The RNA of the embodiment is preferably modified to have degradation resistance. For example, the modification may be a known modification that prevents RNA from being degraded by RNase or the like. Examples of the modification include the use and introduction of naturally modified or unnatural nucleotides into RNA, the use and addition of unnatural sequences, or the addition of natural or unnatural CAP structures, or the addition of poly (A) sequences, or the like.

Examples of the naturally modified nucleotides include pseudouridine, 5-methylcytidine, 1-methyladenosine, and the like. Examples of the unnatural nucleotides include BNA, LNA, PNA, and the like.

Examples of the unnatural sequences include an artificially created non-naturally occurring base sequence, e.g., a random base sequence or a hybrid sequence of natural or unnatural amino acids and a nucleic acid, and the like. The unnatural sequence is preferably added, for example, to the terminus of RNA.

Examples of the natural CAP structure include CAP0 (m7GpppN), CAP1 (m7GpppNm), and the like. Examples of the unnatural CAP structure include an anti-reverse cap analog (ARCA), LNA-guanosine, and the like. The unnatural CAP structure is preferably added, for example, to the 5′ terminus of RNA.

RNA of the embodiment can be synthesized from the DNA sequence of the modified piggyBac transposase described below by using an in vitro transcription method or the like. In vitro transcription can be performed using a commercially available kit, e.g., CUGA (registered trademark) 7 kit or the like. Alternatively, RNA may be artificially synthesized directly.

Subsequently, an example in which the polynucleotide is DNA will be described. The DNA is transcribed and translated, for example, after being introduced into the cell, and supplies the modified piggyBac transposase 1 into a cell.

The DNA of the first embodiment contains at least a region (base sequence) encoding each domain (the NLS domain 2 and the PB domain 3) of the desirable modified piggyBac transposase 1. As the region encoding each domain, for example, a sequence of known DNA encoding the polypeptide of each domain can be used. The region encoding each domain may include a sequence in which uracil (U) of the RNA sequence is changed to thymine (T), or a complementary sequence (cDNA) thereof.

As the region encoding the PB domain 3, for example, a wild-type DNA sequence (Table 5, SEQ ID NO: 5) encoding the amino acid sequence of SEQ ID NO: 1 can be used.

TABLE 5

Wild-type DNA sequence encoding PB domain

of SEQ ID NO: 1 (SEQ ID NO: 5)

ATGGGTAGTT CTTTAGACGA TGAGCATATC CTCTCTGCTC TTCTGCAAAG CGATGACGAG
60

CTTGTTGGTG AGGATTCTGA CAGTGAAATA TCAGATCACG TAAGTGAAGA TGACGTCCAG
120

AGCGATACAG AAGAAGCGTT TATAGATGAG GTACATGAAG TGCAGCCAAC GTCAAGCGGT
180

AGTGAAATAT TAGACGAACA AAATGTTATT GAACAACCAG GTTCTTCATT GGCTTCTAAC
240

AGAATCTTGA CCTTGCCACA GAGGACTATT AGAGGTAAGA ATAAACATTG TTGGTCAACT
300

TCAAAGTCCA CGAGGCGTAG CCGAGTCTCT GCACTGAACA TTGTCAGATC TCAAAGAGGT
360

CCGACGCGTA TGTGCCGCAA TATATATGAC CCACTTTTAT GCTTCAAACT ATTTTTTACT
420

GATGAGATAA TTTCGGAAAT TGTAAAATGG ACAAATGCTG AGATATCATT GAAACGTCGG
480

GAATCTATGA CAGGTGCTAC ATTTCGTGAC ACGAATGAAG ATGAAATCTA TGCTTTCTTT
540

GGTATTCTGG TAATGACAGC AGTGAGAAAA GATAACCACA TGTCCACAGA TGACCTCTTT
600

GATCGATCTT TGTCAATGGT GTACGTCTCT GTAATGAGTC GTGATCGTTT TGATTTTTTG
660

ATACGATGTC TTAGAATGGA TGACAAAAGT ATACGGCCCA CACTTCGAGA AAACGATGTA
720

TTTACTCCTG TTAGAAAAAT ATGGGATCTC TTTATCCATC AGTGCATACA AAATTACACT
780

CCAGGGGCTC ATTTGACCAT AGATGAACAG TTACTTGGTT TTAGAGGACG GTGTCCGTTT
840

AGGATGTATA TCCCAAACAA GCCAAGTAAG TATGGAATAA AAATCCTCAT GATGTGTGAC
900

AGTGGTACGA AGTATATGAT AAATGGAATG CCTTATTTGG GAAGAGGAAC ACAGACCAAC
960

GGAGTACCAC TCGGTGAATA CTACGTGAAG GAGTTATCAA AGCCTGTGCA CGGTAGTTGT
1020

CGTAATATTA CGTGTGACAA TTGGTTCACC TCAATCCCTT TGGCAAAAAA CTTACTACAA
1080

GAACCGTATA AGTTAACCAT TGTGGGAACC GTGCGATCAA ACAAACGCGA GATACCGGAA
1140

GTACTGAAAA ACAGTCGCTC CAGGCCAGTG GGAACATCGA TGTTTTGTTT TGACGGACCC
1200

CTTACTCTCG TCTCATATAA ACCGAAGCCA GCTAAGATGG TATACTTATT ATCATCTTGT
1260

GATGAGGATG CTTCTATCAA CGAAAGTACC GGTAAACCGC AAATGGTTAT GTATTATAAT
1320

CAAACTAAAG GCGGAGTGGA CACGCTAGAC CAAATGTGTT CTGTGATGAC CTGCAGTAGG
1380

AAGACGAATA GGTGGCCTAT GGCATTATTG TACGGAATGA TAAACATTGC CTGCATAAAT
1440

TCTTTTATTA TATACAGCCA TAATGTCAGT AGCAAGGGAG AAAAGGTTCA AAGTCGCAAA
1500

AAATTTATGA GAAACCTTTA CATGAGCCTG ACGTCATCGT TTATGCGTAA GCGTTTAGAA
1560

GCTCCTACTT TGAAGAGATA TTTGCGCGAT AATATCTCTA ATATTTTGCC AAATGAAGTG
1620

CCTGGTACAT CAGATGACAG TACTGAAGAG CCAGTAATGA AAAAACGTAC TTACTGTACT
1680

TACTGCCCCT CTAAAATAAG GCGAAAGGCA AATGCATCGT GCAAAAAATG CAAAAAAGTT
1740

ATTTGTCGAG AGCATAATAT TGATATGTGC CAAAGTTGTT TCTGA
1785

When used in human cells, the region encoding the PB domain 3 is preferably, for example, a sequence obtained using a triplet selected to provide codon-optimized RNA for humans as described above. The DNA sequence is codon-optimized using a triplet in which the U of the preferred codon corresponding to each of the amino acids described above is changed to T or a complementary sequence thereof. For example, it is preferable to use the sequence shown in Table 6 (SEQ ID NO: 6) as the DNA sequence obtained by codon optimization of the PB domain 3 of SEQ ID NO: 1.

TABLE 6

DNA sequence codon-optimized for humans that

encodes PB domain of SEQ ID NO: 1 (SEQ ID NO: 6)

