insulator have the nucleotide sequences of SEQ ID NO: 24 and SEQ ID NO: 25, respectively. [Invention 20] the non-naturally occurring nucleic acid molecule comprises the first insulator upstream of the modified AAV rep gene and further comprises a first spacer sequence and a second spacer sequence upstream and downstream, respectively, of the transgene, the first spacer sequence and the second spacer sequence being independently a) the nucleotide sequence of SEQ ID NO: 67, and b) A non-naturally occurring nucleic acid molecule according to invention 19, selected from the group consisting of the nucleotide sequence of SEQ ID NO: 68. [Invention 21] 21. The non-naturally occurring nucleic acid molecule according to any one of inventions 18 to 20, wherein 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, preferably wherein the promoter has the nucleotide sequence of SEQ ID NO: 21 and the polyadenylation signal has the nucleotide sequence of SEQ ID NO: 23. [Invention 22] A non-naturally occurring nucleic acid molecule comprising, in 5' to 3' order: (A) a first insulator, preferably having a nucleotide sequence of SEQ ID NO: 24; (B) a modified AAV rep gene, comprising, in 5' to 3' order: (i) a 5' portion of the AAV rep gene, preferably having the nucleotide sequence of SEQ ID NO: 55; (ii) an artificial intron, in the 5' to 3' order: (a) a 5' intron fragment, preferably having the nucleotide sequence of SEQ ID NO: 14; (b) a stop cassette comprising, in 5' to 3' order: (1) an attP site having the nucleotide sequence of SEQ ID NO:7; (2) a splice acceptor, preferably having 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 having the nucleotide sequence of SEQ ID NO: 19; and (5) a stop cassette comprising an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9; and (c) an artificial intron, comprising a 3' intron fragment, preferably having the nucleotide sequence of SEQ ID NO: 15; and (iii) a modified AAV rep gene comprising a 3' portion of the AAV rep gene, preferably having the nucleotide sequence of SEQ ID NO: 56; and (C) an AAV cap gene, preferably comprising the nucleotide sequence of SEQ ID NO: 57; and (D) a transgene flanked by a pair of AAV ITRs, preferably the AAV ITRs have the nucleotide sequence of SEQ ID NO: 20, the transgene comprising a promoter operably linked to a coding sequence, the coding sequence 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; (E) a second insulator, preferably having the nucleotide sequence of SEQ ID NO:25. [Invention 23] A non-naturally occurring nucleic acid molecule comprising, in 5' to 3' order: (A) a first insulator, preferably having a nucleotide sequence of SEQ ID NO: 24; (B) a modified AAV rep gene, comprising, in 5' to 3' order: (i) a 5' portion of the AAV rep gene, preferably having the nucleotide sequence of SEQ ID NO: 73; (ii) an artificial intron, in the 5' to 3' order: (a) a 5' intron fragment, preferably having the nucleotide sequence of SEQ ID NO: 14; (b) a stop cassette comprising, in 5' to 3' order: (1) an attP site having the nucleotide sequence of SEQ ID NO:7; (2) a splice acceptor, preferably having 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 having the nucleotide sequence of SEQ ID NO: 19; and (5) a stop cassette comprising an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9; and (c) an artificial intron, comprising a 3' intron fragment, preferably having the nucleotide sequence of SEQ ID NO: 66; and (iii) a modified AAV rep gene comprising a 3' portion of the AAV rep gene, preferably having the nucleotide sequence of SEQ ID NO: 56; and (C) AAV cap gene, (D) a transgene, (1) a pair of AAV ITRs, preferably the AAV ITRs have the nucleotide sequence of SEQ ID NO:20, the transgene comprising a promoter operably linked to a coding sequence, the coding sequence 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 (2) a transgene flanked by a pair of spacer sequences, preferably having the nucleotide sequences of SEQ ID NO:67 and SEQ ID NO:68. [Invention 24] A vector comprising the non-naturally occurring nucleic acid molecule according to any one of Inventions 1 to 22, preferably the vector is a plasmid, more preferably the plasmid comprises the nucleotide sequence of SEQ ID NO: 12. [Invention 25] A vector comprising the non-naturally occurring nucleic acid molecule according to any one of inventions 1 to 21 or 23, preferably wherein said vector is a plasmid, more preferably wherein said plasmid comprises the nucleotide sequence of SEQ ID NO: 70. [Invention 26] A method for producing a non-naturally occurring nucleic acid molecule according to any one of inventions 1 to 23. [Invention 27] 26. A method for producing the vector according to invention 24 or 25. [Invention 28] 1. A cell comprising a non-naturally occurring nucleic acid molecule comprising a modified adeno-associated virus (AAV) rep gene, the 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 the four Rep proteins, the artificial intron comprising a stop cassette inserted downstream of the 5' splice site and upstream of the branch site of the artificial intron, the stop cassette comprising, in 5' to 3' order: (a) an attP site having a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical 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) a cell comprising an