CN107630006A - It is a kind of to prepare TCR and the method for the T cell of the dual-gene knockouts of HLA - Google Patents
It is a kind of to prepare TCR and the method for the T cell of the dual-gene knockouts of HLA Download PDFInfo
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Abstract
The invention discloses a kind of T cell method for preparing TCR and the dual-gene knockouts of HLA, by the CRISPR/Cas9 HLA and CRISPR/Cas9 TCR common transfecting T cells of plasmid, carry out TCR and the dual-gene knockouts of HLA, TCR and the dual-gene knockouts of HLA T cell are obtained, the invention can be used for heteroplastic transplantation without producing immunological rejection.
Description
Technical field
The present invention relates to field of biological genes, is a kind of T cell for preparing TCR and the dual-gene knockouts of HLA in particular
Method.
Background technology
Tumour is all the global major disease of puzzlement all the time, at least to there is over one hundred kind of tumour at present, is seriously endangered
Human health.At present, treatment means mainly include operative treatment, chemotherapy, radiotherapy, monoclonal antibody, traditional Chinese medicine etc., but
Its therapeutic effect is limited, and side effect is obvious.Tumour cell immunization therapy in recent years turns into most active, most promising one kind
Treatment means, immunization therapy include immunotherapy medicaments and cellular immunotherapy.At present, Chimeric antigen receptor cell CAR-T is controlled
Treat and bring hope to oncotherapy, its general principle is the T cell being separately cultured from tumor patient own bodies, utilizes base
Because technology studies it transformation, basic process is by the single-chain antibody (scFv) and T cell of identification tumor associated antigen (TAA)
Activation sequences carry out genetic recombination in vitro, form recombinant plasmid, in vitro by rotaring dyeing technology, expand T cell on a large scale.So
Patient's body is fed back to after the cell that these genetic engineerings are transformed is carried out into amplification in vitro afterwards, makes immunocyte that there is specificity to know
Other and killing tumour ability, so as to reach the effect for the treatment of tumour.However, CAR-T also has some to lack on treatment tumour
Point, it on the one hand can cause the side effects such as cytokine storm, on the other hand, siberian crabapple of the patient after by chemotherapy and radiation
System is destroyed, and its own T cell quantity, activity and multiplication capacity are extremely limited, and it is thin to limit autologous peripheral blood mononuclear
The use of born of the same parents, this just loses best therapeutic scheme for the patient of advanced cancer.So sight is turned to one by people
Kind can the Chimeric antigen receptor cell that feeds back of allosome, on the one hand the contributor of health possess strong immune system enough effectively killing be swollen
Oncocyte, on the other hand, its blood are easy to collect to isolate T cell, but allosome, which is fed back, has the strong anti-place of graft
Main reaction.
φt cell receptor (T cell receptor, TCR) is T cell surface specific identification antigen and mediated immunity response
Molecule, decide how the immune system of people adapts to the change of environment.TCR can be divided into TCR α/βs and TCR gamma/delta two types,
Periphery blood T cell is mainly the T cell of TCR α/βs, is the main cell for mediating body specific cell immunoreaction.
HLA (Human leukocyte antigen, HLA), encoding gene is the Main Tissues of the mankind
Histocompatibility complex (MHC), on No. 6 chromosomes (6p21.31), include a series of locus of close linkages, with the mankind
Function of immune system it is closely related.MHC which part gene code cell surface antigens, turning into everyone cell can not mix
" feature " confused, it is basis of the immune system differentiation with allosome material in itself.The HLA when carrying out transfer operation
Determine histocompatbility.The HLA of contributor and recipient are more similar, and rejection is with regard to smaller.It is only double with ovum
Born of the same parents' tire or the HLA of clone are duplicate.In addition many diseases and certain HLA phase
Association, therefore they are relevant for the possibility of certain disease.HLA includes I class II classes and Group III Gene Partial.HLA I
Antigen expressed by class and II genoids is located on cell membrane, is MHC-I (A, B, C site coding) and MHC-II (D areas coding),
I classes are almost distributed in body whole cell surface, are a heterodimers, are made up of heavy chain (α chains) with β2-microglobulin
(B2M), II classes are mainly positioned at macrophage and the glycoprotein on bone-marrow-derived lymphocyte surface.
φt cell receptor and human leucocyte antigen (HLA) are the main reason for causing allosome to feed back graft versus host disease(GVH disease), therefore, are sunk
The TCR albumen and HLA albumen of silent T cell, the allosome that T cell can be achieved are fed back.
