Retroviral vector expressing CAR and shRNA and application thereof
阅读说明：本技术 表达CAR和shRNA的逆转录病毒载体及其应用 (Retroviral vector expressing CAR and shRNA and application thereof ) 是由 王建勋 张静 朱晶晶 冯娅茹 李晓瑞 尚凤琴 于 2021-07-05 设计创作，主要内容包括：本发明提供一种表达CAR和shRNA的逆转录病毒载体,该病毒载体中靶向LSD1的shRNA与CAR共表达。本发明中将LSD1shRNA与anti-CD19CAR共表达于CAR-T细胞,实现LSD1shRNA对CAR-T细胞功能的同步调节,构建联合治疗方法。并对LSD1shRNA anti-CD19CAR-T细胞进行体内、外功能验证,证明LSD1shRNA anti-CD19CAR-T细胞在体内、外均具有较好的细胞活性、增殖能力与抗肿瘤能力。(The invention provides a retroviral vector for expressing CAR and shRNA, wherein shRNA targeting LSD1 is co-expressed with CAR in the viral vector. In the invention, the LSD1shRNA and the anti-CD19CAR are co-expressed in the CAR-T cell, so that the LSD1shRNA can synchronously regulate the functions of the CAR-T cell, and a combined treatment method is constructed. And in-vivo and in-vitro function verification is carried out on the LSD1shRNA anti-CD19CAR-T cell, and the LSD1shRNA anti-CD19CAR-T cell is proved to have better cell activity, proliferation capacity and anti-tumor capacity in vivo and in vitro.)
1. A retroviral vector for expressing a CAR and an shRNA, wherein the shRNA targeting LSD1 is co-expressed with the CAR in the viral vector.
2. The retroviral vector of claim 1, wherein the U6 promoter and the EF 1a promoter are integrated into the CAR expression vector separately, and instead of the long terminal repeat to drive expression, the U6 promoter drives the expression of the LSD1shRNA EF 1a promoter drives the expression of an anti-CAR, preferably an anti-CD19CAR and/or a CD38 CAR.
3. The retrovirus vector of claim 1, wherein the retrovirus vector comprises a U6 promoter, an LSD1shRNA, an EF 1a promoter, an upstream signal peptide and a myc tag for detection which are connected in series in sequence; a CD19CAR antigen binding region; CD8 hinge-transmembrane domain; CD28 or 4-1BB co-activate domain and CD3 ζ intracellular signaling domain.
4. The retroviral vector of claim 1, wherein the clone ID of the shRNA targeting LSD1 is TRCN0000046068, the clone name is NM-015013.1-1812 s1c1, and the sequence is SEQ ID NO:2: GCCTAGACATTAAACTGAATA.
5. The retroviral vector of claim 1, wherein the clone ID of the shRNA targeting LSD1 is TRCN0000046069, the clone name is NM-015013.1-2168 s1c1, and the sequence is SEQ ID NO:3: GCTCCAATACTGTTGGCACTA.
6. A targeted chimeric antigen receptor T cell comprising a targeted chimeric antigen receptor expressed by the retroviral vector of any one of claims 1-5.
7. A drug for treating tumor, which comprises the chimeric antigen receptor T cell according to claim 6.
8. The medicament of claim 7, wherein the tumor is multiple myeloma.
9. Use of a retroviral vector according to any one of claims 1 to 5 wherein the chimeric antigen receptor T cells are prepared by inserting a gene segment encoding said chimeric antigen receptor into said vector, packaging into a viral vector, infecting human T cells and treating a tumor that is surface CD19 positive and/or CD38 positive.
Technical Field
The invention relates to the technical field of tumor treatment, in particular to an expressed Chimeric Antigen Receptor (CAR) and shRNA retrovirus vector and application thereof.
Background
Short hairpin RNA (shRNA) belongs to the category of RNA interference (RNAi), after cells are transduced or transfected, precursor shRNA is synthesized in cell nuclei, a sense strand and an antisense strand of the shRNA form a stem region through base pairing, unpaired nucleotides in the middle form a ring, and the whole structure is in a hairpin structure. After being processed by ribonuclease IIIDrosha and DGCR8, the siRNA is transported into cytoplasm by Exportin-5 protein, and then is cut by ribonuclease IIIdicer and TRBP/PACT enzyme to remove the sequence of a ring structure, thus forming siRNA. After recognition and integration of siRNA with RISC, unwinding takes place and removes one RNA strand of the double strand, which then recognizes and occupies the target mRNA by the base complementary sequence, leading to its degradation. The shRNA can be stably expressed in a transduction mode, specifically and targetedly inhibits the expression of target mRNA, and has wide application in treatment, diagnosis and scientific research. At present, shRNA of a specific targeting immune checkpoint receptor and CAR-T cells are co-expressed to inhibit the expression of the immune checkpoint receptor and inhibit a tumor microenvironment by a lentivirus vector transduction mode, so that immune response is regulated.
LSD1 is a flavin-dependent monoamine oxidase, and is also the first discovered histone lysine specific demethylase, which is involved in the demethylation of H3K4 or H3K9, which is monomethylated or dimethylated in the presence of xanthine-adenine dinucleotide, regulates the activation or inhibition of gene transcription under different environments from the aspect of epigenetic modification, is involved in different physiological processes, including hematopoiesis, lipogenesis, developmental processes and the like, and plays an important role in the occurrence of tumors, and the LSD1 inhibitor is a potential anti-tumor immunosuppressant. Inhibition of LSD1 can stimulate and increase tumor immunogenicity, stimulate interferon-dependent anti-tumor immunity, and promote T cell infiltration. There are many LSD1 inhibitors currently undergoing clinical evaluation for cancer treatment.