ATGGGCTCCT CCCTCGATGA CGAGCACATT CTGTCCGCTC TGCTGCAGTC CGACGATGAG
60

CTGGTCGGAG AAGACAGCGA TAGCGAGATC AGCGACCACG TCTCCGAGGA CGACGTCCAA
120

AGCGACACAG AGGAGGCCTT TATCGACGAG GTCCATGAAG TGCAGCCCAC ATCCAGCGGC
180

AGCGAGATTC TGGACGAGCA GAACGTGATC GAACAGCCCG GCAGCTCCCT CGCCAGCAAT
240

AGAATTCTGA CACTGCCCCA GAGAACCATT AGAGGCAAGA ACAAGCACTG TTGGAGCACC
300

AGCAAGAGCA CAAGAAGATC CAGAGTCAGC GCCCTCAACA TTGTGAGAAG CCAGAGGGGC
360

CCTACAAGAA TGTGTAGAAA CATCTATGAC CCTCTGCTGT GTTTCAAGCT GTTCTTCACC
420

GACGAGATCA TCAGCGAGAT CGTGAAGTGG ACCAACGCTG AGATCTCTCT GAAGAGGAGA
480

GAAAGCATGA CCGGCGCCAC CTTTAGGGAC ACCAACGAGG ACGAAATCTA TGCTTTTTTT
540

GGAATTCTGG TGATGACAGC CGTGAGGAAA GACAACCACA TGTCCACAGA TGATCTGTTT
600

GATAGATCTC TGTCCATGGT GTATGTGAGC GTCATGTCCA GAGATAGATT CGATTTCCTC
660

ATTAGATGTC TGAGGATGGA CGATAAGTCC ATCAGACCCA CACTGAGAGA GAACGACGTC
720

TTTACCCCCG TGAGAAAAAT CTGGGACCTC TTCATCCACC AGTGCATCCA AAATTATACA
780

CCCGGCGCTC ACCTCACCAT CGACGAGCAG CTCCTCGGCT TCAGAGGAAG ATGCCCCTTT
840

AGAATGTACA TTCCCAACAA GCCCTCCAAG TACGGCATCA AGATCCTCAT GATGTGTGAC
900

AGCGGCACCA AGTACATGAT CAACGGCATG CCCTATCTGG GAAGAGGCAC CCAGACCAAC
960

GGAGTGCCCC TCGGCGAATA TTACGTGAAG GAACTGAGCA AACCCGTGCA CGGCAGCTGC
1020

AGAAATATTA CATGCGATAA CTGGTTCACC AGCATCCCTC TGGCCAAAAA TCTGCTGCAA
1080

GAGCCTTACA AGCTCACAAT CGTGGGAACC GTGAGGAGCA ACAAGAGGGA GATTCCCGAG
1140

GTGCTCAAAA ACTCTAGATC TAGACCCGTG GGAACCTCCA TGTTCTGTTT CGACGGCCCT
1200

CTGACACTCG TCTCCTATAA GCCCAAGCCC GCCAAGATGG TGTATCTGCT CAGCAGCTGC
1260

GACGAAGACG CCAGCATCAA TGAATCCACC GGCAAGCCCC AGATGGTCAT GTACTACAAC
1320

CAGACCAAGG GAGGCGTCGA TACACTGGAC CAGATGTGTT CCGTCATGAC ATGCTCTAGA
1380

AAGACCAATA GATGGCCCAT GGCTCTGCTG TACGGCATGA TCAACATCGC TTGCATTAAC
1440

TCCTTTATCA TTTACTCCCA TAACGTCAGC TCCAAGGGCG AGAAGGTGCA GAGCAGAAAG
1500

AAATTCATGA GAAATCTGTA CATGAGCCTC ACCAGCAGCT TCATGAGAAA GAGGCTGGAG
1560

GCCCCCACAC TGAAAAGATA TCTGAGAGAT AATATCTCCA ACATTCTGCC TAACGAGGTC
1620

CCCGGCACAA GCGATGATAG CACAGAGGAG CCCGTGATGA AGAAGAGAAC ATACTGCACA
1680

TACTGCCCCA GCAAGATTAG AAGGAAGGCC AACGCCAGCT GCAAGAAGTG CAAGAAGGTC
1740

ATCTGCAGAG AGCACAACAT CGACATGTGC CAGAGCTGTT TTTGA
1785

Further, as the DNA sequence for humans obtained by codon optimization of the PB domain 3 of SEQ ID NO: 2, for example, the sequence shown in Table 7 (SEQ ID NO: 7) is preferably used.

TABLE 7

DNA sequence codon-optimized for humans that

encodes PB domain of SEQ ID NO: 2 (SEQ ID NO: 7)

ATGGGCAGCA GCCTGGACGA CGAGCACATC CTGAGCGCCC TGCTGCAGAG CGACGACGAG
60

CTGGTCGGCG AGGACAGCGA CAGCGAGGTG AGCGACCACG TGAGCGAGGA CGACGTGCAG
120

TCCGACACCG AGGAGGCCTT CATCGACGAG GTGCACGAGG TGCAGCCTAC CAGCAGCGGC
180

TCCGAGATCC TGGACGAGCA GAACGTGATC GAGCAGCCCG GCAGCTCCCT GGCCAGCAAC
240

AGGATCCTGA CCCTGCCCCA GAGGACCATC AGGGGCAAGA ACAAGCACTG CTGGTCCACC
300

TCCAAGCCCA CCAGGCGGAG CAGGGTGTCC GCCCTGAACA TCGTGAGAAG CCAGAGGGGC
360

CCCACCAGGA TGTGCAGGAA CATCTACGAC CCCCTGCTGT GCTTCAAGCT GTTCTTCACC
420

GACGAGATCA TCAGCGAGAT CGTGAAGTGG ACCAACGCCG AGATCAGCCT GAAGAGGCGG
480

GAGAGCATGA CCTCCGCCAC CTTCAGGGAC ACCAACGAGG ACGAGATCTA CGCCTTCTTC
540

GGCATCCTGG TGATGACCGC CGTGAGGAAG GACAACCACA TGAGCACCGA CGACCTGTTC
600

GACAGATCCC TGAGCATGGT GTACGTGAGC GTGATGAGCA GGGACAGATT CGACTTCCTG
660

ATCAGATGCC TGAGGATGGA CGACAAGAGC ATCAGGCCCA CCCTGCGGGA GAACGACGTG
720

TTCACCCCCG TGAGAAAGAT CTGGGACCTG TTCATCCACC AGTGCATCCA GAACTACACC
780

CCTGGCGCCC ACCTGACCAT CGACGAGCAG CTGCTGGGCT TCAGGGGCAG GTGCCCCTTC
840

AGGGTCTATA TCCCCAACAA GCCCAGCAAG TACGGCATCA AGATCCTGAT GATGTGCGAC
900

AGCGGCACCA AGTACATGAT CAACGGCATG CCCTACCTGG GCAGGGGCAC CCAGACCAAC
960

GGCGTGCCCC TGGGCGAGTA CTACGTGAAG GAGCTGTCCA AGCCCGTCCA CGGCAGCTGC
1020

AGAAACATCA CCTGCGACAA CTGGTTCACC AGCATCCCCC TGGCCAAGAA CCTGCTGCAG
1080

GAGCCCTACA AGCTGACCAT CGTGGGCACC GTGAGAAGCA ACAAGAGAGA GATCCCCGAG
1140

GTCCTGAAGA ACAGCAGGTC CAGGCCCGTG GGCACCAGCA TGTTCTGCTT CGACGGCCCC
1200

CTGACCCTGG TGTCCTACAA GCCCAAGCCC GCCAAGATGG TGTACCTGCT GTCCAGCTGC
1260

GACGAGGACG CCAGCATCAA CGAGAGCACC GGCAAGCCCC AGATGGTGAT GTACTACAAC
1320

CAGACCAAGG GCGGCGTGGA CACCCTGGAC CAGATGTGCA GCGTGATGAC CTGCAGCAGA
1380

AAGACCAACA GGTGGCCCAT GGCCCTGCTG TACGGCATGA TCAACATCGC CTGCATCAAC
1440

AGCTTCATCA TCTACAGCCA CAACGTGAGC AGCAAGGGCG AGAAGGTGCA GAGCCGGAAA
1500

AAGTTCATGC GGAACCTGTA CATGGGCCTG ACCTCCAGCT TCATGAGGAA GAGGCTGGAG
1560

GCCCCCACCC TGAAGAGATA CCTGAGGGAC AACATCAGCA ACATCCTGCC CAAAGAGGTG
1620

CCCGGCACCA GCGACGACAG CACCGAGGAG CCCGTGATGA AGAAGAGGAC CTACTGCACC
1680

TACTGTCCCA GCAAGATCAG AAGAAAGGCC AGCGCCAGCT GCAAGAAGTG TAAGAAGGTC
1740

ATCTGCCGGG AGCACAACAT CGACATGTGC CAGAGCTGTT TCTGA
1785

When the NLS of SV40 large T antigen of SEQ ID NO: 3 is used as the NLS domain 2, the DNA sequence (SEQ ID NO: 8) shown in Table 8 below can be used as the region encoding the NLS domain 2.

TABLE 8

DNA sequence of NLS of large T antigen

protein of SV40 (SEQ ID NO: 8)

AAGAAGAAGA CAAAGGTC
18

When the TAT protein of HIV (SEQ ID NO: 4) is used as the NLS domain 2, the DNA sequence (SEQ ID NO: 9) shown in Table 9 below can be used as the region encoding the NLS domain 2.

TABLE 9

DNA sequence of TAT protein of

HIV (SEQ ID NO: 9)

GGCAGAAAGA AGAGAAGACA GAGAAGAAGA
30

Further, in the case of using the NLS domain 2 obtained by linking the TAT protein of HIV to the NLS of SV40 large T antigen, the DNA sequence (SEQ ID NO: 10) shown in Table 10 below can be used.

