A virus-independent engineered induced pluripotent stem cell and its preparation method and application

Abstract

The present invention belongs to the field of cellular immunity technology and relates to a non-viral dependent engineered induced pluripotent stem cell and its preparation method and application. A gene encoding a chimeric antigen receptor is inserted into the SIRPA gene on the genome of the engineered induced pluripotent stem cell. The present invention edits iPSC cells by site-directed insertion of the SIRPA gene position, thereby achieving simultaneous knockout of SIRPA and integration of exogenous chimeric antigen receptor CAR, which can further induce differentiation into CAR-iMac, causing it to differentiate into monocyte / macrophage tumor-targeted cells, enhance its phagocytic ability, further present tumor antigens, improve the tumor immune microenvironment, enhance anti-tumor immunity, effectively overcome the corresponding problems of directly extracting natural macrophages from the human body, reduce production costs and time costs, and facilitate clinical applications. In addition, the use of non-viral mediated gene editing methods significantly improves the genetic silencing phenomenon.

Description

Technical Field

[0001] The present invention belongs to the field of cellular immunity technology, and relates to a virus-independent engineered induced pluripotent stem cell and its preparation method and application, and in particular to its application in the preparation of chimeric antigen receptor-macrophages. Background Art

[0002] Macrophages are the most numerous immune cells in the tumor microenvironment. Their interactions with tumor cells have a significant impact on tumor progression, metastasis, and treatment outcomes. Depending on the microenvironment, macrophages can polarize into either the M1 (anti-tumor) or M2 (pro-tumor) phenotype. This plasticity allows macrophages to be repolarized to the anti-tumor M1 phenotype, enhancing their anti-tumor effects. Macrophages possess potent phagocytic abilities, enabling early elimination of tumor cells. Genetically engineered macrophages can maintain their anti-tumor M1 phenotype despite the immunosuppressive microenvironment, effectively killing cancer cells. Macrophages can ingest, process, and present tumor antigens, activating T cell-mediated immune responses and enhancing immune surveillance against tumors. Monocytes in the blood are continuously recruited to tumor sites, providing a continuous supply of macrophages for immunotherapy. For example, alveolar macrophages effectively clear allergens from the lungs, while liver macrophages (Kupffer cells) help eliminate circulating pathogens and toxins from the blood. Macrophages can also phagocytose immunogenic dead tumor cells and present tumor antigens to T cells, exerting anti-tumor immunity. Furthermore, chimeric antigen receptor-macrophages can be developed, modifying macrophages with specific chimeric antigen receptors (CARs) to enhance their phagocytic activity and antigen presentation against tumors.

[0003] However, there are currently many challenges in directly obtaining natural macrophages from the human body for treatment: limited cell numbers, high in vivo heterogeneity leading to unstable efficacy, complex in vitro culture and requiring specific conditions such as M-CSF, the tumor microenvironment will cause them to promote tumor growth and immune escape, and allogeneic cells may also trigger rejection reactions; the risk of random viral insertion: viral vectors are usually used for modification, especially retroviral and lentiviral vectors, which may randomly insert exogenous genes into the genome of host cells. This random insertion may lead to changes in tumor genes and increase the risk of patients developing cancer. Viral vectors may cause an immune response in the host, resulting in limited therapeutic effects or adverse reactions; genes integrated into primary cells using viral vectors may decrease in expression over time or even be completely lost, which limits the effectiveness of complex cell engineering and treatment.

[0004] Induced pluripotent stem cells (iPSCs)-derived macrophages have certain advantages over natural macrophages extracted from the human body as cell therapy in cellular immunotherapy. iPSCs can be expanded indefinitely, providing a continuous source of cells; iPSCs are easy to gene-edit, and macrophages can be customized to enhance their anti-tumor function or avoid immune rejection, while gene editing of natural macrophages is more complicated; using macrophages differentiated from the patient's own iPSCs can avoid immune rejection, while allogeneic macrophages may cause an immune response in the host; iPSC-derived macrophages are genetically and phenotypically homogeneous, which makes treatment more standardized and reliable. It is predicted that the heterogeneity of natural macrophages may lead to inconsistent therapeutic effects; iPSC-derived macrophages have been used to model diseases, screen drugs and study immune responses, providing powerful research tools and application potential; iPSC-derived macrophages have a longer survival time in the body and can continuously exert anti-tumor effects, while the survival and function of natural macrophages in the body may be affected by multiple factors; iPSCs can be generated from any donor and are not restricted by genetic background, which increases the availability of treatment; therefore, iPSC-derived macrophages provide a customizable, scalable, and potentially safer and more effective treatment in cellular immunotherapy.

[0005] In summary, the development of new iPSC-derived macrophages and the expansion of tumor treatment tools are of great significance to the field of tumor treatment. Summary of the Invention

[0006] In response to the deficiencies of the existing technology and actual needs, the present invention provides a non-viral-dependent engineered induced pluripotent stem cell and its preparation method and application. Induced pluripotent stem cells are transformed through non-viral-dependent site-directed gene editing technology, and are further differentiated into function-enhanced chimeric antigen receptor-macrophages to improve the tumor targeting, killing efficiency and safety and effectiveness of clinical applications of chimeric antigen receptor-macrophages.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a virus-independent engineered induced pluripotent stem cell, wherein a gene encoding a chimeric antigen receptor is inserted into the SIRPA gene on the genome of the engineered induced pluripotent stem cell.

[0009] In the present invention, iPSC-derived macrophages (iMac) are used as carrier cells for cellular immunotherapy. Combining the advantages of the current booming chimeric antigen receptor technology and the increasingly mature gene optimization technology, iPSC cells are edited at the SIRPA gene site at the molecular level, and the CAR coding gene is inserted to obtain engineered iPSC cells, which inhibit SIRPA gene expression while stably expressing CAR molecules. This has broad application prospects, such as further inducing differentiation into iPSC-derived chimeric antigen receptor-macrophages (CAR-iMac), blocking the Sirpα-CD47 pathway while utilizing its efficient infiltration to enhance its phagocytic ability and antigen presentation ability, significantly improving the ability to recognize and kill tumor cells, and providing a new strategy for tumor treatment.