The content of the invention
In order to make up above deficiency, TCR and the method for the T cell of the dual-gene knockouts of HLA are prepared the invention provides a kind of.
The solution of the present invention is:
A kind of T cell method for preparing TCR and the dual-gene knockouts of HLA, it is characterised in that comprise the following steps:
1) expressing gene the carrier pX330A and pX330S, under BbsI digestions effect, the skeleton for forming linearisation carries
The body pX330A and skeleton carrier pX330S of linearisation;
2) TRAC and TRBC2 and B2M sgRNA are located at the extron of gene, and the target sequence on gene be it is unique,
Positive oligonucleotides 5' add CACCG respectively on design TRAC and TRBC2 and B2M sgRNA, reverse oligonucleotide 3' with
5' adds C and AAAC respectively, by sgRNA double strand oligonucleotides, mixes and anneals 5 minutes and then be slowly cooled to after 100 DEG C
Room temperature, obtain TRAC-sgRNA and TRBC2-sgRNA and B2M-sgRNA;
3) skeleton of the TRAC-sgRNA obtained in step 2) and TRBC2-sgRNA respectively with linearisation in step 1) is carried
Body pX330A and the skeleton carrier pX330S of linearisation are attached, and obtain the recombinant vector containing TRAC-sgRNA respectively
The pX330A-TRAC and recombinant vector pX330S-TRBC2 containing TRBC2-sgRNA;Pass through BsaI digestions and T4DNA ligases
Recombinant vector pX330A-TRAC and pX330S-TRBC2 are built to a carrier, obtained containing TRAC and two genes of TRBC2
SgRNA recombinant vector, is named as CRISPR/Cas9-TCR;
4) B2M-sgRNA obtained in step 2) is connected with the skeleton carrier pX330S linearized in step 1)
Connect, obtain the recombinant vector pX330S-B2M containing B2M-sgRNA, life of laying equal stress on is CRISPR/Cas9-HLA;
5) using the fresh PMBC of density gradient separation, activated by CD3, CD28 monoclonal antibody, IL-2 cultures,
T cell is activated into, then by CRISPR/Cas9-TCR made from CRISPR/Cas9-HLA made from step 3) and step 4)
The common transfecting T cells of plasmid, TCR and the dual-gene knockouts of HLA are carried out, obtains TCR and the dual-gene knockouts of HLA T cell.
As preferable technical scheme, the method for the common transfecting T cells of step 5) plasmid:CRISPR/ will be contained
Cas9-TCR and CRISPR/Cas9-HLA plasmid is separately added into electric shock cup, cell is added into electric shock cup, top, which turns electric shock cup, to be made
Mixing after, will electric shock cup be put into electric shock tank, cell once, is then transferred in the hole containing culture medium by pulse electric shock,
Orifice plate is gently rocked, mixes cell, after electricity turns 24h-48h, gene transient expression.
As preferable technical scheme, the sgRNA sequences of TRAC described in target gene are selected from chr14 in the step 2):
22550620 3 exons, the sgRNA sequences of TRBC2 described in target gene are selected from chr7:142801040 2 extras show
Son, the sgRNA sequences of B2M described in target gene are selected from chr15:44711557 1 exon.
As preferable technical scheme, TCR and the T cell amplification and sequencing of the dual-gene knockouts of HLA is made in step 5), will
The hla antibody of TCR antibody and biotin labeling with the transfectional cell biotin labeling for knocking out effect after sequencing is through magnetic bead
Sub-elect TCR and the dual-gene knockouts of HLA T cell.
As preferable technical scheme, the T cell of the TCR and the dual-gene knockouts of HLA can be used to treat inhomogeneity
The CAR of type tumour passes through slow-virus infection.
As preferable technical scheme, it can be that the high leukaemia for expressing CD19, CD20, BCMA is therein that the tumour, which is,
It is a kind of, or expression mesothelin, GD2, TNFR2, ROR1, EGFR, CEAmAb, Mucin1, HER1, VEGF solid tumor its
In one kind.
The present invention compared with prior art, has the following advantages that:
It is 1. of the invention by TCR α chains constant region domains corresponding encoded gene TRAC and and β chains constant region domains corresponding encoded gene
The extron of B2M genes knock out simultaneously in TRBC2 and HLA I genoids.One kind is based on CRISPR/Cas9 system structures
TCR and the dual-gene knockouts of HLA universal T cell are built, target gene TRAC sgRNA sequences are selected from chr14:22550620
3 exons, target gene TRBC2 sgRNA sequences are selected from chr7:142801040 2 exons, target gene B2M's
SgRNA sequences are selected from chr15:44711557 1 exon.
