WO2017054647A1 - 一种高效安全的转座子整合系统及其用途 - Google Patents

一种高效安全的转座子整合系统及其用途 Download PDF

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WO2017054647A1
WO2017054647A1 PCT/CN2016/099005 CN2016099005W WO2017054647A1 WO 2017054647 A1 WO2017054647 A1 WO 2017054647A1 CN 2016099005 W CN2016099005 W CN 2016099005W WO 2017054647 A1 WO2017054647 A1 WO 2017054647A1
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cell
vector
recombinant
nucleic acid
sequence
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钱其军
金华君
李林芳
刘韬
左明辉
吴红平
吴孟超
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上海细胞治疗研究院
上海细胞治疗工程技术研究中心集团有限公司
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Priority to US15/764,840 priority Critical patent/US20180265890A1/en
Publication of WO2017054647A1 publication Critical patent/WO2017054647A1/zh

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Definitions

  • the invention belongs to the field of molecular biology and relates to an efficient and safe transposon integration system and its use.
  • the invention also relates to a nucleic acid construct and uses thereof.
  • the nucleic acid construct can be used to mediate efficient integration of a foreign gene in a host cell, and to efficiently and stably express, and the integration site is mainly concentrated in three intergenic segments within the genome of the host cell, which can largely avoid The risk of random insertion.
  • the invention also relates to recombinant vectors and recombinant host cells comprising the nucleic acid construct.
  • the expression pattern of the foreign gene in the host cell can be divided into transient expression and stable expression, wherein stable expression refers to: (1) expression of the foreign gene after transfection into the eukaryotic cell and integration into the genome.
  • the stable expression level of recombinant genes is generally 1 to 2 orders of magnitude lower than transient expression. (2) Although the host cells undergo multiple passages or conditions, the expression level remains stable.
  • CAR-T Chimeric Antigen Receptor T-Cell Immunotherapy
  • CAR-T against CD19 can efficiently kill B cell lymphoma expressing CD19 surface antigen, and the effective remission rate is 90% in patients with advanced refractory B cell lymphoma (Maude SL, Frey N, Shaw PA, Aplenc R, Barrett DM, Bunin NJ, Chew A, Gonzalez VE, Zheng Z, Lacey SF, Mahnke YD, Melenhorst JJ, Rheingold SR, Shen A, Teachey DT, Levine BL, June CH, Porter DL, Grupp SA. Chimeric antigen receptor T cells For sustained remissions in leukemia.N Engl J Med. 2014;371(16):1507-17).
  • Retroviral system can effectively infect host cells and mediate the expression of foreign genes. The genome is efficiently integrated, but its loading capacity is limited, and the preparation process of recombinant virus particles is complicated.
  • Eukaryotic expression plasmid system The preparation process is relatively simple, but it is inserted into the host genome by random DNA recombination, and the integration efficiency is extremely low.
  • Transposition subsystem The plasmid system is adopted, and the preparation process is relatively simple. The foreign gene is integrated into the genome through the transposase, and the integration efficiency is relatively low.
  • the earliest applied mammalian transposon system was derived from the fish "Sleeping Beauty", but the “Sleeping Beauty” transposon has excessive inhibition effect and small fragments (about 5kb). To make it limited in GM applications.
  • the piggyBac (PB) transposon derived from Lepidoptera is currently the most active transposon in mammals. Its host range is extremely wide, from single-celled organisms to mammals; it can carry large foreign DNA fragments, and the transposition efficiency does not decrease significantly when the transposition fragment is within 14 kb.
  • the PB transposon mainly adopts the "cut-paste" mechanism to transpose, and after the transposition fragment is excised, it will not leave a footprint in the in situ spot, and the genome can be accurately repaired after excision, in the application of reversible genes. Has an important role.
  • the PB transposase has high plasticity. By fusing with other functional proteins or changing the functional region of the transposase, not only can the activity and mode of action of the transposase be changed, but also the targeting of the transposition of the foreign gene can be improved.
  • the traditional PB transposon system uses a binary plasmid consisting of a donor plasmid (containing a terminal repeat that can be recognized by PB integrase at both ends of the exogenous gene expression cassette) and a transposase helper plasmid (providing PB transposase). system.
  • a binary transposition system in order to achieve efficient integration of the foreign gene expression cassette, it is necessary to satisfy that two plasmids are transfected into the same cell, and only a part of the cells can be realized during the transfection process (other cells or one plasmid) It has not been successfully transfected, or only one of the plasmids has been transfected, and no effective integration has been achieved.
  • the donor plasmid and the transposase helper plasmid are included in the same plasmid, and the self-inactivation of the transposase is set (Self-inactivating).
  • the mechanism ensures that the expression of the transposase can be shut down in time after the integration of the foreign gene. It has been reported in the literature that one of the strategies is to place a promoter that controls PB expression between the exogenous expression cassette and one of the transposase ITRs.
  • the PB expression box will Defective promoter, transcription is aborted, expression is closed in time (Urschitz J, Kawasumi M, Owens J, Morozumi K, Yamashiro H, Stoytchev I, Marh J, Dee JA, Kawamoto K, Coates CJ, Kaminski JM, Pelczar P, Yanagimachi R, Moisyadi S. Helper-independent piggyBac plasmids for gene delivery approaches: strategies for avoiding potential genotoxic effects. Proc Natl Acad Sci USA. 2010; 107(18): 8117-22.).
  • the shortcoming of this strategy is that the integration of a strong promoter that originally controls the expression of the PB gene into the genome may activate the expression of the flanking gene at the host cell integration site, posing a potential safety risk.
  • Another strategy is to place the PB expression cassette in the same direction as the foreign gene expression cassette and share the same PolyA tailing signal sequence.
  • the PolyA tailing signal sequence causes the transcribed mRNA to be unstable and rapidly degraded, and the expression of PB is turned off (Chakraborty S, Ji H, Chen J, Gersbach CA, Leong KW. Vector modifications to eliminate transposase expression following piggyBac-mediated transgenesis. Sci Rep. 2014; 4:7403).
  • the shortcoming of this strategy is that the mRNA product transcribed from the expression cassette of the PB gene actually covers the entire foreign gene expression frame sequence. If the foreign gene expression frame is large, the mRNA length will be too large, and the PB transcription efficiency will be lowered. It is difficult to achieve the amount of PB expression required for integration.
  • the unary transposition system needs to insert the foreign gene expression cassette and the PB expression cassette into the same vector, but the coding sequence of PB is longer, close to 2 kb, resulting in a larger plasmid fragment, which will greatly reduce the transfection efficiency.
  • the integration site of the existing PB transposon system tends to insert coding genes.
  • Nene (Woodard LE, Wilson MH. Trends Biotechnol. piggyBac-ing models and new therapeutic strategies. 2015; 33(9): 525-33. See Table 1 on page 4).
  • the coding gene described herein is a gene that encodes a corresponding functional protein relative to a non-coding gene; if the tumor-associated gene is inserted inactivated or abnormally activated, there may be a risk of carcinogenesis.
  • the inventors constructed an integrated system based on PiggyBac transposon, which can mediate the efficient integration of foreign genes in host cells and efficiently and stably express them.
  • the inventors have surprisingly found that the system-mediated exogenous gene integration site is mainly concentrated in three intergenic segments within the host cell genome, which can largely avoid the risk of random insertion.
  • nucleic acid construct comprising, in order, the following elements:
  • transposon 5' end repeat a polyA tailing signal sequence, a transposon 3' end repeat, a transposase coding sequence, and a promoter that controls expression of the transposase;
  • the polyA tailing signal sequence has a polyA tail signal function in both the forward and reverse directions;
  • the orientation of the expression cassette of the transposase is opposite to the orientation of the foreign gene expression cassette.
  • nucleic acid construct comprising, in order, six elements as follows:
  • transposon 5' end repeat a polyclonal insertion site, a polyA tailing signal sequence, a transposon 3' end repeat, a transposase coding sequence, and a promoter that controls expression of the transposase;
  • the polyclonal insertion site is for operably inserting a foreign gene coding sequence and optionally a promoter for controlling expression of the foreign gene;
  • the polyA tailing signal sequence has a polyA tail signal function in both the forward and reverse directions;
  • the orientation of the expression cassette of the transposase is opposite to the orientation of the foreign gene expression cassette.
  • the orientation of the exogenous gene expression cassette is positive, and the orientation of the transposase expression cassette is reversed.
  • the direction and/or order referred to by "sequential" in the above-mentioned “in order to include the following elements” means from upstream to downstream.
  • the direction along the above “forward direction” is from upstream to downstream, and the direction along the above “reverse” is from downstream to upstream unless otherwise specified.
  • the six elements described above are each independently a single copy or multiple copies.
  • the above six elements may be directly connected to each other, or may contain other sequences such as a linker or a cleavage site.
  • the nucleic acid construct wherein the polyA tailing signal sequence is a polyA tailing signal sequence having a polyA tailing signal function in both forward and reverse directions; or A polyA tailing signal sequence having a unidirectional polyA tailing signal is connected in opposite directions.
  • polyA tailing signal sequence has a polyA tailing signal function in both the forward and reverse directions
  • polyA tailing signal function includes, but is not limited to, the following cases, unless otherwise specified:
  • Two polyA tailing signal sequences one with a polyA tail signal function and one with a polyA tail signal function.
  • the solution in 1) above is employed.
  • the exogenous gene expression cassette and the PiggyBac transposase expression cassette can share a polyA tailing signal sequence, thereby reducing a polyA tailing signal sequence, embodying the intensive principle, reducing the size of the plasmid, and contributing to Under the premise of ensuring transfection efficiency, increase the capacity of the exogenous gene expression cassette.
  • the PB expression cassette is placed in the same direction as the exogenous gene expression cassette, and two polyA tailing signal sequences are used, wherein the PB expression frame is preceded, and the polyA tailing signal sequence is placed therein.
  • An ITR is linked to a foreign gene promoter.