Disclosure of Invention
In order to solve the technical problem, the invention provides a combined treatment method for constructing a novel shRNA and CAR to be co-expressed in CAR-T cells.
In one embodiment, a retroviral vector is provided that expresses a CAR and an shRNA, wherein the shRNA targeting LSD1 is co-expressed with the CAR.
In one embodiment, the U6 promoter and EF 1a promoter are separately integrated into the CAR expression vector in place of the long terminal repeat to drive expression, and the EF 1a promoter driving expression of the LSD1shRNA by the U6 promoter drives expression of an anti-CAR, preferably an anti-CD19CAR and/or a CD38 CAR.
In one embodiment, the retroviral vector comprises a U6 promoter, an LSD1shRNA, an EF1 alpha promoter, an upstream signal peptide and a myc label for detection which are connected in series in sequence; a CD19CAR antigen binding region; CD8 hinge-transmembrane domain; CD28 or 4-1BB co-activate domain and CD3 ζ intracellular signaling domain.
In one embodiment, the clone ID of the shRNA targeting LSD1 is TRCN0000046068, the clone name is NM-015013.1-1812 s1c1, and the sequence is SEQ ID NO:2: GCCTAGACATTAAACTGAATA.
In one embodiment, the clone ID of the shRNA targeting LSD1 is TRCN0000046069, the clone name is NM-015013.1-2168 s1c1, and the sequence is SEQ ID NO:3: GCTCCAATACTGTTGGCACTA.
In one embodiment, a targeted chimeric antigen receptor T cell is provided that includes a targeted chimeric antigen receptor expressed by a retroviral vector as described above.
In one embodiment, a medicament for treating a tumor is provided, which comprises the chimeric antigen receptor T cell described above.
In one embodiment, the tumor is multiple myeloma.
In one embodiment, there is provided the use of a retroviral vector as described above, wherein the chimeric antigen receptor T cells are prepared by inserting a gene segment encoding the chimeric antigen receptor into the vector, packaging into viral vector particles, infecting human T cells, and treating a tumor that is surface CD19 positive and/or CD38 positive.
In the invention, an MFG (retroviral vector plasmid) -LSD1 shRNA-anti-CD19 CAR retroviral vector plasmid is constructed by adopting a genetic engineering means, a U6 promoter, an LSD1shRNA and an EF1 alpha promoter are integrated into a retroviral vector for expressing CAR, the U6 promoter drives the expression of the LSD1shRNA, and the EF1 alpha promoter drives the expression of the anti-CD19 CAR. And (3) packaging the retrovirus vector to obtain two LSD1shRNA anti-CD19CAR retrovirus vectors with higher titer, and transducing human primary T cells, wherein the transduction efficiency detected by flow cytometry reaches more than 50%, and the LSD1shRNA anti-CD19CAR-T cells are successfully constructed. The expression level of LSD1 in LSD1shRNA anti-CD19CAR-T cells is obviously reduced through qPCR detection, which shows that LSD1shRNA can be simultaneously expressed with CAR genes, and the expression of anti-CD19CAR is not influenced by the simultaneous expression of LSD1 shRNA.
LSD1shRNA enhances anti-tumor function of anti-CD19CAR-T cells in vitro. The LSD1shRNA can enhance the function of anti-CD19CAR-T cells for killing tumor cells in vitro through apoptosis detection, luciferase detection and RTCA detection in flow cytometry; the LSD1shRNA can promote the long-term anti-tumor function of anti-CD19CAR-T cells under repeated antigen stimulation through a pressure test experiment; an ELISA (enzyme-Linked immuno sorbent assay) detection cytokine experiment proves that the LSD1shRNA can promote the release levels of IFN-gamma, TNF-alpha and IL-2 when anti-CD19CAR-T cells kill tumor cells, and promote the anti-tumor function of the anti-CD19CAR-T cells; the LSD1shRNA can enhance the in vitro proliferation capacity of anti-CD19CAR-T cells through cell counting calculation and CFSE proliferation detection.
LSD1shRNA enhances anti-tumor function of anti-CD19CAR-T cells in vivo. Use of immunodeficient NOD-Prkdcscid Il2rgnullAn NPG mouse constructs a Raji-Luc cell tumor animal model, and after LSD1shRNA anti-CD19CAR-T cells are treated, the LSD1shRNA anti-CD19CAR-T cells are proved to show good anti-tumor function in vivo through methods such as in vivo imaging of the small animal, weight monitoring, peripheral blood T cell flow cytometry detection and the like, so that tumors are completely eliminated, and the survival period of the tumor model mouse is obviously prolonged, although the LSD1shRNA anti-CD19CAR-T cell treatment group and the RNAu6 anti-CAR-T cell treatment group have no tumor bioluminescence signal and survival rate in vivo imaging of the small animalThe difference is obvious, but ELISA detection of mouse serum IFN-gamma 7 days after the second CAR-T cell injection finds that the serum IFN-gamma level of anti-CD19CAR-T cell treatment group mice co-expressed by LSD1shRNA is remarkably increased, and LSD1shRNA enhances the anti-tumor function of CAR-T cells in vivo. At the termination of the study on day 52, flow cytometry detected that the number of T cells in peripheral blood of mice in the LSD1 shRNA-2 anti-CD19CAR-T cell treatment group was significantly higher than that in the control group, indicating that LSD1shRNA promotes the proliferative capacity of CAR-T cells in vivo. Therefore, the LSD1shRNA overexpression promotes the anti-tumor activity and the proliferation capacity of anti-CD19CAR-T cells in vivo.
