CN106222145A

CN106222145A - A kind of for gene recombinaton hematopoietic stem cell treating tumor and preparation method thereof

Info

Publication number: CN106222145A
Application number: CN201610698890.7A
Authority: CN
Inventors: 侯昌禾; 任沁沁
Original assignee: Individual
Current assignee: Individual
Priority date: 2016-08-22
Filing date: 2016-08-22
Publication date: 2016-12-14

Abstract

The invention discloses a kind of gene recombinaton hematopoietic stem cell for treating tumor, chronic infection or autoimmune disease etc. and preparation method thereof.Described restructuring hematopoietic stem cell is to insert Car gene and the special promoter of CD3+CD8+CD4 T cell and enhancer in the genome of patient's hematopoietic stem cell.Section of DNA sequence containing Car is actually knocked in patient's hematopoietic stem cell by the present invention, and control the Car specific differential period being expressed in CD8+CD4 T cell by gene recombination technology, simultaneously, also include and pass through gene recombination technology, making the high efficiency directed differentiation of hematopoietic stem cell is CD8+CD4 T cell, reducing myeloid differentiation, B cell differentiation and the differentiation of CD4+CD8 T cell, CAR T is in the concentration of peripheral blood in increase, improves its efficiency.

Description

A kind of for gene recombinaton hematopoietic stem cell treating tumor and preparation method thereof

Technical field

The present invention relates to the treatment technology field of tumor, chronic infection or autoimmune disease etc., in particular it relates to a kind of Gene recombinaton hematopoietic stem cell and preparation method thereof.

Background technology

Car-T is a kind of new Therapeutic Method, the CAR of the most artificial constructed specific antigen for target cell Molecule, and the DNA sequence encoding this molecule is inserted restructuring T cell, restructuring T cell is fed back in the patient, kills target cell. The CAR molecule of the restructuring T cell surface expression built, its extracellular domain is scFv (single-chain variable Fragment), intracellular territory comprises CD3 intracellular territory part, and the former can be independent of MHC I antigen and offer, direct and target cell table Face mark combines (such as ALL and CLL, acute and chronic Lymphocytic leukemia, surface marker CD19), and lives Change the intracellular territory of the i.e. CD3 of the latter, thus start the T cell lethal effect for target cell.In addition, the second filial generation and the third generation The CAR that CAR-T is used also comprises CD28, the intracellular territory of the costimulatory signals such as 4-1BB, can promote Car-T cell proliferation.Research Showing, in hematological system tumor ALL and CLL, the Car-T effect for CD19 is notable.

But, Car-T therapy still suffers from following problems: 1. Car-T is remarkable to the lethal effect of non-solid tumors, When carrying tumor amount in the patient and being bigger, massive tumor necrocytosis can be caused, cause serious inflammatory reaction and cytokine wind Cruelly, even death, there is no the method determined at present and avoid or eliminate this untoward reaction.2. Car-T is the one-tenth of terminal differentiation Ripe T cell, multiplication capacity and life-span are the most limited, it is necessary to after the most once building amplification in whole infusion ex vivos, it is in vivo Cannot Long-term Proliferation, effective time is shorter, thus short run effect is notable and still have the palindromia may for a long time.Research shows, Car-T adoptive therapy CD19+ALL, after 1 month, remission rate reaches 90%, and has 25.9% recurrence after 8.5 months, and its recurrence is all sent out Raw after peripheral blood Car-T vanished cell, and be still CD19+ tumor cell (Maude, S. L. and N. Frey, et al. (2014). "Chimeric antigen receptor T cells for sustained remissions in Leukemia. " N Engl J Med 371 (16): 1507-17.).Therefore, the endurance extending Car-T peripheral blood is conducive to Reduce recurrence.

Summary of the invention

The invention aims to overcome the above-mentioned deficiency of prior art, it is provided that a kind of gene recombinaton hematopoietic stem cell, Making its Self-differentiation is Car-T, in the existence of the most lasting maintenance Car-T of peripheral blood, it is to avoid the most too high Car-T concentration Causing massive tumor downright bad and cytokine storm, the existence of maintenance peripheral blood Car-T " forever ", makes human body the most in theory Obtain the permanent immune surveillance for target cell, reduce risk of recurrence.The inventive point of the present invention is that design is a kind of based on HSCs Rather than the Car system of the T cell of terminal differentiation, named Car-HSCs, so that its continuous proliferation is divided into Car-T, and slowly It is discharged into peripheral blood, within the longer time, progressively kills target cell, and play long-term immunosurveillance, reduce long-term multiple The rate of sending out.