TABLE 10

DNA sequence of TAT protein of HIV + NLS of

large T antigen protein of SV40 (SEQ ID NO: 10)

GGCAGAAAGA AGAGAAGACA GAGAAGAAGA CCCCCCGCCG GCACCAGCGT GAGCCTGAAG
60

AAGAAGAGAA AGGTGCCCCC CGCC
84

It is also possible to use the sequence in which nucleotides contained in the base sequences shown in Tables 5 to 10 have mutations such as substitutions, additions, insertions, and deletions. For example, when such mutations are present, it is preferable to use the sequence having 90% or more of homology with the amino acid sequence encoded by the sequences shown in Tables 5 to 10.

The DNA of the embodiment is preferably used in the configuration of a piggyBac transposase expression unit in which a promoter sequence is further linked to the 5′ terminus of the DNA and a transcription termination sequence is linked to the 3′ terminus. As the promoter sequence, an early enhancer/promoter of cytomegalovirus (CMV), a promoter of simian virus 40 (SV40), or the like can be used. As the transcription termination sequence, the transcription termination sequence of bovine growth hormone (BGH) gene or the transcription termination sequence of simian virus 40 (SV40) can be used. The expression unit may be single-stranded or double-stranded, and may be linear or circular. The termini of the DNA may be labeled or modified with functional groups. Further, the expression unit may be incorporated into a known plasmid vector or viral vector and the resulting expression unit may be used.

Method of Incorporating Target Sequence into Genome of Cell and Method of Producing Cell

Hereinafter, the method of incorporating a target sequence into a cell genome using the modified piggyBac transposase of the first embodiment will be described. As illustrated in FIG. 2, the method of incorporating a target sequence into a cell genome includes introducing donor DNA into a cell with the modified piggyBac transposase 1 (S1: introduction step).

First, donor DNA will be described. As illustrated in FIG. 3, a donor DNA 20 contains a target sequence 21. The donor DNA 20 is, for example, double-stranded DNA, and may be linear or circular. For example, the donor DNA 20 may be a plasmid or viral vector in which the target sequence 21 is incorporated.

The target sequence 21 is a base sequence of DNA incorporated into the cell genome, and is selected according to the purpose of performing the present method. The target sequence 21 includes, for example, a base sequence encoding a specific gene or a part of a gene, a gene expression cassette containing a promoter sequence, a specific gene, and a transcription termination sequence, or a natural or unnatural base sequence that is not a gene. Alternatively, the target sequence 21 may contain a base sequence encoding one to several amino acids, a sequence including three to several dozens of nucleotides, and the like.

The target sequence 21 contains a first transposase recognition sequence 22a and a second transposase recognition sequence 22b at both ends thereof, respectively. The first transposase recognition sequence 22a and the second transposase recognition sequence 22b are sequences by which the transposase recognizes the position of the target sequence 21. The first transposase recognition sequence 22a and the second transposase recognition sequence 22b are sequences, also referred to as “inverted repeat sequences (IRs)” containing identical inverted sequences.

The region excluding the first transposase recognition sequence 22a and the second transposase recognition sequence 22b of the target sequence 21 has, for example, a length of about 3 to about 20000 bases.

The term “introducing the modified piggyBac transposase 1 into a cell” includes when introducing the modified piggyBac transposase 1 in the form of a polypeptide and when introducing the modified piggyBac transposase 1 in the form of RNA or DNA to express the modified piggyBac transposase 1 in a cell.

An example of the process by which the target sequence 21 is introduced into the cell genome when the modified piggyBac transposase 1 is incorporated in the form of RNA will be described with reference to FIG. 4. First, as illustrated in part (a) of FIG. 4, when the donor DNA 20 and an RNA 30 of the modified piggyBac transposase 1 are introduced into a cell 40, the modified piggyBac transposase 1 (“PB” in the figure) is expressed from the RNA 30 by the protein synthesis function of the cell 40 as illustrated in part (b) of FIG. 4. As illustrated in part (c) of FIG. 4, the donor DNA 20 and the modified piggyBac transposase 1 can translocate into a nucleus 41.

Then, the modified piggyBac transposase 1 binds to the donor DNA 20 in the nucleus (part (d) of FIG. 4). For example, two modified piggyBac transposases 1 can bind to the first transposase recognition sequence 22a and the second transposase recognition sequence 22b of the donor DNA 20, respectively. The modified piggyBac transposase 1 then excises the target sequence 21 of the donor DNA 20, and the target sequence 21 is incorporated into a genome 42 of the cell 40 as illustrated in part (e) of FIG. 4.

In the case of using the DNA of the modified piggyBac transposase 1, after the DNA is introduced into the cell 40, the DNA is transcribed and translated, and the modified piggyBac transposase 1 is expressed in a similar manner to part (b) of FIG. 4, followed by the subsequent process. When the modified piggyBac transposase 1 is introduced in the form of a polypeptide, it can translocate directly into the nucleus 41.

Since the modified piggyBac transposase 1 of the embodiment has the NLS domain 2, the translocation efficiency into the nucleus 41 is further improved, and the target sequence 21 can be incorporated more efficiently.

Further, when the region encoding the PB domain 3 of the RNA 30 is codon-optimized according to the species of the cell, the modified piggyBac transposase 1 can be expressed more efficiently and the incorporation efficiency can be further improved.

The form in which the modified piggyBac transposase 1 is introduced may be suitably selected according to the type of introduction method, availability, and the like. However, it is preferable to use the form of RNA. In that case, since the transcription step can be omitted as compared to the case of using the form of DNA, the modified piggyBac transposase 1 can be expressed more quickly and with high efficiency, and the incorporation efficiency can be improved. Further, the modified piggyBac transposase 1 in the form of RNA is not incorporated into the genome 42 of the cell 40, so this is convenient.

The introduction step (S1) can be performed using any of the known methods used for introducing a polypeptide or polynucleotide into a cell. For example, it is preferable to use a liposome method, a lipofection method, an electroporation method, a sonoporation method, a magnetofection method, or the like. As the introduction method, a suitable method is selected according to the type of the cell 40, the type of the target sequence 21, the use of the cell 40 after introduction, and the like.

The liposome method is a more preferable introduction method, and refers to a method in which the donor DNA 20 and the modified piggyBac transposase 1 or a polynucleotide encoding the modified piggyBac transposase 1 are encapsulated in liposomes (lipid particles) and brought into contact with the cell 40. The embodiment of the liposome method will be described in detail later.

The lipofection method refers to a method in which, for example, the donor DNA 20 and the modified piggyBac transposase 1 in the form of a complex with lipids are brought into contact with the cell 40.

It is preferable to introduce about 1 to 100 molecules of each of the donor DNA 20 and the RNA 30.

The cell 40 is preferably an in vitro cell. The in vitro cell 40 can be, for example, an isolated cell, a cultured cell or tissue, or an established cell line. The cell 40 is preferably a mammalian cell, more preferably a human cell.

A target cell in which the target sequence 21 is incorporated into the genome can be produced by the method of incorporating a target sequence into a cell genome of the embodiment using the in vitro cell 40. Therefore, according to the embodiment, there is provided a method of producing a cell that includes the introduction step (S1). The method of producing a cell may further include a step of culturing the cell 40 under conditions suitable for survival after the introduction step (S1). Additionally, the method may further include a step of screening a target cell into which the target sequence 21 is incorporated after culturing.

In the method of producing a cell of the embodiment, the incorporation efficiency is improved by the modified piggyBac transposase 1, so that it is possible to produce the target cell more efficiently. The method of producing a cell of the embodiment can be used, for example, for the production of a target-cell containing pharmaceutical composition, the production of a substance-producing cell, or the like, without limitation.

Alternatively, the cell 40 may be an in vivo cell. In this case, the introduction step (S1) can be performed by administering the donor DNA 20 and the modified piggyBac transposase 1 to the living body. The administration can be performed, for example, by intravenous, subcutaneous, intramuscular, intraarterial, epidural, cerebrospinal, thoracic, intraperitoneal or local intralesional injection or infusion.

Liposome Method

Hereinafter, an example of an embodiment of the liposome method using RNA of the modified piggyBac transposase 1 will be described. However, in the liposome method, it is not always necessary to use the modified piggyBac transposase 1 in the form of RNA. The form of DNA and other forms: the form of a polynucleotide or the form of a polypeptide may be used. However, the form of a polynucleotide is preferred.