In the invention, the LSD1shRNA and the anti-CD19CAR are co-expressed in the CAR-T cell, so that the LSD1shRNA can synchronously regulate the functions of the CAR-T cell, and a combined treatment method is constructed. And in-vivo and in-vitro function verification is carried out on the LSD1shRNA anti-CD19CAR-T cell, and the LSD1shRNA anti-CD19CAR-T cell is proved to have better cell activity, proliferation capacity and anti-tumor capacity in vivo and in vitro.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a schematic of different CAR expression vectors, wherein FIG. 1A is a schematic of an unmodified anti-CD19CAR expression vector, FIG. 1B is a schematic of an engineered control U6 anti-CD19CAR expression vector, and FIG. 1C is a schematic of U6-LSD1 shRNA-anti-CD19 CAR expression vector;
FIG. 2 is a graph showing the results of the detection of the efficiency of transfection of Phoenix-ECO cells with retroviral vector expression plasmids, wherein FIG. 2A is pMFG-U6-LSD1 shRNA-1-EF1 alpha-anti-CD 19CAR, and FIG. 2B is pMFG-U6-LSD1 shRNA-2-EF1 alpha-anti-CD 19 CAR;
FIG. 3 is a graph showing the results of the measurement of the cell efficiency of PG13 transduced by an ecotropic retroviral vector, wherein FIG. 3A is LSD1 shRNA-1 anti-CD19 CAR; FIG. 3B is LSD1 shRNA-2 anti-CD19 CAR;
fig. 4 is a graph of the results of LSD1 expression level assay (n-3) in LSD1shRNA anti-CD19CAR-T cells, wherein fig. 4A is LSD1 shRNA-1 anti-CD19CAR-T cell LSD1 expression level assay; figure 4B is a measurement of LSD1 shRNA-2 anti-CD19CAR-T cell LSD1 expression levels, P <0.05, P <0.01 compared to RNAU6 anti-CD19CAR-T cells;
figure 5 is a graph of LSD1 shRNA-1 anti-CD19CAR-T cells killing Raji cell efficiency assay in vitro (n ═ 3) versus RNAU6 anti-CD19CAR-T cells, P < 0.05;
figure 6 is a graph of the results of stress test measurements of CAR-T cell killing after repeated antigen stimulation in vitro, where 6A: detecting the cell killing efficiency after the first co-culture period; 6B: detecting the cell killing efficiency after the second co-culture period; 6C: detecting the cell killing efficiency after the third co-culture period; p <0.05,. P <0.01 as compared to RNAU6 anti-CD19CAR-T cells;
FIG. 7 is a graph of the results of the assay of the efficiency of LSD1shRNA anti-CD19CAR-T cells to kill Raji-Luc cells as compared to RNAU6 anti-CD19CAR-T cells,. P < 0.05;
fig. 8 is a graph of results of an LSD1shRNA anti-CD19CAR-T cell killing SW620 cell efficiency assay (n-3), wherein fig. 8A is SW620 cell index change; figure 8B is the SW620 cell index at the experimental end time cut-off,
(P <0.001 compared to RNAU6 anti-CD19CAR-T cells);
FIG. 9 is a graph of the results of an assay for LSD1shRNA anti-CD19CAR-T cell cytokine release levels (n-3), where 9A is the IFN- γ release level in the supernatant of co-cultured cells; 9B is the TNF- α release level in the supernatant of the co-cultured cells; 9C is the level of IL-2 release in the supernatant of the co-cultured cells (P <0.05, P <0.01 compared to RNAU6 anti-CD19CAR-T cells);
FIG. 10 is a record of LSD1shRNA anti-CD19CAR-T cell proliferation where 10A is the cell proliferation curve and 10B is the cell viability curve;
FIG. 11 is a graph of CD4+ T cell vs CD8+ T cell detection results in CAR-T cells;
fig. 12 is a graph of the results of tumor bioluminescence signal intensity monitoring (n-6) for in vivo imaging of small animals, with 12A being the change in tumor area and 12B being the overall signal intensity of tumor bioluminescence;
figure 13 is an NPG mouse survival curve (n ═ 6);
fig. 14 is a weight change curve of NPG mice (n ═ 6);
figure 15 results of serum IFN- γ release level assay (n-6) vs RNAU6 anti-CD19CAR-T cell treatment group P <0.05 and P < 0.01.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be further described with reference to the following examples, and it is obvious that the described examples are only a part of the examples of the present application, and not all examples. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Example a CAR Structure and plasmid map of the invention
In the invention, LSD1shRNA is applied to CAR-T cell engineering to construct a novel combined treatment method, LSD1shRNA is integrated into a retrovirus vector for expressing CAR, and gene expression is regulated. To ensure effective inhibition of the target mRNA, two shrnas targeting different sequence targets of LSD1 mRNA were selected. To enable better expression of LSD1shRNA and CAR, the U6 promoter and EF 1A promoter were integrated into CAR expression vectors, respectively, instead of Long Terminal Repeats (LTRs) to drive expression, the U6 promoter driving expression of LSD1shRNA, the EF 1A promoter driving expression of anti-CD19CAR to prolong expression of CAR in T cells, see fig. 1, where fig. 1A is a schematic of an unmodified anti-CD19CAR expression vector, fig. 1B is a schematic of an engineered control U6 anti-CD19CAR expression vector, and fig. 1C is a schematic of a U6-LSD1 shRNA-anti-CD19 CAR expression vector. MMLV (truncated) in FIG. 1 is the retroviral helper DNA encoded by MMLV. The anti-tumor function of the LSD1shRNA anti-CD19CAR-T cell is evaluated by in vivo and in vitro function detection. Provides a new research idea for exploring CAR-T cell activity improvement and function optimization, provides a new method for treating multiple myeloma, and lays an experimental foundation for the treatment of other blood system tumors and solid tumors CAR-T cells. ScFv amino acid sequence of anti-CD19 CAR:
DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS(SEQ ID NO:1)
example construction of the two pMFG-U6-LSD1 shRNA-EF1 alpha-anti-CD 19CAR plasmid
Base sequence search for LSD1shRNA
Human LSD1shRNA target sequences were obtained according to database and literature search, and two LSD1shRNA target sequences were selected for constructing pMFG-U6-LSD1 shRNA-EF1 alpha-anti-CD 19CAR plasmid, pMFG represents retroviral vector plasmid, see Table 1. And (3) referring to a website: https:// ports.broadinstitute.org/gpp/public/trans/details? tname — 015013.4.
