WO2025213672A1 - Cellule modifiée permettant d'exprimer hautement une protéine sécrétoire et utilisation associée - Google Patents

Cellule modifiée permettant d'exprimer hautement une protéine sécrétoire et utilisation associée

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Publication number
WO2025213672A1
WO2025213672A1 PCT/CN2024/113685 CN2024113685W WO2025213672A1 WO 2025213672 A1 WO2025213672 A1 WO 2025213672A1 CN 2024113685 W CN2024113685 W CN 2024113685W WO 2025213672 A1 WO2025213672 A1 WO 2025213672A1
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Prior art keywords
cells
site
engineered
expression
sequence
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PCT/CN2024/113685
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English (en)
Chinese (zh)
Inventor
梁德生
王霈云
周妙金
唐齐玉
田芷欣
赵俊雅
陈艳
王祖佳
周淇
刘亚婷
周代星
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Shanghai Pinpoint Medical Technology Co Ltd
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Shanghai Pinpoint Medical Technology Co Ltd
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Publication of WO2025213672A1 publication Critical patent/WO2025213672A1/fr
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the technical field of DNA recombination, and in particular to an engineered cell for highly expressing secretory proteins and applications thereof.
  • MSCs Mesenchymal stem cells
  • MSCs Mesenchymal stem cells
  • MSCs tumor homing ability, which allows them to migrate to the tumor site and penetrate into the tumor stroma and microenvironment. This characteristic may be related to the presence of receptors on the surface of MSCs that can bind to chemokines in the tumor microenvironment (such as CXCL12/CXCR4, etc.). Numerous studies have shown that natural MSCs can inhibit tumor cell growth and induce apoptosis in the treatment of liver cancer, leukemia, and Kaposi's sarcoma. However, using MSCs alone to combat tumors and other diseases is far from achieving the desired therapeutic effect. Based on the inherent properties of mesenchymal stem cells, loading exogenous therapeutic factors into MSCs to treat diseases has become a relatively effective treatment method.
  • PD-1 programmed death-protein 1
  • CD279 is a type I transmembrane glycoprotein with a molecular weight of approximately 55 kDa. It is expressed on activated T cells, natural killer cells, B cells, macrophages, dendritic cells, and monocytes.
  • the ligands of PD-1 include PD-L1 and PD-L2, of which PD-L1 is the main ligand.
  • PD-L1 is widely expressed in a variety of cells including T cells, dendritic cells, macrophages, vascular endothelial cells, and keratinocytes. After binding to the ligand, PD-1 can inhibit T cell proliferation, activation, and cytokine secretion, thereby inhibiting the immune response and effectively maintaining immune stability in normal organisms.
  • PD-L1 is also widely expressed on a variety of tumor cells. Tumor cells can exploit the immunosuppressive function of PD-1/PD-L1 and achieve immune escape by binding to PD-1 molecules on the surface of lymphocytes.
  • the present invention provides a safer and more effective strategy for engineered cell therapy.
  • This strategy involves constructing engineered cells that incorporate a polynucleotide encoding a secretory protein through a non-viral approach and, combined with an exogenous signal peptide, yielding therapeutic engineered cells that highly express the secretory protein.
  • the polynucleotide encoding the secretory protein is preferably site-specifically integrated into the B2M locus.
  • the present invention provides an engineered cell, wherein a nucleic acid encoding a secretory protein is site-specifically integrated into the engineered cell.
  • the engineered cells include engineered mesenchymal stem cells and/or engineered IPSC cells and cells derived therefrom.
  • the engineered mesenchymal stem cells are derived from adult cells or stem cells.
  • the engineered mesenchymal stem cells are derived from pluripotent stem cells. More preferably, the pluripotent stem cells are selected from induced pluripotent stem cells (iPSCs).
  • iPSCs induced pluripotent stem cells
  • the engineered mesenchymal stem cells are derived from bone marrow, fat, muscle, heart, umbilical cord blood or umbilical cord.
  • the derived cells include CAR-iNK, dopaminergic neural precursor cells, CAR-iMac, cardiomyocytes, endothelial progenitor cells, iNK cells, retinal cells, neurons, osteoblasts, hematopoietic stem cells, blood cells, B cells, fibroblasts, hair cells, monocytes, macrophages, Treg cells, renal progenitor cells, lung epithelial cells, endothelial cells, megakaryocytes, smooth muscle cells, skeletal muscle cells, chondrocytes, bone cells, adipocytes, hepatocytes, pancreatic islet cells, keratinocytes, melanocytes, and dendritic cells.
  • the site of site-directed integration is located at the B2M locus.
  • the secretory protein comprises a dipeptide, an oligopeptide, a polypeptide, or a short protein, wherein the secretory protein is composed of less than 2500 amino acids, preferably less than 2400, 2000, 1500, 1000, 900, 800, 700, 600, 500, 400, 300, or 200 amino acids.
  • the secretory protein includes one or more of immune checkpoint inhibitors, TNF- ⁇ inhibitors, GLP-1 receptor agonists, growth hormones, coagulation factors, interleukins, insulin, interferons, tumor necrosis factors, enzymes, and growth factors.
  • the immune checkpoint is selected from one or more of PD1, PD-L1, CTLA-4, TIGIT, LAG-3, and TIM-3, preferably PD1.
  • the immune checkpoint inhibitor is an antibody or an antibody fragment, preferably a full-length antibody or a single-chain antibody.
  • the immune checkpoint inhibitor is a full-length antibody or a single-chain antibody against PD1, preferably a single-chain antibody against PD1.
  • the TNF- ⁇ inhibitor is selected from TNF- ⁇ receptors or TNF- ⁇ antibodies, such as etanercept, adalimumab, secukinumab, infliximab, golimumab, and benzaluzumab.
  • the interleukin is selected from members of the leukocyte family, such as IL-2, IL-7, IL-10, IL-11, IL-12, IL-15, IL-23 and IL-24.
  • the tumor necrosis factor family member is selected from, for example, TNF, LTA, LTB, FASLG, TNFSF8, TNFSF9, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF14, TNFSF15, TNFSF18, EDA, and TRAIL.
  • the interferon is selected from, for example, interferon ⁇ , ⁇ , and ⁇ .
  • the GLP-1 receptor agonist is a polypeptide GLP-1 receptor agonist, selected from polypeptide GLP-1 receptor agonists such as exenatide, benaglutide, dulaglutide, and albiglutide, whose polypeptide chains are not chemically modified or are chemically modified.
  • the growth hormone is preferably natural or recombinant human growth hormone (rhGH), which is divided into short-acting recombinant human growth hormone or long-acting recombinant human growth hormone.
  • rhGH natural or recombinant human growth hormone
  • the coagulation factor is selected from, for example, prothrombin complex, fibrinogen, antifibrinolytic, recombinant factor VIIa, recombinant factor VIII, recombinant factor IX, and recombinant factor X.
  • the growth factor is selected from, for example, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), GM-CSF, and G-CSF.
  • EGF epidermal growth factor
  • PDGF platelet-derived growth factor
  • FGF fibroblast growth factor
  • GM-CSF GM-CSF
  • G-CSF G-CSF
  • the enzyme is selected from, for example, lipase, amylase, trypsin, chymotrypsin, lysozyme, urokinase, L-asparaginase, glutaminase, neuraminidase, and the like.
  • the nucleic acid encoding the secretory protein is not limited to the functional region, and may include at least one of a coding region, a leader sequence, a signal peptide sequence, exons, introns and an expression frame.
  • the nucleic acid encoding the secretory protein is an expression cassette encoding the secretory protein.
  • the expression cassette comprises an operably linked promoter sequence, a signal peptide sequence, and a nucleic acid sequence encoding a secretory protein. In one embodiment of the present invention, the expression cassette comprises an operably linked promoter sequence, a signal peptide sequence, a nucleic acid sequence encoding a secretory protein, and a screening marker or tag. In one embodiment of the present invention, the expression cassette comprises an operably linked promoter sequence, a signal peptide sequence, a nucleic acid sequence encoding a secretory protein, and a screening marker or tag, and a poly(A) tail.
  • the signal peptide is an exogenous signal peptide. In one embodiment of the present invention, the signal peptide is used to guide the secretion of a secretory protein.
  • the signal peptide is a strong secretory signal peptide suitable for secretory proteins.
  • the strong secretory signal peptide suitable for a secretory protein may be a combination of one or more.
  • the strong secretory signal peptide is selected from secrecon, Gaussia luciferase (Gluc), Mouse Ig Kappa, Human IgG V, Human IgK VIII, Ig heavy chain signal peptide 7 (H7), and Ig ⁇ light chain signal peptide 1 (L1).
  • the promoter sequence is located upstream of the nucleic acid sequence, and the promoter controls the expression of the secretory protein.
  • the promoter is selected from CMV promoter, EF1 ⁇ promoter, SV40 promoter, CAG promoter, PGK promoter or UBC promoter.
  • the selection marker is selected from, for example, ampicillin (Ampr), chloramphenicol (Camr), karatomycin (Kanr), tetracycline (Tetr), puromycin (Puro), G418, hygromycin ⁇ (Hygr), Zeocin, and blasticidin.
  • the tag is selected from, for example, FLAG, His, GST, HA, c-Myc, HSV, V5, SUMO, eGFP/eCFP/eYFP/mCherryeGFP.
  • the engineered cells further comprise one or more of the following:
  • MHC-I and/or MHC-II human leukocyte antigens are selected from one or more of HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, B2M, TAP1, TAP2, LMP2, LMP7, TNFRSF1A, CD8B, CD8A, CCR7, CXCR6, IL2RA, GZMB, GNLY, CKB, P2RY6, ADCY2, CHRM1, ERAP1, ERAP2, Tapasin, CD74, HLA-DO, HLA-DM, CLIP, Cathepsins, RFX5, RFXAP, RFXANK, and CIITA; more preferably, regulated expression of CIITA is included.