Part (a) of FIG. 5 illustrates an example of an introducing carrier 51 obtained by encapsulating the donor DNA 20 and the RNA 30 of the modified piggyBac transposase 1 in lipid particles 50. The introducing carrier 51 is brought into contact with the cell 40, whereby the lipid particles 50 and the cell membrane can be fused by, for example, endocytosis, and the donor DNA 20 and the RNA 30 can be released into the cell 40.

Part (b) of FIG. 5 illustrates an example of introducing carriers 52 and 53 obtained by encapsulating the donor DNA 20 and the RNA 30 in separate lipid particles 50, respectively. The introducing carriers 52 and 53 are used together. These introducing carriers 52 and 53 may be contacted with the cell 40 at the same time, or either one of them may be contacted first.

The lipid particle 50 is an approximately spherical hollow body including a lipid membrane obtained by arranging a plurality of lipid molecules by a non-covalent bond. The donor DNA 20 and/or the RNA 30 is encapsulated in a central lumen of the hollow body. The lipid particle 50 may be a lipid monolayer membrane or a lipid bilayer membrane. Further, the lipid particle 50 may include a single-layer membrane or a multi-layer membrane.

As the material of the lipid particle 50, the base lipids exemplified below can be used. As the base lipid, for example, a lipid that is the main component of the biological membrane can be used. The base lipid is a phospholipid or sphingolipid, such as diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin or cerebroside, or a combination thereof. The base lipid is easy to fuse with the cell membrane. Particularly, in the case of using diacylphosphatidylcholine and diacylphosphatidylethanolamine, the structure and particle size of the lipid particle 50 are easily controlled, and the base lipid is likely to be fused with the cell membrane, which is preferable. The hydrocarbon chain of the acyl group in the lipid preferably has a length of C10 to C20. This hydrocarbon chain may be a saturated hydrocarbon group or an unsaturated hydrocarbon group.

As the base lipid, it is preferable to use

1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),

1,2-stearoyl-sn-glycero-3-phosphoethanolamine (DSPE),

1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC),

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC),

1,2-di-O-octadecyl-3-trimethylammonium propane (DOTMA),

1,2-dioleoyl-3-dimethylammonium propane (DODAP),

1,2-dimyristoyl-3-dimethylammonium propane (14: 0 DAP),

1,2-dipalmitoyl-3-dimethylammonium propane (16: 0 DAP),

1,2-distearoyl-3-dimethylammonium propane (18: 0 DAP),

N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan (DOBAQ),

1,2-dioleoyl-3-trimethylammonium propane (DOTAP),

1,2-dioleoyl-sn-glycero-3-phosphochlorine (DOPC),

1,2-dilinoleoyl-sn-glycero-3-phosphochlorine (DLPC),

1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), or cholesterol,

or a combination of any of these base lipids. As the base lipid, it is particularly preferable to use a cationic lipid or a neutral lipid, and the acid dissociation constant of the lipid particle 50 can be adjusted by the content of the cationic lipid or the neutral lipid. It is preferable to use DOTAP as the cationic lipid, and it is preferable to use DOPE as the neutral lipid.

The base lipid may be contained in an amount close to 100% of the total lipid molecules contained in the lipid particle 50. Preferably, in addition to the base lipid, a first lipid compound and/or a second lipid compound as exemplified below are further contained. When these lipid compounds are contained, the base lipid is preferably contained in an amount of about 30% to about 80% (mole ratio) with respect to the total lipid molecules.

The first lipid compound is, for example, biodegradable. The first lipid compound can be represented by the formula Q-CHR2, wherein Q is a nitrogen-containing aliphatic group containing two or more tertiary nitrogen but containing no oxygen, and each R is independently an aliphatic group of C12 to C24 provided that at least one R has, in the main chain or side chain thereof, a linking group LR selected from the group consisting of —C(═O)—O—, —O—C(═O)—, —O—C(═O)—O—, —S—C(═O)—, —(═O)—S—, —C(═O)—NH—, and —NHC(═O)—.

When the lipid particle 50 contains the first lipid compound, the surface of the lipid particle 50 becomes non-cationic, because of which the obstacle in the cell introduction can be reduced and the introduction efficiency of the encapsulated substance can be increased.

For example, when lipid having structures represented by the following formulas is used as the first lipid compound, the introduction efficiency is more excellent, and this is preferred.

[Image Omitted]

In particular, the lipid particle 50 preferably contains a lipid compound of Formula (1-01) and/or a lipid compound of Formula (1-02) as a constituent component thereof.

The second lipid compound is, for example, biodegradable. The second lipid compound can be represented by the formula P—[X—W—Y—W—Z]2.

- (wherein P is an alkyleneoxy having one or more ether bonds in the main chain,
- each X is independently a divalent linking group having a tertiary amine structure,
- each W is independently a C1 to C6 alkylene,
- each Y is independently a divalent linking group selected from the group consisting of single bond, ether bond, carboxylic ester bond, thiocarboxylic ester bond, thioester bond, amide bond, carbamate bond, and urea bond,
- each W′ is independently a single bond or a C1 to C6 alkylene, and
- each Z is independently a liposoluble vitamin residue, a sterol residue, or a C12 to C22 aliphatic hydrocarbon group.)

When the second lipid compound is contained, the amount of nucleic acid encapsulated in the lipid particle 50 may be large.

For example, when the second lipid compound having the following structures are used, the amount of nucleic acid encapsulated is more excellent, and this is preferred.

[Image Omitted]

When the lipid particle 50 containing the first and second lipid compounds described above is used, it is possible to increase the encapsulation amount and increase the introduction efficiency. Moreover, the cell death of the introduced cells can be reduced. In particular, when the compounds of Formulas (1-01), (1-02) and/or (2-01) are used, the encapsulation amount and the introduction efficiency are particularly excellent, and this is preferred.

The first and second lipid compounds are preferably contained in an amount of about 20% to about 70% (mole ratio) with respect to the total materials of the lipid particle 50.

It is also preferable that the lipid particle 50 contains a lipid that prevents the lipid particle 50 from aggregating. For example, the lipid that prevents aggregation preferably further contains a PEG-modified lipid, such as polyethylene glycol (PEG) dimyristylglycerol (DMG-PEG), polyamide oligomer derived from ω-amino (oligoethyleneglycol) alkanoic acid monomers (U.S. Pat. No. 6,320,017 B), or monosialoganglioside. Such lipids are preferably contained in an amount of about 1% to about 5% (mole ratio) with respect to the total materials of the lipid particle 50.

The lipid particle 50 may contains lipids such as relatively low-toxic lipids for regulating toxicity; lipids having a functional group that binds a ligand to the lipid particle 50; and lipids for suppressing leakage of an encapsulated substance such as sterol (e.g., cholesterol). In particular, cholesterol is preferably contained.

The type and composition of the lipid used for the lipid particle 50 are suitably selected by taking into consideration the acid dissociation constant (pKa) of the lipid particle 50 to be targeted, the size of the lipid particle 50, the type of the encapsulated substance, the stability of the cells to be introduced, and the like. The acid dissociation constant (pKa) is preferably in a range of 6.5 to 8.0. When the acid dissociation constant is a value in this range, it is possible to increase the introduction efficiency.

For example, when the lipid particle 50 contains a compound of Formula (1-01) or Formula (1-02) and/or a compound of Formula (2-01), DOPE and/or DOTAP, cholesterol, and DMG-PEG, the encapsulation amount of nucleic acid and the introduction efficiency of nucleic acid are particularly excellent, and this is preferred.

Additional components may be encapsulated in an introducing carrier, if necessary. Examples of the additional components include pH adjusters, osmotic pressure regulators, gene activators or other therapeutic agents for T-cell tumor cells, other diagnostic agents, and the like. Examples of the pH regulators include organic acids such as a citric acid and a salt thereof. Examples of the osmotic pressure regulators include sugar, amino acids, and the like. The gene activators will be described later.

The introducing carrier can be produced, for example, by using a known method used for encapsulating small molecules in lipid particles, such as a Bangham method, an organic solvent extraction method, a surfactant removal method, or a freezing and thawing method. For example, a lipid mixture obtained by immersing the material of the lipid particles 50 in an organic solvent such as alcohol in a desired ratio and an aqueous buffer solution containing a component to be encapsulated are prepared, and the aqueous buffer solution is added to the lipid mixture. The resulting mixture is stirred and suspended to form an introducing carrier.