  • a promoter that controls expression of PB transposase, a PB transposase coding sequence, and a 5' end of a transposon Multiple sequence polyA tailing signal sequence 1, foreign gene promoter and foreign gene (polyclonal insertion site), polyA tailing signal sequence 2, transposon 3' terminal repeat; and expression of PB transposase
  • the orientation of the box is the same as the orientation of the foreign gene expression cassette.
  • nucleic acid construct according to any of the preceding claims, wherein the 5' terminal repeat of the transposon and the position of the 3' end repeat of the transposon are interchangeable.
  • the 5' terminal repeat of the transposon is a 5' terminal repeat of the PiggyBac transposon; the 3' terminal repeat of the transposon is a 3' terminal repeat of the PiggyBac transposon; the transposase is PiggyBac Enzyme.
  • the nucleotide sequence of the 5' terminal repeat of the PiggyBac transposon is set as shown in SEQ ID NO: 1; and/or the nucleotide sequence of the 3' terminal repeat of the PiggyBac transposon is SEQ ID NO: 4 Shown.
  • the amino acid sequence of the PiggyBac transposase is set forth in SEQ ID NO: 17; preferably, the nucleotide sequence encoding the PiggyBac transposase is set forth in SEQ ID NO: 5.
  • the transposase coding sequence contains or is operably linked to a single copy or multiple copies of a nuclear localization signal coding sequence; preferably a c-myc nuclear localization signal coding sequence, such as the sequence set forth in SEQ ID NO: 18.
  • the nuclear localization signal can guide the transfer of the transposase in the nucleus, thereby improving the transposition efficiency.
  • a nucleic acid construct according to any one of the present invention which is characterized by any one or more of the following items (1) to (3):
  • nucleotide sequence of the polyclonal insertion site is as shown in SEQ ID NO: 2;
  • sequence shown in SEQ ID NO: 3 has a polyA tail signal function in both the forward and reverse directions.
  • the promoter is selected from the group consisting of a CMV promoter (for example, as shown in SEQ ID NO: 6), an EF1 ⁇ promoter, an SV40 promoter, a Ubiquitin B promoter, a CAG promoter, an HSP70 promoter, and PGK-1. Promoter, ⁇ -actin promoter, TK promoter and GRP78 promoter.
  • a nucleic acid construct according to any of the invention operably linked or operably inserted (e.g., at a multiple cloning site) with one or more foreign genes of the same or different and optionally a control foreign source
  • the promoter of gene expression, or a multiple cloning site thereof is replaced with one or more identical or different foreign gene coding sequences and, optionally, a promoter that controls expression of the foreign gene; the foreign gene is independently a single Copy or multiple copies;
  • the foreign gene is selected from a fluorescein reporter gene (eg, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.), a luciferase gene (eg, firefly luciferase, Renilla luciferase, etc.), natural Functional protein genes (eg, TP53, GM-CSF, OCT4, SOX2, Nanog, KLF4, c-Myc), RNAi genes, and artificial chimeric genes (eg, chimeric antigen receptor genes such as CAR19, Fc fusion protein genes, full length antibodies) One or more of the genes;
  • a fluorescein reporter gene eg, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.
  • a luciferase gene eg, firefly luciferase, Renilla luciferase, etc.
  • natural Functional protein genes eg, TP53, GM-CSF, OCT4, SOX2, Nanog, KLF4, c-Myc
  • sequence of the foreign gene is as shown in any one or more of SEQ ID Nos: 9-11 or 16.
  • a further aspect of the invention relates to a recombinant vector comprising the nucleic acid construct of any of the invention
  • the recombinant vector is a recombinant cloning vector, a recombinant eukaryotic expression plasmid or a recombinant viral vector;
  • the recombinant cloning vector is obtained by recombining the nucleic acid construct according to any one of the invention with pUC18, pUC19, pMD18-T, pMD19-T, pGM-T vector, pUC57, pMAX or pDC315 series vectors.
  • the recombinant expression vector is the nucleic acid construct according to any one of the invention, and the pCDNA3 series vector, the pCDNA4 series vector, the pCDNA5 series vector, the pCDNA6 series vector, the pRL series vector, the pUC57 vector, the pMAX vector or the pDC315 series.
  • a recombinant vector obtained by recombinant production is the nucleic acid construct according to any one of the invention, and the pCDNA3 series vector, the pCDNA4 series vector, the pCDNA5 series vector, the pCDNA6 series vector, the pRL series vector, the pUC57 vector, the pMAX vector or the pDC315 series.
  • the recombinant viral vector is a recombinant adenovirus vector, a recombinant adeno-associated virus vector, a recombinant retroviral vector, a recombinant herpes simplex virus vector or a recombinant vaccinia virus vector.
  • a further aspect of the invention relates to a recombinant host cell comprising the nucleic acid construct of any of the invention or the recombinant vector of the invention; preferably, the recombinant host cell is a recombinant mammalian cell; Recombinant primary cultured T cells, Jurkat cells, K562 cells, stem cells, tumor cells, HEK293 cells or CHO cells; preferably, the stem cells are embryonic stem cells.
  • a further aspect of the invention relates to the use of the nucleic acid construct of any of the invention, the recombinant vector of the invention or the recombinant host cell of the invention, selected from any one of the following (1) to (4) item:
  • the host cell is a mammalian cell, such as a primary cultured T cell, a Jurkat cell, or a K562 cell.
  • the host cell is a mammalian cell, such as a primary cultured T cell, a Jurkat cell, a K562 cell, a stem cell, a tumor cell, a HEK293 cell or a CHO cell; preferably, the stem cell is an embryonic stem cell;
  • the stem cells are embryonic stem cells
  • the stem cell is an embryonic stem cell.
  • the above uses can be achieved by insertion of a foreign gene having a corresponding function having a function corresponding to a specific use, such as a therapeutic function or an inducing function.
  • a further aspect of the invention relates to a method of introducing a nucleic acid construct or recombinant vector of the invention into a mammalian cell, the method comprising viral mediated transformation, microinjection, particle bombardment, gene gun transformation and electroporation, and the like.
  • the method is electroporation.
  • a further aspect of the invention relates to a method of integrating a foreign gene expression cassette into a host cell genome comprising expressing a foreign gene using the nucleic acid construct, recombinant vector or recombinant host cell of any of the invention The step of integrating into the host cell genome.
  • the method of the present invention wherein the nucleic acid construct of any one of the present invention or the recombinant vector of the present invention is introduced into a host cell such as a mammalian cell, the method of introduction is selected from the group consisting of Virus-mediated transformation, microinjection, particle bombardment, gene gun transformation, and electroporation.
  • expression cassette refers to the entire element required for expression of a gene, including a promoter, a gene coding sequence, and a PolyA tailing signal sequence.
  • nucleic acid construct is a single- or double-stranded nucleic acid molecule, preferably an artificially constructed nucleic acid molecule.
  • the nucleic acid construct further comprises one or more regulatory sequences operably linked, which, under compatible conditions, direct expression of the coding sequence in a suitable host cell. 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.
  • operably inserted/ligated is defined herein as a conformation wherein the regulatory sequences are located at appropriate positions relative to the coding sequence of the DNA sequence such that the regulatory sequences direct the egg Expression of white or polypeptide.
  • a foreign gene promoter and a foreign gene coding sequence are placed at the multiple cloning site by DNA recombination techniques.
  • the "operably linked" can be achieved by means of DNA recombination, preferably the nucleic acid construct is a recombinant nucleic acid construct.
  • coding sequence is defined herein as a portion of a nucleic acid sequence that directly determines the amino acid sequence of its protein product.
  • the boundaries of the coding sequence are typically determined by a ribosome binding site (for prokaryotic cells) immediately upstream of the open reading frame of the 5' end of the mRNA and a transcription termination sequence immediately downstream of the open reading frame of the 3' end of the mRNA.
  • a coding sequence can include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
  • regulatory sequence as used herein is defined to include all components necessary or advantageous for expression of a peptide of the invention.
  • Each regulatory sequence may be naturally or foreign to the nucleic acid sequence encoding the protein or polypeptide.
  • These regulatory sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal sequence, and a transcription terminator.
  • the regulatory sequences include promoters as well as termination signals for transcription and translation.
  • a linker-containing regulatory sequence can be provided in order to introduce a specific restriction site to link a regulatory sequence to the coding region of a nucleic acid sequence encoding a protein or polypeptide.
  • the control sequence may be a suitable promoter sequence, ie, a nucleic acid sequence that is recognized by a host cell that expresses the nucleic acid sequence.
  • the promoter sequence contains transcriptional regulatory sequences that mediate the expression of the protein or polypeptide.
  • the promoter may be any nucleic acid sequence that is transcriptionally active in the host cell of choice, including mutated, truncated and hybrid promoters, which may be obtained from an extracellular or intracellular encoding homologous or heterologous to the host cell.
  • a gene of a protein or polypeptide polypeptide may be a suitable promoter sequence, ie, a nucleic acid sequence that is recognized by a host cell that expresses the nucleic acid sequence.
  • the promoter sequence contains transcriptional regulatory sequences that mediate the expression of the protein or polypeptide.
  • the promoter may be any nucleic acid sequence that is transcriptionally active in the host cell of choice, including mutated, truncated and hybrid promoter
  • the control sequence may also be a suitable transcription termination sequence, a sequence that is recognized by the host cell to terminate transcription.
  • the termination sequence is operably linked to the 3' end of the nucleic acid sequence encoding the protein or polypeptide. Any terminator that can function in the host cell of choice can be used in the present invention.
  • the control sequence may also be a suitable leader sequence, an untranslated region of mRNA that is important for translation of the host cell.
  • the leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the polypeptide. Any leader sequence that can function in the host cell of choice can be used in the present invention.
  • the control sequence may also be a signal peptide coding region, which encodes a stretch of protein or polypeptide ammonia.
  • the amino acid sequence at the basal end can direct the coding polypeptide into the cell secretory pathway.
