CN112239759A

CN112239759A - MicroRNA for knocking down PD1 gene expression and construction of chimeric antigen receptor-T/NK cell thereof

Info

Publication number: CN112239759A
Application number: CN202011173416.5A
Authority: CN
Inventors: 高基民; 魏成; 赵爱; 尹晓光; 聂代邦; 段德明; 龙婧; 熊华强
Original assignee: Chongqing Sidemu Biological Technology Co ltd
Current assignee: Chongqing Sidemu Biological Technology Co ltd
Priority date: 2020-10-28
Filing date: 2020-10-28
Publication date: 2021-01-19

Abstract

The invention discloses a microRNA for efficiently knocking down PD1 gene expression and construction of a chimeric antigen receptor-T/NK cell thereof, wherein the microRNA is coded by a nucleotide sequence shown in SEQ ID No.1 or SEQ ID No.2, and the nucleotide sequence coding the microRNA is inserted into an EF1 alpha promoter, so that the packaging efficiency of a lentiviral vector cannot be obviously reduced. The PD1 gene in the CAR-T/NK cell is knocked down, the gene knocking efficiency is high, the knocking-down rate can reach more than 85%, and the immune cell exhaustion and apoptosis induced by a PD1-PDL1/2 channel can be effectively inhibited, so that the curative effect of CAR-T/NK therapy is improved, and the CAR-T/NK cell gene knock-down method has a good application prospect.

Description

MicroRNA for knocking down PD1 gene expression and construction of chimeric antigen receptor-T/NK cell thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a microRNA for efficiently knocking down PD1 gene expression and construction of a chimeric antigen receptor-T/NK cell thereof.

Background

CAR-T/NK therapy is a chimeric antigen receptor-based cellular immunotherapy regimen. By in vitro gene transfer techniques, a gene sequence encoding a Chimeric Antigen Receptor (CAR) is transferred into T/NK cells, generating tumor-specific T/NK cells that can bind to a target antigen. In recent years, CART cell immunotherapy has shown great potential in preclinical and clinical trials of hematological tumors, but the efficacy of CART cells in the treatment of solid tumors has been unsatisfactory due to the inhibition of immune checkpoints in the tumor microenvironment.

PD-1 (programmed death receptor) is a protein molecule expressed on the surface of T cells, and can inhibit the transcription of a downstream NF-kB gene and the secretion of interferon-gamma when PD-1 is combined with a ligand PD-L1 or PD-L2 thereof, thereby inhibiting the killing activity of the T cells. Among them, the PD-1/PD-L1 pathway has been proved to be related to T cell exhaustion, which leads to the problems of CART cell function reduction, tumor clearance capacity impairment and the like. The PD-1 antibody or the PD-L1 antibody is adopted to block the combination of PD-1, PD-L1 and PD-L2, so that the tumor killing activity of the T cell is not inhibited, and the tumor killing effect of the T cell is improved.

In order to solve the problems, the invention patent 201711196439.6 discloses a PD1 knockout CD 20-targeted chimeric antigen receptor T cell and a preparation method and application thereof, wherein CRISPR/Cas9 knockout CART cell PD-1 is adopted, although the method shows a high-efficiency anti-tumor effect, the research shows that the unexpected deletion of a programmed cell death factor 1 gene (PDCD1) in an initial CD8+ T cell can increase the exhaustion of the cell and damage the viability and the function of the cell, and the gene knockout can generate unexpected damage and influence on other genes.

RNA interference (RNAi) refers to a phenomenon of highly conserved during evolution, induced by double-stranded RNA (dsRNA), that mediates post-transcriptional gene silencing by inhibiting mRNA translation or by highly specific degradation of mRNA. The process of dsRNA-mediated gene silencing typically requires three steps to be performed. Firstly, dsRNA enters cells, is combined with enzyme protein Dicer and is cut into small segments with the length of 21-23nt by the Dicer, and the small segments are called siRNA; then the siRNA is combined with some enzymes (including endonuclease, exonuclease, helicase and the like) in vivo to form an RNA-induced silencing complex (RISC); finally, the activated RISC binds to the target mRNA under the direction of the antisense strand, mediating post-transcriptional gene silencing by inhibiting translation or degradation of mRNA. Gene silencing is therefore a molecular mechanism that reduces the expression of a target gene, and unlike gene knock-outs that eliminate the expression of the entire target gene, only reduces the expression of the target gene by at least 70%. Compared with the traditional gene knockout means, the RNAi technology has the advantages of high speed, high efficiency, strong specificity, simple operation and the like, has become a tool for revealing gene functions in different species in recent years, and is widely applied to plants, nematodes, fruit flies and mammals. We therefore hypothesized that if the means of siRNA were used to reduce PD-1 expression in CART cells, T/NK cells could not only evade inhibitory signals in the tumor microenvironment, but also avoid the damage and risk of off-target associated with a complete knock-out of the PD1 gene.

