阅读说明：本技术 制备过表达外源基因的细胞的方法 (Method for preparing cell over expressing exogenous gene ) 是由 金华君 许馥慧 黄晨 马星明 于 2021-04-30 设计创作，主要内容包括：本发明制备过表达外源基因的细胞的方法,具体涉及cGAS-STING信号通路抑制剂在培养电转细胞中的应用、培养电转细胞的方法、电转制备过表达外源基因的细胞的方法以及添加有cGAS-STING信号通路抑制剂的细胞培养基。本发明通过使用含有cGAS-STING信号通路抑制剂的培养基对电转后的细胞、尤其是免疫效应细胞进行培养,能够明显地降低电转后细胞的死亡率,提高细胞电转后的活细胞数量与活细胞比例。(The invention discloses a method for preparing a cell over-expressing an exogenous gene, and particularly relates to application of a cGAS-STING signal pathway inhibitor in culturing an electrotransfer cell, a method for culturing the electrotransfer cell, a method for preparing the cell over-expressing the exogenous gene by electrotransfer, and a cell culture medium added with the cGAS-STING signal pathway inhibitor. The invention can obviously reduce the death rate of the cells after the cell electro-transformation and improve the number of the live cells and the proportion of the live cells after the cell electro-transformation by using the culture medium containing the cGAS-STING signal pathway inhibitor to culture the cells after the cell electro-transformation, especially immune effector cells.)

The application of cGAS-STING signal pathway inhibitor in culturing electrotransfer cells; preferably, the cGAS-STING signaling pathway inhibitor is selected from either or both of a small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential and a small molecule compound capable of inhibiting or antagonizing cGAS; preferably, the cell is an immune effector cell; preferably, the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential is compound H-151 shown in formula 5; preferably, the small molecule compound capable of inhibiting or antagonizing cGAS is a compound G150 shown as a formula 22,

2. a method for culturing an electrotransfer cell, comprising the step of culturing the electrotransfer cell in a medium comprising a cGAS-STING signaling pathway inhibitor; preferably, the cGAS-STING signaling pathway inhibitor is selected from either or both of a small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential and a small molecule compound capable of inhibiting or antagonizing cGAS; preferably, the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential is compound H-151 shown in formula 5; the small molecular compound capable of inhibiting or antagonizing cGAS is a compound G150 shown as a formula 22; preferably, the cell is an immune effector cell.

3. The method according to claim 2, wherein the method comprises, after the electroporation is completed, transferring the electroporated cells into a culture medium for culturing for 0-5 hours, adding the cGAS-STING signaling pathway inhibitor to the culture medium, and continuing the culturing; preferably, the final concentration of the cGAS-STING signaling pathway inhibitor in the culture medium after addition is in the range of 0.5-5 μ M; more preferably, in the range of 0.5-1. mu.M or in the range of 2.5-5. mu.M.

4. The method of claim 2, wherein the immune effector cells are selected from one or more of T cells, TILs, NK cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages; preferably, selected from one or more of T cells, TILs and CAR-T cells.

5. The method according to claim 2, wherein the culture medium is a cell culture medium, preferably a culture medium for culturing immune effector cells; preferably, is selected fromCTS^TMAny one of serum-free cell culture medium, DMEM medium, and RPMI1640 medium; more preferably CTS^TMSerum-free cell culture media.

6. A method of electrotransformation of a cell that overexpresses a foreign gene, the method comprising:

1) introducing a nucleic acid containing an expressed foreign gene into a cell by electrotransfer;

2) culturing the cells transfected with the foreign gene in step 1) with a medium containing a cGAS-STING signaling pathway inhibitor;

wherein, the electrically transferred cells are cultured for 0-5 h; preferably 0-1h, then adding said cGAS-STING signaling pathway inhibitor to said medium; preferably, the final concentration of the cGAS-STING signaling pathway inhibitor in the culture medium after addition is in the range of 0.5-5 μ M; more preferably, in the range of 0.5-1. mu.M or in the range of 2.5-5. mu.M;

preferably, the cGAS-STING signaling pathway inhibitor is selected from either or both of a small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential and a small molecule compound capable of inhibiting or antagonizing cGAS;

preferably, the cell is an immune effector cell.

7. The method according to claim 6, wherein the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential is compound H-151 according to formula 5;

and/or the small molecular compound capable of inhibiting or antagonizing cGAS is a compound G150 shown as a formula 22;

and/or, the immune effector cell is selected from one or more of a T cell, TIL, NK cell, NK T cell, CAR-T cell, CIK cell, TCR-T cell, and macrophage;

and/or the medium is a medium for culturing immune effector cells, e.g.selected from CTS^TMSerum-free cell culture medium, DMEM medium, and RPMI1640 medium.

8. A cell culture medium supplemented with an inhibitor of the cGAS-STING signaling pathway; preferably, the cGAS-STING signaling pathway inhibitor is selected from either or both of a small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential and a small molecule compound capable of inhibiting or antagonizing cGAS.

9. The cell culture medium according to claim 8, wherein the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential is compound H-151 according to formula 5; and/or the small molecular compound capable of inhibiting or antagonizing cGAS is a compound G150 shown as a formula 22;

and/or, the concentration of the cGAS-STING signaling pathway inhibitor in the cell culture medium is in the range of 0.5-5 μ Μ;

and/or the cell culture medium is a culture medium for culturing immune effector cells.

10. The cell culture medium of claim 9, wherein the concentration of the cGAS-STING signaling pathway inhibitor in the cell culture medium is in the range of 0.5-2.5 μ Μ or in the range of 2.5-5 μ Μ; and/or the cell culture medium is selected fromCTS^TMSerum-free cell culture medium, DMEM medium, and RPMI1640 medium.

Technical Field

The present invention relates to the culture of electrically transferred cells, and is especially the process of preparing cell over expressing exogenous gene.

Background

Adoptive Cell Therapy (ACT) using immune effector cells expressing Chimeric Antigen Receptors (CARs), such as CAR-T cells, for treating tumors has the potential to permanently alter the current state of tumor Therapy. Such therapies rely on highly efficient, stable and safe gene transfer platforms. The transfer of synthetic genes encoding chimeric antigen receptors into immune effector cells, such as T cells, is the first step in achieving tumor therapy. Gene transfer techniques include mainly viral and non-viral methods. The viral approach mainly involves the use of retroviral or lentiviral vectors to express the CAR gene, the introduction of the CAR gene into immune effector cells by packaged viral particles, and integration into the cell genome by the retroviral or lentiviral self-integrating system. The advantage of viral vector systems is that the viral particles are able to efficiently transduce immune effector cells, such as T cells, and to efficiently and stably integrate into the host cell genome. However, the preparation and production process of the virus is high in cost, time-consuming and labor-consuming, and in order to meet clinical safety standards, immune effector cells modified by a virus system need to show no virus replication, low genotoxicity and low immunogenicity, and long-term monitoring is needed after the virus is returned to a human body, so that certain potential safety hazards exist.

Electrotransformation (or electroporation) is already a well established method in some areas of medicine, but its use in biotechnology has only recently emerged. By applying a certain high electric field pulse to the cell or tissue instantaneously, permeability is formed on the surface of the cell membrane instantaneously, and charged molecules enter the cell. Classical electroporation will lead to enhanced transport of cells across membranes and altered conductivity. The effect of this process on the cell membrane is related to the intensity, repetition, duration and number of electrical pulses of the electrical transduction. Artificial bilayers, cells or tissues, by their very nature, are already accessible by several common electrotransfer procedures. In electroporation-based transgenic procedures, exogenous DNA is introduced intracellularly by reversible electroporation, and the exogenous gene is expressed in its new host cell and inherited as the cell divides. The use of the method of electric transfer in combination with a non-viral gene modification system capable of inducing stable expression of transgenes, such as the transposon system, is another effective method for modifying immune effector cells in addition to viral vector systems. The transposable subsystem, such as Sleeping Beuty or PiggyBac transposable system, comprises a transposase coding sequence, wherein the coded transposase recognizes repetitive sequences on two sides and can efficiently mediate and integrate into a host cell genome. Transposon systems have enjoyed widespread therapeutic application in combination with electrotransfer and have achieved clinical-level requirements (Kebrieii P, Huls H, Jena B, Munsell M, Jackson R, et al (2012) Infusing CD19-Directed T Cells to automatic Disease Control in Patients Underying automated genetic Stem-Cell transformation for Advanced B-Cell amplification strategies human Gene Therapy 23: 444-450), which have higher efficiency in T Cells. ACT using a transposon system relies on electrotransformation of T cells and Tumor Infiltrating Lymphocytes (TIL), for which there are currently well established commercial electrotransformation machines and buffers (e.g., Lonza Nuclear organisms), recently there are also reports of methods for successfully constructing CAR-T cells using SB transpose systems via commercial electrotransformation systems (Jin Z, Maiti S, Huls H, Singh H, Olivares S, et al (2011) The genetic modification of T cells to expression cells, Gene Therapy 18: 849;. PePD, Cohen S, Yang S, C, J. Pat. No. 4. et al.: Japanese cell culture 100X) genetic engineering et al.: Japanese cell culture medium, Japanese cell culture 16), and the ACT method using the SB transposon system has been used in clinical trials (Kebriaiei P, Huls H, Jena B, Munsell M, Jackson R, et al (2012) Infusng CD19-Directed T Cells to automatic Disease Control in Patients Underg Autologous animal Stem-Transplantation for Advanced B-lysine Maliganisics. human Gene Therapy 23: 444-450).