TABLE 1 LSD1shRNA target sequences and primers
Preparation of LSD1 shRNA-1 fragment
Designing a primer, introducing a 5 'end of an LSD1 shRNA-1 primer into a restriction endonuclease site AgeI, introducing a 3' end into a restriction endonuclease site EcoRI, synthesizing a corresponding primer sequence, and completing primer synthesis by a biological engineering (Shanghai) corporation. And gradually annealing the upstream and downstream primers of the LSD1 shRNA-1 to obtain an LSD1 shRNA-1 fragment, wherein the tail end of the LSD1 shRNA-1 DNA is a cohesive end. Reaction conditions are as follows: the temperature of 95 ℃ for 5min, the temperature of 92.5 ℃ for 3min, the temperature of 90 ℃ for 3min and the temperature of 87.5 ℃ for 3min … … are sequentially decreased by 2.5 ℃ until the temperature is reduced to 10 ℃.
Preparation of 3U6-LSD1 shRNA-2 fragment
Synthesizing a U6-LSD1 shRNA-2 sequence, introducing a restriction enzyme site Mlu I at the 5 'end of the DNA sequence, introducing a restriction enzyme site Pac I at the 3' end of the DNA sequence, and cloning the whole DNA sequence into a PUC57 vector.
Verification of expression of pMFG-U6-LSD1 shRNA-EF1 alpha-anti-CD 19CAR plasmid
The HEK-293T cells were recovered and seeded in T75 cell culture flasks, and when the cells reached about 80% confluence in T75 flasks, the HEK-293T cells were passaged and seeded into 6-well cell culture plates at 1X 10 per well6Cells were co-seeded in 3 wells. The respective markers are pMFG-U6-RNA-EF1 alpha-anti-CD 19CAR control hole, pMFG-U6-LSD1 shRNA-1-EF1 alpha-anti-CD 19CAR experimental hole, pMFG-U6-LSD1 shRNA-2-EF1 alpha-anti-CD 19CAR experimental hole, and the experimental holes are put into a CO at 37 DEG C2An incubator. When the cell confluence reaches about 80%, the expression plasmid is transfected into HEK-293T cells by using a transfection reagent FuGene HD, and the cells are placed in a CO atmosphere at 37 DEG C2Incubate in incubator for 24 h.
After 24h, the media supernatant from each set of cell culture plates was carefully removed, discarded, and each well was supplemented with 3mL of fresh DMEM complete media. After 24h, the culture supernatant was carefully removed from each group of cell culture plates, discarded, and after washing the cells with 1 × PBS, the cells were trypsinized and collected in 1.5mL EP tubes for cell counting. Each group is 1 × 106The single cell is used for detecting the transfection efficiency by flow cytometry, and 2 x 10 is taken6The cells were used for total RNA extraction and RT-qPCR was used to detect the expression level of LSD 1.
Detection of expression level of LSD1
Extracting total RNA of transfected or transduced cells, detecting the expression level of LSD1 by a qPCR dye method, taking a GAPDH gene as an internal reference gene, obtaining primer sequences shown in table 2.12, and obtaining target fragment amplification primers of the LSD1 by jw389 and jw390, which are shown in table 2. Data processing fold changes in LSD1 expression levels were calculated using the 2- Δ Δ ct method.
TABLE 2 primer sequences for qPCR detection of LSD1 gene expression level
6. Results
6.1pMFG-U6-LSD1 shRNA-1-EF1 alpha-anti-CD 19CAR plasmid construction
The pMD18S-T-U6-RNA plasmid is subjected to double digestion by restriction enzyme Age I and EcoR I, and the RNA sequence in the plasmid is replaced by LSD1 shRNA-1 sequence to obtain the pMD18S-T-U6-LSD1 shRNA-1 plasmid. Then, the plasmid pMD18S-T-U6-LSD1 shRNA-1 is subjected to double digestion by using restriction enzymes Pac I and Mlu I-HF to obtain a U6-LSD1 shRNA-1 segment. The U6-LSD1 shRNA-1 fragment was replaced with U6-RNA in the pMFG-U6-RNA-EF1 alpha-anti-CD 19CAR plasmid to obtain the pMFG-U6-LSD1 shRNA-1-EF1 alpha-anti-CD 19CAR plasmid.