attB site having a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical 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. [Invention 29] 29. The cell according to claim 28, wherein the splice acceptor comprises the nucleotide sequence of SEQ ID NO:17. [Invention 30] 30. The cell according to claim 28 or 29, wherein the terminator comprises a polyadenylation signal. [Invention 31] 31. The cell according to claim 30, wherein the terminator further comprises the nucleotide sequence of SEQ ID NO:19. [Invention 32] 32. The cell according to any one of claims 28 to 31, wherein said termination cassette comprises a gene encoding a selectable marker, preferably a neomycin phosphotransferase expression cassette having the nucleotide sequence of SEQ ID NO: 18. [Invention 33] 33. The cell according to any one of inventions 28 to 32, wherein the artificial intron comprises, in the 5' to 3' order, a nucleotide sequence of SEQ ID NO: 14, the stop cassette, and a nucleotide sequence of SEQ ID NO: 15. [Invention 34] 33. The cell according to any one of Inventions 28 to 32, wherein the artificial intron comprises, in 5' to 3' order, a nucleotide sequence of SEQ ID NO: 14, the stop cassette, and a nucleotide sequence of SEQ ID NO: 66. [Invention 35] 34. The cell according to any one of Inventions 28 to 33, wherein the AAV rep gene comprises one of the rep genes of AAV1 to AAV8, or a hybrid thereof. [Invention 36] 36. The cell according to invention 35, wherein the AAV rep gene comprises the rep gene of human AAV2 having nucleotide numbers 190 to 2202 of the nucleotide sequence of GenBank Accession No. NC_001401.2. [Invention 37] 37. The cell according to claim 36, wherein the artificial intron is inserted between nucleotide numbers 996 to 1905 of the nucleotide sequence of GenBank Accession No. NC_001401.2. [Invention 38] 38. The cell according to invention 37, wherein the artificial intron is inserted immediately downstream of nucleotide number 1052, 1061, 1712, 1906, 1022, 1112, 1475, 1514, 1700, 1742, 1784, or 1340, preferably immediately downstream of nucleotide number 1052, of the nucleotide sequence of GenBank Accession No. NC_001401.2. [Invention 39] 1. A cell comprising a non-naturally occurring nucleic acid molecule comprising a modified AAV rep gene, said 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, in the 5' to 3' order: (i) a 5' intron fragment having the nucleotide sequence of SEQ ID NO: 14; (ii) a stop 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) a stop cassette comprising an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9; and (iii) an artificial intron comprising a 3' intron fragment having the nucleotide sequence of SEQ ID NO: 15; (c) a 3' portion of the AAV rep gene having the nucleotide sequence of SEQ ID NO:56. [Invention 40] 1. A cell comprising a non-naturally occurring nucleic acid molecule comprising a modified AAV rep gene, said 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, in the 5' to 3' order: (i) a 5' intron fragment having the nucleotide sequence of SEQ ID NO: 14; (ii) a stop 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) a stop cassette comprising 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. [Invention 41] 41. The cell according to claim 30 or 40, wherein the termination cassette comprises the nucleotide sequence of SEQ ID NO: 16. [Invention 42] 42. The cell according to any one of claims 28 to 41, further comprising an AAV cap gene encoding the three capsid proteins VP1, VP2, and VP3. [Invention 43] 43. The cell according to claim 42, wherein the AAV cap gene comprises one of the cap genes of AAV1 to AAV9 and AAVDJ, or a hybrid thereof. [Invention 44] 44. The cell according to claim 43, wherein the AAV cap gene comprises the human AAV9 cap gene having the nucleotide sequence of GenBank Accession No. AY530579.1. [Invention 45] 44. The cell of claim 43, wherein the AAV cap gene comprises a hybrid cap gene of AAV9. [Invention 46] 46. ​​The cell according to any one of inventions 42 to 45, wherein the AAV cap gene further comprises a polyadenylation signal, preferably an AAV2 polyadenylation signal having nucleotide numbers 4411 to 4466 of the nucleotide sequence of GenBank Accession No. NC_001401.2, and an enhancer, preferably an AAV2 rep P5 promoter having nucleotide numbers 190 to 313 of the nucleotide sequence of GenBank Accession No. NC_001401.2, wherein the polyadenylation signal and the enhancer are both downstream of the coding sequence of the cap gene. [Invention 47] 47. The cell according to any one of Inventions 42 to 46, further comprising a transgene flanked by a pair of AAV inverted terminal repeats (ITRs) downstream of the AAV cap gene. [Invention 48] a first insulator upstream of the modified AAV rep gene, and optionally a second insulator downstream of the transgene flanked by the ITRs, preferably wherein the first insulator and the second insulator are independently: (a) a human anti-repressor element 40 having the nucleotide sequence of SEQ ID NO: 24; (b) a mouse anti-repressor element 40 having the nucleotide sequence of SEQ ID NO: 25; (c) anti-repressor element 04 having the nucleotide sequence of GenBank accession number AY190749.1; (d) anti-repressor element 06 having the nucleotide sequence of GenBank accession