Compared with a viral vector system, the electrotransport and transposon system has the advantages of simple operation, low immunogenicity and genotoxicity and low safety risk, and is an important method for ACT. But the problems existing in the method are also obvious: excessive cell death is easily caused at a transient high voltage, and transfection efficiency is low, particularly in relation to the cell type and the electrotransfer conditions (including voltage, waveform, pulse time and electrotransfer buffer composition). Particularly, the difficulty of electrotransformation of immune effector cells, such as PBMC and TIL cells, is relatively higher, and although mature electrotransformation instruments and matched buffer systems are already available in the market at present, the problem of high mortality rate of the immune effector cells after electrotransformation is still more prominent. There is therefore still a need for a method of increasing the survival rate of immune effector cells after electrotransformation.

Disclosure of Invention

In a first aspect, the present invention provides the use of an inhibitor of the cGAS-STING signaling pathway in the culture of electrotransport cells.

In a second aspect, the present invention provides a method for culturing an electrotransferred cell, the method comprising the step of culturing the electrotransferred cell in a medium comprising a cGAS-STING signaling pathway inhibitor.

In one or more embodiments, the cGAS-STING signaling pathway inhibitor includes proteins, nucleic acids, and small molecule compounds capable of inhibiting the cGAS-STING signaling pathway.

In one or more embodiments, the proteins capable of inhibiting the cGAS-STING signaling pathway include proteins that inhibit STING expression and/or binding activity or that reduce or eliminate mitochondrial membrane potential, including but not limited to ULK1, ULK2, caspase3, caspase7, and caspase 9.

In one or more embodiments, the nucleic acid capable of inhibiting the cGAS-STING signaling pathway comprises an siRNA, antisense RNA, ribozyme, gene editing vector such as CRISPR-CAS9 gene editing vector or TALEN gene editing vector that targets the cGAS-STING signaling pathway or reduces or eliminates mitochondrial membrane potential.

In one or more embodiments, the small molecule compounds capable of inhibiting the cGAS-STING signaling pathway include small molecule compounds capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential, and small molecule compounds capable of inhibiting or antagonizing other member molecules than STING in the cGAS-STING signaling pathway.

In one or more embodiments, the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential comprises a carbonylcyano 3-chlorophenylhydrazone (CCCP) of formula 1, a compound of formula 2 coupled with NO₂The linoleic acid is a Compound C-176 with a structural formula shown as a formula 3, a Compound C-178 with a structural formula shown as a formula 4, a Compound H-151 with a structural formula shown as a formula 5, a Compound INHIB1X with a structural formula shown as a formula 6, a Compound INHIB2 with a structural formula shown as a formula 7, a Compound INHIB9 with a structural formula shown as a formula 8, a Compound Compound18 with a structural formula 9, a Compound cyclopeptide astin C with a structural formula 10, a Compound Screening Hit 1 with a structural formula shown as a formula 11, a Compound Compound13 with a structural formula 12, a Compound C-170 with a structural formula 13 and a Compound C-171 with a structural formula 14.

In one or more embodiments, the small molecule compounds that are capable of inhibiting or antagonizing molecules that are members of the cGAS-STING signaling pathway other than STING include small molecule compounds that are capable of inhibiting or antagonizing cyclic GMP-AMP synthase (cGAS). Preferably, the compound comprises a compound quinacrine (quinacrine) with a structural formula shown as a formula 15, a compound hydroxychloroquine (hydroxychloroquine) with a structural formula shown as a formula 16, a compound X6 with a structural formula shown as a formula 17, a compound PF-06928215 with a structural formula shown as a formula 18, a compound RU.365 with a structural formula shown as a formula 19, a compound RU.521 with a structural formula shown as a formula 20, a compound suramin (suramin) with a structural formula 21, a compound G150 with a structural formula shown as a formula 22 and a compound VIII with a structural formula shown as a formula 23.

In one or more embodiments, the small molecule compounds that inhibit or antagonize molecules that are members of the cGAS-STING signaling pathway other than STING include small molecule compounds that inhibit or antagonize TANK-binding kinase 1 (TBK 1). Preferably, the Compound comprises a Compound BX795 with a structural formula shown as a formula 24, a Compound Tozasertib with a structural formula shown as a formula 25, a Compound Tozasertib-15a with a structural formula shown as a formula 26, a Compound 20b with a structural formula shown as a formula 27, a Compound 4-azabenzimidazole hit 1a (azabenzimidazole hit 1a) with a structural formula shown as a formula 28, a Compound CYT387 with a structural formula 29, a Compound Domainex with a structural formula shown as a formula 30, a Compound Amgen Compound II with a structural formula 31, a Compound MRT67307 with a structural formula 32 and a Compound AZ13102909 with a structural formula 33.

In one or more embodiments, the use is of the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential in culturing an electroporated immune effector cell. Preferably, the application of any one or more small molecule compounds in the formulas 1-14 in culturing the electro-transferred immune effector cells.

In one or more embodiments, the use is of the small molecule compound capable of inhibiting or antagonizing molecules of other members of the cGAS-STING signaling pathway other than STING in culturing transduced immune effector cells.

In one or more embodiments, the use is of the small molecule compound capable of inhibiting or antagonizing cyclic GMP-AMP synthase (cGAS) in culturing transduced immune effector cells. Preferably, the use of a small molecule compound of any one or more of formulae 15-23 in the culture of an electroporated immune effector cell.

In one or more embodiments, the use is of the small molecule compound capable of inhibiting or antagonizing TANK-binding kinase 1 (TBK 1) in culturing an electroporated immune effector cell. Preferably, the use of any one or more of the small molecule compounds of formulae 24-33 in the culture of transduced immune effector cells.

In one or more embodiments, the use is of the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential in combination with the small molecule compound capable of inhibiting or antagonizing cGAS in the culture of an electroporated immune effector cell.

In one or more embodiments, the use is of any one or more small molecule compounds selected from formulas 1-14 in combination with any one or more small molecule compounds selected from formulas 15-23 in culturing an electroporated immune effector cell. Preferably, the application is the application of the small molecule compound selected from any one of formulas 1 to 14 and the small molecule compound selected from any one of formulas 15 to 23 in the culture of the electro-transferred immune effector cells.

In one or more embodiments, the use is of the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential in combination with the small molecule compound capable of inhibiting or antagonizing TBK1 in culturing an electroporated immune effector cell.

In one or more embodiments, the use is of any one or more small molecule compounds selected from formulas 1-14 in combination with any one or more small molecule compounds selected from formulas 24-33 in culturing an electroporated immune effector cell. Preferably, the application is the application of the small molecule compound selected from any one of formulas 1 to 14 and the small molecule compound selected from any one of formulas 24 to 33 in the culture of the electro-transferred immune effector cells.

In one or more embodiments, the use is of the small molecule compound capable of inhibiting or antagonizing cGAS in combination with the small molecule compound capable of inhibiting or antagonizing TBK1 in culturing transduced immune effector cells.

In one or more embodiments, the use is of any one or more small molecule compounds selected from formulas 15-23 in combination with any one or more small molecule compounds selected from formulas 24-33 in culturing an electroporated immune effector cell. Preferably, the application is the application of the small molecule compound selected from any one of formulas 15 to 23 and the small molecule compound selected from any one of formulas 24 to 33 in the culture of the electro-transferred immune effector cells.

In one or more embodiments, the use is of the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential, the small molecule compound capable of inhibiting or antagonizing cGAS, and the small molecule compound capable of inhibiting or antagonizing TBK1 together in culturing an electroporated immune effector cell.

In one or more embodiments, the use is of any one or more small molecule compounds selected from formulas 1-14, any one or more small molecule compounds selected from formulas 15-23, and any one or more small molecule compounds selected from formulas 24-33 in the culture of an electroporated immune effector cell. Preferably, the application is the application of the small molecule compound selected from any one of formulas 1 to 14, the small molecule compound selected from any one of formulas 15 to 23 and the small molecule compound selected from any one of formulas 24 to 33 in the culture of the electro-transferred immune effector cells.

In one or more embodiments, the method is a method of culturing an electroporated immune effector cell, the method comprising the step of culturing the electroporated immune effector cell in a medium comprising the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential.

In one or more embodiments, the method comprises the step of culturing the transduced immune effector cells in a medium containing any one or more small molecule compounds selected from formulas 1-14.

In one or more embodiments, the method is a method of culturing a transduced immune effector cell, comprising the step of culturing the transduced immune effector cell in a medium containing the small molecule compound capable of inhibiting or antagonizing cGAS.

In one or more embodiments, the method comprises the step of culturing the transduced immune effector cells in a medium comprising a small molecule compound selected from any one or more of formulas 15-23.

In one or more embodiments, the method is a method of culturing an electroporated immune effector cell, comprising the step of culturing the electroporated immune effector cell in a medium comprising the small molecule compound capable of inhibiting or antagonizing TBK 1.

In one or more embodiments, the method comprises the step of culturing the electroporated immune effector cells in a medium comprising any one or more small molecule compounds selected from formulas 24-33.

In one or more embodiments, the method comprises the step of culturing the electroporated immune effector cells in a culture medium containing the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential and the small molecule compound capable of inhibiting or antagonizing cGAS.

