WO2018103501A1 - 敲减人PD-1的siRNA、重组表达CAR-T载体及其构建方法和应用 - Google Patents

敲减人PD-1的siRNA、重组表达CAR-T载体及其构建方法和应用 Download PDF

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WO2018103501A1
WO2018103501A1 PCT/CN2017/110653 CN2017110653W WO2018103501A1 WO 2018103501 A1 WO2018103501 A1 WO 2018103501A1 CN 2017110653 W CN2017110653 W CN 2017110653W WO 2018103501 A1 WO2018103501 A1 WO 2018103501A1
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lentiviral
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祁伟
俞磊
康立清
余宙
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Shanghai Unicar Therapy Bio Medicine Technology Co Ltd
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Shanghai Unicar Therapy Bio Medicine Technology Co Ltd
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Priority to KR1020197007198A priority patent/KR102266751B1/ko
Priority to US16/331,147 priority patent/US11242530B2/en
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Definitions

  • the invention belongs to the technical field of tumor immunotherapy, and particularly relates to a siRNA for knocking down human programmed death receptor 1 (PD-1) and a recombinant expression CAR-T vector (especially a method for eliminating or alleviating by knocking down PD-1) CAR-T transgenic vector for tumor immune escape mechanism and its construction method and application.
  • PD-1 human programmed death receptor 1
  • CAR-T vector especially a method for eliminating or alleviating by knocking down PD-1) CAR-T transgenic vector for tumor immune escape mechanism and its construction method and application.
  • tumor immunotherapy The theoretical basis of tumor immunotherapy is that the immune system has the ability to recognize tumor-associated antigens and regulate the body's attack on tumor cells (highly specific cell lysis).
  • Burnet and Thomas proposed the theory of "immuno-monitoring", which believed that the mutant tumor cells that often appear in the body can be cleared by the immune system and lay a theoretical foundation for tumor immunotherapy [Burnet FM.Immunological aspects of malignant disease.Lancet, 1967; 1:171-4].
  • various tumor immunotherapy including cytokine therapy, monoclonal antibody therapy, adoptive immunotherapy, vaccine therapy, and the like are successively applied to the clinic.
  • CAR-T In 2013, a more advanced tumor immunotherapy---CAR-T therapy was successfully used in clinical practice and showed unprecedented clinical efficacy.
  • CAR-T the full name is Chimeric Antigen Receptor T-Cell Immunotherapy, chimeric antigen receptor T cell immunotherapy.
  • CAR-T therapy is the most clinically advanced Novartis CLT019.
  • CLT019 is used to treat patients with relapsed and refractory acute lymphoblastic leukemia.
  • the progression-free survival rate of tumors is 67% for six months, and the longest response time is more than two years. .
  • CAR-T therapy is effective, it encounters many difficulties in the treatment of solid tumors.
  • One of the most important reasons is the PD1/PDL1 inhibitory immune checkpoint (as shown in Figure 1A), which combines the transmission of inhibition signals. It inhibits the immune activity of T cells, plays an important role in immune tolerance, and is also an important reason for the escape of tumor cells.
  • PD-1 (also known as CD279) is an immunosuppressive receptor, a type I transmembrane protein belonging to the CD28 family member, and a programmed cell death molecule-1 receptor in 1992 by Ishida et al [Ishida Y, Agata Y, Shibahara K, et al. Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death [J].
  • the human PD-1 gene is located on chromosome 2q37.35 and encodes a transmembrane glycoprotein of approximately 55 kD.
  • PD-1 is widely expressed on the surface of activated T cells, B cells, monocytes and dendritic cells.
  • PD-1 is structurally 30% homologous to CTLA-4, and two tyrosines are present in the intracellular region.
  • Residues which are involved in an immunoreceptor tyrosine-based inhibitory motif (ITIM) at the N-terminus and an immunoreceptor tyrosine-dependent translation motif at the C-terminus (immunoreceptor tyrosin-based switch) Motif, ITSM);
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • ITSM immunoreceptor tyrosine-based switch
  • the extracellular domain is composed of an IgV-like domain, contains multiple glycosylation sites and is heavily glycosylated, which binds to the ligand and functions to inhibit T cell activation.
  • PD-L1 is overexpressed in most cancer tissues, including NSCLC, melanoma, breast cancer, glioma, lymphoma, leukemia and various urological tumors, digestive tract tumors, germline tumors, etc. [Intlekofer AM, Thompson CB. At the bench: preclinical rationale for CTLA-4 and PD-1 blockade as cancer immunotherapy [J]. J Leukoc Biol, 2013, 94(1): 25-39.]. Parsa is found in mouse and human tumor cells. IFN- ⁇ and IFN- ⁇ secreted by T cells can induce high expression of PD-L1 on tumor cells [Ding H, Wu X, Wu J, et al.
  • PD-1 inhibitory signal concomitant with blocking ICOS co-stimulation suppresses Lupus-like syndrome in autoimmune BXSB mice [J]. Clin Immunol, 2006, 118(2/3): 258-267.].
  • High expression of PD-L1 can inhibit the expression of cell cycle checkpoint proteins and cell proliferation-related proteins by inhibiting RAS and PI3K/AKT signaling pathways, ultimately leading to inhibition of T cell proliferation [11].
  • Dong et al. and mouse models also found that activation of PD-1/PD-L1 signaling pathway can induce specific CTL regulation.
  • the object of the present invention is to overcome the deficiencies of the prior art, and provide a siRNA for knocking down human PD-1, a recombinant expression CAR-T vector, a construction method thereof and application thereof
  • a first object of the present invention is to provide an siRNA which knocks down human PD-1, which is selected from any of the following a-g:
  • nucleotide sequence represented by a. SEQ ID NO. 41 and SEQ ID NO. 42 is preferred.
  • a second object of the present invention is to provide the use of the above siRNA for the preparation of a medicament for eliminating or reducing the immune escape mechanism of tumors.
  • a third object of the present invention is to provide a recombinant expression vector comprising the above siRNA.
  • the expression vector is a lentiviral expression vector, a retroviral expression vector, an adenovirus expression vector, an adeno-associated virus expression vector or a plasmid; preferably, a lentiviral expression vector comprising the above siRNA.
  • the lentiviral expression vector comprises: a prokaryotic replicon pUC Ori sequence for plasmid replication, as shown in SEQ ID NO. 2; an ampicillin resistance gene AmpR sequence for large-scale amplification of a target strain, As shown in SEQ ID NO. 1; a viral replicon SV40Ori sequence for enhancing replication in eukaryotic cells, as set forth in SEQ ID NO. 3; a lentiviral packaging cis element for lentiviral packaging; Nuclear cells express green fluorescent ZsGreen1 green fluorescent protein as shown in SEQ ID NO. 11; IRES ribosomal binding sequences for co-transcription of expressed proteins, as shown in SEQ ID NO.
  • chimeric antigen receptor genes Eukaryotic transcribed human EF1 ⁇ promoter, as shown in SEQ ID NO. 15; a gene encoding an anti-BCMA chimeric antigen receptor for a second generation CAR or a third generation CAR that constitutes a collection recognition, delivery, initiation; eWPRE-enhanced woodchuck hepatitis B virus post-transcriptional regulatory element for enhancing transgene expression efficiency, as shown in SEQ ID NO. 13; human RNA polymerase III promoter hU6 for intracellular transcription of siRNA, such as SEQ ID NO. 14 is shown.
  • the lentiviral packaging cis element comprises a second generation lentiviral vector comprising: lentiviral 5terminal LTR as shown in SEQ ID NO. 5, lentivirus 3terminal Self-Inactivating LTR as shown in SEQ ID NO. a Gag cis element as set forth in SEQ ID NO. 7, an RRE cis element as set forth in SEQ ID NO. 8, an env cis element as set forth in SEQ ID NO. 9, as set forth in SEQ ID NO. Shown as a cPPT cis component.
  • the lentiviral packaging cis element comprises a third generation lentiviral vector comprising: lentiviral 5terminal LTR as shown in SEQ ID NO. 5, lentivirus 3terminal Self-Inactivating LTR as shown in SEQ ID NO. a Gag cis element as shown in SEQ ID NO. 7, an RRE cis element as shown in SEQ ID NO. 8, an env as shown in SEQ ID NO.
  • the cis element, the lentiviral packaging cis element of the cPPT cis element as set forth in SEQ ID NO. 10, and the RSV promoter as set forth in SEQ ID NO.
  • the eWPRE-enhanced woodchuck hepatitis B virus post-transcriptional regulatory element has a 6-nucleotide enhancing mutation, specifically: g.396G>A, g.397C>T, g.398T>C, g. 399G>A, g.400A>T, g.411A>T.
  • the anti-BCMA chimeric antigen receptor comprises a CD8 leader chimeric receptor signal peptide as shown in SEQ ID NO. 16 in tandem, BCMA as shown in SEQ ID NO. Single-chain antibody light chain VL, Optimal Linker C as shown in SEQ ID NO. 18, BCMA single-chain antibody heavy chain VH as shown in SEQ ID NO. 19, CD8 Hinge chimeric as shown in SEQ ID NO. a receptor hinge, a CD8 Transmembrane chimeric receptor transmembrane region as set forth in SEQ ID NO. 21, a CD137 chimeric receptor co-stimulatory factor as set forth in SEQ ID NO. 22, and as set forth in SEQ ID NO.
  • the TCR chimeric receptor T cell activation domain comprises a CD8 leader chimeric receptor signal peptide as shown in SEQ ID NO. 16 in tandem, BCMA as shown in SEQ ID NO. Single-chain antibody light chain VL, Optimal Linker C as shown in SEQ ID NO. 18, BCMA single-chain antibody
  • the anti-BCMA chimeric antigen receptor (third generation CAR) comprises a CD8 leader chimeric receptor signal peptide as shown in SEQ ID NO. 16 in tandem, a BCMA single as shown in SEQ ID NO. Chain antibody light chain VL, Optimal Linker C as set forth in SEQ ID NO. 18, BCMA single chain antibody heavy chain VH as set forth in SEQ ID NO. 19, CD8 Hinge chimeric as shown in SEQ ID NO. a body hinge, a CD8 Transmembrane chimeric receptor transmembrane region as set forth in SEQ ID NO. 21, a CD28 chimeric receptor co-stimulatory factor as set forth in SEQ ID NO. 24, CD137 as set forth in SEQ ID NO. Chimeric receptor costimulatory factor and TCR chimeric receptor T cell activation domain as set forth in SEQ ID NO.
  • a fourth object of the present invention is to provide a method for constructing a lentiviral expression vector comprising the above siRNA, comprising the steps of:
  • an ampicillin resistance gene AmpR sequence (as shown in SEQ ID NO. 1), a prokaryotic replicon pUC Ori sequence (as shown in SEQ ID NO. 2), a viral replicon SV40Ori sequence (such as SEQ ID NO) .3), lentiviral packaging cis-element for lentiviral packaging, ZsGreen1 green fluorescent protein (as shown in SEQ ID NO. 11), IRES ribosome binding sequence (shown as SEQ ID NO. 12), The eWPRE-enhanced woodchuck hepatitis B virus post-transcriptional regulatory element (as shown in SEQ ID NO. 13) and the human RNA polymerase III promoter hU6 (shown as SEQ ID NO. 14) are stored in a lentiviral backbone plasmid (pLenti-). 3G silencer);
  • the recombinant lentiviral plasmid (pCARbcma-1453 to pCARbcma-1460) obtained in the step (3) was co-transfected into the HEK293T/17 cells together with the lentiviral packaging plasmids pPac-GP, pPac-R and membrane protein granule pEnv-G. After gene transcriptional expression in HEK293T/17 cells, the packaged recombinant recombinant lentiviral vector is released into the cell culture supernatant, and the supernatant of the recombinant lentiviral vector contained is collected;
  • the obtained recombinant lentiviral supernatant was purified by ion filtration using suction filtration, adsorption and elution, respectively, to obtain a recombinant lentiviral vector.
  • the lentiviral packaging cis element adopts a second generation lentiviral vector comprising: the lentivirus 5terminal LTR as shown in SEQ ID NO. 5, and the slow as shown in SEQ ID NO. Virus 3terminal Self-Inactivating LTR, a Gag cis element as shown in SEQ ID NO. 7, an RRE cis element as shown in SEQ ID NO. 8, an env cis element as shown in SEQ ID NO. 9, such as a cPPT cis element as set forth in SEQ ID NO. 10; the lentiviral packaging cis element employing a third generation lentiviral vector comprising: a lentiviral 5terminal LTR as set forth in SEQ ID NO.
  • the lentivirus 3terminal Self-Inactivating LTR shown the Gag cis element as shown in SEQ ID NO. 7, as set forth in SEQ ID NO.
  • the RRE cis element shown the env cis element as set forth in SEQ ID NO. 9, the lentiviral packaging cis element as shown in SEQ ID NO. 10, and the SEQ ID NO. 4
  • the RSV promoter shown the RSV promoter shown.
  • the anti-BCMA chimeric antigen receptor for the second generation CAR which is composed of the set recognition, transmission and initiation is integrated, and the tandem sequence is as shown in SEQ ID NO. CD8 leader chimeric receptor signal peptide, BCMA single chain antibody light chain VL as shown in SEQ ID NO. 17, Optimal Linker C as shown in SEQ ID NO. 18, BCMA single as shown in SEQ ID NO. Chain antibody heavy chain VH, CD8 Hinge chimeric receptor hinge as set forth in SEQ ID NO. 20, CD8 Transmembrane chimeric receptor transmembrane region as set forth in SEQ ID NO. 21, as set forth in SEQ ID NO.
  • an antigen receptor comprising a CD8 leader chimeric receptor signal peptide as shown in SEQ ID NO. 16 in tandem, a BCMA single chain antibody light chain VL as set forth in SEQ ID NO. 17, as SEQ ID NO. Optimal Linker C shown, BCMA single-chain antibody heavy chain VH as shown in SEQ ID NO. 19, CD8 Hinge chimeric receptor as set forth in SEQ ID NO.
  • CD8 Transmembrane chimeric receptor transmembrane region as set forth in SEQ ID NO. 21, CD28 chimeric receptor co-stimulatory factor as set forth in SEQ ID NO. 24, CD137 as shown in SEQ ID NO.
  • the eWPRE-enhanced woodchuck hepatitis B virus post-transcriptional regulatory element has a 6-nucleotide enhancing mutation, specifically: g.396G>A, g.397C>T, g. 398T>C, g.399G>A, g.400A>T, g.411A>T.
  • step (2) the entire CAR gene expression is initiated by the human EF1 ⁇ promoter; the CD8 leader chimeric receptor signal peptide is located at the N-terminus of the CAR coding sequence, and is used to guide the CAR protein localization to the cell membrane; the CD19 single-chain antibody is light.
  • Optimal Linker C CD19 single-chain antibody heavy chain VH combined into a scfv region for recognition of CD19 antigen; CD8 Hinge chimeric receptor hinge for anchoring scfv to the outer side of the cell membrane; CD8 Transmembrane chimeric receptor transmembrane The region is used to immobilize the entire chimeric receptor on the cell membrane; the CD137 chimeric receptor co-stimulatory factor is used to stimulate T cell proliferation and cytokine secretion; the TCR chimeric receptor T cell activation domain is used to activate expression of downstream signaling pathway When the scfv region binds to the CD20 antigen, the signal is transmitted to the cell through the chimeric receptor, thereby producing a one including T cell proliferation, increased cytokine secretion, increased secretion of anti-apoptotic protein, delayed cell death, and lysis of target cells. A series of biological effects.
  • the lentiviral vector has a version with a fluorescent tag zsGreen1 and a version without a fluorescent tag zsGreen1, a version with a fluorescent tag is used for in vitro experiments, and a version without a fluorescent tag is used for clinical experiments.