number AY190750.1; (e) anti-repressor element 07 having the nucleotide sequence of GenBank accession number AY190751.1; (f) anti-repressor element 12 having the nucleotide sequence of GenBank accession number AY190752.1; (g) anti-repressor element 13 having the nucleotide sequence of GenBank accession number AY190753.1; (h) anti-repressor element 35 having the nucleotide sequence of GenBank accession number AY190754.1; (i) anti-repressor element 36 having the nucleotide sequence of GenBank accession number AY190755.1; (j) anti-repressor element 52 having the nucleotide sequence of GenBank accession number AY190757.1; (k) anti-repressor element 53 having the nucleotide sequence of GenBank accession number AY190758.1, and (l) a chicken HS4 insulator from the globin locus having the nucleotide sequence of AY040835.1 in two or more copies; More preferably, the cell according to invention 47, wherein the first insulator and the second insulator have the nucleotide sequences of SEQ ID NO: 24 and SEQ ID NO: 25, respectively. [Invention 49] the cell comprises the first insulator upstream of the modified AAV rep gene and further comprises a first spacer sequence and a second spacer sequence upstream and downstream, respectively, of the transgene, the first spacer sequence and the second spacer sequence independently comprising: a) the nucleotide sequence of SEQ ID NO: 67, and b) The cell according to invention 48, selected from the group consisting of the nucleotide sequence of SEQ ID NO: 68. [Invention 50] 50. The cell according to any one of inventions 47 to 49, wherein the ITR has the nucleotide sequence of SEQ ID NO: 20, the transgene comprises a promoter operably linked to a coding sequence, the coding sequence being operably linked to a polyadenylation signal, 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. [Invention 51] 1. A cell comprising a non-naturally occurring nucleic acid molecule, the non-naturally occurring nucleic acid molecule comprising, in 5' to 3' order: (A) a first insulator, preferably having a nucleotide sequence of SEQ ID NO: 24; (B) a modified AAV rep gene, comprising, in 5' to 3' order: (i) a 5' portion of the AAV rep gene, preferably having the nucleotide sequence of SEQ ID NO: 55; (ii) an artificial intron, in the 5' to 3' order: (a) a 5' intron fragment, preferably having the nucleotide sequence of SEQ ID NO: 14; (b) a stop cassette comprising, in 5' to 3' order: (1) an attP site having the nucleotide sequence of SEQ ID NO:7; (2) a splice acceptor, preferably having 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 having the nucleotide sequence of SEQ ID NO: 19; and (5) a stop cassette comprising an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9; and (c) an artificial intron, comprising a 3' intron fragment, preferably having the nucleotide sequence of SEQ ID NO: 15; and (iii) a modified AAV rep gene comprising a 3' portion of the AAV rep gene, preferably having the nucleotide sequence of SEQ ID NO: 56; and (C) an AAV cap gene, preferably comprising the nucleotide sequence of SEQ ID NO: 57; and (D) a transgene flanked by a pair of AAV ITRs, preferably the AAV ITRs have the nucleotide sequence of SEQ ID NO: 20, the transgene comprising a promoter operably linked to a coding sequence, the coding sequence 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; (E) a second insulator, preferably having the nucleotide sequence of SEQ ID NO: 25. [Invention 52] 1. A cell comprising a non-naturally occurring nucleic acid molecule, the non-naturally occurring nucleic acid molecule comprising, in 5' to 3' order: (A) a first insulator, preferably having a nucleotide sequence of SEQ ID NO: 24; (B) a modified AAV rep gene, comprising, in 5' to 3' order: (i) a 5' portion of the AAV rep gene, preferably having the nucleotide sequence of SEQ ID NO: 73; (ii) an artificial intron, in the 5' to 3' order: (a) a 5' intron fragment, preferably having the nucleotide sequence of SEQ ID NO: 14; (b) a stop cassette comprising, in 5' to 3' order: (1) an attP site having the nucleotide sequence of SEQ ID NO:7; (2) a splice acceptor, preferably having 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 having the nucleotide sequence of SEQ ID NO: 19; and (5) a stop cassette comprising an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9; and (c) an artificial intron, comprising a 3' intron fragment, preferably having the nucleotide sequence of SEQ ID NO: 66; and (iii) a modified AAV rep gene comprising a 3' portion of the AAV rep gene, preferably having the nucleotide sequence of SEQ ID NO: 56; and (C) AAV cap gene, (D) a transgene, (i) a pair of AAV ITRs, preferably the AAV ITRs have the nucleotide sequence of SEQ ID NO: 20, the transgene comprising a promoter operably linked to a coding sequence, the coding sequence 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 transgene flanked by a pair of spacer sequences, preferably the spacer sequences having the nucleotide sequences of SEQ ID NO:67 and SEQ ID NO:68. [Invention 53] 52. The cell according to any one of inventions 28 to 51, wherein the non-naturally occurring nucleic acid molecule is episomal and has the nucleotide sequence of SEQ ID NO:12. [Invention 54] 53. The cell according to any one of inventions 28 to 50 or 52, wherein