6.1.1pMD18S-T-U6-LSD1 shRNA-1 plasmid construction
The pMD18S-T-U6-RNA plasmid is double digested by restriction enzyme Age I and EcoR I to obtain a pMD18S-T-U6(Age I/EcoR I) vector. The digested product was subjected to agarose gel electrophoresis and gel purified and the DNA recovered, the size of the target vector was about 3 kb.
The LSD1 shRNA-1 fragment was ligated into pMD18S-T-U6(Age I/EcoR I) vector to obtain pMD18S-T-U6-LSD1 shRNA-1. The connection, transformation and plasmid minim extraction processes are the same as before. And (3) selecting a plasmid with correct enzyme digestion identification For sequencing, wherein a sequencing primer is a universal primer M13-For. Sequencing results show that the base sequence of U6-LSD1 shRNA-1 is completely correct.
6.1.2pMFG-U6-LSD1 shRNA-1-EF1 alpha-anti-CD 19CAR plasmid construction
The plasmid pMD18S-T-U6-LSD1 shRNA-1 and the plasmid pMFG-U6-RNA-EF1 alpha-anti-CD 19CAR are subjected to double digestion by restriction enzymes Pac I and Mlu I-HF respectively to obtain a U6-LSD1 shRNA-1 fragment and a pMFG-EF1 alpha-anti-CD 19CAR vector. And (3) carrying out agarose gel electrophoresis on the enzyme digestion product, carrying out gel purification and recovering DNA, wherein the size of a target fragment of U6-LSD1 shRNA-1 is 337bp, and the size of the pMFG-EF1 alpha-anti-CD 19CAR vector is about 9.2 kb.
The U6-LSD1 shRNA-1 target fragment was ligated into pMFG-EF1 alpha-anti-CD 19CAR vector to obtain pMFG-U6-LSD1 shRNA-1-EF1 alpha-anti-CD 19 CAR. The connection, transformation and plasmid minim extraction processes are the same as before. And (4) selecting a plasmid with correct enzyme digestion identification, and sequencing, wherein the result shows that the plasmid is successfully constructed.
Construction of pMFG-U6-LSD1 shRNA-2-EF1 alpha-anti-CD 19CAR plasmid
The plasmid PUC57-LSD1 shRNA-2 is digested by restriction enzymes Pac I and Mlu I-HF to obtain a U6-LSD1 shRNA-2 fragment. The U6-LSD1 shRNA-2 fragment was replaced with U6-RNA in the pMFG-U6-RNA-EF1 alpha-anti-CD 19CAR plasmid to obtain the pMFG-U6-LSD1 shRNA-2-EF1 alpha-anti-CD 19CAR plasmid.
7.1U6-LSD1 shRNA-2 fragment and pMFG-EF1 alpha-anti-CD 19CAR vector
The PUC57-LSD1 shRNA-2 plasmid and pMFG-U6-RNA-EF1 alpha-anti-CD 19CAR plasmid are respectively digested by restriction enzymes Pac I and Mlu I-HF to obtain a U6-LSD1 shRNA-2 fragment and a pMFG-EF1 alpha-anti-CD 19CAR vector. And (3) carrying out agarose gel electrophoresis on the enzyme digestion product, carrying out gel purification and recovering DNA, wherein the size of the target fragment of U6-LSD1 shRNA-2 is 324bp, and the size of the pMFG-EF1 alpha-anti-CD 19CAR vector is about 9.2 kb.
7.2pMFG-U6-LSD1 shRNA-2-EF1 alpha-anti-CD 19CAR plasmid construction and identification
The U6-LSD1 shRNA-2 fragment was ligated into pMFG-EF1 α -anti-CD19 CAR vector to obtain pMFG-U6-LSD1 shRNA-2-EF1 α -anti-CD19 CAR. The connection, transformation and plasmid minim extraction processes are the same as before. And (4) selecting a plasmid with correct enzyme digestion identification, and sequencing, wherein the result shows that the plasmid is successfully constructed.
7.3pMFG-U6-LSD1 shRNA-anti-CD19 CAR plasmid expression assay
HEK-293T cells were transfected with pMFG-U6-LSD1 shRNA-1-anti-CD19 CAR and pMFG-U6-LSD1 shRNA-2-anti-CD19 CAR plasmids, and transfection efficiency was examined by flow cytometry after 48 h. The results show that: has higher transfection efficiency. RT-qPCR detected changes in the expression level of LSD 1. The results show that: the expression level of LSD1 in HEK-293T cell group (0.49 +/-0.044: 1) transfected by MFG-U6-LSD1 shRNA-1-anti-CD19 CAR and the expression level of LSD1 in HEK-293T cell group (0.79 +/-0.003: 1) transfected by MFG-U6-LSD1 shRNA-2-anti-CD19 CAR are obviously lower than those in a control group, so that LSD1shRNA can be normally expressed and can play a function. Therefore, the next retroviral vector packaging experiment can be carried out to construct LSD1shRNA anti-CD19CAR-T cells and carry out in vivo and in vitro functional verification.
Example construction of TriLSD 1shRNA anti-CD19CAR-T cells
LSD1shRNA anti-CD19CAR-T cell construction
The transduction efficiency of the CAR-T cells is detected by flow cytometry by preparing a retrovirus vector, transducing the T cells, constructing LSD1shRNA anti-CD19CAR-T cells and using the RNAU6 anti-CD19CAR-T cells as a control group.