  • the suction filtration step controls the supernatant volume to be 200 ml to 2000 ml, and the vacuum degree is controlled at -0.5 MPA to -0.9 MPA to prevent carrier loss due to plugging; the adsorption step is controlled The pH of the solution is 6-8, which prevents the change of pH from causing the carrier to be inactivated; the elution step controls the ionic strength of the eluent to be 0.5M-1.0M, preventing the change of ionic strength from causing incomplete elution or carrier loss. live.
  • a fifth object of the present invention is to provide a use of a recombinant expression vector comprising the above siRNA for the preparation of a drug for eliminating or reducing the immune escape mechanism of a tumor during the treatment of a tumor by CAR-T.
  • a sixth object of the present invention is to provide a CART cell which is a T lymphocyte modified by the above siRNA.
  • a further object of the present invention is to provide the use of the above CAR-T cells for the preparation of a medicament for treating multiple myeloma, pancreatic cancer, glioma, and myeloma.
  • RNAi has very good sequence specificity [Kisielow, M. et al. (2002) Isoform-specific knockdown and expression of adaptor protein ShcA using small interfering RNA, J. of Biochemistry 363: 1-5].
  • the RNAi system can specifically knock down one type of transcript without affecting other mRNAs with similar sequences. These properties make the siRNA system show potential and value in inhibiting gene expression, gene function research, and drug target validation.
  • the siRNA system can be used to treat related diseases, including (1) diseases caused by gene overexpression or misexpression; (2) diseases caused by gene mutations.
  • the invention relates to human RNA polymerase III promoter hU6, human EF1 ⁇ promoter, CD8 leader chimeric receptor signal peptide, BCMA single chain antibody light chain VL, Optimal Linker C, BCMA single chain antibody heavy chain VH, CD8 Hinge Receptor hinge
  • the chain, CD8 Transmembrane chimeric receptor transmembrane region, CD137 chimeric receptor co-stimulatory factor, TCR chimeric receptor T cell activation domain were constructed into a recombinant lentiviral vector, and the entire CAR gene expression was initiated by the human EF1 ⁇ promoter.
  • the CAR protein localizes on the surface of the cell membrane, recognizes BCMA antigen, stimulates T cell proliferation and cytokine secretion, and activates the expression of downstream signaling pathways.
  • the scfv region binds to the BCMA antigen, the signal is transmitted to the cell through the chimeric receptor, thereby producing a series of organisms such as T cell proliferation, increased cytokine secretion, increased secretion of anti-apoptotic protein, delayed cell death, and lysis of target cells. Learning effect.
  • PD1 siRNA was initiated by the human RNA polymerase III promoter hU6, which degraded PD-1 mRNA, inhibited PD-1 protein synthesis, blocked PD1 binding to PDL1, and blocked PD1/PDL1 signaling.
  • the pathway achieves the effect of suppressing immune escape.
  • the expression vector used in the present invention includes a prokaryotic replicon (pUC ori) for plasmid replication; a prokaryotic selection marker (AmpR) for large-scale amplification of a target strain; and a viral replicon (SV40Ori) for enhancing replication in eukaryotic cells; Lentiviral packaging cis-elements (RSV, 5terminal LTR, 3terminal Self-Inactivating LTR, Gag, RRE, env, cPPT) for lentiviral packaging; human RNA polymerase III promoter (hU6) for intracellular transcription of siRNA; Nuclear fluorescent tagged protein (ZsGreen1) is used to express green fluorescence in eukaryotic cells; co-expression element (IRES) is used for co-transcription and expression of proteins; eukaryotic promoter (EF1 ⁇ ) is used for eukaryotic transcription of chimeric antigen receptor genes; Anti-receptor (CD8 leader, BCMA VL, Optimal Link
  • the invention relates to a pharmaceutical preparation containing a peptide, in particular to:
  • the lentiviral vector can achieve the expression of the BCMA chimeric antigen receptor on human T lymphocytes, and induce and activate the killing effect of T lymphocytes on BCMA positive cells, and is clinically used for the treatment of multiple myeloma (MM).
  • MM multiple myeloma
  • siRNA that expresses programmed death receptor 1 (PD-1) in human T lymphocytes, effectively reduces the expression level of T cell surface programmed death receptor 1 and blocks PD-1/PD-L1 immunoregulatory signaling pathway It can be used clinically to inhibit the immune escape of tumors and improve the efficacy of CAR-T cell immunotherapy.
  • PD-1 programmed death receptor 1
  • the present invention employs siRNA gene silencing technology for PD-1, and since the 1990s, researchers have discovered that double-stranded RNA (“dsRNA”) can be used to inhibit protein expression. This ability to silence genes has broad potential for treating human diseases, and many researchers and commercial entities are currently investing considerable resources in the development of treatments based on this technology.
  • dsRNA double-stranded RNA
  • RNA interference-2001 Genes Dev. 2001, 15: 485
  • the type III ribonuclease Dicer decomposes the dsRNA into a 19-23 bp siRNA with a 2 base bulging 3' cohesive terminus.
  • Bosset, Caudy, Hammond, & Hannon Role for a bidentate ribonuclease in the initiation step of RNA interference, Nature 2001, 409: 363].
  • the siRNA is integrated with an RNA-induced silencing complex (RISC) in which one or more helicases unwind the double-stranded siRNA, allowing the complementary antisense strand to direct target recognition.
  • RISC RNA-induced silencing complex
  • one or more endonucleases in the RISC complex cleave the target mRNA to cause mRNA silencing.
  • endonucleases in the RISC complex cleave the target mRNA to cause mRNA silencing.
  • RISC RNA-induced silencing complex
  • the PD1 siRNA sequence designed by the invention contains 21 bp nucleotides, adopts an oligonucleotide arrangement pattern of N2[CG]N8[AU]N8[AU]N2, and adopts a stem-loop hairpin structure between complementary sequences. Screening of conditions such as siRNA pattern, GC percentage, T or A or G in a row, consecutiver GC, 3'end nt pattern, thermodynamic value, siRNA target, identity, alignment, etc., greatly improved the interference success rate of siRNA design.
  • the human RNA polymerase III promoter hU6 used in the present invention transcribes PD1 siRNA and inhibits the expression of PD-1.
  • QPCR detection can effectively inhibit the transcription level of PD-1 mRNA and effectively reduce T cells.
  • the expression level of PD-1 receptor on the surface, thereby blocking the signaling pathway of PD-1/PDL-1, can inhibit the immune escape; in the future, it can be used clinically to inhibit the expression of PD-1 in CAR-T cells. Enhance the killing effect of CAR-T cells on tumors in vivo.
  • the present invention delivers siRNA using a lentiviral vector (as shown in Figure 2). First, it saves costs and avoids the expensive cost of in vitro synthesis of PD1 siRNA. Secondly, the problem of inefficient delivery of PD1 siRNA is avoided. Third, the expression of PD1 siRNA using the human RNA polymerase III promoter hU6 can effectively utilize the intracellular RNA transcription system and express a large amount of corresponding PD1 siRNA. After a series of enzymatic actions, a good gene silencing effect can be achieved.
  • the vector backbone used in the present invention is a third-generation lentiviral vector (as shown in FIG. 3A), and the 3'SIN LTR removes the U3 region, eliminating the possibility of self-replication of the lentiviral vector, greatly improving safety; cPPT and WPRE elements improve transduction efficiency and transgene expression efficiency; using RSV promoter ensures sustained and efficient transcription of core RNA during lentiviral vector packaging; using human's own EF1 ⁇ promoter, the CAR gene can be long in human body Time continues to be expressed.
  • the siRNA knockdown scheme adopted by the present invention can also be applied to the third generation CAR design scheme.
  • the third generation CAR design increased the CD28 chimeric receptor costimulatory factor (SEQ ID NO. 24) compared to the second generation design.
  • the lentiviral vector column purification system (shown in Figure 8) used in the present invention is a lentivirus large-scale production process developed by the company.
  • the commonly used ultracentrifugation method or high-speed centrifugation method uses the principle of centrifugal sedimentation to separate lentivirus particles, and inevitably, many impurities with similar sedimentation coefficients remain, which adversely affects subsequent experiments.
  • the tube loading process is complicated, the operation is cumbersome, and multiple conversion containers bring more pollution opportunities.
  • the purification process of the lentiviral vector column of the invention is semi-automatic operation, and all processes are completed in the 100-level experimental area, thereby avoiding the cumbersome and polluting probability of manual operation, and the recovered lentiviral vector completely reaches the clinical endotoxin and mycoplasma indexes. standard.
  • Follow-up can be followed up to develop a fully automated purification instrument.
  • the CAR design adopted in the present invention can also be applied to the second generation lentiviral vector structure.
  • the structural difference between the second and third generation lentiviral vectors (as shown in Figure 3B), mainly the third generation of lentiviral vectors replacing the U3 region of the second generation vector 5'LTR with the RSV promoter, thus Elimination of Tat protein dependence during U3 transcription can remove Tat sequences in lentiviral structural genes and increase lentiviral genome transcription levels and transcriptional persistence.
  • the second and third generation lentiviral vectors are mainly distinguished by the transcript pattern of the genome, and thus the CAR design scheme used in the present invention can be applied to the two generations of lentiviral vectors.
  • the third generation lentiviral backbone plasmid of the present invention adopts the enhancedWPRE element and the University of Pennsylvania Carl H. June et al. (Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor modified T cells in chronic lymphoid leukemia .N Engl J Med 2011;365:725-33.)
  • the WPRE element used there are 6 nucleotides of enhanced mutations (g.396G>A, g.397C>T, g.398T>C, g.399G>A, g.400A>T, g.411A>T), can enhance the polyadenylation of primary transcription products, increase the content of mRNA in cells, and enhance the expression efficiency of transgenes.
  • the Lentival packaging system of the present invention is a four-plasmid packaging system without helper virus, which is co-transfected into HEK293T/17 cells by four plasmids to produce a recombinant lentiviral vector.
  • the recombinant lentiviral vector is a replication-defective vector, which can integrate the exogenous fragment into the host gene, can be used once, cannot be replicated and propagated, and the safety is greatly improved.
  • the lentiviral vectors employed in the present invention are available in fluorescently labeled version zsGreen1 and in the absence of fluorescent labeling zsGreen1, fluorescently labeled versions for in vitro experiments, and fluorescently labeled versions for clinical trials.
  • the Linker design of the scfv segment used in the present invention can significantly increase the secretion of cytokines, the killing effect of CAR-T cells in vitro and the clinical therapeutic effect.
  • the recombinant lentiviral vector of the present invention provides a reliable transgene for CAR-T treatment of multiple myeloma (MM). At the same time of protection, it can block the immune escape mechanism of tumors, improve the efficacy of CAR-T cell therapy, and greatly reduce the medical costs incurred by patients.
  • the PD-1 knockdown siRNA expression cassette and the siRNA expression product thereof of the invention can be applied not only to the immune escape mechanism for eliminating or alleviating tumors in CAR-T treatment of multiple myeloma (MM), but also It inhibits the immune escape mechanism of CAR-T in the treatment of tumors such as pancreatic cancer, glioma, myeloma and the like.
  • Figure 1 is a PD-1/PD-L1 signal pathway of the present invention
  • Figure 2 is a schematic illustration of the manner in which the lentivirus of the present invention delivers PD1 siRNA
  • FIG. 3 is a schematic diagram showing the structure of a lentiviral vector of the present invention.
  • FIG. 3(A) is a schematic diagram showing the structure of a third generation lentiviral vector used in the present invention
  • FIG. 3(B) is a second generation and a third generation lentiviral vector structure.
  • Figure 4 is a flow chart showing the construction of the recombinant lentiviral vector of the present invention.
  • A is a schematic diagram of the structure of the lentiviral backbone plasmid pLenti-3G silencer
  • B is a schematic diagram of the structure of the pCARbcma-silencer plasmid
  • the figure is a schematic diagram of the pCARbcma-1453 ⁇ pCARbcma-1460 plasmid
  • D is a schematic diagram of the lentiviral packaging plasmid pPac-GP
  • E is a schematic diagram of the lentiviral packaging plasmid pPac-R
  • F is Schematic diagram of the membrane protein pEnv-G;
  • Figure 5 is a diagram showing the restriction enzyme digestion and enzymatic cleavage agarose gel electrophoresis of the lentiviral backbone plasmid pLenti-3G silencer;
  • Figure 5A is a schematic diagram of the restriction enzyme digestion of the lentiviral backbone plasmid pLenti-3G silencer, wherein lane1 is 1 kb DNA Ladder Marker: The bands are from top to bottom: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp; lane2 is pLenti-3G silencer Cla I+BamH I digestion prediction: the bands were: 7381 bp, 23 bp from top to bottom;
  • Figure 5B is the enzymatic cleavage agarose gel electrophoresis map of the lentiviral backbone plasmid pL
  • Figure 6 is a restriction diagram of restriction endonuclease digestion and enzymatic cleavage agarose gel electrophoresis of recombinant lentiviral plasmid pCARbcma-silencer; wherein, Figure 6A is a schematic diagram of restriction enzyme digestion of recombinant lentiviral plasmid pCARbcma-silencer, wherein lane1 is 1 kb DNA ladder Marker : The bands are: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp; Lane2 is the Pvu I enzyme of pCARbcma-silencer Cut prediction: the bands were: 8988 bp, 896 bp, and 249 bp from top to bottom; Figure 6B is an enzymatic cleavage agarose
  • Figure 7 is a sequence alignment of PD-1 knockdown recombinant lentiviral plasmids pCARbcma-1453 to pCARbcma-1460; wherein A is the sequencing alignment of pCARbcma-1453; B is the sequencing alignment of pCARbcma-1454; Is the sequencing alignment of pCARbcma-1455; D is the sequencing alignment of pCARbcma-1456; E is the sequencing alignment of pCARbcma-1457; F is the sequencing alignment of pCARbcma-1458; G is the sequencing of pCARbcma-1459 Alignment results; H is the sequencing alignment of pCARbcma-1460;
  • Figure 8 is a flow chart of purification of recombinant lentiviral vector by ion exchange chromatography
  • Figure 9 is a titer detection result of a recombinant lentiviral vector
  • Figure 10 shows the results of mycoplasma detection in different purification methods of recombinant lentiviral vector.
  • Lane1 is DL2000marker, and the bands from top to bottom are from top to bottom: 2kb, 1kb, 750bp, 500bp, 250bp, 100bp; lane2 is positive.
  • lane3 is a negative control
  • lane4 is PBS
  • lane5 is water
  • lane6 is lvCARbcma-1453
  • lane7 is lvCARbcma-1454
  • lane8 is lvCARbcma-1455
  • lane9 is lvCARbcma-1456
  • lane10 is lvCARbcma-1457
  • lane11 is lvCARbcma-1458
  • lane12 is lvCARbcma-1459
  • lane13 is lvCARbcma-1460;
  • Figure 11 is a bar graph of relative expression levels of mRNA
  • Figure 12 is a WB detection map of CAR protein expression; wherein, Figure 12A, lane1 is a PBMC empty cell, lane2 is a control virus MOCK, lane3 is lvCARbcma-1453, lane4 is lvCARbcma-1454, lane5 is lvCARbcma-1455, and lane6 is lvCARbcma -1456, lane7 is lvCARbcma-1457, lane8 is lvCARbcma-1458, lane9 is lvCARbcma-1459, lane10 is lvCARbcma-1460; Figure 12B is beta-actin internal reference strip;
  • Figure 13 shows PD-1 knockdown recombinant lentivirus lvCARbcma-1453 ⁇ lvCARbcma-1460 transduced CAR-T cells. After incubation with target cells for 24 hours, RT-QPCR was used to detect PD-1 mRNA transcription level.
  • Figure 14 shows the killing of target cells after 24 h of different effector cells and target cells in a ratio of 10:1, E is an effector cell, and T is a target cell.