Disclosure of Invention

Aiming at the defects in the prior art, the technical problems to be solved by the invention are as follows: how to provide a microRNA for knocking down PD1 gene expression and construction of a chimeric antigen receptor-T/NK cell thereof, and solve the problems of escape of tumor cells and off-target risk of gene knockout.

In order to solve the technical problems, the invention adopts the following technical scheme: a microRNA for efficiently knocking down PD1 gene expression is coded by a nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO. 2.

Another objective of the invention is to provide a recombinant vector containing a nucleotide sequence encoding the microRNA.

The invention also aims to provide a recombinant lentivirus, which is obtained by co-transfecting a host cell with a recombinant plasmid containing a nucleotide sequence for coding the microRNA and a helper plasmid.

Preferably, the host cell used for lentiviral packaging is a 293T cell.

It is another object of the invention to provide a chimeric antigen receptor-T/NK cell, the Chimeric Antigen Receptor (CAR) -encoding structure comprising a first signal, a second signal, and a third signal; the first signal is an antigen binding domain, the second signal is a transmembrane domain and an intracellular transduction domain, the third signal is the microRNA of claim 1, and the microRNA is inserted into the EF1 alpha promoter. Thus, the nucleotide sequence encoding the microRNA is positioned at the downstream of the promoter of the lentiviral vector EF1 alpha, and the transport and the processing of the endogenous microRNA of the host cell are interfered, so that the packaging titer of the lentiviral vector is remarkably reduced.

The other purpose of the invention is to provide a construction method of the chimeric antigen receptor-T/NK cell, wherein the recombinant lentivirus is transfected into positive T/NK lymphocytes, and the cells are harvested after culture, so that the chimeric antigen receptor-T/NK cell capable of knocking down the expression of endogenous PD-1 is prepared.

Further, the positive T lymphocyte is obtained by separating from human source peripheral blood mononuclear cells.

Another object of the present invention is to provide a cytostatic agent (T/NK killer effector cell) comprising the chimeric antigen receptor-T/NK cell as an active ingredient.

The invention also aims to provide the application of the cytostatic agent in preparing antitumor drugs.

Drawings

FIG. 1 is a schematic diagram of the plenti-anti-CD19(PD1 knockdown) plasmid.

FIG. 2 shows the effect of microRNA insertion position into EF1a promoter on viral packaging titer.

FIG. 3 is the effect of PD 1-targeted microRNA on the expression level of PD1 in CAR-T cells.

FIG. 4 is the killing activity of PD1 knocked-down CAR-T cells on target cells expressing GFP Raji; a is flow cytometry analysis chart, B is column analysis chart.

Detailed Description

The present invention will be described in further detail with reference to the following specific embodiments and the accompanying drawings. The experimental procedures, for which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

The main experimental materials: EcoR V-HF, MluI-HF, NdeI restriction enzymes (NEB), seamless cloning enzymes (and metabiol), high fidelity Prime GXL STAR enzyme (TAKARA), TransStbl3 competent cells (Takara Biotech Co., Ltd.), Plasmid Mini Kit I (OMEGA),

plasmid Maxi Kit (QIAGEN), DMEM, RPMI-1640, Opti-MEM medium, Gibco FBS (Thermo Fisher Scientific), Sanger sequencing (Shanghai Sangnie Biotech Co., Ltd.), NaCl, yeast powder, peptone, EDTA, NaOH (Shanghai Biotechnology Co., Ltd.), primers (Jiangsu jin Zhi Biotechnology Co., Ltd.).

