CN108866004A - The T cell and its construction method that SHP-1 is knocked out - Google Patents
The T cell and its construction method that SHP-1 is knocked out Download PDFInfo
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Abstract
The invention discloses the SHP-1 T cell knocked out and its construction methods.Inventors have found that can effectively improve T cell by blocking the SHP-1 endogenous in T cell to the lethal effect of tumour, new target spot provided for tumour immunotherapy.
Description
Technical field
The present invention relates to tumor immunology, molecular biology, cell biology and gene editing technical fields, more particularly to
A method of T cell is knocked out using CRISPR/Cas9 system preparation SHP-1.
Background technique
Immunologic test point blocking treatment shows surprising curative effect in the treatment of tumour.Clinical test discovery, is immunized inspection
The effector function for exhausting T cell can be reactivated by making an inventory of blocking treatment, improve the anti-tumor activity of T cell.Immunologic test point
Blocking treatment mainly improves T cell using antibody come interaction between blocking immunity checkpoint molecule and its corresponding antibodies
Anti-tumor capacity.The most common immunologic test point molecule is PD-1 and CTLA-4.Immunologic test point blocking treatment is more
Remarkable effect is obtained in the treatment of kind tumour, including melanoma, lung cancer in non-cellule type, Hodgkin lymphoma, neck
Cancer, oophoroma, kidney, the tumour [1] of bladder cancer and mis-match repair deficient.Target the antibody ipilimumab of CTLA-4
In the granted treatment for advanced melanoma patient, wherein 20% received the patient of Antybody therapy, life cycle 3 years with
On.Recently, the antibody atezolizumab of the antibody nivolumab and pembrolizumab and PD-L1 of PD-1 are also obtained
FDA ratifies, in the treatment for advanced melanoma, lung cancer in non-cellule type and kidney.Some patientss are receiving immune inspection
After making an inventory of blocking treatment, show part and alleviate either complete incidence graph, however, not all patient to CTLA-4 and
The treatment of PD-1 inhibitor has reaction.
The presence of phosphatase has important adjustment effect to the Function of T cell in T cell.T cell passes through TCR-
After MHC compound identifies antigen, a large amount of signaling molecules intracellular can be activated by tyrosine phosphorylations such as Lck, ZAP70, participate in letter
Number transmitting.However the excessive activation in order to maintain stable state, prevent T cell, largely inhibit acid phosphatase that can also be swashed in T cell
It is living, it causes the dephosphorylation of signaling molecule and inactivates, affect the transmitting of t cell activation signal, finally weaken killing for T cell
Hurt function [2].There are more than 100 kinds of PTP phosphatases in cell, at least 45 kinds is contained human T cells, and more and more
Experimental evidence show these phosphatases often and have the function of independence and can not be substituted.Therefore, although theoretically suitably inhibiting T
The activity of phosphatase is possible to that the ability that T cell removes tumour cell can be improved in cell, but likewise, improper inhibition T is thin
The activity of phosphatase in born of the same parents, the activity of especially crucial phosphatase, may result in unforeseeable negative consequence, to siberian crabapple
The influence of system is unknown, or even causes the generation of autoimmune disease.Selecting any target spot as us therein is also
One problem [2] recognized altogether.In addition, since PTP phosphatase is intracellular protein, it can not be by resisting as PD-1, CTLA-4
Body blocking agent realizes the blocking to its function；The micromolecular inhibitor of phosphatase also tends to lack to the special of enzyme or cell
Property, cause clinically toxic side effect it is very big.These reasons also both increase the difficulty that people select and operate.
T cell is the primary cell of terminal differentiation, is difficult the genetic manipulations such as to be transfected or infected, along with it is proliferated energy
Power is limited, and which limits the knockouts for carrying out genetic manipulation, especially gene to T cell in vitro.In recent years, gene editing skill
The revolutionary development of art provides opportunity for T cell immunization therapy.Gene editing, which is carried out, using CRISPR/Cas9 technology operates letter
Single, high-efficient, high specificity, this to carry out gene editing gradually by that may come true to T cell in vitro.