2. the knockout that present invention detection display sgRNA corresponds to gene is respectively provided with zero off-target effects, can be efficient to base
Because improving safety.
3. use CRISPR/Cas9 gene editing technologies.Cas9 albumen contains two nuclease domains, can cut respectively
It is single-stranded to cut DNA two.Cas9 is combined into compound with sgRNA first, by PAM recognition sequences and combines, and then to target DNA
Double-strand is cut, and the purpose for knocking out gene expression is finally reached using the non-homogeneous recombinantal repair of cell itself.
4. the T cell of xenogenic origin is transformed by genetic engineering, this T cell is set to can be used for heteroplastic transplantation without producing
Raw immunological rejection.In addition, engineered T cell combination third generation CAR-T technologies be prepared into it is a kind of can be with the general of heteroplastic transplantation
The Chimeric antigen receptor cell of type, enables CAR-T cells to carry out killing tumor cell applied to Different Individual simultaneously, in order to swollen
Knurl is treated.
5. the combination CAR-T of the universal T cell of present invention therapy target, can be high expression CD19, CD20, BCMA
Leukaemia, or expression mesothelin, GD2, TNFR2, ROR1, EGFR, CEAmAb, Mucin1, HER1, VEGF etc. reality
Body knurl.
Brief description of the drawings
Fig. 1 is pX330A Vector maps;
Fig. 2 is pX330S Vector maps;
Fig. 3 is that plasmid order-checking verifies that the sgRNA of TRA and TRB and B2M genes is successively inserted into;
Fig. 4 is that T7E1 digestion identification of cell group knocks out efficiency chart;
Fig. 5 is PCR sequence verifications TRA and TRB and B2M gene successful knockouts;
Fig. 6 is TCR and the T cell of the dual-gene knockouts of HLA kills knurl experimental result in vitro.
Embodiment
Case study on implementation 1CRISPR/Cas9-TCR and CRISPR/Cas9-HLA vector constructions
1. recombinant expression carrier
The carrier used in the present invention is pX330A and pX330S (collection of illustrative plates such as Fig. 1, shown in 2), and pX330A expressing genes carry
Body contains original copy, the promoter sequence (CMV) of a cytomegalovirus, and restriction endonuclease sites (Bbs
I, Bsa I etc.), resistance screening gene (anti-ampicillin);PX330S is similar with pX330A structures, and its difference is mainly enzyme
Enzyme site is different and resistance screening gene is different (spectinomycin).By artificial synthesized sgRNA genes, make in T4 ligases
Under, it is connected with the skeleton carrier DNA of linearisation, forms recombinant vector.
2.TRAC-sgRNA and TRBC2-sgRNA and B2M-sgRNA synthesis
TRAC and TRBC2 and B2M sgRNA is located at the extron (SEQ ID NO.1~3) of gene, and on gene
Target sequence is unique, and CACCG is added in the sgRNA forward direction oligonucleotides 5' of design, is distinguished in reverse oligonucleotide 3' and 5'
Plus C and AAAC, by sgRNA double strand oligonucleotides, mix and annealed 5 minutes after 100 DEG C and then be slowly cooled to room temperature, obtain
To TRAC-sgRNA and TRBC2-sgRNA and B2M-sgRNA.
3.CRISPR/Cas9-TCR vector constructions:
The skeleton carrier pX330A and pX330S of TRAC-sgRNA and TRBC2-sgRNA respectively with linearisation are connected
Connect, obtain the recombinant vector pX330A-TRAC containing TRAC-sgRNA and the recombinant vector containing TRBC2-sgRNA respectively
pX330S-TRBC2.Recombinant vector pX330A-TRAC and pX330S-TRBC2 is built by BsaI digestions and T4DNA ligases
To a carrier, the recombinant vector containing TRAC Yu two gene sgRNA of TRBC2 is obtained, is named as CRISPR/Cas9-TCR.
4.CRISPR/Cas9-HLA vector constructions:
B2M-sgRNA is attached with the skeleton carrier pX330S linearized, the restructuring containing B2M-sgRNA is obtained and carries
Body pX330S-B2M, life of laying equal stress on is CRISPR/Cas9-HLA.