the non-naturally occurring nucleic acid molecule is episomal and has the nucleotide sequence of SEQ ID NO: 70. [Invention 55] 55. The cell according to any one of inventions 52 to 54, further comprising a nucleic acid molecule encoding a recombinase having an amino acid sequence 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 wherein said nucleic acid comprises a nucleotide sequence at least 85%, 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 wherein said cell comprises a recombinant ΔE1 / ΔE3 adenovirus serotype 5 (Ad5) virus encoding a recombinase having the amino acid sequence of SEQ ID NO:2. [Invention 56] 56. The cell according to any one of inventions 52 to 55, further comprising adenovirus E1A and E1B genes, and preferably the 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. [Invention 57] 1. A method for producing a recombinant AAV comprising a transgene, comprising: (A) obtaining a first host cell, the first host cell comprising: (i) a modified AAV rep gene, comprising, in 5' to 3' order: (a) a 5' portion of the AAV rep gene, preferably having the nucleotide sequence of SEQ ID NO: 55; (b) an artificial intron, in the 5' to 3' order: (1) a 5' intron fragment, preferably having the nucleotide sequence of SEQ ID NO: 14; (2) a stop 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 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 having the nucleotide sequence of SEQ ID NO: 19, and (ee) a stop cassette comprising an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9, and (3) an artificial intron, including a 3' intron fragment, preferably having the nucleotide sequence of SEQ ID NO: 15; (c) a modified AAV rep gene comprising a 3' portion of the AAV rep gene, preferably having the nucleotide sequence of SEQ ID NO: 56; (ii) an AAV cap gene, preferably comprising the nucleotide sequence of SEQ ID NO: 57; (iii) obtaining a first host cell comprising a transgene flanked by a pair of AAV ITRs, preferably the ITRs have the nucleotide sequence of SEQ ID NO: 20, the transgene comprising a promoter operably linked to a coding sequence, the coding sequence 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; (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 harvesting the recombinant AAV. [Invention 58] 1. A method for producing a recombinant AAV comprising a transgene, comprising: (A) obtaining a first host cell, the first host cell comprising: (i) a modified AAV rep gene, comprising, in 5' to 3' order: (a) a 5' portion of the AAV rep gene, preferably having the nucleotide sequence of SEQ ID NO: 73; (b) an artificial intron, in the 5' to 3' order: (1) a 5' intron fragment, preferably having the nucleotide sequence of SEQ ID NO: 14; (2) a stop 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 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 having the nucleotide sequence of SEQ ID NO: 19, and (ee) a stop cassette comprising an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9, and (3) an artificial intron, including a 3' intron fragment, preferably having the nucleotide sequence of SEQ ID NO: 66; (c) a modified AAV rep gene comprising a 3' portion of the AAV rep gene, preferably having the nucleotide sequence of SEQ ID NO: 66; (ii) the AAV cap gene, and (iii) a transgene, (a) a pair of AAV ITRs, preferably the ITRs have the nucleotide sequence of SEQ ID NO: 20, the transgene comprising a promoter operably linked to a coding sequence, the coding sequence 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) obtaining a first host cell comprising a transgene flanked by a pair of spacer sequences, preferably said spacer sequences having the nucleotide sequences of SEQ ID NO: 67 and SEQ ID NO: 68; (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 harvesting the recombinant AAV. [Invention 59] 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 the ITRs, preferably the first insulator and the second insulator independently: (a) a human anti-repressor element 40 having the nucleotide sequence of SEQ ID NO: 24; (b) a mouse anti-repressor element 40 having the nucleotide sequence of SEQ ID NO: 25; (c) anti-repressor element 04 having the nucleotide sequence of GenBank accession number AY190749.1; (d) anti-repressor element 06 having the nucleotide sequence of GenBank accession number AY190750.1; (e) anti-repressor element 07 having the nucleotide sequence of GenBank accession number AY190751.1; (f) anti-repressor element 12 having the nucleotide sequence of GenBank accession number AY190752.1; (g) anti-repressor element 13 having the nucleotide sequence of GenBank accession number AY190753.1; (h) anti-repressor element 35 having the nucleotide sequence of GenBank accession number AY190754.1; (i) anti-repressor element 36 having the nucleotide sequence of GenBank accession number AY190755.1; (j) anti-repressor element 52 having the nucleotide sequence of GenBank accession number AY190757.1; (k) anti-repressor element 53 having the nucleotide sequence of GenBank accession number AY190758.1, and (l) a chicken HS4 insulator from the globin locus having the nucleotide sequence of AY040835.1 in two or more copies; More preferably, the method according to invention 57 or 58, wherein the first insulator and the second insulator have the nucleotide sequences of SEQ ID NO: 