Bibliography：
1.Topalian SL,Drake CG,Pardoll DM.(2015)Immune checkpoint blockade:a
common denominator approach to cancer therapy.Cancer Cell.27(4):450-461
2.Mustelin T,Vang T,Bottini N.(2005)Protein tyrosine phosphatases and
the immune response.Nat Rev Immunol.5(1):43-57.。
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of SHP-1 T cell knocked out and its building sides
Method.
It is another object of the present invention to provide CAR-T cells and its construction method that a kind of SHP-1 is knocked out.
The technical solution adopted by the present invention is that：
The T cell of Efficient killing effect tumour cell, the SHP-1 gene of T cell is by silent endogenous.
As the further improvement of above-mentioned T cell, the SHP-1 gene or its controlling gene of T cell are at least partly knocked out.
As the further improvement of above-mentioned T cell, the 3rd exon of the SHP-1 gene of T cell is knocked.
As the further improvement of above-mentioned T cell, T cell is CAR-T cell.
As the further improvement of above-mentioned T cell, CAR-T cell includes CD133 CAR T, CD19 CAR T, CD20
CAR T, BMSA CAR T, MSLN CAR T, EGFRVIII CAR T, Her2 CAR T, GD2 CAR T, CEA CAR T.
The construction method of above-mentioned T cell, Plasmid DNA from expression Cas9 and sgRNA to T cell, Cas9 albumen including importing
Or sgRNA-Cas9 protein complexes, knock out SHP-1 at least partly sequence or its at least partly expression regulation sequence makes SHP-1
Gene silent endogenous.
The further improvement of construction method as above-mentioned T cell, sgRNA sequence target aobvious outside the 3rd of SHP-1 gene
Son.
The further improvement of construction method as above-mentioned T cell, CAR-T cell include CD19 CAR T, CD20 CAR
T, BMSA CAR T, MSLN CAR T, EGFRVIII CAR T, Her2 CAR T, GD2 CAR T, CEA CAR T.
The beneficial effects of the invention are as follows：
The present invention breaks through the limitation of the prior art, creatively blocks the SHP-1 endogenous in T cell, effectively improves
Lethal effect of the T cell to tumour.Particularly, by CAR-T cell, such as CD133 CAR-T, CD19 CAR T, CD20 CAR
In T, BMSA CAR T, MSLN CAR T, EGFRVIII CAR T, Her2 CAR T, GD2 CAR T, CEA CAR T cell
SHP-1 is knocked out, and can effectively improve the ability of CAR-T cell killing tumour cell, new target is provided for tumour immunotherapy
Point.
By turning by Plasmid DNA electricity, the plasmid electricity for expressing Cas9 and sgRNA is gone in T cell, is realized efficient
The knockout of SHP-1 gene carries out electricity by Cas9 albumen or Cas9 mRNA and the sgRNA of in-vitro transcription with what is reported before
Phase inversion ratio has saved time and the cost of preparation.
Detailed description of the invention
Fig. 1 is the structure for the three generations CAR that the present invention uses, including CSF2RA Chimerical receptor signal peptide, after birth exoantigen knot
Close area (scFv), c-Myc label peptide fragment, CD8 hinge area, intracellular signal transduction area；
Fig. 2 is SHP-1 in T cell after turning T cell by the plasmid electricity of expression Cas9 and sgRNA in the embodiment of the present invention 1
The knockout situation of gene；
Fig. 3 is that by plasmid electricity turn, realization is overexpressed CD133 CAR and knocks out SHP-1 gene in T cell in the present invention
As a result, having detected the knockout situation of the expression of CAR gene and SHP-1 gene in T cell respectively；
Fig. 4 is that the efficiency of different sgRNA compares；
The CD133 CAR-T cell killing tumour cell ability that Fig. 5 knocks out for SHP-1 obtained in the embodiment of the present invention 3
Detection and cytokine secretion profile detection；
Fig. 6 is the safety experiment result for the cell in vitro SHP-1 gene knockout that CRISPR/Cas9 is mediated.