  • the regulated expression includes no expression or reduced expression.
  • the engineered mesenchymal stem cells have a higher expression level of secretory proteins compared to wild-type mesenchymal cells.
  • the engineered mesenchymal stem cells have a higher expression level of secretory proteins compared to mesenchymal cells with site-specific integration of rDNA regions.
  • the expression level of the engineered mesenchymal stem cells is that the weight of the secretory protein secreted by one million mesenchymal stem cells every 24 hours is greater than 20 ng, preferably greater than 30 ng, preferably greater than 40 ng, preferably greater than 50 ng, preferably greater than 100 ng, preferably greater than 200 ng, preferably greater than 300 ng, preferably greater than 400 ng, preferably greater than 500 ng.
  • the present invention provides a method for preparing the engineered cells, comprising introducing a nucleic acid encoding a secretory protein into a site-specific integration site of the cells.
  • an expression cassette encoding a secretory protein is introduced into the site of site-directed integration of the cell by means of a meganuclease, zinc finger nuclease (ZFNs), transcription activator-like effector nuclease (TALEN) and/or CRISPER/Cas system; more preferably, the CRISPER/Cas system.
  • ZFNs zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • CRISPER/Cas system more preferably, the CRISPER/Cas system.
  • an expression cassette encoding a secretory protein is introduced into the site of site-directed integration of the cell via a targeting vector, an sgRNA vector and/or a nuclease or a nuclease expression vector;
  • the targeting vector comprises a 5' homology arm-an expression cassette encoding a secretory protein-a 3' homology arm.
  • the 5' homology arm and the 3' homology arm are respectively homologous to the sequence in the site of the above-mentioned site-directed integration. In one embodiment of the present invention, the 5' homology arm and the 3' homology arm are respectively homologous to the sequence in the B2M locus.
  • the lengths of the 5' homology arm and the 3' homology arm are 2-1000 bp, for example, 2, 10, 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 bp, respectively.
  • the 5' homology arm and the 3' homology arm are respectively homologous to sequences with a length of 2-1000 bp upstream and downstream of the PAM sequence in the site of site-directed integration.
  • the expression cassette encoding the secretory protein is as described above.
  • the sgRNA is used to bind to a nuclease and guide the nuclease to cleave the target gene fragment by recognizing the PAM sequence of the target sequence.
  • the sgRNA is partially or fully complementary to the target sequence. In one embodiment of the present invention, the sgRNA is partially or fully complementary to the target sequence in the B2M locus.
  • the nuclease includes a meganuclease, a zinc finger nuclease (ZFNs), a transcription activator-like effector nuclease (TALEN), a CRISPER/Cas system, etc., preferably a CRISPER/Cas system.
  • ZFNs zinc finger nuclease
  • TALEN transcription activator-like effector nuclease
  • CRISPER/Cas system preferably a CRISPER/Cas system.
  • the nuclease of the CRISPER/Cas system is selected from Cas12a, Cas12b, Cas13, Cas14, Cas9, CasX, CasY, C2c2, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Cscl , Csc2, Csa5, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3 and Csf4.
  • the nuclease is used for PAM sequence-dependent recognition of the site of site-specific integration and initiates DNA cleavage at a specific site 3 bp upstream of the PAM sequence.
  • the vector is a non-viral vector suitable for expression in mesenchymal stem cells, such as pUC, pET, pGEX, etc.
  • a nucleic acid encoding a secretory protein is introduced into iPSCs for site-specific integration, followed by directed differentiation to obtain derived cells.
  • the derived cells are engineered mesenchymal stem cells (iMSCs).
  • the site-directed integration includes double-copy integration or single-copy integration.
  • the introduction method is a non-viral method.
  • the introduction method is selected from: vector transformation, transfection, heat shock, electroporation, transduction, and microinjection.
  • the present invention provides a preparation comprising the above-mentioned engineered cells and pharmaceutically acceptable excipients.
  • the excipients include a buffer selected from, for example, acetate, Tris, phosphate, citrate and other organic acids; an antioxidant including ascorbic acid and methionine; a preservative selected from, for example, octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzalkonium chloride, phenol, butylbenzyl alcohol, alkyl parabens, such as methyl or propyl parabens, catechol, resorcinol, cyclohexanol; a protein selected from, for example, serum albumin, gelatin or immunoglobulin; a hydrophilic polymer selected from, for example, polyethylene glycol.
  • a buffer selected from, for example, acetate, Tris, phosphate, citrate and other organic acids
  • an antioxidant including ascorbic acid and methionine
  • a preservative selected from, for example, oct
  • Pyrrolidone amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates selected from, for example, glucose, mannose or dextrin; chelating agents selected from, for example, EDTA; sugars selected from, for example, sucrose, mannitol, trehalose or sorbitol; surfactants selected from, for example, polysorbates; metal complexes selected from, for example, zinc protein complexes; non-ionic surfactants selected from, for example, Tween or polyethylene glycol (PEG); liposomes, albumin microspheres, polyesters, micelles, sustained-release matrices, etc.
  • amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine
  • monosaccharides, disaccharides and other carbohydrates selected from, for example, glucose, mannose or dextrin chelating
  • the present invention provides a pharmaceutical composition comprising the above-mentioned engineered cells.
  • the pharmaceutical composition further comprises other therapeutic agents, and the other therapeutic agents include antipyretics, antiasthmatics, antibiotics, antidepressants, antidiabetics, anti-inflammatory drugs, antitumor drugs, antianxiety drugs, immunomodulators, sedatives and hypnotics, antianginal drugs, antipsychotics, antimanics, antiarthritis drugs, antiarrhythmic drugs, antigout drugs, anticoagulants, thrombolytics, antifibrinolytics, hemorheology drugs, antiplatelet drugs, anticonvulsants, antiparkinsonian drugs, antihistamines, drugs for calcium regulation, antivirals, bronchodilators, hormones, lipid-lowering drugs, proteins, polypeptides, nucleic acids, antiulcer or antireflux drugs, antiemetics, diagnostic agents, and nutritional drugs.
  • the other therapeutic agents include antipyretics, antiasthmatics, antibiotics, antidepressants, antidiabetics, anti-inflammatory drugs, antitum
  • anti-tumor drugs are included, specifically including but not limited to paclitaxel and its derivatives, docetaxel, camptothecin and its derivatives, etoposide, teniposide, doxorubicin hydrochloride, cyclophosphamide, dactinomycin, bleomycin, daunomycin, doxorubicin, epirubicin, mitomycin, methotrexate, 5-fluorouracil, carboplatin, carmustine, lomustine, cisplatin, vinblastine, vincristine, tamoxifen, sulfamethoxazole, and phenylephrine.
  • paclitaxel and its derivatives specifically including but not limited to paclitaxel and its derivatives, docetaxel, camptothecin and its derivatives, etoposide, teniposide, doxorubicin hydrochloride, cyclophosphamide, dactinomycin
  • the one or more other therapeutic agents are administered in combination with the engineered cells.
  • Such combined administration includes sequential administration in any order or at any interval, such that the two or more therapeutic agents simultaneously exert their biological activities.
  • such combined administration produces a synergistic therapeutic effect.
  • the present invention provides the use of the above-mentioned engineered cells, preparations, and pharmaceutical compositions in the preparation of drugs for diagnosing, preventing, and treating diseases;
  • the diseases include but are not limited to cell proliferative diseases, such as tumors, melanoma, non-small cell lung cancer, renal cell carcinoma, colorectal cancer, breast cancer, pancreatic cancer, head and neck cancer, and other solid tumors; blood system diseases, such as leukemia, anemia, lymphoma, hemophilia, leukopenia, thrombocytopenia, angiogenesis disorders, Kaposi's sarcoma, etc.; autoimmune diseases, such as Crohn's disease, ulcerative colitis, allergies, inflammatory bowel disease, arthritis, psoriasis, and respiratory inflammation, asthma, and organ transplant rejection, etc.; metabolic diseases, such as diabetes, growth hormone deficiency, and growth retardation in children, etc.; infections, including viral infections, bacterial infections, fungal infections, and parasitic infections,
  • the drug is administered intravenously, intramuscularly, intraperitoneally, cerebrospinal, subcutaneously, intrathecally, orally, topically or by inhalation.
  • the present invention provides the use of the B2M locus in increasing the expression of secretory proteins by mesenchymal stem cells.
  • the present invention provides a method for preparing a secretory protein, comprising expressing the secretory protein using the engineered cells described above.
  • the secretory proteins include immune checkpoint inhibitors, TNF- ⁇ inhibitors, GLP-1 receptor agonists, growth hormones, coagulation factors, interleukins, insulin, interferons, tumor necrosis factors, enzymes, and growth factors.
  • the present invention provides a method for increasing the expression level of a secretory protein, comprising culturing the engineered cells to obtain the secretory protein.
  • the secretory proteins include immune checkpoint inhibitors, TNF- ⁇ inhibitors, GLP-1 receptor agonists, growth hormones, coagulation factors, interleukins, insulin, interferons, tumor necrosis factors, enzymes, and growth factors.
  • iPSCs induced pluripotent stem cells
  • polynucleotides encoding secretory proteins are site-specifically integrated into the B2M locus of iPSCs by a non-viral method.
  • therapeutic iMSCs that can highly secrete the desired secretory proteins are obtained, which achieves better integration and expression effects than other integration sites.
  • the technical solution in the embodiments of the present invention adopts a non-viral approach, overcoming the safety risks of viral vector-mediated gene transfer methods. It avoids the immunogenicity of viral vectors and the random integration of exogenous therapeutic genes into the MSC genome, thereby preventing the expression of exogenous therapeutic genes or the normal function of MSCs.
  • the random integration method can cause differences in the exogenous therapeutic genes between different therapeutic cells, making it difficult to ensure the consistency of product quality.