24 and SEQ ID NO: 25, respectively. [Invention 60] the first host cell comprises the first insulator upstream of the modified AAV rep gene and further comprises a first spacer sequence and a second spacer sequence upstream and downstream, respectively, of the transgene, the first spacer sequence and the second spacer sequence independently comprising: a) the nucleotide sequence of SEQ ID NO: 67, and b) The method according to claim 59, wherein the nucleotide sequence is selected from the group consisting of the nucleotide sequence of SEQ ID NO: 68. [Invention 61] 60. The method according to any one of inventions 57 to 59, wherein the first host cell is obtainable by introducing into a cell one or more nucleic acid molecules comprising the modified AAV rep gene, the AAV cap gene, the transgene flanked by the ITRs, the first insulator, and the second insulator. [Invention 62] 62. The method of claim 61, wherein the first host cell is obtained by introducing into the cell a nucleic acid molecule comprising, in 5' to 3' order, the first insulator, the modified AAV rep gene, the AAV cap gene, the transgene flanked by the ITRs, the first insulator, and the second insulator, preferably a plasmid comprising the nucleotide sequence of SEQ ID NO: 12. [Invention 63] 61. The method of claim 57, 58, or 60, wherein the first host cell is obtained by introducing into a cell one or more nucleic acid molecules comprising the modified AAV rep gene, the AAV cap gene, the transgene flanked by the ITRs, the first insulator, the first spacer sequence, and the second spacer sequence. [Invention 64] 64. The method of claim 63, wherein the first host cell is obtained by introducing into a cell one or more nucleic acid molecules, preferably a plasmid comprising the nucleotide sequence of SEQ ID NO: 70, comprising the modified AAV rep gene, the AAV cap gene, the transgene flanked by the ITRs, the first insulator, the first spacer sequence, and the second spacer sequence. [Invention 65] 63. The method according to any one of Inventions 57 to 62, wherein the recombinant adenovirus is a recombinant ΔE1 / ΔE3 adenovirus serotype 5 (Ad5) virus comprising the nucleotide sequence of SEQ ID NO:3. [Invention 66] 66. The method according to any one of inventions 57 to 65, wherein the host cell contains adenovirus E1A and E1B genes, and 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. [Invention 67] 67. The method according to any one of claims 57 to 66, wherein said conditions for growing said second host cells comprise culturing said second cells with 2-aminopurine. [Invention 68] 68. The method of claim 67, wherein the 2-aminopurine concentration is less than about 1.25 mM. [Invention 69] 69. The method according to Invention 67 or 68, wherein the 2-aminopurine concentration is about 1 μM to about 1.25 mM. [Invention 70] 69. The method according to Invention 67 or 68, wherein the 2-aminopurine concentration is about 10 μM to about 1.25 mM. [Invention 71] 69. The method according to Invention 67 or 68, wherein the 2-aminopurine concentration is about 100 μM to about 1.25 mM. [Invention 72] 69. The method of claim 67 or 68, wherein the 2-aminopurine concentration is about 1.25 mM. [Invention 73] 73. The method according to any one of claims 67 to 72, wherein culturing said second cells with 2-aminopurine is initiated about 24 hours after infection of said first host cells with the recombinant adenovirus. [Invention 74] 56. A composition comprising the cell of invention 55 and 2-aminopurine. [Invention 75] 75. The composition of claim 74, wherein the 2-aminopurine concentration is less than about 1.25 mM. [Invention 76] The composition according to Invention 74, wherein the 2-aminopurine concentration is from about 1 μM to about 1.25 mM. [Invention 77] The composition according to Invention 74, wherein the 2-aminopurine concentration is about 10 μM to about 1.25 mM. [Invention 78] The composition according to Invention 74, wherein the 2-aminopurine concentration is about 100 μM to about 1.25 mM. [Invention 79] 75. The composition according to claim 74, wherein the 2-aminopurine concentration is about 1.25 mM. [Invention 80] A non-naturally occurring nucleic acid molecule comprising a nucleotide sequence encoding a serine recombinase having an amino acid sequence having 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. [Invention 81] 75. A non-naturally occurring nucleic acid molecule according to claim 74, comprising 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. [Invention 82] A vector comprising a non-naturally occurring nucleic acid according to invention 80 or 81. [Invention 83] 83. The vector according to invention 82, further comprising a promoter, preferably a cytomegalovirus (CMV) promoter, operably linked to said nucleotide sequence encoding said serine recombinase. [Invention 84] 84. The vector according to claim 82 or 83, further comprising a polyadenylation signal, such as the Simian Virus 40 (SV40) polyadenylation signal, operably linked to the nucleotide sequence encoding the serine recombinase. [Invention 85] 85. The vector according to any one of Inventions 82 to 84, which is a DNA plasmid. [Invention 86] 86. The vector according to any one of Inventions 82 to 85, which is a recombinant adenovirus vector. [Invention 