The donor DNA 20 and the RNA 30 may be encapsulated in a condensed state with a nucleic acid condensing peptide 60. The nucleic acid condensing peptide 60 is a peptide having a function of condensing nucleic acids into small particles. By using the nucleic acid condensing peptide 60, a large amount of nucleic acids can be encapsulated in the lipid particles 50, and the particle size of the lipid particles 50 can be reduced. Further, the amount of nucleic acids remaining outside the lipid particles 50 is reduced, thereby preventing aggregation of introducing carriers. As a result, the delivery efficiency of nucleic acids can be improved.

The preferred nucleic acid condensing peptide 60 is, for example, a peptide containing 45% or more of the total cationic amino acids. The more preferred nucleic acid condensing peptide 60 has a sequence RRRRRR (SEQ ID NO: 23) (first amino acid sequence) at one end and a sequence RQRQR (SEQ ID NO: 22) (second amino acid sequence) at the other end. Zero or one or more intermediate sequences of RRRRRR (SEQ ID NO: 23) or RQRQR (SEQ ID NO: 22) are contained between the first amino acid sequence and the second amino acid sequence. Further, two or more neutral amino acids are contained between two adjacent sequences of the first amino acid sequence, the second amino acid sequence, and the intermediate sequence. The neutral amino acids are, for example, G or Y. Alternatively, the other end may have the sequence RRRRRR (SEQ ID NO: 23) (first amino acid sequence) instead of the second amino acid sequence.

The above-described nucleic acid condensing peptides preferably has the following amino acid sequences:

(SEQ ID NO: 11)

RQRQRYYRQRQRGGRRRRRR;

(SEQ ID NO: 12)

RQRQRGGRRRRRR;

and

(SEQ ID NO: 13)

RRRRRRYYRQRQRGGRRRRRR.

Further, a nucleic acid condensing peptide having the following amino acid sequence can be used in combination with any of the above-described nucleic acid condensing peptides. This peptide can further condense a nucleic acid aggregate formed by condensation of the above-described nucleic acid condensing peptides.

(SEQ ID NO: 14)

GNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (M9)

For example, the nucleic acid can be condensed by stirring and mixing the nucleic acid with the nucleic acid condensing peptide 60 before being encapsulated in the lipid particles 50. The donor DNA 20 and the RNA 30 may be condensed together or separately.

It is preferable to use the nucleic acid condensing peptide 60 because the effects described above are obtained. However, the nucleic acid condensing peptide 60 is not necessarily used depending on the type of nucleic acid used, the type of cell, and the like.

Kit

According to the embodiment, there is provided a kit used for incorporating the target sequence 21 into the cell genome. The kit includes at least one of the modified piggyBac transposases 1 or the polynucleotide encoding the modified piggyBac transposase 1.

For example, the modified piggyBac transposase 1 or the polynucleotide encoding the modified piggyBac transposase 1 may be provided as a composition contained in a suitable carrier. Alternatively, the modified piggyBac transposase 1 or the polynucleotide encoding the modified piggyBac transposase 1 may be provided in the form of the introducing carrier as the composition contained in a suitable carrier. Examples of the suitable carrier include water, saline such as physiological saline, glycine aqueous solutions or buffers, and the like.

The composition may be sterilized by common methods. Further, the composition may also be provided as a liquid or as a dry powder. For example, the powder composition can be used by dissolving in a suitable liquid.

The kit may further include substances that improve the storage stability of the composition. The substances that improve the storage stability are not limited, and examples thereof include glycoproteins such as albumin, lipoprotein, apolipoprotein, and globulin: pH adjusters, buffer agents, tension regulators, and the like; pharmaceutically acceptable and involved agents that bring the pharmaceutical composition closer to the physiological state, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride; lipophilic free-radical quenchers that suppress free radical damage, such as α-tocopherol; and lipid protectants such as a water-soluble chelator such as ferrioxamine for suppressing peroxidation damage of lipids and improving storage stability. These substances may be contained in the composition or may be included in the kit separately from the composition.

The kit may further include the donor DNA 20, unless the kit includes an introducing carrier in which donor DNA is encapsulated.

Second Embodiment

According to the second embodiment, there is provided a modified piggyBac transposase that further includes a cell division promoting domain. As illustrated in of part (a) of FIG. 6, a modified piggyBac transposase 10 of the second embodiment further includes a cell division promoting domain 4 between, for example, a nuclear localization signal domain 2 and a piggyBac transposase domain 3.

The cell division promoting domain 4 includes an amino acid sequence of a peptide having a function of promoting cell division. As the cell division promoting domain 4, any known peptide known to have such a function can be used. For example, it is preferable to use a sequence having a region of the N-terminus of the SV40 large T antigen protein to 133 amino acids (SEQ ID NO: 15) as shown in Table 11. This sequence contains the J domain and the Rb family-binding motif.

TABLE 11

Cell division promoting domain including J domain and Rb

family-binding motif of SV40 large T antigen protein (SEQ ID NO: 15)

DKVLNREESL QLMDLLGLER SAWGNIPLMR KAYLKKCKEF HPDKGGDEEK MKKMNTLYKK
60

MEDGVKYAHQ PDFGGFWDAT EIPTYGTDEW EQWWNAFNEE NLFCSEEMPS SDDEATADSQ
120

HSTPPKKKRK VE
132

The cell division promoting domain 4 is not limited to the above, and it is possible to use, for example, a cell division promoting domain of a large T antigen protein of JC virus or BK virus belonging to human polyomavirus.

When the cell division promoting domain 4 is included, the order in which each domain is linked is, for example, the NLS domain 2, the cell division promoting domain 4, and the PB domain 3 from the N-terminal side, as illustrated in part (a) of FIG. 6. However, the order of the domains is not limited to the above. For example, the cell division promoting domain 4, the NLS domain 2, and the PB domain 3 may be linked in this order from the N-terminal side. Alternatively, the domains may be linked in another order.

The second embodiment also provides a polynucleotide encoding the modified piggyBac transposase 10. When the amino acid sequence of SEQ ID NO: 15 is used as the cell division promoting domain 4, it is preferable to use, for example, the sequence shown in Table 12 (SEQ ID NO: 16) as the DNA sequence encoding the amino acid sequence.

TABLE 12

DNA sequence encoding cell division promoting

domain of SEQ ID NO: 15 (SEQ ID NO: 16)

ATGGATAAAG TTTTAAACAG AGAGGAATCT TTGCAGCTAA TGGACCTTCT AGGTCTTGAA
60

AGGAGTGCCT GGGGGAATAT TCCTCTGATG AGAAAGGCAT ATTTAAAAAA ATGCAAGGAG
120

TTTCATCCTG ATAAAGGAGG AGATGAAGAA AAAATGAAGA AAATGAATAC TCTGTACAAG
180

AAAATGGAAG ATGGAGTAAA ATATGCTCAT CAACCTGACT TTGGAGGCTT CTGGGATGCA
240

ACTGAGATTC CAACCTATGG AACTGATGAA TGGGAGCAGT GGTGGAATGC CTTTAATGAG
300

GAAAACCTGT TTTGCTCAGA AGAAATGCCA TCTAGTGATG ATGAGGCTAC TGCTGACTCT
360

CAACATTCTA CTCCTCCAAA AAAGAAGAGA AAGGTAGAA
399

As the amino acid sequence or DNA sequence shown in Tables 11 and 12, the sequences having mutations such as substitutions, additions, insertions, and deletions can also be used. For example, when such mutations are present, it is preferable to use the sequence having a base sequence encoding an amino acid sequence having 90% or more of homology with the amino acid sequence encoded by the sequences shown in Tables 11 and 12.

The modified piggyBac transposase 10 of the second embodiment can also be provided as an introducing carrier similar to the introducing carrier described in the first embodiment. Further, similarly to the first embodiment, the modified piggyBac transposase 10 can be used in the method of incorporating a target sequence into a cell genome and the method of producing a cell.

However, when the modified piggyBac transposase 10 of the second embodiment is introduced into a cell 40, the division of the cell 40 may be temporarily facilitated because of the cell division promoting function of the cell division promoting domain 4. Nuclear membrane loss that occurs during cell division of the cell 40 can further facilitate the translocation of the modified piggyBac transposase 10 and a donor DNA 20 into a nucleus 41. Therefore, according to the modified piggyBac transposase 10 of the second embodiment, the incorporation efficiency of a target sequence 21 can be further improved.

