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 PDF

Info

Publication number
CN107630006A
CN107630006A CN201710917059.0A CN201710917059A CN107630006A CN 107630006 A CN107630006 A CN 107630006A CN 201710917059 A CN201710917059 A CN 201710917059A CN 107630006 A CN107630006 A CN 107630006A
Authority
CN
China
Prior art keywords
hla
tcr
cell
sgrna
dual
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201710917059.0A
Other languages
Chinese (zh)
Other versions
CN107630006B (en
Inventor
刘明录
刘敏
王立新
冯建海
张传鹏
强邦明
金海锋
万磊
韩庆梅
王亮
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Xingrui Yida Biotechnology Co.,Ltd.
Original Assignee
Jinan Xingyi Medical Technology Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jinan Xingyi Medical Technology Co Ltd filed Critical Jinan Xingyi Medical Technology Co Ltd
Priority to CN201710917059.0A priority Critical patent/CN107630006B/en
Publication of CN107630006A publication Critical patent/CN107630006A/en
Application granted granted Critical
Publication of CN107630006B publication Critical patent/CN107630006B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Landscapes

  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

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

It is a kind of to prepare TCR and the method for the T cell of the dual-gene knockouts of HLA
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
<110>Shandong Xing Rui bio tech ltd
<120>It is a kind of to prepare TCR and the method for the T cell of the dual-gene knockouts of HLA
<130> 2017
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213>Ethnic group (Homo sapiens)
<400> 1
caccgcgtca tgagcagatt aaacc 25
<210> 2
<211> 25
<212> DNA
<213>Ethnic group (Homo sapiens)
<400> 2
caccgtatct ggagtcattg agggc 25
<210> 3
<211> 25
<212> DNA
<213>Ethnic group (Homo sapiens)
<400> 3
caccgcgcga gcacagctaa ggcca 25

Claims (6)

  1. 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. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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.
CN201710917059.0A 2017-09-30 2017-09-30 Method for preparing T cell with double knockout genes of TCR and HLA Active CN107630006B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710917059.0A CN107630006B (en) 2017-09-30 2017-09-30 Method for preparing T cell with double knockout genes of TCR and HLA

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710917059.0A CN107630006B (en) 2017-09-30 2017-09-30 Method for preparing T cell with double knockout genes of TCR and HLA

Publications (2)

Publication Number Publication Date
CN107630006A true CN107630006A (en) 2018-01-26
CN107630006B CN107630006B (en) 2020-09-11

Family

ID=61103978

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710917059.0A Active CN107630006B (en) 2017-09-30 2017-09-30 Method for preparing T cell with double knockout genes of TCR and HLA

Country Status (1)

Country Link
CN (1) CN107630006B (en)

Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108913693A (en) * 2018-07-23 2018-11-30 安徽古生物科技有限公司 Strike the siRNA for subtracting people's TRAC/TRBC gene, slow virus carrier, CAR-T cell construction method and application
CN109295097A (en) * 2018-09-30 2019-02-01 北京鼎成肽源生物技术有限公司 A kind of MRFFT2 cell
CN109706121A (en) * 2019-01-25 2019-05-03 苏州茂行生物科技有限公司 A kind of universal CAR-T cell and its preparation method and application based on base editor
CN109722437A (en) * 2018-12-29 2019-05-07 广州百暨基因科技有限公司 A kind of universal CAR-T cell and its preparation method and application
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
CN110616233A (en) * 2018-06-20 2019-12-27 西安桑尼赛尔生物医药有限公司 Method for efficiently knocking out primary T cell gene by CRISPR-Cas9 and application thereof
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
WO2020057666A1 (en) * 2018-09-21 2020-03-26 科济生物医药（上海）有限公司 T-cell expressing chimeric receptor
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
CN112105724A (en) * 2018-08-09 2020-12-18 深圳华大生命科学研究院 Construction method of antigen presenting cell line without endogenous HLA gene background, antigen presenting cell line and application thereof
CN112430575A (en) * 2019-08-26 2021-03-02 深圳市菲鹏生物治疗股份有限公司 Universal CAR-T cell, preparation method and application thereof, and antitumor drug
US10947530B2 (en) 2016-08-03 2021-03-16 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
CN112752767A (en) * 2018-04-27 2021-05-04 克里斯珀医疗股份公司 Methods and compositions for cytotoxic T cell depletion
CN112899273A (en) * 2019-12-03 2021-06-04 甘李药业股份有限公司 Compositions and methods for altering gene sequences
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
WO2021136263A1 (en) * 2020-01-02 2021-07-08 江苏茂行科技有限公司 Modified immune effector cell and preparation method therefor
CN113430202A (en) * 2021-08-26 2021-09-24 山东兴瑞生物科技有限公司 Human PD1 gene sgRNA with high knockout rate, plasmid containing sgRNA and T cell
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
WO2023274387A1 (en) * 2021-07-01 2023-01-05 宁波茂行生物医药科技有限公司 Universal car-t cell targeting gd2, preparation method therefor, and application thereof
WO2023274386A1 (en) * 2021-07-01 2023-01-05 宁波茂行生物医药科技有限公司 Universal car-t cell targeting egfr and preparation method therefor
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105647871A (en) * 2016-01-27 2016-06-08 苏州佰通生物科技有限公司 Chimeric antigen receptor T cell capable of conducting allograft and preparation method
CN106459918A (en) * 2014-04-24 2017-02-22 得克萨斯州大学***董事会 Application of induced pluripotent stem cells to generate adoptive cell therapy products
CN107206024A (en) * 2014-10-31 2017-09-26 宾夕法尼亚大学董事会 Gene expression in change CART cells and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106459918A (en) * 2014-04-24 2017-02-22 得克萨斯州大学***董事会 Application of induced pluripotent stem cells to generate adoptive cell therapy products
CN107206024A (en) * 2014-10-31 2017-09-26 宾夕法尼亚大学董事会 Gene expression in change CART cells and application thereof
CN105647871A (en) * 2016-01-27 2016-06-08 苏州佰通生物科技有限公司 Chimeric antigen receptor T cell capable of conducting allograft and preparation method