  • the exogenous therapeutic gene integrated into the MSC genome is preferably a secreted protein, which can continue to remain in the MSC genome with repeated subculture of MSC cells and achieve long-term stable expression, thereby ensuring the sustained therapeutic effect of MSCs cells and reducing the patient's medication costs.
  • the expression cassette for a secretory protein is site-specifically integrated into the B2M locus of mesenchymal stem cells, overcoming the problems of site-specific integration at other sites (e.g., AAVS1, CCR5, rDNA regions, ROSA26, HTRP, H11, TCR, etc.), which can prevent integration or achieve high expression after integration.
  • the expression cassette for the PD1 antibody is preferably site-specifically integrated into the B2M locus of mesenchymal stem cells, achieving excellent expression.
  • the mesenchymal stem cells prepared in a certain embodiment of the present invention highly express coagulation factor F8, and the secretion amount of one million mesenchymal stem cells per 24 hours is as high as 372.29 nanograms, which is much higher than the secretion amounts of 0.59 ⁇ 0.11ng/( 106 cells per 24h) and 0.68 ⁇ 0.14ng/(106 cells per 24h) obtained in the existing technology ("Restoration of FVIII Function and Phenotypic Rescue in Hemophilia A Mice by Transplantation of MSCs Derived From F8-Modified iPSCs", Liyan Qiu, et al, Frontiers in Cell and Developmental Biology, Volume 9 , 2021.2.11).
  • the existing technology also integrates the F8 gene with the deleted B region into the mesenchymal stem cells, but the integration site is the rDNA region.
  • a scFv expression frame for a PD-1 blocking antibody (hereinafter referred to as aPD1-scFv) is preferably site-specifically integrated into the B2M locus of iPSCs via a non-viral vector plasmid, thereby obtaining site-specifically integrated aPD1 scFv-iPSC stem cells. Subsequently, through in vitro directed differentiation, therapeutic aPD1 scFv-iMSCs that can highly secrete aPD1-scFv are obtained for use in the treatment of various tumors. This approach combines the dual advantages of iMSCs and PD1 antibodies.
  • iMSCs first migrate to the tumor site and then stably and persistently secrete PD1 antibodies at high levels, resulting in high-density concentrated secretion of PD1 antibodies at the tumor site, increasing the concentration and effective dose of the antibody, thereby improving the cellular immunotherapy effect of MSCs and providing a safer, more effective, and more uniform MSC-based tumor treatment strategy.
  • Figure 1-2 is a schematic diagram of targeting by integrating anti-human PD1 scFv into the B2M locus and rDNA region locus in Example 1.
  • Figure 3-4 shows PCR identification of aPD1 iPSCs with site-specific integration of aPD1 scFv.
  • Figure 5-6 is a sequencing diagram of the detection site-specific integration PCR product.
  • Figure 7 Morphology of site-specifically integrated aPD1 iPSCs.
  • Figure 8 shows the differences in protein expression levels between rDNA aPD1 iPSCs and B2M aPD1 iPSCs detected by Western Blot.
  • Figure 9 shows the PCR detection of B2M gene editing.
  • Figure 10 shows the sequencing results of PCR products.
  • Figure 11 shows the morphology of aPD1 iMSCs obtained by differentiation of two aPD1 iPSCs.
  • Figure 12 shows the flow cytometry results of surface markers of two aPD1 iMSCs.
  • Figure 13 shows the identification results of the multi-lineage differentiation potential of two aPD1 iMSCs.
  • Figure 14 shows the protein expression of aPD1 iMSCs detected by Western Blot.
  • Figure 15 shows the ELISA detection of the aPD1 protein level secreted in the supernatant of iMSCs.
  • Figure 16 shows the flow cytometry analysis results of HLA-ABC protein expression on the cell membrane surface of aPD1 iMSCs.
  • Figure 17 shows the flow cytometry detection of the binding of aPD1-FLAG secreted by aPD1 iMSCs to human PD1+293T cells.
  • Figure 18 shows flow cytometry detection of aPD1-FLAG secreted by aPD1 iMSCs blocking the binding of human PD-L1 to human PD1+293T cells.
  • Figure 19 shows that aPD1 iMSCs promote the killing effect of human PBMC on HCC827 cell line.
  • FIG20 is a graph showing the weight change trend of mice after aPD1 iMSCs injection.
  • Figure 21 shows the growth trend of subcutaneous tumor volume in mice after aPD1 iMSCs injection.
  • Figure 22 is a schematic diagram of targeting of Gluc-anti-TNF- ⁇ scFv and IgG V-anti-TNF- ⁇ scFv integrated into the B2M locus in Example 8.
  • Figure 23 is an agarose gel image of the PCR identification of anti-TNF- ⁇ scFv integrated into iPSCs across downstream homology arms in Example 9.
  • Figure 24 is the sequencing diagram of the PCR product of the downstream junction of anti-TNF- ⁇ scFv integrated into iPSCs in Example 9.
  • Figure 25 shows the protein secretion level of anti-TNF- ⁇ scFv iPSCs detected by ELISA in Example 9.
  • Figure 26 shows the protein secretion level of anti-TNF- ⁇ scFv iMSCs detected by ELISA in Example 10.
  • FIG27 is a schematic diagram of the targeting process of integrating the BDDF8-CO expression cassette into the B2M locus in Example 11.
  • Figure 28 shows the results of PCR identification of BDDF8-CO iPSCs with site-specific integration of the BDDF8-CO expression frame.
  • FIG29 shows the sequencing results of the site-specific integrated PCR products.
  • Figure 30 shows the results of PCR detection of B2M gene editing.
  • Figure 31 shows the sequencing results of the PCR detection products of B2M gene editing.
  • Figure 32 shows the ELISA detection of the secreted FVIII protein level in the supernatant of BDDF8-CO iMSCs.
  • Figure 33 shows the coagulation activity of FVIII protein secreted in the supernatant of BDDF8-CO iMSCs detected by a fully automatic coagulation analyzer.
  • FIG34 is a schematic diagram of the targeting process of integrating the GH-HyFc expression cassette into the B2M locus in Example 16.
  • Figure 35 shows the results of PCR identification of GH-HyFc iPSCs with site-specific integration of the GH-HyFc expression frame.
  • FIG36 shows the sequencing results of the site-specific integrated PCR products.
  • Figure 37 shows the results of PCR detection of B2M gene editing.
  • Figure 38 shows the sequencing results of PCR detection of B2M gene editing products.
  • Figure 39 shows the ELISA detection of the GH-HyFc protein level secreted in the supernatant of GH-HyFc iMSCs.
  • Figure 40 The number of Nb2-11 cells was detected by luminescence cell viability detection kit to verify the function of GH-HyFc protein secreted in the supernatant of GH-HyFc iMSCs.
  • Figure 41 is a schematic diagram of targeting integration into the B2M locus in Example 21.
  • FIG42 is an agarose gel image of the PCR identification of GLP-1RA integrated iPSCs across upstream homology arms in Example 22.
  • FIG43 is an agarose gel image of the PCR identification of GLP-1RA integrated iPSCs across downstream homology arms in Example 22.
  • Figure 44 is a diagram showing the sequencing results of PCR detection of B2M gene editing products in Example 22.
  • Figure 45 is a graph showing the results of PCR detection of B2M gene editing in Example 22.
  • Figure 46 is a diagram showing the sequencing results of PCR detection of B2M gene editing products in Example 22.
  • Figure 47 shows the ELISA detection of the GLP-1RA protein level secreted in the supernatant of GLP-1RA iMSCs in Example 24.
  • antibody is used herein in the broadest sense to refer to a protein that contains an antigen binding site and encompasses natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), single-chain antibodies, whole antibodies, and antibody fragments.
  • iPSCs or ipscs Induced pluripotent stem cells refer to induced pluripotent stem cells.
  • iMSCs or iMSCs refer to mesenchymal stem cells differentiated from induced pluripotent stem cells.
  • aPD1 and anti-PD1 are synonymous and refer to antibodies that target the PD1 immune checkpoint.
  • aPD1-FLAG refers to a PD1 antibody with a FLAG detection tag to facilitate detection.
  • Single-chain antibody (scFv): It is composed of the light chain variable region and heavy chain variable region of an antibody connected by a hinge region, retaining the ability to bind to the antigen.
  • PD-1 A member of the immunoglobulin superfamily of receptors, PD-1 inhibits the proliferation of immune cells such as T cells and the secretion of cytokines when bound to its ligands PD-L1/PD-L2. In the tumor microenvironment, PD-1 binding to its ligands can weaken the function of tumor-specific T cells, preventing complete tumor clearance.
  • Tumor necrosis factor- ⁇ is a cytokine that plays a core regulatory role in inflammatory responses, apoptosis, and immune regulation. While it can resist infection and prevent tumor formation, it is also closely related to the progression of various diseases, such as malignant tumors, rheumatoid arthritis (RA), psoriatic arthritis, and diabetes. In RA, excessive production of TNF- ⁇ promotes disease progression and joint destruction. Therefore, anti-TNF- ⁇ drugs are widely used in the treatment of rheumatoid arthritis, thereby reducing inflammatory responses, alleviating joint symptoms, and ameliorating joint destruction.
  • sTNFRII is a tumor necrosis factor receptor
  • anti-TNF- ⁇ scFv is a single-chain antibody against tumor necrosis factor- ⁇ . Both can treat diseases caused by excessive TNF- ⁇ .
  • FVIII refers to coagulation factor VIII, a protein encoded by the F8 gene.
  • Coagulation factors are proteins involved in the blood coagulation process, including coagulation factors I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, etc., of which coagulation factor VIII (i.e., F8 or FVIII) is a common type. Mutations in the F8 gene cause structural defects in the FVIII molecule produced or a reduction in the FVIII content, resulting in an inability to perform normal coagulation function.
  • F8 encodes a precursor polypeptide containing 2351 amino acids, which undergoes a series of processing modifications to form the F8 mature protein containing 2332 amino acids.