In one or more embodiments, the method comprises the step of culturing the electroporated immune effector cells in a medium comprising any one or more small molecule compounds selected from formulas 1-14 and any one or more small molecule compounds selected from formulas 15-23.

In one or more embodiments, the method comprises the step of culturing the electroporated immune effector cells in a medium comprising the small molecule compound capable of inhibiting or antagonizing cGAS and the small molecule compound capable of inhibiting or antagonizing TBK 1.

In one or more embodiments, the method comprises the step of culturing the electroporated immune effector cells in a medium comprising any one or more small molecule compounds selected from the group consisting of formulas 15-23 and any one or more small molecule compounds selected from the group consisting of formulas 24-33.

In one or more embodiments, the method comprises the step of culturing the electroporated immune effector cells in a medium comprising the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential and the small molecule compound capable of inhibiting or antagonizing TBK 1.

In one or more embodiments, the method comprises the step of culturing the electroporated immune effector cells in a medium comprising any one or more small molecule compounds selected from formulas 1-14 and any one or more small molecule compounds selected from formulas 24-33.

In one or more embodiments, the method comprises the step of culturing the electroporated immune effector cells in a culture medium containing the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential, the small molecule compound capable of inhibiting or antagonizing cGAS, and the small molecule compound capable of inhibiting or antagonizing TBK 1.

In one or more embodiments, the method comprises the step of culturing the electroporated immune effector cells in a medium comprising any one or more small molecule compounds selected from formulas 1-14, any one or more small molecule compounds selected from formulas 15-23, and any one or more small molecule compounds selected from formulas 24-33.

In one or more embodiments, the method comprises the step of culturing the transfected immune effector cell in a medium comprising a small molecule compound selected from any one of formulas 1-14, a small molecule compound selected from any one of formulas 15-23, and a small molecule compound selected from any one of formulas 24-33.

In one or more embodiments, the method or use comprises, after the electroporation is completed, transferring the electroporated cells into a culture medium for 0.5 to 8 hours, adding the cGAS-STING signaling pathway inhibitor to the culture medium, and continuing the culture.

In one or more embodiments, the cell is an immune effector cell.

In one or more embodiments, the immune effector cell is selected from one or more of a T cell, a TIL cell, an NK T cell, a CAR-T cell, a CIK cell, a TCR-T cell, and a macrophage.

In one or more embodiments, the immune effector cell is selected from one or more of a T cell, a TIL cell, and a CAR-T cell.

In one or more embodiments, the medium is a medium for culturing immune effector cells.

In one or more embodiments, the medium is a medium of T cells, TIL cells, or CAR-T cells.

In one or more embodiments, the medium is selected fromCTS^TMAny one of serum-free cell culture medium, DMEM medium, and RPMI1640 medium; preferably, isCTS^TMSerum-free cell culture media.

In one or more embodiments, the final concentration of the cGAS-STING signaling pathway inhibitor in the medium is in the range of 0.02-100 μ M, preferably in the range of 0.02-80 μ M, more preferably in the range of 0.5-5 μ M, and even more preferably in the range of 0.5-2.5 μ M.

In one or more embodiments, the cGAS-STING signaling pathway inhibitor in the culture medium comprises the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential at a final concentration in the range of 0.5-5 μ M, preferably 0.5-1 μ M.

In one or more embodiments, the cGAS-STING signaling pathway inhibitor in the culture medium comprises the small molecule compound capable of inhibiting or antagonizing cGAS at a final concentration in the range of 0.5-5 μ M, preferably 2.5-5 μ M.

In one or more embodiments, the cGAS-STING signaling pathway inhibitor in the culture medium comprises the small molecule compound capable of inhibiting or antagonizing TBK1 at a final concentration in the range of 0.02-10 μ M, preferably 0.02-1 μ M.

In one or more embodiments, the cGAS-STING signaling pathway inhibitor in the culture medium includes the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential and the small molecule compound capable of inhibiting or antagonizing cGAS at a final concentration in the range of 0.02-10 μ M, preferably 0.02-5 μ M.

In one or more embodiments, the cGAS-STING signaling pathway inhibitor in the culture medium includes the small molecule compound capable of inhibiting or antagonizing cGAS and the small molecule compound capable of inhibiting or antagonizing TBK1 at a final concentration in the range of 0.02-10 μ M, preferably 0.02-5 μ M.

In one or more embodiments, the cGAS-STING signaling pathway inhibitor in the culture medium includes the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential and the small molecule compound capable of inhibiting or antagonizing TBK1 at a final concentration in the range of 0.02-10 μ M, preferably 0.02-5 μ M.

In one or more embodiments, the cGAS-STING signaling pathway inhibitor in the culture medium includes the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential, the small molecule compound capable of inhibiting or antagonizing cGAS, and the small molecule compound capable of inhibiting or antagonizing TBK1 at a final concentration in the range of 0.02-20 μ M, preferably 0.02-5 μ M.

The present invention also provides a method for preparing a cell overexpressing a foreign gene by electroporation, the method comprising:

1) introducing a nucleic acid containing an expressed foreign gene into a cell by electrotransfer;

2) culturing the cells transfected with the foreign gene in step 1) with a medium containing a cGAS-STING signaling pathway inhibitor;

wherein the electroporated cells are cultured for 0-8h, preferably 0-5h, more preferably 0-3h, more preferably 0-1h, and then the cGAS-STING signaling pathway inhibitor is added to the culture medium; preferably, the final concentration of the cGAS-STING signaling pathway inhibitor in the culture medium after addition is in the range of 0.02-100. mu.M, preferably 0.02-80. mu.M; more preferably, in the range of 0.5-5. mu.M; even more preferably, in the range of 0.5-1. mu.M or in the range of 2.5-5. mu.M.

Preferably, the cGAS-STING signaling pathway inhibitor comprises any one or more of a small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential, a small molecule compound capable of inhibiting or antagonizing cGAS, and a small molecule compound capable of inhibiting or antagonizing TBK 1; preferably, the cell is an immune effector cell.

In one or more embodiments, the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential comprises any one or more small molecule compounds selected from formulas 1-14.

In one or more embodiments, the small molecule compound capable of inhibiting or antagonizing cGAS comprises any one or more small molecule compounds selected from formulas 15-23.

In one or more embodiments, the small molecule compound capable of inhibiting or antagonizing TBK1 comprises any one or more small molecule compounds selected from formulas 24-33.

The invention also provides a cell culture medium containing the cGAS-STING signaling pathway inhibitor.

In one or more embodiments, the cell culture medium is a medium for culturing immune effector cells.

In one or more embodiments, the medium used to culture immune effector cells is a medium of T cells, TIL cells, or CAR-T cells.

In one or more embodiments, the medium used to culture the immune effector cells is selected fromCTS^TMAny one of serum-free cell culture medium, DMEM medium, and RPMI1640 medium; preferably, isCTS^TMSerum-free cell culture media.

In one or more embodiments, the cGAS-STING signaling pathway inhibitor includes any one or more of a small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential, a small molecule compound capable of inhibiting or antagonizing cGAS, and a small molecule compound capable of inhibiting or antagonizing TBK 1.

In one or more embodiments, the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential comprises any one or more small molecule compounds selected from formulas 1-14.

In one or more embodiments, the small molecule compound capable of inhibiting or antagonizing cGAS comprises any one or more small molecule compounds selected from formulas 15-23.

In one or more embodiments, the small molecule compound capable of inhibiting or antagonizing TBK1 comprises any one or more small molecule compounds selected from formulas 24-33.

Drawings

FIG. 1: adding TIL cells of G150 0h after the pNB328-EGFP plasmid is electrically transferred, and culturing for 5 days, and then drawing a fluorescence microscope bright field and a fluorescence visual field;

FIG. 2: adding TIL cells 0h, 1h and 5h after the pNB328-EGFP plasmid is electrically transferred into G150-2.5 mu M, and analyzing the result of the flow cytometry of EGFP positive cells after 5 days of culture;

FIG. 3: viable cell number after 5 days of culture in the treatment groups with addition of G150 to 2.5 mu M and the control group without addition of G150 after 0h, 1h and 5h of transferring the pNB328-EGFP plasmid by TIL;

FIG. 4: the proportion of living cells after adding G150 to 2.5 mu M of treatment groups and control groups without adding G150 to culture for 5 days after TIL electric transfer of pNB328-EGFP plasmid for 0h, 1h and 5 h;

FIG. 5: the EGFP positive cell proportion after adding G150 to 2.5 mu M treatment groups and control groups without adding G150 to culture for 5 days after TIL electric transfer of pNB328-EGFP plasmid for 0h, 1h and 5 h;

FIG. 6: viable cell number after 5 days of culture in the treatment groups with addition of G150 to 5 mu M and the control groups without addition of G150 after the activated T cells are electrically transferred with pNB328-EGFP plasmid for 0h, 1h and 5 h;

FIG. 7: the proportion of the living cells after the activated T cells are electrically transferred with the pNB328-EGFP plasmid for 0h, 1h and 5h, and after the G150-5 mu M treatment group and the control group without the G150 are added for culturing for 5 days;

FIG. 8: the EGFP positive cell proportion after the activated T cells are electrically transferred with pNB328-EGFP plasmids is 0h, 1h and 5h, and the EGFP positive cell proportion is obtained after the G150-5 mu M treatment group and the control group without the G150 are added and cultured for 5 days;