According to a further embodiment, the cell division promoting domain 4 does not necessarily have to be included in the modified piggyBac transposase. As illustrated in part (b) of FIG. 6, the modified piggyBac transposase 1 of the first embodiment and a polypeptide containing the cell division promoting domain 4 separated from the modified piggyBac transposase 1 may be used together. At this time, the polypeptide containing the cell division promoting domain 4 may further contain the NLS domain 2.

EXAMPLES

An example of producing and using the modified piggyBac transposase of the embodiment will be described hereinbelow.

Example 1 Synthesis of DNA Sequence of Modified PiggyBac Transposase
Synthesis of PB Domain DNA Sequence

A DNA sequence of the gene of the wild-type piggyBac transposase (WtPB, SEQ ID NO: 1) (SEQ ID NO: 5) and a DNA sequence of human codon-optimized PB (HuPB) (SEQ ID NO: 6) in which the DNA sequence of SEQ ID NO: 5 was codon-optimized for humans were synthesized. In addition, a DNA sequence (SEQ ID NO: 7) of hyper-active PB (HyPB) having a modified amino acid sequence was synthesized.

Synthesis of NLS Domain DNA Sequence

As the NLS domain, the nuclear transport signal of the TAT protein of HIV (TAT NLS, SEQ ID NO: 4) and the nuclear transport signal of the SV40 large T antigen protein (SV40 NLS, SEQ ID NO: 3) were used. A DNA sequence of TAT NLS codon-optimized for humans (SEQ ID NO: 9), a DNA sequence of SV40 NLS codon-optimized for humans (SEQ ID NO: 8), and a DNA sequence in which TAT NLS and SV40 NLS were linked in this order via a linker sequence (TAT-SV40 NLS, SEQ ID NO: 10) were synthesized.

Synthesis of Cell Division Promoting Domain DNA Sequence

As the cell division promoting domain, the N-terminal 133-amino acid sequence containing the J domain and the Rb domain of the SV40 large T antigen protein (LT-J/Rb, SEQ ID NO: 15) was used. A DNA sequence (SEQ ID NO: 16) encoding the amino acid sequence of SEQ ID NO: 15 was synthesized.

Synthesis of DNA Sequence of Modified PiggyBac Transposase of First Embodiment

There was produced T-HyPB (SEQ ID NO: 17) in which the DNA sequence of TAT NLS was added to an N-terminus of HyPB via a linker sequence, and TS-HyPB (SEQ ID NO: 18) in which a linker sequence was linked to TAT-SV40 NLS and added to an N-terminus of HyPB. These sequences are shown in Tables 13 and 14, respectively.

TABLE 13

T-HyPB (SEQ ID NO: 17)

ATGGGCGGCA GAAAGAAGAG AAGACAGAGA AGAAGACCCC CCGCCTCCTC CCTCGATGAC
60

GAGCACATTC TGTCCGCTCT GCTGCAGTCC GACGATGAGC TGGTCGGAGA AGACAGCGAT
120

AGCGAGATCA GCGACCACGT CTCCGAGGAC GACGTCCAAA GCGACACAGA GGAGGCCTTC
180

ATCGACGAGG TGCACGAGGT GCAGCCTACC AGCAGCGGCT CCGAGATCCT GGACGAGCAG
240

AACGTGATCG AGCAGCCCGG CAGCTCCCTG GCCAGCAACA GGATCCTGAC CCTGCCCCAG
300

AGGACCATCA GGGGCAAGAA CAAGCACTGC TGGTCCACCT CCAAGCCCAC CAGGCGGAGC
360

AGGGTGTCCG CCCTGAACAT CGTGAGAAGC CAGAGGGGCC CCACCAGGAT GTGCAGGAAC
420

ATCTACGACC CCCTGCTGTG CTTCAAGCTG TTCTTCACCG ACGAGATCAT CAGCGAGATC
480

GTGAAGTGGA CCAACGCCGA GATCAGCCTG AAGAGGCGGG AGAGCATGAC CTCCGCCACC
540

TTCAGGGACA CCAACGAGGA CGAGATCTAC GCCTTCTTCG GCATCCTGGT GATGACCGCC
600

GTGAGGAAGG ACAACCACAT GAGCACCGAC GACCTGTTCG ACAGATCCCT GAGCATGGTG
660

TACGTGAGCG TGATGAGCAG GGACAGATTC GACTTCCTGA TCAGATGCCT GAGGATGGAC
720

GACAAGAGCA TCAGGCCCAC CCTGCGGGAG AACGACGTGT TCACCCCCGT GAGAAAGATC
780

TGGGACCTGT TCATCCACCA GTGCATCCAG AACTACACCC CTGGCGCCCA CCTGACCATC
840

GACGAGCAGC TGCTGGGCTT CAGGGGCAGG TGCCCCTTCA GGGTCTATAT CCCCAACAAG
900

CCCAGCAAGT ACGGCATCAA GATCCTGATG ATGTGCGACA GCGGCACCAA GTACATGATC
960

AACGGCATGC CCTACCTGGG CAGGGGCACC CAGACCAACG GCGTGCCCCT GGGCGAGTAC
1020

TACGTGAAGG AGCTGTCCAA GCCCGTCCAC GGCAGCTGCA GAAACATCAC CTGCGACAAC
1080

TGGTTCACCA GCATCCCCCT GGCCAAGAAC CTGCTGCAGG AGCCCTACAA GCTGACCATC
1140

GTGGGCACCG TGAGAAGCAA CAAGAGAGAG ATCCCCGAGG TCCTGAAGAA CAGCAGGTCC
1200

AGGCCCGTGG GCACCAGCAT GTTCTGCTTC GACGGCCCCC TGACCCTGGT GTCCTACAAG
1260

CCCAAGCCCG CCAAGATGGT GTACCTGCTG TCCAGCTGCG ACGAGGACGC CAGCATCAAC
1320

GAGAGCACCG GCAAGCCCCA GATGGTGATG TACTACAACC AGACCAAGGG CGGCGTGGAC
1380

ACCCTGGACC AGATGTGCAG CGTGATGACC TGCAGCAGAA AGACCAACAG GTGGCCCATG
1440

GCCCTGCTGT ACGGCATGAT CAACATCGCC TGCATCAACA GCTTCATCAT CTACAGCCAC
1500

AACGTGAGCA GCAAGGGCGA GAAGGTGCAG AGCCGGAAAA AGTTCATGCG GAACCTGTAC
1560

ATGGGCCTGA CCTCCAGCTT CATGAGGAAG AGGCTGGAGG CCCCCACCCT GAAGAGATAC
1620

CTGAGGGACA ACATCAGCAA CATCCTGCCC AAAGAGGTGC CCGGCACCAG CGACGACAGC
1680

ACCGAGGAGC CCGTGATGAA GAAGAGGACC TACTGCACCT ACTGTCCCAG CAAGATCAGA
1740

AGAAAGGCCA GCGCCAGCTG CAAGAAGTGT AAGAAGGTCA TCTGCCGGGA GCACAACATC
1800

GACATGTGCC AGAGCTGTTT CTGA
1824

TABLE 14

TS-HyPB (SEQ ID NO: 18)