Specific embodiment
The T cell of Efficient killing effect tumour cell, the SHP-1 gene of T cell is by silent endogenous.
Gene silencing refers to the expression quantity decline of gene or does not express substantially.Silent endogenous refers to the expression quantity of gene itself
Decline is not expressed substantially.
As the further improvement of above-mentioned T cell, the SHP-1 gene or its controlling gene of T cell are at least partly knocked out.
So that SHP-1 can not also be unable to get active SHP-1 after normal expression, or expression.
As the further improvement of above-mentioned T cell, the 3rd exon of the SHP-1 gene of T cell is knocked.Test number
It according to showing to be knocked out for this exon, has higher efficiency, targeting is more preferable, and correspondingly safety is also more preferably.
CAR-T cell has specific killing power to specific cell.As the further improvement of above-mentioned T cell, T cell is
CAR-T cell.
As the further improvement of above-mentioned T cell, CAR-T cell includes but is not limited to CD133 CAR T, CD19 CAR
T, CD20 CAR T, BMSA CAR T, MSLN CAR T, EGFRVIII CAR T, Her2 CAR T, GD2 CAR T, CEA
CAR T etc..
The construction method of above-mentioned T cell, Plasmid DNA from expression Cas9 and sgRNA to T cell, Cas9 albumen including importing
Or sgRNA-Cas9 protein complexes, knock out SHP-1 at least partly sequence or its at least partly expression regulation sequence makes SHP-1
Gene silent endogenous.
The further improvement of construction method as above-mentioned T cell, sgRNA sequence target aobvious outside the 3rd of SHP-1 gene
Son.
The further improvement of construction method as above-mentioned T cell, CAR-T cell include CD19 CAR T, CD20 CAR
T, BMSA CAR T, MSLN CAR T, EGFRVIII CAR T, Her2 CAR T, GD2 CAR T, CEA CAR T.
Below with reference to embodiment and experiment, further exemplary illustration technical solution of the present invention.Other CAR T cells
It knocks out and is referred to following method progress.
Embodiment 1
Turn the knockout that SHP-1 gene is realized in T cell by Cas9 albumen and sgRNA electricity
1) experiment PBMC needs first to activate via human anti-CD3/CD28Dynabeads, and culture is containing 10%
In the AIM-V culture medium of FBS and 300U/ml IL-2；
2) Dynabeads is removed two days later and carries out electricity turns：
3) sgRNA for taking 4 μ g to be transcribed in vitro and 16 μ g Cas9 albumen mix, and are incubated at room temperature 20 minutes；
4) 82 μ l electricity are added into above-mentioned system to turn buffer (Lonza, V4XP-3024), 18 μ l supplement 1,
It mixes；
5) it after taking 1million PBMC cell, PBS to clean twice, is resuspended with above-mentioned system, and be transferred in electric revolving cup,
Turned using Lonza-4D electroporation EO-115 program electricity；
6) electricity turns to terminate that cell is transferred in the culture medium preheated in advance immediately to cultivate.
7) electricity turns counting in latter 10 days, and carries out T7EN1 digestion identification and the determining knockout efficiency of Sanger sequencing.
As a result as shown in Figure 1.Fig. 1 a shows position of the sequence of sgRNA targeting on SHP1 gene.T7EN1 digestion
It shows (Fig. 1 b), the cutting of DNA has occurred in sgRNA target site.Sanger sequencing result shows (Fig. 1 c), the base of SHP-1
Because mutation rate is 87.5% (7/8ths).
Embodiment 2：
Turn to realize in T cell by plasmid electricity and is overexpressed CD133CAR and knockout SHP-1 gene.