It is a further object to provide a kind of preparation method for gene recombinaton hematopoietic stem cell.

To achieve these goals, the present invention is achieved by the following technical programs:

A kind of gene recombinaton hematopoietic stem cell, inserts one section of sequence in the genome of patient's hematopoietic stem cell, and this sequence includes One fragment gene X, this fragment gene X can encode one section of peptide chain Y, this peptide chain Y and include energy specific recognition target cells epitope Extracellular domain, wear film territory and the intracellular territory of intracellular signaling pathways can be activated；This sequence also includes regulating above-mentioned by specificity The promoter of peptide chain Y expression and enhancer.

It should be noted that letter X and Y be intended merely to express clearly and to genetic fragment and the name of peptide chain, for base The structure of cause and peptide chain does not has limiting meaning.

The peptide chain Y of said gene X coding, can specific be combined with target cells antigen, starts the life in intracellular territory Thing activity, activates intracellular signaling pathways transmission, and its effect includes but not limited to active cell toxic reaction and promotes T cell propagation Function.

Preferably, a kind of restructuring hematopoietic stem cell, inserts Car in the genome of patient's hematopoietic stem cell Gene and the promoter of CD3+CD8+CD4-T cell-specific and enhancer.The present invention is actually by Car gene and control Car The promoter of the CD3+CD8+CD4-T cell-specific of specific gene expression and enhancer knock in the hematopoietic stem cell of patient simultaneously Genome in, the promoter of CD3+CD8+CD4-T cell-specific and enhancer can control being expressed in of Car gene specific The differential period of CD3+CD8+CD4-T cell.

The promoter that the present invention selects only is activated (such as at CD3+ in the specific differential period of specific differentiation direction CD8+CD4-T cell is activated), include HSCs and other differentiation directions at remaining and the stage is in closed mode.

Preferably, described promoter both can come from natural genomic clone product (opening of such as clone's CD8+ molecule Mover), it is also possible to from the DNA sequence of synthetic, even engineer.

It is highly preferred that described promoter is CD8b1 upstream promoter part (CD8b1 transcripting start point upstream 200bp), increase Hadron is SV40 or TCR-alpha enhancer；Or use CD8a promoter and TG-g enhancer (see Ellmeier, W. and S. Sawada, et al. (1999). "The regulation of CD4 and CD8 coreceptor gene Expression during T cell development. " Annu Rev Immunol 17:523-54.).

Convert it is highly preferred that CD8 b1 upstream promoter part (CD8b1 transcripting start point upstream 200bp) is inserted Car SV40 or TCR-alpha enhancer, adjacent to upstream, is inserted Car encoding gene downstream about 1.0kb by the Car encoding gene of carrier Place.

In order to make the high efficiency directed differentiation of improved hematopoietic stem cell be CD3+CD8+CD4-T cell, reduce medullary system Differentiation, B cell differentiation and the differentiation of CD4+CD8-T cell, CAR-T is in the concentration of peripheral blood in increase, improves its efficiency；Preferably, Described gene recombinaton hematopoietic stem cell can add other genomic modifications, as knocked out/process LAN portion gene, makes HSCs be more prone to In lymphatic system differentiation direction, it is highly preferred that described in knock out/process LAN portion gene can be such as knock out simultaneously GATA1 and PU.1, blocks myeloid differentiation.

Preferably, described gene recombinaton hematopoietic stem cell can add other genomic modifications, as knocked out/process LAN part base Cause, makes HSCs be more likely to T cell differentiation direction.Such as knock out Pax5 and can block B cell differentiation.

Preferably, described gene recombinaton hematopoietic stem cell can add other genomic modifications, as knocked out/process LAN part base Cause, makes HSCs be more likely to CD8+CD4-T cell differentiation direction.Such as, knock out Th-POK and divide to block CD4+CD8-T cell Change direction.

