WO2022078310A1 - 新型PiggyBac转座子系统及其用途 - Google Patents

新型PiggyBac转座子系统及其用途 Download PDF

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WO2022078310A1
WO2022078310A1 PCT/CN2021/123191 CN2021123191W WO2022078310A1 WO 2022078310 A1 WO2022078310 A1 WO 2022078310A1 CN 2021123191 W CN2021123191 W CN 2021123191W WO 2022078310 A1 WO2022078310 A1 WO 2022078310A1
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sequence
cells
transposon
promoter
transposase
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PCT/CN2021/123191
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English (en)
French (fr)
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金华君
许馥慧
黄晨
马星明
刘天怡
郭晓春
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上海君赛生物科技有限公司
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Priority to EP21879354.5A priority Critical patent/EP4227414A1/en
Priority to CN202180069727.3A priority patent/CN116490217A/zh
Priority to KR1020237016093A priority patent/KR20230117726A/ko
Priority to JP2023546379A priority patent/JP2023548957A/ja
Priority to US18/248,865 priority patent/US20230383268A1/en
Publication of WO2022078310A1 publication Critical patent/WO2022078310A1/zh

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Definitions

  • the present invention relates to the field of transposon vectors, in particular to a novel PiggyBac transposon system and uses thereof.
  • transgenic systems include virus-based vectors, eukaryotic expression plasmid vectors, and transposon vectors. Genetic modification of human primary T cells using non-viral vector-based methods has proven to be extremely difficult. Thus, worldwide, most laboratories are still using viral vector systems for transgenic modification of cells, including retroviral vectors, such as lentiviral vector systems. Although the viral vector system has been widely used, it has insurmountable problems such as complicated virus preparation operations, relatively high safety risks, and high production costs.
  • transposon vector systems have been increasingly used to modify immune cells for tumor immunotherapy.
  • the earliest transposon applied to mammals was the "Sleeping Beauty” transposon (Sleeping Beauty) derived from fish, but it has defects such as excessive inhibitory effect and small carrying fragment (about 5kb).
  • the application in transgenic manipulation is severely limited.
  • the PiggyBac transposon is another class of transposon systems derived from lepidopteran insects, which can carry larger fragments and can integrate into a variety of eukaryotic host cells.
  • the PiggyBac(PB) transposon system mainly transposes through the "cut-paste" mechanism, which does not leave a footprint in the original site after transposition, and is increasingly used. After transformation, it can be used in the fields of genome research, gene therapy, cell therapy, stem cell induction and post-induction differentiation.
  • WO2019046815A1 discloses a traditional binary system based on the PiggyBac transposon, which includes a vector containing PiggyBac transposase and a helper vector containing 5'ITR and 3'ITR.
  • the PiggyBac transposon system combined with electroporation can introduce foreign genes into T cells, NK cells and HSPC cells.
  • the binary system requires the PiggyBac transposase vector and the auxiliary vector to be transfected into the cells at the same time before transposition can occur, which requires high transfection and is more difficult.
  • the mechanism of the PiggyBac transposition system PiggyBac transposase inserts the transposable fragment into the genome through a "cut-paste" mechanism, and this process is reversible, so as long as the expression of the PiggyBac enzyme continues , the transposition fragments that have been integrated into the genome may also be re-cut, causing genome instability and substantially reducing the transposition efficiency.
  • the transposition efficiency of the common PiggyBac binary transposition system in T cells is usually around 10%, and the efficiency is low.
  • WO2019046815A1 also records that plasmid DNA is highly toxic to T cells, and its toxicity to T cells is related to the amount of DNA used for electroporation. The binary system undoubtedly expands the amount of plasmid DNA required for electroporation, increases the toxicity to cells, especially T cells, and reduces the survival rate of T cells transfected with plasmid DNA.
  • CN105154473B discloses a one-element PiggyBac transposon vector, which combines the PiggyBac transposase vector and the auxiliary vector in the traditional binary PiggyBac transposition system into one vector, and combines the PiggyBac expression cassette with the external expression in the same expression vector.
  • the source gene expression frame shares the same bidirectional polyA sequence. After integration, the polyA in the PiggyBac transposase expression frame is cut and self-inactivating, which effectively reduces the continuous expression of the constitutive PiggyBac transposase.
  • the binary system is simplified into a single unit vector, which greatly reduces the total amount of DNA and reduces the toxicity of exogenous DNA to T cells.
  • the PiggyBac transposase expression box and the exogenous gene expression box share the same bidirectional polyA sequence, which will cause interaction between the two opposite expression boxes, and in some kinds of cells, the single transposon vector mediates The integration efficiency still needs to be further improved.
  • the inventors constructed an integration system based on the PiggyBac transposon, which can mediate the efficient integration of exogenous genes in host cells, and the efficient and stable expression.
  • nucleic acid construct comprising or consisting of the following elements: a transposon 3' terminal repeat, a first polyA sequence, an insulator sequence with transcription termination function, a transposon 5' terminal repeat .
  • the nucleic acid construct further comprises one or more elements selected from the group consisting of a transposase coding sequence, a promoter that controls expression of the transposase, a polyclonal insertion site, an enhancer sub, 5'UTR, second polyA sequence and exogenous gene of interest.
  • the present invention also provides a nucleic acid construct comprising the following elements: a repeating sequence at the 3' end of the transposon, a first polyA sequence, an insulator sequence with transcription termination function, a repeating sequence at the 5' end of the transposon, a transposase encoding sequence and the promoter that controls the expression of the transposase.
  • the nucleic acid construct further comprises one or more elements selected from the group consisting of a polyclonal insertion site, an enhancer, a 5'UTR, a second polyA sequence, and an exogenous source of interest. Gene.
  • any one or more of the transposase coding sequence, the promoter controlling expression of the transposase, the 5'UTR, and the second polyA sequence are in beyond the region between the 3' terminal repeat of the transposon and the 5' terminal repeat of the transposon.
  • the nucleic acid construct sequentially comprises: a transposon 3' terminal repeat, a first polyA sequence, an insulator sequence with transcription termination function, a transposon 5' terminal repeat, a control transposon The promoter for posase expression, the transposase coding sequence and the second polyA sequence.
  • the nucleic acid construct comprises sequentially: a 3' terminal repeat of a transposon, a polyclonal insertion site, a first polyA sequence, an insulator sequence with transcription termination function, a transposon 5' Terminal repeats, transposase coding sequences, and promoters that control expression of the transposase.
  • the nucleic acid construct comprises sequentially: a 3' terminal repeat of a transposon, a polyclonal insertion site, a first polyA sequence, an insulator sequence with transcription termination function, a transposon 5' Terminal repeats, promoter to control expression of transposase, transposase coding sequence and second polyA sequence.
  • the nucleic acid construct comprises sequentially: a 3' terminal repeat of a transposon, a polyclonal insertion site, a first polyA sequence, an enhancer, an insulator sequence with transcription termination function, a transposition 5' terminal repeats, transposase coding sequences, and promoters that control the expression of the transposase.
  • the nucleic acid construct comprises sequentially: a 3' terminal repeat of a transposon, a polyclonal insertion site, a first polyA sequence, an enhancer, an insulator sequence with transcription termination function, a transposition The 5' terminal repeat of the sub, the promoter that controls the expression of the transposase, the transposase coding sequence and the second polyA sequence.
  • the nucleic acid construct comprises sequentially: a 3' terminal repeat of a transposon, a polyclonal insertion site, a first polyA sequence, an insulator sequence with transcription termination function, a transposon 5' Terminal repeats, transposase coding sequence, 5'UTR, and promoters that control expression of the transposase.
  • the nucleic acid construct comprises sequentially: a 3' terminal repeat of a transposon, a polyclonal insertion site, a first polyA sequence, an insulator sequence with transcription termination function, a transposon 5' Terminal repeats, promoter controlling transposase expression, 5'UTR, transposase coding sequence and second polyA sequence.
  • the nucleic acid construct comprises sequentially: a 3' terminal repeat of a transposon, a polyclonal insertion site, a first polyA sequence, an enhancer, an insulator sequence with transcription termination function, a transposition Sub 5' terminal repeat, transposase coding sequence, 5' UTR and promoter controlling the expression of the transposase.
  • the nucleic acid construct comprises sequentially: a 3' terminal repeat of a transposon, a polyclonal insertion site, a first polyA sequence, an enhancer, an insulator sequence with transcription termination function, a transposition Sub 5' terminal repeat, promoter controlling transposase expression, 5' UTR, transposase coding sequence and second polyA sequence.
  • the nucleic acid construct comprises sequentially: a 3' terminal repeat of the transposon, an insulator sequence with transcription termination function, a polyclonal insertion site, a first polyA sequence, a transposon 5' Terminal repeats, promoter controlling transposase expression, 5'UTR, transposase coding sequence and second polyA sequence.
  • the nucleic acid construct comprises sequentially: a 3' terminal repeat of a transposon, an insulator sequence with transcription termination function, a polyclonal insertion site, a first polyA sequence, an enhancer, a transposition Sub 5' terminal repeat, promoter controlling transposase expression, 5' UTR, transposase coding sequence and second polyA sequence.
  • the nucleic acid construct comprises sequentially: a 3' terminal repeat of a transposon, an enhancer, an insulator sequence with transcription termination function, a polyclonal insertion site, a first polyA sequence, a transposition Sub 5' terminal repeat, promoter controlling transposase expression, 5' UTR, transposase coding sequence and second polyA sequence.
  • the polyclonal insertion site is used to operably insert the coding sequence of the exogenous gene and, optionally, a promoter that controls the expression of the exogenous gene.
  • the tailing signal function of the first polyA sequence and the second polyA sequence are in the same or opposite direction.
  • the orientation of the expression cassette of the transposase is the same or opposite to that of the exogenous gene expression cassette.
  • the orientation of the expression cassette of the transposase is the same or opposite to the orientation of the sequence between the transposon 3' terminal repeat and the transposon 5' terminal repeat.
  • each of the above-described elements is independently a single copy or multiple copies.
  • each of the above-described elements are linked directly or via linkers or cleavage sites.
  • nucleic acid construct according to any one of the present inventions, wherein the positions of the transposon 5' terminal repeat and the transposon 3' terminal repeat are interchangeable.
  • the transposon 3' terminal repeat is a PiggyBac transposon 3' terminal repeat.
  • the nucleotide sequence of the 3' terminal repeat of the transposon is shown in SEQ ID NO:1.
  • sequence of the polyclonal insertion site is set forth in SEQ ID NO:2.
  • the first polyA sequence is set forth in SEQ ID NO: 3, 13 or 16.
  • the second polyA sequence is set forth in SEQ ID NO: 3, 13 or 16.
  • the enhancer is selected from the group consisting of: CMV enhancer sequence, SV40 enhancer, human epsilon globulin 5' HS2 enhancer, chicken beta globulin gene 5' HS4 enhancer.
  • the enhancer sequence is shown in any of SEQ ID NO: 4, 26-28.
  • the insulator sequence with transcription termination function is shown in SEQ ID NO: 5 or 15.
  • the transposon 5' terminal repeat is a PiggyBac transposon 5' terminal repeat.
  • the nucleotide sequence of the 5' terminal repeat of the transposon is shown in SEQ ID NO:6.
  • the transposase is a PiggyBac transposase.
  • the amino acid sequence of the PiggyBac transposase is shown in SEQ ID NO:36; preferably, the coding sequence of the PiggyBac transposase is shown in SEQ ID NO:7.
  • the 5'UTR sequence is selected from the 5'UTR of C3 gene, ORM1 gene, HPX gene, FGA gene, AGXT gene, ASL gene, APOA2 gene, ALB gene.
  • the 5'UTR sequence is shown in any of SEQ ID NO:8, 17-24.
  • the promoter is selected from the group consisting of: CMV promoter, miniCMV promoter, CMV53 promoter, miniSV40 promoter, miniTK promoter, MLP promoter, pJB42CAT5 promoter, YB_TATA promoter, EF1 ⁇ promoter promoter, SV40 promoter, UbiquitinB promoter, CAG promoter, HSP70 promoter, PGK-1 promoter, ⁇ -actin promoter, TK promoter and GRP78 promoter.
  • the promoter is selected from the group consisting of miniCMV promoter, CMV53 promoter, miniSV40 promoter, miniTK promoter, MLP promoter, pJB42CAT5 promoter and YB_TATA.
  • the sequence of the promoter is set forth in any of SEQ ID NOs: 9, 37-42.
  • the promoter is a miniCMV promoter, the sequence of which is shown in SEQ ID NO:9.
  • the transposase coding sequence contains or is operably linked to a single copy or multiple copies of a nuclear localization signal coding sequence.
  • the nuclear localization signal is a c-myc nuclear localization signal, preferably having the sequence set forth in SEQ ID NO:35.
  • the nucleic acid construct comprises the sequence set forth in SEQ ID NO: 10 or 14.
  • the nucleic acid construct is a recombinant vector.
  • the nucleic acid construct is a recombinant cloning vector or a recombinant expression vector.
  • the present invention also provides a host cell comprising: (1) a nucleic acid construct described in any of the embodiments herein, and/or (2) a transposon 3' of the nucleic acid construct described in any of the embodiments herein The sequence between the terminal repeat and the 5' terminal repeat of the transposon.
  • the host cell is a mammalian cell.
  • the host cells are selected from immune cells, Jurkat cells, K562 cells, embryonic stem cells, tumor cells, HEK293 cells, and CHO cells.
  • the immune cells are selected from the group consisting of T cells, B cells, CIK cells, LAK cells, NK cells, cytotoxic T cells (CTL), dendritic cells (DC), tumor infiltrating lymphocytes (TIL), any one or more of macrophages, NK T cells, and ⁇ T cells.
  • the present invention also provides pharmaceutical compositions comprising the nucleic acid constructs or host cells described in any of the embodiments herein and pharmaceutically acceptable excipients.
  • the invention also provides the use of a nucleic acid construct or host cell as described in any of the embodiments herein in the manufacture or use of a medicament, reagent or means for integrating a foreign gene expression cassette into a target cell genome, or for gene therapy, cell therapy, stem cell induction or differentiation.
  • the target cells are mammalian cells.
  • the target cells are selected from T cells, Jurkat cells, K562 cells, embryonic stem cells, tumor cells, HEK293 cells, and CHO cells.
  • the present invention also provides a method of integrating an exogenous gene or its expression cassette into the genome of a cell, comprising introducing into said nucleic acid construct according to any of the embodiments herein comprising the exogenous gene and optionally its promoter cells, and optionally incubating the cells under conditions in which the transposase integrates the foreign gene or its expression cassette into the genome of the cell.
  • the transposase is a PiggyBac transposase
  • the introduction includes virus-mediated transformation, microinjection, particle bombardment, biolistic transformation, electroporation, and the like. In one embodiment of the invention, the introduction is electroporation.
  • the cells are incubated for at least three passages.
  • the present invention also provides cells in which exogenous genes or their expression cassettes are integrated into the genome obtained by the methods described herein.
  • Fig. 2 is a fluorescent photograph of Jurkat cells electroporated with pKB20-EGFP, pKB201-EGFP and pKB202-EGFP respectively.
  • FIG. 3 the flow detection results of Jurkat cells electroporated with pKB20-EGFP, pKB201-EGFP and pKB202-EGFP respectively.
  • Figure 4 the number of viable cells of Jurkat cells electroporated with pKB20-EGFP, pKB201-EGFP, pKB202-EGFP and pKC20-EGFP, respectively.
  • FIG. 5 the expression levels of PB transposase in Jurkat cells electroporated with pKB20-EGFP, pKB201-EGFP and pKB202-EGFP, respectively.
  • Fig. 6 is the fluorescence photos of K562 cells electrotransfected with pKB20-EGFP, pKB201-EGFP, pKB202-EGFP and pKC20-EGFP respectively.
  • Figure 7 shows the number of viable cells of K562 cells electroporated with pKB20-EGFP, pKB201-EGFP, pKB202-EGFP and pKC20-EGFP, respectively.
  • Figure 8 shows the results of flow cytometry of K562 cells electroporated with pKB20-EGFP, pKB201-EGFP and pKB202-EGFP respectively.
  • Figure 9 shows the fluorescence positive rate of K562 cells electroporated with pKB20-EGFP, pKB201-EGFP and pKB202-EGFP respectively.
  • Fig. 10 shows the fluorescence photos of primary T cells electroporated with pKB20-EGFP, pKB201-EGFP, pKB202-EGFP and pKC20-EGF, respectively.
  • Figure 11 shows the results of flow cytometry of T cells electroporated with pKB20-EGFP, pKB201-EGFP and pKB202-EGFP, respectively.
  • Figure 14 Positive rate of Jurkat cells electrotransfected with pKB20-EGFP with reduced plasmid dosage.
  • Figure 15 Intracellular vector residual copy number as a function of time.
  • FIG. 1 Schematic diagram of the genomic integration site mediated by pKB20 vector in K562 sample 1.
  • FIG. 19 Schematic diagram of genomic integration sites mediated by pKB20 vector in K562 sample 2.
  • Figure 20 Schematic diagram of the pKB20 vector-mediated genomic integration site in Jurkat sample 1.
  • Figure 21 Schematic diagram of pKB20 vector-mediated genomic integration sites in Jurkat sample 2.
  • FIG. 22 Flow cytometry results of positive rate of primary T cells electroporated with pKB20-HER2CAR.
  • Figure 23 In vitro RTCA killing results of HER2 CAR-T cells on target cells SKOV-3.
  • FIG. 24 Flow cytometry results of positive rate of primary T cells electroporated with pKB20-NY-ESO-1 TCR.
  • Figure 25 In vitro RTCA killing results of NY-ESO-1 TCR-T on target cell A375.
  • nucleic acid construct defined herein as a single- or double-stranded nucleic acid molecule, preferably refers to an artificially constructed nucleic acid molecule.
  • the nucleic acid construct further comprises operably linked one or more regulatory sequences that direct the expression of the coding sequence in a suitable host cell under compatible conditions. Expression is understood to include any step involved in the production of a protein or polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • the transposition system described herein is preferably a univariate nucleic acid construct, ie a single nucleic acid construct can achieve efficient transposition.
  • the direction of the transposase expression cassette is reversed.
  • the direction and/or sequence referred to in the above-mentioned "in order to include the following elements” means from upstream to downstream.
  • the direction along the above-mentioned “forward” direction is from upstream to downstream, and the direction along the above-mentioned “reverse direction” is from downstream to upstream.
  • expression cassette refers to the complete elements required to express a gene, including promoter, gene coding sequence, and PolyA tailed signal sequence.
  • operably inserted/linked is defined herein as a conformation in which the regulatory sequence is positioned in place relative to the coding sequence of the DNA sequence such that the regulatory sequence directs the expression of a protein or polypeptide.
  • the multiple cloning site is operably inserted with one or more identical or different exogenous genes and an optional promoter for controlling the expression of the exogenous gene by DNA recombination technology, or a polyclonal thereof.
  • the sites are replaced with one or more identical or different coding sequences for the exogenous gene and, optionally, a promoter that controls the expression of the exogenous gene.
  • the "operably linked” can be achieved by means of DNA recombination, specifically, the nucleic acid construct is a recombinant nucleic acid construct.
  • a "foreign gene” as used herein can be any source of nucleic acid molecule that is expressed or functions upon transposition into the genome of a host cell.
  • exogenous genes include luciferin reporter genes (eg, green fluorescent protein, yellow fluorescent protein, etc.), luciferase genes (eg, firefly luciferase, Renilla luciferase, etc.), native functional protein genes, RNAi Genes and artificial chimeric genes (eg, chimeric antigen receptor genes, Fc fusion protein genes, full-length antibody genes).
  • coding sequence is defined herein as the portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product.
  • the boundaries of the coding sequence are usually defined by a ribosome binding site (for prokaryotes) immediately upstream of the 5' open reading frame of the mRNA and a transcription termination sequence immediately downstream of the 3' open reading frame of the mRNA.
  • Coding sequences can include, but are not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
  • regulatory sequences is defined herein to include all components necessary or advantageous for the expression of the peptides of the invention.
  • Each regulatory sequence may be naturally contained or foreign to the nucleic acid sequence encoding the protein or polypeptide.
  • These regulatory sequences include, but are not limited to, leader sequences, polyA sequences, propeptide sequences, promoters, signal sequences, and transcription terminators.
  • the regulatory sequences include a promoter and transcriptional and translational termination signals.
  • Linkered regulatory sequences can be provided for the purpose of introducing specific restriction sites for ligation of the regulatory sequences with the coding region of the nucleic acid sequence encoding the protein or polypeptide.
  • the regulatory sequence may be a suitable promoter sequence, ie, a nucleic acid sequence that is recognized by the host cell in which the nucleic acid sequence is expressed.
  • Promoter sequences contain transcriptional regulatory sequences that mediate protein or polypeptide expression.
  • the promoter sequence is usually operably linked to the coding sequence for the protein to be expressed.
  • the promoter can be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice, including mutated, truncated and hybrid promoters, and can be derived from extracellular coding homologous or heterologous to the host cell. Or gene acquisition of intracellular polypeptides.
  • the regulatory sequence may also be a suitable transcription termination sequence, ie, a sequence recognized by the host cell to terminate transcription. Termination sequences are operably linked to the 3' terminus of the nucleic acid sequence encoding the protein or polypeptide. Any terminator that is functional in the host cell of choice can be used in the present invention.