[0010] In the present invention, the coding gene of CAR is introduced into the SIRPA gene site-specifically to transform iPSC cells. It can be understood that the type of specific CAR can be selected according to actual needs, for example, CAR for anti-tumor use can be selected.

[0011] Preferably, the chimeric antigen receptor comprises a leader peptide, an extracellular recognition region, a hinge region and a transmembrane / intracellular signaling region, a co-stimulatory factor, a signal transduction region and a pattern recognition receptor.

[0012] The transmembrane / intracellular signaling region of the present invention can be selected from the transmembrane region sequences of CD8a and CD3ζ. In the structure of CAR, the main function of the transmembrane region is to anchor the CAR on the cell membrane and ensure that the antigen binding region of the CAR (composed of a single-chain variable fragment scFv) can be stably expressed on the cell surface so as to recognize and bind to specific tumor antigens. The advantages of the transmembrane region of CD8a in CAR design include: Enhanced surface expression: A suitable transmembrane region can enhance the expression of CAR on the cell surface, thereby improving the cell's ability to recognize tumor antigens; Improved fluidity and stability: Use The CD8a-derived transmembrane region can improve the fluidity and stability of CAR, which is crucial for the function of CAR; promote cell activation and reduce nonspecific activation; the signal transduction region can select the CD3ζ signal transduction domain, which can enhance the cell's ability to recognize and kill tumor cells; the pattern recognition receptor can select the TLR4 (Toll-like receptor 4) pattern recognition receptor, and the TIR domain of TLR4 can promote the polarization of macrophages to the M1 type, enhance the anti-tumor effect, enhance the antigen presentation ability, and resist tumor immunosuppression, thereby playing a more effective role in the anti-tumor process.

[0013] Preferably, the amino acid sequence of the leader peptide includes the sequence shown in SEQ ID NO.4.

[0014] Preferably, the extracellular recognition region comprises an antibody.

[0015] Preferably, the antibody comprises an anti-GPC3 antibody.

[0016] Preferably, the amino acid sequence of the anti-GPC3 antibody includes the sequence shown in SEQ ID NO.5.

[0017] Preferably, the amino acid sequence of the hinge region includes the sequence shown in SEQ ID NO.6.

[0018] Preferably, the amino acid sequence of the transmembrane / intracellular signaling region includes the sequence shown in SEQ ID NO.7.

[0019] Preferably, the amino acid sequence of the co-stimulatory factor includes the sequence shown in SEQ ID NO.8.

[0020] Preferably, the amino acid sequence of the signal transduction region includes the sequence shown in SEQ ID NO.9.

[0021] Preferably, the amino acid sequence of the pattern recognition receptor includes the sequence shown in SEQ ID NO.10.

[0022] Preferably, the nucleic acid sequence of the gene encoding the chimeric antigen receptor includes the sequence shown in SEQ ID NO.11.

[0023] SEQ ID NO. 1 (sgRNA): GTCTGATTCGGACGAGGTAGAGG.

[0024] SEQ ID NO.2 (left homology arm):

[0025] TGAGCTGTGCAGAGCCTGCTGGCCGCACTGCCCTTCTTCCAGCTGCTGATTCAGTTTCCAGTGGATCTGGGGTGCCCTCATGACCGCTCTGTGGTTCACTCCAGATGAGAAGGAACGTTTCTGTTCCTCAGGAGTGAGATTTCTGCCTGTATCCTGCAGCAGGGGTTCCTCCGTAGCTGTCAGCTACAAATATATTCCCCCCTGGCACCTCAGCATGGTTTTTGTGCTGTTCATGGATGAGTCTCGTGGGCAAGTGTGTACAGACCCATGAGTGTGCCCATGTGGCTCGAGACATCTAAGAAGGTCCAGCCAGATGTTCTCAGTTAATGATGCCTGCTTAGTGGTGAAAAGCAGTGGTGGGTTTGGTTTTCTGTTTTTAATCTGCATACGTGAAGCCTCTATTCCATGTGGTCCCTAGAGAACACTGGATCTAATGAACGGAACATCTATATTGTGGTGGGTGTGGTGTGCACCTTGCTGGTGGCCCTACTGATGGCGGC。

[0026] SEQ ID NO.3 (Right homologous arm):

[0027] TGGGTGCATTCCCCTCTTCCTCCCTAAGGGTTTGTCCCTGGACTGTCCTCGGAGGGAGACGCCATTAGGTGCTTTGGGTTAAGGACATCAGCTTCTGCCAGTAGCAAGAAGTCCAGAGGTAGTGGTGCTAGGGCTGGGGCAGTGGCCTCACCATGTTAGAGTCTGGGTGGGTCACTCTGTGGCTTCCCCCTCATGGTTACAAGATGTTCATCACTGCCCCAGGCCCCGTGTCCTCAAGGGGCCACTACTGAGCAAGGGAGCTGCCAGCCTGGGAAGAGAGGAGGCAGGGACTCTTCTTACCTGTCTCAGGGAGGACATCGTCTTTCCCAGGAGCCTCCCGGCAGACTTCTGCTTTCCTGGTACAAGTCTAGGTTACATGACCAGTCCCTGGACAGGAGGCCAGAGGGAATGGAGATAATAGCGCCACCATTTGATTGGGAGGACTTAACAAGTGATTGCATGGGAAATGTTAGGGAAGTAACCTGGCACACAAGTGCTCA。

[0028] SEQ ID NO.4 (Leader peptide): MALPVTALLLPLALLLHAARP。

[0029] SEQ ID NO.5 (Anti-GPC3 scFv single-chain variable region):

[0030] QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYEMHWVRQAPGQGLEWMGALDPKTGDTAYSQKFKGRVTLTADESTSTAYMELSSLRSEDTAVYYCTRFYSYTYWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTPGEPASISCRSSQSLVHSNGNTYLHWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQNTHVPPTFGQGTKLEIK。

[0031] SEQ ID NO.6 (CD8 hinge region):

[0032] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD.