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
JIANGTAO REN等: "A versatile system for rapid multiplex genome-edited CAR T cell generation", 《ONCOTARGET》 *
刘静: "《分子生物学实验指导》", 31 October 2015, 中南大学出版社 *
徐畅等: "基于CRISPR-Cas9定向编辑TRAC基因的研究", 《广东药科大学学报》 *
梅钧: "《医学免疫学》", 31 March 2014, 中国医药科技出版社 *
王进: "《病原生物学与免疫学》", 28 February 2007, 郑州大学出版社 *
邵红伟等: "CRISPR-Cas9***定向编辑TCR基因的sgRNA筛选", 《集美大学学报》 *

Cited By (59)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12006520B2 (en) 2011-07-22 2024-06-11 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US10954548B2 (en) 2013-08-09 2021-03-23 President And Fellows Of Harvard College Nuclease profiling system
US11920181B2 (en) 2013-08-09 2024-03-05 President And Fellows Of Harvard College Nuclease profiling system
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US11299755B2 (en) 2013-09-06 2022-04-12 President And Fellows Of Harvard College Switchable CAS9 nucleases and uses thereof
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US10912833B2 (en) 2013-09-06 2021-02-09 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US11053481B2 (en) 2013-12-12 2021-07-06 President And Fellows Of Harvard College Fusions of Cas9 domains and nucleic acid-editing domains
US11124782B2 (en) 2013-12-12 2021-09-21 President And Fellows Of Harvard College Cas variants for gene editing
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US11578343B2 (en) 2014-07-30 2023-02-14 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US12043852B2 (en) 2015-10-23 2024-07-23 President And Fellows Of Harvard College Evolved Cas9 proteins for gene editing
US10947530B2 (en) 2016-08-03 2021-03-16 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11999947B2 (en) 2016-08-03 2024-06-04 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11702651B2 (en) 2016-08-03 2023-07-18 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US11820969B2 (en) 2016-12-23 2023-11-21 President And Fellows Of Harvard College Editing of CCR2 receptor gene to protect against HIV infection
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11932884B2 (en) 2017-08-30 2024-03-19 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
CN112752767A (en) * 2018-04-27 2021-05-04 克里斯珀医疗股份公司 Methods and compositions for cytotoxic T cell depletion
CN110616233A (en) * 2018-06-20 2019-12-27 西安桑尼赛尔生物医药有限公司 Method for efficiently knocking out primary T cell gene by CRISPR-Cas9 and application thereof
CN110616233B (en) * 2018-06-20 2022-02-22 西安桑尼赛尔生物医药有限公司 Method for efficiently knocking out primary T cell gene by CRISPR-Cas9 and application thereof
CN108913693A (en) * 2018-07-23 2018-11-30 安徽古生物科技有限公司 Strike the siRNA for subtracting people's TRAC/TRBC gene, slow virus carrier, CAR-T cell construction method and application
CN108913693B (en) * 2018-07-23 2021-02-02 合肥一兮生物科技有限公司 siRNA for knocking down human TRAC/TRBC gene, lentiviral vector, CAR-T cell construction method and application
CN112105724A (en) * 2018-08-09 2020-12-18 深圳华大生命科学研究院 Construction method of antigen presenting cell line without endogenous HLA gene background, antigen presenting cell line and application thereof
WO2020057666A1 (en) * 2018-09-21 2020-03-26 科济生物医药（上海）有限公司 T-cell expressing chimeric receptor
CN109295097A (en) * 2018-09-30 2019-02-01 北京鼎成肽源生物技术有限公司 A kind of MRFFT2 cell
CN109722437B (en) * 2018-12-29 2020-01-07 广州百暨基因科技有限公司 Universal CAR-T cell and preparation method and application thereof
CN109722437A (en) * 2018-12-29 2019-05-07 广州百暨基因科技有限公司 A kind of universal CAR-T cell and its preparation method and application
CN109706121A (en) * 2019-01-25 2019-05-03 苏州茂行生物科技有限公司 A kind of universal CAR-T cell and its preparation method and application based on base editor
US11643652B2 (en) 2019-03-19 2023-05-09 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11795452B2 (en) 2019-03-19 2023-10-24 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
CN112430575B (en) * 2019-08-26 2024-01-19 深圳市菲鹏生物治疗股份有限公司 Universal CAR-T cell, preparation method and application thereof, and anti-tumor drug
CN112430575A (en) * 2019-08-26 2021-03-02 深圳市菲鹏生物治疗股份有限公司 Universal CAR-T cell, preparation method and application thereof, and antitumor drug
CN112899273A (en) * 2019-12-03 2021-06-04 甘李药业股份有限公司 Compositions and methods for altering gene sequences
CN114929878A (en) * 2019-12-03 2022-08-19 甘李药业股份有限公司 Compositions and methods for altering gene sequences
WO2021110099A1 (en) * 2019-12-03 2021-06-10 甘李药业股份有限公司 Composition and method for altering gene sequence
WO2021136263A1 (en) * 2020-01-02 2021-07-08 江苏茂行科技有限公司 Modified immune effector cell and preparation method therefor
US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
US12031126B2 (en) 2020-05-08 2024-07-09 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
WO2023274387A1 (en) * 2021-07-01 2023-01-05 宁波茂行生物医药科技有限公司 Universal car-t cell targeting gd2, preparation method therefor, and application thereof
WO2023274386A1 (en) * 2021-07-01 2023-01-05 宁波茂行生物医药科技有限公司 Universal car-t cell targeting egfr and preparation method therefor
CN113430202A (en) * 2021-08-26 2021-09-24 山东兴瑞生物科技有限公司 Human PD1 gene sgRNA with high knockout rate, plasmid containing sgRNA and T cell