Preferably, described gene recombinaton hematopoietic stem cell can add other genomic modifications, as added regulatable suicide System, such as, add iCasp-9 or other equivalent systems (Wu, C. and S. G. Hong, et al. (2014). " Development of an inducible caspase-9 safety switch for pluripotent stem Cell-based therapies. " Mol Ther Methods Clin Dev 1:14053.).Inject AP20187 or After the inducers such as AP1903, the iCasp-9 that inducer is expressed in restructuring stem cell is combined, and activates Caspase-9 activity, promotes Restructuring stem cell apoptosis.When untoward reaction occurring after restructuring stem cell transplantation, can be terminated it in aforementioned manners and exist.

Preferably, described gene recombinaton hematopoietic stem cell can add the labels such as other genomic modifications, such as GFP, with Just follow the trail of.

Preferably, described gene recombinaton hematopoietic stem cell can add the resistances such as other genomic modifications, such as G418, in order to Screening.

Knock out described in it is highly preferred that/method of process LAN portion gene can be slow virus, retrovirus, electroporation, turn Stand, Crispr-Cas9 and deriving technology thereof, or other can meet and knock in the method that (knock-in) requires.

The preparation method of a kind of gene recombinaton hematopoietic stem cell, comprises the steps:

(1) establishing the transcription factor that CD8+T cell stage is special, this transcription factor exists only in the T cell of CD8+ terminal differentiation In；Establish the specific promoter corresponding with this transcription factor and enhancer, this transcription factor can activate this promoter and Enhancer, and other stages of other directions of hematopoietic stem cell differentiation, this promoter and enhancer all can not be activated；

(2) with conventional gene engineering clone, amplification, sequential concatenation for target cells antigen scFv encoding gene, Membrane-spanning domain encoding gene and CD3 intracellular territory encoding gene (can add CD28 intracellular territory encoding gene and 4-1BB encoding gene etc. auxiliary The costimulatory signal helped), gene fusion construct Car.Above-mentioned promoter and enhancer are placed in Car upstream or downstream, and it is right to be formed The transcriptional control relation of Car, builds Car conversion carrier, such as slow virus.This Car is by only in the T cell of CD8+ terminal differentiation Express, be in silence in other stages and differentiation direction.Also will be only at CD8+ as used TCR to design replacement Car, same TCR The T cell of terminal differentiation is expressed, is in silence in other stages and differentiation direction.

(3) method (if carrier use slow virus form) of transduction can be used containing specific promoter and enhancer Car(or TCR, below repeats no more) insert HSCs genome in, make restructuring HSCs possess express Car ability, this Car institute The outer scFv of the born of the same parents contained can be combined with target cells mark, activates CD3 and CD28/CD134,4-1BB etc. contained by intracellular territory Costimulatory signal；

(4) in order to improve the efficiency that restructuring HSCs breaks up to CD8+T cell further, can knock out further or process LAN part base Cause, including 3 key nodes: determine medullary system/lymphatic system differentiation, T cell/B cell/differentiation of dendritic cells, and CD4+/CD8+ T cell is broken up.Restructuring HSCs is made optionally to move towards lymphatic system-T cell-CD8+ differentiation direction.

Gene recombinaton hematopoietic stem cell of the present invention treatment tumor, chronic infection or the method for autoimmune disease For: expanding above-mentioned restructuring HSCs, be implanted in the patient, or do not expand above-mentioned restructuring HSCs, direct feedback is transplanted and is entered patient Internal.Restructuring HSCs by long-term existence in the patient, slow and long-term generation Car-T.And other differentiation directions and other The hemocyte in stage, as thin in granulocyte, monokaryon eh, NK cell, erythrocyte, B cell, its Phenotype and wild type zero difference.

Compared with prior art, there is advantages that

Section of DNA sequence containing Car is knocked in patient's hematopoietic stem cell by the present invention, and is controlled by gene recombination technology The CAR specific differential period being expressed in CD8+CD4-T cell, meanwhile, also include by gene recombination technology, make hemopoietic The high efficiency directed differentiation of stem cell is CD8+CD4-T cell, reduces myeloid differentiation, B cell differentiation and CD4+CD8-T cell and divides Changing, CAR-T is in the concentration of peripheral blood in increase, improves its efficiency.