[0033] SEQ ID NO. 7 (CD8a transmembrane / intracellular region): IYIWAPLAGTCGVLLLSLVITLYC.

[0034] SEQ ID NO.8 (4-1BB co-stimulatory molecule):

[0035] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0036] SEQ ID NO.9 (CD3ζ):

[0037] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0038] SEQ ID NO.10 (TLR4 Toll pattern recognition receptor):

[0039] NIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDG.

[0040] SEQ ID NO.11:

[0041]

[0042] Preferably, the GenBank accession number of the SIRPA gene is: NM_080792.2.

[0043] In the present invention, the CAR coding gene can be reversely inserted into exon 6 of the SIRPA gene.

[0044] In a second aspect, the present invention provides a method for preparing the virus-independent engineered induced pluripotent stem cells described in the first aspect, the method comprising:

[0045] The coding gene of the chimeric antigen receptor is site-specifically inserted into the SIRPA gene on the genome of the induced pluripotent stem cell to obtain the engineered induced pluripotent stem cell.

[0046] Preferably, the site-directed insertion method comprises the CRISPR / Cas method.

[0047] In the present invention, gene site-directed insertion is performed based on CRISPR / Cas, which does not rely on vectors such as lentivirus. It can effectively overcome the problem of transgene silencing and ensure the stable expression of the transferred gene, while improving safety and reducing the host's immune response to CAR-iMac cells.

[0048] Preferably, the nucleic acid sequence of the sgRNA of the CRISPR / Cas method includes the sequence shown in SEQ ID NO.1.

[0049] Preferably, the nucleic acid sequence of the homologous arms of the homologous recombination template of the CRISPR / Cas method includes the sequences shown in SEQ ID NO.2 and SEQ ID NO.3.

[0050] Preferably, the Cas enzyme of the CRISPR / Cas method includes Cas9.

[0051] The specific CRISPR / Cas editing system designed in the present invention can achieve efficient gene editing, facilitate large-scale production, and control quality.

[0052] In a third aspect, the present invention provides the use of the non-viral dependent engineered induced pluripotent stem cells described in the first aspect in preparing chimeric antigen receptor macrophages.

[0053] The present invention develops a novel CAR-iMac cell construction method, which uses non-virus-dependent site-directed gene-edited iPSCs as the starting material. Through gene editing, the function of CAR-iMac cells is enhanced, the cell killing ability is improved, and the unlimited proliferation ability of iPSCs is utilized to achieve the off-the-shelf supply of CAR-iMac cell therapy, thereby reducing treatment costs, accelerating the treatment process, and promoting the clinical application of CAR-iMac cell therapy.

[0054] In a fourth aspect, the present invention provides a method for preparing chimeric antigen receptor macrophages, the preparation method comprising:

[0055] The non-virus-dependent engineered induced pluripotent stem cells described in the first aspect are induced and cultured to obtain the chimeric antigen receptor macrophages.

[0056] Preferably, the culture medium for induction culture contains cytokines.

[0057] Preferably, the cytokines include at least one of BMP4 (bone morphogenetic protein 4), bFGF (fibroblast growth factor), VEGF (vascular endothelial growth factor), SCF (stem cell factor) or inhibitors (such as Y-27632).

[0058] In a fifth aspect, the present invention provides a chimeric antigen receptor macrophage, which is obtained by induced differentiation of the non-viral dependent engineered induced pluripotent stem cells described in the first aspect, or prepared by the method for preparing a chimeric antigen receptor macrophage described in the fourth aspect.

[0059] In a sixth aspect, the present invention provides use of the non-viral-dependent engineered induced pluripotent stem cells described in the first aspect and / or the chimeric antigen receptor macrophages described in the fifth aspect in the preparation of anti-tumor preparations.

[0060] The present invention designs non-virus-dependent engineered induced pluripotent stem cells and chimeric antigen receptor macrophages, which can improve the tumor targeting, killing efficiency and safety and effectiveness of clinical applications of CAR-iMac cells, and can be applied to anti-tumor preparations such as tumor treatment drugs.

[0061] Compared with the prior art, the present invention has at least the following beneficial effects:

[0062] The present invention edits iPSC cells by site-specific insertion at the SIRPA gene location, achieving simultaneous knockout of SIRPA and integration of exogenous chimeric antigen receptor CAR, which can further induce differentiation into CAR-iMac, enabling it to differentiate into monocyte / macrophage tumor-targeting cells, enhance its phagocytic ability, further present tumor antigens, improve the tumor immune microenvironment, and enhance anti-tumor immunity. It effectively overcomes the corresponding problems of directly extracting natural macrophages from the human body, reduces production costs and time costs, and is conducive to clinical application. In addition, the use of non-viral-mediated gene editing methods can significantly improve the genetic silencing phenomenon. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 Schematic diagram of the chimeric antigen receptor CAR structure;

[0064] Figure 2 Schematic diagram of site-directed insertion and knockout strategies;

[0065] Figure 3 The figure shows the results of immunoblotting to examine SIRPα protein expression;

[0066] Figure 4 The expression level results of SIRPA gene are shown in FIG.