  • Lentiviral backbone plasmid pLenti-3G silencer lentiviral packaging plasmid pPac-GP, pPac-R and membrane protein granule pEnv-G, HEK293T/17 cells, homologous recombinase, Oligo Annealing Buffer by Shi (Shanghai) Provided by Pharmaceutical Technology Co., Ltd.;
  • Primers designed to amplify DNA fragments and target sites according to primer design principles. The primers were synthesized by Shanghai Biotech Co., Ltd., specifically:
  • CD8 leader-F 5'-GGTGTCGTGAGGATCCGCCACCATGGCCTTACCAGTGACCGC-3' (SEQ ID NO. 27)
  • CD8 leader-R 5'-GTGTCATCTGGATGTCCGGCCTGGCGGCGTG-3' (SEQ ID NO. 28)
  • VL-F 5'-CACGCCGCCAGGCCGGACATCCAGATGACCCAGAGCC-3' (SEQ ID NO. 29)
  • VL-R 5'-ACGCTTGATCTCCAGTTTGGT-3' (SEQ ID NO. 30)
  • OLC-VH-F 5'-ACTGGAGATCAAGCGTGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGG
  • VH-R 5'-GCTGGACACGGTCACTAGTGTG-3' (SEQ ID NO. 32)
  • CD8 Hinge-F 5'-AGTGACCGTGTCCAGCACCACGACGCCAGCGCC-3' (SEQ ID NO. 33)
  • CD8 Hinge-R 5'-GTAGATATCACAGGCGAAGTCCA-3' (SEQ ID NO. 34)
  • CD8 Transmembrane-F 5'-CGCCTGTGATATCTACATCTGGGCGCCCTTGGC-3' (SEQ ID NO. 35)
  • CD8 Transmembrane-R 5'-TCTTTCTGCCCCGTTTGCAGTAAAGGGTGATAACCAGTG-3' (SEQ ID NO. 36)
  • CD137-F 5'-AAACGGGGCAGAAAGAAACTC-3' (SEQ ID NO. 37)
  • CD137-R 5'-TGCTGAACTTCACTCTCAGTTCACATCCTCCTTCTTCTTC-3' (SEQ ID NO. 38)
  • TCR-F 5'-AGAGTGAAGTTCAGCAGGAGCG-3' (SEQ ID NO. 39)
  • TCR-R 5'-GGAGAGGGGCGTCGACTTAGCGAGGGGGCAGGGC-3' (SEQ ID NO. 40)
  • siRNA1453-F 5'-CCGGCTAAACTGGTACCGCATGAGCCTCGAGTCATGCGGTACCAGTTTAGCATTTTTTG-3' (SEQ ID NO. 41)
  • siRNA1453-R 5'-AATTCAAAAAATGCTAAACTGGTACCGCATGACTCGAGGCTCATGCGGTACCAGTTTAG-3' (SEQ ID NO. 42)
  • siRNA1454-F 5'-CCGGCATTGTCTTTCCTAGCGGAATCTCGAGTCCGCTAGGAAAGACAATGGTTTTTTTG-3' (SEQ ID NO. 43)
  • siRNA1454-R 5'-AATTCAAAAAACCATTGTCTTTCCTAGCGGACTCGAGATTCCGCTAGGAAAGACAATG-3' (SEQ ID NO. 44)
  • siRNA 1455-F 5'-CCGGAGGCGCAGATCAAAGAGAGTTCTCGAGCTCTTTGATCTGCGCCTTGTTTTTTG-3' (SEQ ID NO. 45)
  • siRNA 1455-R 5'-AATTCAAAAAACAAGGCGCAGATCAAAGAGAGCTCGAGAACTCTCTTTGATCTGCGCCT-3' (SEQ ID NO. 46)
  • siRNA 1456-F 5'-CCGGCCCTGTGGTTCTATTATATTACTCGAGATATAATAGAACCACAGGGAATTTTTTG-3' (SEQ ID NO. 47)
  • siRNA1456-R 5'-AATTCAAAAAATTCCCTGTGGTTCTATTATATCTCGAGTAATATAATAGAACCACAGGG-3' (SEQ ID NO. 48)
  • siRNA 1457-F 5'-CCGGGGAACCCATTCCTGAAATTATCTCGAGAATTTCAGGAATGGGTCCAATTTTTTG-3' (SEQ ID NO. 49)
  • siRNA 1457-R 5'-AATTCAAAAAATTGGAACCCATTCCTGAAATTCTCGAGATAATTTCAGGAATGGGTTCC-3' (SEQ ID NO. 50)
  • siRNA 1458-F 5'-CCGGCAGGCCTAGAGAAGTTTCAGGCTCGAGTGAAACTTCTCTAGGCCTGCATTTTTTG-3' (SEQ ID NO. 51)
  • siRNA 1458-R 5'-AATTCAAAAAATGCAGGCCTAGAGAAGTTTCACTCGAGCCTGAAACTTCTCTAGGCCTG-3' (SEQ ID NO. 52)
  • siRNA 1459-F 5'-CCGGCAGGACTCATGTCTCAATGCCCTCGAGCATTGAGACATGAGTCCTGTGTTTTTTG-3' (SEQ ID NO. 53)
  • siRNA 1459-R 5'-AATTCAAAAAACACAGGACTCATGTCTCAATGCTCGAGGGCATTGAGACATGAGTCCTG-3' (SEQ ID NO. 54)
  • siRNA 1460-F 5'-CCGGTTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAATTTTTTG-3' (SEQ ID NO. 55)
  • siRNA 1460-R 5'-AATTCAAAAAATTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAA-3' (SEQ ID NO. 56)
  • PD-1-QPCR-F 5'-TGCAGCTTCTCCAACACAT-3' (SEQ ID NO. 57)
  • PD-1-QPCR-R 5'-CTTGTCCGTCTGGTTGCT-3' (SEQ ID NO. 58)
  • WPRE-QPCR-F 5'-CCTTTCCGGGACTTTCGCTTT-3' (SEQ ID NO. 59)
  • WPRE-QPCR-R 5'-GCAGAATCCAGGTGGCAACA-3' (SEQ ID NO. 60)
  • Actin-QPCR-F 5'-CATGTACGTTGCTATCCAGGC-3' (SEQ ID NO. 61)
  • Actin-QPCR-R 5'-CTCCTTAATGTCACGCACGAT-3' (SEQ ID NO. 62)
  • CAR-QPCR-F 5'-GACTTGTGGGGTCCTTCTCCT-3' (SEQ ID NO. 63)
  • the DNA sequence shown in SEQ ID NO. 15 to SEQ ID NO. 64 is synthesized by Shanghai Jierui Bioengineering Co., Ltd. and stored as an oligonucleotide dry powder or a plasmid;
  • the tool enzymes BspE I, EcoR I, BamH I, Pvu I, Cla I, and T4 DNA ligase were purchased from NEB Corporation;
  • the plasmid extraction kit and the agarose gel recovery kit were purchased from MN Company;
  • Competent cells TOP10 were purchased from tiangen;
  • Opti-MEM FBS, DMEM, 1640, Pen-Srep, Hepes, purchased from Invitrogen;
  • Biotinylated protein L was purchased from GeneScript;
  • ECL+plusTM Western blotting system was purchased from Amersham;
  • DNeasy kit was purchased from Shanghai Jierui Company;
  • Lymphocyte separation solution was purchased from Shenzhen Dakco as the company;
  • SA-HRP was purchased from Shanghai Shengsheng Company;
  • Mycoplasma test kit endotoxin test kit, BCMA-K562, BCMA-PDL1-K562 cell line were purchased from Shiyi (Shanghai) Co., Ltd.;
  • the LDH test kit was purchased from Promega.
  • the construction method of the recombinant lentiviral vector of the present invention is as follows:
  • the lentiviral backbone plasmid pLenti-3G silencer was digested with Cla I and BamH I restriction enzymes, and the product was subjected to 1.5% agarose gel electrophoresis to confirm the 7381 bp fragment V1 (shown in Figure 5). And the tapping recovery was placed in an Eppendorf tube, and the corresponding fragment was recovered using MN's agarose gel recovery kit (see Table 1), and the purity and concentration of the product were determined;
  • sol The sol solution was added in a ratio of 200 ⁇ l NTI/100 mg gel, and placed in a water bath at 50 ° C for 5-10 minutes. 2, combined with DNA Centrifuge at 11,000 g for 30 seconds and discard the filtrate. 3, wash the film 700 ⁇ l of NT3 was added and centrifuged at 11,000 g for 30 seconds, and the filtrate was discarded. 4, wash the film Repeat the third step once 5, dry Centrifuge at 11000g for 1 minute, replace with a new collection tube and leave it at room temperature for 1 minute. 6, eluting DNA 15-30 ⁇ l of NE was added, and the mixture was allowed to stand at room temperature for 1 minute, centrifuged at 11,000 g for 1 minute, and the filtrate was collected.
  • the PCR cycle conditions were: 98 ° C for 3 min, (98 ° C for 10 sec, 55 ° C for 15 sec, 72 ° C) 30 sec) * 35 cycles, 72 ° C for 5 min.
  • the product was subjected to 1.5% agarose gel electrophoresis, and the 336 bp fragment c was confirmed, and the gel was collected and placed in an Eppendorf tube, and the corresponding fragment was recovered by MN's agarose gel recovery kit (see Table 1), and the product was determined. Purity and concentration;
  • the product was subjected to 1.5% agarose gel electrophoresis, and the 704 bp fragment j was confirmed, and the gel was recovered and placed in an Eppendorf tube, and the corresponding fragment was recovered by MN's agarose gel recovery kit (see Table 1), and the product was determined. Purity and concentration;
  • the clones were picked for colony PCR identification, and the correct clone was identified as recombinant lentiviral plasmid pCARbcma-silencer, and the correct clones were identified by enzyme digestion (see Figure 6).
  • the recombinant lentiviral plasmid pCARbcma-silencer was digested with BspE I and EcoR I restriction enzymes, and the product was subjected to 1.5% agarose gel electrophoresis to confirm the fragment V2 of 10035 bp, and the tapping recovery was carried out in Eppendorf. In the tube, the corresponding fragment was recovered by MN's agarose gel recovery kit (see Table 1), and the purity and concentration of the product were determined;
  • the synthesized siRNA1453-F/R ⁇ siRNA1460-F/R was dissolved in 20 ⁇ M with oligo annealing buffer, and 30 ⁇ l of each of F and R was mixed.
  • the siRNA1453-F&R ⁇ siRNA1460-F&R mixture was then heated in a water bath at 95 °C for 5 minutes, then the water bath was opened and allowed to cool to room temperature at room temperature to form a double-stranded oligonucleotide fragment.
  • the clones were picked for colony PCR identification, and the correct clones were identified as PD-1 knockdown recombinant lentiviral plasmids pCARbcma-1453 ⁇ pCARbcma-1460, and the correct clones were sequenced and identified (see Figure 7).
  • Trypsin solution Weigh Trypsin 2.5g, EDTA 0.19729g in 1000ml beaker, add 900ml 1XPBS to dissolve, dissolve it, use 1000ml measuring cylinder to make up to 1000ml, 0.22 ⁇ M filter sterilization, long-term use can be saved to -20 ° C refrigerator;
  • a DNA/CaCl 2 solution was prepared in accordance with N + 0.5.
  • the amount of HEK293T/17 cell transfection plasmid per dish was used in the following ratios: recombinant lentiviral plasmid (20 ⁇ g), pPac-GP (15 ⁇ g), pPac-R (10 ⁇ g), pEnv-G (7.5 ⁇ g).
  • the same virus supernatant was collected again, and the two collected viruses could be put together and the culture dish discarded; the supernatant collected at this time contained the recombinant lentiviral vector lvCARbcma-1453 to lvCARbcma. -1460.
  • the eluate is divided into 25 to 50 ⁇ l tubes, frozen and stored in a -80 ° C refrigerator for long-term storage;
  • a 24-well plate was used to inoculate 293T cells.
  • the cell volume per well is 5 ⁇ 10 4
  • the volume of the added medium is 500 ul
  • the growth rate of different kinds of cells is different
  • the cell fusion rate when the virus is infected is 40%-60%;
  • the cells were digested with 0.2 ml of 0.25% trypsin-EDTA solution and allowed to stand at 37 ° C for 1 minute. The entire cell surface was washed with a medium, and the cells were collected by centrifugation. Genomic DNA was extracted according to the instructions of the DNeasy kit. 200 ⁇ l of eluate was added to each sample tube to wash the DNA and quantify;
  • the total number of reactions is 40, and 1 ml of 2 ⁇ TaqMan Universal PCR Master Mix, 4 ⁇ l of forward primer, 4 ⁇ l of reverse primer, 4 ⁇ l of probe and 788 ⁇ l of H 2 O are mixed. Put on the ice after the shock;
  • the total number of reactions is 40, and 1 ml of 2 ⁇ TaqMan Universal PCR Master Mix, 100 ⁇ l of 10 ⁇ RNaseP primer/probe mix and 700 ⁇ l of H 2 O are mixed. Put on the ice after the shock;
  • the quantitative PCR instrument used was the ABI PRISM 7500 quantitative system.
  • the cycle conditions were set to: 50 ° C for 2 minutes, 95 ° C for 10 minutes, then 95 ° C for 15 seconds, 60 ° C for 1 minute of 40 cycles.
  • N number of cells at the time of infection (approximately 1 ⁇ 10 5 )
  • V volume of diluted virus added
  • the endotoxin working standard is 15EU/piece;
  • Step 4 is repeated once;
  • PCR reaction system was: ddH20 6.5 ⁇ l, Myco Mix 1 ⁇ l, 2 ⁇ Taq Plus Mix Master (Dye Plus) 12.5 ⁇ l, template 5 ⁇ l; PCR cycle conditions were: 95 ° C 30 sec, (95 ° C 30 sec, 56 ° C 30 sec, 72 ° C 30 sec) * 30 cycles, 72 ° C 5 min.
  • lvCARbcma-1453 ⁇ lvCARbcma-1460 are free of mycoplasma.
  • the cell separation solution was isolated from human blood) and 500 ul of medium (containing 10% serum, 20 U/ml IL-2, Polybrene 8 ug/ml) was added. The cells were allowed to stand for 20 min, centrifuged at 1000 g for 20 min at 20 ° C, and cultured at 37 ° C for 48 h.
  • RNA of PBMC cells in 6-well plates was extracted by Trizol method, and the cDNA was amplified by reverse transcription.
  • the PCR primers (SEQ ID NO. 63---SEQ ID NO. 64) were used for fluorescence quantitative PCR experiments. See Table 8, using the internal reference Actin as a control group to verify the transcription of mRNA.
  • the total protein extracted from PBMC was separated by relative molecular mass by polyacrylamide gel electrophoresis.
  • the protein was transferred to a PVDF membrane using a wet transfer (4 ° C, 400 mA, 120 min).
  • the PVDF membrane was blocked with a blocking solution (containing 5% skim milk in TBST solution) for 1 h at room temperature, and the blocking solution was diluted 1:1000 with Biotinylated protein L, and then incubated with the blocked PVDF membrane at room temperature for 4 ° C overnight.
  • the membrane was washed 3 times with TBST for 10 min each time.
  • the blocking solution was diluted 1:500 with the corresponding SA-HRP.
  • the PVDF membrane was incubated for 2 h at room temperature, and the membrane was washed 3 times with TBST for 10 min each time. Color development was performed using an Amersham ECL+plusTM Western blotting system kit. X-ray development obtained a film showing the strip.
  • RT-QPCR analysis showed that the transcription level of the CAR gene after infection of PBMC by recombinant lentiviral vector was higher than that of control virus MOCK and empty cells were significantly elevated (as shown in Figure 11 and Table 9), indicating that the transcription level of the CAR gene was successfully expressed.
  • RNA of the above mixed cells was extracted by Trizol method, and the cDNA was reverse transcribed, and the QPCR primer (SEQ ID NO. 57---SEQ ID NO. 58) was used for the fluorescence quantitative PCR experiment.
  • the reaction system is shown in Table 6.
  • the internal reference Actin was used as a control group to verify the transcription of mRNA.
  • RT-QPCR results showed that after PD-1 knockdown recombinant lentiviral vector-transduced PBMC was incubated with target cells, the PD-1 gene mRNA was significantly lower than the control virus lvCARbcma-1460 (Fig. 13 and Table 10 shows that PD1 siRNA has a knockdown effect on the transcription level of PD-1 gene, and lvCARbcma-1453 has the best knockdown effect, reaching more than 70%.