  • the FVIII structure can be divided into several different functional regions, including 3 A regions, 1 B region, and 2 C regions.
  • the A region contains a calcium ion binding site, which plays a role in the intrinsic coagulation pathway.
  • B domain deletion F8 BDD-F8
  • coagulation factor F8 includes, but is not limited to, full-length F8, B domain deletion F8, or other mutant F8 forms, and optionally, an antibody capable of achieving coagulation.
  • F8 with B domain deletion (BDDF8): The complete F8 gene encodes the full-length coagulation factor VIII (FVIII).
  • the full-length FVIII has six domains: A1, A2, B, A3, C1, and C2.
  • the B structure is not necessary for the coagulation catalytic activity of FVIII.
  • the F8 gene with B domain deletion is relatively small and easy to integrate, and also encodes FVIII with coagulation activity.
  • Glucagon-like peptide-1 is a 30/31 amino acid peptide hormone released by intestinal L cells after nutrient consumption. Glucagon-like peptide-1 acts on the GLP-1 receptor (GLP-1R) to lower blood sugar by enhancing glucose-dependent insulin secretion, inhibiting glucagon secretion, and slowing gastric emptying. Generally speaking, glucagon-like peptide-1 receptor agonists (GLP-1RAs) can activate the GLP-1 receptor and inhibit glucagon secretion, thereby achieving blood sugar lowering and weight loss effects. In the prior art, methods for expressing GLP-1RA in cells often use viral methods, which are high-risk and difficult to achieve good uniformity in cell quality.
  • a non-viral method is used to achieve safe, stable and high expression of GLP-1RA in cells by site-directed integration of a polynucleotide sequence encoding GLP-1RA at a specific site in the cell genome.
  • the above-mentioned cells are mesenchymal stem cells.
  • the GLP-1RA secretion rate per million mesenchymal stem cells per 24 hours is above 20 nanograms.
  • GH Human Growth Hormone
  • IGF-1 insulin-like growth factor 1
  • GH Globally stable hormone
  • GH-HyFc Hybrid Fc
  • GH-HyFc Hybrid Fc
  • GH with a Hybrid Fc exhibits a shorter half-life compared to GH with a Hybrid Fc (a mixture of IgD and IgG4).
  • GH with a Hybrid Fc exhibits a longer half-life.
  • GLP-1RA Glucagon-Like Peptide-1 Receptor Agonists
  • GLP-1RA drugs are glucagon-like peptide-1 receptor agonists. This type of drug regulates blood sugar levels for the treatment and research of type 2 diabetes by mimicking the effects of glucagon-like peptide-1 (GLP-1).
  • GLP-1RA drugs are mainly used to treat type 2 diabetes, help control blood sugar, and also have a weight loss effect.
  • Common GLP-1RA drugs include liraglutide, exenatide, and semaglutide. These drugs are usually administered by subcutaneous injection, and due to their good metabolic regulation effects, they are also used in the treatment of metabolic diseases such as obesity.
  • Signal peptide It is a short peptide chain of 5-30 amino acids in the newly synthesized polypeptide chain that guides the transfer of proteins to the secretory pathway; it is located at the N-terminus of the secretory protein and consists of three parts: the N-terminus is a positively charged alkaline amino terminus; the middle is the main functional region, which is a hydrophobic sequence formed by neutral amino acids; the C-terminus is a negatively charged processing region, which is the cleavage site of the signal peptide.
  • the newly synthesized protein is guided into the endoplasmic reticulum cavity by the signal peptide, and the signal peptide sequence is removed under the action of the signal peptidase, and then the protein continues to be translated while being folded and modified.
  • the signal peptide is selected from the signal peptides commonly used in the art, but the inventors of the present application have found through research that for different proteins, when different signal peptides are selected, their secretion levels will show different effects.
  • the signal peptides selected in the specific embodiment of the present application are all strong secretory signal peptides suitable for secretory proteins.
  • the strong secretory signal peptide suitable for a secretory protein may be a combination of one or more to achieve the required expression level.
  • the strong secretory signal peptide is secrecon, Gaussia luciferase (Gluc), Mouse Ig Kappa, Human IgG V, Human IgK VIII, Ig heavy chain signal peptide 7 (H7), and Ig ⁇ light chain signal peptide 1 (L1).
  • the signal peptide can also be selected from other types of signal peptides known in the art, as long as it can achieve the desired expression level.
  • Endogenous signal peptide a signal peptide derived from the corresponding target gene (protein) in the organism.
  • Exogenous signal peptide This is not the signal peptide of the target gene (protein) itself in the organism, but is an additional signal peptide artificially added during the gene expression process. Exogenous signal peptides include any signal peptide that can be used to promote the secretion of secretory proteins from cells.
  • Examples include signal peptides from immunoglobulins (such as IgG heavy chains or IgG-kappa light chains), cytokines (such as interleukin-2 (IL-2) or CD33), serum albumins (such as HSA or albumin), Azurocidin preproprotein, luciferase, trypsinogens (such as trypsinogen or pancreatin), or other signal peptides that can effectively secrete proteins from cells.
  • immunoglobulins such as IgG heavy chains or IgG-kappa light chains
  • cytokines such as interleukin-2 (IL-2) or CD33
  • serum albumins such as HSA or albumin
  • Azurocidin preproprotein such as luciferase
  • trypsinogens such as trypsinogen or pancreatin
  • other signal peptides that can effectively secrete proteins from cells.
  • the B2M locus refers to the location of the Beta-2 Microglobulin gene in the human genome.
  • the B2M locus is located on chromosome 15. This gene encodes Beta-2 Microglobulin, a small protein that normally binds to major histocompatibility complex (MHC I) molecules on the surface of most human cells.
  • MHC I major histocompatibility complex
  • the B2M locus is the coordinates NC_000015.10: 44711391-44721145 of the human reference genome version 38 (GRCh38/hg38).
  • the site-directed integration site of the B2M locus is NC_000015.10:44711496-44711616, NC_000015.10:44712633-44712758, NC_000015.10:44714254-44714367, NC_000015.10:44716166-44716285, NC_000015.10:44717129-44717251 or NC_000015.10:44718022-44718131.
  • Vector A polynucleotide or other molecule capable of transferring at least one nucleic acid fragment into a cell.
  • a vector may optionally contain components/elements that mediate its maintenance and/or its intended use (e.g., an origin of replication, an antibiotic resistance gene, a multiple cloning site, and/or an operably linked promoter/enhancer element that enables expression of the gene of interest).
  • Vectors include plasmids, bacteriophages, and plant or animal viruses.
  • Expression cassette refers to a polynucleotide sequence that is operably linked and capable of expression in a specific host.
  • polynucleotide sequences expressed in prokaryotes include, but are not limited to, a promoter, an operator, a ribosome binding site, and a sequence encoding the gene of interest.
  • Polynucleotide sequences expressed in eukaryotes include, but are not limited to, a promoter, an enhancer, a termination signal, a sequence encoding the gene of interest, and a polyadenylation signal (among other sequences).
  • a promoter is a region of DNA, typically located upstream (5') of a nucleic acid, that enhances transcription of that nucleic acid. Promoters can activate or repress the operably linked nucleic acid, as appropriate. Promoters contain specific sequences recognized by transcription factors. Binding of transcription factors to the promoter DNA sequence leads to the recruitment of RNA polymerase, which synthesizes RNA from the coding region of the nucleic acid.
  • Introduction refers to the introduction of nucleic acid into cells using any method known in the art, including but not limited to transfection, transformation, and transduction. Examples include viral vector transfection; plasmid vector transformation; electroporation (Fromm et al. (1986) Nature 319: 791-3); lipofectamine transfection (Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7); microinjection (Mueller et al. (1978) Cell 15: 579-85); Agrobacterium-mediated transfer (Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80: 4803-7); direct DNA uptake; and microprojectile bombardment (Klein et al. (1987) Nature 327: 70).
  • a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first and second nucleic acid sequences are placed in a functional relationship.
  • a promoter is operably linked to a coding sequence when the promoter affects the transcription or expression of the coding sequence.
  • operably linked nucleic acid sequences are generally contiguous and, when necessary to link two protein coding regions, in the same reading frame.
  • Nucleic acid refers to a polymeric form of nucleotides (i.e., ribonucleotides, deoxyribonucleotides, and/or modified forms of any of the foregoing).
  • nucleotides i.e., ribonucleotides, deoxyribonucleotides, and/or modified forms of any of the foregoing.
  • nucleic acid refers to a polymeric form of nucleotides (i.e., ribonucleotides, deoxyribonucleotides, and/or modified forms of any of the foregoing.
  • nucleic acid refers to a polymeric form of nucleotides (i.e., ribonucleotides, deoxyribonucleotides, and/or modified forms of any of the foregoing).
  • nucleic acid refers to a polymeric form of nucleotides (i.e., ribonucleotides, deoxy
  • Nucleic acids encompass any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular, and padlocked conformations. Nucleic acids can include either or both naturally occurring and modified nucleotides. Modifications include, but are not limited to, labeling, methylation, substitution of one or more naturally occurring nucleotides with an analog, and internucleotide modifications such as methylphosphonates, phosphotriesters, phosphoramidates, and carbamates; such as phosphorothioates and phosphorodithioates.
  • a fragment of a polynucleotide refers to a portion of a polynucleotide that encodes a polypeptide that provides substantially the same function as the polypeptide encoded by the entire polynucleotide.
  • mutants of a specific polynucleotide sequence include naturally occurring allelic mutants, artificial mutants, and polynucleotide sequences obtained by deleting, substituting, adding, and/or inserting one or more nucleotides in the specific polynucleotide sequence. It should be understood that such fragments and/or mutants of a specific polynucleotide sequence encode polypeptides that have substantially the same function as the polypeptide encoded by the original specific polynucleotide sequence.