  • the 5' end of the coding region of the nucleic acid sequence may naturally contain a signal peptide coding region in which the translational reading frame is naturally joined to the coding region fragment 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 that directs the expressed polypeptide into the secretory pathway of the host cell used can be used in the present invention.
  • the control sequence may also be a propeptide coding region that encodes an amino acid sequence at the amino terminus of the polypeptide.
  • the resulting polypeptide is referred to as a zymogen or propolypeptide.
  • a propolypeptide is generally inactive and can be converted to a mature active polypeptide by cleavage of the propeptide from the propolypeptide by catalytic or autocatalytic.
  • the peptide region is immediately adjacent to the amino terminus of the polypeptide, and the signal peptide region is adjacent to the amino terminus of the peptide region.
  • regulatory sequences that modulate the expression of the polypeptide depending on the growth of the host cell.
  • regulatory systems are those that respond to chemical or physical stimuli, including in the presence of a regulatory compound, to open or shut down gene expression.
  • Other examples of regulatory sequences are those that enable gene amplification.
  • the nucleic acid sequence encoding the protein or polypeptide should be operably linked to a regulatory sequence.
  • the present invention provides a highly efficient integration system based on PiggyBac transposon, which can mediate the efficient integration of foreign genes into the host cell genome and stably express them.
  • the inventors have surprisingly found that the insertion of the system into the host genome has a clear tendency, and the mediated foreign gene integration site is mainly concentrated in the three intergenic segments within the host cell genome, which can be larger. To the extent that the risk of random insertion is avoided.
  • Figure 1 Map of the pNB vector.
  • Figure 2 Time course of relative expression of PB gene after pNB transfection of Jurkat cells.
  • Figure 3 Time course of the proportion of EGFP positive cells after pN:328-EGFP transfection of Jurkat cells.
  • Figure 4 Fluorescence detection map of 4 cells transfected with pNB328-EGFP.
  • 4A-4B are Jurkat cells
  • Figures 4C-4D are K562 cells
  • Figures 4E-4F are primary T cells
  • Figures 4G-4H are mouse embryonic stem cells (ES).
  • 4A, 4C, 4E, and 4G on the left side are photographed under white light to show cell morphology; and the right side of FIGS. 4B, 4D, 4F, and 4H are photographed under fluorescence, showing green fluorescence.
  • the field of view taken by the left and right images is the same.
  • Figure 5 Flow cytometry of pNB328-EGFP transfected Jurkat cells (5A), K562 cells (5B), primary T cells (5C), mouse ES cells (5D).
  • Figure 6 Luciferase assay map of pNB328-luc transfected Huh7 cells.
  • Figure 7 Detection map of the fluorescence expression intensity of EGFP gene expression after pNB328-EGFP transfection of primary T cells.
  • 7A, 7C are photographs taken under white light, showing cell morphology;
  • 7B, 7D is a photo taken under fluorescence, showing green fluorescence.
  • FIG. 8 Integration site analysis of pNB328-EGFP after transfection of primary T cells.
  • the circled part is the integration hotspot.
  • 8A, 8B, and 8C represent primary T cell integration site detection of 3 normal humans from different sources.
  • the triangle represents that the integration site belongs to the intergenic segment, the arrow represents the integration site belongs to the intragenic segment, and the circle represents the integration hotspot.
  • Figure 9 Detection of the killing effect of pNB328-CAR19 on Raji cells after transfection of primary T cells.
  • Sequence 1 (SEQ ID NO: 1, 67 bp), PiggyBac transposon 5' end repeat
  • Sequence 3 (SEQ ID NO: 3, 222 bp), polyA tailing signal sequence
  • Sequence 5 (SEQ ID NO: 5, 1815 bp), PiggyBac transposase sequence containing the c-myc nuclear localization signal coding sequence, underlined as the c-myc nuclear localization signal coding sequence.
  • Sequence 7 (SEQ ID NO: 7, 2760 bp), a long sequence spliced in Example 1.
  • Sequence 8 (SEQ ID NO: 8,545 bp), EF1 ⁇ promoter sequence
  • Sequence 9 (SEQ ID NO: 9, 720 bp), EGFP coding sequence
  • Sequence 11 (SEQ ID NO: 11, 435 bp), GM-CSF gene coding sequence
  • Sequence 17 (SEQ ID NO: 17, 604aa), amino acid sequence of PiggyBac transposase
  • Sequence 18 (SEQ ID NO: 18, 27 bp), c-myc nuclear localization signal coding sequence
  • PiggyBac transposon 5' end repeat (SEQ ID NO: 1), the polyclonal insertion site (SEQ ID NO: 2), the polyA tail signal sequence (SEQ ID NO: 3), and the PiggyBac transposon 3 'Terminal repeat sequence (SEQ ID NO: 4), PiggyBac transposase coding sequence containing c-myc nuclear localization signal (SEQ ID NO: 5), CMV promoter sequence (SEQ ID NO: 6), spliced into a length Sequence (SEQ ID NO: 7), wherein the PiggyBac transposase coding sequence containing the c-myc nuclear localization signal and the CMV promoter sequence are reverse-complementary (the reverse complementation herein refers to the foreign gene expression cassette and the PB gene)
  • the expression cassette is in the opposite direction, so the PiggyBac transposase coding sequence and the reverse complement of the CMV promoter sequence are shown. It was commissioned by Shanghai Jerry Biotechnology Co., Ltd
  • Example 2 Construction of a pNB vector containing a foreign gene expression cassette
  • the company was commissioned by Shanghai Jerry Biotechnology Co., Ltd., and XbaI and EcoRI restriction sites were added to both ends, and the pNB vector prepared in the previous Example 1 was loaded and designated as pNB328 vector.
  • the EF1 ⁇ promoter sequence is shown in SEQ ID NO: 8.
  • the company was commissioned by Shanghai Jerry Biotechnology Co., Ltd., and the EcoRI and SalI restriction sites were added at both ends, and the pNB328 vector was inserted into the pNB328-EGFP vector.
  • the EGFP coding sequence is set forth in SEQ ID NO: 9.
  • the company was commissioned by Shanghai Jerry Biotechnology Co., Ltd., and the EcoRI and SalI restriction sites were added at both ends, and the pNB328 vector was inserted into the pNB328-Luc vector.
  • the Luc luciferase coding sequence is set forth in SEQ ID NO: 10.
  • pNB328-GM-CSF vector According to the enzymatic coding sequence of human GM-CSF gene, it was commissioned by Shanghai Jerry Biotechnology Co., Ltd., and added EcoRI and SalI restriction sites at both ends, and loaded into pNB328 vector, named as pNB328-GM-CSF vector. .
  • the GM-CSF gene coding sequence is shown in SEQ ID NO:11.
  • Example 3 PB expression time curve after transfection of JNB cells with pNB328 vector analysis
  • PB transposase expression plasmid purchased from System Bioscience, was transfected into the nucleus and cultured in a 37 ° C, 5% CO 2 incubator. RNA was extracted on the 6th, 12th, 24th, 48th, 96th, and 15th day after transfection, and the relative expression level of PB transposase was detected by RT-PCR. Taking ⁇ -actin as an internal reference, the specific primers are as follows:
  • PB-F as SEQ ID NO: 12
  • PB-R as SEQ ID NO: 13;
  • Actin-F SEQ ID NO: 14
  • Actin-R SEQ ID NO: 15.
  • the polyA tailing signal sequence in the PB transposase expression cassette which we designed in the PB transposase expression cassette, is upstream of the 3' ITR of the transposon, and will function as an ITR-external with the PB transposase.
  • the source gene expression cassette-ITR is excised from the pNB328-EGFP vector and integrated into the host cell gene
  • the polyA tailing signal sequence in the PB transposase expression cassette is also excised, resulting in a PB transposase expression cassette. Complete, the expression is quickly closed.
  • Plasmid (providing expression plasmid for PB transposase) was transfected into the nucleus and cultured in a 37 ° C, 5% CO 2 incubator. After the cells are full, they are subcultured at a ratio of 1:10. At 12 hours (P0), 5 (P0+5) after transfection, after 1 passage (P1), after 2 passages (P2), after 3 passages (P3), using flow cytometry The instrument detects changes in the proportion of EGFP-positive cells.
  • green fluorescent positive cells can be considered to have a stable integration of the green fluorescent expression cassette.
  • the efficiency of integration can be determined by flow detection of the proportion of green fluorescent positive cells.
  • the proportion of EGFP-positive Jurkat cells gradually decreased with successive passages of 1:10 ratio.
  • the Jurkat T cells transfected with the binary system PB transposon (PB513B-1+PB210PA-1) had a ratio of EGFP positive cells of 6.5% (integration efficiency of 6.5%); and the modified monobasic system of PB
  • the transposon pNB328-EGFP transfected Jurkat T cells had a ratio of EGFP positive cells of 36.4% (integration efficiency 36.4%).
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • transfect 6 ⁇ g of pNB328-EGFP and pcDNA3.1-EGFP plasmid into the nucleus by Lonza 2b-Nucleofector instrument. Incubate at 37 ° C, 5% CO 2 incubator; after 6 hours, transfer to a 6-well plate containing 30 ng / mL anti-CD3 antibody, 3000 IU / mL IL-2 (purchased from Novoprotein), set at 37 ° C, 5% CO 2 incubator culture. After the cells are full, they are subcultured at a ratio of 1:10. After 3 passages, the expression of green fluorescence in the cells was recorded by fluorescence microscopy; at the same time, 1 ⁇ 10 5 cells were collected, and the proportion of EGFP-positive cells was detected by flow cytometry.