Also Published As

Publication number Publication date
CN107630006B (en) 2020-09-11

Similar Documents

Publication Publication Date Title
CN107630006A (en) It is a kind of to prepare TCR and the method for the T cell of the dual-gene knockouts of HLA
US20230312675A1 (en) Chimeric antigen receptors (car) and methods for making and using the same
CN106191062B (en) A kind of TCR-/PD-1- double negative t cells and its construction method
AU2014339926B2 (en) Polyclonal gamma delta T cells for immunotherapy
Maus et al. Adoptive immunotherapy for cancer or viruses
CN106544321A (en) Universal CAR T cells and its production and use
CN103502438A (en) Method and compositions for cellular immunotherapy
WO2019052577A1 (en) Gene editing t cell and use thereof
CN107746831A (en) Universal CART/TCRT cells and its construction method with chemotherapeutic drug resistance
CN108409840A (en) The Chimeric antigen receptor of anti-CD123 single-chain antibodies and combinations thereof and application
CN106591363A (en) Preparation method of universal heterologous CAR-T cells and application
CN109797171A (en) Modified T cell, preparation method and the usage
CN107828730A (en) Universal CART/TCRT cells and its construction method with antibody drug resistance
CN109963944A (en) It is external to knock out crRNA used in the method and this method of target gene in T cell
CN109055380A (en) A kind of preparation method of universal CAR-T cell
CN109652378A (en) A kind of universal CAR-T cell and its preparation method and application of function enhancing
CN109055430A (en) A kind of preparation method for co-expressing IL18 and CCL19 albumen and targeting MUC1 gene C AR-T cell
CN109021114A (en) Combine the bispecific chimeric antigen receptor and expression vector of two kinds of single-chain antibodies
WO2023123195A1 (en) Engineered immune cell target gene of which can be regulated, preparation method therefor, and use thereof
JP7043114B2 (en) Methods and kits for activating cells
CN106957822A (en) Cultural method, kit and the application of amplification in vitro gene editing activating T cell
Kath et al. CAR NK-92 cell–mediated depletion of residual TCR+ cells for ultrapure allogeneic TCR-deleted CAR T-cell products
EP4086341A1 (en) Method for purifying ucart cell and use thereof
CN109294998B (en) RFF1 cell
CN109355309B (en) Construction method of CD3E gene modified humanized animal model

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
TR01 Transfer of patent right

Effective date of registration: 20230410

Address after: No.1 building and No.2 building, No.333, Haiyang 1st Road, Lingang New District, China (Shanghai) pilot Free Trade Zone, Pudong New Area, Shanghai, 201208

Patentee after: Shanghai Xingrui Yida Biotechnology Co.,Ltd.

Address before: 261500 high tech Industrial Development Zone, Gaomi City, Weifang City, Shandong Province

Patentee before: SHANDONG XINRUI BIOTECHNOLOGY CO.,LTD.

TR01 Transfer of patent right