Preparation of LSD1shRNA anti-CD19CAR ecotropic retroviral vector
Phoenix-ECO cells are respectively transfected by pMFG-U6-LSD1 shRNA-1-EF1 alpha-anti-CD 19CAR and pMFG-U6-LSD1 shRNA-2-EF1 alpha-anti-CD 19CAR expression plasmids to prepare the tropism retrovirus vector. Detecting the expression level of Myc label of Phoenix-ECO cell by flow cytometry after 48h of transfection, and the result shows that: pMFG-U6-LSD1 shRNA-1-EF1 alpha-anti-CD 19CAR and pMFG-U6-LSD1 shRNA-2-EF1 alpha-anti-CD 19CAR plasmids can express at higher levels in Phoenix-ECO cells, see FIG. 2.
Preparation of LSD1shRNA anti-CD19CAR amphotropic retroviral vector
The ecotropic retroviral vector respectively transduces PG13 cells, establishes PG13 cell line stably producing LSD1 shRNA-1 anti-CD19CAR amphotropic retroviral vector, and PG13 cell line stably producing LSD1 shRNA-2 anti-CD19CAR amphotropic retroviral vector. The flow cytometry tests the transduction efficiency of PG13 cells transduced by the ecotropic retrovirus vector, and the results show that: the cell transduction efficiency of PG13 was over 70%, see FIG. 3, which shows that PG13 cell line stably producing LSD1 shRNA-1 anti-CD19CAR amphotropic retroviral vector and PG13 cell line stably producing LSD1 shRNA-2 anti-CD19CAR amphotropic retroviral vector were successfully constructed.
4. Retroviral vector titer detection
And (3) harvesting the amphotropic retrovirus vector, detecting the titer of the virus vector by RT-qPCR, successfully preparing the retrovirus vector, and carrying out the next experiment.
LSD1shRNA anti-CD19CAR-T cell construction
PBMC is separated from peripheral blood donated by healthy volunteers, and T cells are cultured and activated in vitro. Respectively transducing T cells with LSD1 shRNA-1 anti-CD19CAR amphotropic retroviral vectors and LSD1 shRNA-2 anti-CD19CAR amphotropic retroviral vectors to construct LSD1 shRNA-1 anti-CD19CAR-T cells and LSD1 shRNA-2 anti-CD19CAR-T cells, and detecting the transduction efficiency by flow cytometry. The results show that: the transduction efficiency reaches more than 50%, and the simultaneous expression of the LSD1shRNA does not influence the expression of the anti-CD19 CAR.
LSD1shRNA anti-CD19CAR-T cell integration copy number assay
And (3) detecting the gene integration copy number of the LSD1 shRNA-1 anti-CD19CAR-T cell and the LSD1 shRNA-2 anti-CD19CAR-T cell virus vector by qPCR. The results show that all CAR-T cell retroviral vector integrated copy number is less than 3, anti-CD19CAR copy number is 2.26 ± 0.12, RNAU6 anti-CD19CAR copy number is 1.81 ± 0.03, LSD1 shRNA-1 anti-CD19CAR copy number is 2.27 ± 0.41, LSD1 shRNA-2 anti-CD19CAR copy number is 2.31 ± 0.25.
Detection of expression level of LSD1
LSD1shRNA anti-CD19CAR retrovirus vector transduces human primary T cells to construct LSD1shRNA anti-CD19CAR-T cells, and the expression of anti-CD19CAR is detected by flow cytometry by using FITC labeled CD19 recombinant antigen molecules, and the result shows that: anti-CD19CAR was expressed efficiently. The expression level of LSD1 was then detected by RT-qPCR, and the results showed: the mean level of LSD1 expression was significantly reduced in LSD1 shRNA-1 anti-CD19CAR-T cells and LSD1 shRNA-2 anti-CD19CAR-T cells, see figure 4. This demonstrates that a combination therapeutic approach of integrating shRNA into CAR-expressing retroviral vectors, with simultaneous gene expression and regulation can be achieved, having achieved simultaneous expression of LSD1shRNA and anti-CD19 CAR.
Example in vitro study of four LSD1shRNA to enhance anti-tumor function of anti-i-CD 19CAR-T cells
Function detection of LSD1shRNA for enhancing anti-CD19CAR-T cell in-vitro killing tumor cell
1.1.LSD 1shRNA anti-CD19CAR-T cell in vitro killing Raji cell efficiency detection
And co-culturing the LSD1 shRNA-1 anti-CD19CAR-T cell and a tumor cell Raji with positive CD19 molecule expression for 12h according to the number ratio of 1:16, 1:8, 1:4, 1:2 and 1:1, and detecting the killing efficiency of the LSD1shRNA anti-CD19CAR-T cell on the Raji cell by flow cytometry. The results show that: the killing efficiency of LSD1 shRNA-1 anti-CD19CAR-T cells is obviously enhanced, and PanT refers to untransformed T cells, and is shown in figure 5.
1.2LSD 1shRNA anti-CD19CAR-T cell in vitro killing Raji cell stress test detection CAR-T cells and Raji cells are co-cultured for 48h according to the quantitative ratio of 1: 1. The expression of CD19 molecules of the co-cultured cells is detected by flow cytometry, and the result shows that the LSD1shRNA anti-CD19CAR-T cell group and the RNAU6 anti-CD19CAR-T cell group have no cells with positive CD19 molecule expression. And repeating 3 periods of co-culture, after each co-culture period is finished, carrying out an apoptosis detection experiment for killing Raji cells by the CAR-T cells, and detecting the killing efficiency of the in-vitro killing Raji cells by the CAR-T cells by flow cytometry for 3 times in total.