[0067] Figure 5 This is the result of flow cytometry detection of GPC3-CAR expression;

[0068] Figure 6 This is the result of the killing experiment to detect the killing ability of CAR-iMac cells on Hepeg2 liver cancer cells. DETAILED DESCRIPTION

[0069] The technical solution of the present invention will be further described below with reference to the accompanying drawings and through specific embodiments. However, the following embodiments are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0070] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0071] Example 1

[0072] In this example, a chimeric antigen receptor molecule (GPC3-CAR) targeting GPC3 (glypican 3) was designed, and a homologous recombination (HDR) template was assembled using GPC3-CAR and inserted into the gene encoding signal regulatory protein α (SIRPα) (SIRPA, GenBank accession number: NM_080792.2).

[0073] The homologous recombination template includes left and right homology arms (SEQ ID NO. 2 and SEQ ID NO. 3) and the GPC3-CAR (leader peptide, extracellular recognition region, hinge region, transmembrane / intracellular signaling region, costimulatory factor, signaling domain, and pattern recognition receptor) nucleic acid sequences. From N-terminus to C-terminus, the GPC3-CAR sequence consists of the leader peptide (SEQ ID NO. 4), the anti-GPC3 antibody sequence (scFv amino acid sequence, SEQ ID NO. 5) for the extracellular recognition region, the CD8 hinge region (SEQ ID NO. 6), the transmembrane / intracellular signaling domain (CD8a TM) (SEQ ID NO. 7), the costimulatory factor 4-1BB (SEQ ID NO. 8), the cell antigen receptor CD3ζ signaling domain (SEQ ID NO. 9), and the pattern recognition receptor TLR4 (SEQ ID NO. 10). The homology arms and the GPC3-CAR nucleic acid sequence (SEQ ID NO. 11) were synthesized at GenScript Biotechnology to generate a double-stranded DNA template containing the chimeric antigen receptor sequence for homologous recombination. Figure 1 Schematic diagram of the chimeric antigen receptor structure.

[0074] Specifically, the chimeric antigen receptor is GPC3-CAR, and the obtained CAR sequence base sequence is integrated into the plasmid vector by enzyme cutting and ligation. The method is to cut the vector and the CAR base sequence with EcoRV / EcoRI enzymes, and link the vector puc57 with T4 ligase to obtain the target plasmid.

[0075] Example 2

[0076] This example designs sgRNAs for SIRPA gene knockout and CAR knockin and verifies their efficiency.

[0077] Experimental materials: OPTI-MEM culture medium and Lipofectamine 2000 (lipo2000) were purchased from Invitrogen.

[0078] Experimental steps:

[0079] (I) Schematic diagram of site-directed insertion and knockout strategies Figure 2 As shown, after selecting the targeting sequence and designing the sgRNA and excluding the SIRPA and SIRP homologous sequences, SIRPA exon6 was selected as the targeting position, and the sgRNA (SEQ ID NO. 1) was designed using the IDT tool;

[0080] (2) sgRNA was submitted to GenScript for custom synthesis;

[0081] (3) sgRNA editing efficiency detection;

[0082] (1) Transfection

[0083] iPSCs were electroporated with RNP using 4D-Nucleofector.

[0084] Experimental Materials

[0085] 4D-Nucleofector™ electroporation instrument: 4D-Nucleofector™ Core Unit + 4D-Nucleofector™ X Unit.

[0086] P3 primary cell 4D-Nucleofector™ X kit (Lonza, V4XP-3032). Kit components are as follows:

[0087] Two 20 µL 16-well Nucleocuvette™ electroporation strips, 0.675 mL Nucleofector™ electroporation solution, 0.15 mL supplement solution, 50 µg pmaxGFP™ Yangshen plasmid, and DPBS (LIFE, 14190-144).

[0088] Cas9 protein: GenCRISPR Ultra NLS-Cas9-Research nuclease (GenScript, Z03621-1);

[0089] sgRNA: EasyEdit sgRNA / SafeEdit sgRNA (Genscript, SC1969 / SC1968);

[0090] Homologous recombination template (HDRT): dsDNA (synthesized by GenScript);

[0091] 6-well cell culture plates;

[0092] Vitronectin VTN (Vitronectin, Zhongsheng Tracing Source).

[0093] Experimental procedures

[0094] 1) Set up the 4D-nucleofector program for transfection

[0095] a. Turn on the 4D-nucleofector instrument.

[0096] b. Select X unit;

[0097] c. Select X-16 Strip;

[0098] d. Select the wells to be transfected;

[0099] e. Set the program to CA-137 and P3 primary buffer (or the buffer you are using);

[0100] f. Press OK to open the tray port and proceed with cell preparation.

[0101] 2) Preparing iPSCs for electroporation

[0102] I. Aspirate the culture medium from the wells of the 6-well plate and wash the cells by adding 1 mL of DPBS. Gently shake the plate and then aspirate the DPBS. Add 1 mL of recombinase (TrypLE Select) to the iPSCs and incubate the plate in a 37°C incubator for 7 minutes; gently shaking the plate every 3 minutes will help completely detach the cells.

[0103] II. Add 1 mL of mTeSR Plus medium and 10 µM Y-27632 and gently pipette the cells until the iPSCs are completely detached.

[0104] III. Count cells using a hemocytometer and trypan blue;

[0105] IV. For one electroporation condition, add 1.0 × 10 6 cells. If there are 5 conditions, multiply by 5 (i.e., 5.0 × 10 6 cells);

[0106] V. Centrifuge at 120 × g for 5 min (at room temperature);

[0107] VI. Aspirate the supernatant and set aside.

[0108] 3) Preparation of Lonza RNP electroporation solution

[0109] a. Prepare Lonza P3 electroporation buffer by mixing Nucleofector™ Solution and Supplement Solution in a ratio of 4.5:1 (16.4 µL:3.6 µL). Equilibrate to room temperature. Each reaction requires 20 µL of this mixed P3 electroporation buffer.

