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KR-20260067431-A - Increased efficiency of gene scissors knock-out or knock-in technology by dexamethasone treatment and its uses

KR20260067431AKR 20260067431 AKR20260067431 AKR 20260067431AKR-20260067431-A

Abstract

The present invention relates to increasing the efficiency of gene editing knock-out or knock-in technology by dexamethasone treatment and the use thereof, and more specifically, to a method for producing immune effector cells with increased knock-out or knock-in efficiency of a specific gene or increased expression of a knock-in gene, comprising the step of treating with dexamethasone during the knock-out or knock-in process of a specific gene. In the present invention, it was confirmed that when immune effector cells are prepared by knocking in a CAR gene targeting a cancer cell-related antigen at a site where the TGFBR2 gene has been knocked out using a CRISPR/Cas9 system, treatment with dexamethasone enables the preparation of immune effector cells with increased insertion and expression of the CAR gene. Furthermore, since it was confirmed that immune effector cells prepared by dexamethasone treatment have excellent cancer cell killing effects, immune effector cells prepared by the method of the present invention can be usefully utilized as a composition for the prevention or treatment of various cancers.

Inventors

  • 장미희
  • 이수민

Assignees

  • 한국과학기술연구원

Dates

Publication Date
20260513
Application Date
20241104

Claims (14)