  • the regulatory sequence may also be a suitable leader sequence, an untranslated region of an mRNA that is important for translation by the host cell.
  • the leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice can be used in the present invention.
  • the control sequence can also be a signal peptide coding region, which encodes an amino acid sequence linked to the amino terminus of a protein or polypeptide, which can guide the encoded polypeptide into the cell secretion pathway.
  • the 5' end of the coding region of the nucleic acid sequence may naturally contain a signal peptide coding region naturally linked in translation reading frame with the fragment of the coding region of the secreted polypeptide.
  • the 5' end of the coding region may contain a signal peptide coding region that is foreign to the coding sequence.
  • the coding sequence does not normally contain a signal peptide coding region, it may be necessary to add a foreign signal peptide coding region.
  • the native signal peptide coding region can be simply replaced with a foreign signal peptide coding region to enhance polypeptide secretion.
  • any signal peptide coding region capable of directing the expressed polypeptide into the secretory pathway of the host cell used can be used in the present invention.
  • the nucleic acid constructs of the present invention comprise the following elements: a transposon 3' terminal repeat, a first polyA sequence, an insulator sequence with transcription termination function, a transposon 5' terminal repeat, a transposase coding sequence, and a sequence that controls the transposase. Promoter for locase expression.
  • the nucleic acid construct may also comprise one or more elements selected from the group consisting of a polyclonal insertion site, an enhancer, a 5'UTR, and a second polyA sequence.
  • the nucleic acid construct sequentially comprises: a 3'-terminal repeat of the transposon, a polyclonal insertion site, a first polyA sequence, an enhancer, an insulator sequence with transcription termination function, a transposon
  • the 5' terminal repeat of the transposon, the transposase coding sequence, the 5' UTR, and the promoter that controls the expression of the transposase are shown in Figure 1A.
  • the nucleic acid construct sequentially comprises: a transposon 3' terminal repeat, a polyclonal insertion site, a first polyA sequence, an enhancer, an insulator sequence with transcription termination function, The 5' terminal repeat of the transposon, the promoter controlling the expression of the transposase, the 5' UTR, the transposase coding sequence and the second polyA sequence are shown in Figure IB.
  • Each of the elements in the nucleic acid constructs herein is independently single or multiple copies.
  • the position of the 5' terminal repeat of a transposon and the 3' terminal repeat of the transposon can be interchanged.
  • the repeat sequence at the 5' end of the transposon is the repeat sequence at the 5' end of the PiggyBac transposon; the repeat sequence at the 3' end of the transposon is the repeat sequence at the end of the PiggyBac transposon.
  • the transposase is preferably a PiggyBac transposase, the coding sequence of which contains or is operably linked to a single-copy or multiple-copy nuclear localization signal coding sequence, so as to improve the transposition efficiency.
  • An exemplary PiggyBac transposase coding sequence is set forth in SEQ ID NO:7.
  • An exemplary nuclear localization signal coding sequence is set forth in SEQ ID NO:35.
  • the nucleic acid constructs of the present invention can use polyA sequences for transposases and foreign genes. This design can shorten the full length of the nucleic acid construct to a certain extent, which facilitates the incorporation of longer exogenous genes for transposition. Alternatively, the nucleic acid constructs of the present invention may also use separate polyA sequences for the transposase and the foreign gene, both in the same or opposite direction of the tailing signal function. This design avoids the interaction between two expression cassettes in opposite directions due to sharing the same bidirectional polyA sequence.
  • the polyA sequences described herein may or may not have bidirectional transcription termination function. Preferably, the polyA sequence is independently selected from SEQ ID NO: 3, 13 or 16.
  • the nucleic acid constructs of the invention may also or further use insulator sequences for transcription termination against transposases and foreign genes.
  • an insulator sequence can be included at either end of any polyA sequence.
  • the insulator sequence of the present invention is located between the transposon 5' terminal repeat and the transposon 3' terminal repeat.
  • the insulator sequence described herein has a transcription termination function, and the sequence can be any sequence known in the art that has a transcription termination function.
  • the insulator sequence with transcription termination function is shown in SEQ ID NO: 5 or 15.
  • a transposase can use a polyA sequence, an insulator sequence, or a polyA sequence and an insulator sequence to achieve transcription termination; an exogenous gene can use a polyA sequence, an insulator sequence, or a polyA sequence and an insulator sequence to achieve transcription termination.
  • any of the insulator sequences are located between the transposon 5' terminal repeat and the transposon 3' terminal repeat.
  • a suitable transposase promoter sequence is one capable of driving high-level expression of the transposase operably linked thereto, including but not limited to the simian virus 40 (SV40) early stage Promoter, Mouse Breast Cancer Virus (MMTV), Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) Promoter, MoMuLV Promoter, Avian Leukemia Virus Promoter, Epstein-Barr Virus Immediate Early Promoter, Russell Sarcoma Virus Promoters, as well as human gene promoters such as, but not limited to, the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter.
  • SV40 simian virus 40
  • MMTV Mouse Breast Cancer Virus
  • HIV Human Immunodeficiency Virus
  • LTR Long Terminal Repeat
  • MoMuLV Promoter MoMuLV Promoter
  • Avian Leukemia Virus Promoter Epstein-Barr Virus Immediate Early Promoter
  • the promoter of the transposase is selected from the group consisting of: CMV promoter, miniCMV promoter, CMV53 promoter, miniSV40 promoter, miniTK promoter, MLP promoter, pJB42CAT5 promoter, YB_TATA promoter, EF1 ⁇ promoter, SV40 promoter, UbiquitinB promoter, CAG promoter, HSP70 promoter, PGK-1 promoter, ⁇ -actin promoter, TK promoter and GRP78 promoter.
  • the promoter is selected from the group consisting of miniCMV promoter, CMV53 promoter, miniSV40 promoter, miniTK promoter, MLP promoter, pJB42CAT5 promoter and YB_TATA promoter. More preferably, the promoter is the miniCMV promoter.
  • the miniCMV promoter is much shorter than the CMV promoter, making the vector length smaller and more conducive to the integration of larger foreign genes.
  • a 5'UTR sequence is added between the miniCMV and the transposase to enhance transcription and translation.
  • the 5'UTR sequence can be set forth in any of SEQ ID NOs: 8, 17-24.
  • the nucleic acid constructs of the present invention may contain enhancers, which may be located at any end of any element other than an enhancer in the nucleic acid constructs described herein.
  • the enhancer is located between the transposon 3' terminal repeat and the transposon 5' terminal repeat. More preferably, the enhancer is located downstream of the first polyA sequence.
  • the enhancer sequence can be set forth in any of SEQ ID NOs: 4, 25-28.
  • the nucleic acid construct may not include the 5'UTR sequence and the enhancer sequence, or include either or both, and the resulting nucleic acid construct can efficiently integrate the exogenous gene into the cell genome.
  • regulatory sequences that regulate the expression of the polypeptide in response to the growth conditions of the host cell.
  • regulatory systems are those that turn gene expression on or off in response to chemical or physical stimuli, including in the presence of regulatory compounds.
  • Other examples of regulatory sequences are those that enable gene amplification.
  • the nucleic acid sequence encoding the protein or polypeptide is operably linked to the regulatory sequences.
  • the nucleic acid construct of the present invention sequentially comprises a PiggyBac transposon 3' terminal repeat (3' ITR) (SEQ ID NO: 1), a multiple cloning site (SEQ ID NO: 1) : 2), polyA signal sequence (SEQ ID NO: 3, 13 or 16), optional enhancer motif sequence (any of SEQ ID NO: 4, 25-28), insulator sequence (SEQ ID NO: 5 or 15), the reverse complement of the PiggyBac transposon 5' terminal repeat (5'ITR) (SEQ ID NO:6), the reverse complement of the PiggyBac transposase coding sequence (SEQ ID NO:7), Optionally the reverse complement of the 5'UTR sequence (any of SEQ ID NO:8, 17-24) and the reverse complement of the miniCMV promoter sequence (SEQ ID NO:9).
  • the nucleic acid construct of the invention has the sequence shown in SEQ ID NO: 10.
  • the nucleic acid construct of the present invention sequentially comprises a PiggyBac transposon 3' terminal repeat (3' ITR) (SEQ ID NO: 1), a multiple cloning site (SEQ ID NO: 2), first polyA signal sequence (SEQ ID NO: 3, 13 or 16), optional enhancer motif sequence (any of SEQ ID NO: 4, 25-28), insulator sequence (SEQ ID NO: 4, 25-28) NO: 5 or 15), PiggyBac transposon 5' terminal repeat (5' ITR) (SEQ ID NO: 6), miniCMV promoter sequence (SEQ ID NO: 9), optional 5' UTR sequence (SEQ ID NO: 9) ID NO: 8, any of 17-24), the PiggyBac transposase coding sequence (SEQ ID NO: 7) and the second polyA signal sequence (SEQ ID NO: 3, 13 or 16).
  • the nucleic acid construct of the invention has the sequence set forth in SEQ ID NO:14
  • the nucleic acid construct is a recombinant vector.
  • the recombinant vector can be a recombinant cloning vector or a recombinant expression vector.
  • the elements of the nucleic acid constructs of the present invention can be packaged into many types of vectors, eg, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers.
  • the vector introduced into the cell may also contain either or both of a selectable marker gene or a reporter gene to facilitate the identification and selection of cells from a population of cells.
  • Selectable markers can be carried on a single piece of DNA and used in co-transfection procedures. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell.
  • Useful selectable markers include Flag, HA or V5. Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. After the DNA has been introduced into the recipient cells, the expression of the reporter gene is measured at an appropriate time. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein genes.
  • Recombinant cloning vectors can be used to provide coding sequences containing elements of the nucleic acid constructs of the invention and optionally foreign genes.
  • the recombinant cloning vector can be a recombinant vector obtained by recombining each element of the nucleic acid construct of the present invention with pUC18, pUC19, pMD18-T, pMD19-T, pGM-T vector, pUC57, pMAX or pDC315 series vectors.
  • Recombinant expression vectors can be used to integrate and express foreign gene expression cassettes into the genome by the various elements of the nucleic acid constructs of the invention in suitable host cells.
  • the vector may be suitable for replication and integration in eukaryotic cells.
  • Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.
  • the recombinant expression vector is a recombinant vector obtained by recombining each element of the nucleic acid construct of the present invention with pCDNA3 series vectors, pCDNA4 series vectors, pCDNA5 series vectors, pCDNA6 series vectors, pRL series vectors, pUC57 vectors, pMAX vectors or pDC315 series vectors;
  • the recombinant vector can be a recombinant viral vector, including but not limited to recombinant adenoviral vector, recombinant adeno-associated viral vector, recombinant retroviral vector, recombinant herpes simplex virus vector or recombinant vaccinia virus vector.
  • Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks of virology and molecular biology.
  • the nucleic acid constructs described herein can generally be obtained by PCR amplification.
  • primers can be designed according to the nucleotide sequences disclosed herein, especially the open reading frame sequences, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art is used as a template, amplified sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splicing the amplified fragments together in the correct order.
  • the nucleic acid constructs described herein can also be synthesized directly.
  • the present invention also provides host cells comprising the nucleic acid constructs described in any of the embodiments herein.
  • Host cells include both mammalian cells and various cells used in the production of mammalian cells, such as E. coli cells, for providing nucleic acid constructs or vectors as described herein.
  • a mammalian cell comprising a nucleic acid construct or vector described herein, including but not limited to: T cells, B cells, CIK cells, LAK cells, NK cells, cytotoxic T cells (CTL), dendritic cells (DC), tumor infiltrating lymphocytes (TIL), macrophages, NK T cells, ⁇ T cells, Jurkat cells, K562 cells, embryonic stem cells, tumor cells, HEK293 cells and CHO cells.
  • T cells T cells, B cells, CIK cells, LAK cells, NK cells, cytotoxic T cells (CTL), dendritic cells (DC), tumor infiltrating lymphocytes (TIL), macrophages, NK T cells, ⁇ T cells, Jurkat cells, K562 cells, embryonic stem cells, tumor cells, HEK293 cells and CHO cells.
  • compositions of the present invention comprise the nucleic acid constructs or cells described herein and pharmaceutically acceptable excipients.
  • pharmaceutically acceptable adjuvant is a pharmaceutically or food acceptable carrier, solvent, suspending agent or excipient for delivering the nucleic acid construct or cell of the present invention to animals or humans.
  • pharmaceutically acceptable excipients are nontoxic to recipients of the composition at the doses and concentrations employed.
  • Various types of carriers or excipients commonly used in the delivery of biologics in therapy as known in the art may be included.
  • excipients can be liquid or solid, including but not limited to: pH adjusters, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, glidants, Sweeteners, dyes/colorants, flavor enhancers, wetting agents, dispersing agents, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers.
  • pharmaceutically acceptable excipients may include one or more inactive ingredients including, but not limited to, stabilizers, preservatives, additives, adjuvants, sprays, compressed air, or other suitable gases, or other suitable inactive ingredients in combination with the pharmacologically effective compound.
  • excipients may be those commonly used in the art for administration of transposition systems or cells containing the same.
  • excipients include various lactose, mannitol, oils such as corn oil, buffers such as PiggyBacS, saline, polyethylene glycol, glycerol, polypropylene glycol, dimethyl sulfoxide, amides such as dimethylacetamide, proteins such as white Proteins, and detergents such as Tween 80, monosaccharides and oligopolysaccharides such as glucose, lactose, cyclodextrin and starch.
  • compositions will be apparent to those skilled in the art, including formulations comprising the nucleic acid constructs or cells described herein in sustained or controlled release delivery formulations.
  • Techniques for formulating a variety of other sustained or controlled delivery modes, such as liposomal vehicles, bioerodible microparticles or porous beads, and depot injections, are also known to those of skill in the art.
  • compositions for in vivo administration are usually provided in the form of sterile formulations. Sterilization is achieved by filtration through sterile filtration membranes. When the composition is lyophilized, it can be sterilized using this method before or after lyophilization and reconstitution. Compositions for parenteral administration can be stored in lyophilized form or in solution. Parenteral compositions are usually presented in containers with sterile access ports, such as intravenous solution strips or vials with a hypodermic needle pierceable stopper.
  • a therapeutically effective amount of an agent described herein is included in the composition.
  • a therapeutically effective amount refers to a dose that will effect treatment, prevention, alleviation and/or amelioration of a disease or disorder in a subject. These effects can be achieved by inserting exogenous genes with corresponding functions that have functions corresponding to specific uses, such as therapeutic functions or inductive functions.
  • the therapeutically effective amount can be determined according to factors such as the patient's age, sex, the condition and its severity, and other physical conditions of the patient.
  • a therapeutically effective amount may be administered as a single dose, or may be administered in multiple doses according to an effective therapeutic regimen.
  • a subject or patient generally refers to a mammal, especially a human.
  • the composition contains, for example, 0.001-50% by weight, preferably 0.01-30%, more preferably 0.05-10% by weight of the nucleic acid constructs or cells described herein.
  • compositions described herein can be used in combination with other agents having functions similar or corresponding to those performed by the exogenous gene.
  • agents having functions similar or corresponding to those performed by the exogenous gene.
  • an agent that treats the disease or condition being treated by the exogenous gene in combination with an agent that treats the disease or condition being treated by the exogenous gene.
  • Dosages for other agents to be administered can be determined by those skilled in the art.
  • the dosage form of the pharmaceutical composition of the present invention can be various, as long as the dosage form can make the active ingredient reach the mammalian body effectively, it can be made into the form of unit dosage form.
  • Dosage forms such as can be selected from: gels, aerosols, tablets, capsules, powders, granules, syrups, solutions, suspensions, injections, powders, pills, controlled-release preparations, infusions, suspensions and the like.
  • the preferred compositions are solid compositions, especially tablets and solid- or liquid-filled capsules.
  • nucleic acid constructs or cells or compositions thereof described herein can also be stored in sterile devices suitable for injection or instillation.
  • the nucleic acid constructs or cells or compositions thereof described herein can also be stored in a suitable container and placed in a kit or kit.
  • the exogenous gene When the exogenous gene is integrated into the genome, it can effectively terminate the transcription and expression of the PiggyBac transposase, and at the same time, it can function as an insulator of the exogenous gene expression box, reducing the impact of the integrated exogenous gene expression box on the expression of genes near the integration site.
  • the present invention relates to a method for integrating an exogenous gene or its expression cassette into the genome of a cell, comprising introducing into said cell a nucleic acid construct according to any of the embodiments herein comprising the exogenous gene and optionally its promoter , and optionally incubating the cells under conditions in which the PiggyBac transposase integrates the foreign gene or its expression cassette into the cell genome.
  • the present invention also provides cells in which the exogenous gene or its expression cassette is integrated into the genome obtained by the above method.
  • Vectors can be readily introduced into host cells, eg, mammalian, bacterial, yeast or insect cells, by any method known in the art.
  • a vector can be transferred into a host cell by physical, chemical or biological means.
  • Exemplary physical or chemical methods include calcium phosphate precipitation, lipofection, microinjection, particle bombardment, microinjection, biolistic transformation, electroporation, colloidal dispersion systems, macromolecular complexes, nanocapsules, micro- Spheres, beads, lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes).
  • Biological methods for introducing nucleic acid constructs into host cells include virus-mediated transformation, particularly retroviral vectors.
  • Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, among others.
  • the nucleic acid sequences and selected nucleic acid sequences can be inserted into vectors and packaged into retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to subject cells in vivo or ex vivo.
  • Reagents for viral packaging are well known in the art, such as conventional lentiviral vector systems including pRsv-REV, pMDlg-pRRE, pMD2G and interfering plasmids of interest.
  • the invention also provides the use of a nucleic acid construct or host cell as described in any of the embodiments herein in the manufacture or use of a medicament, reagent or means for integrating a foreign gene expression cassette into a target cell genome, or for gene therapy, cell therapy, stem cell induction or differentiation.
  • the target cells are mammalian cells, including but not limited to T cells, B cells, CIK cells, LAK cells, NK cells, cytotoxic T cells (CTL), dendritic cells cells (DC), tumor infiltrating lymphocytes (TIL), macrophages, NK T cells, ⁇ T cells, Jurkat cells, K562 cells, embryonic stem cells, tumor cells, HEK293 cells and CHO cells.
  • a nucleic acid construct comprising or consisting of the following elements: a transposon 3' terminal repeat, a first polyA sequence, an insulator sequence with transcription termination function, a transposon 5' terminal repeat,
  • the nucleic acid construct further comprises one or more elements selected from the group consisting of a transposase coding sequence, a promoter controlling the expression of the transposase, a polyclonal insertion site, an enhancer, a 5'UTR, The second polyA sequence and the foreign gene of interest,
  • any one or more of the transposase coding sequence, the promoter controlling the expression of the transposase, the 5'UTR and the second polyA sequence are in the transposon 3 outside the region between the 'terminal repeats and the 5' terminal repeats of the transposon.
  • Item 2 The nucleic acid construct as described in item 1, comprising the following elements: a transposon 3'-terminal repeat, a first polyA sequence, an insulator sequence with transcription termination function, a transposon 5'-terminal repeat, a transposon a transposase coding sequence and a promoter controlling the expression of the transposase,
  • the nucleic acid construct further comprises one or more elements selected from the group consisting of a polyclonal insertion site, an enhancer, a 5'UTR, a second polyA sequence, and a foreign gene of interest.
  • the nucleic acid construct sequentially comprises: a repeating sequence at the 3' end of the transposon, a polyclonal insertion site, a first polyA sequence, an insulator sequence with transcription termination function, a repeating sequence at the 5' end of the transposon, and a transposase coding sequence and a promoter that controls the expression of the transposase,
  • the nucleic acid construct sequentially comprises: a repeating sequence at the 3' end of the transposon, a polyclonal insertion site, a first polyA sequence, an enhancer, an insulator sequence with transcription termination function, a repeating sequence at the 5' end of the transposon, a transposition an enzyme coding sequence and a promoter that controls the expression of the transposase,
  • the nucleic acid construct sequentially comprises: a repeating sequence at the 3' end of the transposon, a polyclonal insertion site, a first polyA sequence, an insulator sequence with transcription termination function, a repeating sequence at the 5' end of the transposon, and a transposase coding sequence , 5'UTR and the promoter that controls the expression of the transposase,
  • the nucleic acid construct sequentially comprises: a repeating sequence at the 3' end of the transposon, a polyclonal insertion site, a first polyA sequence, an enhancer, an insulator sequence with transcription termination function, a repeating sequence at the 5' end of the transposon, a transposition an enzyme coding sequence, a 5'UTR, and a promoter that controls the expression of the transposase,
  • the nucleic acid construct sequentially comprises: a repeating sequence at the 3' end of the transposon, a first polyA sequence, an insulator sequence with transcription termination function, a repeating sequence at the 5' end of the transposon, a promoter controlling the expression of the transposase, and a transposition the enzyme coding sequence and the second polyA sequence,
  • the nucleic acid construct sequentially comprises: a repeating sequence at the 3' end of the transposon, a polyclonal insertion site, a first polyA sequence, an insulator sequence with transcription termination function, a repeating sequence at the 5' end of the transposon, and controlling the expression of the transposase. promoter, transposase coding sequence and second polyA sequence,
  • the nucleic acid construct sequentially comprises: a repeating sequence at the 3' end of the transposon, a polyclonal insertion site, a first polyA sequence, an enhancer, an insulator sequence with transcription termination function, a repeating sequence at the 5' end of the transposon, and a control transduction sequence.