Firstly, construction of chimeric antigen receptor-T/NK cells with knocked-down endogenous PD1 gene expression (1) construction of plenti-CD19-microRNA # PD1 recombinant plasmid:

the microRNA (SEQ ID NO.1 and SEQ ID NO.2) targeting PD1 was synthesized by Suzhou Jinzhi Biotech Co., Ltd, and the plenti-EF1a-anti-CD19CAR vector was double-digested with Ndel and MluI-HF under the conditions of 37 ℃ for 3h and 65 ℃ for 20min, and the digestion system is shown in Table 3. And (3) carrying out 1% agarose gel electrophoresis on the enzyme digestion product to obtain a vector fragment, then recovering the Plenti vector fragment and the microRNA targeting PD1 by using a XYGENE gel recovery kit (the operation steps are shown in the following table 1), and detecting the concentration and the purity. The vector fragment and the target fragment were cloned by T4 (system shown in Table 2), 16-24h later, 65 ℃ 10min later, then plasmid transformation was performed (T4 clone product was placed on ice for 5min, then transferred into 50. mu.l TransStbl3 competent, placed on ice for 30min, 42 ℃ 45s, further 5min, added 500. mu.l LB, activated in 37 ℃, 225rpm/min shaker for 1h, then centrifuged at 5000rpm/min 20 ℃ for 5min, supernatant was discarded, the remaining bacterial solution was mixed well and plated, and cultured at 37 ℃ for 12-14 h). Selecting a monoclonal colony, carrying out bacterial liquid amplification at 37 ℃, 250rpm/min, 12-14h, extracting plasmids, finally carrying out enzyme digestion identification by AFLII-HF restriction enzyme, finally carrying out Sanger sequencing, and screening positive clones. Successfully constructing a plenti-CD19-microRNA # PD1 recombinant plasmid shown in figure 1, wherein SEQ ID NO.1 is inserted into an EF1 alpha promoter and is marked as plenti-CD19-miRNA- # PD1# 1; SEQ ID NO.2 was inserted into the EF1 α promoter and designated as plenti-CD19-miRNA- # PD1# 2.

TABLE 1 gum recovery

TABLE 2 cloning System of T4

TABLE 3 restriction enzyme cleavage System

(2) Recombinant lentivirus construction

S1: culturing 293T cells in a 15cm cell dish, and after the 293T cells are fully grown to 70% of the full field, resuspending 60 μ g PEI (PBS-PEI) with 1.5ml PBS, and resuspending the total mass of plenti-CD19-microRNA # PD1 and helper plasmid (PBS-DNA) with 1.5ml PBS; and then adding the PBS-PEI mixed solution into the PBS-DNA to obtain a PEI-DNA-PBS mixed solution, and standing for 20min at room temperature.

S2: preparing OPTI-DMEM full culture in an incubator at 37 ℃ for rewarming, sucking out a DMEM original culture medium in 293T cells, and adding the OPTI-DMEM into the 293T cells along the dish wall; then adding the PEI-DNA-PBS mixed solution into a culture dish, and culturing for 48h at 37 ℃;

s3: the lentivirus in the supernatant is collected in a 50ml centrifuge tube, and then incubated for 24h with 20ml of medium to collect the virus within 72h, centrifuged at 1500rpm for 5min to remove cell debris, or filtered through a 0.45 μm filter using a syringe and centrifuged at 3000 Xg for 12-14h 4 ℃ to concentrate the virus.

S4: after centrifugation is finished, supernatant is discarded, liquid remained on the tube wall is removed as much as possible, Vivo full culture or AIM-V full culture (preferably 1% HEPES) is added at a ratio of 1:200-1:400, heavy suspension virus is blown and beaten repeatedly and gently, the virus is subpackaged in 1.5ml Ep tubes and is stored at-80 ℃, repeated freeze thawing is avoided (the freeze thawing reduces the titer by one order of magnitude), and the recombinant lentivirus is obtained.

(3) Construction of chimeric antigen receptor T cell for knocking down PD1 expression quantity

S1: isolation of PBMC (peripheral blood mononuclear cells)

Taking 10ml of peripheral blood of a healthy person to an EDTA-Na2 anticoagulation tube, and uniformly mixing the anticoagulation tube with DPBS according to the ratio of 1: 1; taking four 15ml sterile centrifuge tubes, respectively adding 5ml Ficoll separating medium, slowly adding the mixture of peripheral blood and DPBS onto the surface of the Ficoll separating medium, taking care not to damage the liquid surface, horizontally centrifuging at 800g for 20min, 25 ℃, adjusting the speed of acceleration and deceleration to 0, sucking out the white flocculent layer (PBMC layer) in the centrifuge tube by a Pasteur pipette after centrifugation, placing in a new sterile centrifuge tube, adding PBS, centrifuging and washing the PBMC twice, centrifuging at 1500rpm/min for 5min, discarding the supernatant, adding 1ml Buffer1(DPBS contains 5% FBS), counting and re-suspending the PBMC to obtain the PBMC.