  • PBMC peripheral blood mononuclear cells
  • Example 7 Integration analysis of pNB328-EGFP vector in mouse embryonic stem cells
  • a 5 ⁇ 10 6 mouse H9 embryonic stem cell strain (purchased from ATCC) was prepared, and 6 ⁇ g of the pNB328-EGFP plasmid was transfected into the nucleus by a Lonza 2b-Nucleofector instrument, and cultured at 37 ° C in a 5% CO 2 incubator. After the cells are full, they are subcultured at a ratio of 1:10. After 3 passages, the expression of green fluorescence in the cells was recorded by fluorescence microscopy; at the same time, 1 ⁇ 10 5 cells were collected, and the proportion of EGFP-positive cells was detected by flow cytometry.
  • Example 8 Integration analysis of pNB328-luc vector in tumor cells
  • Example 9 Integration analysis of pNB328-GM-CSF vector in HEK293 cells
  • HEK293 cells 5 ⁇ 10 6 HEK293 cells (purchased from ATCC) were prepared, and 6 ⁇ g of pNB328-GM-CSF plasmid was transfected into the nucleus by Lonza 2b-Nucleofector instrument, and cultured at 37 ° C in a 5% CO 2 incubator. After the cells are full, they are subcultured at a ratio of 1:10. After 3 passages, the supernatant of 1 ⁇ 10 6 cells cultured for 2 days was collected, and after dilution of a certain number, HEK293 cells transfected with pNB328-GM-CSF plasmid by human GM-CSF ELISA MAX Deluxe assay kit (purchased from Biolegend) were used. Secretion of GM-CSF protein.
  • HEK293 cells transfected with pNB328-GM-CSF could still express GM-CSF protein (1253.7 ng/ml) at high level after 3 passages, indicating that the GM-CSF expression cassette has been integrated into the genome of the cell. Cell division is stable and expressed.
  • Example 10 Exogenous gene of pNB328-EGFP vector after primary T cell integration Comparative analysis of expression levels
  • Group 1 Prepare 1 x 10 7 freshly isolated peripheral blood mononuclear cells (PBMC). 6 ⁇ g of pNB328-EGFP and pcDNA3.1-EGFP plasmids were transfected into the nucleus by Lonza 2b-Nucleofector instrument, and cultured in 37 ° C, 5% CO 2 incubator; after 6 hours, transferred to 30 ng/mL anti-CD3 The antibody, 3000 IU/mL IL-2 (purchased from Novoprotein) was placed in a 6-well plate and cultured at 37 ° C in a 5% CO 2 incubator.
  • PBMC peripheral blood mononuclear cells
  • Group 2 Prepare 1 ⁇ 10 6 PBMC cells of the same healthy human origin, stimulate culture for 3 days under the conditions of 30 ng/mL anti-CD3 antibody and 3000 IU/mL IL-2, and then take 5 ⁇ 10 6 activated T cells.
  • the treated cells in the two groups were subcultured at a ratio of 1:10. After 3 passages, the expression of green fluorescence was observed by a fluorescence microscope. At the same time, 1 ⁇ 10 5 cells were collected, and the mean fluorescence intensity (MFI) in EGFP-positive cells was detected by flow cytometry.
  • MFI mean fluorescence intensity
  • the results showed that the T cells of the pNB328-EGFP vector integrated had high fluorescence intensity (Fig. 7A, 7B) and the MFI reached 1507.63. However, the T cells after lentivirus infection had lower green fluorescence intensity of 50.34 (Fig. 7C, 7D). ), the difference between the two is nearly 29 times.
  • the results showed that pNB328-EGFP vector can promote the efficient expression of foreign genes after the integration of foreign genes into transfected primary T cells.
  • Example 11 Integration site analysis of pNB328-EGFP vector in primary T cells
  • bioinformatics can be used to determine whether the interval is an intergenic sequence or a genetic spacer sequence. After analysis, the above three intervals belong to the intergenic segment, and the insertion of the exogenous gene expression frame does not constitute the inactivation and insertion mutation of the related gene.
  • Example 12 Construction of pNB328-CAR19 vector and genetic modification of primary T cells
  • CAR chimeric antigen receptor
  • the CAR19 coding sequence is set forth in SEQ ID NO: 16.
  • Example 13 Detection of in vitro killing effect of CAR19-T cells on target cells
  • CAR19-T and unmodified T cells at different target ratios (8:1, 4:1, 2:1, 1:1, 0.5:1, 0.25:1, 0.125:1, 0.0625:1)
  • Co-culture with Raji cells purchased from ATCC
  • LDH-Cytotoxicity Assay Kit Biovision
  • Target cells were plated in 96-well plates (5 ⁇ 10 3 /well), medium background, volume correction, spontaneous LDH release from target cells, maximum LDH release from target cells, control cells spontaneous LDH release control wells, treatment group wells, each group The 3 wells were repeated, and the final volume of each well was the same and not less than 100 ⁇ L.
  • Cytotoxicity (%) [(D experimental well-D medium background well) - (D effector cell spontaneous LDH release well - D medium background well) - (D target cell spontaneous LDH release well - D medium background well) ] / [(D target cell maximum LDH release well - D volume correction well) - (D target cell spontaneous LDH release well - D medium background well)] ⁇ 100%.
  • Raji cells are representative of CD19 positive cells. Therefore, p19328-CAR19 mediated modification of CAR19-T can be highly efficient against Raji cells. Killing can also effectively kill CD19-positive tumor cells, and has clinical application value, such as high-efficiency killing of B cell lymphoma expressing CD19 surface antigen, especially for advanced refractory B cell lymphoma. .