FIG. 9: viable cell number after 5 days of culture in the treatment group with H-151 to 0.5 mu M and the control group without H-151 after 0H, 1H and 5H of transferring the pNB328-EGFP plasmid by TIL;

FIG. 10: the proportion of living cells after the treatment group with H-151 to 0.5 mu M and the control group without H-151 are added for 0H, 1H and 5H after the pNB328-EGFP plasmid is transformed by TIL;

FIG. 11: the EGFP positive cell proportion after adding H-151 to 0.5 mu M treatment groups and control groups without adding H-151 to culture for 5 days after TIL electric transfer of pNB328-EGFP plasmid for 0H, 1H and 5H;

FIG. 12: the number of living cells after the H-151 to 1 mu M treatment group and the H-151-free control group are added for 0H, 1H and 5H after the pNB328-EGFP plasmid is transformed by the activated T electricity after 5 days of culture;

FIG. 13: the proportion of living cells after the addition of H-151 to 1 mu M of treatment groups and the addition of H-151 control groups for 5 days after the activation of T electric transfer pNB328-EGFP plasmid for 0H, 1H and 5H;

FIG. 14: the EGFP positive cell proportion after adding H-151 to 1 mu M treatment groups and control groups without adding H-151 after activating T electric transfer pNB328-EGFP plasmids for 0H, 1H and 5H;

FIG. 15: viable cell number after the TIL cells are electrically transferred to the pNB328-EGFP plasmid for 0h, 1h and 5h, and after the IRAK-IN-4 is added into the treated group of 2 mu M and the control group without adding the IRAK-IN-4 is cultured for 5 days;

FIG. 16: the proportion of living cells after the cells are cultured for 5 days by adding IRAK-IN-4 to 2 mu M treatment groups and control groups without adding IRAK-IN-4 after the TIL cells are electrically transferred to the pNB328-EGFP plasmid for 0h, 1h and 5 h;

FIG. 17: the proportion of EGFP positive cells after adding IRAK-IN-4 to 2 mu M treatment groups and control groups without adding IRAK-IN-4 to culture for 5 days after the TIL cells are electrically transferred to the pNB328-EGFP plasmid for 0h, 1h and 5 h;

FIG. 18: viable cell number after 5 days of culture in the treatment groups with addition of C-170 to 2 mu M and the control groups without addition of C-170 after the activated T cells are electrically transferred to the pNB328-EGFP plasmid at 0h, 1h and 5 h;

FIG. 19: the proportion of the living cells after the activated T cells are electrically transferred with pNB328-EGFP plasmids is 0h, 1h and 5h, and the living cells are cultured for 5 days after C-170 to 2 mu M treatment groups and control groups without C-170 are added;

FIG. 20: the EGFP positive cell proportion after the addition of C-170 to 2 mu M of the treatment group and the control group without the addition of C-170 after the electric transfer of pNB328-EGFP plasmid to activated T cells for 0h, 1h and 5 h.

Detailed Description

It is to be understood that within the scope of the present invention, the above-described technical features of the present invention and the technical features described in detail below (e.g., the embodiments) may be combined with each other to constitute a preferred embodiment.

Interferon activating protein (STING) is an important molecule in innate immune response and plays an important role in defense against viral and intracellular bacterial infections and in mediating the production of type I interferons. STING, an important component of the signal transduction cascade, can exert immune defense effects in the case of infection with pathogens (viruses, bacteria, parasites, etc.) as well as anti-tumor immune responses in the case of tumor development. When the cGAS-STING signaling pathway is over-activated, it may also cause a range of autoimmune diseases.

The invention discovers that the survival rate of cells after electrotransfer can be obviously improved by adding a cGAS-STING signal pathway inhibitor for culturing after the cells, particularly immune effector cells, are electrically transformed and transferred into a culture medium for a period of time, thereby completing the invention.

Herein, the cell may be any cell of interest, particularly cells conventionally used in the art for electroporation to express foreign genes, and may be eukaryotic cells (e.g., animal cells and plant cells) and prokaryotic cells (e.g., E.coli and other bacteria, etc.). For example, the cell can be a human cell. Examples of cells include, but are not limited to, HEK293 cells, MDCK cells, Hela cells, and the like. In certain embodiments, the cell is an immune cell. Herein, immune effector cells refer to immune cells involved in the clearance of foreign antigens and the functioning of effectors in an immune response, including but not limited to one or more of T cells, TIL cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages. Preferably, in certain embodiments, the immune effector cells suitable for use in the methods of the invention are selected from one or more of T cells, TILs and CAR-T cells.

Herein, the cGAS-STING signaling pathway inhibitor can be any of various agents known in the art that inhibit the cGAS-STING signaling pathway, including but not limited to proteins, nucleic acids, and small molecule compounds that inhibit the cGAS-STING signaling pathway. For example, proteins capable of inhibiting the cGAS-STING signaling pathway include proteins that inhibit STING expression and/or binding activity or that reduce or eliminate mitochondrial membrane potential, including but not limited to ULK1, ULK2, caspase3, caspase7, and caspase 9. Nucleic acids capable of inhibiting the cGAS-STING signaling pathway include, but are not limited to, sirnas, antisense RNAs, ribozymes, and gene editing vectors that target the cGAS-STING signaling pathway or reduce or eliminate mitochondrial membrane potential, such as CRIPR-CAS9 gene editing vectors or TALEN gene editing vectors. Small molecule compounds capable of inhibiting the cGAS-STING signaling pathway include, but are not limited to, small molecule compounds capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential, small molecule compounds capable of inhibiting or antagonizing cGAS, and small molecule compounds capable of inhibiting or antagonizing TBK 1. The small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential includes, but is not limited to, the small molecule compounds in formulas 1-14. The small molecule compounds capable of inhibiting or antagonizing cGAS include, but are not limited to, the small molecule compounds in formulas 15-23. The small molecule compound capable of inhibiting or antagonizing TBK1 includes, but is not limited to, the small molecule compounds in formulas 24-33.

In certain embodiments, the invention relates to culturing the electroporated cells using a small molecule compound capable of inhibiting STING expression and/or binding activity or a small molecule compound that reduces or eliminates mitochondrial membrane potential, e.g., using any one or more of the small molecule compounds of formulas 1-14.

In certain embodiments, the electrotransfer is cultured using a compound of formula 1 CCCP (carbonylcyano 3-chlorophenylhydrazone). In certain embodiments, the transfected cells are cultured using compound H-151 as shown in formula 5. In certain embodiments, the electroporated cells are cultured using the small molecule Compound18 as shown in formula 9. In certain embodiments, the electroporated cells are cultured using Compound13 as shown in formula 12. In certain embodiments, the electroporated cells are cultured using a compound of formula 1 CCCP (carbonylcyano 3-chlorophenylhydrazone) and a compound of formula 5H-151. In certain embodiments, the electroporated cells are cultured using a combination of compound H-151, as shown in formula 5, and small molecule compound18, as shown in formula 9. In certain embodiments, the electroporated cells are cultured using Compound H-151, as shown in formula 5, and Compound13, as shown in formula 12.

In certain embodiments, the invention relates to the use of small molecule compounds capable of inhibiting or antagonizing cGAS for culturing transduced cells, e.g., using any one or more of the small molecule compounds of formulas 15-23.

In certain embodiments, the electroporated cells are cultured using the compound quinacrine, as shown in formula 15. In certain embodiments, the transfected cells are cultured using hydroxychloroquine, a compound as shown in formula 16. In certain embodiments, the compound ru.365 as shown in formula 19 is used to culture the transfected cells. In certain embodiments, the transfected cells are cultured using the compound ru.521, as shown in formula 20. In certain embodiments, the electroporated cells are cultured using a compound quinacrine, as shown in formula 15, in combination with hydroxychloroquine, as shown in formula 16. In certain embodiments, the compound quinacrine, as shown in formula 15, is used in combination with the compound RU.365, as shown in formula 19, to culture the electroporated cells. In certain embodiments, the compound quinacrine, as shown in formula 15, is used in combination with the compound ru.521, as shown in formula 20, to culture the electroporated cells.

In certain embodiments, the invention relates to the use of small molecule compounds capable of inhibiting or antagonizing TBK1 for culturing transduced cells, e.g., using any one or more of the small molecule compounds of formulas 24-33.

In certain embodiments, the transfected cells are cultured using a compound BX795 as shown in formula 24. In certain embodiments, the transfected cells are cultured using Tozasertib, a compound shown in formula 25. In certain embodiments, the transfected cells are cultured using compound 20b as shown in formula 27. In certain embodiments, the transfected cells are cultured using compound CYT387, as shown in formula 29. In certain embodiments, the transfected cells are cultured using a compound of formula 24 BX795 in combination with a compound of formula 25 Tozasertib. In certain embodiments, the electroporated cells are cultured using a compound of formula 24 BX795 in combination with a compound of formula 27 BX 20 b. In certain embodiments, the transfected cells are cultured using compound 20b, as shown in formula 27, in combination with compound CYT387, as shown in formula 29.

In certain embodiments, the invention relates to culturing the electroporated cells using a small molecule compound capable of inhibiting STING expression and/or binding activity or a small molecule compound that reduces or eliminates mitochondrial membrane potential in combination with the small molecule compound capable of inhibiting or antagonizing cGAS, e.g., using any one of the small molecule compounds of formulas 1-14 in combination with any one of the small molecule compounds of formulas 15-23.