S2: separation of antigen specific T lymphocyte by immunomagnetic bead method

The proportion of CD3 positive cells in PBMCs was determined by flow cytometry. CD3/CD28 beads (106 CD3 positive cells plus 30. mu.l beads) were added to the cell suspension at a ratio of CD3/CD28 dynabeads: CD3 positive cells to 3:1 and spun at 1rpm at 4 ℃Shaking for 30min to make the magnetic strain and cell contact and combine; after 30 minutes, adding enough Buffer1 (more than 1ml) into the test tube, then placing the test tube on a magnetic frame, rotating left and right for 1-2 minutes, and sucking and removing the supernatant; preparing a Vivo complete culture medium: vivo air culture + 5% FBS + 1% HEPES + 1% sodium pyruvate + 1% non-essential amino acids +1:30 glutamine +1:10000IL-2+1:2000IL-7+1:2000IL-15, and Vivo whole culture heavy suspension cell and magnetic beads, counting; the medium was added to give a concentration of CD3 positive cells between 0.5 and 1X 106/ml. The concentration of the plated cells is 0.5-1.0 × 106/ml, and the plated cells are placed in 5% CO₂Culturing in an incubator at 37 ℃ for 24-36h, and screening the cells incubated with the magnetic beads by adopting a magnet; after washing with PBS and removal of immunomagnetic beads, CD 3-positive T lymphocytes were obtained.

S3: preparation of antigen-specific T lymphocytes by virus transfection method

CD3 positive T lymphocytes obtained by immunomagnetic bead separation were transduced with recombinant lentiviruses at MOI values of 5, 20, 40, 80, MOI (number of viral infections) ([ viral titer x viral volume (ml) ]/cell number; 1200xg, 90min, centrifuging at 4 ℃, incubating in an incubator at 37 ℃ until a 96-well plate is full of cells, transferring to a 24-well plate, measuring the CAR transduction rate at 5-7 days, and collecting the cells to obtain the chimeric antigen receptor T cells with the endogenous PD1 knocked down.

Second, performance verification

1. The packaged lentiviruses were transfected into Jurkat Cell to calculate virus titers.

Centrifuging Jurkat cells at 1500rpm for 5min, discarding the supernatant, resuspending in 1ml 1640 medium, and counting; then add 0.5 × 10 in 96-well plate⁶Jurkat cells, adding viruses in a gradient proportion of 1:50, 1:500, 1:1000, 1:2000 and the like, and supplementing a culture medium until the total volume of each hole is 200 mu l; add 0.1. mu.l polybrene B protein per well to promote transduction (0.1. mu.l/200. mu.l system); centrifuging a 96-well plate at the temperature of 32 ℃ for 90min at 1200g, and incubating the plate in an incubator at the temperature of 37 ℃ for 4 h; and blowing and uniformly mixing the jurkat cell suspension of each hole of the 96-hole plate, transferring the well to a 1.5ml Ep tube, centrifuging the mixture at 1500rpm for 5min, discarding the supernatant, fully culturing and re-suspending the mixture by using 1ml 1640, transferring the mixture to a 24-hole plate, and performing amplification culture for 48h at 37 ℃. The results are shown in FIG. 2.

As can be seen from the figure, the sequences shown as SEQ ID No.1 and SEQ ID No.2 which respectively target PD1 are inserted into an EF1a promoter to obtain anti-CD19(miRNA- # PD1#1) CAR-T and anti-CD19(miRNA- # PD1#2) CAR-T virus infected Jurkat cells, the expression is about 80%, the capability of the virus infected Jurkat cells without miRNA (anti-CD19CAR-T) is equivalent, and the packaging titer is not obviously changed. However, the virus (anti-CD19CAR-T-miRNA # PD1#2) obtained by inserting the sequence described in SEQ ID NO.2 behind the EF1a promoter only expresses 60% of infected jurkat cells, and has obvious difference with the virus without miRNA, which indicates that the virus packaging titer is not influenced when microRNA is inserted into the EF1a promoter.

2. The expression level of PD1 of the PD1 knockout CD19 targeted chimeric antigen receptor T cell is measured by a flow cytometer.