Case study on implementation 2TCR and the acquisition of the T cell of the dual-gene knockouts of HLA
Using the PMBC that density gradient separation is fresh, activated by CD3, CD28 monoclonal antibody, IL-2 (final concentrations
300IU/ml) cultivate, activate into T cell, then by recombinant vector CRISPR/Cas9-HLA made from case study on implementation 1 with
The common transfecting T cells of CRISPR/Cas9-TCR plasmids, carry out TCR and the dual-gene knockouts of HLA.Transfected and be referred to as:
Electric revolving cup will be separately added into containing plasmid (CRISPR/Cas9-TCR, CRISPR/Cas9-HLA), electricity turns every time
Plasmid is 200ng, and cell (1 × 10 is added into electric shock cup7), after top turn electric shock cup is allowed to mixing, electric shock cup is put into electric shock
In groove, cell once (2.0KV, 25uFD), is then transferred in the hole of the culture medium containing 0.5mlDMEM, gently by pulse electric shock
Orifice plate is rocked, mixes cell, after electricity turns 24h-48h, gene transient expression.
Further, after plasmid transfection is completed, continue to cultivate T cell and further expand.Transfection is collected after 72h respectively
PX330A, pX330S, CRISPR/Cas9-TCR, CRISPR/Cas9-HLA T cell, utilize kit extraction genome
DNA, then detected using T7E1 enzymes and knock out efficiency, and PCR primer is further verified into TCR and HLA bases by TA cloning and sequencings
Because whether being knocked, as illustrated in figures 4-5.
Further, TCR antibody and biotin mark with the transfectional cell biotin labeling for knocking out effect will be detected
The hla antibody of note goes out TCR and the dual-gene knockouts of HLA T cell through magnetic bead sorting.
Case study on implementation 3TCR treats tumour with the T cell combination CAR of the dual-gene knockouts of HLA
It will be struck for the CAR for treating different type tumour by the way that the above-mentioned TCR the being separated to and HLA of slow-virus infection is dual-gene
The T cell removed, its detailed process are:Leader-scFv-CD8-CD137-CD3 ζ-T2A-HSV-TK nucleotides synthesizes, and will synthesize
Leader-scFv-CD8-CD137-CD3 ζ-T2A-HSV-TK genetic fragment be inserted into pLent-C-GFP carriers NotI-
AsiSI sites, after being sequenced correctly, plasmid is built into, by the plasmid transfection 293T cells, is packaged into and carries encoding gene
Slow virus, by the slow virus 1mL for carrying Leader-scFv-CD8-CD137-CD3 ζ-T2A-HSV-TK encoding genes
Add culture TCR and the T cell (1 × 10 of the dual-gene knockouts of HLA7) culture dish in, be well mixed, renew after 24 hours fresh
Nutrient solution.
Further, after infection is completed, the T cell Large scale in vitro of the CAR dual-gene knockout of TCR and HLA will be expressed
Amplification, is collected to cell, after finally cleaning three times with phosphate buffer, is fed back for allosome, for different tumours
Treated.
Case study on implementation 4TCR and the T cell of the dual-gene knockouts of HLA kill knurl experimental result in vitro
This experiment is using LDH method for releasing measure cell killing activity, and with the T cell of autologous patient, as comparing.
Target cell and each 100ul of effector cell are taken, and different effect targets are set respectively than 5:1、10:1、20:1、40:1, add U-shaped 96 hole
In culture plate, target cell Spontaneous release hole and nutrient solution each 100ul, target cell maximum release aperture and 2.5%Triton are each
100ul, above-mentioned three are all provided with three parallel holes, and 4h is cultivated in 37 DEG C, 5%CO2 incubators.Then by 96 orifice plate porous plates
Centrifuge 1500rpm centrifuges 5min, and each hole takes supernatant 100ul respectively, adds in flat 96 orifice plate respective aperture, while each hole point
Jia Ru not lactic dehydrogenase (LDH) matrix liquid 100ul, reaction 10min, the addition 30ul HCl (1mol/L) per each hole, in enzyme mark
Absorbance (A) is determined at instrument 490nm.Cell killing activity is calculated according to below equation:Killing activity (%)=[(reacting hole A
Value-Spontaneous release hole A values)/(maximum release group A values-Spontaneous release hole A values)] × 100%.
Shown in testing result (such as Fig. 6), TCR and the tumor-killing energy of the T cell of the dual-gene knockouts of HLA prepared by the present invention
Power is significantly stronger than the T cell in autologous patient source, is that the T of the dual-gene knockouts of TCR of the present invention and HLA is thin when it is 40 to imitate target ratio
The tumor-killing ability of born of the same parents is of about 56%, and the tumor-killing ability of autologous patient T cell is only about 37%.