ATGGGCGGCA GAAAGAAGAG AAGACAGAGA AGAAGACCCC CCGCCGGCAC CAGCGTGAGC
60

CTGAAGAAGA AGAGAAAGGT GCCCCCCGCC TCCTCCCTCG ATGACGAGCA CATTCTGTCC
120

GCTCTGCTGC AGTCCGACGA TGAGCTGGTC GGAGAAGACA GCGATAGCGA GGTGAGCGAC
180

CACGTCTCCG AGGACGACGT CCAAAGCGAC ACAGAGGAGG CCTTCATCGA CGAGGTGCAC
240

GAGGTGCAGC CTACCAGCAG CGGCTCCGAG ATCCTGGACG AGCAGAACGT GATCGAGCAG
300

CCCGGCAGCT CCCTGGCCAG CAACAGGATC CTGACCCTGC CCCAGAGGAC CATCAGGGGC
360

AAGAACAAGC ACTGCTGGTC CACCTCCAAG CCCACCAGGC GGAGCAGGGT GTCCGCCCTG
420

AACATCGTGA GAAGCCAGAG GGGCCCCACC AGGATGTGCA GGAACATCTA CGACCCCCTG
480

CTGTGCTTCA AGCTGTTCTT CACCGACGAG ATCATCAGCG AGATCGTGAA GTGGACCAAC
540

GCCGAGATCA GCCTGAAGAG GCGGGAGAGC ATGACCTCCG CCACCTTCAG GGACACCAAC
600

GAGGACGAGA TCTACGCCTT CTTCGGCATC CTGGTGATGA CCGCCGTGAG GAAGGACAAC
660

CACATGAGCA CCGACGACCT GTTCGACAGA TCCCTGAGCA TGGTGTACGT GAGCGTGATG
720

AGCAGGGACA GATTCGACTT CCTGATCAGA TGCCTGAGGA TGGACGACAA GAGCATCAGG
780

CCCACCCTGC GGGAGAACGA CGTGTTCACC CCCGTGAGAA AGATCTGGGA CCTGTTCATC
840

CACCAGTGCA TCCAGAACTA CACCCCTGGC GCCCACCTGA CCATCGACGA GCAGCTGCTG
900

GGCTTCAGGG GCAGGTGCCC CTTCAGGGTC TATATCCCCA ACAAGCCCAG CAAGTACGGC
960

ATCAAGATCC TGATGATGTG CGACAGCGGC ACCAAGTACA TGATCAACGG CATGCCCTAC
1020

CTGGGCAGGG GCACCCAGAC CAACGGCGTG CCCCTGGGCG AGTACTACGT GAAGGAGCTG
1080

TCCAAGCCCG TCCACGGCAG CTGCAGAAAC ATCACCTGCG ACAACTGGTT CACCAGCATC
1140

CCCCTGGCCA AGAACCTGCT GCAGGAGCCC TACAAGCTGA CCATCGTGGG CACCGTGAGA
1200

AGCAACAAGA GAGAGATCCC CGAGGTCCTG AAGAACAGCA GGTCCAGGCC CGTGGGCACC
1260

AGCATGTTCT GCTTCGACGG CCCCCTGACC CTGGTGTCCT ACAAGCCCAA GCCCGCCAAG
1320

ATGGTGTACC TGCTGTCCAG CTGCGACGAG GACGCCAGCA TCAACGAGAG CACCGGCAAG
1380

CCCCAGATGG TGATGTACTA CAACCAGACC AAGGGCGGCG TGGACACCCT GGACCAGATG
1440

TGCAGCGTGA TGACCTGCAG CAGAAAGACC AACAGGTGGC CCATGGCCCT GCTGTACGGC
1500

ATGATCAACA TCGCCTGCAT CAACAGCTTC ATCATCTACA GCCACAACGT GAGCAGCAAG
1560

GGCGAGAAGG TGCAGAGCCG GAAAAAGTTC ATGCGGAACC TGTACATGGG CCTGACCTCC
1620

AGCTTCATGA GGAAGAGGCT GGAGGCCCCC ACCCTGAAGA GATACCTGAG GGACAACATC
1680

AGCAACATCC TGCCCAAAGA GGTGCCCGGC ACCAGCGACG ACAGCACCGA GGAGCCCGTG
1740

ATGAAGAAGA GGACCTACTG CACCTACTGT CCCAGCAAGA TCAGAAGAAA GGCCAGCGCC
1800

AGCTGCAAGA AGTGTAAGAA GGTCATCTGC CGGGAGCACA ACATCGACAT GTGCCAGAGC
1860

TGTTTCTGA
1869

These DNA sequences of modified piggyBac transposase were produced by chemically synthesizing the N-terminal DNA sequence of TAT NLS or TAT-SV40 NLS and HyPB, and then linking the resulting synthetic DNA sequence to the DNA sequence of HyPB from which the synthetic N-terminal sequence had been removed.

Synthesis of DNA Sequence of Modified PiggyBac Transposase of Second Embodiment

A DNA sequence shown in Table 15 (SEQ ID NO: 19), in which TAT-SV40 NLS (SEQ ID NO: 10) and LT-J/Rb (SEQ ID NO: 16) were linked, was added to an N-terminus of HyPB via a linker sequence, whereby TS-LTJ/Rb-HyPB (SEQ ID NO: 20) shown in Table 16 was produced.

TABLE 15

TAT-SV40LT-NLS-added T-J/Rb (SEQ ID NO: 19)

ATGGGCGGCA GAAAGAAGAG AAGACAGAGA AGAAGACCCC CCGCCGGCAC CAGCGTGAGC
60

CTGAAGAAGA AGAGAAAGGT GCCCCCCGCC GATAAAGTTT TAAACAGAGA GGAATCTTTG
120

CAGCTAATGG ACCTTCTAGG TCTTGAAAGG AGTGCCTGGG GGAATATTCC TCTGATGAGA
180

AAGGCATATT TAAAAAAATG CAAGGAGTTT CATCCTGATA AAGGAGGAGA TGAAGAAAAA
240

ATGAAGAAAA TGAATACTCT GTACAAGAAA ATGGAAGATG GAGTAAAATA TGCTCATCAA
300

CCTGACTTTG GAGGCTTCTG GGATGCAACT GAGATTCCAA CCTATGGAAC TGATGAATGG
360

GAGCAGTGGT GGAATGCCTT TAATGAGGAA AACCTGTTTT GCTCAGAAGA AATGCCATCT
420

AGTGATGATG AGGCTACTGC TGACTCTCAA CATTCTACTC CTCCAAAAAA GAAGAGAAAG
480

GTAGAA
486

TABLE 16

TS-LTJ-HyPB (SEQ ID NO: 20)