1) antigen for the CAR targeting that the present embodiment uses is CD133, and the structure of CAR is as shown in Fig. 2, successively include
CSF2RA Chimerical receptor signal peptide (SEQ ID NO：1), after birth exoantigen combined area (scFv, SEQ ID NO：
2), c-Myc label peptide fragment (SEQ ID NO：3), CD8 hinge area, intracellular signal transduction area (SEQ ID NO：
4) the resistant gene puromycin connected with T2A small peptide.Entire CAR gene is loaded in PiggyBac plasmid,
Constitute PiggyBac-CD133 CAR.The expression of CAR gene initial gene under the driving of EF1 α promoter.CAR gene integration
What is relied on into genome is the transposition of transposons；
2) by PiggyBac-CD133 CAR plasmid, transposase plasmid, Cas9 plasmid (Addgene plasmid#
44758) and knock out SHP-1 gene the sgRNA plasmid (sequence of sgRNA such as SEQ ID NO：Shown in 5) each 5 μ g and Amaxa electricity
Turn the electricity in kit and turn reagent mixing, and is resuspended 2 × 10 with this mixed liquor7A PBMC cell is simultaneously transferred in electric revolving cup, benefit
Turned with Lonza-2B electroporation U-014 program electricity；
3) electricity turns to terminate that cell is transferred in the culture medium preheated in advance immediately to cultivate；
4) after one day, activate PBMC using human anti-CD3/CD28 Dynabeads, culture containing 10%FBS and
In the AIM-V culture medium of 300U/ml IL-2, after cell-stimulating 5 days, removes Dynabeads and continue to cultivate, and with 0.5 μ g/ml
Puromycin medicine sieve, be enriched with the CAR positive T cell；
5) it when fortnight, determines by T7EN1 digestion identification and Sanger sequencing and knocks out efficiency；It is examined by flow cytometer showed
Survey the expression of CAR.
As a result as shown in Figure 3.The sgRNA sequence knocked out for SHP-1 is consistent with Fig. 1 a.T7EN1 digestion shows (Fig. 3 a),
The cutting of DNA has occurred in sgRNA target site.Sanger sequencing result shows (Fig. 3 b) that the gene mutation rate of SHP-1 is
87.5% (7/8ths).The expression of CAR is in knockout group and does not knock out in group in 95% or more (Fig. 3 c).
Opposite with above-mentioned sgRNA used, inventor has selected the sgRNA of some other site targetings to compare simultaneously
(Fig. 4 a and Fig. 4 b).When being detected using T7EN1 digestion and the cutting effect of more different sgRNA, it can be found that above-mentioned used
Sg1 effect it is best, and other the 2nd exons of targeting or are equally the sgRNA (sg2-4) of the 3rd exon, editor
Efficiency is lower than sg1 (editorial efficiency is directly proportional to the brightness of smaller band generated, Fig. 4 b).Experimental data shows this hair
The bright middle sequence after selecting, optimizing has better targeting and shear efficiency, has unexpected effect.
Embodiment 3：
Experiment in vitro proves the fragmentation effect for the CD133 CAR-T cells against tumor that SHP-1 is knocked out.
By killing experiments in vitro, cytokine secretion experiments have shown that SHP-1 is knocked out to CD133 CAR-T cell anti-tumor
The influence of ability.