  • the Blank group was not up-regulated due to its inability to be activated by target cells.
  • step 15 The corrected values obtained in step 15 were taken to the following formula to calculate the percentage of cytotoxicity produced by each of the target ratios.
  • the results are shown in Figure 14.
  • the killing efficiency of PBMC cells transduced with PD-1 knockdown recombinant lentiviral vector was significantly higher than that of PBMC empty cells at 10:1 target ratio, and lvCARbcma-1453-PBMC cells were targeted to target cells.
  • the killing efficiency is the highest, higher than 40%.
  • the killing efficiency of lvCARbcma-1454-PBMC cells against target cells is second.
  • the killing efficiency of the remaining lvCARbcma-1455-PBMC ⁇ lvCARbcma-1458-PBMC cells was similar to that of the control lvCARbcma-1460-PBMC cells.
  • the results of Figure 13 can be seen, due to the presence of PD-L1 in the target cells.
  • the inhibitory signal is transmitted, and the immune activity of the T cell is inhibited, resulting in a significant decrease in the killing efficiency of the T cell to the target cell.
  • the expression level of PD-1 molecule is greatly reduced, it can effectively block the activation of PD-1/PDL-1 signaling pathway, so that T cells can play a normal killing effect on target cells.
  • lvCARbcma-1453 vector and its transduced T cells can be used clinically, inhibit the expression of PD-1 in CAR-T cells, enhance the killing effect of CAR-T cells on tumors, and achieve the effect of inhibiting immune escape.
  • Killing efficiency (experimental well - effector cell hole - target cell well) / (target cell maximum pore - target cell well) X100%

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Abstract

提供了一种敲减人PD-1的siRNA、重组表达CAR-T载体及其构建方法和应用。其中PD-1敲减siRNA表达框及其siRNA表达产物，不仅可以应用于CAR-T治疗多发性骨髓瘤(MM)中用于消除或减轻肿瘤的免疫逃脱机制，还可应用于抑制CAR-T治疗诸如胰腺癌、脑胶质瘤、骨髓瘤等等类型肿瘤时的免疫逃脱机制。

Description

敲减人PD-1的siRNA、重组表达CAR-T载体及其构建方法和应用 技术领域
本发明属于肿瘤免疫治疗技术领域，具体涉及一种敲减人程序性死亡受体1(PD-1)的siRNA、重组表达CAR-T载体(尤其是一种通过敲减PD-1消除或减轻肿瘤的免疫逃脱机制的CAR-T转基因载体)及其构建方法和应用。
背景技术
肿瘤免疫治疗的理论基础是免疫系统具有识别肿瘤相关抗原、调控机体攻击肿瘤细胞(高度特异性的细胞溶解)的能力。1950年代，Burnet和Thomas提出了“免疫监视”理论，认为机体中经常会出现的突变的肿瘤细胞可被免疫系统所识别而清除，为肿瘤免疫治疗奠定了理论基础[Burnet FM.Immunological aspects of malignant disease.Lancet,1967；1:1171-4]。随后，各种肿瘤免疫疗法包括细胞因子疗法、单克隆抗体疗法、过继免疫疗法、疫苗疗法等相继应用于临床。
2013年一种更先进的肿瘤免疫疗法---CAR-T疗法成功用于临床，并表现了前所未有的临床疗效。CAR-T，全称是Chimeric Antigen Receptor T-Cell Immunotherapy，嵌合抗原受体T细胞免疫疗法。CAR-T疗法在临床上最领先的有诺华的CLT019，采用CLT019治疗复发难治急性淋巴细胞白血病患者，六个月的肿瘤无进展生存率达到67％，其中最长的应答时间达到两年多。总部位于中国上海的上海优卡迪生物医药科技有限公司与医院合作，共治疗复发难治急性淋巴细胞白血病患者36例，其中完全24例，缓解比例达到66.6％。这是抗癌研究的颠覆性突破。CAR-T细胞疗法可能是最有可能治愈癌症的手段之一，并被《Science》杂志评为2013年度十大科技突破之首。
CAR-T疗法虽然效果显著，但是在治疗实体肿瘤的过程中遇到很多困难，其中一个很重要的原因就是PD1/PDL1抑制性免疫检查点(如图1A所示)，它们结合传递抑制信号，抑制了T细胞的免疫活性，在免疫耐受中发挥了重要作用，同时也是促使肿瘤细胞逃逸的重要原因。
PD-1(又称CD279)是一种免疫抑制性受体，属于CD28家族成员的Ⅰ型跨膜蛋白，程序性细胞死亡分子-1受体1992年由Ishida等[Ishida Y,Agata Y,Shibahara K,et al.Induced expression of PD-1,a novel member of the immunoglobulin gene superfamily,upon programmed cell death〔J〕.EMBO J,1992,11(11):3887-3895.]采用消减杂交方法于凋亡的T细胞杂交瘤中得到并命名。人PD-1基因位于2q37.35染色体上，编码一个约55kD的跨膜糖蛋白。PD-1在激活的T细胞、B细胞、单核细胞和树突状细胞表面广泛表达，PD-1结构上与CTLA-4有30％的同源性，胞内区存在两个酪氨酸残基，分别参与构成了N端的一个免疫受体酪氨酸抑制基序(immunoreceptor tyrosine-based inhibitory motif，ITIM)和C端的一个免疫受体酪氨酸依赖的转换基序(immunoreceptor tyrosin-based switch motif，ITSM)；胞外区则是由一个IgV样结构域组成，含有多个糖基化位点并被重度糖基化，该结构域可以与配体结合，从而发挥抑制T细胞活化的功能[李瑛,焦顺昌等.PD-1/PD-L1信号通路在肿瘤免疫逃逸中的作用及临床意义[J].Acad J Chin PLA Med Sch,Jul 2015,36(7).]。
PD-L1在多数癌症组织中过量表达，包括NSCLC、黑色素瘤、乳腺癌、胶质瘤、淋巴瘤、白血病及各种泌尿系肿瘤、消化道肿瘤、生殖系肿瘤等[Intlekofer AM,Thompson CB.At the bench:preclinical rationale for CTLA-4 and PD-1 blockade as cancer immunotherapy[J].J Leukoc Biol,2013,94(1):25-39.].Parsa在鼠和人的肿瘤细胞中，发现T细胞异常分泌的IFN-γ，IFN-γ可以诱导肿瘤细胞上的PD-L1高表达[Ding H,Wu X,Wu J,et al.Delivering PD-1 inhibitory signal concomitant with blocking ICOS co-stimulation suppresses lupus-like syndrome in autoimmune BXSB mice[J].Clin Immunol,2006,118(2/3):258-267.]。PD-L1高表达，可以通过抑制RAS及PI3K/AKT信号通路，进而调控细胞周期检查点蛋白和细胞增殖相关蛋白表达，最终导致T细胞增殖的抑制[11]。Dong等体外实验和小鼠模型还发现，PD-1/PD-L1信号通路的激活可以诱导特异性CTL调 亡，使CTL的细胞毒杀伤效应敏感性下降，促使肿瘤细胞发生免疫逃逸[Dong H,Strome SE,Salomao DR,et al.Tumor-associated B7-H1 promotes T-cell apoptosis:a potential mechanism of immune evasion[J].Nat Med,2002,8(8):793-800.]。
目前临床上主要是是使用商品化的PD-1单抗，作为免疫检查点抑制剂，来抑制肿瘤细胞的免疫逃脱。2014年9月3日，百时美施贵宝公司抗PD-1药物Opdivo(Nivolumab)在日本正式上市，在日本国内此药物目前仍然只限于黑色素瘤患者。药物被设定为每100mg，729849日元(约合人民币43000元)的价格。体重每1kg就需要2mg的使用量，因此体重50kg的人则需要100mg的使用量。并且，体重每增加10kg用药剂量需要增加20mg(150200日元，约合人民币8778元)，每3周为一个疗程。价格相当昂贵，普通家庭难以承受。
发明内容
本发明的目的在于克服现有技术的不足，提供了一种敲减人PD-1的siRNA、重组表达CAR-T载体及其构建方法和应用
本发明的目的通过以下技术方案来实现：
本发明的第一目的在于提供一种敲减人PD-1的siRNA，所述siRNA选自下述中的a-g中的任一对：
a.SEQ ID NO.41和SEQ ID NO.42所示的核苷酸序列；
b.SEQ ID NO.43和SEQ ID NO.44所示的核苷酸序列；
c.SEQ ID NO.45和SEQ ID NO.46所示的核苷酸序列；
d.SEQ ID NO.47和SEQ ID NO.48所示的核苷酸序列；
e.SEQ ID NO.49和SEQ ID NO.50所示的核苷酸序列；
f.SEQ ID NO.51和SEQ ID NO.52所示的核苷酸序列；
g.SEQ ID NO.53和SEQ ID NO.54所示的核苷酸序列。
进一步的，优选a.SEQ ID NO.41和SEQ ID NO.42所示的核苷酸序列。
本发明的第二目的在于提供上述siRNA在制备消除或减轻肿瘤的免疫逃脱机制药物中的应用。
本发明的第三目的在于提供一种包含上述siRNA的重组表达载体。
进一步的，所述表达载体为慢病毒表达载体、逆转录病毒表达载体、腺病毒表达载体、腺相关病毒表达载体或质粒；优选的，包含上述siRNA的慢病毒表达载体。
进一步的，所述的慢病毒表达载体包括：用于质粒复制的原核复制子pUC Ori序列，如SEQ ID NO.2所示；用于目的菌株大量扩增的含氨苄青霉素抗性基因AmpR序列，如SEQ ID NO.1所示；用于增强真核细胞内的复制的病毒复制子SV40Ori序列，如SEQ ID NO.3所示；用于慢病毒包装的慢病毒包装顺式元件；用于真核细胞表达绿色荧光的ZsGreen1绿色荧光蛋白，如SEQ ID NO.11所示；用于共同转录表达蛋白质的IRES核糖体结合序列，如SEQ ID NO.12所示；用于嵌合抗原受体基因的真核转录的人EF1α启动子，如SEQ ID NO.15所示；用于组成集识别、传递、启动于一体的二代CAR或三代CAR的抗BCMA嵌合抗原受体的编码基因；用于增强转基因的表达效率的eWPRE增强型土拨鼠乙肝病毒转录后调控元件，如SEQ ID NO.13所示；用于胞内转录siRNA的人RNA聚合酶III启动子hU6，如SEQ ID NO.14所示。
进一步的，所述慢病毒包装顺式元件采用第二代慢病毒载体包括：如SEQ ID NO.5所示的慢病毒5terminal LTR、如SEQ ID NO.6所示的慢病毒3terminal Self-Inactivating LTR、如SEQ ID NO.7所示的Gag顺式元件、如SEQ ID NO.8所示的RRE顺式元件、如SEQ ID NO.9所示的env顺式元件、如SEQ ID NO.10所示的cPPT顺式元件。
进一步的，所述慢病毒包装顺式元件采用第三代慢病毒载体包括：如SEQ ID NO.5所示的慢病毒5terminal LTR、如SEQ ID NO.6所示的慢病毒3terminal Self-Inactivating LTR、如SEQ ID NO.7所示的Gag顺式元件、如SEQ ID NO.8所示的RRE顺式元件、如SEQ ID NO.9所示的env 顺式元件、如SEQ ID NO.10所示的cPPT顺式元件所述慢病毒包装顺式元件，以及如SEQ ID NO.4所示的RSV启动子。
进一步的，所述eWPRE增强型土拨鼠乙肝病毒转录后调控元件有6个核苷酸的增强突变，具体为：g.396G>A、g.397C>T、g.398T>C、g.399G>A、g.400A>T、g.411A>T。
进一步的，所述抗BCMA嵌合抗原受体(二代CAR)，包括依次串联的如SEQ ID NO.16所示的CD8 leader嵌合受体信号肽、如SEQ ID NO.17所示的BCMA单链抗体轻链VL、如SEQ ID NO.18所示的Optimal Linker C、如SEQ ID NO.19所示的BCMA单链抗体重链VH、如SEQ ID NO.20所示的CD8 Hinge嵌合受体铰链、如SEQ ID NO.21所示的CD8 Transmembrane嵌合受体跨膜区、如SEQ ID NO.22所示的CD137嵌合受体共刺激因子，以及如SEQ ID NO.23所示的TCR嵌合受体T细胞激活域。
进一步的，所述抗BCMA嵌合抗原受体(三代CAR)，包括依次串联的如SEQ ID NO.16所示的CD8 leader嵌合受体信号肽、如SEQ ID NO.17所示的BCMA单链抗体轻链VL、如SEQ ID NO.18所示的Optimal Linker C、如SEQ ID NO.19所示的BCMA单链抗体重链VH、如SEQ ID NO.20所示的CD8 Hinge嵌合受体铰链、如SEQ ID NO.21所示的CD8 Transmembrane嵌合受体跨膜区、如SEQ ID NO.24所示的CD28嵌合受体共刺激因子、如SEQ ID NO.22所示的CD137嵌合受体共刺激因子以及如SEQ ID NO.23所示的TCR嵌合受体T细胞激活域。
本发明的第四目的在于提供一种包含上述siRNA的慢病毒表达载体的构建方法，包括以下步骤：
(1)将含氨苄青霉素抗性基因AmpR序列(如SEQ ID NO.1所示)、原核复制子pUC Ori序列(如SEQ ID NO.2所示)、病毒复制子SV40Ori序列(如SEQ ID NO.3所示)、用于慢病毒包装的慢病毒包装顺式元件、ZsGreen1绿色荧光蛋白(如SEQ ID NO.11所示)、IRES核糖体结合序列(如SEQ ID NO.12所示)、eWPRE增强型土拨鼠乙肝病毒转录后调控元件(如SEQ ID NO.13所示)、人RNA聚合酶III启动子hU6(如SEQ ID NO.14所示)存储于慢病毒骨架质粒(pLenti-3G silencer)上；
(2)将人EF1α启动子(如SEQ ID NO.15所示)、用于组成集识别、传递、启动于一体的二代CAR或三代CAR的抗BCMA嵌合抗原受体组合成二代CAR或三代CAR设计方案，经过酶切、连接、重组反应克隆至慢病毒骨架质粒中，得到二代CAR或三代CAR设计的重组慢病毒质粒pCARbcma-silencer；
(3)将上述siRNA以及如SEQ ID NO.55和SEQ ID NO.56所示的negative control序列，分别克隆至步骤(2)所得的重组慢病毒质粒中，得到PD-1敲减重组慢病毒质粒(pCARbcma-1453～pCARbcma-1459和negative control pCARbcma-1460)；