87] 87. The vector according to invention 86, which is a recombinant ΔE1 / ΔE3 adenovirus serotype 5 (Ad5) virus comprising a nucleotide sequence encoding a serine recombinase having the amino acid sequence of SEQ ID NO:2 under the control of a CMV promoter, said nucleotide sequence being further operably linked to an SV40 polyadenylation signal (NC_001669.1, nt 2550 to 2774). [Invention 88] A cell comprising a non-naturally occurring nucleic acid molecule comprising a nucleotide sequence encoding a serine recombinase having an amino acid sequence having at least 85%, for example 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. [Invention 89] 89. The cell of claim 88, comprising a nucleotide sequence having at least 85%, 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. [Invention 90] 89. A cell comprising a vector comprising a non-naturally occurring nucleic acid according to claim 88 or 89. [Invention 91] 91. The cell according to invention 90, further comprising a promoter, preferably a cytomegalovirus (CMV) promoter, operably linked to said nucleotide sequence encoding said serine recombinase. [Invention 92] 92. The cell of claim 90 or 91, further comprising a polyadenylation signal, such as the Simian Virus 40 (SV40) polyadenylation signal, operably linked to the nucleotide sequence encoding the serine recombinase. [Invention 93] 93. The cell according to any one of inventions 90 to 92, wherein the vector is a DNA plasmid. [Invention 94] 94. The cell according to any one of Inventions 90 to 93, wherein the vector is a recombinant adenovirus vector. [Invention 95] The cell according to invention 94, wherein the recombinant adenovirus vector is a recombinant ΔE1 / ΔE3 adenovirus serotype 5 (Ad5) virus comprising a nucleotide sequence encoding a serine recombinase having the amino acid sequence of SEQ ID NO:2 under the control of a CMV promoter, the nucleotide sequence being further operably linked to an SV40 polyadenylation signal (NC_001669.1, nt 2550-2774). [Invention 96] 96. The cell according to any one of inventions 88 to 95, comprising adenovirus E1A and E1B genes, and preferably, the 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. [Invention 97] 1. A method for performing site-specific recombination in a cell, comprising: (a) obtaining a cell comprising a nucleic acid molecule having an attP site having a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7, preferably an attP site having the nucleotide sequence of SEQ ID NO:7, and an attB site having a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical 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 allow the serine recombinase to catalyze the site-specific recombination between the attP site and the attB site. [Invention 98] 1. A product produced by a process for performing site-specific recombination in a cell, said process comprising: (a) obtaining a cell comprising a nucleic acid molecule having an attP site having a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7, preferably an attP site having the nucleotide sequence of SEQ ID NO:7, and an attB site having a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical 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 allow the serine recombinase to catalyze the site-specific recombination between the attP site and the attB site. [Invention 99] 1. A process for obtaining a product from a cell, comprising: (a) obtaining a cell comprising a nucleic acid molecule having an attP site having a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7, preferably an attP site having the nucleotide sequence of SEQ ID NO:7, and an attB site having a nucleotide sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical 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 cells under conditions that allow the serine recombinase to catalyze the site-specific recombination between the attP and attB sites; (d) producing and recovering a product from the cells. [Invention 100] A non-naturally occurring system, A non-naturally occurring system comprising a means for AAV-mediated recombination, said means optionally comprising transgenic elements. [Invention 101] 101. A means for transferring a non-naturally occurring system according to claim 100. [Invention 102] A non-naturally occurring system, 102. A non-naturally occurring system comprising recombinant means for engineering the system of invention 100, said recombinant means comprising the use of at least one serine residue in catalysis. [Invention 103] 103. A means for transferring a non-naturally occurring system according to claim 102. [Invention 104] A means for producing a molecule, the means comprising any one of the means according to inventions 100 to 103, and capable of being replicated. [Invention 105] 1. A process for AAV-mediated site-specific recombination, comprising: (a) a step for carrying out the function of obtaining a cell, comprising the means according to invention 100; (b) growing said cell under conditions that permit site-specific recombination that uses at least one serine residue in catalysis. [Invention 106] 106. A process for AAV-mediated site-specific recombination according to invention 105, comprising obtaining a product, optionally wherein said product is a therapeutic product.