  • Site-directed integration refers to the insertion or integration of all or part of the desired sequence (e.g., target sequence) into the desired site or locus (e.g., target sequence) within the genome.
  • Methods for site-directed integration are various methods well known to those skilled in the art. For example, calcium phosphate-mediated integration is used: by combining the exogenous gene with a calcium ion carrier (such as CaPO 4 ), the exogenous gene is integrated into the cell using electric shock or ultraviolet activation.
  • Transposon-mediated integration using transposons (such as Tn7, Tn5) to integrate the exogenous gene into the cell chromosome.
  • CRISPR/Cas9-mediated integration using the CRISPR/Cas9 system to integrate the exogenous gene into the cell chromosome.
  • Direct DNA ligation using DNA ligase to directly connect the exogenous gene to a specific location on the cell chromosome.
  • the CRISPR/Cas9 system is used for site-directed integration.
  • Homology arm refers to a sequence that is substantially identical or substantially complementary to a sequence at or near a target site (or target sequence) in a genome.
  • the number of homology arms is at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 100, at least about 150, at least about 155, at least about 160, at least about 165, at least about 170, at least about 175, at least about 180, at least about 190, at least about 200, at least about 25 ...
  • the homology arms can integrate the target sequence into the target site (or target sequence) in the genome by homologous recombination, wherein the homology arms are substantially identical or substantially complementary to the sequence at or near the target site (or target sequence) in the genome.
  • the 5′ homology arm is homologous to the sequence present in the nucleotide sequence of SEQ ID NO: 1
  • the 3′ homology arm is homologous to the sequence present in the nucleotide sequence of SEQ ID NO: 2.
  • the PAM sequence is located next to the target sequence that is recognized and cut by the CRISPR RNA-guided Cas protein (such as Cas9).
  • the PAM sequence is usually a short nucleic acid sequence that is not directly recognized by the CRISPR RNA-guided Cas protein, but serves as a necessary auxiliary sequence to help the Cas protein determine the binding position on the target sequence.
  • the PAM sequence is 5'-NGG-3', and the N is A, T, C or G.
  • the PAM sequence is TGG.
  • Target sequence A region that is recognized by integrase and inserted into a foreign DNA sequence. It is the binding site for integrase and is typically located near the coding region or promoter of a gene.
  • the target sequence can be a naturally occurring sequence or a sequence that is artificially designed or synthesized.
  • sgRNA sgRNA
  • guide RNA sgRNA of the present invention
  • sgRNA sgRNA that targets a target sequence in a site-directed integration site.
  • the sgRNA targets a target sequence in the B2M gene.
  • the sgRNA is the nucleotide sequence set forth in SEQ ID NO: 3.
  • AAVS1, B2M, CCR5, CIITA, and rDNA regions were constructed.
  • CRISPR/Cas9 gene editing tools were used to site-specifically integrate the anti-human PD1 scFv sequence into the AAVS1, B2M, CCR5, or CIITA loci, respectively.
  • TALENickase editing tools were used to site-specifically integrate the anti-human PD1 scFv sequence into the rDNA region.
  • the targeting vector for the B2M locus as an example, the main components and the site-specific integration process are shown in Figure 1.
  • FIG. 2 The main components of the rDNA region anti-human PD1 scFv targeting vector and the process of combined site-specific integration are shown in Figure 2.
  • the upstream homology arm was 935 bp long, and the downstream homology arm was 596 bp long.
  • the screening gene NEO was located between the two homology arms to assist in the selection of site-specific integration clones (see prior art document: IL24 gene targeting in the ribosomal gene region of human iPSCs and its anti-tumor effect on differentiated MSCs, Liu Bo, graduation thesis, Central South University, 2017: 19-25; the TALENickase editing tool used in the specific embodiments of the present invention was constructed with reference to this prior art document).
  • the targeting vector sequence was synthesized by Sangon Biotech (i.e., Sangon Biotech (Shanghai) Co., Ltd.), and the endotoxin-free target plasmid was obtained for subsequent experiments.
  • sgRNAs were screened, and approximately 600 bp upstream and downstream of the PAM site were used as upstream and downstream homology arms.
  • the aPD1 scFv was driven by the EF1 ⁇ promoter and a FLAG tag was added for detection.
  • nucleotide sequence of the sgRNA used in the CRISPR/Cas9 gene editing tool in the specific embodiment of the present invention is as follows:
  • AAVS1 sgRNA GTCCCCTCCACCCCACAGTG (SEQ ID NO: 4)
  • B2M sgRNA GGCCGAGATGTCTCGCTCCG (SEQ ID NO: 3)
  • CCR5 sgRNA GACTATGCTGCCGCCCAGT (SEQ ID NO: 5)
  • CIITA sgRNA GAGATTGAGCTCTACTCAGG (SEQ ID NO: 6)
  • the corresponding sgRNA was connected to the PX330 plasmid (source: Addgene) to form a CRISPR/Cas9 gene editing tool plasmid targeting each site.
  • AAVS1 LHA (SEQ ID NO: 7)
  • AAVS1 RHA (SEQ ID NO: 8)
  • nucleotide sequence of the homology arms of the rDNA region targeting vector is as follows:
  • amino acid sequence of the aPD1 scFv (derived from WO2018/020476A1) used in the specific embodiments of the present invention is as follows:
  • Hinge region (SEQ ID NO: 16)
  • nucleotide sequence of aPD1 scFv (derived from WO2018/020476A1) used in the specific embodiments of the present invention is as follows:
  • Hinge region (SEQ ID NO: 19)
  • aPD1 scFv can also adopt the following amino acid sequence (derived from Keytruda monoclonal antibody):
  • Hinge region (SEQ ID NO: 22)
  • the nucleotide sequence of the aPD1 scFv (derived from Keytruda monoclonal antibody) is as follows:
  • Hinge region (SEQ ID NO: 25)
  • the amino acid sequence of the FLAG tag is: DYKDDDDK; (SEQ ID NO: 27)
  • the nucleotide sequence of the FLAG tag is: GACTACAAGGACGACGACGACAAG. (SEQ ID NO: 28)
  • SP is a signal sequence, the amino acid sequence of which is: DMRVPAQLLGLLLLWLRGARC; (SEQ ID NO: 29)
  • the nucleotide sequence of the SP signal sequence is: (SEQ ID NO: 30)
  • the nuclear transfection method for AAVS1, B2M, CCR5, CIITA, and rDNA regions is as follows:
  • iPSCs in good condition to be targeted were seeded into Matrigel (Corning)-coated 6-well plates and cultured using mTeSR TM Plus, with the medium changed daily;
  • PCR amplification was performed using primers LHA-F/R across the upstream homology arm region and primers RHA-F/R across the downstream homology arm region.
  • primers LHA-F/R across the upstream homology arm region
  • RHA-F/R across the downstream homology arm region.
  • AAVS1, B2M, CCR5, and CIITA if the site-specific integration was a monoclonal clone, a product of approximately 1100 bp in length could be amplified by PCR.
  • the rDNA region if the site-specific integration was a monoclonal clone, a product of approximately 1600 bp in length could be amplified by PCR.
  • nucleotide sequences of the primers used in the examples of the present invention are:
  • AAVS1 LHA-F gccattgtcactttgcgctgc (SEQ ID NO: 31)
  • AAVS1 RHA-F aagagcctgatcttcagcgcc (SEQ ID NO: 33)
  • AAVS1 RHA-R ggcatgagatggtggacgagg (SEQ ID NO: 34)
  • B2M LHA-F tagagggcgctggaagctctaa (SEQ ID NO: 35)
  • B2M LHA-R gggaaccacacacggcacttac (SEQ ID NO: 32)
  • B2M RHA-F aagagcctgatcttcagcgcc (SEQ ID NO: 33)
  • B2M RHA-R gcaaagcacataaagtccttggcac (SEQ ID NO: 36)
  • CCR5 LHA-F aattagcttggtgtggtggcg (SEQ ID NO: 37)
  • CCR5 LHA-R gggaaccacacacggcacttac (SEQ ID NO: 32)
  • CCR5 RHA-F aagagcctgatcttcagcgcc (SEQ ID NO: 33)
  • rDNA LHA-F cctgagaaacggctaccaca (SEQ ID NO: 41)
  • rDNA LHA-R gaactgcttccttcacgacat (SEQ ID NO: 42)
  • 96 iPSC clones were selected from each of the AAVS1, B2M, CCR5, and CIITA loci for expansion culture and genomic DNA (gDNA) extraction. PCR amplification and identification were performed using primers LHA-F/R spanning the upstream homology arm region and RHA-F/R spanning the downstream homology arm region. In the rDNA region targeting experiment, a total of 10 G418-resistant clones were selected, of which 5 produced product fragments of approximately 1600 bp in size ( Figure 3). PCR products of the correct fragment size were sequenced to confirm that the product sequence was consistent with the expected one ( Figure 5), confirming successful site-specific integration and obtaining 5 rDNA aPD1 iPSCs.
  • PCR identification of iPSC clones targeting the AAVS1, CCR5, and CIITA loci confirmed that no iPSC clones with site-specific integration of the anti-PD1 scFv were obtained.
  • Two iPSC clones at the B2M locus produced products of the corresponding size by PCR ( Figure 4), and sequencing confirmed that the product sequence matched the theoretical sequence ( Figure 6).
  • Two iPSCs expressing B2M-anti-PD1 scFv were identified by PCR and site-specifically integrated. Their clonal morphology is shown in Figure 7 and designated aPD1 iPSC-1 and aPD1 iPSC-2.
  • the gene expressing the anti-PD1 scFv did not integrate into the AAVS1, CCR5, or CIITA sites, while it successfully integrated into the B2M locus and rDNA region.
  • Sample loading Place the prepared SDS-PAGE gel in an electrophoresis tank and prepare fresh 1 ⁇ running buffer. Add the prepared protein samples to the wells in order. Run at a constant current of 80 V for 30 min, then at 120 V for 60 min.