  • the nucleic acid construct sequentially comprises: a repeating sequence at the 3' end of the transposon, a polyclonal insertion site, a first polyA sequence, an insulator sequence with transcription termination function, a repeating sequence at the 5' end of the transposon, and controlling the expression of the transposase. promoter, 5'UTR, transposase coding sequence and second polyA sequence, or
  • the nucleic acid construct sequentially comprises: a repeating sequence at the 3' end of the transposon, a polyclonal insertion site, a first polyA sequence, an enhancer, an insulator sequence with transcription termination function, a repeating sequence at the 5' end of the transposon, and a control transduction sequence.
  • the nucleic acid construct sequentially comprises: a repeating sequence at the 3' end of the transposon, an insulator sequence with transcription termination function, a polyclonal insertion site, a first polyA sequence, a repeating sequence at the 5' end of the transposon, and a control transposase expression.
  • promoter, 5'UTR, transposase coding sequence and second polyA sequence
  • the nucleic acid construct sequentially comprises: a repeating sequence at the 3' end of the transposon, an insulator sequence with transcription termination function, a polyclonal insertion site, the first polyA sequence, an enhancer, a repeating sequence at the 5' end of the transposon, and a control transposon sequence.
  • the nucleic acid construct sequentially comprises: a repeating sequence at the 3' end of the transposon, an enhancer, an insulator sequence with transcription termination function, a polyclonal insertion site, the first polyA sequence, a repeating sequence at the 5' end of the transposon, and a control transposon sequence.
  • Promoter for posase expression 5'UTR, transposase coding sequence and second polyA sequence.
  • nucleic acid construct according to any one of items 1-3, wherein the nucleic acid construct has one or more features selected from the following:
  • the orientation of the expression cassette of the transposase is the same or opposite to the orientation of the expression cassette of the exogenous gene
  • the orientation of the expression cassette of the transposase is the same or opposite to the orientation of the sequence between the 3' terminal repeat of the transposon and the 5' terminal repeat of the transposon,
  • the repeat sequence at the 3' end of the transposon is the repeat sequence at the 3' end of the PiggyBac transposon
  • the repeat sequence at the 5' end of the transposon is the repeat sequence at the 5' end of the PiggyBac transposon
  • Described enhancer is selected from: CMV enhancer sequence, SV40 enhancer, human epsilon globulin 5' HS2 enhancer, chicken beta globulin gene 5' HS4 enhancer,
  • the transposase is PiggyBac transposase
  • the 5'UTR is selected from the 5'UTR of C3 gene, ORM1 gene, HPX gene, FGA gene, AGXT gene, ASL gene, APOA2 gene, ALB gene,
  • the promoter is selected from: CMV promoter, miniCMV promoter, CMV53 promoter, miniSV40 promoter, miniTK promoter, MLP promoter, pJB42CAT5 promoter, YB_TATA promoter, EF1 ⁇ promoter, SV40 promoter, UbiquitinB promoter , CAG promoter, HSP70 promoter, PGK-1 promoter, ⁇ -actin promoter, TK promoter and GRP78 promoter,
  • the transposase coding sequence contains or is operably linked to a single or multiple copies of a nuclear localization signal coding sequence.
  • nucleic acid construct according to any one of items 1-3, wherein the nucleic acid construct has one or more features selected from the following:
  • the nucleotide sequence of the repeat sequence at the 3' end of the transposon is shown in SEQ ID NO: 1,
  • the nucleotide sequence of the repeat sequence at the 5' end of the transposon is shown in SEQ ID NO: 6,
  • the first polyA sequence is shown in SEQ ID NO: 3, 13 or 16,
  • the second polyA sequence is shown in SEQ ID NO: 3, 13 or 16,
  • the enhancer sequence is shown in any of SEQ ID NO:4, 26-28,
  • the insulator sequence is shown in SEQ ID NO: 5 or 15,
  • the amino acid sequence of the PiggyBac transposase is shown in SEQ ID NO:36; preferably, the coding sequence of the PiggyBac transposase is shown in SEQ ID NO:7,
  • the 5'UTR sequence is shown in any of SEQ ID NO:8, 17-24,
  • sequence of the promoter is shown in any one of SEQ ID NO:9 and SEQ ID NO:37-42;
  • the nuclear localization signal is c-myc nuclear localization signal; preferably, the nuclear localization signal has the sequence shown in SEQ ID NO:35.
  • the nucleic acid construct comprises the sequence shown in SEQ ID NO: 10 or 14, or
  • the nucleic acid construct is a recombinant vector, preferably, the nucleic acid construct is a recombinant cloning vector or a recombinant expression vector.
  • a host cell comprising
  • nucleic acid construct described in any one of items 1-6, and/or
  • the host cell is a mammalian cell
  • the host cells are selected from T cells, Jurkat cells, K562 cells, embryonic stem cells, tumor cells, HEK293 cells and CHO cells.
  • Item 8 A pharmaceutical composition, comprising the nucleic acid construct according to any one of items 1-6 or the host cell according to item 8 and a pharmaceutically acceptable adjuvant.
  • nucleic acid construct described in any one of items 1-6 or the host cell described in item 7 in the preparation of medicines, reagents or tools or use as medicines, reagents or tools, the medicines, reagents or tools. or tools for integrating foreign gene expression cassettes into target cell genomes, or for gene therapy, cell therapy, stem cell induction or differentiation,
  • the target cells are mammalian cells
  • the target cells are selected from immune cells, Jurkat cells, K562 cells, embryonic stem cells, tumor cells, HEK293 cells and CHO cells,
  • the immune cells are selected from T cells, B cells, CIK cells, LAK cells, NK cells, cytotoxic T cells (CTL), dendritic cells (DC), tumor infiltrating lymphocytes (TIL), Any one or more of macrophages, NK T cells, and ⁇ T cells.
  • Item 10 a method for integrating an exogenous gene or its expression cassette into a cell genome, comprising introducing the nucleic acid construct described in any one of items 1-6 containing an exogenous gene and optionally its promoter into The cell, and optionally incubating the cell under conditions in which a transposase integrates an exogenous gene or its expression cassette into the genome of the cell, the exogenous gene and optionally its promoter located in the nucleic acid construct in the polyclonal insertion site of
  • the transposase is a PiggyBac transposase.
  • the transposon vector system of the present invention has multiple regulatory elements to regulate the expression of PiggyBac and the integration of exogenous genes mediated by multiple levels.
  • the promoter used herein to drive expression of the PiggyBac transposase reduces the strength of expression and reduces DNA length.
  • an enhancer element is introduced to transiently enhance the expression of the transposase in the state of the complete plasmid, and play the cutting and integration functions of the PiggyBac transposase; when the exogenous gene expression cassette is cut from the complete plasmid, the enhancer loses its effect on the promoter.
  • the enhancement effect of the gene can realize the efficient opening and closing of the PiggyBac transposase, so that the level of the transposase decreases after the expression reaches the peak in a short time, and the cytotoxicity is greatly reduced while mediating the integration of the exogenous gene;
  • the PiggyBac expression box adopts an insulator sequence with transcription termination function, which can effectively terminate transcription and express transposase genes before the integration of exogenous genes; Efficiently terminates the transcriptional expression of the PiggyBac transposase.
  • the insulator sequence with transcription termination function is integrated into the genome together with the exogenous gene expression box, the insulator can also block the influence of exogenous gene expression on adjacent regions in the genome, reducing the impact of the integrated exogenous gene expression box on the integration site. Effects of nearby gene expression;
  • the transposon vector system of the present invention has fewer insertion sites in the genome, and fewer insertion sites in the gene, and has less impact on the stability of the genome;
  • pKB20 PiggyBac transposon 3' terminal repeat (3'ITR) (SEQ ID NO:1), multiple cloning site (SEQ ID NO:2), bGH polyA signal sequence (SEQ ID NO:3) ), enhancer motif sequence (SEQ ID NO:4), insulator sequence with transcription termination function (C2 transcriptional pause site, SEQ ID NO:5), PiggyBac transposon 5' terminal repeat (5'ITR) (SEQ ID NO:6), reverse complement of PiggyBac transposase coding sequence (SEQ ID NO:7), reverse complement of 5'UTR sequence (SEQ ID NO:8) and miniCMV
  • the reverse complementary sequence of the promoter sequence (SEQ ID NO:9) was spliced into a long sequence (SEQ ID NO:10), which was entrusted to Shanghai Jierui Biotechnology Co., Ltd. to synthesize, and AgeI and AscI were added at both ends for digestion The site was loaded into pUC57 (purchased from Shanghai Jier
  • pKB20-EGFP insert the EF1A promoter sequence (SEQ ID NO: 11) with NFAT motif between the XbaI and EcoRI sites of the multiple cloning site of pKB20, and insert the EGFP coding sequence between the EcoRI and SalI sites ( SEQ ID NO: 12), named pKB20-EGFP, the EF1A promoter sequence with NFAT motif and the EGFP coding sequence were entrusted to Shanghai Jerry Biotechnology Co., Ltd. to synthesize.
  • pKB205 PiggyBac transposon 3' terminal repeat (3'ITR) (SEQ ID NO: 1), multiple cloning site MCS (SEQ ID NO: 2), bGH polyA signal sequence (SEQ ID NO: 2) in order 3), enhancer motif sequence (SEQ ID NO: 4), insulator sequence with transcription termination function (C2 transcription stop site, SEQ ID NO: 5), PiggyBac transposon 5' terminal repeat sequence (5' ITR ) (SEQ ID NO:6), miniCMV promoter sequence (SEQ ID NO:9), 5'UTR sequence (SEQ ID NO:8), PiggyBac transposase coding sequence (SEQ ID NO:7) and SV40 polyA signal
  • the sequence (SEQ ID NO: 13) was spliced into a long sequence (SEQ ID NO: 14), which was entrusted to Shanghai Jierui Biotechnology Co., Ltd. From Shanghai Jierui Bio), named pKB205.
  • a schematic diagram of the plasmid is
  • pKB205-EGFP insert the EF1A promoter sequence (SEQ ID NO: 11) with NFAT motif between the XbaI and EcoRI sites of the multiple cloning site of pKB205, and insert the EGFP coding sequence between the EcoRI and SalI sites ( SEQ ID NO: 12), named pKB205-EGFP, the EF1A promoter sequence with NFAT motif and the EGFP coding sequence were entrusted to Shanghai Jerry Biotechnology Co., Ltd. to synthesize.
  • pKB201-EGFP The pKB20-EGFP sequence was obtained by deleting the enhancer motif sequence
  • pKB202-EGFP The pKB20-EGFP sequence was obtained by deleting the 5'UTR sequence
  • pKB2003-EGFP The pKB20-EGFP sequence was obtained by deleting the enhancer motif sequence and the 5'UTR sequence;
  • pKC20-EGFP The pKB20-EGFP sequence was obtained by deleting the miniCMV promoter sequence, 5' UTR sequence, and the PiggyBac transposase coding sequence containing the nuclear localization signal;
  • pK201-PB The pKB20 sequence deletes the 5'UTR, 5'ITR, enhancer motif, bGH polyA signal sequence, multiple cloning site and 3'ITR, leaving only miniCMV, the PiggyBac transposase coding sequence with nuclear localization signal and Obtained insulator sequences with transcription termination function;
  • pKB20I1-EGFP obtained by replacing the insulator sequence with transcription termination function in pKB20-EGFP with the transcriptional stop site of human ⁇ 2globin gene (SEQ ID NO: 15);
  • pKB20A1-EGFP obtained by replacing bGH polyA in pKB20-EGFP with SEQ ID NO: 16;
  • pKB20A2-EGFP obtained by replacing the bGH polyA in pKB20-EGFP with the SV40 polyA signal sequence (SEQ ID NO: 13);
  • pKB20U1-EGFP obtained by replacing the 5'UTR sequence in pKB20-EGFP with the 5'UTR of the C3 gene (SEQ ID NO: 17);
  • pKB20U2-EGFP obtained by replacing the 5'UTR sequence in pKB20-EGFP with the 5'UTR of ORM1 gene (SEQ ID NO: 18);
  • pKB20U3-EGFP obtained by replacing the 5'UTR sequence in pKB20-EGFP with the 5'UTR of HPX gene (SEQ ID NO: 19);
  • pKB20U4-EGFP obtained by replacing the 5'UTR sequence in pKB20-EGFP with the 5'UTR of the FGA gene (SEQ ID NO:20);
  • pKB20U5-EGFP obtained by replacing the 5'UTR sequence in pKB20-EGFP with the 5'UTR of the AGXT gene (SEQ ID NO: 21);
  • pKB20U6-EGFP obtained by replacing the 5'UTR sequence in pKB20-EGFP with the 5'UTR of the ASL gene (SEQ ID NO: 22);
  • pKB20U7-EGFP obtained by replacing the 5'UTR sequence in pKB20-EGFP with the 5'UTR of APOA2 gene (SEQ ID NO:23);
  • pKB20U8-EGFP obtained by replacing the 5'UTR sequence in pKB20-EGFP with the 5'UTR of the ALB gene (SEQ ID NO: 24);
  • pKB20E1-EGFP obtained by replacing the enhancer motif sequence in pKB20-EGFP with the CMV enhancer sequence (SEQ ID NO: 25);
  • pKB20E2-EGFP obtained by replacing the enhancer motif sequence in pKB20-EGFP with the SV40 enhancer sequence (SEQ ID NO: 26);
  • pKB20E3-EGFP obtained by replacing the enhancer motif sequence in pKB20-EGFP with the human ⁇ -globin 5'HS2 enhancer sequence (SEQ ID NO: 27);
  • pKB20E4-EGFP obtained by replacing the enhancer motif sequence in pKB20-EGFP with the chicken ⁇ -globin gene 5'HS4 enhancer sequence (SEQ ID NO: 28).
  • pKB20P1-EGFP obtained by replacing the miniCMV sequence in pKB20-EGFP with the CMV53 promoter (SEQ ID NO: 37).
  • pKB20P2-EGFP obtained by replacing the miniCMV sequence in pKB20-EGFP with the miniSV40 promoter (SEQ ID NO: 38).
  • pKB20P3-EGFP obtained by replacing the miniCMV sequence in pKB20-EGFP with the miniTK promoter (SEQ ID NO: 39).
  • pKB20P4-EGFP obtained by replacing the miniCMV sequence in pKB20-EGFP with the MLP promoter (SEQ ID NO: 40).
  • pKB20P5-EGFP obtained by replacing the miniCMV sequence in pKB20-EGFP with the pJB42CAT5 promoter (SEQ ID NO: 41).
  • pKB20P6-EGFP obtained by replacing the miniCMV sequence in pKB20-EGFP with YB_TATA (SEQ ID NO: 42).
  • green fluorescence positive cells can be considered to have stably integrated the green fluorescence expression cassette.
  • the efficiency of integration can be determined by measuring the proportion of green fluorescence positive cells by flow cytometry.
  • Figure 2 shows that a large number of cells with high fluorescence brightness were still seen in Jurkat cells electroporated with pKB20-EGFP, pKB201-EGFP and pKB202-EGFP on the 10th day after electroporation.
  • pKC20-EGFP without the PiggyBac transposase expression cassette, almost no fluorescence can be seen.
  • the vector with the PiggyBac transposase expression cassette has successfully integrated EGFP into the genome of Jurkat cells.
  • the vector without the PiggyBac transposase expression cassette cannot effectively mediate the integration of the foreign gene EGFP.
  • Figure 3 shows the results of flow cytometry of Jurkat cells electroporated with pKB20-EGFP, pKB201-EGFP and pKB202-EGFP on day 7 after electroporation.
  • the results showed that the proportion of positive cells in Jurkat cells electrotransfected with pKB20-EGFP was as high as 67% on the 7th and 10th days, and the positive rate remained at a high level of 65% on the 14th day after 3 passages of cell culture.
  • the positive rate of electrotransfected cells with pKB201-EGFP and pKB202-EGFP was about 48% on the 7th day, and exceeded 27% and 26% on the 14th day after culturing for 3 passages, respectively.
  • FIG. 4 shows that Jurkat cells electrotransfected with plasmids pKB20-EGFP, pKB201-EGFP and pKB202-EGFP containing the PB transposase expression cassette compared with Jurkat cells electrotransfected with pKC20-EGFP without the PB transposase expression cassette. There were no significant differences in the number of viable cells on days 5, 7, 10, and 14 after electroporation. It was shown that the introduction of the cassette containing the PB transposase expression did not affect the proliferation of Jurkat cells.
  • pKB20-EGFP has a very high integration rate and exogenous gene positive expression rate after electroporation into Jurkat cells
  • pKB201-EGFP and pKB202-EGFP also have a high integration rate and exogenous gene positive expression rate after electroporation into Jurkat cells
  • the expression rate was lower than that after pKB20-EGFP electrotransformation.
  • the integration of pKB series vector has no effect on cell proliferation.
  • Example 3 PB transposase expression time curve after the pKB vector was electroporated into Jurkat cells
  • Figure 5 shows that the expression levels of PB transposase in Jurkat cells electroporated with pKB20-EGFP, pKB201-EGFP and pKB202-EGFP reached a peak at 6h, and then began to decrease significantly.
  • the expression level of PB transposase in the electrotransfected cells of pKB20-EGFP and pKB201-EGFP at the peak of 6h was significantly higher than that of the electrotransfected pKB202-EGFP cells.
  • the expression levels of PB transposase in the electrotransfected cells with pKB20-EGFP and pKB201-EGFP decreased sharply at the 24h time point.
  • the expression level of PB transposase in all cells dropped to a very low level at 96 hours and was undetectable by day 15.
  • green fluorescence positive cells can be considered to have stably integrated the green fluorescence expression cassette.
  • the efficiency of integration can be determined by measuring the proportion of green fluorescence positive cells by flow cytometry.
  • Figure 6 shows that on the 10th day after electrotransformation, a large number of cells with high fluorescence brightness were still seen in K562 cells electroporated with pKB20-EGFP, pKB201-EGFP and pKB202-EGFP.
  • K562 cells electroporated with pKC20-EGFP that lacks the PiggyBac transposase expression cassette no cells with high fluorescence can be seen.
  • the vector with the PiggyBac transposase expression cassette has successfully integrated EGFP into the genome of K562 cells.
  • the vector without the PiggyBac transposase expression cassette cannot effectively mediate the integration of the foreign gene EGFP.
  • Figure 7 shows that cells electrotransfected with plasmids pKB20-EGFP, pKB201-EGFP, and pKB202-EGFP containing the PB transposase expression cassette were electroporated with pKC20-EGFP without the PB transposase expression cassette. There were no significant differences in the number of viable cells on days 5, 7, 10, and 14. It was shown that the introduction of the PB transposase expression cassette had no effect on the proliferation of K562 cells.
  • Figure 8 shows the results of flow cytometry of the cells electroporated with pKB20-EGFP, pKB201-EGFP and pKB202-EGFP on the 10th day and the 14th day after electroporation, respectively.
  • the results showed that the positive rates of cell fluorescence in the cells transfected with pKB20-EGFP and pKB201-EGFP exceeded 75% and 73% respectively on the 10th day, and the fluorescence positive rates remained at 70% on the 14th day (cultured for more than 3 passages) after electroporation. % or more, showing a very high integration efficiency.
  • the fluorescence positive rate of the electrotransfected pKB202-EGFP cells was also close to 70% on the 10th day after electroporation, and the fluorescence positive rate was still maintained at a level close to 70% on the 14th day after electroporation (cultured for more than 3 passages).
  • Figure 9 shows that on the 10th to 14th day after electroporation, the fluorescence positive rate of K562 cells electroporated with pKB20-EGFP and pKB201-EGFP vectors did not change much, both were above 70%, close to 75%; K562 cells electroporated with pKB202-EGFP vector The positive rate of cell fluorescence decreased significantly between the 10th and 14th day after electroporation, which was slightly lower than 70%.
  • PBMCs peripheral blood mononuclear cells
  • FIG. 10 shows that, on the 10th day after electrotransformation, a large number of cells with high fluorescence brightness were still seen in the T cells electroporated with pKB20-EGFP, pKB201-EGFP and pKB202-EGFP.
  • T cells electroporated with pKC20-EGFP deficient in the expression cassette of PiggyBac transposase can hardly see cells with high fluorescence brightness. It shows that the vector with the PiggyBac transposase expression cassette has successfully integrated EGFP into the genome of T cells. The vector without the PiggyBac transposase expression cassette cannot effectively mediate the integration of the foreign gene EGFP.
  • Figure 11 shows the results of flow cytometry of T cells electrotransfected with pKB20-EGFP, pKB201-EGFP, and pKB202-EGFP on days 7-14 after electroporation.
  • the results showed that the positive rate of T cells electroporated with pKB20-EGFP reached nearly 74% on the 7th day, and remained close to 72% on the 14th day after electroporation (3 passages of culture).
  • the positive rate of T cells electrotransfected with pKB201-EGFP and pKB202-EGFP was as high as nearly 70% on the 7th day after electroporation, and the positive rate remained above 66% and 62% on the 14th day after electroporation (cultured for 3 generations), respectively. .