In certain embodiments, the electroporated cells are co-cultured using a compound of formula 1 CCCP (carbonylcyano 3-chlorophenylhydrazone) and a compound of formula 15 quinacrine. In certain embodiments, the electroporated cells are co-cultured using a compound of formula 1 CCCP (carbonylcyano 3-chlorophenylhydrazone) and a compound of formula 15 quinacrine. In certain embodiments, the electroporated cells are co-cultured using a compound of formula 5, H-151, and a compound of formula 15, quinacrine. In certain embodiments, the electroporated cells are co-cultured using the small molecule Compound18, as shown in formula 9, and the Compound quinacrine, as shown in formula 15. In certain embodiments, the electroporated cells are co-cultured using the small molecule Compound13, as shown in formula 12, and the Compound quinacrine, as shown in formula 15.

In certain embodiments, the electroporated cells are co-cultured using a compound of formula 1 CCCP (carbonylcyano 3-chlorophenylhydrazone) and a compound of formula 16 hydroxychloroquine. In certain embodiments, the electroporated cells are co-cultured using a compound of formula 5, H-151, and a compound of formula 16, hydroxychloroquine. In certain embodiments, the electroporated cells are co-cultured using the small molecule Compound18, as shown in formula 9, and the hydroxychloroquine Compound, as shown in formula 16. In certain embodiments, the electroporated cells are co-cultured using the small molecule Compound13, as shown in formula 12, and the hydroxychloroquine Compound, as shown in formula 16.

In certain embodiments, the electroporated cells are co-cultured using the compound CCCP (carbonylcyano 3-chlorophenylhydrazone) as shown in formula 1 and the compound RU.365 as shown in formula 19. In certain embodiments, the electroporated cells are co-cultured using compound H-151, as shown in formula 5, and compound RU.365, as shown in formula 19. In certain embodiments, the electroporated cells are co-cultured using the small molecule Compound18, as shown in formula 9, and the Compound ru.365, as shown in formula 19. In certain embodiments, the electroporated cells are co-cultured using the small molecule Compound13 as shown in formula 12 and the Compound ru.365 as shown in formula 19.

In certain embodiments, the electroporated cells are co-cultured using the compound CCCP (carbonylcyano 3-chlorophenylhydrazone) as shown in formula 1 and the compound RU.521 as shown in formula 20. In certain embodiments, the electroporated cells are co-cultured using compound H-151, as shown in formula 5, and compound RU.521, as shown in formula 20. In certain embodiments, the electroporated cells are co-cultured using the small molecule Compound18, as shown in formula 9, and the Compound ru.521, as shown in formula 20. In certain embodiments, the electroporated cells are co-cultured using the small molecule Compound13 as shown in formula 12 and the Compound ru.521 as shown in formula 20.

In certain embodiments, the invention relates to culturing the electroporated cells using a small molecule compound capable of inhibiting STING expression and/or binding activity or a small molecule compound that reduces or eliminates mitochondrial membrane potential in combination with a small molecule compound capable of inhibiting or antagonizing TBK1, e.g., using any one of the small molecule compounds of formulas 1-14 in combination with any one of the small molecule compounds of formulas 24-33.

In certain embodiments, the electroporated cells are co-cultured using a compound of formula 1 CCCP (carbonylcyano 3-chlorophenylhydrazone) and a compound of formula 24 BX 795. In certain embodiments, the transfected cells are co-cultured with a compound of formula 5, H-151, and a compound of formula 24, BX 795. In certain embodiments, the electroporated cells are co-cultured using the small molecule Compound18, as shown in formula 9, and the Compound BX795, as shown in formula 24. In certain embodiments, the electroporated cells are co-cultured using the small molecule Compound13, as shown in formula 12, and the Compound BX795, as shown in formula 24.

In certain embodiments, the electroporated cells are co-cultured using a compound of formula 1 CCCP (carbonylcyano 3-chlorophenylhydrazone) and a compound of formula 25 Tozasertib. In certain embodiments, the transfected cells are co-cultured with a compound of formula 5, H-151, and Tozasertib, as shown in formula 25. In certain embodiments, the electroporated cells are co-cultured using the small molecule Compound18, as shown in formula 9, and the Compound Tozasertib, as shown in formula 25. In certain embodiments, the electroporated cells are co-cultured using the small molecule Compound13, as shown in formula 12, and the Compound Tozasertib, as shown in formula 25.

In certain embodiments, the electroporated cells are co-cultured using a compound of formula 1 CCCP (carbonylcyano 3-chlorophenylhydrazone) and a compound of formula 27 b. In certain embodiments, the electroporated cells are co-cultured using compound H-151, shown in formula 5, and compound 20b, shown in formula 27. In certain embodiments, the electroporated cells are co-cultured using the small molecule Compound18 as shown in formula 9 and the Compound 20b as shown in formula 27. In certain embodiments, the electroporated cells are co-cultured using the small molecule Compound13 as shown in formula 12 and the Compound 20b as shown in formula 27.

In certain embodiments, the electroporated cells are co-cultured using a compound of formula 1 CCCP (carbonylcyano 3-chlorophenylhydrazone) and a compound of formula 29 CYT 387. In certain embodiments, the transfected cells are co-cultured with a compound of formula 5, H-151, and a compound of formula 29, CYT 387. In certain embodiments, the transfected cells are co-cultured using the small molecule Compound18, as shown in formula 9, and the Compound CYT387, as shown in formula 29. In certain embodiments, the transfected cells are co-cultured using the small molecule Compound13, as shown in formula 12, and the Compound CYT387, as shown in formula 29.

In certain embodiments, the invention relates to culturing the electroporated cells using a small molecule compound capable of inhibiting or antagonizing cGAS in combination with a small molecule compound capable of inhibiting or antagonizing TBK1, e.g., using any of the small molecule compounds of formulas 15-23 in combination with any of the small molecule compounds of formulas 24-33.

In certain embodiments, the transfected cells are co-cultured with a compound of quinacrine, as shown in formula 15, and a compound of BX795, as shown in formula 24. In certain embodiments, the electroporated cells are co-cultured using hydroxychloroquine, a compound represented by formula 16, and BX795, a compound represented by formula 24. In certain embodiments, the transfected cells are co-cultured with compound ru.365, as shown in formula 19, and compound BX795, as shown in formula 24. In certain embodiments, the transfected cells are co-cultured with compound ru.521, shown in formula 20, and compound BX795, shown in formula 24.

In certain embodiments, the transfected cells are co-cultured with a compound quinacrine, as shown in formula 15, and a compound Tozasertib, as shown in formula 25. In certain embodiments, the electroporated cells are co-cultured using hydroxychloroquine, a compound shown as formula 16, and Tozasertib, a compound shown as formula 25. In certain embodiments, the transfected cells are co-cultured with a compound of formula 19 ru.365 and a compound of formula 25 Tozasertib. In certain embodiments, the transfected cells are co-cultured with a compound of formula 20 ru.521 and a compound of formula 25 Tozasertib.

In certain embodiments, the electroporated cells are co-cultured using a compound quinacrine, as shown in formula 15, and a compound 20b, as shown in formula 27. In certain embodiments, the electroporated cells are co-cultured with hydroxychloroquine, a compound represented by formula 16, and 20b, a compound represented by formula 27. In certain embodiments, the transfected cells are co-cultured with compound ru.365, as shown in formula 19, and compound 20b, as shown in formula 27. In certain embodiments, the transfected cells are co-cultured with compound ru.521, as shown in formula 20, and compound 20b, as shown in formula 27.

In certain embodiments, the transfected cells are co-cultured with a compound quinacrine, as shown in formula 15, and a compound CYT387, as shown in formula 29. In certain embodiments, the transfected cells are co-cultured with hydroxychloroquine, a compound represented by formula 16, and CYT387, a compound represented by formula 29. In certain embodiments, the transfected cells are co-cultured with compound ru.365, as shown in formula 19, and compound CYT387, as shown in formula 29. In certain embodiments, the transfected cells are co-cultured with compound ru.521, as shown in formula 20, and compound CYT387, as shown in formula 29.

In certain embodiments, the invention relates to the use of a small molecule compound capable of inhibiting STING expression and/or binding activity or a small molecule compound that reduces or eliminates mitochondrial membrane potential, a small molecule compound capable of inhibiting or antagonizing TBK1, and a small molecule compound capable of inhibiting or antagonizing cGAS together to culture an electroporated cell, for example, the electroporated cells are cultured using any one or more of the small molecule compounds of formulae 1 to 14, any one or more of the small molecule compounds of formulae 15 to 23, and any one or more of the small molecule compounds of formulae 24 to 33, preferably, the electroporated cells are cultured using any one of the small molecule compounds of formulae 1 to 14, any one of the small molecule compounds of formulae 15 to 23, and any one of the small molecule compounds of formulae 24 to 33.

Electroporation, also known as electroporation, is used to introduce the DNA of interest into the host cell. The invention can be practiced using various electrotransformation methods and agents well known in the art, e.g., LONZA can be usedI Device and electrotransformation reagent provided by it. The electrotransformation liquid can be prepared according to the instruction of a commercially available electrotransformation reagent, and electrotransformation plasmid is added. When the cells are electroporated, the cells are resuspended in an electroporation solution containing an electroporation plasmid, and electroporation is carried out in an electroporation apparatus.