Taking T cells after transduction of lentivirus, demagnetizing, centrifuging at 2500rpm for 3min, and removing supernatant; then, the cells were resuspended by FACS, centrifuged at 2500rpm for 3min, the supernatant was discarded, and then flow antibody was added, protected from light, incubated for 20min, washed twice by FACS, and analyzed by flow-based machine for expression of PD1 on the cell surface, the results are shown in FIG. 3.

Compared with CAR-T cells which do not express miRNA of targeted PD1, the viruses expressing miRNA # PD1#1 and miRNA # PD1#2 have the advantages that the amount of PD1 expressed by the CAR-T cells is obviously reduced, the efficient knockdown rate can reach more than 85%, and therefore two screened miRNA sequences aiming at PD1 efficient knockout are proved.

3. The in vitro tumor cell killing of the chimeric antigen receptor T cells knockdown PD1 was examined by flow cytometry.

Culturing Raji-Luc-GFP cells to logarithmic growth state, taking a certain number of cells, centrifuging, precipitating and counting; adding 10 into a 96-hole flat-bottom opaque white board⁴Raji-Luc-GFP cells, medium supplemented to 100. mu.L; respectively setting the cell ratio of anti-CD19CAR-T, anti-CD19(microRNA # PD1#1) CAR-T, anti-CD19(microRNA # PD1#2) CAR-T cells to Raji-Luc-GFP cells as 5:1, 10:1 and 20:1, and adding the corresponding CAR-T cells into each well for mixed culture; setting a Mock cell group, wherein the number of T cells is the same as that of the CAR-T cells; two controls were set simultaneously, negative control was raji-Luc-GFP cellsCulturing in a culture medium; the positive control was the addition of 2.5% Triton-X100 to the culture medium, neither Mock cells nor CAR-T cells, as the minimum and maximum background values for cell killing, i.e., Kmin and Kmax. After 4 hours of culture, the 96-well plate was centrifuged at 1500rpm for 5min, the supernatant was discarded, the cells were resuspended after one wash with medium, and the killing results were detected after surface staining of the flow cells. The results are shown in FIG. 4.

As can be seen from the figure, after incubating anti-CD19CAR-T with target cells respectively, only 1.22% and 3.07% of target cells remained in the incubation solutions of anti-CD19(microRNA # PD1#1) CAR-T and anti-CD19(microRNA # PD1#2) CAR-T compared to the control group, which was enhanced by about 4-fold compared to the original. The insertion of SEQ ID NO.1 or SEQ ID NO.2 in the EF1 alpha promoter can obviously enhance the killing power of anti-CD19CAR-T on tumor cells. And because the targeting positions of miRNA designs are different, the knocking efficiency of PD1 is possibly different, and the killing condition is different. The results show that the chimeric antigen receptor T cell targeting CD19 after the PD1 gene expression is knocked down, prepared by the method, has high-efficiency and specific tumor killing capacity, and can prevent tumor cells from escaping immune surveillance.

The above is only a preferred embodiment of the present invention, and it should be noted that several modifications and improvements made by those skilled in the art without departing from the technical solution should also be considered as falling within the scope of the claims.

Sequence listing

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Claims

1. A microRNA for efficiently knocking down PD1 gene expression is characterized in that the microRNA is coded by a nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO. 2.

2. A recombinant vector comprising a nucleotide sequence encoding the microRNA of claim 1.

3. A recombinant lentivirus, wherein the recombinant lentivirus is obtained by co-transfecting a host cell with the recombinant plasmid of claim 2 and a helper plasmid.

4. The recombinant lentivirus of claim 3, wherein the host cell used for packaging the lentivirus is a 293T cell.

5. A chimeric antigen receptor-T/NK cell, wherein said encoding chimeric antigen receptor structure comprises a first signal, a second signal, and a third signal; the first signal is an antigen binding domain, the second signal is a transmembrane domain and an intracellular transduction domain, the third signal is the microRNA of claim 1, and the microRNA is inserted into the EF1 alpha promoter.

6. The method for constructing chimeric antigen receptor-T/NK cells according to claim 5, wherein the chimeric antigen receptor-T/NK cells capable of knocking down the expression of endogenous PD-1 are prepared by transfecting the recombinant lentivirus according to claim 4 into positive T/NK lymphocytes and harvesting the cells after culturing.

7. The method according to claim 6, wherein the positive T/NK cells are isolated from human peripheral blood mononuclear cells.

8. A T/NK killer effector cell comprising the chimeric antigen receptor-T/NK cell according to claim 3 as an active ingredient.

9. Use of the T/NK killer effector cells of claim 8 for the preparation of antitumor drugs.