Accompanying drawing explanation

Fig. 1 is the Car structural representation built as a example by the scFv of targeting CD19.

Fig. 2 is the Car structural representation built as a example by the scFv of targeting HER2.

Detailed description of the invention

Making the present invention with specific embodiment below in conjunction with the accompanying drawings and elaborating further, described embodiment is served only for Explain the present invention, be not intended to limit the scope of the present invention.Test method used in following embodiment if no special instructions, It is conventional method；The material that used, reagent etc., if no special instructions, for the reagent commercially obtained and material.

Embodiment 1

HCD19 Positive Acute Lymphoblastic Leukemia or chronic lymphocytic leukemia patients undergoing subcutaneous injecting G-SCF 10ug/kg, 5 days, with mobilizing hematopoietic stem cells.Or do not mobilize hematopoietic cell.

Special equipment, circulation absorption, it is thus achieved that mononuclearcell is gathered with PERIPHERAL BLOOD MONONUCLEAR CELL.

To be marked with anti-hCD34 magnetic bead screening mononuclearcell.Obtain hCD34+ mononuclearcell after eluting, be HSCs。

Select specific scFv, such as anti human CD 19 monoclonal antibody, intercept its Fab end encoding gene as scFv.Choose CD8 membrane-spanning domain encoding gene.Choose CD137 intracellular territory (4-1BB) encoding gene.Choose CD3 intracellular territory encoding gene.Conventional base Because engineering is linked in sequence above-mentioned encoding gene, constitute the Car fusion gene (structure is shown in Fig. 1) of anti-hCD19.

Choose CD8b1 upstream promoter part (transcripting start point upstream 200bp), choose SV40 enhancer；According to following Order is spliced with Car fusion gene: CD8b1 promoter-Car fusion gene-1.0kb-SV40 enhancer；Wherein, promoter sequence Row are as shown in SEQ ID NO:1, and SV40 enhancer sequence is as shown in SEQ ID NO:2, and 1.0kb is that insignificant arbitrary sequence is done Spacer, Car fusion gene can be anti-hCD19 molecule, it is possible to for other target spots, the present embodiment is lifted with anti-hCD19 Example, the sequence of Car fusion gene is as shown in SEQ ID NO:3.This splicing product is inserted plasmid, conventional packaging slow virus.

By above-mentioned lentiviruses transduction hCD34+ mononuclearcell.Insertion point is searched in order-checking, cultivates the detection rate of increase with row Except cancerating.

This patient is returned in the hCD34+ mononuclearcell intravenous injection again of above-mentioned restructuring.The hematopoietic stem cell of restructuring will be from Dynamic being divided into CD3+CD8+ Car-T cell, specificity kills CD19 positive lymphocyte leukaemia, and Hematopoietic Stem of recombinating Car molecule is not expressed in other differentiation (granulocyte, mononuclear cell, B cell etc.) of cell.

Embodiment 2

HER2 positive breast cancer patient, routine bone marrow punctures, and bone marrow extraction liquid is centrifuged with lymphocyte separation medium method, in taking Interbed mononuclearcell.

Select specific scFv, the most anti-human HER2 monoclonal antibody, intercept its Fab end encoding gene as scFv.Choose CD8 membrane-spanning domain encoding gene.Choose CD137 intracellular territory (4-1BB) encoding gene.Choose CD3 intracellular territory encoding gene.Conventional base Because engineering is linked in sequence above-mentioned encoding gene, constitute the Car fusion gene (structure is shown in Fig. 2) of anti-hHER2.

Choose CD8b1 upstream promoter part (transcripting start point upstream 200bp), choose SV40 enhancer, according to following Order is spliced with Car fusion gene: CD8b1 promoter-Car fusion gene-1.0kb-SV40 enhancer, promoter sequence is such as Shown in SEQ ID NO:1, SV40 enhancer sequence is as shown in SEQ ID NO:2, and 1.0kb is that insignificant arbitrary sequence is done Spacer, Car fusion gene is the nucleotide sequence obtained according to the design of Fig. 2 structure.

The genome that this splicing product inserts above-mentioned hCD34+ mononuclearcell with Crispr-Cas9 similar technique is specified Site.Insertion point is searched in order-checking, cultivates the detection rate of increase and cancerates to get rid of.