General principle, principal character and the advantages of the present invention of the present invention has been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the simply explanation described in above-described embodiment and specification is originally
The principle of invention, without departing from the spirit and scope of the present invention, various changes and modifications of the present invention are possible, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent circle.
Sequence table
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Claims (6)
- A kind of 1. T cell method for preparing TCR and the dual-gene knockouts of HLA, it is characterised in that comprise the following steps:1) expressing gene carrier pX330A and pX330S, under BbsI digestions effect, the skeleton carrier pX330A of linearisation is formed With the skeleton carrier pX330S of linearisation;2) TRAC and TRBC2 and B2M sgRNA is located at the extron of gene, and the target sequence on gene is unique, is being set Positive oligonucleotides 5' adds CACCG respectively on meter TRAC and TRBC2 and B2M sgRNA, divides in reverse oligonucleotide 3' and 5' Not Jia Shang C and AAAC, by sgRNA double strand oligonucleotides, mix and annealed 5 minutes after 100 DEG C and then be slowly cooled to room temperature, Obtain TRAC-sgRNA and TRBC2-sgRNA and B2M-sgRNA;3) by the TRAC-sgRNA obtained in step 2) and TRBC2-sgRNA respectively with step 1) in linearisation skeleton carrier PX330A and the skeleton carrier pX330S of linearisation are attached, and obtain the recombinant vector containing TRAC-sgRNA respectively The pX330A-TRAC and recombinant vector pX330S-TRBC2 containing TRBC2-sgRNA;Pass through BsaI digestions and T4DNA ligases Recombinant vector pX330A-TRAC and pX330S-TRBC2 are built to a carrier, obtained containing TRAC and two genes of TRBC2 SgRNA recombinant vector, is named as CRISPR/Cas9-TCR;4) B2M-sgRNA obtained in step 2) is attached with the skeleton carrier pX330S linearized in step 1), The recombinant vector pX330S-B2M containing B2M-sgRNA is obtained, life of laying equal stress on is CRISPR/Cas9-HLA;5) using the fresh PMBC of density gradient separation, activated by CD3, CD28 monoclonal antibody, IL-2 cultures, activation Into T cell, then by the plasmid of CRISPR/Cas9-TCR made from CRISPR/Cas9-HLA made from step 3) and step 4) Common transfecting T cells, TCR and the dual-gene knockouts of HLA are carried out, obtains TCR and the dual-gene knockouts of HLA T cell.
- 2. a kind of T cell method for preparing TCR and the dual-gene knockouts of HLA as claimed in claim 1, it is characterised in that described The method of the common transfecting T cells of step 5) plasmid:By the plasmid containing CRISPR/Cas9-TCR and CRISPR/Cas9-HLA point Jia Ru not shocked by electricity cup, and cell is added into electric shock cup, after top turn electric shock cup is allowed to mixing, electric shock cup is put into electric shock tank, arteries and veins Cell once, is then transferred in the hole containing culture medium by punching electric shock, gently rocks orifice plate, mixes cell, and electricity turns 24h-48h Afterwards, gene transient expression.
- A kind of 3. T cell method for preparing TCR and the dual-gene knockouts of HLA as claimed in claim 1, it is characterised in that:It is described The sgRNA sequences of TRAC described in target gene are selected from chr14 in step 2):22550620 3 exons, described in target gene TRBC2 sgRNA sequences are selected from chr7:142801040 2 exons, the sgRNA sequences of B2M described in target gene are selected from chr15:44711557 1 exon.
- A kind of 4. T cell method for preparing TCR and the dual-gene knockouts of HLA as claimed in claim 1, it is characterised in that:Will step It is rapid that TCR and the T cell amplification and sequencing of the dual-gene knockouts of HLA 5) is made, will there is the transfectional cell for knocking out effect to use after sequencing The T that the TCR antibody of biotin labeling and the hla antibody of biotin labeling go out the dual-gene knockouts of TCR and HLA through magnetic bead sorting is thin Born of the same parents.
- A kind of 5. T cell method for preparing TCR and the dual-gene knockouts of HLA as claimed in claim 1, it is characterised in that:It is described The CAR that the T cell of TCR and the dual-gene knockouts of HLA can be used to treat different type tumour passes through slow-virus infection.
- A kind of 6. T cell method for preparing TCR and the dual-gene knockouts of HLA as claimed in claim 5, it is characterised in that:It is described Leukaemia one kind therein that it can be high expression CD19, CD20, BCMA that tumour, which is, or expression mesothelin, GD2, TNFR2, ROR1, EGFR, CEAmAb, Mucin1, HER1, VEGF solid tumor one kind therein.
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