(4)将步骤(3)得到的重组慢病毒质粒(pCARbcma-1453～pCARbcma-1460)分别与慢病毒包装质粒pPac-GP、pPac-R以及膜蛋白质粒pEnv-G共同转染HEK293T/17细胞，在HEK293T/17细胞中进行基因转录表达后，包装成功重组慢病毒载体会释放到细胞培养上清中，收集包含的重组慢病毒载体的上清液；
(5)将得到的重组慢病毒上清采用抽滤、吸附、洗脱的离子交换方式进行纯化，分别得到重组慢病毒载体。
进一步的，步骤(1)中，所述慢病毒包装顺式元件采用第二代慢病毒载体包括：如SEQ ID NO.5所示的慢病毒5terminal LTR、如SEQ ID NO.6所示的慢病毒3terminal Self-Inactivating LTR、如SEQ ID NO.7所示的Gag顺式元件、如SEQ ID NO.8所示的RRE顺式元件、如SEQ ID NO.9所示的env顺式元件、如SEQ ID NO.10所示的cPPT顺式元件；所述慢病毒包装顺式元件采用第三代慢病毒载体包括：如SEQ ID NO.5所示的慢病毒5terminal LTR、如SEQ ID NO.6所示的慢病毒3terminal Self-Inactivating LTR、如SEQ ID NO.7所示的Gag顺式元件、如SEQ ID NO.8所 示的RRE顺式元件、如SEQ ID NO.9所示的env顺式元件、如SEQ ID NO.10所示的cPPT顺式元件所述慢病毒包装顺式元件，以及如SEQ ID NO.4所示的RSV启动子。
进一步的，步骤(2)中，所述用于组成集识别、传递、启动于一体的二代CAR的所述抗BCMA嵌合抗原受体，包括依次串联的如SEQ ID NO.16所示的CD8 leader嵌合受体信号肽、如SEQ ID NO.17所示的BCMA单链抗体轻链VL、如SEQ ID NO.18所示的Optimal Linker C、如SEQ ID NO.19所示的BCMA单链抗体重链VH、如SEQ ID NO.20所示的CD8 Hinge嵌合受体铰链、如SEQ ID NO.21所示的CD8 Transmembrane嵌合受体跨膜区、如SEQ ID NO.22所示的CD137嵌合受体共刺激因子，以及如SEQ ID NO.23所示的TCR嵌合受体T细胞激活域；所述用于组成集识别、传递、启动于一体的三代CAR的抗BCMA嵌合抗原受体，包括依次串联的如SEQ ID NO.16所示的CD8 leader嵌合受体信号肽、如SEQ ID NO.17所示的BCMA单链抗体轻链VL、如SEQ ID NO.18所示的Optimal Linker C、如SEQ ID NO.19所示的BCMA单链抗体重链VH、如SEQ ID NO.20所示的CD8 Hinge嵌合受体铰链、如SEQ ID NO.21所示的CD8 Transmembrane嵌合受体跨膜区、如SEQ ID NO.24所示的CD28嵌合受体共刺激因子、如SEQ ID NO.22所示的CD137嵌合受体共刺激因子以及如SEQ ID NO.23所示的TCR嵌合受体T细胞激活域。
进一步的，步骤(1)中，所述eWPRE增强型土拨鼠乙肝病毒转录后调控元件有6个核苷酸的增强突变，具体为：g.396G>A、g.397C>T、g.398T>C、g.399G>A、g.400A>T、g.411A>T。
进一步的，步骤(2)中，由人EF1α启动子启动整个CAR基因表达；CD8 leader嵌合受体信号肽位于CAR编码序列的N端，用于引导CAR蛋白定位于细胞膜；CD19单链抗体轻链VL、Optimal Linker C、CD19单链抗体重链VH组合成scfv区域，用于识别CD19抗原；CD8 Hinge嵌合受体铰链用于将scfv锚定于细胞膜外侧；CD8 Transmembrane嵌合受体跨膜区用于将整个嵌合受体固定于细胞膜上；CD137嵌合受体共刺激因子用于刺激T细胞增殖和细胞因子分泌；TCR嵌合受体T细胞激活域用于激活下游信号通路的表达；当scfv区域与CD20抗原结合时，信号通过嵌合受体传递至细胞内，从而产生包括T细胞增殖、细胞因子分泌增加、抗细胞凋亡蛋白分泌增加、细胞死亡延迟、裂解靶细胞的一系列生物学效应。
进一步的，步骤(4)中，所述慢病毒载体有带荧光标签zsGreen1的版本和不带荧光标签zsGreen1版本，带荧光标签的版本用于体外实验，不带荧光标签的版本用于临床实验。
进一步的，步骤(5)中，所述抽滤步骤控制上清体积在200ml～2000ml，真空度控制在-0.5MPA～-0.9MPA，防止由于堵孔带来的载体损失；所述吸附步骤控制溶液的PH值在6～8，防止PH的变化导致载体失活；所述洗脱步骤控制洗脱液的离子强度在0.5M～1.0M，防止离子强度的变化导致洗脱不完全或者载体失活。
本发明的第五目的在于提供包含上述siRNA的重组表达载体在制备CAR-T治疗肿瘤过程中消除或减轻肿瘤免疫逃脱机制药物中的应用。
本发明的第六目的在于提供一种CART细胞，所述的CAR-T细胞是由上述siRNA修饰的T淋巴细胞。
本发明的再一目的在于提供上述CAR-T细胞在制备治疗多发性骨髓瘤、胰腺癌、脑胶质瘤、骨髓瘤药物中的应用。
这种干扰效应可以持续很久，而且在细胞分裂后仍然有效。并且，RNAi具有非常好的序列特异性【Kisielow,M.et al.(2002)Isoform-specific knockdown and expression of adaptor protein ShcA using small interfering RNA,J.of Biochemistry 363:1-5】。因此，RNAi系统可以特异性敲减一种类型的转录本，而不影响序列相近的其它mRNA。这些特性使siRNA系统在抑制基因表达、基因功能研究和药物靶点验证方面表现出潜力和价值。此外，siRNA系统可以用来治疗相关疾病，包括(1)基因过表达或错误表达引起的疾病；(2)的基因突变引起的疾病。
本发明是将人RNA聚合酶III启动子hU6、人EF1α启动子、CD8 leader嵌合受体信号肽、BCMA单链抗体轻链VL、Optimal Linker C、BCMA单链抗体重链VH、CD8 Hinge嵌合受体铰 链、CD8 Transmembrane嵌合受体跨膜区、CD137嵌合受体共刺激因子、TCR嵌合受体T细胞激活域构建进入重组慢病毒载体，由人EF1α启动子启动整个CAR基因表达。CAR蛋白定位于细胞膜表面，识别BCMA抗原，刺激T细胞增殖和细胞因子分泌，激活下游信号通路的表达。当scfv区域与BCMA抗原结合时，信号通过嵌合受体传递至细胞内，从而产生T细胞增殖、细胞因子分泌增加、抗细胞凋亡蛋白分泌增加、细胞死亡延迟、裂解靶细胞等一系列生物学效应。由人RNA聚合酶III启动子hU6启动PD1siRNA的表达，通过RISC complex的作用，降解PD-1mRNA，抑制PD-1的蛋白质合成，阻断PD1的与PDL1的结合，从而阻断PD1/PDL1的信号通路，达到抑制免疫逃脱的效果。
本发明所采用的表达载体包括原核复制子(pUC ori)用于质粒复制；原核筛选标记(AmpR)用于目的菌株大量扩增；病毒复制子(SV40Ori)用于增强真核细胞内的复制；慢病毒包装顺式元件(RSV、5terminal LTR、3terminal Self-Inactivating LTR、Gag、RRE、env、cPPT)用于慢病毒包装；人RNA聚合酶III启动子(hU6)用于胞内转录siRNA；真核荧光标签蛋白(ZsGreen1)用于真核细胞表达绿色荧光；共表达元件(IRES)用于共同转录表达蛋白质；真核启动子(EF1α)用于嵌合抗原受体基因的真核转录；嵌合抗原受体(CD8 leader、BCMA VL、Optimal Linker C、BCMA VH、CD8 Hinge、CD8 Transmembrane、CD28(SEQ ID NO.24)、CD137、TCR)用于组成集识别、传递、启动于一体的二代和三代CAR；转录后调控元件(eWPRE)用于增强转基因的表达效率。
本发明涉及含肽的医药配置品，具体涉及：
一、人RNA聚合酶III启动子hU6、含氨苄青霉素抗性基因AmpR序列、原核复制子pUC Ori序列,、病毒复制子SV40Ori序列、RSV启动子、人EF1α启动子、慢病毒5terminal LTR、慢病毒3terminal Self-Inactivating LTR、Gag顺式元件、RRE顺式元件、env顺式元件、cPPT顺式元件、IRES核糖体结合序列、ZsGreen1绿色荧光蛋白、WPRE土拨鼠乙肝病毒转录后调控元件的重组慢病毒载体骨架，这种重组慢病毒载体骨架可以搭载不同的治疗性基因并广泛的用于过继性细胞治疗领域，搭载不同的siRNA并广泛的用于基因过表达、错误表达和基因突变引起的疾病。
二、重组慢病毒载体骨架、PD1-siRNA、CD8 leader嵌合受体信号肽、BCMA单链抗体轻链VL、单链抗体铰链Linker C、BCMA单链抗体重链VH、CD8 Hinge嵌合受体铰链、CD8Transmembrane嵌合受体跨膜区、CD28嵌合受体共刺激因子、CD137嵌合受体共刺激因子、TCR嵌合受体T细胞激活域构建形成重组慢病毒载体，该方法得到的重组慢病毒载体可以实现在人T淋巴细胞上表达BCMA嵌合抗原受体，引导并激活T淋巴细胞对BCMA阳性细胞的杀伤作用，在临床上用于治疗多发性骨髓瘤(MM)。在人T淋巴细胞内表达程序性死亡受体1(PD-1)的siRNA，有效降低T细胞表面程序性死亡受体1的表达水平，阻断PD-1/PD-L1免疫负调节信号通路，临床上可用于抑制肿瘤的免疫逃脱，提高CAR-T细胞免疫治疗的疗效。
本发明所采用的是针对PD-1的siRNA基因沉默技术，自从20世纪90年代以来，研究人员发现双链RNA(“dsRNA”)可以用来抑制蛋白表达。这种沉默基因的能力在治疗人类疾病方面具有具有广泛的潜力，和许多研究人员和商业实体目前投入相当大的资源在发展中基于这种技术的治疗方法。
从机制的角度来看，当dsRNA进入植物和无脊椎动物细胞后，被III型核酸内切酶Dicer分解成siRNA。【Sharp,RNA interference-2001,Genes Dev.2001,15:485】。III型核糖核酸酶Dicer，将dsRNA分解成带有2碱基凸出3’粘性末端的19-23bp的siRNA。【Bernstein,Caudy,Hammond,&Hannon,Role for a bidentate ribonuclease in the initiation step of RNA interference,Nature 2001,409:363】。siRNA与RNA诱导的沉默复合体(RISC)整合，复合体中一个或多个解旋酶解开双链siRNA，使互补反义链引导靶点识别。【Nykanen,Haley,&Zamore,ATP requirements and small interfering RNA structure in the RNA interference pathway,Cell 2001,107:309】。在结合相应的目标mRNA后，RISC复合物中的一个或多个核酸内切酶切割目标mRNA引起mRNA沉默。【Elbashir， Elbashir,Lendeckel,&Tuschl,RNA interference is mediated by 21-and 22-nucleotide RNAs,Genes Dev 2001,15:188】。
本发明设计的PD1siRNA序列含有21个bp的核苷酸，采用N2[CG]N8[AU]N8[AU]N2的寡核苷酸排列模式，互补序列之间采用stem-loop的发卡结构，通过siRNA pattern、GC percentage、T or A or G in a row、consecutiver GC、3’end nt pattern、thermodynamic value、siRNA target、identity、alignment等条件的筛选，大大提高siRNA设计的干扰成功率。
本发明所采用的人RNA聚合酶III启动子hU6转录PD1siRNA，抑制PD-1表达的系统，在T细胞杀伤实验中，经过QPCR检测，能有效抑制PD-1mRNA的转录水平，能有效减少T细胞表面PD-1受体的表达量，从而阻断PD-1/PDL-1的信号通路，达到抑制免疫逃脱的效果；将来可以应用于临床，抑制CAR-T细胞内的PD-1表达水平，增强CAR-T细胞在体内对肿瘤的杀伤效果。
本发明采用慢病毒载体方式递送siRNA(如图2所示)。首先，节约了成本，避免PD1siRNA体外合成的昂贵费用。其次，避免了PD1siRNA的在体递送效率低的问题。第三，使用人RNA聚合酶III启动子hU6表达PD1siRNA，能够有效利用细胞内的RNA转录体系，大量表达出相应的PD1siRNA，经过一系列的酶促作用，达到良好的基因沉默效果。
本发明所采用的载体骨架为第三代慢病毒载体(如图3A所示)，3’SIN LTR去除了U3区域，消除了慢病毒载体自我复制的可能性，大大提高了安全性；增加了cPPT和WPRE元件，提高了转导效率和转基因的表达效率；采用RSV启动子保证了慢病毒载体包装时核心RNA的持续高效转录；采用人自身的EF1α启动子，使CAR基因能够在人体内长时间持续表达。
本发明所采用的siRNA敲减方案，同样可以应用于第三代CAR设计方案。第三代CAR设计与第二代设计相比，增加了CD28嵌合受体共刺激因子(SEQ ID NO.24)。
本发明所采用的慢病毒载体柱纯化系统(如图8所示)，系本公司开发出的慢病毒规模化生产工艺。常用的超速离心法或者高速离心法，是利用离心沉降原理分离慢病毒颗粒，不可避免的会残留很多沉降系数相近的杂质，对后续实验带来不利影响。并且，装管过程复杂、操作繁琐、多次转换容器带来更多的污染机会。而本发明的慢病毒载体柱纯化工艺为半自动化操作，全部过程在百级实验区域完成，避免人工操作的繁琐和污染几率，所回收的慢病毒载体在内毒素、支原体等指标上完全达到临床标准。后续可跟进开发全自动纯化仪。
本发明所采用CAR设计方案也可以应用于第二代慢病毒载体结构上。第二代和第三代慢病毒载体在结构上的区别(如图3B所示)，主要是第三代慢病毒载体把第二代载体5’LTR的U3区域替换为RSV启动子，这样就消除了U3转录时对Tat蛋白的依赖，既可以在慢病毒的结构基因里去除Tat序列，也提高了慢病毒基因组转录水平和转录持续性。第二代和第三代慢病毒载体主要是基因组转录方式的区别，因此本发明所采用CAR设计方案可以应用于这两代慢病毒载体。
本发明的第三代慢病毒骨架质粒，采用enhancedWPRE元件，与宾州大学Carl H.June等人(Porter DL,Levine BL,Kalos M,Bagg A,June CH.Chimeric antigen receptormodified T cells in chronic lymphoid leukemia.N Engl J Med 2011；365:725-33.)所采用的WPRE元件相比，有6个核苷酸的增强突变(g.396G>A、g.397C>T、g.398T>C、g.399G>A、g.400A>T、g.411A>T)，能够增强初级转录产物的多聚腺苷化，增加细胞内mRNA的含量，增强转基因的表达效率。
本发明的Lentival包装系统是无辅助病毒的四质粒包装系统，通过四种质粒共同转染至HEK293T/17细胞中，产生重组慢病毒载体。重组后的慢病毒载体是复制缺陷型载体，能将外源片段整合入宿主基因，一次性使用，无法复制和增殖，安全性有很大提高。
本发明采用的慢病毒载体有带荧光标签zsGreen1的版本和不带荧光标签zsGreen1版本，带荧光标签的版本用于体外实验，不带荧光标签的版本用于临床实验。
本发明采用的scfv段的Linker设计，能够显著提高细胞因子的分泌、CAR-T细胞的体外杀伤作用以及临床治疗效果。
可见，本发明所述的重组慢病毒载体在给多发性骨髓瘤(MM)的CAR-T治疗提供可靠转基因 保障的同时，可以阻断肿瘤的免疫逃脱机制，提高CAR-T细胞治疗的疗效，大大降低患者承担的医疗成本。
本发明所述的PD-1敲减siRNA表达框及其siRNA表达产物，不仅可以应用于CAR-T治疗多发性骨髓瘤(MM)中用于消除或减轻肿瘤的免疫逃脱机制，还可应用于抑制CAR-T治疗诸如胰腺癌、脑胶质瘤、骨髓瘤等等类型肿瘤时的免疫逃脱机制。
附图说明
图1是本发明的PD-1/PD-L1信号通路；
图2是本发明的慢病毒递送PD1siRNA的方式示意图；
图3是本发明的慢病毒载体结构示意图；其中,图3(A)是本发明采用的第三代慢病毒载体结构示意图，图3(B)是第二代和第三代慢病毒载体结构比较；
图4是本发明的重组慢病毒载体的构建流程图；其中，(A)图是慢病毒骨架质粒pLenti-3G silencer的结构示意图；(B)图是pCARbcma-silencer质粒的结构示意图；(C)图是pCARbcma-1453～pCARbcma-1460质粒的示意图；(D)图是慢病毒包装质粒pPac-GP的结构示意图；(E)图是慢病毒包装质粒pPac-R的结构示意图；(F)图是膜蛋白pEnv-G的结构示意图；