[0110] b. To a sterile, DNase- / RNase-free centrifuge tube, add the following reagents in order: 5 µL of mixed Lonza P3 electroporation buffer, 4 µL (100 pmol, 3.3 µg) of sgRNA, and 0.8 µL (50 pmol, 8 µg) of Cas9 protein. Mix thoroughly and incubate at room temperature for 13 minutes to form the RNP mixture. Then, add 2 µL (5 µg) of HDRT and gently mix with the RNP mixture. Incubate at room temperature for 2 minutes to form the RNP & HDRT mixture.

[0111] 4) Electroporation Experimental Procedure

[0112] c. Carefully resuspend the cell pellet in the prepared Lonza P3 electroporation buffer (15 µL per reaction).

[0113] d. Carefully take 15 µL of cell suspension, add it to the RNP & HDRT mixture, and mix gently;

[0114] e. Transfer the mixed solution to a 16-well Nucleocuvette™ electroporation plate without creating bubbles.

[0115] f. Gently tap the Nucleocuvette™ strips to ensure the sample solution covers the bottom of the wells.

[0116] g. Close the Nucleocuvette™ electroporation strip cover and place the strip into the strip holder of the 4D-Nucleofector™ X Unit, ensuring the strip is oriented correctly.

[0117] h. Click “Start” on the 4D-Nucleofector™ Core Unit screen to begin the electroporation process.

[0118] i. Immediately after the run is complete, carefully remove the Nucleocuvette™ electroporation strip from the machine's strip holder. Add 60 µL of mTeSR medium containing Y-27632 to each well. Gently pipette up and down several times, then transfer all cells to one well of a 6-well plate and incubate in a 37°C / 5% CO2 incubator.

[0119] j. Incubate cells at 37°C for culture. Y-27632 can be removed one day after transfection.

[0120] k. Five days after transfection, when cells become semi-confluent, dissociate the cells and expand a portion of the cells when needed for cell maintenance, cell stock preparation, or genomic DNA extraction.

[0121] (2) Genome extraction

[0122] 1') Extract genomic DNA from SIRPA-edited cell pellets using the FastPure® Cell / Tissue DNA Isolation Mini Kit; prepare genomic DNA from untransfected iPSCs as a negative control;

[0123] 2') Use the T5 Direct PCR Kit to prepare B2M / CIITA / NKG2A cell lysate as a template for genomic DNA PCR.

[0124] (3) PCR and sequencing

[0125] 1') Design a pair of PCR primers to amplify the target site. For Sanger sequencing, the preferred PCR band size is approximately 500-800 bp. The gRNA target site should be located in the middle of the amplified region. Amplify the target site from a genomic sample by PCR. Ensure that amplification from a non-genome-edited sample serves as a negative control.

[0126] 2') Send PCR samples for Sanger sequencing service;

[0127] 3') Use a CRISPR analysis tool (such as Inference of CRISPR Edits, ICE) to decompose the Sanger sequence results. After receiving the Sanger sequencing results as an "ab1" file, visit the ICE website and upload the control sample file and the genome-edited sample file. Alternatively, you can use the TIDE website.

[0128] Example 3

[0129] In this example, an iPSC cell line with GPC3-CAR knocked-in at the SIRPA gene location was constructed.

[0130] Experimental materials: 4D-Nucleofector™ Electroporator (4D-Nucleofector™ Core Unit + 4DNucleofector™ X Unit), P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza, V4XP-3032, kit components: two 20 µL 16-well Nucleocuvette™ electroporation plates, 0.675 mL Nucleofector™ electroporation solution, 0.15 mL supplement solution, 50 µg pmaxGFP™ Yangshen plasmid), DPBS (LIFE, 14190-144), Cas9 protein: GenCRISPR Ultra NLS-Cas9-Research nuclease (GenScript, Z03621-1), sgRNA: EasyEdit sgRNA / SafeEdit sgRNA (GenScript, SC1969 / SC1968), homologous recombination template (HDRT): dsDNA, 6-well cell culture plate, vitronectin VTN (Vitronectin, Zhongsheng Tracing).

[0131] Experimental procedures

[0132] 1) iPSC culture

[0133] iPSCs were prepared and cultured in wells of a 6-well plate using VTN, DMEM / F2 medium, and mTeSR Plus medium.

[0134] 2) Preparation and storage of ribonucleoprotein (RNP) and HDRT (GPC3-CAR homologous recombination template) working solutions formed by Cas9 and sgRNA

[0135] a. Dissolve lyophilized powdered GenScript sgRNA (EasyEdit sgRNA / SafeEdit sgRNA) in RNase- / DNase-free and pyrogen-free water to a final concentration of 100 µM (100 pmol / µL). Aliquot into 8 µL tubes and store at -20°C for short-term storage or at -80°C for long-term storage.

[0136] b. Cas9 protein: Store at the concentration indicated on the product label at -20°C.

[0137] c. Dissolve the lyophilized HDRT powder in RNase- / DNase-free and pyrogen-free water to a final concentration of 2 µg / µL and store at -20°C.

[0138] 3) Culture dish coating

[0139] a. To coat an entire 6-well plate, add 120 µL of VTN to 6 mL of DMEM / F12 and mix, then add 1 mL (final concentration: 1 µg / cm2) to each well of the 6-well plate. 2 ). Incubate at room temperature for 1 hour;

[0140] b. Aspirate the coating buffer and add 2 mL of mTeSR Plus medium plus 2 µL of 10 mM Y-27632 (final 10 µM) to each well. Incubate in a 37°C CO2 incubator to prewarm the medium until iPSC dissociation is complete.

[0141] 4) iPSC cell passaging

[0142] a. Aspirate the culture medium from the wells of a 6-well plate and wash the cells by adding 1 / 2 mL of PBS. Gently shake the plate, then aspirate the PBS. Add 300 µL of animal-origin-free recombinase (TrypLE Select) or 1 mL of cell digestion solution (Relesr) to the iPSCs. Incubate at room temperature for 1 minute, then aspirate the digestion solution.