ATGGGCGGCA GAAAGAAGAG AAGACAGAGA AGAAGACCCC CCGCCGGCAC CAGCGTGAGC
60

CTGAAGAAGA AGAGAAAGGT GCCCCCCGCC GATAAAGTTT TAAACAGAGA GGAATCTTTG
120

CAGCTAATGG ACCTTCTAGG TCTTGAAAGG AGTGCCTGGG GGAATATTCC TCTGATGAGA
180

AAGGCATATT TAAAAAAATG CAAGGAGTTT CATCCTGATA AAGGAGGAGA TGAAGAAAAA
240

ATGAAGAAAA TGAATACTCT GTACAAGAAA ATGGAAGATG GAGTAAAATA TGCTCATCAA
300

CCTGACTTTG GAGGCTTCTG GGATGCAACT GAGATTCCAA CCTATGGAAC TGATGAATGG
360

GAGCAGTGGT GGAATGCCTT TAATGAGGAA AACCTGTTTT GCTCAGAAGA AATGCCATCT
420

AGTGATGATG AGGCTACTGC TGACTCTCAA CATTCTACTC CTCCAAAAAA GAAGAGAAAG
480

GTAGAAGGTG GTGGTGGTTC TGGTGGTGGT GGTTCTTCCT CCCTCGATGA CGAGCACATT
540

CTGTCCGCTC TGCTGCAGTC CGACGATGAG CTGGTCGGAG AAGACAGCGA TAGCGAGGTG
600

AGCGACCACG TCTCCGAGGA CGACGTCCAA AGCGACACAG AGGAGGCCTT TATCATCGAC
660

GAGGTGCACG AGGTGCAGCC TACCAGCAGC GGCTCCGAGA TCCTGGACGA GCAGAACGTG
720

ATCGAGCAGC CCGGCAGCTC CCTGGCCAGC AACAGGATCC TGACCCTGCC CCAGAGGACC
780

ATCAGGGGCA AGAACAAGCA CTGCTGGTCC ACCTCCAAGC CCACCAGGCG GAGCAGGGTG
840

TCCGCCCTGA ACATCGTGAG AAGCCAGAGG GGCCCCACCA GGATGTGCAG GAACATCTAC
900

GACCCCCTGC TGTGCTTCAA GCTGTTCTTC ACCGACGAGA TCATCAGCGA GATCGTGAAG
960

TGGACCAACG CCGAGATCAG CCTGAAGAGG CGGGAGAGCA TGACCTCCGC CACCTTCAGG
1020

GACACCAACG AGGACGAGAT CTACGCCTTC TTCGGCATCC TGGTGATGAC CGCCGTGAGG
1080

AAGGACAACC ACATGAGCAC CGACGACCTG TTCGACAGAT CCCTGAGCAT GGTGTACGTG
1140

AGCGTGATGA GCAGGGACAG ATTCGACTTC CTGATCAGAT GCCTGAGGAT GGACGACAAG
1200

AGCATCAGGC CCACCCTGCG GGAGAACGAC GTGTTCACCC CCGTGAGAAA GATCTGGGAC
1260

CTGTTCATCC ACCAGTGCAT CCAGAACTAC ACCCCTGGCG CCCACCTGAC CATCGACGAG
1320

CAGCTGCTGG GCTTCAGGGG CAGGTGCCCC TTCAGGGTCT ATATCCCCAA CAAGCCCAGC
1380

AAGTACGGCA TCAAGATCCT GATGATGTGC GACAGCGGCA CCAAGTACAT GATCAACGGC
1440

ATGCCCTACC TGGGCAGGGG CACCCAGACC AACGGCGTGC CCCTGGGCGA GTACTACGTG
1500

AAGGAGCTGT CCAAGCCCGT CCACGGCAGC TGCAGAAACA TCACCTGCGA CAACTGGTTC
1560

ACCAGCATCC CCCTGGCCAA GAACCTGCTG CAGGAGCCCT ACAAGCTGAC CATCGTGGGC
1620

ACCGTGAGAA GCAACAAGAG AGAGATCCCC GAGGTCCTGA AGAACAGCAG GTCCAGGCCC
1680

GTGGGCACCA GCATGTTCTG CTTCGACGGC CCCCTGACCC TGGTGTCCTA CAAGCCCAAG
1740

CCCGCCAAGA TGGTGTACCT GCTGTCCAGC TGCGACGAGG ACGCCAGCAT CAACGAGAGC
1800

ACCGGCAAGC CCCAGATGGT GATGTACTAC AACCAGACCA AGGGCGGCGT GGACACCCTG
1860

GACCAGATGT GCAGCGTGAT GACCTGCAGC AGAAAGACCA ACAGGTGGCC CATGGCCCTG
1920

CTGTACGGCA TGATCAACAT CGCCTGCATC AACAGCTTCA TCATCTACAG CCACAACGTG
1980

AGCAGCAAGG GCGAGAAGGT GCAGAGCCGG AAAAAGTTCA TGCGGAACCT GTACATGGGC
2040

CTGACCTCCA GCTTCATGAG GAAGAGGCTG GAGGCCCCCA CCCTGAAGAG ATACCTGAGG
2100

GACAACATCA GCAACATCCT GCCCAAAGAG GTGCCCGGCA CCAGCGACGA CAGCACCGAG
2160

GAGCCCGTGA TGAAGAAGAG GACCTACTGC ACCTACTGTC CCAGCAAGAT CAGAAGAAAG
2220

GCCAGCGCCA GCTGCAAGAA GTGTAAGAAG GTCATCTGCC GGGAGCACAA CATCGACATG
2280

TGCCAGAGCT GTTTCTGA
2298

TS-LTJ/Rb-HyPB was produced by chemically synthesizing DNA sequences of the N-termini of TAT-SV40 NLS, LTJ/Rb, the linker sequence, and HyPB, and then linking the resulting sequence to the DNA sequence of HyPB from which the synthetic N-terminal sequence was removed. Further, a DNA sequence shown in Table 17 (TS-LTJ/Rb, SEQ ID NO: 21) was produced as a polypeptide of the cell division promoting domain alone that does not link to PB.

TABLE 17

TS-LTJ/Rb (SEQ ID NO: 21)

ATGGGCGGCA GAAAGAAGAG AAGACAGAGA AGAAGACCCC CCGCCGGCAC CAGCGTGAGC
60

CTGAAGAAGA AGAGAAAGGT GCCCCCCGCC GATAAAGTTT TAAACAGAGA GGAATCTTTG
120

CAGCTAATGG ACCTTCTAGG TCTTGAAAGG AGTGCCTGGG GGAATATTCC TCTGATGAGA
180

AAGGCATATT TAAAAAAATG CAAGGAGTTT CATCCTGATA AAGGAGGAGA TGAAGAAAAA
240

ATGAAGAAAA TGAATACTCT GTACAAGAAA ATGGAAGATG GAGTAAAATA TGCTCATCAA
300

CCTGACTTTG GAGGCTTCTG GGATGCAACT GAGATTCCAA CCTATGGAAC TGATGAATGG
360

GAGCAGTGGT GGAATGCCTT TAATGAGGAA AACCTGTTTT GCTCAGAAGA AATGCCATCT
420

AGTGATGATG AGGCTACTGC TGACTCTCAA CATTCTACTC CTCCAAAAAA GAAGAGAAAG
480

GTAGAATGA
489

TS-LTJ/Rb was produced through chemical synthesis by adding a stop codon to the terminus of the DNA sequence of TAT-SV40 NLS and LT-J/Rb.

Example 2 Production of Plasmid

As template DNA for RNA preparation of modified PB, a plasmid was produced in which the DNA sequences of PB (WtPB, HuPB, HyPB, T-HyPB, TS-HyPB, and TS-LTJ/Rb-HyPB) obtained in Example 1 were incorporated into pGEM-GL-pA.

pGEM-GL-pA was produced by inserting the leader sequence of human β-globin (Globin leader) and the poly (A) sequence of pSP64 pA vector (Promega Corporation) into the commercially available plasmid DNA for RNA synthesis containing the T7 promoter and the poly (A) sequence: pGEM (registered trademark)-4Z (Promega Corporation). The DNA sequence of PB obtained in Example 1 was incorporated between the human β-globin leader sequence and the poly (A) sequence of pGEM-GL-pA.

Further, there was produced template DNA for RNA preparation of the cell division promoting domain (TS-LTJ/Rb) unlinked to the PB domain. This template DNA plasmid was produced by incorporating the TS-LTJ/Rb DNA sequence between the human β-globin leader sequence and the poly (A) sequence of pGEM-GL-pA.

Example 3 Synthesis of mRNA

The pGEM-GL-pA obtained in Example 2 was used to synthesize mRNA as described below. First, the plasmid DNA was cut with the restriction enzyme EcoRI and purified, and then 20 μL of an in vitro transcription reaction solution containing 1.0 μg of template DNA was prepared and reacted in a constant temperature bath at 37° C. for 2 hours. To the in vitro transcription reaction solution, three types of nucleotides (ATP, CTP, and GTP) supplied in the CUGA (registered trademark) 7 kit and the nucleotide analog ψUTP (Jena Bioscience GmbH) were added. The RNA synthesis procedure was conducted according to the protocol of the CUGA (registered trademark) 7 kit.

After completion of the reaction, synthetic RNA was purified with MEGAclear™ transcription Clean-Up kit (Invitrogen Corporation). The purification of RNA was conducted according to the procedure for the kit.

After purification, a Cap structure and a poly (A) structure were added to the 5′-UTR and 3′-UTR of RNA, respectively, to form mRNA. The Cap structure and the poly (A) structure were added using the ScriptCap™ m7G Capping System (CellScript) and the ScriptCap™ 2′-O-Metyltransferase Kit (CellScript). The operation was performed as follows according to the manual of the kit.

The resulting RNA was incubated at 65° C. for 10 minutes and then immediately placed on ice to disrupt the secondary structure of the RNA. 100 μL of a Capping reaction solution containing 60 μg of this RNA was prepared and reacted in a constant temperature bath at 37° C. for 1 hour in order to add the Cap structure to the 5′-UTR of RNA. The A-Plus™ Poly (A) Polymerase Tailing kit (CellScript) was added to the same solution, and the mixture was reacted in a constant temperature bath at 37° C. for 2 hours in order to add the poly (A) structure to the 3′-UTR. After completion of the reaction, the mRNA in the reaction solution was purified by the ammonium acetate precipitation method according to the manual of the kit. The absorbance (A260) was measured using a spectrophotometer (BioSpec-mini, Shimadzu Corporation) and the RNA concentration was calculated as OD1.0=40.0 μg/mL RNA.

Example 4 Preparation of mRNA-Encapsulating Liposomes

1/20 amount of mRNA solution (1.6 μg/mL) was added to 100 μL of ethanol lipid solution (FFT20 (the compound of Formula (1-02) above)/DOTAP/DOPE/cholesterol/PEG−DMG=37/10.5/5.25/60/4 (mole ratio)) to prepare 105 μL of RNA-containing lipid solution, and then 695 μL of 10 mM HEPES (pH 7.3) was gently added to form a liposome solution. Four sets of this liposome solution were prepared, and these solutions were concentrated by centrifugation at 14,000×g using a centrifugal ultrafiltration tube: Amicon Ultra 0.5 mL, Ultracel-50K (Millipore Corporation). After the concentration, buffer exchange and concentration were carried out by adding 400 μL of 10 mM HEPES (pH 7.3) and the final volume was adjusted to 100 μL. The amount of RNA contained in the liposome was measured by the QuantiFluor™ RNA System (Promega Corporation).