图5是慢病毒骨架质粒pLenti-3G silencer的酶切预测及酶切琼脂糖凝胶电泳图；其中，图5A是慢病毒骨架质粒pLenti-3G silencer的酶切预测示意图，其中，lane1是1kb DNA ladder Marker：条带从上到下依次为：10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp；lane2是pLenti-3G silencer的Cla I+BamH I酶切预测：条带从上到下依次为：7381bp，23bp；图5B是慢病毒骨架质粒pLenti-3G silencer的酶切琼脂糖凝胶电泳图，其中，lane1是1kb DNA ladder Marker的电泳结果；lane2是pLenti-3G silencer的Cla I+BamH I酶切电泳结果；
图6是重组慢病毒质粒pCARbcma-silencer的酶切预测及酶切琼脂糖凝胶电泳图；其中，图6A是重组慢病毒质粒pCARbcma-silencer的酶切预测示意图，其中，lane1是1kb DNA ladder Marker：条带从上到下依次为：10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp；lane2是pCARbcma-silencer的Pvu I酶切预测：条带从上到下依次为：8898bp、896bp、249bp；图6B是重组慢病毒质粒pCARbcma-silencer的酶切琼脂糖凝胶电泳图，其中，lane1是1kb DNA ladder Marker的电泳结果；lane2是pCARbcma-silencer的Pvu I酶切电泳结果；
图7是PD-1敲减重组慢病毒质粒pCARbcma-1453～pCARbcma-1460的测序比对结果；其中，A是pCARbcma-1453的测序比对结果；B是pCARbcma-1454的测序比对结果；C是pCARbcma-1455的测序比对结果；D是pCARbcma-1456的测序比对结果；E是pCARbcma-1457的测序比对结果；F是pCARbcma-1458的测序比对结果；G是pCARbcma-1459的测序比对结果；H是pCARbcma-1460的测序比对结果；
图8是离子交换色谱法纯化重组慢病毒载体的流程图；
图9是重组慢病毒载体的滴度检测结果；
图10是重组慢病毒载体的不同纯化方式的支原体检测结果，lane1为DL2000marker，从上到下条带条带从上到下依次为：2kb、1kb、750bp、500bp、250bp、100bp；lane2为阳性对照；lane3为阴性对照；lane4为PBS；lane5为水；lane6为lvCARbcma-1453；lane7为lvCARbcma-1454；lane8为lvCARbcma-1455；lane9为lvCARbcma-1456；lane10为lvCARbcma-1457；lane11为lvCARbcma-1458；lane12为lvCARbcma-1459；lane13为lvCARbcma-1460；
图11为mRNA相对表达量的柱状图；
图12为CAR蛋白表达量的WB检测图；其中，图12A，lane1为PBMC空细胞，lane2为对照病毒MOCK，lane3为lvCARbcma-1453，lane4为lvCARbcma-1454，lane5为lvCARbcma-1455，lane6为lvCARbcma-1456，lane7为lvCARbcma-1457，lane8为lvCARbcma-1458，lane9为lvCARbcma-1459，lane10为lvCARbcma-1460；图12B，是beta-actin内参条带；
图13为PD-1敲减重组慢病毒lvCARbcma-1453～lvCARbcma-1460转导的CAR-T细胞，与靶细胞孵育24h后，RT-QPCR检测PD-1的mRNA转录水平；
图14为不同效应细胞分别与靶细胞以10:1的比例共培养条件下，24h后检测对靶细胞的杀伤情况，E为效应细胞，T为靶细胞。
具体实施方式
以下实施例仅用于说明本发明而不用于限制本发明的范围。实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。
实施例1 构建重组慢病毒载体
一、材料
1、慢病毒骨架质粒pLenti-3G silencer，慢病毒包装质粒pPac-GP、pPac-R以及膜蛋白质粒pEnv-G，HEK293T/17细胞、同源重组酶、Oligo Annealing Buffer由世翱(上海)生物医药科技有限公司提供；
2、引物：根据引物设计原则设计扩增DNA片段和靶位点所需的引物，该引物由上海生物公司合成，具体为：
EF1 α-F：5’-ATTCAAAATTTTATCGATGCTCCGGTGCCCGTCAGT-3’(SEQ ID NO.25)
EF1 α-R：5’-TCACGACACCTGAAATGGAAGA-3’(SEQ ID NO.26)
CD8 leader-F：5’-GGTGTCGTGAGGATCCGCCACCATGGCCTTACCAGTGACCGC-3’(SEQ ID NO.27)
CD8 leader-R：5’-GTGTCATCTGGATGTCCGGCCTGGCGGCGTG-3’(SEQ ID NO.28)
VL-F：5’-CACGCCGCCAGGCCGGACATCCAGATGACCCAGAGCC-3’(SEQ ID NO.29)
VL-R：5’-ACGCTTGATCTCCAGTTTGGT-3’(SEQ ID NO.30)
OLC-VH-F：5’-ACTGGAGATCAAGCGTGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGG
GTGGCGGCGGATCTCAGGTGCAGCTGGTCCAGAG-3’(SEQ ID NO.31)
VH-R：5’-GCTGGACACGGTCACTAGTGTG-3’(SEQ ID NO.32)
CD8 Hinge-F：5’-AGTGACCGTGTCCAGCACCACGACGCCAGCGCC-3’(SEQ ID NO.33)
CD8 Hinge-R：5’-GTAGATATCACAGGCGAAGTCCA-3’(SEQ ID NO.34)
CD8 Transmembrane-F：5’-CGCCTGTGATATCTACATCTGGGCGCCCTTGGC-3’(SEQ ID NO.35)
CD8 Transmembrane-R：5’-TCTTTCTGCCCCGTTTGCAGTAAAGGGTGATAACCAGTG-3’(SEQ ID NO.36)
CD137-F：5’-AAACGGGGCAGAAAGAAACTC-3’(SEQ ID NO.37)
CD137-R：5’-TGCTGAACTTCACTCTCAGTTCACATCCTCCTTCTTCTTC-3’(SEQ ID NO.38)
TCR-F：5’-AGAGTGAAGTTCAGCAGGAGCG-3’(SEQ ID NO.39)
TCR-R：5’-GGAGAGGGGCGTCGACTTAGCGAGGGGGCAGGGC-3’(SEQ ID NO.40)
siRNA1453-F：5’-CCGGCTAAACTGGTACCGCATGAGCCTCGAGTCATGCGGTACCAGTTTAGCATTTTTTG-3’(SEQ ID NO.41)
siRNA1453-R：5’-AATTCAAAAAATGCTAAACTGGTACCGCATGACTCGAGGCTCATGCGGTACCAGTTTAG-3’(SEQ ID NO.42)
siRNA1454-F：5’-CCGGCATTGTCTTTCCTAGCGGAATCTCGAGTCCGCTAGGAAAGACAATGGTTTTTTTG-3’(SEQ ID NO.43)
siRNA1454-R：5’-AATTCAAAAAAACCATTGTCTTTCCTAGCGGACTCGAGATTCCGCTAGGAAAGACAATG-3’(SEQ ID NO.44)
siRNA1455-F：5’-CCGGAGGCGCAGATCAAAGAGAGTTCTCGAGCTCTCTTTGATCTGCGCCTTGTTTTTTG-3’(SEQ ID NO.45)
siRNA1455-R：5’-AATTCAAAAAACAAGGCGCAGATCAAAGAGAGCTCGAGAACTCTCTTTGATCTGCGCCT-3’(SEQ ID NO.46)
siRNA1456-F：5’-CCGGCCCTGTGGTTCTATTATATTACTCGAGATATAATAGAACCACAGGGAATTTTTTG-3’(SEQ ID NO.47)
siRNA1456-R：5’-AATTCAAAAAATTCCCTGTGGTTCTATTATATCTCGAGTAATATAATAGAACCACAGGG-3’(SEQ ID NO.48)
siRNA1457-F：5’-CCGGGGAACCCATTCCTGAAATTATCTCGAGAATTTCAGGAATGGGTCCAATTTTTTG-3’(SEQ ID NO.49)
siRNA1457-R：5’-AATTCAAAAAATTGGAACCCATTCCTGAAATTCTCGAGATAATTTCAGGAATGGGTTCC-3’(SEQ ID NO.50)
siRNA1458-F：5’-CCGGCAGGCCTAGAGAAGTTTCAGGCTCGAGTGAAACTTCTCTAGGCCTGCATTTTTTG-3’(SEQ ID NO.51)
siRNA1458-R：5’-AATTCAAAAAATGCAGGCCTAGAGAAGTTTCACTCGAGCCTGAAACTTCTCTAGGCCTG-3’(SEQ ID NO.52)
siRNA1459-F：5’-CCGGCAGGACTCATGTCTCAATGCCCTCGAGCATTGAGACATGAGTCCTGTGTTTTTTG-3’(SEQ ID NO.53)
siRNA1459-R：5’-AATTCAAAAAACACAGGACTCATGTCTCAATGCTCGAGGGCATTGAGACATGAGTCCTG-3’(SEQ ID NO.54)
siRNA1460-F：5’-CCGGTTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAATTTTTTG-3’(SEQ ID NO.55)
siRNA1460-R：5’-AATTCAAAAAATTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAA-3’(SEQ ID NO.56)
PD-1-QPCR-F：5’-TGCAGCTTCTCCAACACAT-3’(SEQ ID NO.57)
PD-1-QPCR-R：5’-CTTGTCCGTCTGGTTGCT-3’(SEQ ID NO.58)
WPRE-QPCR-F：5’-CCTTTCCGGGACTTTCGCTTT-3’(SEQ ID NO.59)
WPRE-QPCR-R：5’-GCAGAATCCAGGTGGCAACA-3’(SEQ ID NO.60)
Actin-QPCR-F：5’-CATGTACGTTGCTATCCAGGC-3’(SEQ ID NO.61)
Actin-QPCR-R：5’-CTCCTTAATGTCACGCACGAT-3’(SEQ ID NO.62)
CAR-QPCR-F：5’-GACTTGTGGGGTCCTTCTCCT-3’(SEQ ID NO.63)
CAR-QPCR-R：5’-GCAGCTACAGCCATCTTCCTC-3’(SEQ ID NO.64)
3、SEQ ID NO.15～SEQ ID NO.64所示的DNA序列由上海捷瑞生物工程有限公司合成，并以寡核苷酸干粉或者质粒形式保存；
4、工具酶BspE I、EcoR I、BamH I、Pvu I、Cla I、T4DNA连接酶均购自NEB公司；
5、高保真酶PrimeSTAR、RN购自Takara公司；
6、0.22μm-0.8μm PES滤器购自millipore公司；
7、质粒抽提试剂盒、琼脂糖凝胶回收试剂盒均购自MN公司；
8、感受态细胞TOP10购自tiangen公司；
9、NaCl、KCl、Na2HPO4.12H2O、KH2PO4、Trypsin、EDTA、CaCl2、NaOH、PEG6000均购自上海生工；
10、Opti-MEM、FBS、DMEM、1640、Pen-Srep、Hepes、购自invitrogen公司；
11、Biotinylated protein L购自GeneScript公司；
12、辣根过氧化物酶标记的二抗、DAB工作液均购自北京中杉金桥；
13、ECL+plusTM Western blotting system购自Amersham公司；
14、DNeasy试剂盒购自上海捷瑞公司；
15、淋巴细胞分离液购自深圳达科为公司；
16、SA-HRP购自上海翊圣公司；
17、支原体检测试剂盒、内毒素检测试剂盒、BCMA-K562、BCMA-PDL1-K562细胞株购自世翱(上海)公司；
18、LDH检测试剂盒购自promega公司。
二、重组慢病毒载体lvCARbcma-1453～lvCARbcma-1460的构建方法
参见图4，本发明所述重组慢病毒载体的构建方法如下：
1、将人EF1α启动子、CD8 leader嵌合受体信号肽、BCMA单链抗体轻链VL、Optimal Linker C、BCMA单链抗体重链VH、CD8 Hinge嵌合受体铰链、CD8 Transmembrane嵌合受体跨膜区、CD137嵌合受体共刺激因子、TCR嵌合受体T细胞激活域片段克隆至慢病毒骨架质粒pLenti-3G silencer，得到重组慢病毒质粒pCARbcma-silencer，再将siRNA片段分别连接到pCARbcma-silencer中，得到PD-1敲减重组慢病毒质粒pCARbcma-1453～pCARbcma-1460。
(1)将慢病毒骨架质粒pLenti-3G silencer使用Cla I和BamH I限制性内切酶进行双酶切，产物经过1.5％的琼脂糖凝胶电泳，确认7381bp的片段V1(图5所示)，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
1、溶胶 按200μl NTI/100mg gel比例加入溶胶液，50℃水浴放置5-10分钟。
2、结合DNA 11000g离心30秒，弃去滤液。
3、洗膜 加入700μl NT3，11000g离心30秒，弃去滤液。
4、洗膜 重复第三步一次
5、晾干 11000g离心1分钟，换新的收集管，室温放置1分钟。
6、洗脱DNA 加入15-30μl NE，室温放置1分钟，11000g离心1分钟，收集滤液。
表1 琼脂糖凝胶回收步骤
(2)用引物EF1α-F和EF1α-R以合成的SEQ ID NO.15为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃2min)*35cycle，72℃10min。产物经过1.5％的琼脂糖凝胶电泳，确认1208bp的片段a，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
试剂 体积(μl)
H2O 32.5
5×Buffer(with Mg2+) 10
dNTP(各2.5mM) 4
Primer1(+)(10μM) 1
Primer2(－)(10μM) 1
Template 1
PrimeSTAR 0.5
表2 50μl PCR反应体系
(3)用引物CD8 leader-F和CD8 leader-R以合成的SEQ ID NO.16为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认101bp的片段b，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(4)用引物VL-F和VL-R以合成的SEQ ID NO.17为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认336bp的片段c，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(5)用引物OLC-VH-F和VH-R以合成的SEQ ID NO.19为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认421bp的片段d，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(6)用引物CD8 Hinge-F和CD8 Hinge-R以合成的SEQ ID NO.20为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认147bp的片段e，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(7)用引物CD8 Transmembrane-F和CD8 Transmembrane-R以合成的SEQ ID NO.21为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认100bp的片段f，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(8)用引物CD137-F和CD137-R以合成的SEQ ID NO.22为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认142bp的片段g，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(9)用引物TCR-F和TCR-R以合成的SEQ ID NO.23为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认355bp的片段h，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(10)将DNA片段b、c、d各1μl作为模板，使用表3中的体系，除引物外加入Eppendorf管内，PCR循环条件为：98℃3min，(98℃10sec，60℃10sec，72℃30sec)*6cycle，加入引物CD8leader-F/VH-R,(98℃10sec，60℃10sec，72℃40sec)*24cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认814bp的片段i，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