  • Antibody incubation Dilute the primary antibody to an appropriate concentration with TBST or 5% skim milk powder, pour into a small box, incubate overnight at 4°C, and wash three times with TBST on a shaker at room temperature for 10 min each time;
  • the gene expressing the anti-PD1 scFv was successfully integrated into the rDNA region, the rDNA region was unable to express the aPD1-FLAG protein, and no protein expression was observed.
  • the B2M site achieved both successful site-specific integration and significant protein expression.
  • PCR amplification was performed using primers B2M-F/R.
  • Unedited blank iPSCs produced a 513 bp product.
  • aPD1 iPSC-1 and aPD1 iPSC-2 if both copies of the B2M locus were site-directedly integrated with anti-PD1 scFv, no 513 bp product was produced. However, if only one copy was integrated with anti-PD1 scFv, a 513 bp product was still produced.
  • the PCR product size and sequencing results were combined to detect the editing status of the B2M locus in the two iPSCs.
  • nucleotide sequences of the primers used in this example are:
  • B2M-R gagatccagccctggactagc (SEQ ID NO: 44)
  • aPD1 iPSC-1 did not produce a 513 bp product, indicating that this clone had dual-copy integration of the anti-PD1 scFv.
  • aPD1 iPSC-2 produced a 513 bp product, and sequencing results ( Figure 10) revealed a multi-base deletion at the B2M locus, indicating that this clone had single-copy integration of the anti-PD1 scFv; the other copy was only edited, without anti-PD1 scFv integration.
  • the culture medium was aspirated and the wells were washed twice with DPBS.
  • the cells were digested with TrypLE TM Express at room temperature for 90 seconds.
  • the TrypLE TM Express was aspirated and the cells were blown up with 2 ml of complete MesenCult TM -ACF Medium and inoculated into 6-well plates pre-coated with MesenCult TM -ACF Attachment Substrat. 2 ⁇ L of 10 ⁇ M Y-27632 was added and the passage was recorded as P1.
  • P1 cells When the P1 cells are confluent, they are passaged at a ratio of 1:2 into 6-well plates pre-coated with MesenCult TM -ACF Attachment Substrat. They are then cultured with complete MesenCult TM -ACF Medium and Y-27632 at a final concentration of 10 ⁇ M. This is designated as the P2 generation.
  • P4 cells will slowly proliferate in a 6 cm dish. At this time, the medium can be changed every other day. Replace it with complete MesenCult TM -ACF Medium containing Y-27632 at a final concentration of 10 ⁇ M. After about 5 days, the cells in the 6 cm dish can reach 90% confluence.
  • aPD1 iPSC-1 was directed differentiated into aPD1 iMSC-1
  • aPD1 iPSC-2 was directed differentiated into aPD1 iMSC-2.
  • Both iMSCs had typical MSC morphology and exhibited a fibroblast-like morphology that was significantly different from the iPSCs morphology.
  • the surface marker flow cytometric analysis results of aPD1 iMSC-1 and aPD1 iMSC-2 met the ISCT identification criteria, showing CD44+, CD73+, CD90+, CD105+, CD34-/CD45-/HLA-DR- characteristics.
  • iMSCs were seeded into six-well plates at a density of approximately 3 ⁇ 10 5 cells per well. After the cells reached 70% confluence, the wells were washed twice with DPBS and then replaced with 2 mL of MSC osteogenic differentiation medium for differentiation. The medium was changed every 2 days. After 2 weeks of differentiation culture, the cells were stained with Alizarin Red (Cyagen) for identification.
  • APD1 iMSC-1 and aPD1 iMSC-2 have the potential to differentiate into osteoblasts, adipocytes, and chondrocytes.
  • iMSCs were collected, 100 ⁇ L of cell lysis buffer was added, and lysed on ice for 30 min;
  • Sample loading Place the prepared SDS-PAGE gel in an electrophoresis tank and prepare fresh 1 ⁇ running buffer. Add the prepared protein samples to the wells in order. Run at a constant current of 80 V for 30 min, then at 120 V for 60 min.
  • Antibody incubation Dilute the primary antibody to an appropriate concentration with TBST or 5% skim milk powder, pour into a small box, incubate overnight at 4°C, and wash three times with TBST on a shaker at room temperature for 10 min each time;
  • the operation method is the same as step 1.
  • aPD1-FLAG protein expression was detected in the lysates of both aPD1 iMSC-1 and aPD1 iMSC-2 cells, while B2M protein expression was absent.
  • Significant aPD1-FLAG protein secretion was detected in the supernatants of both aPD1 iMSC-1 and aPD1 iMSC-2 cells.
  • aPD1-FLAG protein expression levels were higher in aPD1 iMSC-1 cells and in the supernatant.
  • Dual-copy integrated aPD1 iMSC-1 cells expressed higher levels of aPD1-FLAG protein than single-copy integrated aPD1 iMSC-2 cells, both intracellularly and in the supernatant.
  • Blank iMSCs, aPD1 iMSC-1, and aPD1 iMSC-2 were seeded into 6-well plates at a density of 10 6 cells, respectively, with a total culture medium volume of 2 mL. After 24 h, the supernatant of the iMSCs culture medium was collected and centrifuged at 1000 g for 5 min to remove cell debris.
  • the standard substance used was aPD1 scFv-FLAG protein synthesized and quantified by Bio-Ying Biotechnology, and the coating solution (bioss, #C04-01001) was used to prepare the required concentrations for the standard curve: 200 ng/ ⁇ L, 100 ng/ ⁇ L, 50 ng/ ⁇ L, 25 ng/ ⁇ L, 12.5 ng/ ⁇ L;
  • the ELISA results are shown in Figure 15. High aPD1-FLAG protein expression levels were detected in the supernatants of both aPD1 iMSC-1 and aPD1 iMSC-2 cells.
  • the secretion level of double-copy integrated aPD1 iMSC-1 cells was 1674 ng/10 6 cells/24 h, and the secretion level of single-copy integrated aPD1 iMSC-1 cells was 1212 ng/10 6 cells/24 h.
  • PD-1 293T cells Construction of HEK293T cell line overexpressing PD-1 protein (hereinafter referred to as PD-1 293T cells)
  • a HEK293T cell line overexpressing PD-1 protein (hereinafter referred to as PD-1 293T cells) was constructed using a lentivirus carrying PD1-GFP (purchased from Shanghai Heyuan Biotechnology).
  • the HEK293T cell line can be constructed using conventional operating methods known in the art, and no special restrictions are imposed herein.
  • the PD-1 nucleotide sequence carried by the lentivirus is: (SEQ ID NO: 45)
  • the upper chamber was inoculated with a 1:1 mixed population of 293T:PD-1 293T cells, totaling 2 ⁇ 10 5 cells;
  • aPD1-FLAG secreted by aPD1 iMSCs binds to PD-1 protein
  • aPD1-FLAG is detected on the surface of PD-1 293T cells ( Figure 17, center).
  • aPD1-FLAG binding was detected on the surface of approximately 80% of PD-1 293T cells
  • aPD1-FLAG binding was detected on the surface of approximately 60% of PD-1 293T cells.
  • no aPD1-FLAG binding was detected on the surface of 293T cells ( Figure 17, right), indicating that aPD1-FLAG secreted by aPD1 iMSCs specifically binds to PD-1 protein.
  • iMSCs supernatant was added to each well and incubated with PD-1 293T cells for 1 hour.
  • the left panel of Figure 18 shows the peak results obtained by flow cytometry detection of PDL1-IgG1 protein bound to the surface of PD-1 293T cells.
  • the statistical results in the right panel show that when blank iMSCs supernatant was added, PDL1-IgG1 protein could normally bind to PD-1 protein on the surface of PD-1 293T cells.
  • aPD1 iMSCs-1 and aPD1 iMSCs-2 When the culture supernatant of aPD1 iMSCs-1 and aPD1 iMSCs-2 was added, the level of binding of PDL1-IgG1 protein to PD-1 protein on the surface of PD-1 293T cells decreased, indicating that the aPD1-FLAG protein in the supernatant of aPD1 iMSCs can block the binding of PDL1-IgG1 to PD-1 protein, with aPD1 iMSCs-1 having a better blocking effect. This also indicates that aPD1 iMSCs-1 have a higher level of secreted expression of PD1 antibodies. Based on the experimental results in this example, aPD1 iMSCs-1 were selected for subsequent functional experiments.
  • HCC827 human lung adenocarcinoma cells (Wuhan Punosai Life Science Co., Ltd.) were seeded in the upper chamber of a six-well Transwell plate, and 1.5 ⁇ 10 5 iMSCs were seeded in the lower chamber;
  • HCC827 cells were collected and stained with PE Annexin V Apoptosis Detection Kit (BD biosciences, #559763), and the apoptosis ratio of HCC827 cells was detected by flow cytometry.
  • Figure 19 shows the flow cytometry analysis results of the apoptosis ratio of HCC827 cells.
  • the anti-PD1 monoclonal antibody Keytruda (Selleck Bio) was used as a positive control to verify the stability of the co-culture system.
  • An increase in the apoptosis ratio of HCC827 cells was observed in the aPD1 iMSC group, demonstrating that aPD1-FLAG secreted by aPD1 iMSC can promote the killing ability of human PBMCs against HCC827.
  • CT26 cells (Zhejiang Meisen Cell Technology Co., Ltd.) in the logarithmic growth phase were inoculated subcutaneously in the right flank of BALB/chPD-1 humanized mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.);
  • mice were divided into groups and prepared for injection.
  • the solvent DPBS, 3 ⁇ 10 6 blank iMSCs, and 3 ⁇ 10 6 therapeutic cells aPD1 iMSCs were injected through the tail vein respectively.
  • the drugs were administered once every three days for a total of 3 times.
  • the experimental endpoint was 12 days after the last administration, and the mice were sacrificed.