The method that cell killing detection uses is Luciferase-Luciferin chemiluminescence detecting method, and killing target is thin
Luciferase is overexpressed in born of the same parents, after T cell killing, target cell population is reduced, and the luminous detected value of induced chemical reduces.Pass through ratio
Compared with the chemiluminescence detection value before killing with target cell after killing, the killing ability of T cell is estimated.The target cell that the present invention uses
For the U251 cell (U251-CD133-luc) for being overexpressed CD133 antigen and Luciferase, non-target cell is to be overexpressed
The U251 cell (U251-CD133) of luciferase.Concrete operations are as follows：
1) 2 × 10 are taken5A target cell or non-target cell are laid in 48 orifice plates；
2) according to effect target than 1:Isosorbide-5-Nitrae:1,16:1 ratio is separately added into the T cell (T cell) that electricity does not turn over, CAR T
The CAR T cell (SHP-1KO CD133 CAR) that cell (CD133CAR) and gene editing are crossed, wherein it is thin that T is not added in control group
Born of the same parents' culture, 300 hole μ l/ of total volume, every kind of effect target are put in 37 DEG C, 5%CO than three multiple holes of setting2Culture in cell incubator
16-18 hours；Microscope, which is taken pictures, detects T cell killing ability；
3) cell of culture is collected, after PBS cleaning is primary, with 100 μ l PBS resuspension.And cell is transferred to opaque
In 96 orifice plates, the D-luciferin that 100 μ l concentration are 150 μ g/ml is added, microplate reader detects Luminal 560nm luminous value；
4) the killing ability of T cell is calculated：Using the hole for containing only PBS as blank control, reads as N0, contain only tumour
Reading is set as N in the hole of cell, and experiment group number-reading is set as N1, then cell killing efficiency is 1- (N1-N0)/(N-N0) × 100%.
Cytokine release experiment is detected using AlphaLISA reagents series box：
1) by 2 × 105A target cell U251-CD133-luc is laid in 48 orifice plates, is 16 according to effect target ratio:1 ratio,
The CAR-T cell that a certain amount of CAR-T cell or SHP-1 are knocked out is added into every hole, every kind of T cell is divided into three multiple holes, altogether
300 μ l AIM-V culture solutions are added to cultivate in 37 DEG C of incubators；
2) after 16 hours, 100 μ l supernatants are drawn, 500g is centrifuged 5 minutes, is collected supernatant and is used for subsequent detection.It uses
The AlphaLISA reagents series box detection IFN-γ and TNF-α cytokine release situation of PerkinElmer company.
As a result as shown in Figure 5.SHP-1 knock out after significantly enhance CAR T cell killing tumor cell ability (Fig. 5 a,
5b), the ability (Fig. 5 c) of CAR T cell secrete cytokines TNF-α and IFN-γ is enhanced.
Embodiment 4：
In vitro cell experiment proves the safety for the SHP-1 gene knockout that CRISPR/Cas9 is mediated.
T cell electricity turns the form turned using plasmid electricity, is likely to result in the radom insertion of genome, Cas9 and sgRNA's
Insertion will cause the multiple cutting of genome simultaneously, improve undershooting-effect.Because Cas9 protein fusion has GFP fluorescin, lead to
Cross the integration for detecting whether that GFP signal can substantially judge Cas9.Turn to turn with electricity one day after by flow cytometer detection electricity
The insertion situation (Fig. 6 a) of 24 days Cas9 afterwards.The result shows that and Cas9 gene integration has been not detected into genome.
Another safety, that is, mop effect of CRISPR/Cas9 system progress gene editing.According to targeting SHP-1's
SgRNA sequence finds the highest site of missing the target of marking on the website www.crispr.mit.edu.It is final to choose preceding ten marking
Highest site, which is used as, potentially misses the target site (Fig. 6 b).Further potentially missed the target site by PCR and T7EN1 to first four
It is detected.T7EN1 restriction analysis shows (Fig. 6 c), does not occur undershooting-effect in corresponding site.Experimental data table
Bright system of the invention has good safety.
Other CAR T cells can also use similar method, and building obtains without creative efforts.
SEQUENCE LISTING
<110>Profound biotechnology（Guangzhou）Co., Ltd
<120>The T cell and its construction method that SHP-1 is knocked out
<130>
<140> CN2018106655486
<141> 2018-06-26
<160> 5
<170> PatentIn version 3.5
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Claims (8)
1. the T cell of Efficient killing effect tumour cell, it is characterised in that：The SHP-1 gene of T cell is by silent endogenous.