By the hCD34+ mononuclearcell cultured and amplified in vitro of above-mentioned restructuring, this patient is returned in intravenous injection again.Restructuring Hematopoietic stem cell will be divided into CD3+CD8+Car-T cell automatically, and specificity kills hHER2 breast cancer cell, and hemopoietic of recombinating Car molecule is not expressed in other differentiation (granulocyte, mononuclear cell, B cell etc.) of stem cell.

Embodiment 3

Choose CD8a promoter and CD8-TG-g enhancer, splice with Car fusion gene according to following order: CD8a starts Son-Car fusion gene-1.0kb-TG-g enhancer.Wherein, the sequence of CD8a promoter as shown in SEQ ID NO:4, TG-g The sequence of enhancer is as shown in SEQ ID NO:5；1.0kb is that insignificant arbitrary sequence is spacer；Car fusion gene be by The nucleotide sequence obtained according to the design of Fig. 2 structure.

Choose human Caspase-9, replace it with human FK506-binding protein (FKBP12) Recruitment domain (CARD), is spliced into fusion gene iCapsp9.This fusion gene is similar with Crispr-Cas9 Technology is inserted the genome of above-mentioned Car-HSCs and is specified site.Insertion point is searched in order-checking, cultivates the detection rate of increase to get rid of evil Become.

By the hCD34+ mononuclearcell cultured and amplified in vitro of above-mentioned restructuring, this patient is returned in intravenous injection again.Restructuring Hematopoietic stem cell will be divided into CD3+CD8+ Car-T cell automatically, and specificity kills hHER2 breast cancer cell, and hemopoietic of recombinating Car molecule is not expressed in other differentiation (granulocyte, mononuclear cell, B cell etc.) of stem cell.

When there is the untoward reaction that this restructuring hematopoietic stem cell any causes in patient, all it is contemplated that give AP20187 or AP1903, inducer is combined with the FKBP12 territory of iCapsp9 after entering restructuring stem cell, activates Caspase-9 activity, induction weight Group hematopoietic stem cell apoptosis.

SEQUENCE LISTING

<110>Hou Changhe, Ren Qinqin

<120>a kind of for gene recombinaton hematopoietic stem cell treating tumor and preparation method thereof

<130>

<160> 5

<170> PatentIn version 3.3

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tgtaaatgaa gaatggtcaa attcatggga acagaaaaca gaatagtggt ttccgggctt 1320

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tcctacgctt ttttttcccc ttttatatac attgtttctt cttgtgaatt atctttaaat 1740

tttgaagttt ctttaaaaat aatcagaaca aacatcaggc atggtggtga gagccttaaa 1800

ctcagcactg ggccagtgga ggcaggagag tcagaaattc aatattatgg gccattttaa 1860

ggccagccag ggttacctga gatcctgtct caaaagaaag aaaatgtata ttattaatca 1920

taataatata cattttatgt cattgtagaa gcttagaaat aggaacaaaa gagcaaggga 1980

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tgtatgtatc cataaaatat accgtagcca tttcccagct ccgtacatgt ttttcataat 2100