  • Example 7 Detection of integration efficiency after electroporation of primary T cells by dual-plasmid PB transposition system
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • Lonza Nucleofector-2b electroporator to electroporate into the nucleus (according to the instrument’s operating instructions)
  • cells were placed in AIM-V medium and cultured in a 37°C, 5% CO 2 incubator after electroporation. After 6 hours, it was transferred to a 6-well plate containing 30ng/mL anti-CD3 antibody and 3000IU/mL IL-2 (purchased from Novoprotein), and cultured in a 37°C, 5% CO 2 incubator. After the cells were confluent, they were diluted and passaged at a ratio of 1:10, and the cells were detected by flow cytometry on the 7th and 14th days after electroporation, respectively.
  • PBMC peripheral blood mononuclear cells
  • Example 8 Detection of integration efficiency after electroporation of the pKB vector with reduced plasmid usage into Jurkat cells
  • Embodiment 9 detection of plasmid copy number of pKB vector residues in cells
  • Jurkat cells and K562 were electroporated with 5 ⁇ g of pKB20-EGFP, respectively, and the cells were harvested on the 10th, 14th and 20th days after electroporation, respectively; according to the method of Example 8, Jurkat cells were electroporated with 3 ⁇ g of pKB20 -EGFP, all were carried out according to the operating instructions of the instrument, and the cells were harvested on the 10th, 12th and 14th days after electroporation; according to the method of Example 5, 5 ⁇ g of pKB20-EGFP was electroporated into fresh PBMCs, and 5 ⁇ g of pKB20-EGFP was electroporated on the 10th, 12th and 14th days after electroporation. Cells were harvested separately at 14 days. All operations were repeated 3 times. All the above harvested cells containing the PB transposase expression cassette were used to detect the amount of residual plasmid at different time points using the Taqman probe fluorescence quantitative PCR method:
  • PB-F 5' ggacgagatctacgccttct (SEQ ID NO: 29)
  • PB-R 5' ctcatcacgctcacgtacac (SEQ ID NO:30)
  • Actin-F 5'gggacctgactgactacctc (SEQ ID NO:32)
  • Actin-probe 5'caccgagcgcggctacagct (SEQ ID NO:34)
  • the amount of electroporated plasmid was reduced to 3 ⁇ g, the residual plasmid content in Jurkat cells after electroporation was further significantly reduced compared with 5 ⁇ g of electroporated plasmid, and the average number of plasmid copies per cell was less than 10 on the 10th day. , and by the 14th day the copy number was less than 1.
  • the vector of the present invention has a very low residual level in the host cell while fully exerting its genome integration function.
  • the pKB series vectors of the present invention can reduce the amount of plasmid DNA under the premise of ensuring high integration efficiency after electroporation, and further reduce the residue of plasmid DNA in cells after electroporation.
  • Jurkat cells and K562 cells were electro-transformed with pKB2003-EGFP plasmid according to the methods described in above-mentioned Example 2 and Example 4, respectively, and the positive rate of cells was detected by flow cytometry on the 7th, 10th, and 14th days after electroporation.
  • the results are shown in Figure 16.
  • the proportion of positive cells in Jurkat cells electrotransfected with pKB2003-EGFP reached 49% and 48% on day 7 and day 10, respectively, and the positive rate remained at a high level of nearly 48% on day 14 after 3 passages of cell culture.
  • the proportion of positive cells in K562 cells electrotransfected with pKB2003-EGFP reached nearly 70% on the 7th and 10th days, and the positive rate remained at a high level of more than 66% on the 14th day after 3 passages of cell culture.
  • Example 4 and Example 5 the amount of plasmid in electroporation was reduced to 3 ⁇ g, and Jurkat cells, K562 cells and PBMCs from healthy human blood were electroporated with pKB20I1-EGFP, pKB20A1-EGFP, pKB20A2-EGFP, pKB20U1-EGFP, pKB20U2-EGFP, pKB20U3-EGFP, pKB20U4-EGFP, pKB20U5-EGFP, pKB20U6-EGFP, pKB20U7-EGFP, pKB20U8-EGFP, pKB20E1-EGFP, pKB20E2-EGFP, pKB20E3-EGFP, pKB20E4 EGFP, pKB20P1-EGFP, pKB20P2-EGFP, pKB20P3-EGFP, pKB20P4-EGFP, pKB20P5-EGFP and p
  • Table 2 show that the pKB series of plasmid vectors replaced by the above-mentioned regulatory original sequences can be efficiently integrated into the genomes of different cells, and the integration rate of pKB20-EGFP in the above-mentioned types of cells is at the same level.
  • the amount of plasmid in electroporation was reduced to 3 ⁇ g
  • PBMCs from healthy human blood were electroporated with pKB205-EGFP, and the positive rate of cells was detected by flow cytometry on the 7th and 14th days after electroporation.
  • Example 14 Analysis of mRNA expression profile after the integration of pKB vector in Jurkat cells and K562 cells
  • Jurkat cells and K562 cells were electroporated with the pKB20-EGFP plasmid according to the methods described in the above examples 2 and 4, respectively, and the plasmid dosage was 4 ⁇ g.
  • Cells were harvested 14 days after electroporation for mRNA sequencing and expression profiling, and compared with the mRNA expression profiling of Jurkat cells and K562 cells without plasmid transfection. Both K562 and Jurkat sent 2 samples for analysis.
  • the EF1A promoter sequence (SEQ ID NO: 11) with NFAT motif was inserted between the XbaI and EcoRI sites of the multiple cloning site of pKB20, and the coding sequence of HER2CAR (SEQ ID NO: 11) was inserted between the EcoRI and SalI sites :43), named pKB20-HER2CAR, the EF1A promoter sequence with NFAT motif and the coding sequence of HER2CAR were entrusted to Shanghai Jereh Biotechnology Co., Ltd. to synthesize.
  • the PBMCs isolated from peripheral blood were electroporated with the pKB20-HER2CAR vector according to the following steps to prepare CAR-T cells targeting HER2.
  • the PBMCs used were purchased from AllCells and were obtained from the peripheral blood of healthy adults.
  • the cells cultured in a 37°C, 5% CO2 incubator after electroporation were transferred into a six-well plate coated with HER2 extracellular domain antigen and CD28 antibody, and IL- 2.
  • Example 15 The in vitro killing activity of the HER2CAR-T cells obtained in Example 15 was detected by the real-time label-free cell function analyzer (RTCA) of Essen, and the specific steps were as follows:
  • Target cell plating human ovarian cancer cell SKOV-3 (purchased from the American Culture Collection Center ATCC, positive for HER2 expression), 10 4 cells/50 ⁇ L per well were plated in a plate containing detection electrodes, and placed for several minutes , after the cells are stable, put them into the instrument, and start step 2 to culture the cells;
  • the killing curve of the control Mock-T cell group is similar to the change trend of the SKOV-3 tumor cell curve, indicating that the killing effect of Mock-T cells on SKOV-3 cells is small.
  • the killing effect of HER2CAR-T on SKOV-3 cells was very obvious at the three effector-target ratios of 1:1, 2:1, and 4:1, and the killing effect was also significantly improved with the increase of effector-target ratio.
  • the gene DNA sequences encoding the ⁇ chain and the ⁇ chain of the TCR that recognize the NY-ESO-1 antigen peptide SLLMWITQC (HLA-*02:01) were synthesized, and the two were linked by the DNA sequence encoding the P2A peptide segment, and the spliced sequence was as follows shown in SEQ ID NO:44. Then connect the DNA sequence encoding EGFP at the 3' end of SEQ ID NO:44 by the DNA encoding the P2A peptide segment to obtain the NY-ESO-1-TCR gene covalently connected to the EGFP reading frame, and the resulting sequence is as SEQ ID NO. :45.
  • the EF1A promoter sequence (SEQ ID NO: 11) with NFAT motif was inserted between the XbaI and EcoRI sites of the multiple cloning site of pKB20, and the NY covalently linked EGFP reading frame was inserted between the EcoRI and SalI sites - Coding sequence of ESO-1-TCR gene (SEQ ID NO: 45), named pKB20-NY-ESO-1-TCR, EF1A promoter sequence with NFAT motif and NY-ESO covalently linked to EGFP reading frame
  • the coding sequence of the -1-TCR gene was synthesized by Shanghai Jereh Biotechnology Co., Ltd.
  • AIM-V medium was added to 2 wells of a 12-well plate in advance, 2 mL per well, and then transferred to a cell culture incubator at 37°C and 5% CO 2 to preheat for 1 hour;
  • Embodiment 19 NY-ESO-1 TCR-T cell NY-ESO-1 TCR expression positive cell detection
  • the proportion of cells positive for EGFP expression was 37.57%.
  • the gene DNA sequences of the ⁇ chain and ⁇ chain of NY-ESO-1 TCR and the coding DNA sequence of EGFP are connected by the P2A peptide coding sequence, and cells with positive EGFP expression can indirectly reflect the expression of NY-ESO-1 TCR gene. Express. It can be speculated that the proportion of NY-ESO-1 TCR-positive cells is about 37%.
  • Embodiment 20 the cell killing function test of NY-ESO-1 TCR-T
  • the in vitro killing activity of the NY-ESO-1 TCR-T cells obtained in Example 18 was detected by the real-time label-free cell function analyzer (RTCA) of Essen, and the specific steps were as follows:
  • Target cell plating human malignant melanoma cell line A375 (purchased from American Type Culture Collection ATCC, positive for NY-ESO-1 expression), 10 4 cells/50 ⁇ L per well were plated in a plate containing detection electrodes , leave it for a few minutes, and after the cells are stable, put them into the instrument, and start step 2, culturing the cells;
  • the killing curve of the control Mock-T cell group basically overlapped with the A375 tumor cell curve, indicating that Mock-T cells have basically no killing effect on A375 cells.
  • the killing effect of NY-ESO-1 TCR-T on A375 cells was very obvious at two effector-target ratios of 0.25:1 and 0.5:1, and the killing effect was also significantly improved with the increase of effector-target ratio.
  • pNB vector and pNB328-EGFP were constructed respectively.
  • pNB328-EGFP was used to prepare K562 stably integrated expressing EGFP by electroporation, and the cells positive for EGFP expression were detected by flow cytometry on the 14th day after electroporation (3 passages of culture).
  • pNB328-EGFP was used to prepare primary T cells that stably integrate and express EGFP by electroporation, and EGFP-positive cells were detected by flow cytometry on the 14th day after electroporation (3 passages of cell culture).
  • the PBMC used for electroporation and the PBMC used in Example 5 were the same batch of PBMC.
  • EGFP-positive cells were detected by flow cytometry 14 days after electroporation (3 passages of culture).