2. T cell according to claim 1, it is characterised in that：The SHP-1 gene or its controlling gene of T cell are by least
Part knocks out.
3. T cell according to claim 1, it is characterised in that：3rd exon of the SHP-1 gene of T cell is struck
It removes.
4. described in any item T cells according to claim 1~3, it is characterised in that：T cell is CAR-T cell.
5. T cell according to claim 4, it is characterised in that：CAR-T cell includes CD133 CAR T, CD19 CAR
T, CD20 CAR T, BMSA CAR T, MSLN CAR T, EGFRVIII CAR T, Her2 CAR T, GD2 CAR T, CEA
CAR T。
6. the construction method of T cell described in claim 1, including imported to T cell expression Cas9 and sgRNA Plasmid DNA,
Cas9 albumen or sgRNA-Cas9 protein complexes knock out at least partly sequence or its at least partly expression regulation sequence of SHP-1
Column make SHP-1 gene silent endogenous.
7. according to the method described in claim 6, it is characterized in that：3rd exon of sgRNA sequence targeting SHP-1 gene.
8. method according to claim 6 or 7, it is characterised in that：CAR-T cell includes CD19 CAR T, CD20 CAR
T, BMSA CAR T, MSLN CAR T, EGFRVIII CAR T, Her2 CAR T, GD2 CAR T, CEA CAR T.
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| PCT/CN2018/095635 WO2020000526A1 (en) | 2018-06-26 | 2018-07-13 | Shp-1-knockout t-cell and construction method therefor |
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| CN116334077A (en) * | 2022-07-29 | 2023-06-27 | 江苏省人民医院（南京医科大学第一附属医院） | Construction method and application of airway epithelial cell SHP-1 gene specific knockout mouse model |
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| CN107109421A (en) * | 2014-10-09 | 2017-08-29 | 国立大学法人山口大学 | CAR expression vectors and CAR expression T cells |
| CN107164407A (en) * | 2017-07-04 | 2017-09-15 | 王小平 | Gene knockout and gene overexpression are carried out simultaneously without the eucaryote that species are limited |
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| US20170335281A1 (en) * | 2014-03-15 | 2017-11-23 | Novartis Ag | Treatment of cancer using chimeric antigen receptor |
| EP3132030B1 (en) * | 2014-04-18 | 2020-08-26 | Editas Medicine, Inc. | Crispr-cas-related methods, compositions and components for cancer immunotherapy |
| EP3274454B1 (en) * | 2015-03-25 | 2021-08-25 | Editas Medicine, Inc. | Crispr/cas-related methods, compositions and components |
| GB201620070D0 (en) * | 2016-11-28 | 2017-01-11 | Autolus Ltd | Signal transduction modifying protein |
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| CN107109421A (en) * | 2014-10-09 | 2017-08-29 | 国立大学法人山口大学 | CAR expression vectors and CAR expression T cells |
| CN107164407A (en) * | 2017-07-04 | 2017-09-15 | 王小平 | Gene knockout and gene overexpression are carried out simultaneously without the eucaryote that species are limited |
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| INGUNN M.STROMNES等: "Abrogation of SHP-1 in tumor-specific T cells improves efficacy of adoptive immunotherapy by enhancing the effector function and accumulation of short-lived effector T cells in vivo", 《J IMMUNOL.》 * |
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| CN116334077A (en) * | 2022-07-29 | 2023-06-27 | 江苏省人民医院（南京医科大学第一附属医院） | Construction method and application of airway epithelial cell SHP-1 gene specific knockout mouse model |
| CN116334077B (en) * | 2022-07-29 | 2024-02-02 | 江苏省人民医院（南京医科大学第一附属医院） | Construction method and application of airway epithelial cell SHP-1 gene specific knockout mouse model |
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