  • mice All experimental animals maintained normal activity and feeding patterns. Mice in the DPBS and blank iMSCs groups showed significant weight loss near the endpoint, but their weight increased somewhat due to tumor weight. After injection of the therapeutic cells, the mice maintained a relatively stable weight, with no abnormalities observed (Figure 20).
  • the average tumor volume in the DPBS solvent group was 1776.53 mm 3
  • the average tumor volume in the blank iMSCs group was 1418.14 mm 3
  • the tumor growth inhibition rate (TGI) was 20.17%.
  • the average tumor volume in the aPD1 iMSCs-treated group was 444.88 mm 3 , with a TGI of 74.96%.
  • the aPD1 iMSCs-treated group exhibited a significant inhibitory effect on CT26 subcutaneous xenograft tumors.
  • site-directed integration of the PD1 antibody expression frame into the B2M locus of iPSCs overcomes the issues of inability to integrate or difficulty achieving high expression after integration that arise when integrating at other sites. Furthermore, site-directed integration of the PD1 antibody expression frame into the B2M locus of iPSCs achieved excellent expression and enhanced anti-tumor efficacy.
  • a gene target entry site was designed at exon 1 of human B2M, its upstream and downstream sequences were selected as homology arms, EF-1 ⁇ was used as the promoter for exogenous gene expression, and the target gene sequence was synthesized by Sangon Biotech (Shanghai) Co., Ltd.
  • amino acid sequence of the anti-TNF- ⁇ scFv (derived from CN1300173C) used is as follows:
  • Hinge region (SEQ ID NO: 47)
  • nucleotide sequence of the anti-TNF- ⁇ scFv (derived from CN1300173C) used is as follows:
  • Hinge region (SEQ ID NO: 50)
  • amino acid sequence of the signal peptide Gluc is: MGVKVLFALICIAVAEA; (SEQ ID NO: 52)
  • the nucleotide sequence of the signal peptide Gluc is: (SEQ ID NO: 53)
  • the amino acid sequence of the signal peptide IgG V is: MDWTWRFLFVVAAATGVQS; (SEQ ID NO: 54)
  • the nucleotide sequence of the signal peptide IgG V is: (SEQ ID NO: 55)
  • the targeting vector sequence of Gluc-anti-TNF- ⁇ scFv is: (SEQ ID NO: 56)
  • the targeting vector sequence of IgG V-anti-TNF- ⁇ scFv is: (SEQ ID NO: 57)
  • the B2M transfer method is as follows:
  • iPSCs in good condition to be targeted were seeded into Vitronectin (Nuwacell)-coated 6-well plates and cultured using ncEpic, with the medium changed daily.
  • primers B2M RHA-F/R can be used to amplify a product with a length of approximately 1000 bp across the downstream homology arm by PCR.
  • nucleotide sequences of the primers used in the examples of the present invention are:
  • B2M RHA-F gactacaaggacgacgacgacaag (SEQ ID NO: 28)
  • B2M RHA-R gcaaagcacataaagtccttggcac (SEQ ID NO: 36)
  • iPSC clones For Gluc-anti-TNF- ⁇ scFv and IgG V-anti-TNF- ⁇ scFv integrated at the B2M locus, 25 and 58 iPSC clones, respectively, were selected. Five iPSC clones from each site amplified products of corresponding sizes by PCR ( Figure 23), designated G1, G2, G3, G4, G5, V1, V2, V3, V4, and V5, respectively. Sequencing confirmed that the product sequences matched the theoretical sequences ( Figure 24). Five B2M-Gluc-anti-TNF- ⁇ scFv iPSCs and five B2M-IgG V-anti-TNF- ⁇ scFv iPSCs were obtained by PCR.
  • the anti-TNF- ⁇ scFv protein level in the cell supernatant was detected using a FLAG ELISA kit (Cayman, #501560). The operation steps were carried out according to the kit instructions.
  • Figure 25 shows the results of the anti-TNF- ⁇ scFv protein secretion level test.
  • the expression levels of G2 and G5 in the five clones of Gluc-anti-TNF- ⁇ scFv iPSCs were relatively higher, about 0.35ug/106cells/24h, while only V1 in the five clones of IgG V-anti-TNF-a scFv iPSCs could be detected with weak expression, and the measured values of other monoclonal clones were all lower than the background value. Therefore, the Gluc signal peptide has a stronger promoting effect on the secretion of anti-TNF- ⁇ scFv in iPSCs than IgG V.
  • iPSC clones G5 and V1 were directed to differentiate into G5 iMSCs and V1 iMSCs.
  • the differentiation steps were as described in step 1 of Example 3 of the PD1 protocol.
  • 10 6 cells of blank iMSCs, G5 iMSCs, and V1 iMSCs were seeded in 6-cm dishes in a total volume of 3 mL of culture medium. After 24 h, the supernatant of the iMSC culture medium was collected and centrifuged at 1000 g for 5 min to remove cell debris.
  • the ELISA results are shown in Figure 26.
  • the relative concentrations of FLAG tags in G5 iMSCs and V1 iMSCs samples were measured to be 311.3 ng/ml and 34.8 ng/ml.
  • the secretion levels of the target protein anti-TNF- ⁇ scFv in the cell culture supernatant of G5 iMSCs and V1 iMSCs were 2.63 ug/10 6 cells/24 h and 0.29 ug/10 6 cells/24 h, respectively.
  • Example 12 Construction of a targeting vector carrying the BDDF8-CO expression cassette and nuclear transfection into iPSCs
  • the B2M region was used as the site of site-directed integration of exogenous genes.
  • a corresponding targeting vector carrying a codon-optimized B-domain deleted F8 (hereinafter referred to as BDDF8-CO) expression cassette was constructed.
  • the CRISPR/Cas9 gene editing tool was used to site-directedly integrate the BDDF8-CO expression cassette into the B2M locus.
  • the main components of this targeting vector and a schematic diagram of site-directed integration are shown in Figure 27.
  • the nucleotide sequence encoding the BDDF8-CO expression cassette was inserted into the multiple cloning site of the pUC57-kan expression vector by Beijing Qingke Biotechnology Co., Ltd. to construct a targeting vector plasmid carrying the BDDF8-CO expression cassette.
  • the B2M sgRNA nucleotides used in this example are:
  • nucleotide sequence of the homology arms of the targeting vector in this example is as follows:
  • amino acid sequence of BDDF8-CO used in the specific embodiment of the present invention is as follows: (SEQ ID NO: 59)
  • the BDDF8-CO nucleotide sequence used in the specific embodiment of the present invention is as follows: (SEQ ID NO: 60)
  • SP Signal peptide
  • MGVKVLFALICIAVAEA amino acid sequence
  • the nucleotide sequence of the SP signal peptide sequence is: (SEQ ID NO: 53)
  • nucleotide sequence of the complete targeting vector in this example is as follows: (SEQ ID NO: 61)
  • iPSCs in good condition to be targeted were seeded into Matrigel (Corning, #354277)-coated 6-well plates and cultured using mTeSR TM Plus (Stemcell, #05825), with the medium changed daily.
  • the cells were first analyzed using a WOLF cell sorter (Nanocellect), and HLA-A/B/C-negative cells were selected for single-cell inoculation in a 96-well plate. A small number of cells were directly inoculated into well B2, which was the positive well.
  • nucleotide sequences of the primers used in the examples of the present invention are:
  • BDDF8-CO iPSC-2 cells harboring dual site-specific integration of the BDDF8-CO expression cassette, were differentiated to generate BDDF8-CO iMSC-2 cells.
  • BDDF8-CO iMSC-2 cells were characterized by typical MSC fibroblast-like morphology, cell surface markers consistent with MSC characteristics, and trilineage differentiation potential.
  • the prior art also integrates BDD-F8 into iPSCs, but the site-specific integration site is the rDNA region, and the site-specifically integrated cells are differentiated into iMSC cells.
  • the F8 secretion amount of the obtained iMSC cells is 0.59 ⁇ 0.11 ng/(10 6 cells every 24 hours) and 0.68 ⁇ 0.14 ng/(10 6 cells every 24 hours).
  • the B2M locus is an integration site suitable for site-specific integration of nucleotide sequences encoding secretory proteins, and can achieve a high secretion effect. In mesenchymal stem cells, its secretion of secretory proteins is much higher than that of other integration sites.
  • FVIII coagulation activity detection The results of FVIII coagulation activity detection are shown in Figure 33. FVIII coagulation activity is almost undetectable in the culture supernatant of Wild-type iMSC, while the FVIII coagulation activity in the culture supernatant of BDDF8-CO iMSC-2 cells is very high, exceeding 200%.
  • Example 17 Construction of a targeting vector carrying a GH-HyFc expression cassette and nuclear transfection into iPSCs
  • the B2M locus was used as the site for site-directed integration of the exogenous gene.
  • the B2M locus is the coordinates NC_000015.10:44711391-44721145 of the human reference genome, version 38 (GRCh38/hg38).
  • a targeting vector carrying a codon-optimized GH with Hybrid Fc (hereinafter referred to as GH-HyFc) expression cassette was first constructed.
  • the nucleotide sequence of this targeting vector is shown in SEQ ID NO: 64.
  • the GH-HyFc expression cassette was site-directedly integrated into the B2M locus using the CRISPR/Cas9 gene editing tool.
  • FIG. 34 A schematic diagram of the main components of this targeting vector and its site-directed integration is shown in Figure 34.
  • the nucleotide sequence encoding the GH-HyFc expression cassette was inserted into the multiple cloning site of the pUC57-kan expression vector to construct a targeting vector plasmid carrying the GH-HyFc expression cassette (synthesized by Beijing Qingke Biotechnology Co., Ltd.).
  • the B2M sgRNA nucleotide sequence is shown in SEQ ID NO: 3
  • the homology arm B2M LHA nucleotide sequence of the targeting vector is shown in SEQ ID NO: 65
  • the B2M RHA nucleotide sequence is shown in SEQ ID NO: 2.