[0143] b. Place the plate in a 37°C incubator and incubate for 7 minutes. Gently rock the plate every 4 minutes to help completely separate the cells.

[0144] c. Add 1 mL of mTeSR Plus medium and 10 µM Y-27632 and gently pipette the cells until the iPSCs are completely detached.

[0145] d. Count the cells and transfer 1.0 × 10 4 ~1.5 × 10 4 Cells were seeded into VTN-coated 6-well plates containing 2 mL of StemFit medium containing 10 µM Y-27632, as described in step c;

[0146] e. Incubate the cells in a CO2 incubator at 37°C overnight.

[0147] f. The next day, replace the culture medium with 2 mL of mTeSR medium. If there are a large number of floating dead cells, continue to add Y-27632 to the culture medium at a final concentration of 10 µM.

[0148] j. Change the culture medium every 2 days during culture;

[0149] h.iPSCs will be semi-confluent on day 6-8.

[0150] 5) Electroporate iPSCs using the 4D-Nucleofector according to the instrument's operating instructions. The steps and dosages are as described in Example 2.

[0151] a. Immediately after the run is complete, carefully remove the Nucleocuvette™ electroporation strip from the machine strip holder. Add 50-80 µL of mTeSR medium containing Y-27632 to each well. Gently pipette up and down several times, then transfer all cells to a well of a 6-well plate containing 2 mL of 10 µM Y-27632 pre-coated with VTN and incubate in a 37°C / 5% CO2 incubator.

[0152] b. Incubate cells at 37°C for culture. Y-27632 can be removed 1-2 days after transfection.

[0153] c. 4-7 days after transfection, when cells become semi-confluent, dissociate the cells and expand a portion of the cells when needed to maintain the cells, prepare cell stock solution, or extract genomic DNA.

[0154] 6) Assessing genome editing efficiency in bulk iPSCs by Sanger sequencing

[0155] Assessing overall genome editing efficiency in electroporated iPSCs. This is an important step in validating that the genome editing procedure is working and estimating how many subclones are needed for genotyping from a bulk of cells.

[0156] a. Genome extraction

[0157] a') Genomic DNA was extracted from SIRPA-edited cell pellets using the FastPure® Cell / Tissue DNA Isolation Mini Kit. Genomic DNA was prepared from untransfected iPSCs as a negative control.

[0158] b') B2M / CIITA / NKG2A cell lysate was prepared using the T5 Direct PCR Kit and used as a template for genomic DNA PCR.

[0159] b. PCR and sequencing

[0160] Design a pair of PCR primers to amplify the target site. For Sanger sequencing, the preferred PCR band size is approximately 500-800 bp. The gRNA target site should be located in the middle of the amplified region. Amplify the target site by PCR from a genomic sample and amplify from a non-genome-edited sample as a negative control.

[0161] c. Send PCR samples for Sanger sequencing service;

[0162] d. Use the ICE (Inference of CRISPR Edits) CRISPR analysis tool to decompose the Sanger sequence results. After receiving the Sanger sequencing results as an "ab1" file, visit the ICE website and upload the control sample file and the genome-edited sample file. Alternatively, you can use the TIDE website.

[0163] e. Select the optimal conditions that show the highest knock-in efficiency (i.e., select the amount of gRNA or dsODN);

[0164] f. Expand iPSCs that have been confirmed to have successfully undergone genome editing and make some frozen stocks as needed.

[0165] Example 4

[0166] In this example, the iPSC subclones were screened by inserting GPC3-CAR at the SIRPA gene locus.

[0167] Experimental materials: TrypLE Select digestion solution (Thermo Fisher), mTeSR Plus complete medium (Stem Cell), DPBS buffer (Thermo Fisher), 15 mL centrifuge tubes, VTN (Zhongsheng Shuoyuan), cell culture 96-well plates, and cell culture six-well plates.

[0168] Experimental procedures

[0169] 1) Coat three 96-well plates with VTN for each edited sample;

[0170] 2) Subcloning by limiting dilution

[0171] a. Digest the edited cells with Accutase solution, discard the culture medium in the culture dish with a pipette, and rinse the dish once with calcium- and magnesium-free PBS (DPBS).

[0172] b. Discard the DPBS and add the cell dissociation reagent (StemPro Accutase solution). Adjust the volume of StemPro Accutase solution according to the size of the culture dish.

[0173] c. Incubate the culture dish in a 37°C, 5% CO2 incubator for 10 minutes, until individual cells begin to round up. Gently tap the side of the culture vessel every 3-4 minutes to dissociate most of the cells from the surface of the culture vessel.

[0174] d. Add an appropriate amount of mTeSR Plus medium to each dish to terminate the dissociation reaction. Gently pipette the dissociated mTeSR Plus medium up and down to disperse the cell colonies into a single-cell suspension. Ensure that the pipetting is gentle to reduce bubble formation.

[0175] e. Transfer the cell suspension to a 15 mL conical tube and centrifuge at 250 × g for 5 minutes. Carefully aspirate and discard the supernatant from the cell pellet.

[0176] f. Resuspend the pellet in an appropriate amount of culture medium and mix the cell suspension thoroughly using a 10-mL pipette tip.

[0177] g. Count cells using a hemocytometer and trypan blue;

[0178] h. Convert 1.0 × 10 4 Transfer the cells to a 15 mL tube containing 10 mL of StemFit medium containing 10 μM Y-27632;

[0179] i. Further dilute the cell suspension by transferring 10 μL, 30 μL, and 100 μL into three 15 mL tubes containing 10 mL of mTeSR plus medium and 10 μM Y-27632. The final concentration of cells should be 10 cells, 30 cells, and 100 cells per 10 mL.

[0180] j. Use a multichannel pipette to add 100 μL of cell suspension to one well of a 96-well plate.

[0181] k. Culture in a 5% CO2 incubator at 37°C;

[0182] On the next day, observe each well of the 96-well plate under a microscope and mark the wells with single cell seeding. Wells with more than one cell should be discarded.