tctaatattt cacagtaatg tttaaaggtg tgctggctgt tccttttgga gaacagttca 2160

gtctcaccca tgttcacgtc tctgtgcctc cctcccctgc ctcccttctg agctgacctt 2220

aagattacac ccatgacagc agatttccac cttggaatga ctgagcatat gggtgcaaac 2280

attttaatag atcggcctct ttttataatt ttttttttta cttttcattg gtataatcat 2340

aatttatggg atacagggtg tttcccccct tctccaaaag ttgcgtgtat cctagactgg 2400

tctctaactc atcatgtagc caaggatgac gttaaaccct tgaagctcct gcttccacct 2460

gccccgtgct gggaatgcag gtttggcacc accacgcctg gcctggtttt cccttggctt 2520

atttgctggt cggttgtgtt cttactgctt tggatattca tccctccctg gattctctgt 2580

agttattttc tccctcttgg cagactgcct catcactgtg tctgtcgaag ctaatttgtt 2640

tgaaatagtt catttgtcaa ttttgggttt tgttccctgt gttctgggga tgatattcaa 2700

aagatatttc cccacttttg tattatagat ttttgttggt gtgtggggag gtggtatgca 2760

tgtgtgtatg tgcaagtgtg ccatggagtc cagaagataa gcttatagcg ttggttcttt 2820

ccgttcacct ttatgtgggt tttggggatc aaacccaggc taccaggctt gcacagcaag 2880

tacctgctaa ggcatctcac tggtcctaag ttacagcttt ttaatatctg atgtgagatt 2940

gctttctggg acactgccgc tccctcccac agtaagtacc atcctgtccc atgttgtgtt 3000

acaagtggga gctccttttc tgcactggtg ctgctctata caagattgct gcatctttct 3060

cccattttat aaatgatcaa atgcgcattg tgcaaagtga cagatttcca cgtggcattt 3120

ttatgtttgt atcgtgttct tgtatcatcg tcacatcctc cattatcctt tctgtctcct 3180

atacaccccc ttccattttc atgttatata tttccttctt tgtaaaaata tagattccat 3240

atatgggcac aaacatctgg caccatcttc ccaagtctgg ctcattcctt tgaacatgat 3300

ggtccaaggc cccttgtaat aaaatccata gtcataagta agatggagtc ttctgtttgt 3360

tttgttttgt tttttgtttt tggagataga atttctctgt atagccctgg ctgtcctgga 3420

actcactctg tagaccaggc tggcctcaaa ctcagaaatc tgcctgcctc tgcctcccaa 3480

gttctgggat taaaggcatg cgtcaccact gcccggatgt aagatggagt ctgaatacag 3540

ctttgataga ttggcaaaat cttcttaaaa ggcatgcatt tcctaagtga acacctttac 3600

tttgaacaca cagtccagtg tttacagagc aaaagcagag ggtttgggct ttgatgctgt 3660

aaacatggca gaaactcaaa acccaagacc caaaatgctc agggacaaat caaggcgtga 3720

cagttctagg tacaatccaa ggaatggatt gtgcagagtt gctgaatcca acattggaga 3780

ggcaaggact ctgatctgag ccatggacag agcctgacat cgtgcccaag tcagcatcaa 3840

ccttcccatg aggaacagag ctggaaaagc ccatcagaag ggcagccgac agggagaaga 3900

gacttttgaa caagcccagc cacccaagga ttcaccattc agagagacct gtctatgtgg 3960

gggctacctc tgtctcccac agctgtgctg gggtcttgtt aaaagtgaaa ctgactttca 4020

gagggttctg ggggcgtttg ctacaggaaa gtcgatgttc cacttcaggg tccaactagc 4080

ctcaaacaaa gcctcttgct tgctttcagc ttctggggcc ccttggttgc ctctgctact 4140

tctagatgtt tctgaggtta cagcaggcca cagcatgttc cagtctaggc agaggaaata 4200

taaaaaccaa tgcgaatgtg actcaagacc ccctcgtgct tctacagcac cacacagctg 4260

gaaatggcaa agtctgccgg cttccgggtt ccaccattgc cgggaagcca cttccttttc 4320

tcccaaccaa atgagatcgg catctgagat ccattcagta gatgaggaat aagatcagaa 4380

aggctcaggt catactgatc acaccgcatt aataaagtcc tgagctagga ctcagcccca 4440

gcggcctgac ctgacttaac ctatgagtgg gatgtgacag agctcagtgg tccattcaca 4500

agcttcatgg acgtagaaaa ggaacctggg agcaggccag tgtagcctta tacaaacctg 4560

gggttattgg atttgagggg gataagacaa caatatagtt cattatagtt gagtctgtag 4620

ttcaaagcag ataaagccag agagtcagaa gctgagtatg gtacacaaga cacttggggg 4680

gggggggaga ttgagagaaa gcgctaagga gataactcag tagttaaaac acttgccact 4740

cagaagtgag gaccagaatt tggatcttca gaaacataaa tattgagtgg accttctggc 4800

ctgactgtaa aagcaggcag agtcctcagc aagatccacc ttggtgagta aggttgaaga 4860

gtcagagagg acgattcctg acttcgagtg tcatgcatac cccactctgg aaaaacaaag 4920

tgaaaaaagc catttaaaaa ggcacaaaag tataaagcac tggagcataa ctaaatgtta 4980

ttatattggt ttatttttag tatatatatt tttttggtgt ggctggggct atagctctgt 5040

aggtcaaaca tacatggagc ccttgattcc atctcccagg gctggtttac aggatgctga 5100

gtatg 5105

Claims

1., for treating a gene recombinaton hematopoietic stem cell for tumor, autoimmune disease, chronic infection etc., its feature exists In, the genome of patient's hematopoietic stem cell inserts one section of sequence, this sequence includes that a fragment gene X, this fragment gene X can encode One section of peptide chain Y, this peptide chain Y include outside the born of the same parents of the epitope of energy specific recognition tumor cell or autoimmune response cell Territory, wear film territory and the intracellular territory of intracellular signaling pathways can be activated；This sequence also includes to regulate above-mentioned peptide chain Y table by specificity The promoter reached and enhancer.