  1. A method for producing immune effector cells with increased knock-out or knock-in efficiency of a specific gene, or increased expression of a knock-in gene, comprising the step of treating with dexamethasone during the knock-out or knock-in process of a specific gene.
  2. In paragraph 1, The specific genes knocked out include TGFBR2 (Transforming Growth Factor Beta Receptor II), PD-1 (Programmed cell death protein 1), CTLA-4 (Cytotoxic T-lymphocyte-associated protein 4), Fas (CD95), Cbl-b, LAG-3 (Lymphocyte-activation gene 3), SHP-2 (SH2-containing protein tyrosine phosphatase 2), SOCS1 (Suppressor of cytokine signaling 1), AREG (Amphiregulin), HSPA1B (Heat Shock Protein Family A (Hsp70) Member 1B), NK4A3 (Nuclear Receptor Subfamily 4 Group A Member 3), HES4 (Hes Family BHLH Transcription Factor 4), NR4A1 (Nuclear Receptor Subfamily 4 Group A Member 1), CREM (CAMP) Responsive Element Modulator, EGR2(Early Growth Response) A method for producing immune effector cells, characterized by being any one gene selected from the group consisting of 2), RASGEF1B (RasGEF Domain Family Member 1B), RGCC (regulator of cell cycle), HSPA6 (Heat Shock Protein Family A (Hsp70) Member 6), TNFSF9 (TNF Superfamily Member 9), HIC1 (Hypermethylated-in-Cancer 1), ZNF331 (Zinc Finger Protein 331), ATF3 (Activating Transcription Factor 3), and BAG3 (BAG Cochaperone 3).
  3. In paragraph 1, The specific gene knocked in above is a chimeric antigen receptor (CAR) gene specific to cancer cell antigens, The above-mentioned cancer cell antigens are mesothelin (MSLN), ErbB2 (HER2/neu), EphA2 (EPH receptor A2), CEA (carcinoembryonic antigen), EpCAM (epithelial cell adhesion molecule), EGFR (epidermal growth factor receptor), EGFRvIII (EGFR variant III), GD2 (disialoganglioside), ductal-epithelial mucine, gp36, TAG-72 (Tumor-associated glycoprotein), glycosphingolipids, glioma-associated antigen, AFP (B-human chorionic gonadotropin, alphafetoprotein), lectin-reactive AFP, thyroglobulin, RAGE-1 (multiligand member of the immunoglobulin superfamily), MN/CA9, and hTERT (human telomerase reverse transcriptase), RU1, RU2(AS), intestinal carboxyl esterase, mut-hsp70-2, M-CSF (Macrophage colony-stimulating factor), protease, PSA (prostate specific antigen), PAP (Poly(A) polymerase), NY-ESO-1 (cancer/testis antigen 1B), LAGA-1a (Suppressor of hairless protein homolog), p53, mutant-p53, prostein, PSMA (Prostate specific membrane antigen), survivin, telomerase, PCTA-1 (prostate-carcinoma tumor antigen-1), MAGE (Melanoma-associated antigen), MAGE-A1 (MAGE Family Member A1), ELF2M (elongation factor 2 mutant), neutrophil elastase, ephrin B2, CD22, IGF-I (insulin growth factor-I), IGF-II (insulin growth factor-II), IGF-I receptor, 5T4 (Trophoblast glycoprotein), ROR1 (receptor tyrosine kinase like orphan receptor 1), Nkp30 (natural cytotoxicity triggering receptor 3), NKG2D (natural killer group 2D), tumor stromal antigen stromal antigen), FN-EDA (the extra domain A of fibronectin), FN-EDB (the extra domain B of fibronectin), TnC A1 (the Al domain of tenascin-C), fap (fibroblast associated protein), CD3, CD4, CD8, CD19, CD20, CD23, CD24, CD25, CD30, CD33, CD34, CD38, CD40, CD44, CD56, CD123, CD133, CD138, CTLA-4 (Cytotoxic T-lymphocyte-associated protein 4), B7-1 (CD80), B7-2 (CD86), endoglin, MHC molecule (a major histocompatibility complex molecule), HIV-specific antigen, HIV gp120, EBV-specific antigen (Epstein-Barr virus-specific antigen), CMV-specific antigen (cytomegalovirus-specific antigen), HPV-specific antigen (human papillomavirus-specific antigen), HBV-specific antigen (hepatitis B virus-specific antigen), HCV-specific antigen (hepatitis C virus-specific antigen), Lassa virus-specific antigen, Influenza virus-specific antigen, CA-125 (cancer antigen 125), MUC-1 (Mucin-1), surface adhesion molecule, Characterized as being any one selected from the group comprising VEGFR2 (vascular endothelial growth factor receptor-2), HMW-MAA (high molecular weight-melanoma associated antigen), epithelial tumor antigen, IL-13R-a2 (Interleukin-13 receptor subunit alpha-2), GD2, GD3, melanoma-associated antigen, mutated RAS, folate binding protein, folate receptor, HIV-1 gp41, c-Met, HERV-K (Human endogenous retrovirus K), IL-11Ralpha (interleukin 11 receptor subunit alpha), kappa chain, lambda chain, CSPG4 (Chondroitin sulfate proteoglycan 4), HER2 (human epidermal growth factor receptor type 2), HER1-HER2 combinations, and HER2-HER3 combinations. A method for manufacturing immune effector cells.
  4. In paragraph 1, The above manufacturing method is a method for manufacturing immune effector cells, characterized by knocking out a specific gene that is knocked out in immune effector cells using gene scissors, and then knocking in a specific gene for knock-in at the knocked-out site.