试剂 体积(μl)
H2O 33.5-1*模板数
5×Buffer(with Mg2+) 10
dNTP(各2.5mM) 4
Primer1(+)(10μM) 1
Primer2(－)(10μM) 1
Template 1*模板数
PrimeSTAR 0.5
表3 50μl重叠PCR反应体系
(11)将DNA片段e、f、g、h各1μl作为模板，使用表3中的体系，除引物外加入Eppendorf管内，PCR循环条件为：98℃3min，(98℃10sec，60℃10sec，72℃30sec)*6cycle，加入引物CD8 Hinge-F/TCR-R,(98℃10sec，60℃10sec，72℃30sec)*24cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认704bp的片段j，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(12)将DNA片段V1、a、i、j以5μl总体积且摩尔比1:1:1:1的比例加入Eppendorf管内，加入同源重组酶反应液15μl，混匀后在42℃孵育30分钟，转移至冰上放置2-3分钟，将反应液加入50μl TOP10中，轻轻旋转以混匀内容物，在冰中放置30分钟，将管放到预加温到42℃的恒温水浴锅中热激90秒，快速将管转移到冰浴中，使细胞冷却2-3分钟，每管加900μl LB培养液，然后将管转移到37℃摇床上，温育1小时使细菌复苏，取100μl的转化菌液涂布于Amp LB琼脂平 板上，倒置平皿，于恒温培养箱中37℃培养，16小时。
挑取克隆进行菌落PCR鉴定，鉴定正确的克隆即为重组慢病毒质粒pCARbcma-silencer，对正确的克隆进行酶切鉴定(见图6)；
(13)将重组慢病毒质粒pCARbcma-silencer使用BspE I和EcoR I限制性内切酶进行双酶切，产物经过1.5％的琼脂糖凝胶电泳，确认10035bp的片段V2，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(14)将合成好的siRNA1453-F/R～siRNA1460-F/R分别用oligo annealing buffer溶解成20μM，对应的F和R各取30μl混合。然后将siRNA1453-F&R～siRNA1460-F&R混合物在水浴锅中95℃加热5分钟，然后水浴锅开盖置室温中自然冷却至室温，形成双链寡核苷酸片段。取1μl用于的连接反应(见表4)，4℃连接16h，转移至冰上放置2-3分钟，将反应液加入50μl TOP10中，轻轻旋转以混匀内容物，在冰中放置30分钟，将管放到预加温到42℃的恒温水浴锅中热激90秒，快速将管转移到冰浴中，使细胞冷却2-3分钟，每管加900μl LB培养液，然后将管转移到37℃摇床上，温育1小时使细菌复苏，取100μl的转化菌液涂布于Amp LB琼脂平板上，倒置平皿，于恒温培养箱中37℃培养，16小时。
挑取克隆进行菌落PCR鉴定，鉴定正确的克隆即为PD-1敲减重组慢病毒质粒pCARbcma-1453～pCARbcma-1460，对正确的克隆进行测序鉴定(见图7)。
试剂 体积(μl)
H2O 13
V2 3
10×T4DNA ligase Buffer 2
T4DNA ligase 1
退火的双链寡核苷酸 1
表4 20μl连接反应体系
2、重组慢病毒载体lvCARbcma-1453～lvCARbcma-1460的包装。
(1)完全培养基：取出预热好的新鲜培养基，加入10％FBS+5ml Pen-Srep，上下颠倒混匀即可；
(2)1XPBS溶液：称量NaCl 8g，KCl 0.2，Na2HPO4.12H2O 3.58g，KH2PO4 0.24g置于1000ml烧杯中，加入900ml Milli-Q grade超纯水溶解，溶解完成后，使用1000ml量筒定容至1000ml，121℃高温湿热灭菌20min；
(3)0.25％Trypsin溶液:称量Trypsin 2.5g，EDTA 0.19729g置于1000ml烧杯中，加入900ml1XPBS溶解，溶解完成后，使用1000ml量筒定容至1000ml，0.22μM过滤除菌，长期使用可保存至-20℃冰箱；
(4)0.5M CaCl2溶液：称量36.75g CaCl2用400ml Milli-Q grade超纯水溶解；用Milli-Q grade超纯水将总体积定容至500ml，混匀；0.22μm过滤除菌，分装保存到50ml离心管中，每管45ml左右，4℃保存。
(5)2XHBS溶液：称量4.09g NaCl，0.269g Na2HPO4，5.96g Hepes，用400ml Milli-Q grade超纯水溶解；校准pH仪后，用2M NaOH溶液将HBS溶液的pH调到7.05。调整每瓶HBS的PH消耗2M NaOH为3ml左右；
(6)从液氮罐中取出冻存的HEK293T/17细胞，迅速转移到37℃水浴中，1～2min后转移到超净台中，无菌操作将冻存管中的液体全部转移至10cm2培养皿中，补足含10％FBS的DMEM至8mL/10cm2dish，24h后显微镜观察细胞，细胞汇合的程度大于80％进行传代；
(7)选择细胞状态良好、无污染的HEK293T/17细胞，每2-6个培养皿为一组，将细胞胰酶消化后，用电动移液器吸取4-12ml完全培养基，向每个消化后的培养皿中加2ml，避免培养皿变干；使用1ml移液器将所有细胞吹打成单细胞悬液，转移到培养基瓶中；
(8)将上述2-6个培养皿中的剩余细胞转移到培养基瓶中，并用培养基再冲洗一便培养皿；
(9)盖紧培养基瓶盖，上下颠倒10次左右充分混匀细胞悬液，将细胞传到8-24个10cm2培养皿中，每皿的细胞密度应当约4×106个/10ml完全培养基左右。如果细胞密度和预期的相差较大，则需要对细胞进行计数，然后按照4×106个/皿的量接种；
(10)每6个培养皿整理为一摞，注意保持上下皿之间的配合。将培养皿左右，前后晃动数次，使细胞充分铺开，然后放入5％CO2培养箱。剩余细胞做同样处理；
(11)检查所传代细胞，细胞汇合度应当为70-80％，轮廓饱满，贴壁良好，在细胞培养皿中均匀分布；
(12)为细胞换液，将培养基替换为新鲜完全培养基,每皿9ml，并将培养箱的CO2浓度设定值提高到8％；
(13)按照N+0.5配DNA/CaCl2溶液。每皿HEK293T/17细胞转染质粒量按照下列比例使用：重组慢病毒质粒(20μg),pPac-GP(15μg)，pPac-R(10μg),pEnv-G(7.5μg)。取一个新的5ml离心管，加入0.5M CaCl2：0.25ml，重组慢病毒质粒20μg：pPac-GP 15μg：pPac-R 10μg：pEnv-G 7.5μg，补充超纯水至0.5ml盖上盖子，充分混匀；
(14)另取一支5ml离心管，加入0.5ml DNA/CaCl2溶液。打开涡旋振荡器，一只手拿住5ml离心管的上端，使管底接触振荡头，使液体在管壁上散开流动，另一只手拿一把1mL移液枪，吸取0.5mL 2×HBS溶液，缓慢滴加进入离心管，控制流速，以半分钟滴完为宜。2×HBS加入后，继续振荡5秒钟，停止振荡，可直接加入需要转染的细胞中；
(15)取一皿细胞，将离心管中的1mL钙转液滴加进去，尽可能使钙转试剂分布到整个培养皿中；
(16)钙转液加入后，在皿盖上做好标记，将培养皿放还到另一个5％CO2培养箱中。确保培养皿水平放置，每摞培养皿不要超过6个。在5％CO2培养箱中放置(6–8h)；
(17)将第一个培养箱的CO2浓度设定值调回到5％；
(18)24小时后，检查细胞状态。细胞汇合度应当为80–85％左右，状态良好。将培养基吸走，更换10ml新鲜的DMEM完全培养基；
(19)48小时后，观察转染效率。绝大多数细胞仍然是贴壁的。可以看到超过95％细胞都会带有绿色荧光。将同一个病毒包装上清液收集到一起，并向培养皿中继续添加10mL新鲜培养基；
(20)72小时后，再次将同一个病毒上清液收集到一起，两次收集的病毒可以放在一起，丢弃培养皿；此时收集的上清里包含了重组慢病毒载体lvCARbcma-1453～lvCARbcma-1460。
实施例2 重组慢病毒载体的浓缩及检测
一、离子交换色谱法纯化重组慢病毒载体(如图8所示)；
(1)将收集的上清液使用Thermo真空泵，经0.22μm-0.8μm的PES滤器抽滤，除去杂质；
(2)按1:1～1:10的比例往上清中加入1.5M NaCl 250mM Tris-HCl(pH 6-8)；
(3)将2个离子交换柱串联放置，用4ml 1M NaOH、4ml 1M NaCl、5ml 0.15M NaCl 25mM Tris-HCl(pH 6-8)溶液依次过柱；
(4)将步骤2中获得的溶液通过蠕动泵以1-10ml/min的速度给离子交换柱上样；
(5)全部上清液过柱后，使用10ml 0.15M NaCl 25mM Tris-HCl(pH 6-8)溶液清洗一遍；
(6)根据上样量使用1-5ml 1.5M NaCl 25mM Tris-HCl(pH 6-8)进行洗脱，收集洗脱液；
(7)将洗脱液分成25到50μl一管，冻存到-80℃冰箱，进行长期保存；
二、滴度测定
(1)取24孔板接种293T细胞。每孔细胞为5×104个，所加培养基体积为500ul,不同种类的细胞生长速度有所差异，进行病毒感染时的细胞融合率为40％-60％；
(2)准备3个无菌EP管，在每个管中加入90ul的新鲜完全培养基(高糖DMEM+10％FBS)接种细胞24小时后，取两个孔的细胞用血球计数板计数，确定感染时细胞的实际数目，记为N；
(3)取待测定的病毒原液10ul加入到第一个管中，轻轻混匀后，取10ul加入到第二个管中，然后依 次操作直到最后一管；在每管中加入410ul完全培养基(高糖DMEM+10％FBS),终体积为500ul；
(4)感染开始后20小时，除去培养上清，更换为500μl完全培养基(高糖DMEM+10％FBS)，5％CO2继续培养48小时；
(5)72小时后，观察荧光表达情况，正常情况下，荧光细胞数随稀释倍数增加而相应减少,并拍照；
(6)用0.2ml 0.25％胰酶-EDTA溶液消化细胞，在37℃放置1分钟。用培养基吹洗整个细胞面，离心收集细胞。按照DNeasy试剂盒的说明抽提基因组DNA。每个样品管中加入200μl洗脱液洗下DNA并定量；
(7)准备目的DNA检测qPCRmix总管Ⅰ(QPCR引物序列为SEQ ID NO.59---SEQ ID NO.60)：
Figure PCTCN2017110653-appb-000001
n＝number of reactions.例如：总反应数为40，将1ml 2×TaqMan Universal PCR Master Mix，4μl forward primer，4μl reverse primer，4μl probe和788μl H2O混和。震荡后放在冰上；
(8)准备内参DNA检测qPCRmix管Ⅱ(QPCR引物序列为SEQ ID NO.61---SEQ ID NO.62)：
Figure PCTCN2017110653-appb-000002
n＝number of reactions.例如：总反应数为40，将1ml 2×TaqMan Universal PCR Master Mix，100μl10×RNaseP primer/probe mix和700μl H2O混和。震荡后放在冰上；
(9)在预冷的96孔PCR板上完成PCR体系建立。从总管Ⅰ中各取45μl加入到A-D各行的孔中，从总管Ⅱ中各取45μl加入到E-G各行的孔中。
(10)分别取5μl质粒标准品和待测样品基因组DNA加入到A-D行中，每个样品重复1次。另留1个孔加入5μl的水做为无模板对照(no-template control)。
(11)分别取5μl基因组标准品和待测样品基因组DNA加入到E-G行中，每个样品重复1次。另留1个孔加入5μl的水做为无模板对照(no-template control)。
(12)所使用定量PCR仪为ABI PRISM 7500定量系统。循环条件设定为：50℃2分钟，95℃10分钟，然后是95℃15秒，60℃1分钟的40个循环。
数据分析：测得的DNA样品中整合的慢病毒载体拷贝数用基因组数加以标定，得到每基因组整合的病毒拷贝数。
滴度(integration units per ml，IU ml-1)的计算公式如下：
IU ml-1＝(C×N×D×1000)/V
其中：C＝平均每基因组整合的病毒拷贝数
N＝感染时细胞的数目(约为1×105)
D＝病毒载体的稀释倍数
V＝加入的稀释病毒的体积数
(13)重组慢病毒载体lvCARbcma-1453～lvCARbcma-1460的滴度结果(如图9所示)；
三、内毒素测定
(1)、内毒素工作标准品为15EU/支；
(2)、鲎试剂灵敏度λ＝0.25EU/ml,0.5ml/管
(3)、内毒素标准品稀释：取内毒素标准品一支，分别用BET水按比例稀释成4λ和2λ的溶解，封口膜封口，震荡溶解15min；稀释时每稀释一步均应在漩涡混合器上混匀30s；
(4)、加样：取鲎试剂若干支，每支加入BET水0.5ml溶解，分装至若干支无内毒素试管中，每管0.1ml。 其中2支为阴性对照管，加入BET水0.1ml；
2支为阳性对照管，加入2λ浓度的内毒素工作标准品溶液0.1ml；
2支为样品阳性对照管，加入0.1ml含2λ内毒素标准品的样品溶液(稀释20倍的待测样品1ml+4λ的内毒素标准品溶液1ml＝2ml含2λ内毒素标准品的稀释40倍样品)。
样品管中加入0.1ml样品，稀释比例见表5，37±1℃水浴(或培养箱)保温60±1min；
稀释倍数 原液 5 10 20 40 80 160
对应EU/ml 0.25 1.25 2.5 5 10 20 40
结果              
表5 内毒素稀释比例及对应内毒素含量
(5)、重组慢病毒载体lvCARbcma-1453～lvCARbcma-1460的内毒素检测结果(如表6所示)，内毒素含量在0～2.5EU/ml之间，符合要求；
稀释倍数 原液 5 10 20 40 80 160
对应EU/ml 0.25 1.25 2.5 5 10 20 40
lvCARbcma-1453 (+) (-) (-) (-) (-) (-) (-)
lvCARbcma-1454 (+) (-) (-) (-) (-) (-) (-)
lvCARbcma-1455 (+) (-) (-) (-) (-) (-) (-)
lvCARbcma-1456 (+) (+) (-) (-) (-) (-) (-)
lvCARbcma-1457 (+) (-) (-) (-) (-) (-) (-)
lvCARbcma-1458 (+) (+) (-) (-) (-) (-) (-)
lvCARbcma-1459 (+) (+) (-) (-) (-) (-) (-)
lvCARbcma-1460 (+) (-) (-) (-) (-) (-) (-)
表6 内毒素检测结果
四、支原体测定及比较
(1)在实验前三日，细胞样品用无抗生素培养基进行培养；
(2)收集1ml细胞悬浮液(细胞数大于1*105)，置于1.5ml离心管中；
(3)13000×g离心1min，收集沉淀，弃去培养基；
(4)加入500ul PBS用枪头吹吸或涡旋振荡，重悬沉淀。13000×g离心5min；
(5)步骤4重复一次；
(6)加入50μl Cell Lysis Buffer，用枪头吹吸，充分混匀后,在55℃水浴中孵育20min；
(7)将样品置于95℃中加热5min；
(8)13000×g离心5min后，取5μl上清作为模板，25μlPCR反应体系为：ddH20 6.5μl、Myco Mix 1μl、2x Taq Plus Mix Master(Dye Plus)12.5μl、模板5μl；PCR循环条件为：95℃ 30sec，(95℃ 30sec，56℃ 30sec，72℃ 30sec)*30cycle，72℃ 5min。
(9)支原体检测结果显示(如图10和表7所示)，重组慢病毒载体
lvCARbcma-1453～lvCARbcma-1460均不含支原体。
Figure PCTCN2017110653-appb-000003