Abstract

提供了一种酸构建物，其包含以下元件：转座子3'末端重复序列、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5'末端重复序列、转座酶编码序列以及控制该转座酶表达的启动子。还提供了包含所述核酸构建物的宿主细胞或药物组合物及其用途。

Description

新型PiggyBac转座子系统及其用途 技术领域
本发明涉及转座子载体领域，具体地涉及一种新型PiggyBac转座子系统及其用途。
背景技术
将外源基因导入目的细胞内稳定表达从而实现对目的细胞的转基因改造是包括免疫细胞治疗在内的众多细胞治疗技术实现的必要条件。相较于瞬时表达，稳定表达的外源基因能够整合进入目的细胞基因组中，在细胞多次传代或培养条件变化的情况下仍能保持长时间持续稳定表达。常用的转基因系统包括基于病毒的载体、真核表达质粒载体和转座子载体。使用基于非病毒载体的方法对人原代T细胞进行遗传改造修饰被证明是及其困难的。因而在全世界范围内，大部分实验室仍然在使用病毒载体系统进行细胞的转基因修饰，包括逆转录病毒载体，如慢病毒载体系统。病毒载体系统虽然一直被广泛使用，但其存在病毒制备操作复杂、安全风险相对较高、生产成本较高等难以克服的问题。
近年来，转座子载体系统越来越多地被用于修饰免疫细胞进而进行肿瘤免疫治疗。最早应用于哺乳动物的转座子是源于鱼类的“睡美人”转座子(Sleeping Beauty)，但其存在过量抑制效应和携带片段偏小(5kb左右)等缺陷，在哺乳动物细胞的转基因操作中应用受到严重的限制。PiggyBac转座子是另一类源于鳞翅目昆虫的转座子系统，其能够携带较大的片段，能够整合多种真核宿主细胞。PiggyBac(PB)转座子系统主要通过“切割-黏贴”(cut-paste)机制发生转座，不会在转座发生后在原位点留下印迹(footprint)，被越来越多地改造后用于基因组研究、基因治疗、细胞治疗、干细胞诱导和诱导后分化等领域。
WO2019046815A1公开了一种传统的基于PiggyBac转座子的二元系统，其包括含有PiggyBac转座酶的载体与含有5’ITR和3’ITR的辅助载体。该PiggyBac转座子系统配合电转法能够将外源基因导入T细胞、NK细胞和HSPC细胞。但二元系统需要PiggyBac转座酶载体与辅助载体同时转进细胞内以后才能发生转座，对转染的 要求较高，难度较大。同时，PiggyBac转座系统的机制PiggyBac转座酶通过“切割-黏贴”(cut-paste)机制将转座片段插入基因组，而这一过程是可逆的，因此只要当PiggyBac酶的表达在持续进行，已经整合到基因组上的转座片段还可能被重新切割下来，造成基因组不稳定，实质上降低了转座效率。普通的PiggyBac二元转座系统在T细胞中的转座效率通常在10％左右的水平，效率较低。并且WO2019046815A1同时也记载了质粒DNA对T细胞的毒性较大，且其对T细胞的毒性与电转所用DNA的量有关。二元系统无疑扩大了电转所需的质粒DNA的用量，增加了对细胞特别是T细胞的毒性，降低了转染质粒DNA的T细胞的存活率。
CN105154473B公开了一种一元PiggyBac转座子载体，该载体将传统二元PiggyBac转座系统中的PiggyBac转座酶载体与辅助载体合并到一个载体中，并通过在同一表达载体中PiggyBac表达框与外源基因表达框共用同一段双向polyA序列，设置整合后PiggyBac转座酶表达框中的polyA即被切割而自失活(self-inactivating)的机制，有效减少了组成型PiggyBac转座酶的持续表达，提升了PiggyBac转座酶的转座效率，同时二元体系精简为一元单载体大幅度减少了DNA总量，降低了外源DNA对T细胞产生的毒性。但PiggyBac转座酶表达框与外源基因表达框共用同一段双向polyA序列会使两个方向相对的表达框之间产生相互影响，并且在某些种类的细胞中该一元转座子载体介导的整合效率仍有待进一步提高。
因此，目前仍然缺乏能够在高效整合外源基因的同时及时关闭整合功能的高效一元转座子体系。
发明内容
发明人构建了一种基于PiggyBac转座子的整合系统，该系统能介导外源基因在宿主细胞内高效整合，并高效稳定表达。
本发明一方面涉及一种核酸构建物，其包含以下元件或由其组成：转座子3’末端重复序列、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列。在一个或多个实施方案中，所述核酸构建物还包含选自以下的一种或多种元件：转座酶编码序列、控制该转座酶表达的启动子、多克隆插入位点、增强子、5’UTR、第二polyA序列和感兴趣的外源基因。
本发明还提供一种核酸构建物，其包含以下元件：转座子3’末端重复序列、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、转座酶编码序列以及控制该转座酶表达的启动子。
在一个或多个实施方案中，所述核酸构建物还包含选自以下的一种或多种元件：多克隆插入位点、增强子、5’UTR、第二polyA序列和感兴趣的外源基因。
在一个或多个实施方案中，所述转座酶编码序列、所述控制该转座酶表达的启动子、所述5’UTR和所述第二polyA序列中的任一种或多种在所述转座子3’末端重复序列和所述转座子5’末端重复序列之间的区域以外。
在一个或多个实施方案中，所述核酸构建物依次包含：转座子3’末端重复序列、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、控制转座酶表达的启动子、转座酶编码序列和第二polyA序列。
在一个或多个实施方案中，所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、转座酶编码序列以及控制该转座酶表达的启动子。
在一个或多个实施方案中，所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、控制转座酶表达的启动子、转座酶编码序列和第二polyA序列。
在一个或多个实施方案中，所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、增强子、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、转座酶编码序列以及控制该转座酶表达的启动子。
在一个或多个实施方案中，所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、增强子、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、控制转座酶表达的启动子、转座酶编码序列和第二polyA序列。
在一个或多个实施方案中，所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、转座酶编码序列、5’UTR以及控制该转座酶表达的启动子。
在一个或多个实施方案中，所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、控制转座酶表达的启动子、5’UTR、转座酶编码序列和第二polyA序列。
在一个或多个实施方案中，所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、增强子、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、转座酶编码序列、5’UTR以及控制该转座酶表达的启动子。
在一个或多个实施方案中，所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、增强子、具有转录终止功能的绝缘子序列、转座 子5’末端重复序列、控制转座酶表达的启动子、5’UTR、转座酶编码序列和第二polyA序列。
在一个或多个实施方案中，所述核酸构建物依次包含：转座子3’末端重复序列、具有转录终止功能的绝缘子序列、多克隆插入位点、第一polyA序列、转座子5’末端重复序列、控制转座酶表达的启动子、5’UTR、转座酶编码序列和第二polyA序列。
在一个或多个实施方案中，所述核酸构建物依次包含：转座子3’末端重复序列、具有转录终止功能的绝缘子序列、多克隆插入位点、第一polyA序列、增强子、转座子5’末端重复序列、控制转座酶表达的启动子、5’UTR、转座酶编码序列和第二polyA序列。
在一个或多个实施方案中，所述核酸构建物依次包含：转座子3’末端重复序列、增强子、具有转录终止功能的绝缘子序列、多克隆插入位点、第一polyA序列、转座子5’末端重复序列、控制转座酶表达的启动子、5’UTR、转座酶编码序列和第二polyA序列。
在一个或多个实施方案中，所述多克隆插入位点用于可操作地插入所述外源基因的编码序列以及任选的控制外源基因表达的启动子。
在一个或多个实施方案中，第一polyA序列与第二polyA序列的加尾信号功能的方向相同或相反。
在一个或多个实施方案中，所述转座酶的表达框的方向与外源基因表达框的方向相同或相反。
在一个或多个实施方案中，所述转座酶的表达框的方向与转座子3’末端重复序列和转座子5’末端重复序列之间的序列的方向相同或相反。
在一个或多个实施方案中，上述的各元件各自独立地为单拷贝或者多拷贝。
在一个或多个实施方案中，上述的各元件直接连接或通过接头或酶切位点连接。
根据本发明任一项所述的核酸构建物，其中，所述转座子5’末端重复序列与所述转座子3’末端重复序列的位置能够互换。
在一个或多个实施方案中，所述转座子3’末端重复序列为PiggyBac转座子3’末端重复序列。在优选实施方案中，所述转座子3’末端重复序列的核苷酸序列如SEQ ID NO:1所示。
在一个或多个实施方案中，所述多克隆插入位点的序列如SEQ ID NO:2所示。
在一个或多个实施方案中，所述第一polyA序列如SEQ ID NO:3、13或16所示。
在一个或多个实施方案中，所述第二polyA序列如SEQ ID NO:3、13或16所示。
在一个或多个实施方案中，增强子选自：CMV增强子序列、SV40增强子、人ε球蛋白5’HS2增强子、鸡β球蛋白基因5’HS4增强子。优选地，增强子序列如SEQ ID NO:4、26-28中任一所示。
在一个或多个实施方案中，具有转录终止功能的绝缘子序列如SEQ ID NO:5或15所示。
在一个或多个实施方案中，所述转座子5’末端重复序列为PiggyBac转座子5’末端重复序列。在优选实施方案中，所述转座子5’末端重复序列的核苷酸序列如SEQ ID NO:6所示。
在一个或多个实施方案中，所述转座酶为PiggyBac转座酶。在一个或多个实施方案中，所述PiggyBac转座酶的氨基酸序列如SEQ ID NO:36所示；优选地，所述PiggyBac转座酶的编码序列如SEQ ID NO:7所示。
在一个或多个实施方案中，5’UTR序列选自C3基因、ORM1基因、HPX基因、FGA基因、AGXT基因、ASL基因、APOA2基因、ALB基因的5’UTR。优选地，5’UTR序列如SEQ ID NO:8、17-24中任一所示。
在一个或多个实施方案中，所述启动子选自：CMV启动子、miniCMV启动子、CMV53启动子、miniSV40启动子、miniTK启动子、MLP启动子、pJB42CAT5启动子、YB_TATA启动子、EF1α启动子、SV40启动子、UbiquitinB启动子、CAG启动子、HSP70启动子、PGK-1启动子、β-actin启动子、TK启动子和GRP78启动子。优选地，所述启动子选自miniCMV启动子、CMV53启动子、miniSV40启动子、miniTK启动子、MLP启动子、pJB42CAT5启动子和YB_TATA。在一个或多个实施方案中，所述启动子的序列如SEQ ID NO:9、37-42中任一所示。优选地，所述启动子是miniCMV启动子，其序列如SEQ ID NO:9所示。
在一个或多个实施方案中，所述转座酶编码序列含有或者可操作地连接单拷贝或者多拷贝的核定位信号编码序列。在一个或多个实施方案中，核定位信号为c-myc核定位信号，优选具有SEQ ID NO:35所示的序列。
在一个或多个实施方案中，所述核酸构建物包含SEQ ID NO:10或14所示序列。
在一个或多个实施方案中，所述核酸构建物是重组载体。
在一个或多个实施方案中，所述核酸构建物是重组克隆载体或重组表达载体。
本发明还提供宿主细胞，其包含：(1)本文任一实施方案中所述的核酸构建物，和/或(2)本文任一实施方案中所述的核酸构建物的转座子3’末端重复序列与转座子5’末端重复序列之间的序列。
在一个或多个实施方案中，所述宿主细胞为哺乳动物细胞。
在一个或多个实施方案中，所述宿主细胞选自免疫细胞、Jurkat细胞、K562细胞、胚胎干细胞、肿瘤细胞、HEK293细胞和CHO细胞。
在一个或多个实施方案中，所述免疫细胞选自T细胞、B细胞、CIK细胞、LAK细胞、NK细胞、细胞毒性T细胞(CTL)、树突状细胞(DC)、肿瘤浸润淋巴细胞(TIL)、巨噬细胞、NK T细胞和γδT细胞中的任一种或多种。
本发明还提供药物组合物，包含本文任一实施方案中所述的核酸构建物或宿主细胞以及药学上可接受的辅料。
本发明还提供本文任一实施方案所述的核酸构建物或宿主细胞在制备或作为药物、试剂或工具中的用途，所述药物、试剂或工具用于将外源基因表达框整合到靶细胞基因组中，或用于基因治疗、细胞治疗、干细胞诱导或分化。
在一个或多个实施方案中，所述靶细胞为哺乳动物细胞。
在一个或多个实施方案中，所述靶细胞选自T细胞、Jurkat细胞、K562细胞、胚胎干细胞、肿瘤细胞、HEK293细胞和CHO细胞。
本发明还提供一种将外源基因或其表达框整合到细胞基因组中的方法，包括将含有外源基因和任选的其启动子的本文任一实施方案所述的核酸构建物导入所述细胞，和任选的在转座酶将外源基因或其表达框整合到细胞基因组的条件下孵育所述细胞。
在一个或多个实施方案中，所述转座酶是PiggyBac转座酶
在一个或多个实施方案中，所述导入包括病毒介导的转化、显微注射、粒子轰击、基因枪转化和电穿孔等。在本发明的一个实施方案中，所述导入为电穿孔。
在一个或多个实施方案中，所述细胞孵育至少三代。
本发明还提供本文所述方法获得的基因组中整合有外源基因或其表达框的细胞。
附图说明
图1A和图1B，本文所述核酸构建物的示意图。
图2，分别电转了pKB20-EGFP、pKB201-EGFP、pKB202-EGFP的Jurkat细胞的荧光照片。
图3，分别电转了pKB20-EGFP、pKB201-EGFP与pKB202-EGFP的Jurkat细胞的流式检测结果。
图4，分别电转了pKB20-EGFP、pKB201-EGFP、pKB202-EGFP和pKC20-EGFP的Jurkat细胞的活细胞数。
图5，分别电转了pKB20-EGFP、pKB201-EGFP和pKB202-EGFP的Jurkat细胞中PB转座酶的表达水平。
图6，分别电转了pKB20-EGFP、pKB201-EGFP、pKB202-EGFP和pKC20-EGFP的K562细胞的荧光照片。
图7，分别电转了pKB20-EGFP、pKB201-EGFP、pKB202-EGFP和pKC20-EGFP的K562细胞的活细胞数。
图8，分别电转了pKB20-EGFP、pKB201-EGFP与pKB202-EGFP的K562细胞的流式检测结果。
图9，分别电转了pKB20-EGFP、pKB201-EGFP和pKB202-EGFP的K562细胞的荧光阳性率。
图10，分别电转了pKB20-EGFP、pKB201-EGFP、pKB202-EGFP和pKC20-EGF的原代T细胞荧光照片。
图11，分别电转了pKB20-EGFP、pKB201-EGFP与pKB202-EGFP的T细胞的流式检测结果。
图12，双质粒PB转座系统电转Jurkat细胞的阳性率结果。
图13，双质粒PB转座系统电转原代T细胞的阳性率结果。
图14，用降低的质粒用量电转pKB20-EGFP的Jurkat细胞的阳性率。
图15，细胞内载体残留拷贝数随时间的变化。
图16，电转pKB2003-EGFP的Jurkat细胞的阳性率。
图17，电转pKB205-EGFP的原代T细胞的阳性率。
图18，K562样本1中pKB20载体介导的基因组整合位点示意图。
图19，K562样本2中pKB20载体介导的基因组整合位点示意图。
图20，Jurkat样本1中pKB20载体介导的基因组整合位点示意图。
图21，Jurkat样本2中pKB20载体介导的基因组整合位点示意图。
图22，电转了pKB20-HER2CAR的原代T细胞的阳性率流式结果。
图23，HER2CAR-T细胞对靶细胞SKOV-3的体外RTCA杀伤结果。
图24，电转了pKB20-NY-ESO-1 TCR的原代T细胞的阳性率流式结果。
图25，NY-ESO-1 TCR-T对靶细胞A375的体外RTCA杀伤结果。
图26，电转了pNB328-EGFP的K562细胞的阳性率流式结果。
图27，电转了pNB328-EGFP的原代T细胞的阳性率流式结果。
具体实施方式
应理解，在本发明范围中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成优选的技术方案。
本文和所附权利要求书所用的单数形式“一个”、“一种”和“所述”包括复数含义，除非上下文另有明确说明。同样，术语“一个”(或“一种”)、“一个或多个”和“至少一个”在本文中可互换使用。术语“包括”及其变化形式不具有限制意义，其中这些术语出现在本说明书和权利要求中。因此，术语“包括”、“包含”、和“含有”可互换使用。
核酸构建物和细胞
术语“核酸构建物”，在文中定义为单链或双链核酸分子，优选是指人工构建的核酸分子。可选地，所述核酸构建物还包含有可操作地连接的1个或多个调控序列，所述调控序列在其相容条件下能指导编码序列在合适的宿主细胞中进行表达。表达应理解为包括蛋白或多肽生产中所涉及的任何步骤，包括，但不限于转录、转录后修饰、翻译、翻译后修饰和分泌。本文所述的转座系统优选是一元核酸构建物，即一个核酸构建物即可实现高效转座。
在本发明中，如果没有特别说明，以转座酶表达框的方向为反向。上述的“依次包含如下元件”中的“依次”所指的方向和/或顺序是指从上游至下游。在本发明中，如果没有特别说明，沿着上述“正向”的方向为从上游至下游，沿着上述“反向”的方向为从下游至上游。
在本发明中，术语“表达框”是指表达一个基因所需的完整元件，包括启动子、基因编码序列、PolyA加尾信号序列。
术语“可操作地插入/连接”在文中定义为这样一种构象，其中调控序列位于相对DNA序列之编码序列的适当位置，以使调控序列指导蛋白或多肽的表达。在本发 明的核酸构建物中，多克隆位点通过DNA重组技术可操作地插入有一个或多个相同或不同的外源基因以及可选的控制外源基因表达的启动子，或者其多克隆位点被替换为一个或多个相同或不同的外源基因编码序列以及可选的控制外源基因表达的启动子。所述“可操作地连接”可以通过DNA重组的手段实现，具体地，所述核酸构建物为重组核酸构建物。
本文所述“外源基因”可以是任何来源的核酸分子，其在转座到宿主细胞基因组后表达或行使功能。外源基因的非限制性示例包括荧光素报告基因(例如绿色荧光蛋白、黄色荧光蛋白等)、荧光素酶基因(例如萤火虫荧光素酶、海肾荧光素酶等)、天然功能蛋白基因、RNAi基因以及人工嵌合基因(例如嵌合抗原受体基因、Fc融合蛋白基因、全长抗体基因)。
术语“编码序列”在文中定义为核酸序列中直接确定其蛋白产物的氨基酸序列的部分。编码序列的边界通常是由紧邻mRNA 5’端开放读码框上游的核糖体结合位点(对于原核细胞)和紧邻mRNA 3’端开放读码框下游的转录终止序列确定。编码序列可以包括，但不限于DNA、cDNA和重组核酸序列。
本文中术语“调控序列”定义为包括表达本发明肽所必需或有利的所有组分。每个调控序列对于编码蛋白或多肽的核酸序列可以是天然含有的或外来的。这些调控序列包括，但不限于，前导序列、polyA序列、前肽序列、启动子、信号序列和转录终止子。最低限度，调控序列要包括启动子以及转录和翻译的终止信号。为了导入特定的限制位点以便将调控序列与编码蛋白或多肽的核酸序列的编码区进行连接，可以提供带接头的调控序列。
调控序列可以是合适的启动子序列，即可被表达核酸序列的宿主细胞识别的核酸序列。启动子序列含有介导蛋白或多肽表达的转录调控序列。启动子序列通常与待表达蛋白的编码序列操作性连接。启动子可以是在所选择的宿主细胞中显示转录活性的任何核苷酸序列，包括突变的、截短的和杂合启动子，并且可以从编码与该宿主细胞同源或异源的胞外或胞内多肽的基因获得。
调控序列还可以是合适的转录终止序列，即能被宿主细胞识别从而终止转录的一段序列。终止序列可操作连接在编码蛋白或多肽的核酸序列的3’末端。在选择的宿主细胞中有功能的任何终止子都可用于本发明。
调控序列还可以是合适的前导序列，即对宿主细胞的翻译十分重要的mRNA非翻译区。前导序列可操作连接于编码多肽的核酸序列的5’末端。在选择的宿主细胞中有功能的任何终止子都可用于本发明。
调控序列还可以是信号肽编码区，该区编码一段连在蛋白或多肽氨基端的氨基酸序列，能引导编码多肽进入细胞分泌途径。核酸序列编码区的5’端可能天然含有翻译读框一致地与分泌多肽的编码区片段自然连接的信号肽编码区。或者，编码区的5’端可含有对编码序列是外来的信号肽编码区。当编码序列在正常情况下不含有信号肽编码区时，可能需要添加外来信号肽编码区。或者，可以用外来的信号肽编码区简单地替换天然的信号肽编码区以增强多肽分泌。但是，任何能引导表达后的多肽进入所用宿主细胞的分泌途径的信号肽编码区都可以用于本发明。
本发明的核酸构建物包含以下元件：转座子3’末端重复序列、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、转座酶编码序列以及控制该转座酶表达的启动子。所述核酸构建物还可包含选自以下的一种或多种元件：多克隆插入位点、增强子、5’UTR和第二polyA序列。在一种特别优选的实施方案中，所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、增强子、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、转座酶编码序列、5’UTR以及控制该转座酶表达的启动子，如图1A所示。在另一种特别优选的实施方案中，所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、增强子、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、控制转座酶表达的启动子、5’UTR、转座酶编码序列以及第二polyA序列，如图1B所示。本文核酸构建物中的各元件各自独立地为单拷贝或者多拷贝。
本文中，转座子5’末端重复序列与所述转座子3’末端重复序列的位置能够互换。优选地所述转座子5’末端重复序列为PiggyBac转座子5’末端重复序列；所述转座子3’末端重复序列为PiggyBac转座子3’末端重复序列。
本文中，转座酶优选为PiggyBac转座酶，其编码序列含有或者可操作地连接单拷贝或者多拷贝的核定位信号编码序列，从而提高转座效率。示例性PiggyBac转座酶编码序列如SEQ ID NO:7所示。示例性核定位信号编码序列如SEQ ID NO:35所示。
本发明的核酸构建物针对转座酶和外源基因可以使用polyA序列。这种设计可以一定程度上缩短核酸构建物的全长，有助于纳入更长的外源基因用于转座。或者，本发明的核酸构建物针对转座酶和外源基因也可以使用分开的polyA序列，二者的加尾信号功能的方向可以相同或相反。这种设计避免了共用同一段双向polyA序列会使两个方向相对的表达框之间的相互影响。本文所述polyA序列可以具有或不具有双向转录终止功能。优选地，polyA序列独立地选自SEQ ID NO:3、13或16。
本发明的核酸构建物针对转座酶和外源基因也可以或进一步使用绝缘子序列用 于转录终止。因此，在任意polyA序列的任意端可包含绝缘子序列。优选地，本发明的绝缘子序列位于转座子5’末端重复序列和转座子3’末端重复序列之间。本文所述绝缘子序列具有转录终止功能，该序列可以是本领域已知的任何具有转录终止功能的序列。优选地，具有转录终止功能的绝缘子序列如SEQ ID NO:5或15所示。
因此，转座酶可以使用polyA序列、绝缘子序列、或polyA序列和绝缘子序列实现转录终止；外源基因可以使用polyA序列、绝缘子序列、或polyA序列和绝缘子序列实现转录终止。优选地，任意所述绝缘子序列位于所述转座子5’末端重复序列和转座子3’末端重复序列之间。