The electrotransport plasmid can be any plasmid of interest. The amount of electrotransport plasmid may be an amount conventional in the art, for example, every 5X 10⁶Each cell was electroporated with 3-8. mu.g of plasmid.

After the electrotransfer is complete, the cell suspension is removed, preheated cell culture medium is added, and the mixture is incubated under conventional conditions (e.g., 37 ℃ C., 5% CO)₂) And (5) culturing. After at least 0.5 hour of culture, a cGAS-STING signaling pathway inhibitor is added to the cell culture medium containing the transfected cells. Addition of cGAS-STING signaling pathway inhibitors either too early or too late may not increase the total number and survival of the electrotransport cells. Therefore, it is preferable to add the cGAS-STING signaling pathway inhibitor to the culture medium within 8 hours, more preferably within 5 hours, and even more preferably within 3 hours of culturing the transfected cells. For example, in certain particularly preferred embodiments, the cGAS-STING signaling pathway inhibitor is added at 0.5-3 hours of culturing the electrotransfer cell, preferably at 45min to 2 hours of culturing, and more preferably at 1-2 hours of culturing.

In general, the final concentration of the cGAS-STING signaling pathway inhibitor in the medium after addition is in the range of 0.02-100. mu.M, preferably in the range of 0.02-80. mu.M, more preferably in the range of 0.5-5. mu.M; even more preferably, in the range of 0.5-1. mu.M or in the range of 2.5-5. mu.M.

Herein, the cell culture medium may be various media suitable for the culture of cells, particularly immune cells, particularly media conventionally used in the art for culturing various electroporated cells. In certain embodiments, the medium is a medium for culturing immune cells, including but not limited to a medium for culturing one or more of T cells, TIL cells, NK T cells, CAR-T cells, CIK cells, TCR-T cells, and macrophages. In certain embodiments, the culture medium is a culture medium of T cells, TILs and/or CAR-T cells, in particular a culture medium that cultures transduced T cells, TILs and/or CAR-T cells. Exemplary culture media include, but are not limited toCTS^TMAny one of serum-free cell culture medium, DMEM medium, and RPMI1640 medium; preferably, isCTS^TMSerum-free cell culture media.

After the electrotransfer cells are cultured according to a conventional culture process, compared with the conventional electrotransfer method in the field, the culture added with the cGAS-STING signaling pathway inhibitor can obviously improve the total number and/or the survival rate of the electrotransfer cells.

Therefore, the present invention provides a method for culturing an electrotransferred cell (particularly, an immune effector cell), which comprises adding a cGAS-STING signaling pathway inhibitor to a culture medium and continuing the culture, after the electrotransferred cell is transferred to the culture medium and cultured for 0 to 8 hours after the end of the electrotransferred cell. Preferably, the cGAS-STING signaling pathway inhibitor is added for culture when the culture medium is cultured for 0-5h, 0-3h, 0-1h or 1-2 h.

In certain embodiments, the present invention provides a method of electrotransformation of cells expressing a foreign gene (particularly immune effector cells), the method comprising:

1) introducing a nucleic acid containing an expressed foreign gene into a cell by electrotransfer;

2) culturing the cells transfected with the foreign gene in 1) with a culture medium containing a cGAS-STING signal pathway inhibitor;

wherein the cGAS-STING signaling pathway inhibitor is added after the electro-transformation is completed and the electro-transformed cells are cultured for 0.5-8h, preferably 0.5-5h, more preferably 0.5-3h, more preferably 0.5-2h, more preferably 1-2 h.

The invention also provides the use of an inhibitor of the cGAS-STING signaling pathway in the culture of transduced cells, particularly immune effector cells.

The invention also provides a cell culture medium containing the cGAS-STING signaling pathway inhibitor. Preferably, the cell culture medium is a medium for immune effector cells. More preferably, the cGAS-STING signaling pathway inhibitor is selected from one or more of a small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential, a small molecule compound capable of inhibiting or antagonizing cGAS, and a small molecule compound capable of inhibiting or antagonizing TBK 1.

Thus, in certain embodiments, the cell culture medium of the invention is a medium for culturing immune effector cells comprising the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential; preferably, the medium contains one or more small molecule compounds selected from any one of formulas 1-14.

In certain embodiments, the cell culture medium of the invention is a medium for culturing immune effector cells comprising the small molecule compound capable of inhibiting or antagonizing cGAS; preferably, the medium contains one or more small molecule compounds selected from any one of formulas 15-23.

In certain embodiments, the cell culture medium of the invention is a medium for culturing immune effector cells comprising the small molecule compound capable of inhibiting or antagonizing TBK 1; preferably, the medium contains one or more small molecule compounds selected from any one of formulas 24-33.

In certain embodiments, the cell culture medium of the invention is a medium for culturing immune effector cells comprising the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential and the small molecule compound capable of inhibiting or antagonizing cGAS; preferably, the medium contains any one or more small molecule compounds selected from formulas 1 to 14 and any one or more small molecule compounds selected from formulas 15 to 23.

In certain embodiments, the cell culture medium of the invention is a medium for culturing immune effector cells comprising the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential and the small molecule compound capable of inhibiting or antagonizing TBK 1; preferably, the culture medium contains one or more small molecule compounds selected from any one of formulas 1 to 14 and one or more small molecule compounds selected from any one of formulas 24 to 33.

In certain embodiments, the cell culture medium of the invention is a medium for culturing immune effector cells comprising the small molecule compound capable of inhibiting or antagonizing cGAS and the small molecule compound capable of inhibiting or antagonizing TBK 1; preferably, the medium contains one or more small molecule compounds selected from any one of formulas 15 to 23 and one or more small molecule compounds selected from any one of formulas 24 to 33.

In certain embodiments, the cell culture medium of the present invention is a medium for culturing immune effector cells comprising the small molecule compound capable of inhibiting STING expression and/or binding activity or reducing or eliminating mitochondrial membrane potential, the small molecule compound capable of inhibiting or antagonizing cGAS, and the small molecule compound capable of inhibiting or antagonizing TBK 1; preferably, the medium contains any one or more small molecule compounds selected from formulas 1 to 14, any one or more small molecule compounds selected from formulas 15 to 23, and any one or more small molecule compounds selected from formulas 24 to 33.

Conventional use for culturing T cells, TIL cells, NK T cells, CAOne or more of R-T cells, CIK cells, TCR-T cells and macrophages, in particular media conventionally used for culturing T cells, TIL cells and/or CAR-T cells. More preferably, the medium is a medium conventionally used for culturing electroporated immune effector cells. The cGAS-STING signal pathway inhibitor is added into the culture medium and used for culturing the immune effector cells after electrotransformation, so that the total number and the survival rate of the cells can be obviously improved. Preferably, the final concentration of the cGAS-STING signaling pathway inhibitor in the medium is in the range of 0.02-100. mu.M, preferably in the range of 0.02-80. mu.M, more preferably in the range of 0.5-5. mu.M; even more preferably, in the range of 0.5-1. mu.M or in the range of 2.5-5. mu.M. An exemplary immune effector cell culture medium of the invention is one containing an inhibitor of the cGAS-STING signaling pathwayCTS^TMSerum-free cell culture medium, DMEM medium or RPMI1640 medium; preferably, it contains cGAS-STING signal pathway inhibitorCTS^TMSerum-free cell culture media; more preferably containing the cGAS-STING signaling pathway inhibitor at the concentrationCTS^TMSerum-free cell culture media.

The invention has the advantages that the death rate of the cells after electrotransformation, especially immune effector cells, can be obviously reduced and the number of live cells and the proportion of the live cells after the cells are electrotransformed can be improved by culturing the cells after electrotransformation, especially the immune effector cells, by using a culture medium containing one or more of a cGAS-STING signaling pathway inhibitor, such as a small molecular compound capable of inhibiting the expression and/or the binding activity of STING or reducing or eliminating the mitochondrial membrane potential, the small molecular compound capable of inhibiting or antagonizing the cGAS and the small molecular compound capable of inhibiting or antagonizing TBK 1.

The present invention will be illustrated below by way of specific examples. Should be takenIt is understood that these examples are illustrative only and are not intended to limit the scope of the present invention. The electrotransformation instrument used in the examples was a LONZA Nucleofector from Lonza^TM2 b; other methods and reagents used in the examples are conventional in the art.

The structural formula of the compounds used in this application is shown below:

the pNB328-EGFP used in the examples below is described in Chinese patent CN105154473B, which is hereby incorporated by reference in its entirety.

Small molecule compounds G150, H-151 and IRAK-IN-4 used IN the following examples were all available from MCE under the accession numbers HY-128583, HY-112693 and HY-114181, respectively. C-170 was purchased from Cayman Chemical, cat # 30157. Other reagents used are commercially available.

Example 1: isolated culture of human liver cancer tissue-derived TIL cells

Freshly excised liver cancer specimens were collected and immediately processed under sterile conditions. The specific method comprises the following steps: removing normal tissue and necrotic area around the liver cancer specimen, and removing the normal tissue and necrotic area from the specimenThe size of the taken-down part is 1-2mm³One for each well of a 24-well plate. Add 2mL of complete medium (AIM-V medium with 10% FBS) and 3000IU/mL IL-2 per well. The 24-well plate was placed at 37 ℃ in 5% CO₂Culturing in an incubator. Half-volume changes were made for all wells on days 5-6 after initiation of culture. And then, according to the growth condition of the TIL, half-amount liquid change is carried out every 1-2 days. Once the wells are full of TIL and all adherent cells have been removed, the TIL from each full well is collected.