  • the amino acid sequence of GH-HyFc used in the specific embodiments of the present invention is shown in SEQ ID NO: 66, and the nucleotide sequence is shown in SEQ ID NO: 67.
  • Signal peptide (abbreviated as SP) is a signal peptide, and its amino acid sequence is: MGVKVLFALICIAVAEA (SEQ ID NO: 52); the nucleotide sequence of the SP signal peptide sequence is: ATGGGCGTGAAGGTGCTGTTTGCCCTGATTTGCATCGCCGTGGCCGAGGCC (SEQ ID NO: 53).
  • iPSCs in good condition to be targeted were seeded into Matrigel (Corning, #354277)-coated 12-well plates and cultured using mTeSR TM Plus (Stemcell, #05825), with the medium changed daily.
  • the cells were first analyzed using a WOLF cell sorter (Nanocellect), and HLA-A/B/C-negative cells were selected for single-cell inoculation in a 96-well plate. A small number of cells were directly inoculated into well B2, which was the positive well.
  • PCR amplification was performed using primers TYB-GSH-UP-F/R across the upstream homology arm region and primers 3-191-down-F/R across the downstream homology arm region. If it is a monoclonal clone with site-specific integration, products of 1060 bp and 915 bp in length can be amplified by PCR, respectively.
  • nucleotide sequences of the primers used in the examples of the present invention are:
  • TYB-GSH-UP-F tagagggcgctggaagctctaa (SEQ ID NO: 35)
  • nucleotide sequences of the primers used in the examples of the present invention are:
  • TYB-GSH-down-R gcaaagcacataaagtccttggcac(SEQ ID NO:36)
  • GH-HyFc iPSC-3 and GH-HyFc iPSC-9 cells harboring a single copy of the GH-HyFc expression cassette were then differentiated to obtain GH-HyFc iMSC-3 and GH-HyFc iMSC-9 cells (using the same differentiation method as described in the PD1 protocol, step 1a, in Example 3).
  • GH-HyFc iMSC-3 and GH-HyFc iMSC-9 cells were characterized by typical MSC fibroblast-like morphology and cell surface markers consistent with MSC characteristics, demonstrating trilineage differentiation potential.
  • GH-HyFc expression is almost undetectable in the culture supernatant of wild-type iMSC cells, while higher GH-HyFc protein expression levels of 410.72 ng/10 6 cells/24 h and 607.28 ng/10 6 cells/24 h were detected in the culture supernatants of GH-HyFc iMSC-3 and GH-HyFc iMSC-9 cells, respectively.
  • B2M locus is a suitable integration site for site-specific integration of nucleotide sequences encoding secretory proteins in iMSC cells, and can achieve high secretion effects.
  • Nb2-11 cells in the logarithmic growth phase by pipetting, centrifuge at 350g for 5 minutes, collect the cells, wash them twice with Nb2-11 basal medium (DMEM (HG) + 1% NEAA + 1% HS), count the number of living cells, and dilute them with Nb2-11 basal medium to a suspension containing 1.0 ⁇ 10 5 cells per milliliter.
  • a luminescent cell viability assay kit (Promega, #G7570) was used to detect the number of Nb2-11 cells in each well according to the method in the kit instructions. The more cell proliferation there is, the stronger the proliferation-promoting ability of GH-HyFc is.
  • site-specific integration of the GH-HyFc expression frame into the B2M locus of iMSCs achieved excellent high expression and high secretion effects.
  • Cells with site-specific integration of the B2M locus secreted high levels of GH-HyFc protein with a proliferation-promoting effect.
  • the present invention constructed a GLP-1RA targeting vector with codon optimization and signal peptide modification.
  • the codon-optimized and signal peptide-modified GLP-1RA expression cassette was targeted into the B2M locus of iPSCs.
  • the main components of the constructed targeting vector and a schematic diagram of the targeting are shown in Figure 41.
  • a gene target entry site was designed at exon 1 of human B2M, and its upstream and downstream sequences were selected as homology arms.
  • EF-1 ⁇ was used as the promoter for exogenous gene expression.
  • the GLP-1RA expression cassette was designed to contain Gaussia luciferase signal peptide (Gaussi in Figure 41) and forin protease cleavage site (F in Figure 41).
  • the nucleotide sequence of the targeting vector (its nucleotide sequence is shown in SEQ ID NO: 70) was synthesized by Sangon Biotech (Shanghai) Co., Ltd.
  • the B2M sgRNA nucleotides used in this example are:
  • nucleotide sequence of the homology arms of the targeting vector in this example is as follows:
  • LHA is shown as SEQ ID NO: 65; RHA is shown as SEQ ID NO: 2.
  • the amino acid sequence of GLP-1RA used in the embodiments of the present invention is shown in SEQ ID NO: 71, which is derived from CN101974090B.
  • the nucleotide sequence of GLP-1RA used in the embodiments of the present invention is shown in SEQ ID NO: 72.
  • Gaussia luciferase signal peptide (SP) is a signal peptide sequence, and its amino acid sequence is shown in SEQ ID NO: 52.
  • the nucleotide sequence of the SP signal peptide sequence is shown in SEQ ID NO: 53.
  • GLP-1RA amino acid sequence (SEQ ID NO: 71)
  • GLP-1RA nucleotide sequence (SEQ ID NO: 72)
  • iPSCs in good condition to be targeted were seeded into Matrigel (Corning, #354277)-coated 6-well plates and cultured using mTeSR TM Plus (Stemcell, #05825), with the medium changed daily.
  • the cells were first analyzed using a WOLF cell sorter (Nanocellect), and HLA-A/B/C-negative cells were selected for single-cell inoculation in a 96-well plate. A small number of cells were directly inoculated into well B2, which was the positive well.
  • PCR amplification was performed using primers TYB-GSH-2-UP-F & TYB-GSH-UP-R across the upstream homology arm region and primers 6-31-R-F4 & 6-31-R-R4 across the downstream homology arm region. If it is a monoclonal clone with site-specific integration, products of 1053 bp and 989 bp in length can be amplified by PCR, respectively.
  • nucleotide sequences of the primers used in the examples of the present invention are:
  • 6-31-R-F4 aacaactacaagaccacgcc(SEQ ID NO: 73)
  • GLP-1RA iPSC-11 and GLP-1RA iPSC-51 cells which harbor dual site-specific integration of the GLP-1RA expression cassette, were then differentiated to obtain GLP-1RA iMSC-11 and GLP-1RA iMSC-51 cells (using the same differentiation method as described in the PD1 protocol, step 1a, in Example 3).
  • GLP-1RA iMSC-11 and GLP-1RA iMSC-51 cells were identified to possess typical MSC-like fibroblast-like morphology, with cell surface markers consistent with MSC characteristics, indicating trilineage differentiation potential.
  • the ELISA results show that GLP-1 expression is almost undetectable in the culture supernatant of wild-type iMSC cells, while high GLP-1RA protein expression is detected in the culture supernatant of GLP-1RA iMSC-11 and GLP-1RA iMSC-51 cells, reaching 1.77 ⁇ 104 ng/ 106 cells/24h/mL and 1.90 ⁇ 104 ng/ 106 cells/24h/mL, respectively.
  • the total GLP-1RA protein expression per 106 iMSC cells over 24h is 7.08 ⁇ 104 ng and 7.6 ⁇ 104 ng, respectively.
  • the B2M locus is a suitable site for the targeted integration of nucleotide sequences encoding secretory proteins in iMSC cells, achieving high secretion efficiency.
  • the secretion of secretory GLP-1 receptor agonists is significantly higher than that of other integration sites.
  • the site-specific integration of the GLP-1RA expression frame into the B2M locus of iMSCs achieved excellent expression and secretion effects, and the cells with site-specific integration were able to secrete GLP-1RA molecules at levels exceeding the expected expression level.

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Abstract

L'invention concerne une cellule modifiée et une utilisation associée. La cellule modifiée est intégrée, de manière dirigée sur un site, avec un acide nucléique pour coder et exprimer une protéine sécrétoire ; et le site de l'intégration dirigée sur un site est situé dans un locus B2M. L'invention concerne une cellule CSM permettant d'obtenir une expression à long terme et stable de la protéine sécrétoire, de telle sorte que l'effet thérapeutique continu de la cellule CSM est assuré et le coût de médicament pour un patient est réduit.
PCT/CN2024/113685 2024-04-10 2024-08-21 Cellule modifiée permettant d'exprimer hautement une protéine sécrétoire et utilisation associée Pending WO2025213672A1 (fr)

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CN110564764A (zh) * 2019-08-07 2019-12-13 佛山科学技术学院 一种外源基因定点整合的打靶载体及其应用
US20220184123A1 (en) * 2020-12-03 2022-06-16 Century Therapeutics, Inc. Genetically Engineered Cells and Uses Thereof
WO2024050349A2 (fr) * 2022-08-30 2024-03-07 Emendobio Inc. Stratégies pour knock-ins au niveau de sites safe harbor b2m

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CN110564764A (zh) * 2019-08-07 2019-12-13 佛山科学技术学院 一种外源基因定点整合的打靶载体及其应用
US20220184123A1 (en) * 2020-12-03 2022-06-16 Century Therapeutics, Inc. Genetically Engineered Cells and Uses Thereof
WO2024050349A2 (fr) * 2022-08-30 2024-03-07 Emendobio Inc. Stratégies pour knock-ins au niveau de sites safe harbor b2m

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JUSTIN EYQUEM, JORGE MANSILLA-SOTO, THEODOROS GIAVRIDIS, SJOUKJE J. C. VAN DER STEGEN, MOHAMAD HAMIEH, KRISTEN M. CUNANAN, ASHLESH: "Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection", NATURE, vol. 543, no. 7643, pages 113 - 117, XP055397283, DOI: 10.1038/nature21405 *
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