本发明的核酸构建物中，合适的转座酶的启动子序列是能够驱动可操作地连接至其上的转座酶高水平表达的启动子序列，包括但不限于类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、EB病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。在一个或多个实施方案中，转座酶的启动子选自：CMV启动子、miniCMV启动子、CMV53启动子、miniSV40启动子、miniTK启动子、MLP启动子、pJB42CAT5启动子、YB_TATA启动子、EF1α启动子、SV40启动子、UbiquitinB启动子、CAG启动子、HSP70启动子、PGK-1启动子、β-actin启动子、TK启动子和GRP78启动子。优选地，所述启动子选自miniCMV启动子、CMV53启动子、miniSV40启动子、miniTK启动子、MLP启动子、pJB42CAT5启动子和YB_TATA启动子。更优选地，所述启动子是miniCMV启动子。miniCMV相比CMV启动子短了很多，使得载体长度更小，更有利于整合更大的外源基因。在一个或多个实施方案中，在miniCMV和转座酶之间添加5’UTR序列以增强转录和翻译。5’UTR序列可如SEQ ID NO:8、17-24中任一所示。
本发明的核酸构建物可含有增强子，该增强子可位于本文所述核酸构建物中除了增强子以外的任何元件的任意端。优选地，增强子位于转座子3’末端重复序列和转座子5’末端重复序列之间。更优选地，增强子位于第一polyA序列的下游。增强子序列可如SEQ ID NO:4、25-28中任一所示。本文中，核酸构建物可不包含所述5’UTR序列和增强子序列，或包含二者之一，或包含二者，所得核酸构建物均能将外源基因高效整合入细胞基因组。
添加能根据宿主细胞的生长情况来调节多肽表达的调控序列可能也是需要的。调控系统的例子是那些能对化学或物理刺激物(包括在有调控化合物的情况下)作出 反应，从而开放或关闭基因表达的系统。调控序列的其他例子是那些能使基因扩增的调控序列。在这些例子中，应将编码蛋白或多肽的核酸序列与调控序列可操作地连接在一起。
在含一个polyA信号序列的具体实施方案中，本发明的核酸构建物依次包含PiggyBac转座子3’末端重复序列(3’ITR)(SEQ ID NO:1)、多克隆位点(SEQ ID NO:2)、polyA信号序列(SEQ ID NO:3、13或16)、任选的增强子基序序列(SEQ ID NO:4、25-28中任一)、绝缘子序列(SEQ ID NO:5或15)、PiggyBac转座子5’末端重复序列(5’ITR)(SEQ ID NO:6)的反向互补序列、PiggyBac转座酶编码序列(SEQ ID NO:7)的反向互补序列、任选的5’UTR序列(SEQ ID NO:8、17-24中任一)的反向互补序列和miniCMV启动子序列(SEQ ID NO:9)的反向互补序列。在一个或多个实施方案中，本发明的核酸构建物SEQ ID NO:10所示的序列。
在含两个polyA信号序列的具体实施方案中，本发明的核酸构建物依次包含PiggyBac转座子3’末端重复序列(3’ITR)(SEQ ID NO:1)、多克隆位点(SEQ ID NO:2)、第一polyA信号序列(SEQ ID NO:3、13或16)、任选的增强子基序序列(SEQ ID NO:4、25-28中任一)、绝缘子序列(SEQ ID NO:5或15)、PiggyBac转座子5’末端重复序列(5’ITR)(SEQ ID NO:6)、miniCMV启动子序列(SEQ ID NO:9)、任选的5’UTR序列(SEQ ID NO:8、17-24中任一)、PiggyBac转座酶编码序列(SEQ ID NO:7)和第二polyA信号序列(SEQ ID NO:3、13或16)。在一个或多个实施方案中，本发明的核酸构建物具有SEQ ID NO:14所示的序列。
在某些实施方案中，所述核酸构建物是重组载体。重组载体可以是重组克隆载体，也可以是重组表达载体。本发明的核酸构建物的各元件可被装入许多类型的载体，例如，质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。通常，合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记。
为了评估所携带基因的表达，被引入细胞的载体也可包含可选择的标记基因或报道基因中的任一个或两者，以便于从细胞群中鉴定和选择细胞。可选择的标记可被携带在单独一段DNA上并用于共转染程序。可选择的标记和报道基因两者的侧翼都可具有适当的调节序列，以便能够在宿主细胞中表达。有用的可选择标记包括Flag、HA或V5。报道基因用于鉴定潜在转染的细胞并用于评价调节序列的功能性。在DNA已经被引入受体细胞后，报道基因的表达在合适的时间下进行测定。合适的报道基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌型碱性磷酸酶或绿 色萤光蛋白基因的基因。
重组克隆载体可用于提供含有本发明核酸构建物的各元件和可选的外源基因的编码序列。重组克隆载体可为本发明核酸构建物的各元件与pUC18、pUC19、pMD18-T、pMD19-T、pGM-T载体、pUC57、pMAX或pDC315系列载体经重组得到的重组载体。
重组表达载体可用于在合适的宿主细胞中通过本发明核酸构建物的各元件将外源基因表达框整合到基因组中并表达。该载体对于复制和整合真核细胞可为合适的。典型的克隆载体包含可用于调节期望核酸序列表达的转录和翻译终止子、起始序列和启动子。重组表达载体为本发明核酸构建物的各元件与pCDNA3系列载体、pCDNA4系列载体、pCDNA5系列载体、pCDNA6系列载体、pRL系列载体、pUC57载体、pMAX载体或pDC315系列载体经重组得到的重组载体；
重组载体(重组克隆载体或重组表达载体)可以是重组病毒载体，包括但不限于重组腺病毒载体、重组腺相关病毒载体、重组逆转录病毒载体、重组单纯疱疹病毒载体或重组痘苗病毒载体。病毒载体技术在本领域中是公知的并在例如Sambrook等(2001，Molecular Cloning:A Laboratory Manual，Cold Spring Harbor Laboratory，New York)和其他病毒学和分子生物学手册中进行了描述。
本文所述的核酸构建物通常可以用PCR扩增法获得。具体而言，可根据本文所公开的核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。或者，也可直接合成本文所述的核酸构建物。
本发明还提供包含本文任一实施方案中所述的核酸构建物的宿主细胞。宿主细胞既包括哺乳动物细胞，也包括生产哺乳动物细胞过程中使用到的各种细胞，如大肠杆菌细胞，以用于如提供本文所述的核酸构建物或载体。在某些实施方案中，本文提供一种含有本文所述的核酸构建物或载体的哺乳动物细胞，包括但不限于：T细胞、B细胞、CIK细胞、LAK细胞、NK细胞、细胞毒性T细胞(CTL)、树突状细胞(DC)、肿瘤浸润淋巴细胞(TIL)、巨噬细胞、NK T细胞、γδT细胞、Jurkat细胞、K562细胞、胚胎干细胞、肿瘤细胞、HEK293细胞和CHO细胞。
药物组合物
本发明的药物组合物包含本文所述的核酸构建物或细胞和药学上可接受的辅料。本发明中，“药学上可接受的辅料”是用于将本发明的核酸构建物或细胞传送给动物 或人的药学上或食品上可接受的载体、溶剂、悬浮剂或赋形剂。本文中，药学上可接受的辅料在所采用的剂量和浓度对所述组合物的接受者是无毒的。可包括本领域周知的治疗中常用于递送康生物的各种类型的载体或赋形剂。示例性的辅料可以是液体或固体，包括但不限于：pH调节剂，表面活性剂，碳水化合物，佐剂，抗氧化剂，螯合剂，离子强度增强剂、防腐剂、载剂、助流剂、甜味剂、染料/着色剂、增味剂、润湿剂、分散剂、悬浮剂、稳定剂、等渗剂、溶剂或乳化剂。在一些实施方案中，药学上可接受的辅料可以包括一种或多种非活性成分，包括但不限于：稳定剂、防腐剂、添加剂、佐剂、喷雾剂、压缩空气或其它适宜的气体，或其它适宜的与药效化合物合用的非活性成分。更具体而言，合适的辅料可以是本领域常用于转座系统或含有其的细胞给药的辅料。辅料的示例包括各种乳糖、甘露醇，油类如玉米油，缓冲剂如PiggyBacS、盐水、聚乙二醇、甘油、聚丙二醇、二甲亚砜，酰胺如二甲基乙酰胺，蛋白质如白蛋白，和去污剂如吐温80，单糖和低聚多糖如葡萄糖、乳糖、环糊精和淀粉。
其它药物组合物将为本领域技术人员显而易见，包括在持续或控制释放递送配制物中包含本文所述的核酸构建物或细胞的配制物。用于配制多种其它持续或可控传递方式的技术(诸如脂质体载剂、生物易蚀微粒或多孔珠粒和积存注射)也为本领域技术人员所知。
用于体内施用的药物组合物通常以无菌制剂的形式提供。通过经无菌过滤膜过滤来实现灭菌。在组合物冻干时，可在冻干和复水之前或之后使用此方法进行灭菌。用于肠胃外施用的组合物可以冻干形式或在溶液中储存。肠胃外组合物通常放在具有无菌进入孔的容器中，例如具有皮下注射针可刺穿的塞子的静脉内溶液带或小瓶。
通常，组合物中含有治疗有效量的本文所述试剂。治疗有效量是指可在受试者中实现治疗、预防、减轻和/或缓解疾病或病症的剂量。实现这些效果可以通过插入具有相应功能的外源基因实现的，所述相应功能的外源基因具有相应于具体用途的功能，例如治疗功能或者诱导功能。可根据患者年龄、性别、所患病症及其严重程度、患者的其它身体状况等因素确定治疗有效量。治疗有效量可作为单一剂量施用，或者可依据有效的治疗方案在多个剂量中给药。本文中，受试者或患者通常指哺乳动物，尤其指人。示例性地，所述组合物含有按照重量比例为例如0.001-50％，优选0.01-30％，更优选0.05-10％的本文所述的核酸构建物或细胞。
本文所述组合物可与具有所述外源基因所实现功能相似或相应的功能的其他试剂联用。例如与治疗所述外源基因所治疗的疾病或病症的试剂联用。本领域技术人员 可确定其他试剂的给药剂量。
本发明所述的药物组合物的剂型可以是多种多样的，只要是能够使活性成分有效地到达哺乳动物机体的剂型都是可以的，可以被制成单位剂型的形式。剂型比如可选自：凝胶剂、气雾剂、片剂、胶囊、粉末、颗粒、糖浆、溶液、悬浮液、注射剂、散剂、丸剂、控速释剂、输液剂、混悬剂等等。根据本文所述的核酸构建物或细胞所预防和治疗的疾病类型，本领域人员可以选择方便应用的剂型。从易于制备和储存的立场看，优选的组合物是固态组合物，尤其是片剂和固体填充或液体填充的胶囊。本文所述的核酸构建物或细胞或其组合物也可储存在适宜于注射或滴注的消毒器具中。本文所述的核酸构建物或细胞或其组合物也可储存在适当的容器，并置于试剂盒或药盒中。
方法和用途
本发明人在研究中发现，本文核酸构建物可以以可控的方式高效实现外源基因的基因组整合。当外源基因整合到基因组以后，能够有效终止PiggyBac转座酶的转录表达，同时能够发挥外源基因表达框绝缘子功能，减少整合的外源基因表达框对整合位点附近基因表达产生的影响。因此，本发明涉及将外源基因或其表达框整合到细胞基因组中的方法，包括将含有外源基因和任选的其启动子的本文任一实施方案所述的核酸构建物导入所述细胞，和任选的在PiggyBac转座酶将外源基因或其表达框整合到细胞基因组的条件下孵育所述细胞。本发明还提供上述方法获得的基因组中整合有外源基因或其表达框的细胞。
将核酸构建物引入细胞的方法在本领域中是已知的。载体可通过在本领域中的任何方法容易地引入宿主细胞，例如，哺乳动物、细菌、酵母或昆虫细胞。例如，载体可通过物理、化学或生物学手段转移入宿主细胞。示例性的物理或化学方法包括：包括磷酸钙沉淀、脂质转染法、显微注射、粒子轰击、微注射、基因枪转化、电穿孔、胶体分散系统、大分子复合物、纳米胶囊、微球、珠、基于脂质的系统(包括水包油乳剂、胶束、混合胶束和脂质体)。将核酸构建物引入宿主细胞的生物学方法包括病毒介导的转化，特别是逆转录病毒载体。其他病毒载体可源自慢病毒、痘病毒、单纯疱疹病毒I、腺病毒和腺伴随病毒等等。可利用本领域中已知的技术将和选择的核酸序列插入载体并包装入逆转录病毒颗粒。该重组病毒可随后被分离和传递至体内或离体的对象细胞。用于病毒包装的试剂为本领域所周知，如常规的慢病毒载体系统包括pRsv-REV、pMDlg-pRRE、pMD2G和目的干扰质粒。
本发明还提供本文任一实施方案所述的核酸构建物或宿主细胞在制备或作为药 物、试剂或工具中的用途，所述药物、试剂或工具用于将外源基因表达框整合到靶细胞基因组中，或用于基因治疗、细胞治疗、干细胞诱导或分化。在一个或多个实施方案中，所述靶细胞为哺乳动物细胞，包括但不限于选自T细胞、B细胞、CIK细胞、LAK细胞、NK细胞、细胞毒性T细胞(CTL)、树突状细胞(DC)、肿瘤浸润淋巴细胞(TIL)、巨噬细胞、NK T细胞、γδT细胞、Jurkat细胞、K562细胞、胚胎干细胞、肿瘤细胞、HEK293细胞和CHO细胞。
示例性实施方案
项目1、一种核酸构建物，其包含以下元件或由其组成：转座子3’末端重复序列、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列，
优选地，所述核酸构建物还包含选自以下的一种或多种元件：转座酶编码序列、控制该转座酶表达的启动子、多克隆插入位点、增强子、5’UTR、第二polyA序列和感兴趣的外源基因，
优选地，所述转座酶编码序列、所述控制该转座酶表达的启动子、所述5’UTR和所述第二polyA序列中的任一种或多种在所述转座子3’末端重复序列和所述转座子5’末端重复序列之间的区域以外。
项目2、如项目1所述的核酸构建物，其包含以下元件：转座子3’末端重复序列、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、转座酶编码序列以及控制该转座酶表达的启动子，
优选地，所述核酸构建物还包含选自以下的一种或多种元件：多克隆插入位点、增强子、5’UTR、第二polyA序列和感兴趣的外源基因。
项目3、如项目2所述的核酸构建物，其特征在于，
所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、转座酶编码序列以及控制该转座酶表达的启动子，
所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、增强子、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、转座酶编码序列以及控制该转座酶表达的启动子，
所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、转座酶编码序列、5’UTR以及控制该转座酶表达的启动子，
所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、增强子、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、转座酶编码序列、5’UTR以及控制该转座酶表达的启动子，
所述核酸构建物依次包含：转座子3’末端重复序列、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、控制转座酶表达的启动子、转座酶编码序列和第二polyA序列，
所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、控制转座酶表达的启动子、转座酶编码序列和第二polyA序列，
所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、增强子、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、控制转座酶表达的启动子、转座酶编码序列和第二polyA序列，
所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、控制转座酶表达的启动子、5’UTR、转座酶编码序列和第二polyA序列，或
所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、增强子、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、控制转座酶表达的启动子、5’UTR、转座酶编码序列和第二polyA序列，
所述核酸构建物依次包含：转座子3’末端重复序列、具有转录终止功能的绝缘子序列、多克隆插入位点、第一polyA序列、转座子5’末端重复序列、控制转座酶表达的启动子、5’UTR、转座酶编码序列和第二polyA序列，
所述核酸构建物依次包含：转座子3’末端重复序列、具有转录终止功能的绝缘子序列、多克隆插入位点、第一polyA序列、增强子、转座子5’末端重复序列、控制转座酶表达的启动子、5’UTR、转座酶编码序列和第二polyA序列，或
所述核酸构建物依次包含：转座子3’末端重复序列、增强子、具有转录终止功能的绝缘子序列、多克隆插入位点、第一polyA序列、转座子5’末端重复序列、控制转座酶表达的启动子、5’UTR、转座酶编码序列和第二polyA序列。
项目4、如项目1-3中任一项所述的核酸构建物，其特征在于，所述核酸构建物具有选自以下的一项或多项特征：
所述转座酶的表达框的方向与外源基因的表达框的方向相同或相反，
所述转座酶的表达框的方向与转座子3’末端重复序列和转座子5’末端重复序列之间的序列的方向相同或相反，
所述转座子5’末端重复序列与所述转座子3’末端重复序列的位置能够互换，
所述转座子3’末端重复序列为PiggyBac转座子3’末端重复序列，
所述转座子5’末端重复序列为PiggyBac转座子5’末端重复序列，
所述增强子选自：CMV增强子序列、SV40增强子、人ε球蛋白5’HS2增强子、鸡β球蛋白基因5’HS4增强子，
所述转座酶为PiggyBac转座酶，
所述5’UTR选自C3基因、ORM1基因、HPX基因、FGA基因、AGXT基因、ASL基因、APOA2基因、ALB基因的5’UTR，
所述启动子选自：CMV启动子、miniCMV启动子、CMV53启动子、miniSV40启动子、miniTK启动子、MLP启动子、pJB42CAT5启动子、YB_TATA启动子、EF1α启动子、SV40启动子、UbiquitinB启动子、CAG启动子、HSP70启动子、PGK-1启动子、β-actin启动子、TK启动子和GRP78启动子，
所述转座酶编码序列含有或者可操作地连接单拷贝或者多拷贝的核定位信号编码序列。
项目5、如项目1-3中任一项所述的核酸构建物，其特征在于，所述核酸构建物具有选自以下的一项或多项特征：
所述转座子3’末端重复序列的核苷酸序列如SEQ ID NO:1所示，
所述转座子5’末端重复序列的核苷酸序列如SEQ ID NO:6所示，
所述多克隆插入位点的序列如SEQ ID NO:2所示，
所述第一polyA序列如SEQ ID NO:3、13或16所示，
所述第二polyA序列如SEQ ID NO:3、13或16所示，
所述增强子序列如SEQ ID NO:4、26-28中任一所示，
所述绝缘子序列如SEQ ID NO:5或15所示，
所述PiggyBac转座酶的氨基酸序列如SEQ ID NO:36所示；优选地，所述PiggyBac转座酶的编码序列如SEQ ID NO:7所示，
所述5’UTR序列如SEQ ID NO:8、17-24中任一所示，
所述启动子的序列如SEQ ID NO:9、SEQ ID NO:37-42中任一项所示；
所述核定位信号为c-myc核定位信号；优选地，所述核定位信号具有SEQ ID NO:35所示的序列。
项目6、如项目1-3中任一项所述的核酸构建物，其特征在于，
所述核酸构建物包含SEQ ID NO:10或14所示序列，或
所述核酸构建物是重组载体，优选地，所述核酸构建物是重组克隆载体或重组表达载体。
项目7、一种宿主细胞，包含
(1)项目1-6中任一项所述的核酸构建物，和/或
(2)项目1-6中任一项所述的核酸构建物的转座子3’末端重复序列与转座子5’末端重复序列之间的序列，
优选地，所述宿主细胞为哺乳动物细胞，
更优选地，所述宿主细胞选自T细胞、Jurkat细胞、K562细胞、胚胎干细胞、肿瘤细胞、HEK293细胞和CHO细胞。
项目8、一种药物组合物，包含项目1-6中任一项所述的核酸构建物或项目8所述的宿主细胞以及药学上可接受的辅料。
项目9、项目1-6中任一项所述的核酸构建物或项目7所述的宿主细胞在制备药物、试剂或工具中的用途或作为药物、试剂或工具的用途，所述药物、试剂或工具用于将外源基因表达框整合到靶细胞基因组中，或用于基因治疗、细胞治疗、干细胞诱导或分化，
优选地，所述靶细胞为哺乳动物细胞，
更优选地，所述靶细胞选自免疫细胞、Jurkat细胞、K562细胞、胚胎干细胞、肿瘤细胞、HEK293细胞和CHO细胞，
进一步更优选地，所述免疫细胞选自T细胞、B细胞、CIK细胞、LAK细胞、NK细胞、细胞毒性T细胞(CTL)、树突状细胞(DC)、肿瘤浸润淋巴细胞(TIL)、巨噬细胞、NK T细胞和γδT细胞中的任一种或多种。
项目10、一种将外源基因或其表达框整合到细胞基因组中的方法，包括将含有外源基因和任选的其启动子的项目1-6中任一项所述的核酸构建物导入所述细胞，和任选的在转座酶将外源基因或其表达框整合到细胞基因组的条件下孵育所述细胞，所 述外源基因和任选的其启动子位于所述核酸构建物的多克隆插入位点中，
优选地，所述转座酶是PiggyBac转座酶。
本发明的优点：
1)本发明转座子载体系统具有多重调控元件，多层次调控PiggyBac的表达及其介导的外源基因整合。本文所用的驱动PiggyBac转座酶表达的启动子降低了表达强度且减少了DNA长度。同时引入增强子元件，在完整质粒状态，瞬时增强转座酶的表达，发挥PiggyBac转座酶的切割与整合功能；当外源基因表达框从完整质粒上被切开后，增强子失去对启动子的增强作用，实现PiggyBac转座酶的高效开启与关闭，使转座酶水平在短时间表达达到峰值后降低，介导外源基因完成整合的同时大幅减少细胞毒性；
2)在启动子下游加上5’UTR序列，与增强子元件相配合瞬时提升转座酶的表达强度，使PiggyBac酶表达强度足够高，提高整体整合效率。如下实施例所示，本发明转座子载体系统的整合效率有非常显著的提升；
3)PiggyBac表达框采用具有转录终止作用的绝缘子序列，外源基因整合发生前能有效终止转录，表达转座酶基因；当外源基因整合到基因组以后，具有转录终止作用的绝缘子序列的缺失能够有效终止PiggyBac转座酶的转录表达。同时，具有转录终止作用的绝缘子序列与外源基因表达框一起整合到基因组中以后绝缘子还能阻隔外源基因的表达对基因组中邻近区域的影响，减少整合的外源基因表达框对整合位点附近基因表达产生的影响；
4)本发明转座子载体系统在基因组中插入位点较少，且基因内插入位点少，对基因组稳定性影响较小；
5)在mRNA水平对基因表达谱影响较小。
下面将结合实施案例对本发明所涉及的实施方案进行详细描述。本领域技术人员将会理解，下面的实施案例仅用于说明本发明，而不应视为限定本发明的范围。实施案例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。以下实施例中所用细胞系均购自ATCC。
实施例
实施例1，载体的构建
pKB20：按顺序依次将PiggyBac转座子3’末端重复序列(3’ITR)(SEQ ID NO:1)、多克隆位点(SEQ ID NO:2)、bGH polyA信号序列(SEQ ID NO:3)、增强子基序序列(SEQ ID NO:4)、具有转录终止功能的绝缘子序列(C2转录停顿位点，SEQ ID NO:5)、PiggyBac转座子5’末端重复序列(5’ITR)(SEQ ID NO:6)的反向互补序列、PiggyBac转座酶编码序列(SEQ ID NO:7)的反向互补序列、5’UTR序列(SEQ ID NO:8)的反向互补序列和miniCMV启动子序列(SEQ ID NO:9)的反向互补序列，拼接成一段长序列(SEQ ID NO:10)，委托上海捷瑞生物科技有限公司合成，并在两端分别加入AgeI与AscI酶切位点，装入pUC57(购自上海捷瑞生物)，命名pKB20。质粒示意图见图1A。
pKB20-EGFP：在pKB20的多克隆位点的XbaI与EcoRI位点间插入带有NFAT基序的EF1A启动子序列(SEQ ID NO:11)，在EcoRI和SalI位点之间插入EGFP编码序列(SEQ ID NO:12)，命名为pKB20-EGFP，带有NFAT基序的EF1A启动子序列与EGFP编码序列委托上海杰瑞生物科技有限公司合成。
pKB205：按顺序依次将PiggyBac转座子3’末端重复序列(3’ITR)(SEQ ID NO:1)、多克隆位点MCS(SEQ ID NO:2)、bGH polyA信号序列(SEQ ID NO:3)、增强子基序序列(SEQ ID NO:4)、具有转录终止功能的绝缘子序列(C2转录停顿位点，SEQ ID NO:5)、PiggyBac转座子5’末端重复序列(5’ITR)(SEQ ID NO:6)、miniCMV启动子序列(SEQ ID NO:9)、5’UTR序列(SEQ ID NO:8)、PiggyBac转座酶编码序列(SEQ ID NO:7)和SV40 polyA信号序列(SEQ ID NO:13)拼接成一段长序列(SEQ ID NO:14)，委托上海捷瑞生物科技有限公司合成，并在两端分别加入AgeI与AscI酶切位点，装入pUC57(购自上海捷瑞生物)，命名pKB205。质粒示意图见图1B。