Claims

1. A non-naturally occurring nucleic acid molecule comprising a modified adeno-associated virus (AAV) rep gene, the AAV rep gene encoding four Rep proteins, Rep78, Rep68, Rep52, and Rep40, and an artificial intron inserted into a coding sequence of the rep gene shared by the four Rep proteins, the artificial intron comprising a stop cassette inserted downstream of a 5' splice site and upstream of a branch site of the artificial intron, the stop cassette comprising, in 5' to 3' order: (a) an attP site having the nucleotide sequence of SEQ ID NO:7; (b) a splice acceptor; and (c) a terminator; and (d) an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:

9.

2. The non-naturally occurring nucleic acid molecule described in claim 1, wherein the splice acceptor comprises a nucleotide sequence of SEQ ID NO:

17.

3. A non-naturally occurring nucleic acid molecule described in claim 1 or 2, wherein the terminator includes a polyadenylation signal.

4. The non-naturally occurring nucleic acid molecule described in claim 3, wherein the terminator further comprises a nucleotide sequence of SEQ ID NO:

19.

5. The non-naturally occurring nucleic acid molecule of any one of claims 1 to 4, wherein the termination cassette comprises a gene encoding a selectable marker.

6. A non-naturally occurring nucleic acid molecule described in any one of claims 1 to 5, wherein the artificial intron comprises, in 5' to 3' order, a nucleotide sequence of SEQ ID NO: 14, the stop cassette, and a nucleotide sequence of SEQ ID NO:

15.

7. The non-naturally occurring nucleic acid molecule of claim 1, wherein the AAV rep gene comprises a rep gene of one of AAV1 to AAV8, or a hybrid thereof.

8. The non-naturally occurring nucleic acid molecule of claim 7, wherein the AAV rep gene comprises a human AAV2 rep gene having nucleotide numbers 190 to 2202 of the nucleotide sequence of GenBank Accession No. NC_001401.

2.

9. The non-naturally occurring nucleic acid molecule described in claim 8, wherein the artificial intron is inserted between any two nucleotides from nucleotide numbers 996 to 1905 of the nucleotide sequence of the AAV rep gene of GenBank Accession No. NC_001401.

2.

10. The non-naturally occurring nucleic acid molecule of claim 9, wherein the artificial intron is inserted immediately downstream of nucleotide numbers 1052, 1061, 1712, 1906, 1022, 1112, 1475, 1514, 1700, 1742, 1784, or 1340 of the nucleotide sequence of the AAV rep gene having GenBank Accession No. NC_001401.

2.

11. A 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) a 5' portion of the AAV rep gene having the nucleotide sequence of SEQ ID NO:55; (b) an artificial intron, which, in 5' to 3' order, is (i) a 5' intron fragment having the nucleotide sequence of SEQ ID NO: 14; (ii) a stop 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) a stop cassette comprising an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9; and (iii) an artificial intron comprising a 3' intron fragment having the nucleotide sequence of SEQ ID NO: 15; (c) a 3' portion of the AAV rep gene having the nucleotide sequence of SEQ ID NO:56; or (B) (a) a 5' portion of the AAV rep gene having the nucleotide sequence of SEQ ID NO:73; (b) an artificial intron, which, in 5' to 3' order, is (i) a 5' intron fragment having the nucleotide sequence of SEQ ID NO: 14; (ii) a stop 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) a stop cassette comprising 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.

12. The non-naturally occurring nucleic acid molecule described in claim 11, wherein the termination cassette comprises the nucleotide sequence of SEQ ID NO:

16.

13. The non-naturally occurring nucleic acid molecule of any one of claims 1 to 12, further comprising an AAV cap gene encoding three capsid proteins VP1, VP2, and VP3.

14. The non-naturally occurring nucleic acid molecule of claim 13, wherein the AAV cap gene comprises any one of the cap genes of AAV1 to AAV9 and AAVDJ, or a hybrid thereof.

15. The non-naturally occurring nucleic acid molecule of claim 14, wherein the AAV cap gene comprises a human AAV9 cap gene having a nucleotide sequence of GenBank Accession No. AY530579.

1.

16. The non-naturally occurring nucleic acid molecule of any one of claims 13 to 15, wherein the AAV cap gene further comprises a polyadenylation signal.