[0183] m. After seeding single cells into a 96-well plate, there is a risk of cell loss during medium changes. Add 50 μL of fresh StemFit medium with 10 μM Y-27632 to the wells of the 96-well plate every 2 or 3 days instead of completely removing the medium.

[0184] n. On day 6 or 7, replace the medium with mTeSR Plus (without Y-27632).

[0185] o. Colonies can be seen around day 8. Single iPS colonies should be marked and retained. If more than two colonies are observed, these wells should be discarded.

[0186] p. Add culture medium to wells containing a single iPSC colony every 2 days (50 μL for a 96-well plate).

[0187] q. Around day 10, once the iPSC colonies reach 100-300 μm in diameter, transfer all cells from one well of the 96-well plate to a well of a 24-well plate.

[0188] r. Change the culture medium every 2 days until iPSCs become semi-confluent;

[0189] s. Day 17: Derivation of colonies from single cells and passage of cells;

[0190] t. When the passaged cells become semi-confluent, extract genomic DNA from half of the cells using a genomic DNA extraction kit. Since the starting cell number is small, elute the genomic DNA in a smaller volume (i.e., 40 μL) of elution buffer. Prepare a vial of frozen stock solution from the other half of the cells using a STEM-CELLBANKER.

[0191] u. When the number of samples accumulates to a certain level, perform PCR amplification of the target region and Sanger sequencing. Repeat this step for all isolated subclones, or until the desired subclone is identified.

[0192] v. Select the subclone containing the desired knockout or knockin sequence.

[0193] Example 5

[0194] In this example, FACS was used to detect cell CAR expression.

[0195] Experimental materials: cell digestion solution Accutase (Stem cell), digestion enzyme TrypLE (Stem cell), HumanTruStain FcX isotype control flow cytometry antibody (Bipsys), flow cytometry antibody (Thermo).

[0196] Experimental steps:

[0197] 1) Remove the cells to be tested, discard the original culture medium, wash once with DPBS, add 1 mL / well of TrypLE, and incubate at 37°C for about 5 minutes. If the cells are MAC cells, add 1 mL / well of Accutase and incubate at 37°C for about 20-30 minutes.

[0198] 2) After digestion, gently scrape and pipette the cells to remove them, collect the cells into a 15 mL centrifuge tube, add 4 mL of DPBS to the tube, centrifuge at 300 g for 5 minutes, and discard the supernatant;

[0199] 3) Determine whether flow cytometry reagent (Human TruStain FcX (FcReceptor Blocking Solution)) is needed for blocking according to the cell type. If necessary, block as follows: 6 The cells were resuspended in 100 μL DPBS + 5 μL Human TruStain FcX, incubated at room temperature for 10 min, added with 2 mL DPBS, centrifuged at 300 g for 5 min, and the supernatant was removed;

[0200] 4) If the experiment requires differentiation of live and dead cells, add a zombie NIR dye. If not, proceed directly to step 5).

[0201] 5) Add 2 μL of the corresponding antibody to 100 μL of DPBS, mix well, resuspend the cells, and incubate at 4°C for 15 min;

[0202] 6) Wash the cells with 2 mL of DPBS, centrifuge at 300 g for 5 min, and discard the supernatant.

[0203] 7) Resuspend the cells in 300-400 μL of DPBS depending on the cell volume and transfer them to the appropriate flow cytometry tube for loading.

[0204] Example 6

[0205] In this example, iPSC cells were differentiated into iMac.

[0206] Experimental materials: TryPLE digestion solution, 96-well round-bottom culture plates, APEL II medium (Stem cells), BMP4, bFGF (5 ng / mL), VEGF, SCF, Rock inhibitor (Sigma).

[0207] Experimental procedures

[0208] 1. On Day 0, add 300 μL TryPLE to one well of iPSC and digest it at 37°C for 5 minutes to make single cells. Count and dilute to 8×10 4 One round-bottom 96-well plate requires 10 mL of culture medium, a total of 8×10 5 cells.

[0209] Day 0 culture medium:

[0210] Basal medium: APEL II medium;

[0211] BMP4 (10 ng / mL);

[0212] bFGF (5 ng / mL);

[0213] VEGF (50 ng / mL);

[0214] SCF (100 ng / mL);

[0215] Rock inhibitor (1:1000).

[0216] Add 100 μL of cell suspension to each well using a dispenser, centrifuge at 300 g for 5 min, and return to the incubator.

[0217] 2. Embryoid bodies (EBs) in good condition can be observed to have obvious vacuoles on day 3-day 4. In addition, EBs that mature faster can be observed to have a noticeable yellowing of the culture medium on day 4. The culture medium needs to be replaced with 60 μL, 80 μL, or 100 μL of medium depending on the color change.

[0218] The culture medium used for the medium change was the same as that used on day 0 (but without the Rock inhibitor).

[0219] Fluid exchange method:

[0220] 1) The waste liquid can be aspirated into a centrifuge tube one hole at a time. The advantage is that it is easy to observe and will not aspirate EB. Once EB is aspirated, it can be quickly returned to the well.

[0221] 2) Use a pistol to suck the liquid into a 10 cm dish. After sucking, observe whether EB is sucked out. If an empty hole is found, put it back.

[0222] 3) On Day 6-7 (Day 6-Day 7), the EB can be removed and directly attached to the wall (VTN needs to be laid in advance);

[0223] 4) EBs in good condition will see single cores appear in about 10 days, and subsequent production will increase.

[0224] Example 7

[0225] In this example, a CAR-iMac cell killing experiment was performed.

[0226] Experimental materials: Accutase digestion solution, DMEM (High Glucose) medium (Thermo Fisher), Penicillin-Streptomyci (Thermo Fisher), NEAA glutamine-free (Thermo Fisher), serum (Gibco).