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Abstract

提供了一种核酸构建体及其用途。所述的核酸构建体，其依次包含如下元件：转座子5'末端重复序列、多克隆插入位点、polyA加尾信号序列、转座子3'末端重复序列、转座酶基因编码序列以及控制该转座酶表达的启动子；其中，所述多克隆插入位点用于可操作地插入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polyA加尾信号序列正反向均具有polyA加尾信号功能；所述转座酶的表达框的方向与外源基因表达框的方向相反。所述核酸构建体能够用于介导外源基因在宿主细胞内的表达。

Description

一种高效安全的转座子整合系统及其用途 技术领域
本发明属于分子生物学领域，涉及一种高效安全的转座子整合系统及其用途。本发明还涉及一种核酸构建体及其用途。所述核酸构建体能够用于介导外源基因在宿主细胞内高效整合，并高效稳定表达，且整合位点主要集中在宿主细胞基因组内的3个基因间区段，能较大程度上避免随机插入引发的风险。本发明还涉及含有该核酸构建体的重组载体和重组宿主细胞。
背景技术
外源基因在宿主细胞内的表达形式可分为瞬时表达与稳定表达，其中稳定表达是指：(1)外源基因转染真核细胞并整合入基因组后的表达。重组基因的稳定表达水平一般要比短暂表达低1～2个数量级。(2)宿主细胞虽经过多次传代或条件变化，但表达水平仍然保持稳定。
鉴于稳定表达能随着细胞分裂维持外源基因长时间持续表达，在离体细胞修饰(ex vivo)，如转基因嵌合抗原受体T细胞(Chimeric Antigen Receptor T-Cell Immunotherapy，CAR-T)治疗研究中具有重要意义。CAR-T细胞能特异识别并高效杀伤表达特定细胞表面抗原的肿瘤细胞，已取得显著的临床疗效。如针对CD19的CAR-T能高效杀伤表达CD19表面抗原的B细胞淋巴瘤，对晚期难治性B细胞淋巴瘤患者，有效缓解率达到90％(Maude SL,Frey N,Shaw PA,Aplenc R,Barrett DM,Bunin NJ,Chew A,Gonzalez VE,Zheng Z,Lacey SF,Mahnke YD,Melenhorst JJ,Rheingold SR,Shen A,Teachey DT,Levine BL,June CH,Porter DL,Grupp SA.Chimeric antigen receptor T cells for sustained remissions in leukemia.N Engl J Med.2014；371(16):1507-17)。
为实现外源基因在宿主细胞内的稳定表达，常用的载体系统包括：1.逆转录病毒系统：能有效感染宿主细胞，并介导外源基因表达框的 基因组高效整合，但其装载容量有限，且重组病毒颗粒制备工艺复杂。2.真核表达质粒系统：制备工艺相对简单，但其通过随机DNA重组的方式插入宿主基因组，整合效率极低。3.转座子系统：采用质粒系统，制备工艺相对简单，通过转座酶将外源基因整合入基因组，整合效率相对较低。
最早应用的哺乳动物转座子系统是源于鱼类的“睡美人”转座子(Sleeping Beauty)，但是“睡美人”转座子存在过量抑制效应和携带片段偏小(5kb左右)等缺陷，使其在转基因应用上受到限制。来源于鳞翅目昆虫的piggyBac(PB)转座子是目前哺乳动物中活性最高的转座子。其宿主范围极其广泛，从单细胞生物到哺乳动物都能够发挥作用；能够携带较大的外源DNA片段，当转座片段在14kb以内时，转座效率不会显著下降。PB转座子主要采取“cut-paste”机制发生转座，在转座片段被切除后不会在原位点留下印迹(footprint)，基因组可以实现切除后精确修复，在可逆转基因的应用中具有重要作用。此外，PB转座酶可塑性高，通过与其它功能蛋白融合或改变转座酶的功能区域，不仅能够改变转座酶的活性和作用方式，也可以提高外源基因转座的靶向性。近年来，通过密码子优化、特定位点氨基酸定点突变、相应核定位标签的引入等，使PB在哺乳动物细胞内的整合效率进一步提高，使得该系统在基因组研究、基因治疗、细胞治疗、干细胞诱导和诱导后分化等领域获得了广泛的应用。
传统的PB转座子系统采用供体质粒(在外源基因表达框两端含有可以为PB整合酶识别的末端重复序列)和转座酶辅助质粒(提供PB转座酶)构成的二元转座系统。在该二元转座系统中，为实现外源基因表达框的有效整合，必须满足两个质粒被转染到同一细胞内，这在转染过程中只有一部分细胞能够实现(其它细胞要么一个质粒也没成功转染，要么只转染了其中一个质粒，都不能实现有效整合)，一定程度上降低了整合效率。同时，由于PB转座子系统采用完全可逆的“cut-paste”形式发生，只要整合酶维持表达，其仍具有将已整合入基因组的外源基因表达框重新剪切掉的可能，导致基因组不稳定，且实 际上降低了整合效率。
因此，为提高PB转座子系统的整合效率，非常有必要对该载体系统进行改造，将供体质粒与转座酶辅助质粒纳入同一质粒，同时设置转座酶的自我失活(Self-inactivating)机制，保证在外源基因实现整合后，转座酶的表达能被及时关闭。有文献报道，其中一个策略是将控制PB表达的启动子放置在外源表达框与其中一个转座酶ITR之间，一旦外源基因表达框被从质粒上切除并整合到基因组，PB表达框将缺损启动子，转录被中止，表达被及时关闭(Urschitz J,Kawasumi M,Owens J,Morozumi K,Yamashiro H,Stoytchev I,Marh J,Dee JA,Kawamoto K,Coates CJ,Kaminski JM,Pelczar P,Yanagimachi R,Moisyadi S.Helper-independent piggyBac plasmids for gene delivery approaches:strategies for avoiding potential genotoxic effects.Proc Natl Acad Sci USA.2010；107(18):8117-22.)。然而这种策略的缺陷是，将原先控制PB基因表达的强启动子一并整合到基因组中，有可能激活宿主细胞整合位点侧翼基因的表达，具有潜在的安全风险。
另一种策略是使PB表达框与外源基因表达框同向放置，并共用同一个PolyA加尾信号序列，一旦外源基因表达框被从质粒上切除并整合到基因组，PB表达框将缺损PolyA加尾信号序列，导致转录的mRNA不稳定而被快速降解，PB的表达被关闭(Chakraborty S,Ji H,Chen J,Gersbach CA,Leong KW.Vector modifications to eliminate transposase expression following piggyBac-mediated transgenesis.Sci Rep.2014；4:7403)。这种策略的缺陷是，PB基因的表达框所转录的mRNA产物实际上覆盖了整个外源基因表达框序列，如果外源基因表达框较大，将导致其mRNA长度过大，降低PB转录效率，难以达到整合所需的PB表达量。
一元转座系统需要将外源基因表达框与PB表达框装入同一载体，但PB的编码序列较长，接近2kb，导致质粒片段较大，将大幅降低转染效率。
另外，目前已有的PB转座子系统的整合位点倾向于插入编码基因 内内(Woodard LE,Wilson MH.Trends Biotechnol.piggyBac-ing models and new therapeutic strategies.2015；33(9):525-33.见第4页表1)。这里所述的编码基因是相对于非编码基因而言，即能编码产生相应功能蛋白质的基因；如果是肿瘤相关基因被插入失活，或者异常激活，则可能有致癌风险。
发明内容
本发明人经过大量的试验和创造性的劳动，构建了一种基于PiggyBac转座子的整合型系统，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。本发明人惊奇地发现，该系统介导的外源基因整合位点主要集中在宿主细胞基因组内的3个基因间区段，能较大程度上避免随机插入引发的风险。由此提供了下述发明：
本发明的一个方面涉及一种核酸构建体，其依次包含如下元件：
转座子5’末端重复序列、polyA加尾信号序列、转座子3’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；
其中，所述polyA加尾信号序列正反向均具有polyA加尾信号功能；
所述转座酶的表达框的方向与外源基因表达框的方向相反。
本发明的一个方面涉及一种核酸构建体，其依次包含如下6种元件：
转座子5’末端重复序列、多克隆插入位点、polyA加尾信号序列、转座子3’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；
其中，
所述多克隆插入位点用于可操作地插入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polyA加尾信号序列正反向均具有polyA加尾信号功能；
所述转座酶的表达框的方向与外源基因表达框的方向相反。
在本发明中，如果没有特别说明，以外源基因表达框的方向为正向，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。
在本发明的一个实施方案中，上述的6种元件各自独立地为单拷贝或者多拷贝。
上述的6个元件之间可以直接相连接，也可以包含有其它的序列例如连接序列(linker)或者酶切位点。
在本发明的一个实施方案中，所述的核酸构建体，其中，所述polyA加尾信号序列为1个polyA加尾信号序列，其正反向均具有polyA加尾信号功能；或者由两个具有单向polyA加尾信号的polyA加尾信号序列以相反方向连接组成。
在本发明中，如果没有特别说明，上述的“所述polyA加尾信号序列正反向均具有polyA加尾信号功能”包括但不限于如下的情形：
1)一种polyA加尾信号序列，其正反向均具有polyA加尾信号功能；
2)两种polyA加尾信号序列，一个正向具有polyA加尾信号功能，一个反向具有polyA加尾信号功能。
优选地，采用上面1)中的方案。不拘于理论的限制，这样外源基因表达框与PiggyBac转座酶表达框可以共用一个polyA加尾信号序列，从而减少了一个polyA加尾信号序列，体现集约原则，缩小质粒大小，有助于在保证转染效率的前提下，增加外源基因表达框的容量。
本发明另外一个非优选的技术方案中，PB表达框与外源基因表达框同向放置，用两个polyA加尾信号序列,其中PB的表达框在前，其polyA加尾信号序列放在其中一个ITR与外源基因启动子之间。例如：控制PB转座酶表达的启动子、PB转座酶编码序列、转座子5’末端重 复序列、polyA加尾信号序列1、外源基因启动子和外源基因(多克隆插入位点)、polyA加尾信号序列2、转座子3’末端重复序列；并且PB转座酶的表达框的方向与外源基因表达框的方向相同。
根据本发明任一项所述的核酸构建体，其中，所述转座子选自PiggyBac、sleeping beauty、frog prince、Tn5和Ty中的一种或多种；优选为PiggyBac转座子。
根据本发明任一项所述的核酸构建体，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。
根据本发明任一项所述的核酸构建体，其中，
所述转座子5’末端重复序列为PiggyBac转座子5’末端重复序列；所述转座子3’末端重复序列为PiggyBac转座子3’末端重复序列；所述转座酶为PiggyBac转座酶。
根据本发明任一项所述的核酸构建体，其中
所述PiggyBac转座子5’末端重复序列的核苷酸序列如SEQ ID NO:1所示；和/或所述PiggyBac转座子3’末端重复序列的核苷酸序列如SEQ ID NO:4所示。