After 48 hours of 1 st co-culture, the apoptosis detection experiment of killing Raji cells by the CAR-T cells finds that: the killing efficiency of LSD1shRNA anti-CD19CAR-T cells and RNAU6 anti-CD19CAR-T cells on Raji cells is enhanced compared with that under the condition of no antigen stimulation, and the killing efficiency of LSD1shRNA anti-CD19CAR-T cells on Raji cells is obviously enhanced compared with that of RNAU6 anti-CD19CAR-T cells, as shown in figure 6A.
After 48h of 2 nd co-culture, the apoptosis detection experiment of killing Raji cells by the CAR-T cells finds that: the killing efficiency of the LSD1shRNA anti-CD19CAR-T cell and the RNAU6 anti-CD19CAR-T cell for killing Raji cells is not obviously different from that of the Raji cells after the 1 st co-culture period, and the killing efficiency of the LSD1shRNA anti-CD19CAR-T cell is still obviously improved compared with that of the RNAU6 anti-CD19CAR-T cell for killing Raji cells, as shown in figure 6B.
After 48 hours of 3 rd co-culture, the apoptosis detection experiment of killing Raji cells by the CAR-T cells finds that: the killing efficiency of LSD1shRNA anti-CD19CAR-T cells and RNAU6 anti-CD19CAR-T cells to kill Raji cells was reduced compared to the killing efficiency after 2 nd co-culture period, which indicates that CAR-T cells may be gradually depleted under long-term tumor antigen stimulation, while LSD1shRNA anti-CD19CAR-T cells still have significantly enhanced killing efficiency to Raji cells compared to RNAU6 anti-CD19CAR-T cells, which indicates that LSD1shRNA may be beneficial to the long-term anti-tumor function of anti-CD19CAR-T cells, see fig. 6C and the following table.
1.3 detection of efficiency of LSD1shRNA anti-CD19CAR-T cells in killing Raji-Luc cells in vitro
LSD1shRNA anti-CD19CAR-T cells were co-cultured with Raji-Luc cells stably expressing luciferase, which detected CAR-T cell killing efficiency. The results show that: two groups of LSD1shRNA anti-CD19CAR-T cells have obviously enhanced killing efficiency on Raji-Luc cells compared with RNAU6 anti-CD19CAR-T cells, and are shown in a figure 7 and a table below.
1.4LSD 1shRNA anti-CD19CAR-T cell killing SW620 cell efficiency test.
Inoculating 1X 10 to an E-plate 96 detection plate matched with RTCA4After detection and recording for 48h, 2500 PanT or RNAU6 anti-CD19CAR-T or LSD1 shRNA-1 anti-CD19CAR-T cells (effective target ratio 1:4) were added and recording was continued for 3 days. And after the detection is finished, displaying a data analysis result: the SW620 cell index of the LSD1 shRNA-1 anti-CD19CAR-T cell group is obviously lower than that of the RNAU6 anti-CD19CAR-T cell control group, and is statistically different, as shown in the figure 8 and the following table, which shows that the SW620 cell killing efficiency of the LSD1 shRNA-1 anti-CD19CAR-T cell is obviously enhanced.
1.5LSD 1shRNA anti-CD19CAR-T cell cytokine release level assay
ELISA detects the cytokine release level in the supernatant of the co-cultured cells after LSD1shRNA anti-CD19CAR-T cells and target cells Raji are co-cultured for 12 h. As a result, it was found that: the mean levels of IFN-. gamma.TNF-. alpha.and L-2 release were significantly increased in the cell supernatants, see FIG. 9 and the following table.
Detection of LSD1 shRNA-enhanced anti-CD19CAR-T cell proliferation capacity in vitro
2.1CFSE proliferation assay
RNAU6 anti-CD19CAR-T cells, LSD1 shRNA-1 anti-CD19CAR-T cells and LSD1 shRNA-2 anti-CD19CAR-T cells were stained for CFSE, and the stained cells were taken for flow cytometry analysis. The results show that: CFSE staining among groups was uniform, and the mean fluorescence intensity of FITC signal was not significantly different.
And (3) culturing the CFSE uniformly-stained CAR-T cells for 24h under the condition of antigen stimulation of target cells (the number ratio of effector cells to target cells is 1:2), and detecting the proliferation condition of the CAR-T cells by flow cytometry. The results show that: compared with the two groups of LSD1shRNA anti-CD19CAR-T cells and the RNAU6 anti-CD19CAR-T cells, the average fluorescence intensity of FITC signals is obviously reduced, namely the CFSE signal intensity is obviously reduced, and the proliferation speed of the LSD1shRNA anti-CD19CAR-T cells is obviously higher.
2.2 cell proliferation records.
The cells were cultured for 20 days, counted every 48 hours, and passaged to a concentration of 1X 106one/mL. According to cell counting, cell growth fold is calculated, and an LSD1shRNA anti-CD19CAR-T cell proliferation curve and a survival rate curve are prepared. It can be seen that LSD1shRNA anti-CD19CAR-T cells have enhanced proliferative capacity and persistence after day 15 as compared to RNAU6 anti-CD19CAR-T cells as cell culture time is extended, see figure 10 and table below.
2.3 CD4 in CAR-T cells+T cells and CD8+T cell assay
CD4 in RNAU6 anti-CD19CAR-T cells and LSD1shRNA anti-CD19CAR-T cells when only effector cells are present+T cells and CD8+There was no significant difference in T cell ratios, and LSD1shRNA was not on CD4+T cells and CD8+Changes in the proportion of T cells have a significant effect; when effector cells and target cells Raji are co-cultured for 12h, RNAU6 anti-CD19CAR-T cells and LSD1shRNA anti-CD19CAR-T cells have CD8+T cells were significantly increased, and there was no significant difference between groups, as shown in FIG. 11.