Example 2: preparation of human activated T cells

Coating a six-well plate with a coating solution containing 5 mu g/ml of anti-CD 3 antibody and 5 mu g/ml of anti-CD 28 antibody for 2-4 hours at room temperature, sucking out the coating solution, washing the plate with physiological saline for 1-3 times, and adding an AIM-V culture medium containing 2% FBS for later use; resuscitating human peripheral blood PBMC (purchased from ALLCELLS) in a water bath at 37 ℃, culturing the PBMC for 2-4h in an adherent manner, wherein nonadherent suspension cells are initial T cells, collecting the suspension cells into a 15ml centrifuge tube, centrifuging for 3min at 1200rmp, discarding supernatant, adding physiological saline, centrifuging for 3min at 1200rmp, discarding the physiological saline, and repeating the steps; the washed primary T cells were then transferred to antibody-coated wells containing the ready-to-use medium at 37 ℃ with 5% CO₂And carrying out subsequent experiments after culturing for 3-4 days.

Example 3 application of cGAS small molecule inhibitor G150 in electrotransformation of TIL cells overexpressing EGFP

Electronically transforming the TIL expressing EGFP by the following steps:

1) AIM-V medium was added to 4 wells of a 12-well plate in advance at 2mL per well, and then transferred to a cell incubator at 37 ℃ with 5% CO₂Preheating for 1 hour;

2) the electrotransfer liquid ratio of the single dosage of each hole is carried out according to the following table:

	100μL NucleocuvetteTMStrip(μL)
		NucleofectorTMvolume of solution	82
Electrotransformation make-up solution	18

3) The TIL obtained in example 1 was taken out into 4 EP tubes, and 5X 10 tubes were placed in each EP tube⁶Centrifuging the cells at 1200rpm for 5min, discarding the supernatant, then resuspending the cells in 500. mu.L of physiological saline, and repeating the centrifugation step to wash the cell pellet;

4) adding 5 mu g of plasmid pNB328-EGFP into the electrotransformation liquid prepared in the step 2), and then standing the mixture at room temperature for less than 30 min;

5) resuspending 4 tubes of TIL with 100. mu.L of plasmid-containing electrotransfer prepared in 4), carefully pipetting the cell resuspension solution, transferring the cell resuspension solution into a LONZA 100. mu.L electric rotor, and placing the electric rotor into a LONZA Nucleofector^TM2b, starting an electrotransfer program in the electrotransfer tank, wherein the electrotransfer program selects T-020;

6) after completion of the electrotransfer, the cuvette was carefully removed, the cell suspension was aspirated and transferred to an EP tube, 200. mu.L of preheated AIM-V medium was added to each tube, and then transferred to a 12-well plate of 1) containing preheated AIM-V medium at 37 ℃ with 5% CO₂Culturing;

7) adding compound G150 represented by formula 22 to 3 wells to a final concentration of 2.5. mu.M after culturing for 0h, 1h and 5h, respectively; the remaining wells were not added with G150 as control wells, and the culture was continued.

The culturing operation was repeated 3 times, and the number of cells and the cell viability in each well after 5 days of culturing were measured with a cell counter and averaged.

The results are shown in FIGS. 1 to 5. FIG. 1 shows EGFP-positive cells observed by fluorescence microscopy after culturing TIL cells added with G150 at 0h after electroporation for 5 days. FIG. 2 shows the results of flow cytometric analysis of EGFP-positive cells after 0h, 1h and 5h post-electroporation with addition of G150 to 2.5. mu.M for 5 days. FIGS. 3 to 5 show the number of viable cells, the ratio of viable cells and the ratio of EGFP-positive cells in the TIL-transfected pNB 328-EGFP-treated group at 0h, 1h and 5h after 5 days of culture in the G150-2.5. mu.M-added group and the G150-added control group, respectively. The results show that the number of living cells, the proportion of the living cells and the proportion of the cells with positive EGFP expression after the electric transformation of the TIL cells can be obviously improved by adding G150 molecules 0h, 1h and 5h after the electric transformation of the TIL to the EGFP expression plasmid. This shows that the addition of cGAS inhibitor G150 can obviously improve the survival level of the TIL cells after electrotransformation and improve the transformation efficiency of the exogenous gene.

Example 4 application of cGAS small molecule inhibitor G150 in electrotransformation of T cells overexpressing EGFP

T cells expressing EGFP were electroporated as follows:

1) AIM-V medium was added to 4 wells of a 12-well plate in advance at 2mL per well, and then transferred to a cell incubator at 37 ℃ with 5% CO₂Preheating for 1 hour;

2) the electrotransfer liquid ratio of the single dosage of each hole is carried out according to the following table:

	100μL NucleocuvetteTM Strip(μL)
		NucleofectorTMvolume of solution	82
Electrotransformation make-up solution	18

3) The activated T cells obtained in example 2 were dispensed into 4 EP tubes, and 5X 10 cells were added to each EP tube⁶Centrifuging the cells at 1200rpm for 5min, discarding the supernatant, then resuspending the cells in 500. mu.L of physiological saline, and repeating the centrifugation step to wash the cell pellet;

4) adding 5 mu g of plasmid pNB328-EGFP into the electrotransformation liquid prepared in the step 2), and then standing the mixture at room temperature for less than 30 min;

5) resuspending 4 tubes of T cells with 100. mu.L of plasmid-containing electrotransfer prepared in 4), carefully pipetting the cell resuspension solution, transferring the cell resuspension solution into a LONZA 100. mu.L electric rotor, and placing the electric rotor into a LONZA Nucleofector^TM2b, starting an electrotransfer program in the electrotransfer tank, wherein the electrotransfer program selects T-020;

6) after completion of the electrotransfer, the cuvette was carefully removed, the cell suspension was aspirated and transferred to an EP tube, 200. mu.L of preheated AIM-V medium was added to each tube, and then transferred to a 12-well plate of 1) containing preheated AIM-V medium at 37 ℃ with 5% CO₂Culturing;

7) adding compound G150 represented by formula 22 to 3 wells to a final concentration of 5. mu.M after culturing for 0h, 1h and 5h, respectively; the remaining wells were not added with G150 as control wells, and the culture was continued.

The culturing operation was repeated 3 times, and the number of cells and the cell viability in each well after 5 days of culturing were measured with a cell counter and averaged.

The results are shown in FIGS. 6-8. FIGS. 6 to 8 show the number of viable cells, the proportion of viable cells, and the proportion of EGFP-positive cells after 5 days of culture in the treatment groups containing G150 to 5. mu.M and the control groups containing no G150 added, respectively, at 0h, 1h, and 5h after the electric transfer of pNB328-EGFP to activated T cells. The results show that the number of living cells, the proportion of the living cells and the proportion of the cells with positive EGFP expression after T cell electrotransformation can be obviously improved by adding G150 molecules 0h, 1h and 5h after the T electrotransformation of the plasmid expressing EGFP. This indicates that the addition of the cGAS inhibitor G150 can significantly improve the survival level of activated T cells after electrotransfer and improve the efficiency of exogenous gene transfer.

Example 5 application of Small molecule inhibitor of STING H-151 to electrotransformation of EGFP-overexpressing TIL cells

Electronically transforming EGFP-expressing TIL cells as follows:

1) AIM-V medium was previously added to 4 wells of a 12-well plate at 2mL per well, followed byTransferring into cell culture box at 37 deg.C with 5% CO₂Preheating for 1 hour;

2) the electrotransfer liquid ratio of the single dosage of each hole is carried out according to the following table:

	100μL NucleocuvetteTM Strip(μL)
		NucleofectorTMvolume of solution	82
Electrotransformation make-up solution	18

3) The TIL cells obtained in example 1 were taken out into 4 EP tubes, and 5X 10 cells were added to each EP tube⁶Centrifuging the cells at 1200rpm for 5min, discarding the supernatant, then resuspending the cells in 500. mu.L of physiological saline, and repeating the centrifugation step to wash the cell pellet;

4) adding 5 mu g of plasmid pNB328-EGFP into the electrotransformation liquid prepared in the step 2), and then standing the mixture at room temperature for less than 30 min;

5) resuspending 4 tubes of TIL cells in 100. mu.L/tube with the plasmid-containing electrotransfer prepared in 4), carefully pipetting the cell resuspension solution, transferring the cell resuspension solution into a LONZA 100. mu.L electric rotor, and placing the electric rotor into a LONZA Nucleofector^TM2b, starting an electrotransfer program in the electrotransfer tank, wherein the electrotransfer program selects T-020;

6) after completion of the electrotransfer, the cuvette was carefully removed, the cell suspension was aspirated and transferred to an EP tube, 200. mu.L of preheated AIM-V medium was added to each tube, and then transferred to a 12-well plate of 1) containing preheated AIM-V medium at 37 ℃ with 5% CO₂Culturing;

7) adding a compound H-151 shown in formula 5 to 3 holes after culturing for 0H, 1H and 5H respectively to a final concentration of 0.5. mu.M; the remaining wells were left without H-151 as control wells, and the incubation was continued.

The culturing operation was repeated 3 times, and the number of cells and the cell viability in each well after 5 days of culturing were measured with a cell counter and averaged.