pKB205-EGFP：在pKB205的多克隆位点的XbaI与EcoRI位点间插入带有NFAT基序的EF1A启动子序列(SEQ ID NO:11)，在EcoRI和SalI位点之间插入EGFP编码序列(SEQ ID NO:12)，命名为pKB205-EGFP，带有NFAT基序的EF1A启动子序列与EGFP编码序列委托上海杰瑞生物科技有限公司合成。
采用相同方法分别获得以下质粒：
pKB201-EGFP：pKB20-EGFP序列删除增强子基序序列获得；
pKB202-EGFP：pKB20-EGFP序列删除5’UTR序列获得；
pKB2003-EGFP：pKB20-EGFP序列删除增强子基序序列和5’UTR序列获得；
pKC20-EGFP：pKB20-EGFP序列删除miniCMV启动子序列、5’UTR序列、含 核定位信号的PiggyBac转座酶编码序列后获得；
pK201-PB：pKB20序列删除5’UTR、5’ITR、增强子基序、bGH polyA信号序列、多克隆位点和3’ITR，仅保留miniCMV、含核定位信号的PiggyBac转座酶编码序列和具有转录终止功能的绝缘子序列获得；
pKB20I1-EGFP：将pKB20-EGFP中的具有转录终止功能的绝缘子序列替换为人α2globin基因转录停顿位点(SEQ ID NO:15)后获得；
pKB20A1-EGFP：将pKB20-EGFP中的bGH polyA替换为SEQ ID NO:16后获得；
pKB20A2-EGFP：将pKB20-EGFP中的bGH polyA替换为SV40 polyA信号序列(SEQ ID NO:13)后获得；
pKB20U1-EGFP：将pKB20-EGFP中的5’UTR序列替换为C3基因的5’UTR(SEQ ID NO:17)后获得；
pKB20U2-EGFP：将pKB20-EGFP中的5’UTR序列替换为ORM1基因的5’UTR(SEQ ID NO:18)后获得；
pKB20U3-EGFP：将pKB20-EGFP中的5’UTR序列替换为HPX基因的5’UTR(SEQ ID NO:19)后获得；
pKB20U4-EGFP：将pKB20-EGFP中的5’UTR序列替换为FGA基因的5’UTR(SEQ ID NO:20)后获得；
pKB20U5-EGFP：将pKB20-EGFP中的5’UTR序列替换为AGXT基因的5’UTR(SEQ ID NO:21)后获得；
pKB20U6-EGFP：将pKB20-EGFP中的5’UTR序列替换为ASL基因的5’UTR(SEQ ID NO:22)后获得；
pKB20U7-EGFP：将pKB20-EGFP中的5’UTR序列替换为APOA2基因的5’UTR(SEQ ID NO:23)后获得；
pKB20U8-EGFP：将pKB20-EGFP中的5’UTR序列替换为ALB基因的5’UTR(SEQ ID NO:24)后获得；
pKB20E1-EGFP：将pKB20-EGFP中的增强子基序序列替换为CMV增强子序列(SEQ ID NO:25)后获得；
pKB20E2-EGFP：将pKB20-EGFP中的增强子基序序列替换为SV40增强子序列(SEQ ID NO:26)后获得；
pKB20E3-EGFP：将pKB20-EGFP中的增强子基序序列替换为人ε-globin 5’HS2 增强子序列(SEQ ID NO:27)后获得；
pKB20E4-EGFP：将pKB20-EGFP中的增强子基序序列替换为鸡β-globin基因5’HS4增强子序列(SEQ ID NO:28)后获得。
pKB20P1-EGFP：将pKB20-EGFP中的miniCMV序列替换为CMV53启动子(SEQ ID NO:37)后获得。
pKB20P2-EGFP：将pKB20-EGFP中的miniCMV序列替换为miniSV40启动子(SEQ ID NO:38)后获得。
pKB20P3-EGFP：将pKB20-EGFP中的miniCMV序列替换为miniTK启动子(SEQ ID NO:39)后获得。
pKB20P4-EGFP：将pKB20-EGFP中的miniCMV序列替换为MLP启动子(SEQ ID NO:40)后获得。
pKB20P5-EGFP：将pKB20-EGFP中的miniCMV序列替换为pJB42CAT5启动子(SEQ ID NO:41)后获得。
pKB20P6-EGFP：将pKB20-EGFP中的miniCMV序列替换为YB_TATA(SEQ ID NO:42)后获得。
实施例2，含EGFP表达框pKB载体在Jurkat细胞中的整合
准备5×10 6生长旺盛的低代数Jurkat细胞，通过Lonza2b-Nucleofector仪器，将质量均为5μg的pKB20-EGFP、pKB201-EGFP、pKB202-EGFP与pKC20-EGFP分别转染到细胞核中(按仪器操作说明书进行)，置37℃、5％CO 2孵箱培养。待细胞长满后，按1:10的比例传代培养。在电转后的第7天、第10天、第14天荧光显微镜拍照观察；电转后第7天、第10天、第14天(3次传代后)式细胞仪检测EGFP阳性细胞的比例变化，未转染质粒的Jurkat细胞作为流式细胞检测的对照。电转全过程荧光显微镜观察细胞荧光强度。在电转后第5天、第7天、第10天、第14天分别进行活细胞计数，观察电转pKB载体对Jurkat细胞增殖的影响。
按1:10的比例稀释传代，非整合的质粒随着细胞的分裂，质粒很快丢失。3代(约13天)以后，绿色荧光阳性的细胞可以认为绿色荧光表达框已经稳定整合。通过流式检测绿色荧光阳性细胞比例，可确定整合的效率。
结果如图2-4所示。图2显示到电转后第10天，电转了pKB20-EGFP、pKB201-EGFP、pKB202-EGFP的Jurkat细胞中仍可见大量高荧光亮度的细胞。电转了缺失PiggyBac转座酶表达框的pKC20-EGFP的Jurkat细胞已基本看不到荧光。表 明带有PiggyBac转座酶表达框的载体已经成功地将EGFP在Jurkat细胞中进行了基因组整合。而不包括PiggyBac转座酶表达框的载体不能有效地介导外源基因EGFP进行整合。
图3显示电转后第7天分别电转了pKB20-EGFP、pKB201-EGFP与pKB202-EGFP的Jurkat细胞的流式检测结果。结果显示，电转pKB20-EGFP的Jurkat细胞在第7天和第10天的阳性细胞比例都高达67％以上，在细胞培养3代后的第14天阳性率仍维持在65％的高水平。电转pKB201-EGFP和pKB202-EGFP的细胞阳性率在第7天在48％左右，在培养3代后的第14天分别超过27％与26％。
图4显示，电转了包含PB转座酶表达框的质粒pKB20-EGFP、pKB201-EGFP和pKB202-EGFP的Jurkat细胞与电转了不包含PB转座酶表达框的pKC20-EGFP的Jurkat细胞相比，在电转后第5、7、10、14天的活细胞数都没有显著差异。表明包含了PB转座酶表达框的导入不会对Jurkat细胞的增殖产生影响。
以上的结果表明pKB20-EGFP在电转Jurkat细胞后均有非常高的整合率和外源基因阳性表达率，pKB201-EGFP与pKB202-EGFP电转Jurkat细胞后也具有较高的整合率与外源基因阳性表达率，但低于pKB20-EGFP电转后的水平。并且pKB系列载体的整合对细胞的增殖基本不产生影响。
实施例3，pKB载体电转Jurkat细胞后PB转座酶表达时间曲线
取5×10 6生长旺盛的低代数Jurkat细胞，通过Lonza2b-Nucleofector仪器，将质量均为5μg的pKB20-EGFP、pKB201-EGFP和pKB202-EGFP分别转染到细胞核中(按仪器操作说明书进行)，37℃、5％CO 2培养，电转后的第6、12、24、48、96小时及第15天收集细胞后提RNA，以β-actin为内参RT-PCR定量检测PB转座酶的表达水平，结果如图5所示。
图5显示，电转了pKB20-EGFP、pKB201-EGFP和pKB202-EGFP的Jurkat细胞中PB转座酶的表达水平均在6h到达高峰，随后开始显著下降。电转pKB20-EGFP和pKB201-EGFP的细胞在6h高峰时的PB转座酶表达水平明显高于电转pKB202-EGFP细胞的PB转座酶表达水平。其中电转pKB20-EGFP和pKB201-EGFP的细胞在24h时间点PB转座酶的表达水平急剧降低。所有细胞在96小时PB转座酶的表达水平均已降至很低，到第15天均已检测不到。
实施例4，含EGFP表达框pKB载体在K562细胞中的整合
准备5×10 6生长旺盛的低代数K562细胞，通过Lonza2b-Nucleofector仪器，将质量均为5μg的pKB20-EGFP、pKB201-EGFP、pKB202-EGFP与pKC20-EGFP分别转染到细胞核中(按仪器操作说明书进行)，置37℃、5％CO 2孵箱培养。待细胞长满后，按1:10的比例传代培养。在电转后的第1天、第7天、第10天荧光显微镜拍照观察；电转后第10天、第14天(3次传代后)式细胞仪检测EGFP阳性细胞的比例变化，未转染质粒的K562细胞作为流式细胞检测的对照。电转全过程荧光显微镜观察细胞荧光强度。在电转后第5天、第7天、第10天、第14天分别进行活细胞计数，观察电转pKB载体对K562细胞增殖的影响。
按1:10的比例稀释传代，非整合的质粒随着细胞的分裂，质粒很快丢失。3代(约13天)以后，绿色荧光阳性的细胞可以认为绿色荧光表达框已经稳定整合。通过流式检测绿色荧光阳性细胞比例，可确定整合的效率。
结果如图6-9所示。图6显示到电转后第10天，电转了pKB20-EGFP、pKB201-EGFP、pKB202-EGFP的K562细胞中仍可见大量高荧光亮度的细胞。电转了缺失PiggyBac转座酶表达框的pKC20-EGFP的K562细胞已基本看不到高荧光亮度的细胞。表明带有PiggyBac转座酶表达框的载体已经成功地将EGFP在K562细胞中进行了基因组整合。而不包括PiggyBac转座酶表达框的载体不能有效地介导外源基因EGFP进行整合。
图7显示电转了包含PB转座酶表达框的质粒pKB20-EGFP、pKB201-EGFP和pKB202-EGFP的细胞与电转了不包含PB转座酶表达框的pKC20-EGFP的细胞相比，在电转后第5、7、10、14天的活细胞数都没有显著差异。表明包含了PB转座酶表达框的导入不会对K562细胞的增殖产生影响。
图8显示电转后第10天和第14天分别电转了pKB20-EGFP、pKB201-EGFP与pKB202-EGFP的细胞的流式检测结果。结果显示，电转pKB20-EGFP和pKB201-EGFP的细胞在第10天的细胞荧光阳性率分别超过75％和73％，电转后第14天(培养超过3代)的荧光阳性率均仍然维持在70％以上，表现出非常高的整合效率。电转pKB202-EGFP的细胞在电转后第10天的荧光阳性率也接近70％，并且在电转后第14天(培养超过3代)的荧光阳性率也仍然维持在接近70％的水平。
图9显示在电转后的第10-14天，电转了pKB20-EGFP和pKB201-EGFP载体的K562细胞荧光阳性率变化不大，均在70％以上，接近75％；电转pKB202-EGFP载体的K562细胞荧光阳性率在电转后的第10-14天之间下降也不明显，略低于70％。
以上的结果表明pKB20-EGFP、pKB201-EGFP与pKB202-EGFP在电转K562 细胞后均有非常高的整合率和外源基因阳性表达率。
实施例5，含EGFP表达框pKB载体在原代T细胞中的整合
准备四组新鲜分离的外周血单个核细胞(PBMC)，每组5×10 6个，用Lonza Nucleofector-2b电转仪分别将将质量均为5μg的pKB20-EGFP、pKB201-EGFP、pKB202-EGFP和pKC20-EGFP电转到细胞核中(按仪器操作说明书进行)，电转后将细胞置于AIM-V培养基中置37℃、5％CO 2孵箱培养。6小时后转移至含30ng/mL抗CD3抗体、3000IU/mL IL-2(购自Novoprotein)的6孔板中，置37℃、5％CO 2孵箱培养。待细胞长满后，按1:10的比例稀释传代，分别在电转后的第1、7和10天对电转pKB20-EGFP、pKB201-EGFP、pKB202-EGFP和pKC20-EGFP的细胞荧光显微镜下观察并拍照，在电转后第7、10和14天对电转pKB20-EGFP、pKB201-EGFP和pKB202-EGFP的细胞进行流式检测，结果如图10-11所示。
图10显示，到电转后第10天，电转了pKB20-EGFP、pKB201-EGFP、pKB202-EGFP的T细胞中仍可见大量高荧光亮度的细胞。电转了缺失PiggyBac转座酶表达框的pKC20-EGFP的T细胞已基本看不到高荧光亮度的细胞。表明带有PiggyBac转座酶表达框的载体已经成功地将EGFP在T细胞中进行了基因组整合。而不包括PiggyBac转座酶表达框的载体不能有效地介导外源基因EGFP进行整合。
图11显示电转后第7-14天分别电转了pKB20-EGFP、pKB201-EGFP与pKB202-EGFP的T细胞的流式检测结果。结果显示，电转了pKB20-EGFP的T细胞在第7天的细胞阳性率达到接近74％，在电转后的第14天(培养3代)仍然维持在接近72％。电转pKB201-EGFP和pKB202-EGFP的T细胞在电转后第7天的阳性率也都高达近70％，在电转后第14天(培养3代)阳性率仍分别维持在66％和62％以上。
以上的结果表明pKB20-EGFP、pKB201-EGFP与pKB202-EGFP在电转原代T细胞后均有非常高的整合率和外源基因阳性表达率。
实施例6，双质粒PB转座系统电转Jurkat细胞后整合效率检测
准备1×10 7生长旺盛的低代数Jurkat细胞，分为A、B两组，每组5×10 6个细胞。分别准备4μg的pK201-PB+3μg的pKC20-EGFP混合液与3μg的pK201-PB+4μg的pKC20-EGFP的混合液，通过Lonza2b-Nucleofector仪器分别电转到A、B两组细胞的细胞核中(按仪器操作说明书进行)，置37℃、5％CO 2孵箱培养。待细胞 长满后，按1:10的比例传代培养。在电转后的第7天、第10天、第14天荧光显微镜拍照观察；电转后第7天、第10天、第14天(培养3代后)流式细胞仪检测EGFP阳性细胞的比例变化，未转染质粒的Jurkat细胞作为流式细胞检测的对照。
结果如图12所示。结果显示，电转4μg的pK201-PB+3μg的pKC20-EGFP的Jurkat细胞在电转后第7天细胞阳性率超过43％，在电转后第14天(培养3代后)的细胞阳性率降低至13.31％。类似地，电转3μg的pK201-PB+4μg的pKC20-EGFP的Jurkat细胞在电转后第7天细胞阳性率超过52％，在电转后第14天(培养3代后)的细胞阳性率降低至10.57％。
上述结果表明，分别表达PB转座酶和外源基因的双载体转座系统在Jurkat细胞中的整合效率远低于前述单载体pKB系统的整合效率。
实施例7，双质粒PB转座系统电转原代T细胞后整合效率检测
准备5×10 6个新鲜分离的外周血单个核细胞(PBMC)，准备4μg的pK201-PB+3μg的pKC20-EGFP混合液，用Lonza Nucleofector-2b电转仪电转到细胞核中(按仪器操作说明书进行)，电转后将细胞置于AIM-V培养基中置37℃、5％CO 2孵箱培养。6小时后转移至含30ng/mL抗CD3抗体、3000IU/mL IL-2(购自Novoprotein)的6孔板中，置37℃、5％CO 2孵箱培养。待细胞长满后，按1:10的比例稀释传代，分别在电转后的第7和14天对细胞进行流式检测。
结果如图13所示。结果显示，电转4μg的pK201-PB+3μg的pKC20-EGFP的原代细胞在电转后第7天细胞阳性率为16.20％，在电转后第14天(培养3代后)的细胞阳性率降低至15.34％。
上述结果表明，分别表达PB转座酶和外源基因的双载体转座系统在原代T细胞中的整合效率明显低于前述单载体pKB系统的整合效率。
实施例8，质粒用量减少的pKB载体电转Jurkat细胞后整合效率检测
准备5×10 6生长旺盛的低代数Jurkat细胞，准备3μg的pKB20-EGFP质粒，通过Lonza2b-Nucleofector仪器电转到细胞核中(按仪器操作说明书进行)，置37℃、5％CO 2孵箱培养。待细胞长满后，按1:10的比例传代培养。电转后第7天、第10天、第14天(培养3代后)流式细胞仪检测EGFP阳性细胞的比例变化，未转染质粒的Jurkat细胞作为流式细胞检测的对照。
结果如图14所示。结果显示，电转3μg pKB20-EGFP质粒的Jurkat细胞在电 转后第7天细胞阳性率超过66％，在电转后第14天(培养3代后)的细胞阳性率仍保持在接近63％，与实施例2中用6μg pKB20-EGFP质粒电转Jurkat细胞后14天的细胞阳性率接近。
上述结果表明，在实施例2的基础上将电转所用的pKB20-EGFP质粒用量减半后，pKB载体系统在细胞中的整合效率基本不变，说明本发明的pKB载体系统能够在减少DNA量的情况下实现同等效果的整合效率，极大地有利于电转中降低DNA用量进而减少DNA对T细胞产生的毒性以及降低电转后细胞中的残留质粒DNA。
实施例9，细胞内pKB载体残留质粒拷贝数检测
根据实施例2和4的方法，分别对Jurkat细胞和K562电转5μg的pKB20-EGFP，在电转后第10、14和20天分别收获细胞；根据实施例8的方法，对Jurkat细胞电转3μg的pKB20-EGFP，均按仪器操作说明书进行，在电转后第10、12和14天分别收获细胞；根据实施例5的方法，对新鲜PBMC电转5μg的pKB20-EGFP，在电转后的第10、12和14天分别收获细胞。所有操作均重复3次。使用Taqman探针荧光定量PCR法对含有PB转座酶表达框的上述所有收获的细胞进行不同时间点的残留质粒量检测：
1)使用含有PB转座酶表达框的pKB20-EGFP质粒作为标准品，使用含内参基因Actin的质粒作为内参标准品，分别制备10倍梯度稀释的标准品；
2)提取电转pKB20-EGFP后指定天数的细胞总DNA，作为检测PB转座酶基因和Actin基因的样品；
3)配置PCR反应体系，对待检测样品分别进行PB转座酶基因和内参Actin基因扩增；对PB基因梯度稀释标准品和Actin基因梯度稀释标准品进行扩增，准备制作PB基因和Actin基因各自的标准曲线。扩增PB基因与扩增Actin基因的引物、探针序列及反应体系分别如下所示：
PB基因扩增：
PB-F：5’ggacgagatctacgccttct(SEQ ID NO:29)
PB-R：5’ctcatcacgctcacgtacac(SEQ ID NO:30)
PB-探针：5’tgcgcacggcggtcatcacc(SEQ ID NO:31)
Actin基因扩增：
Actin-F：5’gggacctgactgactacctc(SEQ ID NO:32)
Actin-R：5’aatgtcacgcacgatttccc(SEQ ID NO:33)
Actin-探针：5’caccgagcgcggctacagct(SEQ ID NO:34)
表1：探针荧光定量PCR反应体系
Figure PCTCN2021123191-appb-000001
4)实时荧光定量PCR对待检测样品及标准品进行扩增反应，反应体系如表1所示，反应程序为1、50℃2min；2、95℃10min；3、95℃15s，60℃1min，共进行40个循环。制作标准曲线，根据标准曲线计算质粒残留量。拷贝数计算公式为PB拷贝数/Actin拷贝数*2。
结果如图15所示。电转质粒用量为5μg的正常用量情况下，细胞内的残留质粒的拷贝数随着时间急剧降低，在细胞培养至电转后第14天(3代以后)，平均每个Jurkat细胞中的残留质粒拷贝数已小于10，平均每个K562细胞和原代T细胞内的残留质粒拷贝数已小于5；培养至电转后第20天时，Jurkat细胞和K562细胞中平均每个细胞中的残留质粒拷贝数已经小于1。当电转质粒的用量减少为3μg时，电转后Jurkat细胞内残留的质粒含量与电转5μg质粒相比有了进一步的明显下降，在第10天时平均每个细胞内的质粒拷贝数即已低于10，而到第14天拷贝数已不到1。
以上结果表明本发明的载体在充分发挥其基因组整合功能的同时在宿主细胞中的残留水平非常低。同时结合实施例8的结果也表明，本发明的pKB系列载体能够在保证电转后高整合效率的前提下减少质粒DNA的用量，进而进一步减少电转后质粒DNA在细胞内的残留。
实施例10，pKB2003-EGFP载体电转后在细胞中的整合
按上述实施例2和实施例4记载的方法，分别对Jurkat细胞与K562细胞电转pKB2003-EGFP质粒，电转后第7、10、14天流式检测细胞阳性率。结果如图16显 示。电转pKB2003-EGFP的Jurkat细胞在第7天和第10天的阳性细胞比例分别达到49％和48％以上，在细胞培养3代后的第14天阳性率仍维持在接近48％的高水平。电转pKB2003-EGFP的K562细胞在第7天和第10天的阳性细胞比例都达到接近70％，在细胞培养3代后的第14天阳性率仍维持在超过66％的高水平。
以上结果表明，pKB2003载体能够在细胞中高效整合并表达外源基因。
实施例11，调控原件序列替换的pKB系列载体电转后在细胞中的整合
分别按实施例2、实施例4和实施例5记载的方法，将电转中质粒的用量降至3μg，分别对Jurkat细胞、K562细胞和来自健康人血液的PBMC电转pKB20I1-EGFP、pKB20A1-EGFP、pKB20A2-EGFP、pKB20U1-EGFP、pKB20U2-EGFP、pKB20U3-EGFP、pKB20U4-EGFP、pKB20U5-EGFP、pKB20U6-EGFP、pKB20U7-EGFP、pKB20U8-EGFP、pKB20E1-EGFP、pKB20E2-EGFP、pKB20E3-EGFP、pKB20E4-EGFP、pKB20P1-EGFP、pKB20P2-EGFP、pKB20P3-EGFP、pKB20P4-EGFP、pKB20P5-EGFP和pKB20P6-EGFP质粒，电转后第14天流式检测细胞阳性率，结果如表2所示。
表2调控原件序列替换的pKB系列载体在不同细胞的整合率
  Jurkat K562 原代T细胞
pKB20I1-EGFP 62.33％ 70.43％ 69.65％
pKB20A1-EGFP 64.02％ 72.33％ 72.55％
pKB20A2-EGFP 68.58％ 69.27％ 70.64％
pKB20U1-EGFP 70.05％ 68.89％ 68.25％
pKB20U2-EGFP 66.51％ 70.97％ 71.36％
pKB20U3-EGFP 62.11％ 73.29％ 72.01％
pKB20U4-EGFP 57.43％ 71.82％ 70.13％
pKB20U5-EGFP 58.27％ 67.44％ 69.81％
pKB20U6-EGFP 61.89％ 69.49％ 67.07％
pKB20U7-EGFP 59.65％ 72.93％ 69.12％
pKB20U8-EGFP 63.43％ 72.01％ 71.79％
pKB20E1-EGFP 62.72％ 66.95％ 65.23％
pKB20E2-EGFP 58.55％ 68.39％ 69.97％
pKB20E3-EGFP 69.79％ 69.28％ 72.17％
pKB20E4-EGFP 71.37％ 71.03％ 70.02％
pKB20P1-EGFP 66.75％ 72.16％ 70.69％
pKB20P2-EGFP 65.31％ 70.06％ 70.75％
pKB20P3-EGFP 70.03％ 68.24％ 71.53％
pKB20P4-EGFP 59.93％ 69.14％ 68.90％
pKB20P5-EGFP 65.47％ 67.98％ 69.74％
pKB20P6-EGFP 71.01％ 72.09％ 60.58％
表2结果显示，上述调控原件序列替换的pKB系列质粒载体均能够高效整合到不同细胞的基因组中，与pKB20-EGFP在上述各类细胞中的整合率处于同一水平。
实施例12，pKB205-EGFP载体电转原代T细胞后整合效率检测
按实施例5记载的方法，将电转中质粒的用量降至3μg，对来自健康人血液的PBMC电转pKB205-EGFP，电转后第7天和第14天流式检测细胞阳性率。
结果如图17所示。电转了pKB205-EGFP的T细胞在第7天的细胞阳性率超过42％，在电转后的第14天(培养3代)仍然维持在36％以上。说明pKB205-EGFP载体在电转原代T细胞后有较高的整合率和外源基因阳性表达率。