根据本发明任一项所述的核酸构建体，其中，
所述PiggyBac转座酶的氨基酸序列如SEQ ID NO:17所示；优选地，所述PiggyBac转座酶的编码核苷酸序列如SEQ ID NO:5所示。
根据本发明任一项所述的核酸构建体，其中，
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；优选为c-myc核定位信号编码序列，例如为SEQ ID NO:18所示的序列。核定位信号能够引导转座酶在细胞核聚集，从而提高了转座效率。
根据本发明任一项所述的核酸构建体，其特征在于如下的(1)-(3)项中的任意一项或者多项：
(1)所述多克隆插入位点的核苷酸序列如SEQ ID NO:2所示；
(2)所述polyA加尾信号序列的核苷酸序列如SEQ ID NO:3所示；
SEQ ID NO:3所示的序列正反向均具有polyA加尾信号功能。
(3)所述启动子选自和CMV启动子(例如，如SEQ ID NO:6所示)、EF1α启动子、SV40启动子、Ubiquitin B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。
根据本发明任一项所述的核酸构建体，其可操作地连接或者可操作地插入有(例如在多克隆位点)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；所述外源基因独立地为单拷贝或多拷贝；
优选地，所述外源基因选自荧光素报告基因(例如绿色荧光蛋白、红色荧光蛋白、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(例如TP53、GM-CSF、OCT4、SOX2、Nanog、KLF4、c-Myc)、RNAi基因以及人工嵌合基因(例如嵌合抗原受体基因如CAR19、Fc融合蛋白基因、全长抗体基因)中的一种或多种；
优选地，所述外源基因的序列如SEQ ID NO:9-11或16中的任意一个或者多个序列所示。
本发明的再一方面涉及一种重组载体，其含有本发明中任一项所述的核酸构建体；
优选地，所述重组载体为重组克隆载体、重组真核表达质粒或者重组病毒载体；
优选地，所述重组克隆载体为本发明中任一项所述的核酸构建体与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；
优选地，所述重组表达载体为本发明中任一项所述的核酸构建体与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；
优选地，所述重组病毒载体为重组腺病毒载体、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。
本发明的再一方面涉及一种重组宿主细胞，其含有本发明中任一项所述的核酸构建体或者本发明的重组载体；优选地，所述重组宿主细胞为重组的哺乳动物细胞；例如重组的原代培养T细胞、Jurkat细胞、K562细胞、干细胞、肿瘤细胞、HEK293细胞或CHO细胞；优选地，所述干细胞为胚胎干细胞。
根据本发明中任一项所述的核酸构建体、重组载体或者重组宿主细胞，其用于将外源基因表达框整合到宿主细胞基因组中。
本发明的再一方面涉及本发明中任一项所述的核酸构建体、本发明的重组载体或者本发明的重组宿主细胞的用途，其选自如下的(1)-(4)中任一项：
(1)在制备或作为将外源基因表达框整合到宿主细胞基因组的药物或者试剂中的用途；优选地，所述宿主细胞为哺乳动物细胞，例如原代培养T细胞、Jurkat细胞、K562细胞、干细胞、肿瘤细胞、HEK293细胞或CHO细胞；优选地，所述干细胞为胚胎干细胞；
(2)在制备或作为将外源基因表达框整合到宿主细胞基因组的工具的用途；优选地，所述宿主细胞为哺乳动物细胞，例如原代培养T细胞、Jurkat细胞、K562细胞、干细胞、肿瘤细胞、HEK293细胞或CHO细胞；优选地，所述干细胞为胚胎干细胞；
(3)在制备或作为基因组研究、基因治疗、细胞治疗或者干细胞诱导和诱导后分化的药物或制剂中的用途；优选地，所述干细胞为胚胎干细胞；
(4)在制备或作为基因组研究、基因治疗、细胞治疗或者干细胞 诱导和诱导后分化的工具的用途；优选地，所述干细胞为胚胎干细胞。
上述用途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。
本发明的再一方面涉及将本发明的核酸构建体或者重组载体导入哺乳动物细胞的方法，所述方法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，所述方法为电穿孔。
本发明的再一方面涉及一种将外源基因表达框整合到宿主细胞基因组中的方法，包括使用本发明中任一项所述的核酸构建体、重组载体或者重组宿主细胞将外源基因表达框整合到宿主细胞基因组中的步骤。在本发明的一个实施方案中，所述的方法，其中，将本发明中任一项所述的核酸构建体或者本发明的重组载体导入宿主细胞例如哺乳动物细胞，所述导入的方法选自病毒介导的转化、显微注射、粒子轰击、基因枪转化和电穿孔。
下面对本发明涉及的部分术语进行解释。
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“核酸构建体”，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，所述核酸构建体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。
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体中，例如，外源基因启动子与外源基因编码序列通过DNA重组技术被置于所述多克隆位点。所述“可操作地连接”可以通过DNA重组的手段实现，优选地，所述核酸构建体为重组核酸构建体。
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、多聚腺苷酸化序列、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。启动子可以是在所选宿主细胞中有转录活性的任何核酸序列，包括突变的、截短的和杂合的启动子，可以得自编码与宿主细胞同源或异源的胞外或胞内蛋白或多肽多肽的基因。
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。在所选宿主细胞中可发挥功能的任何终止子都可以用于本发明。
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。在所选宿主细胞中可发挥功能的任何前导序列均可用于本发明。
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。
调控序列还可以是肽原编码区，该区编码位于多肽氨基末端的一段氨基酸序列。所得多肽被称为酶原或多肽原。多肽原通常没有活性，可以通过催化或自我催化而从多肽原切割肽原而转化为成熟的活性多肽。
在多肽的氨基末端即有信号肽又有肽原区时，肽原区紧邻多肽的氨基末端，而信号肽区则紧邻肽原区的氨基末端。
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。
发明的有益效果
本发明具有一种或几种下述技术效果：
(1)本发明提供了一种基于PiggyBac转座子的高效整合系统，该系统能介导外源基因高效整合到宿主细胞基因组，并稳定表达。
(2)本发明人惊奇地发现，该系统插入宿主基因组的位置具有明显倾向性，其介导的外源基因整合位点主要集中在宿主细胞基因组内的3个基因间区段，能较大程度上避免随机插入引发的风险。
附图说明
图1:pNB载体图。
图2:pNB转染Jurkat细胞后，PB基因相对表达量的时间曲线。
图3：pN:328-EGFP转染Jurkat细胞后，EGFP阳性细胞比例的时间曲线。
图4:pNB328-EGFP转染4种细胞的荧光检测图。图4A-4B为Jurkat细胞，图4C-4D为K562细胞，图4E-4F为原代T细胞，图4G-4H为小鼠胚胎干细胞(ES)。其中，左侧的图4A、4C、4E、4G为白光下拍照，显示细胞形态；右侧的图4B、4D、4F、4H为荧光下拍照，显示绿色荧光。对于同一种细胞，左右两幅图所拍的视野是相同的。
图5:pNB328-EGFP转染Jurkat细胞(5A)、K562细胞(5B)、原代T细胞(5C)、小鼠ES细胞(5D)的流式检测图。
图6：pNB328-luc转染Huh7细胞的荧光素酶检测图。
图7：pNB328-EGFP转染原代T细胞后EGFP基因表达的荧光表达强度的检测图。7A，7C是白光下的拍照图片，显示细胞形态；7B，7D是荧光下拍照图片，显示绿色荧光。
图8：pNB328-EGFP转染原代T细胞后的整合位点分析图。圆圈标注部分为整合热点。8A,8B,8C,分别代表3个不同来源正常人的原代T细胞整合位点检测。三角形代表的是整合位点属于基因间区段，箭头代表整合位点属于基因内区段，圆圈代表整合热点。
图9：pNB328-CAR19转染原代T细胞后对Raji细胞的杀伤作用检测图。
序列信息：
序列1(SEQ ID NO:1，67bp)，PiggyBac转座子5’末端重复序列
Figure PCTCN2016099005-appb-000001
序列2(SEQ ID NO:2，51bp)，多克隆插入位点
Figure PCTCN2016099005-appb-000002
Figure PCTCN2016099005-appb-000003
序列3(SEQ ID NO:3，222bp)，polyA加尾信号序列
Figure PCTCN2016099005-appb-000004
序列4(SEQ ID NO:4，40bp)，PiggyBac转座子3’末端重复序列
Figure PCTCN2016099005-appb-000005
序列5(SEQ ID NO:5，1815bp)，含c-myc核定位信号编码序列的PiggyBac转座酶序列,其中下划线为c-myc核定位信号编码序列。
Figure PCTCN2016099005-appb-000006
Figure PCTCN2016099005-appb-000007
序列6(SEQ ID NO:6，531bp)，CMV启动子
Figure PCTCN2016099005-appb-000008
序列7(SEQ ID NO:7，2760bp)，实施例1中拼接成的一段长序列
Figure PCTCN2016099005-appb-000009
Figure PCTCN2016099005-appb-000010
序列8(SEQ ID NO:8，545bp)，EF1α启动子序列
Figure PCTCN2016099005-appb-000011
Figure PCTCN2016099005-appb-000012
序列9(SEQ ID NO:9，720bp)，EGFP编码序列
Figure PCTCN2016099005-appb-000013
序列10(SEQ ID NO:10，936bp)，Luc荧光素酶编码序列
Figure PCTCN2016099005-appb-000014
Figure PCTCN2016099005-appb-000015
序列11(SEQ ID NO:11，435bp)，GM-CSF基因编码序列
Figure PCTCN2016099005-appb-000016
序列12(SEQ ID NO:12，20bp)，引物PB-F
Figure PCTCN2016099005-appb-000017
序列13(SEQ ID NO:13，20bp)，引物PB-R
Figure PCTCN2016099005-appb-000018
序列14(SEQ ID NO:14，17bp)，引物Actin-F
Figure PCTCN2016099005-appb-000019
序列15(SEQ ID NO:15，17bp)，引物Actin-R
Figure PCTCN2016099005-appb-000020
序列16(SEQ ID NO:16，1542bp)，CAR19编码序列
Figure PCTCN2016099005-appb-000021