Gene recombinaton hematopoietic stem cell the most according to claim 1, it is characterised in that at the gene of patient's hematopoietic stem cell Inserting one section of sequence in group, this sequence includes Car gene or tcr gene, and the promoter of CD3+CD8+CD4-T cell-specific and Enhancer.

Gene recombinaton hematopoietic stem cell the most according to claim 2, it is characterised in that described promoter is CD8b1 upstream Promoter part, enhancer is SV40 or TCR-alpha enhancer；Or, promoter is CD8a promoter, and enhancer is TG-g Enhancer.

Gene recombinaton hematopoietic stem cell the most according to claim 1, it is characterised in that described gene recombinaton hematopoietic stem cell Other genomic modifications can be added, as knocked out/process LAN portion gene, make HSCs be more likely to lymphatic system differentiation direction, such as Knock out GATA1 and PU.1 simultaneously and can block myeloid differentiation.

Gene recombinaton hematopoietic stem cell the most according to claim 1, it is characterised in that described gene recombinaton hematopoietic stem cell Other genomic modifications can be added, as knocked out/process LAN portion gene, make HSCs be more likely to T cell differentiation direction；Such as strike Except Pax5 can block B cell differentiation.

Gene recombinaton hematopoietic stem cell the most according to claim 1, it is characterised in that described gene recombinaton hematopoietic stem cell Other genomic modifications can be added, as knocked out/process LAN portion gene, make HSCs be more likely to CD8+CD4-T cell differentiation side To；Such as knock out Th-POK to block CD4+CD8-T cell differentiation direction.

Gene recombinaton hematopoietic stem cell the most according to claim 1, it is characterised in that described gene recombinaton hematopoietic stem cell Can add and can induce Suicide systems, in order to when after the transfer untoward reaction occurring, use inducer to kill stem cell, as added ICasp-9 or other equivalent systems.

8. for treating a preparation method for the gene recombinaton hematopoietic stem cell of tumor, autoimmune disease, chronic infection etc., It is characterized in that, comprise the steps:

(1) establishing the transcription factor that CD8+T cell stage is special, this transcription factor exists only in the T cell of CD8+ terminal differentiation In；Establish the specific promoter corresponding with this transcription factor and enhancer, this transcription factor can activate this promoter and Enhancer, and in other stages of other directions of hematopoietic stem cell differentiation, this promoter and enhancer all can not be activated；

(2) this promoter and enhancer are inserted in the carrier of coding Car or TCR, and the upstream becoming Car or TCR is adjusted Control sequence, this Car or TCR, by only expressing in the T cell of CD8+ terminal differentiation, is in sinking in other stages and differentiation direction Silent；

(3) Car containing this promoter and enhancer is inserted in the genome of HSCs, make restructuring HSCs possess and express Car's Ability, the extracellular domain of this Car encoding proteins comprises scFv, can be combined with target cells mark, activates the CD3 of Car coding Intracellular territory and CD28/CD134, the intracellular territory such as 4-1BB, to provide costimulatory signal；

(4) in order to improve the efficiency that restructuring HSCs breaks up to CD8+T cell further, can knock out further or process LAN part base Cause, including 3 key nodes: determine medullary system/lymphatic system differentiation, T cell/B cell/differentiation of dendritic cells, and CD4+/CD8+ T cell is broken up, and makes restructuring HSCs optionally move towards lymphatic system-T cell-CD8+ differentiation direction.