[0227] Experimental steps:

[0228] 1) Cell Preparation: Digest WT-iMac and CAR-iMac cells with Accutase (37°C for 30 min), and digest target HepG2 / Huh7-Luci-RFP cells with TrypLE (37°C for 5 min). Centrifuge at 300 g for 5 min. Discard the supernatant, resuspend the cell pellet in the respective culture medium, and count the cells.

[0229] 2) Luciferase Assay: Cells were seeded into 96-well white plates at an E:T ratio of 20:1 (iMac: 4E4, HepG2 / Huh7: 2E3) or 40:1 (iMac: 8E4, HepG2 / Huh7: 2E3), with six replicates per group. After 3 days of culture, an equal volume of 100 μL / well One-Lite™ Detection Reagent was added. The cells were incubated at room temperature for 3 minutes and then RLU was measured on an M5 microplate reader.

[0230] 3) Incucyte Assay: Cells were seeded in 24-well plates at an E:T ratio of 40:1 (iMac: 4E5, HepG2 / Huh7: 1E4) or 80:1 (iMac: 8E5, HepG2 / Huh7: 1E4), with triplicate wells per group. The 24-well plates were placed in the Incucyte instrument and the following parameters were set: photography at 10x magnification, 16 fields of view per well, imaged every 2 hours, for 8 consecutive days.

[0231] Results and Discussion

[0232] The expression of SIRPα protein was detected by immunoblotting. Figure 3 As shown, WT refers to wild-type iPSC cells, CAR KI monoclonal iPSC cell line refers to the iPSC cell line with GPC3-CAR knocked into the SIRPA gene position (lanes 1 and 2 are two technical replicates), and SIRPA-KO iPSC cell line refers to the monoclonal cell line with SIRPA gene knocked out alone. The results show that whether SIRPA is knocked out alone or GPC3-CAR is knocked into the reverse direction of the SRIRPA gene, the SIRPA gene itself has been destroyed and the SIRPA protein cannot be expressed normally, indicating that the strategy of inserting GPC3-CAR at this site is successful.

[0233] The results of analysis of SIRPA gene expression levels are as follows Figure 4 As shown, the SIRPA gene was knocked out again at the mRNA level.

[0234] The results of flow cytometry detection of GPC3-CAR expression are as follows Figure 5As shown, iPSC-CAR-KI (knock in) indicates that GPC3-CAR has been successfully knocked into the SIRPA exon6 gene position and can express GPC3-CAR protein molecules normally. Whether in the iPSC stage or after differentiation into macrophages (iMac, iPSC derived Macrophage), GPC3-CAR protein expression can be detected by flow cytometry technology.

[0235] The results of CAR-iMac macrophages' killing ability on Hepeg2 liver cancer cells are as follows Figure 6 As shown, it is shown that the killing ability of the CAR-iMac designed and prepared by the present invention is significantly improved.

[0236] In summary, the present invention develops a novel method for constructing CAR-iMac cells, which utilizes a non-viral-dependent site-directed gene editing strategy, iPSCs as the starting material, and site-directed reverse insertion of a chimeric antigen expression receptor CAR encoding gene into the exon 6 region of the SIRPA gene in the iPSCs, thereby inhibiting the expression of the SIRPA gene while expressing the CAR molecule to obtain engineered iPSCs, which are then further induced to differentiate to obtain CAR-iMacs. CAR-iMacs can maintain a higher level and longer duration of M1 polarization during the anti-tumor process, and have significantly enhanced anti-tumor efficacy. Moreover, the construction based on iPSCs effectively overcomes the corresponding problems of directly extracting natural macrophages from the human body, reduces production costs and time costs, and can also avoid immune rejection, which is beneficial for clinical applications.

[0237] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing chimeric antigen receptor macrophages, characterized in that: The preparation method comprises: Inducing and culturing non-virus-dependent engineered induced pluripotent stem cells to obtain the chimeric antigen receptor macrophages; The induction culture medium contains cytokines; the cytokines include at least one of BMP4, bFGF, VEGF, SCF or inhibitors; A chimeric antigen receptor encoding gene is reversely inserted into exon 6 of the SIRPA gene on the genome of the engineered induced pluripotent stem cell; The chimeric antigen receptor comprises a leader peptide, an extracellular recognition region, a hinge region and a transmembrane / intracellular signaling region, a costimulatory factor, a signal transduction region and a pattern recognition receptor; The amino acid sequences of the leader peptide, hinge region, transmembrane / intracellular signaling region, co-stimulatory factor, signal transduction region, and pattern recognition receptor are shown in SEQ ID NO. 4, SEQ ID NO. 6 to SEQ ID NO. 10, respectively; The extracellular recognition region includes an antibody, the antibody includes an anti-GPC3 antibody, and the amino acid sequence of the anti-GPC3 antibody includes the sequence shown in SEQ ID NO.5; The nucleic acid sequence of the gene encoding the chimeric antigen receptor is shown in SEQ ID NO.11; The method for preparing the engineered induced pluripotent stem cells comprises: inserting the gene encoding the chimeric antigen receptor into the SIRPA gene on the genome of the induced pluripotent stem cell to obtain the engineered induced pluripotent stem cell; The method of site-directed insertion is the CRISPR / Cas9 method; The nucleic acid sequence of the sgRNA of the CRISPR / Cas9 method is shown in SEQ ID NO.1; The nucleic acid sequences of the homologous arms of the homologous recombination template of the CRISPR / Cas9 method are shown in SEQ ID NO.2 and SEQ ID NO.

3.

2. A chimeric antigen receptor macrophage, characterized in that The chimeric antigen receptor macrophages are prepared by the method for preparing chimeric antigen receptor macrophages according to claim 1.

3. The use of the chimeric antigen receptor macrophages according to claim 2 in the preparation of an anti-tumor preparation, characterized in that: The tumor is liver cancer.

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