The results are shown in FIGS. 9-11. FIGS. 9 to 11 show the number of viable cells, the ratio of viable cells and the ratio of EGFP-positive cells in the TIL-transfected cells at 0H, 1H and 5H after incubation for 5 days in the H-151-added to 0.5. mu.M-treated group and the H-151-added control group, respectively. Results show that the number of living cells, the proportion of the living cells and the proportion of cells with positive EGFP expression after the electric transformation of the TIL cells can be obviously improved by adding H-151 molecules 0H, 1H and 5H after the electric transformation of the TIL cells to the EGFP expression plasmid. This indicates that the addition of STING inhibitor H-151 can significantly improve the survival level of TIL cells after electrotransformation and improve the transformation efficiency of foreign genes.

Example 6 application of Small molecule inhibitor H-151 for STING in electrotransformation of T cells overexpressing EGFP

T cells expressing EGFP were electroporated as follows:

1) AIM-V medium was added to 4 wells of a 12-well plate in advance at 2mL per well, and then transferred to a cell incubator at 37 ℃ with 5% CO₂Preheating for 1 hour;

2) the electrotransfer liquid ratio of the single dosage of each hole is carried out according to the following table:

3) the activated T cells obtained in example 2 were dispensed into 4 EP tubes, and 5X 10 cells were added to each EP tube⁶Centrifuging the cells at 1200rpm for 5min, discarding the supernatant, then resuspending the cells in 500. mu.L of physiological saline, and repeating the centrifugation step to wash the cell pellet;

4) adding 5 mu g of plasmid pNB328-EGFP into the electrotransformation liquid prepared in the step 2), and then standing the mixture at room temperature for less than 30 min;

5) resuspending 4 tubes of T cells with 100. mu.L of plasmid-containing electrotransfer prepared in 4), carefully pipetting the cell resuspension solution, transferring the cell resuspension solution into a LONZA 100. mu.L electric rotor, and placing the electric rotor into a LONZA Nucleofector^TM2b, starting an electrotransfer program in the electrotransfer tank, wherein the electrotransfer program selects T-020;

6) after completion of the electrotransfer, the cuvette was carefully removed, the cell suspension was aspirated and transferred to an EP tube, 200. mu.L of preheated AIM-V medium was added to each tube, and then transferred to a 12-well plate of 1) containing preheated AIM-V medium at 37 ℃ with 5% CO₂Culturing;

7) adding a compound H-151 shown in formula 5 to 3 wells after culturing for 0H, 1H and 5H respectively to a final concentration of 1 μ M; the remaining wells were left without H-151 as control wells, and the incubation was continued.

The culturing operation was repeated 3 times, and the number of cells and the cell viability in each well after 5 days of culturing were measured with a cell counter and averaged.

The results are shown in FIGS. 12-14. FIGS. 12 to 14 show the number of viable cells, the ratio of viable cells and the ratio of EGFP-positive cells after 5 days of culture in the H-151-1. mu.M-treated group and the H-151-free control group at 0H, 1H and 5H after the electric transfer of pNB328-EGFP to activated T cells, respectively. The results show that the number of living cells after the T cells are electrically transformed into the EGFP expressing plasmid and the proportion of cells with positive EGFP expression can be obviously improved by adding H-151 molecules 0H, 1H and 5H after the T cells are electrically transformed into the EGFP expressing plasmid. This indicates that the addition of STING inhibitor H-151 can significantly increase the survival level of activated T cells after electrotransfer and increase the efficiency of exogenous gene transfer.

Comparative example 1 application of cGAS small molecule inhibitor IRAK-IN-4 IN preparation of TIL cell over-expressing EGFP by electrotransformation

Electronically transforming EGFP-expressing TIL cells as follows:

1) AIM-V medium was added to 4 wells of a 12-well plate in advance at 2mL per well, and then transferred to a cell incubator at 37 ℃ with 5% CO₂Preheating for 1 hour;

2) the electrotransfer liquid ratio of the single dosage of each hole is carried out according to the following table:

	100μL NucleocuvetteTM Strip(μL)
		NucleofectorTMvolume of solution	82
Electrotransformation make-up solution	18

3) The TIL cells obtained in example 1 were taken out into 4 EP tubes, and 5X 10 cells were added to each EP tube⁶Centrifuging the cells at 1200rpm for 5min, discarding the supernatant, then resuspending the cells in 500. mu.L of physiological saline, and repeating the centrifugation step to wash the cell pellet;

4) adding 5 mu g of plasmid pNB328-EGFP into the electrotransformation liquid prepared in the step 2), and then standing the mixture at room temperature for less than 30 min;

5) resuspending 4 tubes of TIL cells in 100. mu.L/tube with the plasmid-containing electrotransfer prepared in 4), carefully pipetting the cell resuspension solution, transferring the cell resuspension solution into a LONZA 100. mu.L electric rotor, and placing the electric rotor into a LONZA Nucleofector^TM2b, starting an electrotransfer program in the electrotransfer tank, wherein the electrotransfer program selects T-020;

6) after completion of the electrotransfer, the cuvette was carefully removed, the cell suspension was aspirated and transferred to an EP tube, 200. mu.L of preheated AIM-V medium was added to each tube, and then transferred to a 12-well plate of 1) containing preheated AIM-V medium at 37 ℃ with 5% CO₂Culturing;

7) adding a cGAS inhibitor IRAK-IN-4 into 3 holes after culturing for 0h, 1h and 5h respectively until the final concentration is 2 mu M; the remaining wells were not added IRAK-IN-4 as control wells, and the culture was continued.

The culturing operation was repeated 3 times, and the number of cells and the cell viability in each well after 5 days of culturing were measured with a cell counter and averaged.

The results are shown in FIGS. 15-17. FIGS. 15 to 17 show the number of viable cells, the ratio of viable cells and the ratio of EGFP-positive cells IN the TIL-transfected cells after incubation for 5 days IN the case of 0h, 1h and 5h after transfection of pNB328-EGFP with IRAK-IN-4 added to the treated group at 2. mu.M and the control group without IRAK-IN-4 added, respectively. The results show that the addition of IRAK-IN-4 molecules at 0h, 1h and 5h after the TIL is electrically transformed into the EGFP expression plasmid does not obviously improve the number of living cells, the proportion of the living cells and the proportion of cells with positive EGFP expression after the TIL cells are electrically transformed. This indicates that the addition of cGAS inhibitor IRAK-IN-4 has no obvious effect on improving the survival level of the TIL cells after electrotransformation and improving the transformation efficiency of exogenous genes.

Comparative example 2 application of small molecule STING inhibitor C-170 in preparation of T cells over-expressing EGFP by electrotransformation

T cells expressing EGFP were electroporated as follows:

1) AIM-V medium was added to 4 wells of a 12-well plate in advance at 2mL per well, and then transferred to a cell incubator at 37 ℃ with 5% CO₂Preheating for 1 hour;

2) the electrotransfer liquid ratio of the single dosage of each hole is carried out according to the following table:

	100μL NucleocuvetteTM Strip(μL)
		NucleofectorTMvolume of solution	82
Electrotransformation make-up solution	18

3) The activated T cells obtained in example 2 were dispensed into 4 EP tubes, and 5X 10 cells were added to each EP tube⁶Centrifuging the cells at 1200rpm for 5min, discarding the supernatant, then resuspending the cells in 500. mu.L of physiological saline, and repeating the centrifugation step to wash the cell pellet;

4) adding 5 mu g of plasmid pNB328-EGFP into the electrotransformation liquid prepared in the step 2), and then standing the mixture at room temperature for less than 30 min;

5) resuspending 4 tubes of T cells with 100. mu.L of plasmid-containing electrotransfer prepared in 4), carefully pipetting the cell resuspension solution, transferring the cell resuspension solution into a LONZA 100. mu.L electric rotor, and placing the electric rotor into a LONZA Nucleofector^TM2b, starting an electrotransfer program in the electrotransfer tank, wherein the electrotransfer program selects T-020;

6) after completion of the electrotransfer, the cuvette was carefully removed, the cell suspension was aspirated and transferred to an EP tube, 200. mu.L of preheated AIM-V medium was added to each tube, and then transferred to a 12-well plate of 1) containing preheated AIM-V medium at 37 ℃ with 5% CO₂Culturing;

7) adding STING inhibitor C-170 to 3 wells after culturing for 0h, 1h and 5h respectively to a final concentration of 2 μ M; the remaining wells were left without C-170 as control wells, and the culture was continued.

The culturing operation was repeated 3 times, and the number of cells and the cell viability in each well after 5 days of culturing were measured with a cell counter and averaged.

The results are shown in FIGS. 18-20. FIGS. 18 to 20 show the number of viable cells, the proportion of viable cells, and the proportion of EGFP-positive cells after 5 days of culture in the treatment group containing C-170 to 2. mu.M and the control group containing no C-170, respectively, at 0h, 1h, and 5h after the electric transfer of pNB328-EGFP to activated T cells. The results show that the addition of C-170 molecules 0h, 1h and 5h after T-cell transformation of the EGFP expression plasmid does not obviously improve the number of live cells, the proportion of the live cells and the proportion of cells with positive EGFP expression after T-cell transformation. This indicates that the addition of the STING inhibitor C-170 does not significantly contribute to the enhancement of the survival level of activated T cells after electrotransfer and the enhancement of the efficiency of transfer of foreign genes.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications can be made by those skilled in the art after reading the above disclosure, and equivalents also fall within the scope of the invention as defined by the appended claims.