2.4TCM cell assays
The TCM cells express that CD45RO and CD62L molecules are positive, and flow cytometry detects that the content of the TCM cells in LSD1shRNA anti-CD19CAR-T cells is changed, namely the proportion of double positive cell populations of CD45RO and CD62L in a CD3 positive cell population is changed, and the result shows that: the content of TCM cells in LSD1shRNA anti-CD19CAR-T cells is not obviously different from that in control group RNAU6 anti-CD19CAR-T cells.
Example in vivo study of pentaLSD 1shRNA to enhance anti-tumor function of anti-i-CD 19CAR-T cells
1. Construction of tumor animal model
After injecting Raji-Luc cells into NPG mouse tail vein for 4 days, the small animal living body images visible tumor signals, and the average light quantum number is (1.53 +/-0.34) multiplied by 104Thus, the success of the tumor animal model construction is demonstrated.
2. Small animal in vivo imaging tumor monitoring
The result of the small animal living body imaging detection shows that: tumor signals of the model group and the PanT treatment group injected with non-transduced CAR were gradually increased and died, while tumor signals of the mice of the RNAU6 anti-CD19CAR-T cell treatment group and the LSD1shRNA anti-CD19CAR-T cell treatment group disappeared and remained until the 52 th day of the experiment end, no tumor signal was detected, and changes of tumor area size and overall signal intensity are shown in FIG. 12.
3.3 record mouse survival curves
Raji-Luc cells were injected into tail vein on day 0, and survival of mice was continuously monitored until day 52, which indicated that survival time of mice in CAR-T cell treated group was significantly prolonged, as shown in FIG. 13 and the following table.
3.4 mouse weight detection
The weight change of the mice was monitored, and it was seen that the body weight of the model group and PanT group mice sharply decreased around day 20 after the injection of tumor cells, and then died. The body weight of the CAR-T cell treated mice varied smoothly with a tendency to increase slowly, as shown in figure 14 and the table below.
Mouse weight change (g)
3.5 in vivo T cell proliferation level assay CD3 was consistently detected in peripheral blood of CAR-T cell treated mice+T to the end of the experiment. On day 52 after injection of tumor cells Raji-Luc into NPG mice, T cell content was examined by flow cytometry using BV 785-labeled anti-human CD3 antibody, anti-CD19CAR-T cell treated mice peripheral blood CD3+The proportion of the T cells in the peripheral blood nucleated cells is (1.29 +/-0.99)%; the RNAU6 anti-CD19CAR-T cell treatment group was (1.82. + -. 0.94)%; the LSD1 shRNA-1 anti-CD19CAR-T cell treatment group is (5.32 +/-1.17)%, and the LSD1 shRNA-2 anti-CD19CAR-T cell treatment group is (9.52 +/-5.23)%. The T cell number was calculated from the standard curve, i.e.: each 100 mu L of peripheral blood of the mouse contains 5187.04 +/-8329.14 anti-CD19CAR-T cells; 7181.21 + -7951.34 RNAu6 anti-CD19CAR-T cells; LSD1 shRNA-1 anti-CD19CAR-T cells 38597.9 +/-9925.14; LSD1 shRNA-2 anti-CD19CAR-T cell 72228.47 +/-44207.59 pieces. The content of T cells in peripheral blood of mice in the LSD1shRNA anti-CD19CAR-T cell treatment group is obviously higher than that of mice in the RNAU6 anti-CD19CAR-T cell treatment group.
3.6 detection of IFN-. gamma.Release levels in mouse serum
ELISA measures IFN- γ release levels in serum from NPG mice 7 days after the second CAR-T cell injection. The results show that the mean level of IFN-gamma release in the serum of mice in the LSD1shRNA anti-CD19CAR-T cell treatment group is obviously increased, and the results are shown in FIG. 15 and the following table.
It is to be understood that the invention disclosed is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Sequence listing
<110> Beijing university of traditional Chinese medicine
<120> retroviral vector expressing CAR and shRNA and use thereof
<130> PF2133
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 242
<212> PRT
<213> Murine adenovirus (Murine adenovirus)
<400> 1
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu
115 120 125
Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys
130 135 140
Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg
145 150 155 160
Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser
165 170 175
Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile
180 185 190
Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln
195 200 205
Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly
210 215 220
Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
225 230 235 240
Ser Ser
<210> 2
<211> 21
<212> DNA
<213> human (Homo sapiens)
<400> 2
gcctagacat taaactgaat a 21
<210> 3
<211> 21
<212> DNA
<213> human (Homo sapiens)
<400> 3
gctccaatac tgttggcact a 21
<210> 4
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ccgggcctag acattaaact gaatactcga gtattcagtt taatgtctag gctttttg 58
<210> 5
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ccgggctcca atactgttgg cactactcga gtagtgccaa cagtattgga gctttttg 58
<210> 6
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
aattcaaaaa gcctagacat taaactgaat actcgagtat tcagtttaat gtctaggc 58
<210> 7
<211> 58
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
aattcaaaaa gctccaatac tgttggcact actcgagtag tgccaacagt attggagc 58
<210> 8
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
acatctgcag tccaaaggat ggga 24
<210> 9
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
gtactgccaa catgcccgaa caaa 24
- 上一篇：一种医用注射器针头装配设备
- 下一篇：一种大片段同源重组修复方法