表7 支原体检测结果
实施例3 重组慢病毒载体lvCARbcma-1453～lvCARbcma-1460的功能检测
一、CAR基因的细胞水平表达检测：
(1)重组慢病毒载体lvCARbcma-1453～lvCARbcma-1460和对照病毒MOCK感染PBMC细胞后，收集细胞采用RT-PCR进行CAR mRNA转录水平的检测，验证CAR基因的表达，如果CAR mRNA转录水平增高，则说明CAR基因的转录水平表达成功；
(2)重组慢病毒载体lvCARbcma-1453～lvCARbcma-1460和对照病毒MOCK感染PBMC细胞后，收集细胞采用western blot进行CAR蛋白表达水平的检测，验证CAR基因的表达，如果CAR蛋白表达水平增高，则说明CAR基因的翻译水平表达成功；
(3)分别将MOI＝15的lvCARbcma-1453～lvCARbcma-1460和对照病毒MOCK感染细胞，48h后提取6孔板中细胞的总RNA和总蛋白分别进行荧光定量PCR实验和免疫印迹实验。具体步骤：包被6孔板的四个孔，每个孔加入相应的PBS和RN，4℃过夜。12小时后按MOI＝15包被病毒，37℃培养箱放置5h；取出的6孔板，弃掉病毒上清，用PBS洗两遍，按1*106/孔，包被PBMC(用淋巴细胞分离液从人血中分离)，加入500ul培养基(含10％血清、20U/ml IL-2、Polybrene 8ug/ml)。静置20min，1000g 20℃离心30min，37℃培养48h。
(4)Trizol法提取6孔板中PBMC细胞的总RNA，逆转录扩增cDNA,用QPCR引物(序列为SEQ ID NO.63---SEQ ID NO.64)进行荧光定量PCR实验，反应体系见表8，以内参Actin为对照组，验证其mRNA的转录情况。
试剂 体积(μl)
SYBR premix ex taq: 10μl
ROX Reverse Dye(50x) 0.4μl
上游引物(2.5μM)： 0.5μl
下游引物(2.5μM)： 0.5μl
cDNA 1.0μl
ddH2O 7.6μl
表8 20μl qPCR反应体系
(5)蛋白免疫印迹(Western Blot)通过聚丙烯酰胺凝胶电泳将从PBMC中提取的总蛋白质按相对分子质量分离。采用湿转(4℃，400mA，120min)，将蛋白转移到PVDF膜上。用封闭液(含5％脱脂牛奶的TBST溶液)室温封闭PVDF膜1h，封闭液1:1000稀释Biotinylated protein L，然后与封闭好的PVDF膜室温孵育4℃过夜。TBST洗膜3次，每次10min。封闭液1:500稀释相应的SA-HRP，室温下孵育PVDF膜2h，TBST洗膜3次，每次10min。采用Amersham公司ECL+plusTM Western blotting system试剂盒进行显色。X光显影获得显示条带的胶片。
(6)RT-QPCR检测显示，重组慢病毒载体感染PBMC后的CAR基因的转录水平比对照病毒 MOCK和空细胞有明显升高（如图11和表9所示），说明CAR基因的转录水平表达成功。
Figure PCTCN2017110653-appb-000004
表9
（7）蛋白免疫印迹(Western Blot)的结果表明，重组慢病毒载体感染PBMC后CAR蛋白的表达水平比对照病毒MOCK和空细胞有明显升高（如图12所示），说明CAR基因的翻译水平表达成功。
二、PD-1敲减效果评估（PD-1mRNA转录水平）
（1）分别培养BCMA-K562细胞和PBMC细胞；
（2）实验开始前4天，MOI=15的lvCARbcma-1453～lvCARbcma-1460的病毒感染PBMC细胞，培养72-96h后可安排开始实验；
（3）收集靶细胞（BCMA-K562）4x105cells和效应细胞（lvCARbcma-1453-PBMC～lvCARbcma-1460-PBMC细胞）2.8x106cells，800g，6min离心，弃上清；
（4）用1ml 1xPBS溶液分别重悬靶细胞和效应细胞，800g，6min离心，弃上清；
（5）重复步骤4一次；
（6）用700ul培养基（1640培养基+10%FBS）重悬效应细胞，用2ml培养基（1640培养基+10%FBS）重悬靶细胞；
（7）设置效靶比为10:1的实验孔，并设置Blank组；
（8）250xg，5min平板离心；
（9）37℃5%CO2培养箱中共培养24小时，1000xg，2min平板离心，收集细胞检测PD-1mRNA转录水平；
（10）Trizol法提取上述混合细胞的总RNA，逆转录扩增cDNA,用QPCR引物（序列为SEQ ID NO.57---SEQ ID NO.58）进行荧光定量PCR实验，反应体系见表6，以内参Actin为对照组，验证其mRNA的转录情况。
（11）RT-QPCR检测结果显示，一部分PD-1敲减重组慢病毒载体转导的PBMC与靶细胞孵育后，PD-1基因的mRNA比对照病毒lvCARbcma-1460有明显降低（如图13和表10所示），说明PD1siRNA对PD-1基因的转录水平有敲减作用，其中lvCARbcma-1453的敲减效果最好，达到70%以上。Blank组由于不会被靶细胞激活，所以PD-1转录水平没有上调。
Figure PCTCN2017110653-appb-000005
表10
三、细胞杀伤实验效果评估
(1)分别培养BCMA-PDL1-K562细胞和PBMC细胞；
(2)实验开始前4天，MOI＝15的lvCARbcma-1453～lvCARbcma-1460的病毒感染PBMC细胞，培养72-96h后可安排开始实验；
(3)收集靶细胞(BCMA-PDL1-K562)4x105cells和效应细胞(CART细胞)2.8x106cells，800g，6min离心，弃上清；
(4)用1ml 1xPBS溶液分别重悬靶细胞和效应细胞，800g，6min离心，弃上清；
(5)重复步骤3一次；
(6)用700ul培养基(1640培养基+10％FBS)重悬效应细胞，用2ml培养基(1640培养基+10％FBS)重悬靶细胞；
(7)设置效靶比为10:1的实验孔，并设置对照组，每组3个复孔；
(8)250xg，5min平板离心；
(9)37℃5％CO2培养箱中培养24小时；
(10)250xg，5min平板离心；
(11)取每个孔的50ul上清到新96孔板中，并且每孔加入50ul底物溶液(避光操作)；
(12)避光孵育25分钟；
(13)每孔加入50ul终止液；
(14)酶标仪检测490nm吸光度；
(15)将3个复孔取平均值；将所有实验孔、靶细胞孔和效应细胞孔的吸光值减去培养基背景吸光值的均值；将靶细胞最大值的吸光值减去体积校正对照吸光值的均值。
(16)将步骤15中获得的经过校正的值带入下面公式，计算每个效靶比所产生的细胞毒性百分比。结果如图14所示，PD-1敲减重组慢病毒载体转导的PBMC细胞在10:1效靶比条件下杀伤效率明显高于PBMC空细胞，其中lvCARbcma-1453-PBMC细胞对靶细胞的杀伤效率最高，高于40％。lvCARbcma-1454-PBMC细胞对靶细胞的杀伤效率次之。其余lvCARbcma-1455-PBMC～lvCARbcma-1458-PBMC细胞对靶细胞的杀伤效率与对照lvCARbcma-1460-PBMC细胞相比相差不多，对比图13的结果可以看出，由于靶细胞中PD-L1的存在，与PD-1结合后传递抑制信号，抑制了T细胞的免疫活性，导致T细胞对靶细胞的杀伤效率大幅度降低。而PD-1分子表达水平被大幅度降低的情况下，可以有效阻断PD-1/PDL-1的信号通路的激活，使得T细胞可以正常发挥对靶细胞的杀伤作用。未来lvCARbcma-1453载体及其转导的T细胞可以应用于临床，抑制CAR-T细胞内的PD-1表达水平，增强CAR-T细胞在体内对肿瘤的杀伤效果，达到抑制免疫逃脱的效果。
杀伤效率＝(实验孔-效应细胞孔-靶细胞孔)/(靶细胞最大孔-靶细胞孔)X100％
以上已对本发明创造的较佳实施例进行了具体说明，但本发明创造并不限于所述实施例，熟悉本领域的技术人员在不违背本发明创造精神的前提下还可作出种种的等同的变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
Figure PCTCN2017110653-appb-000006
Figure PCTCN2017110653-appb-000007
Figure PCTCN2017110653-appb-000008
Figure PCTCN2017110653-appb-000009
Figure PCTCN2017110653-appb-000010
Figure PCTCN2017110653-appb-000011
Figure PCTCN2017110653-appb-000012
Figure PCTCN2017110653-appb-000013
Figure PCTCN2017110653-appb-000014
Figure PCTCN2017110653-appb-000015
Figure PCTCN2017110653-appb-000016
Figure PCTCN2017110653-appb-000017
Figure PCTCN2017110653-appb-000018
Figure PCTCN2017110653-appb-000019
Figure PCTCN2017110653-appb-000020

Claims (15)

  1. 一种敲减人PD-1的siRNA，所述siRNA选自下述中的a-g中的任一对：
    a.SEQ ID NO.41和SEQ ID NO.42所示的核苷酸序列；
    b.SEQ ID NO.43和SEQ ID NO.44所示的核苷酸序列；
    c.SEQ ID NO.45和SEQ ID NO.46所示的核苷酸序列；
    d.SEQ ID NO.47和SEQ ID NO.48所示的核苷酸序列；
    e.SEQ ID NO.49和SEQ ID NO.50所示的核苷酸序列；
    f.SEQ ID NO.51和SEQ ID NO.52所示的核苷酸序列；
    g.SEQ ID NO.53和SEQ ID NO.54所示的核苷酸序列。
  2. 如权利要求1所述的siRNA在制备消除或减轻肿瘤的免疫逃脱机制药物中的应用。
  3. 一种包含权利要求1所述的siRNA的重组表达载体。
  4. 如权利要求3所述的重组表达载体，其特征在于：所述表达载体为慢病毒表达载体、逆转录病毒表达载体、腺病毒表达载体、腺相关病毒表达载体或质粒。
  5. 如权利要求4所述的重组表达载体，其特征在于：所述的慢病毒表达载体包括：用于质粒复制的原核复制子pUC Ori序列，如SEQ ID NO.2所示；用于目的菌株大量扩增的含氨苄青霉素抗性基因AmpR序列，如SEQ ID NO.1所示；用于增强真核细胞内的复制的病毒复制子SV40 Ori序列，如SEQ ID NO.3所示；用于慢病毒包装的慢病毒包装顺式元件；用于真核细胞表达绿色荧光的ZsGreen1绿色荧光蛋白，如SEQ ID NO.11所示；用于共同转录表达蛋白质的IRES核糖体结合序列，如SEQ ID NO.12所示；用于嵌合抗原受体基因的真核转录的人EF1α启动子，如SEQ ID NO.15所示；用于组成集识别、传递、启动于一体的二代CAR或三代CAR的抗BCMA嵌合抗原受体的编码基因；用于增强转基因的表达效率的eWPRE增强型土拨鼠乙肝病毒转录后调控元件，如SEQ ID NO.13所示；用于胞内转录siRNA的人RNA聚合酶III启动子hU6，如SEQ ID NO.14所示。
  6. 如权利要求5所述的重组表达载体，其特征在于：所述慢病毒包装顺式元件采用第二代慢病毒载体包括：如SEQ ID NO.5所示的慢病毒5 terminal LTR、如SEQ ID NO.6所示的慢病毒3 terminal Self-Inactivating LTR、如SEQ ID NO.7所示的Gag顺式元件、如SEQ ID NO.8所示的RRE顺式元件、如SEQ ID NO.9所示的env顺式元件、如SEQ ID NO.10所示的cPPT顺式元件。
  7. 如权利要求5所述的重组表达载体，其特征在于：所述慢病毒包装顺式元件采用第三代慢病毒载体包括：如SEQ ID NO.5所示的慢病毒5 terminal LTR、如SEQ ID NO.6所示的慢病毒3 terminal Self-Inactivating LTR、如SEQ ID NO.7所示的Gag顺式元件、如SEQ ID NO.8所示的RRE顺式元件、如SEQ ID NO.9所示的env顺式元件、如SEQ ID NO.10所示的cPPT顺式元件所述慢病毒包装顺式元件，以及如SEQ ID NO.4所示的RSV启动子。
  8. 如权利要求5所述的重组表达载体，其特征在于：所述eWPRE增强型土拨鼠乙肝病毒转录后调控元件有6个核苷酸的增强突变，具体为：g.396G>A、g.397C>T、g.398T>C、g.399G>A、g.400A>T、g.411A>T。
  9. 如权利要求5所述的重组表达载体，其特征在于：所述抗BCMA嵌合抗原受体，包括依次串联的如SEQ ID NO.16所示的CD8 leader嵌合受体信号肽、如SEQ ID NO.17所示的BCMA单链抗体轻链VL、如SEQ ID NO.18所示的Optimal Linker C、如SEQ ID NO.19所示的BCMA单链抗体重链VH、如SEQ ID NO.20所示的CD8 Hinge嵌合受体铰链、如SEQ ID NO.21所示的CD8 Transmembrane嵌合受体跨膜区、如SEQ ID NO.22所示的CD137嵌合受体共刺激因子，以及如SEQ ID NO.23所示的TCR嵌合受体T细胞激活域。
  10. 如权利要求5所述的重组表达载体，其特征在于：所述抗BCMA嵌合抗原受体，包括依次串联的如SEQ ID NO.16所示的CD8 leader嵌合受体信号肽、如SEQ ID NO.17所示的BCMA单链抗体轻链VL、如SEQ ID NO.18所示的Optimal Linker C、如SEQ ID NO.19所示的BCMA单链 抗体重链VH、如SEQ ID NO.20所示的CD8 Hinge嵌合受体铰链、如SEQ ID NO.21所示的CD8 Transmembrane嵌合受体跨膜区、如SEQ ID NO.24所示的CD28嵌合受体共刺激因子、如SEQ ID NO.22所示的CD137嵌合受体共刺激因子以及如SEQ ID NO.23所示的TCR嵌合受体T细胞激活域。
  11. 一种包含权利要求1所述的siRNA的慢病毒表达载体的构建方法，包括以下步骤：
    (1)将含氨苄青霉素抗性基因AmpR序列(如SEQ ID NO.1所示)、原核复制子pUC Ori序列(如SEQ ID NO.2所示)、病毒复制子SV40 Ori序列(如SEQ ID NO.3所示)、用于慢病毒包装的慢病毒包装顺式元件、ZsGreen1绿色荧光蛋白(如SEQ ID NO.11所示)、IRES核糖体结合序列(如SEQ ID NO.12所示)、eWPRE增强型土拨鼠乙肝病毒转录后调控元件(如SEQ ID NO.13所示)、人RNA聚合酶III启动子hU6(如SEQ ID NO.14所示)存储于慢病毒骨架质粒上；
    (2)将人EF1α启动子(如SEQ ID NO.15所示)、用于组成集识别、传递、启动于一体的二代CAR或三代CAR的抗BCMA嵌合抗原受体组合成二代CAR或三代CAR设计方案，经过酶切、连接、重组反应克隆至慢病毒骨架质粒中，得到二代CAR或三代CAR设计的重组慢病毒质粒；
    (3)将上述siRNA以及如SEQ ID NO.55和SEQ ID NO.56所示的negative control序列，分别克隆至步骤(2)所得的重组慢病毒质粒中，得到PD-1敲减重组慢病毒质粒；
    (4)将步骤(3)得到的重组慢病毒质粒分别与慢病毒包装质粒pPac-GP、pPac-R以及膜蛋白质粒pEnv-G共同转染HEK293T/17细胞，在HEK293T/17细胞中进行基因转录表达后，包装成功重组慢病毒载体会释放到细胞培养上清中，收集包含的重组慢病毒载体的上清液；
    (5)将得到的重组慢病毒上清采用抽滤、吸附、洗脱的离子交换方式进行纯化，分别得到重组慢病毒载体。
  12. 如权利要求11所述的构建方法，其特征在于：步骤(5)中，所述抽滤步骤控制上清体积在200ml～2000ml，真空度控制在-0.5MPA～-0.9MPA，防止由于堵孔带来的载体损失；所述吸附步骤控制溶液的PH值在6～8，防止PH的变化导致载体失活；所述洗脱步骤控制洗脱液的离子强度在0.5M～1.0M，防止离子强度的变化导致洗脱不完全或者载体失活。
  13. 如权利要求3-10任一项所述的重组表达载体在制备CAR-T治疗肿瘤过程中消除或减轻肿瘤免疫逃脱机制药物中的应用。
  14. 一种CART细胞，所述的CAR-T细胞是由上述siRNA修饰的T淋巴细胞。
  15. 根据权利要求14所述的CAR-T细胞在制备治疗多发性骨髓瘤、胰腺癌、脑胶质瘤、骨髓瘤药物中的应用。
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