Figure PCTCN2016099005-appb-000022
序列17(SEQ ID NO:17，604aa)，PiggyBac转座酶的氨基酸序列
Figure PCTCN2016099005-appb-000023
序列18(SEQ ID NO:18，27bp)，c-myc核定位信号编码序列
Figure PCTCN2016099005-appb-000024
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施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。
实施例1：pNB载体的构建
依次按PiggyBac转座子5’末端重复序列(SEQ ID NO:1)、多克隆插入位点(SEQ ID NO:2)、polyA加尾信号序列(SEQ ID NO:3)、PiggyBac转座子3’末端重复序列(SEQ ID NO:4)、含c-myc核定位信号的PiggyBac转座酶编码序列(SEQ ID NO:5)、CMV启动子序列(SEQ ID NO:6)，拼接成一段长序列(SEQ ID NO:7)，其中含c-myc核定位信号的PiggyBac转座酶编码序列、CMV启动子序列序列反向互补(这里的反向互补是指由于外源基因表达框与PB基因表达框方向相反，所以显示的是PiggyBac转座酶编码序列、CMV启动子序列序列的反向互补序列)，委托上海杰瑞生物科技有限公司合成，并在两端分别加入AscI与PacI酶切位点，装入pUC57(购自上海杰瑞生物)，命名为pNB载体(图谱见图1)。
实施例2：含外源基因表达框的pNB载体的构建
1.按EF1α启动子的序列，委托上海杰瑞生物科技有限公司合成，并在两端分别加入XbaI与EcoRI酶切位点，装入前面实施例1制备的pNB载体，命名为pNB328载体。
EF1α启动子序列如SEQ ID NO:8所示。
2.按EGFP的编码序列，委托上海杰瑞生物科技有限公司合成，并在两端分别加入EcoRI与SalI酶切位点，装入pNB328载体，命名为pNB328-EGFP载体。
EGFP编码序列如SEQ ID NO:9所示。
3.按Luc荧光素酶编码序列，委托上海杰瑞生物科技有限公司合成，并在两端分别加入EcoRI与SalI酶切位点，装入pNB328载体，命名为pNB328-Luc载体。
Luc荧光素酶编码序列如SEQ ID NO:10所示。
4.按照人GM-CSF基因的酶编码序列，委托上海杰瑞生物科技有限公司合成，并在两端分别加入EcoRI与SalI酶切位点，装入pNB328载体，命名为pNB328-GM-CSF载体。
GM-CSF基因编码序列如SEQ ID NO:11所示。
实施例3：pNB328载体转染Jurkat细胞后PB的表达时间曲线 分析
准备5×106生长状态良好的低代数Jurkat(购于美国标准生物品收藏中心，ATCC)，通过Lonza 2b-Nucleofector仪器(按仪器操作说明书进行)，分别将6μg的pNB328、PB210PA-1(提供PB转座酶的表达质粒,购自System Bioscience公司)质粒转染到细胞核中，置37℃、5％CO2孵箱培养。分别在转染后的第6、12、24、48、96小时，以及第15天，抽取RNA，利用RT-PCR的方法检测PB转座酶的相对表达量。以β-actin作为内参，具体引物如下：
PB-F：如SEQ ID NO:12，PB-R：如SEQ ID NO:13；
Actin-F:如SEQ ID NO:14，Actin-R:如SEQ ID NO:15。
结果表明，在pNB328转染的Jurkat细胞中，PB基因的mRNA含量在转染后的第12小时达到峰值，随后快速下降，在转染后的第24小时已基本检测不到PB RNA的表达；而对照质粒PB210PA-1转染的Jurkat细胞中，PB基因的mRNA含量也在转染后的第12小时达到峰值，但下降较慢，在转染后的第96小时尚能检测到PB的表达(图2)。
以上结果表明，在我们设计的PB自失活机制，即PB转座酶表达框中的polyA加尾信号序列在转座子3’ITR上游，随着PB转座酶发挥作用将“ITR-外源基因表达框-ITR”从pNB328-EGFP载体中切除并整合到宿主细胞基因中时，PB转座酶表达框中的polyA加尾信号序列也被一并切除，导致PB转座酶表达框不完整，表达被快速关闭。
实施例4：pNB载体在Jurkat细胞内的整合效率定量检测
准备5×106代代数旺盛的Jurkat细胞，通过Lonza2b-Nucleofector仪器，分别将6μg的pNB328-EGFP以及5μg PB513B-1(提供包含ITR元件的EGFP表达质粒,购自System Bioscience公司)+2μg PB210PA-1质粒(提供PB转座酶的表达质粒)转染到细胞核中，置37℃、5％CO2孵箱培养。待细胞长满后，按1:10的比例传代培养。分别在转染后的第12小时(P0)、第5天(P0+5)，1次传代后(P1)、2次传代后(P2)、3次传代后(P3)，利用流式细胞仪检测EGFP阳性细胞的比例变化。
由于T细胞的增殖非常快，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。因而，3代以后，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。
如图3所示，随着连续1:10比例的传代，EGFP阳性的Jurkat细胞比例逐渐下降。3次传代后，二元系统PB转座子(PB513B-1+PB210PA-1)转染的Jurkat T细胞，EGFP阳性细胞比例为6.5％(整合效率6.5％)；而改造过的一元系统的PB转座子pNB328-EGFP转染的Jurkat T细胞，EGFP阳性细胞比例为36.4％(整合效率36.4％)。
以上结果表明，改造过的一元系统的PB转座子--pNB载体系统能高效的介导外源基因的整合。
实施例5：pNB328-EGFP载体在Jurkat、K562细胞内的整合分 析
准备5×106生长状态良好的低代数Jurkat、K562细胞株(购于美国标准生物品收藏中心，ATCC)，通过Lonza 2b-Nucleofector仪器(按仪器操作说明书进行)，分别将6μg的pNB328-EGFP、pcDNA3.1-EGFP(购自Addgene公司)质粒转染到细胞核中，置37℃、5％CO2孵箱培养。待细胞长满后，按1:10的比例传代培养。3代后，应用荧光显微镜记录细胞内绿色荧光的表达情况；收集1×105细胞， 利用流式细胞仪检测EGFP阳性细胞的比例。
结果显示，对照质粒pcDNA3.1-EGFP转染后的Jurkat、K562在3代后，几乎检测不到绿色荧光信号，表明转染到细胞内、处于游离状态存在的非整合质粒随着细胞分裂已经完全丢失；相反，pNB328-EGFP转染后的Jurkat、K562在3代后，仍能检测到强烈的绿色荧光信号(图4A、4B、4C、4D)，表明EGFP表达框已经整合到细胞基因组内，能随着细胞分裂稳定存在并表达。
流式结果表明，pNB328-EGFP质粒转染Jurkat、K562后，整合效率分别为36.4％与40.54％(图5A、5B)。
实施例6：pNB328-EGFP载体在原代T细胞内的整合分析
准备1×107新鲜分离获得的外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)，通过Lonza 2b-Nucleofector仪器，分别将6μg的pNB328-EGFP、pcDNA3.1-EGFP质粒转染到细胞核中，置37℃、5％CO2孵箱培养；6小时后转移到含30ng/mL抗CD3抗体、3000IU/mL IL-2(购自Novoprotein公司)的6孔板中，置37℃、5％CO2孵箱培养。待细胞长满后，按1:10的比例传代培养。3代后，应用荧光显微镜记录细胞内绿色荧光的表达情况；同时，收集1×105细胞，利用流式细胞仪检测EGFP阳性细胞的比例。
结果显示，对照质粒pcDNA3.1-EGFP转染后的原代T细胞3代后，几乎检测不到绿色荧光信号，表明转染到细胞内、处于游离状态存在的非整合质粒随着细胞分裂已经完全丢失；相反，pNB328-EGFP转染后的原代T细胞在3代后，仍能检测到强烈的绿色荧光信号，表明EGFP表达框已经整合到细胞基因组内，能随着细胞分裂稳定存在并表达(图4E、4F)。
流式结果表明，pNB328-EGFP质粒转染原代T细胞后，整合效率分别为56.9％(图5C)。
实施例7：pNB328-EGFP载体在小鼠胚胎干细胞内的整合分析
准备5×106小鼠H9胚胎干细胞株(购于ATCC)，通过Lonza2b-Nucleofector仪器，将6μg的pNB328-EGFP质粒转染到细胞核中，置37℃、5％CO2孵箱培养。待细胞长满后，按1:10的比例传代培养。3代后，应用荧光显微镜记录细胞内绿色荧光的表达情况；同时，收集1×105细胞，利用流式细胞仪检测EGFP阳性细胞的比例。
结果显示，pNB328-EGFP转染后的小鼠胚胎干细胞在3代后，仍能检测到强烈的绿色荧光信号，表明EGFP表达框已经整合到细胞基因组内，能随着细胞分裂稳定存在并表达(图4G、4H)。流式结果表明，pNB328-EGFP质粒转染小鼠ES细胞后，整合效率分别为73.12％(图5D)。
实施例8：pNB328-luc载体在肿瘤细胞内的整合分析
准备5×106人肝癌细胞株Huh7(购于ATCC)，通过Lonza2b-Nucleofector仪器，分别将6μg的pNB328-luc、pGL4.75-CMV(购自Promega公司)质粒转染到细胞核中，置37℃、5％CO2孵箱培养。待细胞长满后，按1:10的比例传代培养。3代后，收集1×105细胞，裂解细胞后使用荧光素酶检测试剂盒(购自Promega公司)测定Luc荧光素酶的活性。
结果表明，对照质粒pGL4.75-CMV转染后的Huh7细胞3代后，几乎检测不到荧光素酶活性，表明转染到细胞内、处于游离状态存在的非整合质粒随着细胞分裂已经完全丢失；相反，pNB328-luc转染后的Huh7细胞在3代后，仍能检测到强烈的荧光素酶活性，表明luc表达框已经整合到细胞基因组内，能随着细胞分裂稳定存在并表达(图6)。
实施例9：pNB328-GM-CSF载体在HEK293细胞内的整合分析
准备5×106人HEK293细胞(购于ATCC)，通过Lonza2b-Nucleofector仪器，分别将6μg的pNB328-GM-CSF质粒转染到细胞核中，置37℃、5％CO2孵箱培养。待细胞长满后，按1:10的比例 传代培养。3代后，收集1×106细胞的培养2天后的上清，稀释一定倍数后用人GM-CSF ELISA MAX Deluxe检测试剂盒(购于Biolegend公司)转染pNB328-GM-CSF质粒后的HEK293细胞中GM-CSF蛋白的分泌情况。
结果表明，pNB328-GM-CSF转染后的HEK293细胞在3代后，仍能高水平表达GM-CSF蛋白(1253.7ng/ml)，表明GM-CSF表达框已经整合到细胞基因组内，能随着细胞分裂稳定存在并表达。
实施例10：pNB328-EGFP载体在原代T细胞整合后的外源基因 表达量的比较分析
组1:准备1×107新鲜分离获得的外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)。通过Lonza 2b-Nucleofector仪器，分别将6μg的pNB328-EGFP、pcDNA3.1-EGFP质粒转染到细胞核中，置37℃、5％CO2孵箱培养；6小时后转移到含30ng/mL抗CD3抗体、3000IU/mL IL-2(购自Novoprotein公司)的6孔板中，置37℃、5％CO2孵箱培养。
组2：准备1×106同一健康人来源的PBMC细胞，在30ng/mL抗CD3抗体、3000IU/mL IL-2条件下刺激培养3天，而后取5×106活化后的T细胞，应用携带绿色荧光蛋白的重组慢病毒rLV-EGFP(购自上海比昂生物医药科技有限公司，MOI＝100)进行病毒感染。