  5. In paragraph 1, The above manufacturing method is (1) A single guide RNA for knock-out of a specific gene to be knocked out; (2) any one of the gene scissors selected from the group consisting of a Cas9 protein, a base sequence encoding the Cas9 protein, a Cpf1 protein, a base sequence encoding the Cpf1 protein and a protein that acts as a gene scissors; and (3) a DNA construct for knock-in of a specific gene to be knocked in or a DNA fragment thereof; a step of treating immune effector cells with the DNA construct and then culturing the immune effector cells in a medium containing dexamethasone, or (a) (1) A single guide RNA for knock-out of a specific gene to be knocked out; and (2) A step of knocking out a target gene of an immune effector cell using any one of the gene scissors selected from the group consisting of a Cas9 protein, a base sequence encoding the Cas9 protein, a Cpf1 protein, a base sequence encoding the Cpf1 protein and a protein acting as a gene scissors; (b) a method for producing immune effector cells, characterized by including the step of treating immune effector cells with the gene knocked out with dexamethasone, then treating the immune effector cells with a gene transfer vector containing a DNA construct for knock-in of a specific gene to be knocked in, and culturing the immune effector cells in a medium containing dexamethasone.
  6. In paragraph 1, A method for producing immune effector cells, characterized by culturing the immune effector cells, in which the above-mentioned specific gene is knocked in, in a medium containing dexamethasone for 10 to 15 days.
  7. In paragraph 1, A method for manufacturing immune effector cells, characterized in that the immune effector cell is selected from the group consisting of T cells, natural killer (NK) cells, macrophages, monocytes, and dendritic cells.
  8. In paragraph 1, The above single guide RNA and gene scissors are delivered to immune effector cells via electroporation or a gene delivery vector, and the electroporation is performed under conditions of 500 V to 2500 V, 5 ms to 100 ms, and 1 pulse to 5 pulses, and A method for producing immune effector cells, characterized in that the above vector is a plasmid, a viral vector, or a non-viral vector.
  9. In paragraph 1, A method for producing immune effector cells, characterized by additionally treating the immune effector cells with one or more cytokines selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and TGF-β1 before or after treating the immune effector cells with the single guide RNA, gene scissors, and DNA fragments.
  10. In paragraph 5, A method for producing immune effector cells, characterized in that the above single guide RNA is one or more single guide RNAs for TGFBR2 knockout selected from the group consisting of single guide RNAs represented by the nucleotide sequences of SEQ ID NOs 1 to 6.
  11. In paragraph 5, The above-mentioned specific gene knock-in DNA construct is A nucleotide sequence encoding a promoter; A nucleotide sequence encoding a signal peptide; A nucleotide sequence encoding a cancer cell antigen-binding domain; A nucleotide sequence encoding a transmembrane domain; A nucleotide sequence encoding a costimulatory domain; and A method for producing an immune effector cell, characterized by being a chimeric antigen receptor knock-inducing DNA construct specific to cancer cell antigens, comprising a nucleotide sequence encoding an intracellular signal transduction domain.
  12. In Paragraph 11, The above promoter is pSFFV, pCMV, or pEF1A, and The above cancer cell antigens are mesothelin (MSLN), CD22, CD19, HER2 (human epidermal growth factor receptor type 2), GD2, folate receptor, MUC1 (Mucin 1), ErbB2 (Erythroblastic oncogene B-2 gene), EphA2 (EPH receptor A2), or EGFR (Epidermal growth factor receptor), and The above-mentioned penetrating domain is any one selected from the group consisting of CD8α, CD4, CD28, CD137, CD80, CD86, CD152, PD1, NKG2D, 41BB, 2B4, NKp30, NKp44, NKp46, NKG2C, and OX40, and The above co-stimulation domain is any one selected from the group consisting of CD28, OX-40, ICOS, NKG2D, 4-1BB, 2B4, DAP10, DAP12, NKp30, NKp44, NKp46, and NKG2C, and The above-mentioned intracellular signaling domain is derived from CD3ζ, and A method for producing immune effector cells with increased knock-in efficiency or expression of a specific gene, characterized by additionally including a nucleotide sequence encoding a HA (homology arm)-tag at the 5'-terminus and 3'-terminus of the above DNA construct.
  13. A composition for increasing gene knock-out or knock-in efficiency based on gene editing tools, comprising dexamethasone as an active ingredient.
  14. In Paragraph 12, A composition for increasing gene knock-out or knock-in efficiency based on gene scissors, characterized by further comprising one or more cytokines selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21, and TGF-β1.