17. The non-naturally occurring nucleic acid molecule of any one of claims 13 to 16, further comprising an introduced gene flanked by a pair of AAV inverted terminal repeats (ITRs) downstream of the AAV cap gene.

18. The modified AAV gene further comprising a first insulator upstream of the modified AAV rep gene and a second insulator downstream of the transgene flanked by the ITRs, wherein the first insulator and the second insulator are independently: (a) a human anti-repressor element 40 having the nucleotide sequence of SEQ ID NO:24; (b) a mouse anti-repressor element 40 having the nucleotide sequence of SEQ ID NO:25; (c) anti-repressor element 04 having the nucleotide sequence of GenBank accession number AY190749.1; (d) anti-repressor element 06 having the nucleotide sequence of GenBank accession number AY190750.1; (e) anti-repressor element 07 having the nucleotide sequence of GenBank accession number AY190751.1; (f) anti-repressor element 12 having the nucleotide sequence of GenBank accession number AY190752.1; (g) an anti-repressor element 13 having the nucleotide sequence of GenBank accession number AY190753.1; (h) an anti-repressor element 35 having the nucleotide sequence of GenBank accession number AY190754.1; (i) an anti-repressor element 36 having the nucleotide sequence of GenBank accession number AY190755.1; (j) anti-repressor element 52 having the nucleotide sequence of GenBank accession number AY190757.1; (k) an anti-repressor element 53 having the nucleotide sequence of GenBank accession number AY190758.1; and (l) a chicken HS4 insulator from the globin locus having the nucleotide sequence of AY040835.1 in two or more copies, 19. The non-naturally occurring nucleic acid molecule, comprising the first insulator upstream of the modified AAV rep gene, and further comprising a first spacer sequence and a second spacer sequence upstream and downstream, respectively, of the transgene, wherein the first spacer sequence and the second spacer sequence are independently a) the nucleotide sequence of SEQ ID NO:67, and b) the nucleotide sequence of SEQ ID NO:

68.

20. The non-naturally occurring nucleic acid molecule of any one of claims 17 to 19, wherein the ITR has the nucleotide sequence of SEQ ID NO:20, the introduced gene comprises a promoter operably linked to a coding sequence, the coding sequence operably linked to a polyadenylation signal, the promoter having the nucleotide sequence of SEQ ID NO:21, and the polyadenylation signal having the nucleotide sequence SEQ ID NO:

23.

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

25.

22. A non-naturally occurring nucleic acid molecule comprising, in 5' to 3' order: (A) a first insulator comprising a nucleotide sequence of SEQ ID NO:24; and (B) a modified AAV rep gene, comprising, in 5' to 3' order: (i) a 5' portion of the AAV rep gene comprising the nucleotide sequence of SEQ ID NO:73; (ii) an artificial intron, which, in 5' to 3' order, is (a) a 5' intron fragment comprising the nucleotide sequence of SEQ ID NO:14; (b) a stop cassette comprising, in 5' to 3' order: (1) an attP site having the nucleotide sequence of SEQ ID NO:7; (2) a splice acceptor comprising the nucleotide sequence of SEQ ID NO: 17; (3) A gene encoding a selectable marker comprising the nucleotide sequence of SEQ ID NO: 18; (4) a terminator comprising the nucleotide sequence of SEQ ID NO: 19; and (5) a stop cassette comprising an attB site having the nucleotide sequence of SEQ ID NO:8 or SEQ ID NO:9; and (c) an artificial intron comprising a 3' intron fragment comprising the nucleotide sequence of SEQ ID NO: 66; and (iii) a 3' portion of the AAV rep gene comprising the nucleotide sequence of SEQ ID NO:56; and (C) an AAV cap gene; and (D) a transgene, (1) a pair of AAV ITRs, the AAV ITRs having the nucleotide sequence of SEQ ID NO:20, the transgene comprising a promoter operably linked to a coding sequence, the coding sequence operably linked to a polyadenylation signal, the promoter having the nucleotide sequence of SEQ ID NO:21, and the polyadenylation signal having the nucleotide sequence SEQ ID NO:23; and (2) A non-naturally occurring nucleic acid molecule comprising an introduced gene flanked by a pair of spacer sequences, the spacer sequences having the nucleotide sequences of SEQ ID NO:67 and SEQ ID NO:

68.

23. A vector comprising a non-naturally occurring nucleic acid molecule described in any one of claims 1 to 22.

24. A vector comprising a non-naturally occurring nucleic acid molecule described in any one of claims 1 to 20 or 22, wherein the vector is a plasmid comprising the nucleotide sequence of SEQ ID NO:70 or SEQ ID NO:

12.

25. A cell comprising a non-naturally occurring nucleic acid molecule described in any one of claims 1 to 22.

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