待细胞长满后，两组处理完的细胞按1:10的比例传代培养。3代后，利用荧光显微镜观察绿色荧光的表达情况。同时，分别收集1×105细胞，利用流式细胞仪检测EGFP阳性细胞中的平均荧光强度(MFI)。结果表明，pNB328-EGFP载体整合后的T细胞，荧光强度高(图7A、7B)，MFI达到1507.63；而慢病毒感染后的T细胞，绿色荧光的强度较低MFI为50.34(图7C、7D)，两者相差近29倍。结果表明，pNB328-EGFP载体介导外源基因整合到转染原代T细胞后，能促进外源基因的高效表达。
实施例11：pNB328-EGFP载体在原代T细胞内的整合位点分析
准备3份不同人来源的新鲜PMBC，通过Lonza 2b-Nucleofector仪器，将6μg的pNB328-EGFP质粒转染到细胞核中，置37℃、5％CO2孵箱培养；6小时后转移到含30ng/mL抗CD3抗体、3000IU/mL IL-2(购自Novoprotein公司)的6孔板中，置37℃、5％CO2孵箱培养。待细胞长满后，按1:10的比例传代培养。收集5×107细胞，提取基因组DNA，委托云健康基因科技(上海)有限公司进行全基因组测序，并分析EGFP插入位点在基因组内的分布情况。结果表明，相对于样品1共检测到18个插入位点，样品2共检测到36个插入位点，样品3共检测到61个插入位点(插入位点是指一个样品中被检出有基因组整合的所有基因组位点；在同一位点被重复检出，就是检出数。)(图8A、8B、8C)。出于意外的是，在第5号染色体5p15.1、第7号染色体7p15.1、第9号染色体9q34.3区间内存在整合热点(图8A、8B、8C，圆圈标出了整合热点。注：通常情况下，一个位点的检出数在1-3之间。)，三个样品的在3个区间内相邻位置上的检出数分别达到50/66/50(注：是指在第5号染色体5p15.1处发生整合的检出数，第一个样品为50，第二个样品为66，第三个样品为50，下面的68/82/64、78/59/54可做类似理解。)、68/82/64、78/59/54。
由于目前人类基因组的注释已经比较完整，可通过生物信息学确定该区间是否为基因间序列，还是基因间隔序列。经分析，上述三个区间均属于基因间区段，外源基因表达框的插入不会构成相关基因的失活、插入突变。
实施例12：pNB328-CAR19载体的构建与原代T细胞的遗传修饰
1.按针对CD19抗原的嵌合抗原受体(chimeric antigen receptor,CAR)序列，委托上海杰瑞生物科技有限公司合成，并在两端分别加入EcoRI与SalI酶切位点，装入pNB328载体，命名为pNB328-CAR19载体。
CAR19编码序列如SEQ ID NO:16所示。
2.准备1×107新鲜分离的人PBMC，通过Lonza2b-Nucleofector仪器，分别将6μg的pNB328-CAR19质粒转染到细胞核中，置37℃、5％CO2孵箱培养；6小时后转移到含30ng/mL抗CD3抗体、3000IU/mL IL-2(购自Novoprotein公司)的6孔板中，置37℃、5％CO2孵箱培养。待细胞稳定生长后，即得到经CAR19遗传修饰的T细胞(CAR19-T)。
实施例13：CAR19-T细胞对靶细胞的体外杀伤作用检测
按不同的效靶比(8:1，4:1，2:1，1:1，0.5:1，0.25:1，0.125:1,0.0625:1)，将CAR19-T以及未修饰的T细胞与Raji细胞(购自ATCC)共培养，应用LDH乳酸脱氢酶-细胞毒性检测分析试剂盒(LDH-Cytotoxicity Assay Kit，Biovision)检测遗传修饰前后的T细胞对Raji细胞的体外杀伤能力，方法如下：靶细胞铺96孔板(5×103/孔)，设培养基背景、体积校正、靶细胞自发LDH释放、靶细胞最大LDH释放、效应细胞自发LDH释放对照孔，治疗组孔，每组重复3孔，每个孔的终体积相同且不少于100μL。250g离心4min，在37℃，5％CO2孵育至少4h。在离心前45min，向靶细胞最大释放孔加入10×裂解液，体积校正孔加入等量的裂解液。再次离心，从每孔转移50μL上清至新的96孔板中，再加入50μL底物溶液，室温避光孵育30min。每孔加入50μL终止液，1h内测定D490。细胞毒性(％)＝[(D实验孔-D培养基背景孔)-(D效应细胞自发LDH释放孔-D培养基背景孔)-(D靶细胞自发LDH释放孔-D培养基背景孔)]/[(D靶细胞最大LDH释放孔-D体积校正孔)-(D靶细胞自发LDH释放孔-D培养基背景孔)]×100％。
结果表明，相对于未经修饰的T细胞，pNB328-CAR19介导修饰获得的CAR19-T对CD19阳性的Raji细胞具有明显的杀伤作用(图9，p<0.001)。
本领域技术人员知悉，Raji细胞可作为CD19阳性的细胞的代表。因此，pNB328-CAR19介导修饰获得的CAR19-T能对Raji细胞高效 杀伤，同样能对CD19阳性的肿瘤细胞进行高效杀伤，具有临床上的应用价值，例如高效杀伤表达CD19表面抗原的B细胞淋巴瘤，特别是对晚期难治性B细胞淋巴瘤具有较高的疗效。
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 (16)

  1. 一种核酸构建体，其依次包含如下元件：
    转座子5’末端重复序列、polyA加尾信号序列、转座子3’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；
    其中，所述polyA加尾信号序列正反向均具有polyA加尾信号功能；
    所述转座酶的表达框的方向与外源基因表达框的方向相反。
  2. 根据权利要求1所述的核酸构建体，其依次包含如下元件：
    转座子5’末端重复序列、多克隆插入位点、polyA加尾信号序列、转座子3’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；
    其中，
    所述多克隆插入位点用于可操作地插入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polyA加尾信号序列正反向均具有polyA加尾信号功能；
    所述转座酶的表达框的方向与外源基因表达框的方向相反。
  3. 根据权利要求1或2所述的核酸构建体，其中，所述转座子选自PiggyBac、sleeping beauty、frog prince、Tn5和Ty中的一种或多种；优选为PiggyBac转座子。
  4. 根据权利要求1或2所述的核酸构建体，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。
  5. 根据权利要求1或2所述的核酸构建体，其中，所述polyA加尾信号序列为1个polyA加尾信号序列，其正反向均具有polyA加尾信号功能；或者由两个具有单向polyA加尾信号的polyA加尾信号序 列以相反方向连接组成。
  6. 根据权利要求1或2所述的核酸构建体，其中，
    所述转座子5’末端重复序列为PiggyBac转座子5’末端重复序列；
    所述转座子3’末端重复序列为PiggyBac转座子3’末端重复序列；
    所述转座酶为PiggyBac转座酶。
  7. 根据权利要求6所述的核酸构建体，其中
    所述PiggyBac转座子5’末端重复序列的核苷酸序列如SEQ ID NO:1所示；和/或
    所述PiggyBac转座子3’末端重复序列的核苷酸序列如SEQ ID NO:4所示；和/或
    所述PiggyBac转座酶的氨基酸序列如SEQ ID NO:17所示；优选地，所述PiggyBac转座酶的编码核苷酸序列如SEQ ID NO:5所示。
  8. 根据根据权利要求1或2所述的核酸构建体，其中，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；优选为c-myc核定位信号编码序列。
  9. 根据权利要求1至8中任一项所述的核酸构建体，其特征在于如下的(1)-(3)项中的任意一项或者多项：
    (1)所述多克隆插入位点的核苷酸序列如SEQ ID NO:2所示；
    (2)所述polyA加尾信号序列的核苷酸序列如SEQ ID NO:3所示；
    (3)所述启动子选自和CMV启动子(例如，如SEQ ID NO:6所示)、EF1α启动子、SV40启动子、Ubiquitin B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。
  10. 根据权利要求1至9中任一项所述的核酸构建体，其可操作地连接或者可操作地插入有(例如在多克隆位点)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；所述外源基因独立地为单拷贝或多拷贝；
    优选地，所述外源基因选自荧光素报告基因(例如绿色荧光蛋白、红色荧光蛋白、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(例如TP53、GM-CSF、OCT4、SOX2、Nanog、KLF4、c-Myc)、RNAi基因以及人工嵌合基因(例如嵌合抗原受体基因如CAR19、Fc融合蛋白基因、全长抗体基因)中的一种或多种；
    优选地，所述外源基因的序列如SEQ ID NO:9-11或16中的任意一个或者多个序列所示。
  11. 一种重组载体，其含有权利要求1至10中任一项所述的核酸构建体；
    优选地，所述重组载体为重组克隆载体、重组真核表达质粒或者重组病毒载体；
    优选地，所述重组克隆载体为权利要求1至10中任一项所述的核酸构建体与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；
    优选地，所述重组表达载体为权利要求1至10中任一项所述的核酸构建体与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；
    优选地，所述重组病毒载体为重组腺病毒载体、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。
  12. 一种重组宿主细胞，其含有权利要求1至10中任一项所述的核酸构建体或者权利要求11所述的重组载体；优选地，所述重组宿主细胞为重组的哺乳动物细胞；例如重组的原代培养T细胞、Jurkat细胞、K562细胞、干细胞、肿瘤细胞、HEK293细胞或CHO细胞；优选地，所述干细胞为胚胎干细胞。
  13. 权利要求1至10中任一项所述的核酸构建体、权利要求11所述的重组载体或者权利要求12所述的重组宿主细胞的用途，其选自如下的(1)-(4)中任一项：
    (1)在制备或作为将外源基因表达框整合到宿主细胞基因组的药物或者试剂中的用途；优选地，所述宿主细胞为哺乳动物细胞，例如原代培养T细胞、Jurkat细胞、K562细胞、干细胞、肿瘤细胞或HEK293细胞或CHO细胞；优选地，所述干细胞为胚胎干细胞；
    (2)在制备或作为将外源基因表达框整合到宿主细胞基因组的工具的用途；优选地，所述宿主细胞为哺乳动物细胞，例如原代培养T细胞、Jurkat细胞、K562细胞、干细胞、肿瘤细胞、HEK293细胞或CHO细胞；优选地，所述干细胞为胚胎干细胞；
    (3)在制备或作为基因组研究、基因治疗、细胞治疗或者干细胞诱导和诱导后分化的药物或制剂中的用途；优选地，所述干细胞为胚胎干细胞；
    (4)在制备或作为基因组研究、基因治疗、细胞治疗或者干细胞诱导和诱导后分化的工具的用途；优选地，所述干细胞为胚胎干细胞。
  14. 根据权利要求1至10中任一项所述的核酸构建体、权利要求11所述的重组载体或者权利要求12所述的重组宿主细胞，其用于将外源基因表达框整合到宿主细胞基因组中。
  15. 一种将外源基因表达框整合到宿主细胞基因组中的方法，包括使用权利要求1至10中任一项所述的核酸构建体、权利要求11所述的重组载体或者权利要求12所述的重组宿主细胞将外源基因表达框整合到宿主细胞基因组中的步骤。
  16. 根据权利要求15所述的方法，其中，将权利要求1至10中任一项所述的核酸构建体或者权利要求11所述的重组载体导入宿主细胞例如哺乳动物细胞，所述导入的方法选自病毒介导的转化、显微注射、粒子轰击、基因枪转化和电穿孔。
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