Description

Increased efficiency of gene scissors knock-out or knock-in technology by dexamethasone treatment and its uses The present invention relates to increasing the efficiency of gene editing knock-out or knock-in technology by dexamethasone treatment and the use thereof, and more specifically, to a method for producing immune effector cells with increased knock-out or knock-in efficiency of a specific gene or increased expression of a knock-in gene, comprising the step of treating with dexamethasone during the knock-out or knock-in process of a specific gene. Over the past few years, the development of cancer therapies using chimeric antigen receptor-T cells (CAR-T cells) has been underway. Anti-CD19 CAR-T cells have demonstrated significant therapeutic effects in the treatment of hematological malignancies, and two products received FDA approval in 2017. However, despite numerous attempts, CAR-T cells targeting various tumor-associated antigens have not demonstrated favorable clinical outcomes in patients with solid tumors (Na Tang et al. , JCI Insight , 27;5(4):e133977, 2020). In 2021, B cell maturation antigen (BCMA)-specific CAR-T cells were approved as a treatment for multiple myeloma (MM), but they have problems such as relatively high costs, time-consuming production, insufficient introduction into solid tumors, and induced cytotoxic effects including immune effector cell-associated neurologic syndrome (ICANS) and cytokine release syndrome (CRS). Therefore, it is important to address these problems while protecting and enhancing CAR activity, and among other immune cell platforms (e.g., γ/δ T cells, NKT cells), natural killer (NK) cells are considered an alternative to genetic engineering using CARs. CAR-NK cells have demonstrated several advantages over CAR-T cells in terms of improved efficiency and safety, and the first clinical use of CD19 CAR-NK cells in patients with relapsed/refractory lymphoid malignancies demonstrated the persistence of CAR-NK cells (Ali Bashiri Dezfouli et. al. , Cells , 1;10(12):3390,2021). Meanwhile, the immunosuppressive tumor microenvironment (TME) present in solid tumors includes not only cancer cells but also numerous cell types, extracellular matrix components, and inflammatory mediators. In this complex microenvironment, immune effector cells with introduced CAR genes are affected by many suppressor cells and molecules that can impair survival, activation, proliferation, and effector functions. Recent studies have shown that natural killer cells with the TGFBR2 gene knocked out using CRISPR gene editing technology exhibited increased anticancer effects in glioblastoma multiforme (GBM) (Hila Shaim et al. , J Clin Invest , 131(14):e142116, 2021). However, CAR-introduced immune effector cells have problems with low CAR expression efficiency and survival rates, and improvements are required in this regard. Figure 1 is a schematic diagram showing the process of gene editing-based TGFBR2 gene knock-out and mesothelin-specific CAR gene knock-in. In the diagram, the One-step CAR-NK KO/KI is a method of delivering gene scissors, a single guide RNA, and knock-in DNA at once using electroporation, and the Two-step CAR-NK KO/KI is a method of delivering gene scissors and a single guide RNA first using electroporation, and then delivering knock-in DNA using a gene delivery vector (AAV). Figure 2 is data confirming the increase in knock-in efficiency following dexamethasone (Dex) treatment using FACS analysis. Figure 3 is data confirming the increase in knock-in efficiency following dexamethasone (Dex) treatment using Junction PCR. Figure 4 is data evaluating knock-in efficiency through DNA sequencing. Figure 5 is data confirming the activity of immune effector cells (NK cells) following treatment with dexamethasone (Dex). The present invention will be described in detail below. Using gene editing tools allows for the removal of unwanted genes and the precise knock-in of CAR genes within specific genomic locations, thereby enabling the production of CAR-immune effector cells with enhanced anticancer therapeutic efficacy. However, when producing CAR-immune effector cells using gene editing tools, there are problems such as low CAR expression efficiency and low survival rates. Accordingly, the present invention confirmed that when dexamethasone is applied during the production of gene editing-based CAR-immune effector cells, not only are the knock-in efficiency and expression of the CAR gene increased, but the activity of the immune effector cells is also increased. Method for producing immune effector cells with increased knock-out/knock-in efficiency or expression of a specific gene In one aspect, the present invention relates to a method for producing immune effector cells with increased knock-out or knock-in efficiency of a specific gene or increased expression of a knock-in gene, comprising the step of treating with dexamethasone during the knock-out or knock-in process of a specif