实施例13，pKB载体在Jurkat细胞和K562细胞中的整合位点分析
分别准备Jurkat细胞与K562细胞各两份，分别按照实施例2和实施例4记载的方法对两种细胞电转pKB20-EGFP质粒，电转后培养14天，收集细胞提取基因组DNA，委托晶能生物技术(上海)有限公司进行全基因组测序，分析EGFP在基因组上的插入位点分布情况。结果如图18-21和表3所示。图18、19、20和21分别为K562样本1、K562样本2、Jurkat样本1和Jurkat样本2中pKB20载体介导的基因组整合位点示意图。
表3 Jurkat和K562细胞中pKB20载体介导的整合位点位置分类统计
Figure PCTCN2021123191-appb-000002
图18-21和表3中的结果显示，由本发明pKB20载体介导的外源基因细胞基因组整合主要集中发生在基因间区域和内含子区域，在外显子区和基因表达调控相关区域(如3’UTR)中整合位点较少。这表明由pKB20载体介导的外源基因在细胞基因组上的整合对细胞自身基因表达的影响非常小。
实施例14，pKB载体在Jurkat细胞与K562细胞中整合后mRNA表达谱分析
按上述实施例2和实施例4记载的方法，分别对Jurkat细胞与K562细胞电转pKB20-EGFP质粒，质粒用量4μg。电转后14天收获细胞进行mRNA测序与表达谱分析，并与未电转质粒的Jurkat细胞与K562细胞的mRNA表达谱进行比较。K562与Jurkat均送2份样本进行分析。
测序与分析结果显示，与各自未电转质粒的对照细胞相比，电转pKB20-EGFP后pKB20整合位点的相邻基因mRNA表达变化不大，相关基因差异表达情况如表4-7所示。这表明本发明pKB载体在基因组上的整合对细胞基因组的稳定性和基因表达谱的影响较小。
表4 K562样本1整合位点相邻基因的差异表达情况
相关基因名称 插入位点位置 是否差异表达
DENND1B；C1orf53 基因间 否
GNPAT；EXOC8 基因间 否
ELK4 基因上游 表达下调
MTRNR2L5；ZWINT 基因间 否
LPXN 基因下游 否
CCDC179；MIR8054 基因间 否
LINC01995；ATP11B 基因间 否
LINC01267 基因上游 否
NFKB1 基因下游 否
LCORL；SLIT2 基因下游 否
LINC01378；LINC02264 基因间 否
LINC01378；LINC02264 基因间 否
ATP10B 基因下游 否
CRHBP；AGGF1 基因间 否
CRHBP；AGGF1 基因间 否
TULP1；FKBP5 基因间 否
ADGB 基因下游 否
LOC105374972；NRSN1 基因间 否
LMX1B；ZBTB43 基因间 否
PCDH11X；MIR4454 基因间 否
表5 K562样本2整合位点相邻基因的差异表达情况
相关基因名称 插入位点位置 是否差异表达
TSPAN2 基因上游 否
KHDRBS1 基因下游 否
LINC00900；LOC101929011 基因间 否
LINC01309；DAOA-AS1 基因间 否
KIF2B；TOM1L1 基因间 否
MIB1；GATA6-AS1 基因间 否
TWSG1；RALBP1 基因间 否
MCM5；RASD2 基因间 否
MIR4465；NMBR 基因间 否
FNDC1；LOC102724053 基因间 否
MGC4859；NDUFA4 基因间 否
STOM；GGTA1P 基因间 否
FAM9A；FAM9B 基因间 否
表6 Jurkat样本1整合位点相邻基因的差异表达情况
相关基因名称 插入位点位置 是否差异表达
TMEM167B 基因上游 否
LOC441666 基因上游 否
LINC01831；LOC100287010 基因上游 否
PLSCR5；LINC02010 基因间 否
MIR7641-2；KIAA1211 基因间 否
CD83 基因上游 否
LINC00972；GRM3 基因间 否
STC1；ADAM28 基因间 否
GPR174；ITM2A 基因间 否
NLGN4Y；NONE 基因间 否
表7 Jurkat样本2整合位点相邻基因的差异表达情况
相关基因名称 插入位点位置 是否差异表达
SFRP5；LINC00866 基因间 否
LINC00558；LINC00458 基因间 否
LINC01551；PRKD1 基因间 否
EGLN3；SPTSSA 基因间 否
NFATC1；LOC284241 基因间 否
IPO5P1；ZNF91 基因间 否
AP2S1；ARHGAP35 基因间 否
实施例15，pKB20-HER2CAR载体及HER2CAR-T细胞的制备
在pKB20的多克隆位点的XbaI与EcoRI位点间插入带有NFAT基序的EF1A启动子序列(SEQ ID NO:11)，在EcoRI和SalI位点之间插入HER2CAR的编码序列(SEQ ID NO:43)，命名为pKB20-HER2CAR，带有NFAT基序的EF1A启动子序列与HER2CAR的编码序列委托上海杰瑞生物科技有限公司合成。
按以下步骤用pKB20-HER2CAR载体对从外周血中分离的PBMC进行电转，制备靶向HER2的CAR-T细胞，所用PBMC购自AllCells公司，来自健康成年人外周血。
1)将悬浮细胞收集到50ml离心管中，1200rmp，离心3min；
2)弃上清，加入生理盐水重悬，1200rmp，离心3min，弃生理盐水，并重复此步骤，细胞计数；
3)取2个1.5ml离心管，每管加入5×10 6个细胞，1200rmp，离心3min；
4)弃上清，取电转试剂盒(购买于Lonza公司)，加入18μL的solution I试剂和82l的solution II试剂，第1管加入5μg pKB20空载质粒用作对照，第2管加入5μg pKB20-HER2CAR质粒；
5)将离心管中混合有质粒的细胞悬液转移至电转杯中，放入电转仪，选取程序T020，进行电击；
6)使用试剂盒中的微量吸管将电转好的细胞悬液转移到加好AIM-V培液的十二孔板中(含2％FBS的AIM-V培液)，混匀，置于37℃，5％CO2培养箱培养；同时用含有5μg/mL HER2胞外区抗原(义翘神州10004-H08H)和5μg/mL CD28抗体(ThermoFisher 14-0281-82)的混合液包被6孔板2个孔，每孔加入1mL，放置6孔板于37℃孵育。
7)6小时后，将电转后在37℃，5％CO2培养箱培养的细胞转入用HER2胞外区抗原和CD28抗体包被的六孔板中，并加入终浓度为100IU/mL IL-2，补加培液直3ml，培养4～5天后，观察T细胞的生长情况，将激活后的细胞转入含2％FBS的AIM-V培液继续培养至所需量，获得HER2CAR-T细胞；转入pKB20空载的细胞为作为对照的Mock-T细胞。
实施例16，HER2CAR-T细胞中CAR表达阳性细胞的检测
1)收集实施例15制备的HER2CAR-T细胞1×10 6个，1000rpm，离心3min；
2)弃上清，各加入生理盐水重悬细胞，1000rpm，离心3min；
3)弃上清，各加入100μL生理盐水重悬细胞，每管各加1μL生物素标记的HER2抗原(购自恺佧生物货号：HER-HM402)，4℃孵育30分钟；
4)各加入适量生理盐水，1000rpm，离心3min，洗涤两遍，弃上清；
5)各加入100μL生理盐水重悬细胞，加1μL PE标记的链霉亲和素(购自ThermoFisher货号：S20982)，混匀，4℃孵育30min；
6)各加入适量生理盐水，1000rpm，离心3min，洗涤两遍，弃上清；
7)用400μL生理盐水重悬，上流式细胞仪检测。
结果如图22所示，有高达93.41％的细胞显示为PE阳性，表明通过pKB20-HER2CAR质粒电转PBMC所制备获得的HER2CAR-T细胞中CAR表达阳性的细胞所占的比例在90％以上。
实施例17，HER2-CAR-T的细胞杀伤功能测试
应用艾森公司的实时无标记细胞功能分析仪(RTCA)检测实施例15获得的HER2CAR-T细胞的体外杀伤活性，具体步骤如下：
(1)调零：每孔加入50μL DMEM培养液，放入仪器中，选择步骤1，调零；
(2)靶细胞铺板：人卵巢癌细胞SKOV-3(购买于美国菌种保藏中心ATCC，HER2表达阳性)，按每孔10 4个细胞/50μL铺在含有检测电极的板中，放置数分钟，待细胞稳定后，再放入仪器中，开始步骤2，培养细胞；
(3)加入效应细胞：靶细胞培养18h后，分别加入对照细胞Mock-T和效应细胞HER2CAR-T，每孔50μL，HER2CAR-T分别设置1:1、2:1、4:1三个效靶比，Mock-T设置效靶比为4:1，效靶比的设置均按活细胞总数计算，开始共培养，超过60h后，观察细胞增殖曲线。
结果如图23所示，对照Mock-T细胞组的杀伤曲线与SKOV-3肿瘤细胞曲线的变化趋势接近，表明Mock-T细胞对SKOV-3细胞的杀伤效果较小。HER2CAR-T在1:1、2:1、4:1三个效靶比下对SKOV-3细胞的杀伤效果均十分明显，且杀伤效果随着效靶比的提升也有明显提升。
实施例18，pKB20-NY-ESO-1-TCR载体及NY-ESO-1 TCR-T细胞的制备
pKB20-NY-ESO-1-TCR载体的构建：
合成编码识别NY-ESO-1抗原肽SLLMWITQC(HLA-*02:01)的TCR的α链和β链的基因DNA序列，两者通过编码P2A肽段的DNA序列链接，所拼接成的序列如SEQ ID NO:44所示。再在SEQ ID NO:44的3’端通过编码P2A肽段的DNA连接编码EGFP的DNA序列，获得共价连接EGFP阅读框的NY-ESO-1-TCR基因，所得到的序列如SEQ ID NO:45所示。在pKB20的多克隆位点的XbaI与EcoRI位点间插入带有NFAT基序的EF1A启动子序列(SEQ ID NO:11)，在EcoRI和SalI位点之间插入共价连接EGFP阅读框的NY-ESO-1-TCR基因的编码序列(SEQ ID NO:45)，命名为pKB20-NY-ESO-1-TCR，带有NFAT基序的EF1A启动子序列与共价连接EGFP阅读框的NY-ESO-1-TCR基因的编码序列委托上海杰瑞生物科技有限公司合成。
NY-ESO-1 TCR-T细胞的制备：
用含有5μg/ml的抗CD3抗体(ThermoFisher 14-0037-82)和5μg/ml的抗CD28抗体(ThermoFisher 14-0281-82)的包被液包被六孔板室温2-4小时，吸去包被液后用生理盐水洗涤孔板1-3次，加入含2％FBS AIM-V培养基待用；人外周血PBMC(HLA-*02:01，购买自ALLCELLS)37℃水浴复苏，将PBMC贴壁培养2-4h，其中未贴壁的悬浮细胞即为初始T细胞，将悬浮细胞收集到15ml离心管中，1200rmp离心3min，弃上清，加入生理盐水，1200rmp离心3min，弃生理盐水，并重复此步骤；再将洗涤后的初始T细胞转移至盛有待用培养基的抗体包被孔中，37℃，5％CO2培养3～4天后进行后续实验。
电转制备表达NY-ESO-1 TCR-T细胞，步骤如下
1)预先将AIM-V培养基加入12孔板的2孔内，每孔2mL，随后转入细胞培养箱内37℃5％CO 2预热1小时；
2)按下表8进行每孔单次用量的电转液配比，配制2孔：
表8
  100μL Nucleocuvette TM Strip(μL)
Nucleofector TM溶液的体积 82
电转补充溶液 18
3)取获得的活化T细胞至2个EP管内，每个EP管内加入5×10 6个细胞，1200rpm离心5min，弃上清，随后用500μL生理盐水重悬细胞，重复离心步骤洗涤细胞沉淀；
4)向2)中配置好的2个孔电转液体系分别加入质粒pKB20-NY-ESO-1-TCR和pKB20-EGFP载体各4μg，随后室温静置30min以内；
5)用4)中配置好的含质粒的电转液重悬的2管活化T细胞，每管100μL，小心吸取细胞重悬液转入LONZA 100μL电转杯内，将电转杯放入LONZA Nucleofector TM 2b电转槽内，启动电转程序，电转程序选择T-020；
6)电转完成后小心取出电转杯，吸取细胞悬液转移至EP管中，每管加入预热的AIM-V培养基200μL，随后转入1)中12孔板内含预热AIM-V培养基的孔中，37℃、5％CO 2培养，培养1h后加入化合物G150(购自MedChemExpress)至终浓度为5μM，随后继续培养13天，期间根据细胞增殖情况进行传代，13天后对各电转样品的细胞数量和细胞成活率进行检测，分别制得NY-ESO-1 TCR-T细胞和Mock-T细 胞。
实施例19，NY-ESO-1 TCR-T细胞NY-ESO-1 TCR表达阳性细胞的检测
1)收集实施例18制备的NY-ESO-1 TCR-T细胞1×10 6个，1000rpm，离心3min；
2)弃上清，各加入生理盐水重悬细胞，1000rpm，离心3min；
3)上流式细胞仪检测。
结果如图24所示，EGFP表达阳性的细胞比例为37.57％。NY-ESO-1 TCR的α链和β链的基因DNA序列以及EGFP的编码DNA序列三者间通过P2A肽段编码序列连接，EGFP表达阳性的细胞即可间接反映NY-ESO-1 TCR基因的表达。可以推测NY-ESO-1 TCR表达阳性的细胞比例在37％左右。
实施例20，NY-ESO-1 TCR-T的细胞杀伤功能测试
应用艾森公司的实时无标记细胞功能分析仪(RTCA)检测实施例18获得的NY-ESO-1 TCR-T细胞的体外杀伤活性，具体步骤如下：
(1)调零：每孔加入50μL DMEM培养液，放入仪器中，选择步骤1，调零；
(2)靶细胞铺板：人恶性黑色素瘤细胞系A375(购买于美国菌种保藏中心ATCC，NY-ESO-1表达阳性)，按每孔10 4个细胞/50μL铺在含有检测电极的板中，放置数分钟，待细胞稳定后，再放入仪器中，开始步骤2，培养细胞；
(3)加入效应细胞：靶细胞培养18h后，观察细胞指数，当细胞指数为1时，分别加入对照细胞Mock-T和效应细胞NY-ESO-1 TCR-T，每孔50μL，NY-ESO-1TCR-T设置0.25:1和0.5:1两个效靶比，Mock-T设置效靶比为0.5:1，效靶比的设置均按NY-ESO-1 TCR表达阳性细胞数计算，开始步骤3共培养，超过70h后，观察细胞增殖曲线。
结果如图25所示，对照Mock-T细胞组的杀伤曲线与A375肿瘤细胞曲线基本重叠，表明Mock-T细胞对A375细胞的基本没有杀伤效果。NY-ESO-1 TCR-T在0.25:1和0.5:1两个效靶比下对A375细胞的杀伤效果均十分明显，且杀伤效果随着效靶比的提升也有明显提升。
对比例1，含EGFP表达框pNB载体在K562细胞中的整合
按照中国专利CN105154473B说明书第15页实施例1和说明书第16页实施例2记载的方法，分别构建pNB载体和pNB328-EGFP。按本申请实施例4记载的方法，用pNB328-EGFP通过电转法制备稳定整合表达EGFP的K562，电转后第14天(培养3代)流式检测EGFP表达阳性的细胞。
结果如图26所示，电转后14天EGFP表达阳性的K562细胞比例为45.54％。
对比例2，含EGFP表达框pNB载体在原代T细胞中的整合
按本申请实施例5记载的方法，用pNB328-EGFP通过电转法制备稳定整合表达EGFP的原代T细胞，电转后第14天(细胞培养3代)流式检测EGFP表达阳性的细胞。电转所用PBMC与实施例5所用PBMC为同一批PBMC。电转后14天(培养3代)流式检测EGFP表达阳性的细胞。
结果如图27所示，电转后14天EGFP表达阳性的T细胞比例为34.60％。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (10)

  1. 一种核酸构建物，其包含以下元件或由其组成：转座子3’末端重复序列、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列，
    优选地，所述核酸构建物还包含选自以下的一种或多种元件：转座酶编码序列、控制该转座酶表达的启动子、多克隆插入位点、增强子、5’UTR、第二polyA序列和感兴趣的外源基因，
    优选地，所述转座酶编码序列、所述控制该转座酶表达的启动子、所述5’UTR和所述第二polyA序列中的任一种或多种在所述转座子3’末端重复序列和所述转座子5’末端重复序列之间的区域以外。
  2. 如权利要求1所述的核酸构建物，其包含以下元件：转座子3’末端重复序列、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、转座酶编码序列以及控制该转座酶表达的启动子，
    优选地，所述核酸构建物还包含选自以下的一种或多种元件：多克隆插入位点、增强子、5’UTR、第二polyA序列和感兴趣的外源基因。
  3. 如权利要求2所述的核酸构建物，其特征在于，
    所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、转座酶编码序列以及控制该转座酶表达的启动子，
    所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、增强子、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、转座酶编码序列以及控制该转座酶表达的启动子，
    所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、转座酶编码序列、5’UTR以及控制该转座酶表达的启动子，
    所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第 一polyA序列、增强子、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、转座酶编码序列、5’UTR以及控制该转座酶表达的启动子，
    所述核酸构建物依次包含：转座子3’末端重复序列、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、控制转座酶表达的启动子、转座酶编码序列和第二polyA序列，
    所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、控制转座酶表达的启动子、转座酶编码序列和第二polyA序列，
    所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、增强子、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、控制转座酶表达的启动子、转座酶编码序列和第二polyA序列，
    所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、控制转座酶表达的启动子、5’UTR、转座酶编码序列和第二polyA序列，或
    所述核酸构建物依次包含：转座子3’末端重复序列、多克隆插入位点、第一polyA序列、增强子、具有转录终止功能的绝缘子序列、转座子5’末端重复序列、控制转座酶表达的启动子、5’UTR、转座酶编码序列和第二polyA序列，
    所述核酸构建物依次包含：转座子3’末端重复序列、具有转录终止功能的绝缘子序列、多克隆插入位点、第一polyA序列、转座子5’末端重复序列、控制转座酶表达的启动子、5’UTR、转座酶编码序列和第二polyA序列，
    所述核酸构建物依次包含：转座子3’末端重复序列、具有转录终止功能的绝缘子序列、多克隆插入位点、第一polyA序列、增强子、转座子5’末端重复序列、控制转座酶表达的启动子、5’UTR、转座酶编码序列和第二polyA序列，或
    所述核酸构建物依次包含：转座子3’末端重复序列、增强子、具有转录终止功能的绝缘子序列、多克隆插入位点、第一polyA序列、转座子5’末端重复序列、控制转座酶表达的启动子、5’UTR、转座酶编码序列和第二polyA序列。
  4. 如权利要求1-3中任一项所述的核酸构建物，其特征在于，所述核酸构建物具有选自以下的一项或多项特征：
    所述转座酶的表达框的方向与外源基因的表达框的方向相同或相反，
    所述转座酶的表达框的方向与转座子3’末端重复序列和转座子5’末端重复序列之间的序列的方向相同或相反，
    所述转座子5’末端重复序列与所述转座子3’末端重复序列的位置能够互换，
    所述转座子3’末端重复序列为PiggyBac转座子3’末端重复序列，
    所述转座子5’末端重复序列为PiggyBac转座子5’末端重复序列，
    所述增强子选自：CMV增强子序列、SV40增强子、人ε球蛋白5’HS2增强子、鸡β球蛋白基因5’HS4增强子，
    所述转座酶为PiggyBac转座酶，
    所述5’UTR选自C3基因、ORM1基因、HPX基因、FGA基因、AGXT基因、ASL基因、APOA2基因、ALB基因的5’UTR，
    所述启动子选自：CMV启动子、miniCMV启动子、CMV53启动子、miniSV40启动子、miniTK启动子、MLP启动子、pJB42CAT5启动子、YB_TATA启动子、EF1α启动子、SV40启动子、UbiquitinB启动子、CAG启动子、HSP70启动子、PGK-1启动子、β-actin启动子、TK启动子和GRP78启动子，
    所述转座酶编码序列含有或者可操作地连接单拷贝或者多拷贝的核定位信号编码序列。
  5. 如权利要求1-3中任一项所述的核酸构建物，其特征在于，所述核酸构建物具有选自以下的一项或多项特征：
    所述转座子3’末端重复序列的核苷酸序列如SEQ ID NO:1所示，
    所述转座子5’末端重复序列的核苷酸序列如SEQ ID NO:6所示，
    所述多克隆插入位点的序列如SEQ ID NO:2所示，
    所述第一polyA序列如SEQ ID NO:3、13或16所示，
    所述第二polyA序列如SEQ ID NO:3、13或16所示，
    所述增强子序列如SEQ ID NO:4、26-28中任一所示，
    所述绝缘子序列如SEQ ID NO:5或15所示，
    所述PiggyBac转座酶的氨基酸序列如SEQ ID NO:36所示；优选地，所述PiggyBac转座酶的编码序列如SEQ ID NO:7所示，
    所述5’UTR序列如SEQ ID NO:8、17-24中任一所示，
    所述启动子的序列如SEQ ID NO:9、SEQ ID NO:37-42中任一项所示；
    所述核定位信号为c-myc核定位信号；优选地，所述核定位信号具有SEQ ID NO:35所示的序列。
  6. 如权利要求1-3中任一项所述的核酸构建物，其特征在于，
    所述核酸构建物包含SEQ ID NO:10或14所示序列，或
    所述核酸构建物是重组载体，优选地，所述核酸构建物是重组克隆载体或重组表达载体。
  7. 一种宿主细胞，包含
    (1)权利要求1-6中任一项所述的核酸构建物，和/或
    (2)权利要求1-6中任一项所述的核酸构建物的转座子3’末端重复序列与转座子5’末端重复序列之间的序列，
    优选地，所述宿主细胞为哺乳动物细胞，
    更优选地，所述宿主细胞选自T细胞、Jurkat细胞、K562细胞、胚胎干细胞、肿瘤细胞、HEK293细胞和CHO细胞。
  8. 一种药物组合物，包含权利要求1-6中任一项所述的核酸构建物或权利要求8所述的宿主细胞以及药学上可接受的辅料。
  9. 权利要求1-6中任一项所述的核酸构建物或权利要求7所述的宿主细胞在制备药物、试剂或工具中的用途或作为药物、试剂或工具的用途，所述药物、试剂或工具用于将外源基因表达框整合到靶细胞基因组中，或用于基因治疗、细胞治疗、干细胞诱导或分化，
    优选地，所述靶细胞为哺乳动物细胞，
    更优选地，所述靶细胞选自免疫细胞、Jurkat细胞、K562细胞、胚胎干细胞、肿瘤细胞、HEK293细胞和CHO细胞，
    进一步更优选地，所述免疫细胞选自T细胞、B细胞、CIK细胞、LAK细胞、NK细胞、细胞毒性T细胞(CTL)、树突状细胞(DC)、肿瘤浸润淋巴细胞(TIL)、巨噬细胞、NK T细胞和γδT细胞中的任一种或多种。
  10. 一种将外源基因或其表达框整合到细胞基因组中的方法，包括将含有外源基因和任选的其启动子的权利要求1-6中任一项所述的核酸构建物导入所述细胞，和任选的在转座酶将外源基因或其表达框整合到细胞基因组的条件下孵育所述细胞，所述外源基因和任选的其启动子位于所述核酸构建物的多克隆插入位点中，
    优选地，所述转座酶是PiggyBac转座酶。
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