Universal cell system, construction method therefor, and use thereof

Abstract

Provided are a universal cell system, a construction method therefor, and use thereof. Universal mesenchymal stem cells are constructed by means of the combination of electroporation and CRISPRoff gene editing, and the cells can survive stably in a pulmonary inflammatory pathological environment to provide sustained treatment, significantly improve multiple indicators of acute pneumonia, solve immune rejection, and provide new cell therapies and ideas for acute pneumonia. CRISPRoff is introduced into universal CTL cells by means of electroporation technology, targeting a specific region and inhibiting the expression of HLA-I in T cells; thus, T cells with low immunogenicity are constructed, and the cells can avoid allogeneic immune rejection. The universal cell system combines the advantages of both transient transfection and CRISPRoff gene editing, has great significance in the field of cell therapy, provides a more comprehensive and effective solution for the treatment of various diseases, and has a wide application prospect.

Description

A universal cell system, its construction method and application Technical Field

[0001] This invention belongs to the medical field, specifically relating to a universal cell system, its construction method, and its application. Background Technology

[0002] The description of the background art is merely a general description for the purpose of facilitating understanding of the content of the present invention and does not constitute any limitation on the present invention.

[0003] Acute lung injury (ALI) is a common and life-threatening lung disease characterized by increased alveolar and capillary permeability, damage to lung tissue structure, and lung inflammation. Currently, there is no specific drug for treating ALI. The pathogenesis of ALI is complex, but the most widely accepted mechanism is that various etiologies induce an increase in pro-inflammatory factors in the lungs. Therefore, controlling the inflammatory response is a key measure in the treatment of ALI.

[0004] Currently, the main clinical treatments for ALI include: (1) Mechanical ventilation: This is the classic treatment for ALI. However, improper mechanical ventilation may lead to alveolar over-inflation, which in turn causes mechanical ventilation-related lung injury. In clinical practice, individualized ventilation strategies should be adopted for different patients to prevent and reduce mechanical ventilation-related toxic side effects as much as possible. How to avoid respiratory complications while giving full play to the therapeutic effect of mechanical ventilation is still a major problem that needs to be solved in the field of ALI treatment; (2) Chemotherapy: Commonly used chemical drugs for treating acute lung injury include glucocorticoids, antibiotics, traditional Chinese medicine preparations, protease inhibitors, anticoagulants, vasodilators, etc. Although these drugs have certain efficacy in the treatment of ALI, they have side effects.

[0005] In recent years, stem cell therapy, represented by mesenchymal stem cells (MSCs), has emerged as a potential clinical approach for anti-inflammatory treatment. MSCs are self-renewing stem cells with multi-lineage differentiation, immunomodulatory, and anti-inflammatory capabilities. They tend to migrate to sites of inflammation and exert immunomodulatory functions through paracrine pathways, promoting tissue repair and regeneration. They can alleviate multi-organ diseases and viral lung complications, thereby achieving therapeutic effects. However, allogeneic immune rejection remains a bottleneck hindering the widespread clinical application of MSCs. Although MSCs are generally considered to have low immunogenicity, in pathological inflammatory environments, pro-inflammatory factors such as IFNγ can stimulate MSCs to upregulate HLA-I, increasing their immunogenicity and leading to immune rejection, thus preventing them from sustaining their therapeutic effects. Furthermore, autologous MSCs are limited by insufficient cell sources, batch instability, high prices, and long preparation cycles, making their widespread clinical use difficult. While allogeneic MSCs can address these bottlenecks to some extent, they are still subject to allogeneic immune rejection, preventing them from sustaining their therapeutic effects in the recipient and resulting in poor efficacy.

[0006] Meanwhile, although the efficacy of T-cell therapies such as CAR-T is well-established, their clinical application is still limited by bottlenecks such as insufficient autologous cell sources, high cost, and long preparation cycles. Universal cell therapies can overcome the drawbacks of allogeneic cell immune rejection, insufficient autologous cell sources, and personalized customization, helping to improve the clinical accessibility of T-cell therapies such as CAR-T and benefit a wider range of patients. However, the current construction of universal T cells mostly relies on multiple gene editing technologies such as gene knockout, gene mutation, and gene overexpression mediated by lentiviral systems, which inevitably increases the technical difficulty and safety risks.

[0007] Researchers have created several universal T cells that can evade immune rejection, mainly including the following strategies: (1) avoiding allogeneic T cell immune responses by gene editing to knock out the B2M gene, and then avoiding allogeneic NK cell immune responses by gene knock-in technology to overexpress the HLA-E gene; (2) avoiding allogeneic T cell immune responses by gene editing to knock out the B2M and CIITA genes, and then avoiding allogeneic NK cell immune responses by gene knock-in technology to overexpress the CD47 gene; (3) avoiding allogeneic T cell immune responses by gene editing to knock out the B2M and CIITA genes, and then avoiding allogeneic NK cell immune responses by gene knocking out the CD155 gene and overexpressing the HLA-E gene by gene knock-in technology.

[0008] Current methods for constructing universal T cells still require multiple gene editing techniques, such as gene knockout and gene knock-in, which inevitably introduces permanent alterations to the DNA sequence and foreign DNA sequences, leading to safety risks such as instability in the cell genome and karyotype. Furthermore, these gene editing methods are not only cumbersome but also largely rely on viral vector transduction, resulting in the random integration of the viral genome into the host cell genome, further increasing the risks of insertional mutations and regulatory dysregulation. In addition, the long-term stable expression of gene editing systems within host cells significantly increases the risk of off-target editing.

[0009] In the invention patent application with publication number CN113846063A, the inventors constructed a universal human stem cell suitable for allogeneic transplantation in previous experiments. By knocking down or silencing the enhancer gene sequence of the B2M gene in the stem cells, the stem cells can maintain low immunogenicity under inflammatory conditions. However, the construction method uses lentiviral transfection, which is complex, highly selective for cell types, and requires skilled operators, making it difficult to implement in general laboratories. Furthermore, viral DNA can permanently integrate into the MSC genome, potentially posing safety risks. Additionally, the application and mechanism of action of the proposed universal mesenchymal stem cells in lung injury have not been thoroughly investigated.

[0010] Therefore, there is an urgent need to develop a safe, stable, and universal cell system construction method with lower immunogenicity. This method should be safer and simpler, and should be able to successfully construct cells with permanently reduced immunogenicity, avoid allogeneic immune rejection, and clarify the mechanism of action and related applications of universal mesenchymal stem cells constructed using the aforementioned method in acute lung injury, in order to provide new strategies for the clinical treatment of acute lung injury and other diseases.

[0011] Summary of the Invention

[0012] To address the problems existing in the prior art, this invention provides a universal cell system, its construction method, and its application. A novel universal cell system is constructed by combining electroporation and CRISPR-off gene editing methods, including universal mesenchymal stem cells and universal CTL cells. In the described construction method, a novel epigenetic editor, CRISPRoff, is delivered into CTL cells via electroporation. This editor targets a super-enhancer region of the B2M gene within the cells. With only a single, transient epigenetic edit, T cell HLA-I expression is suppressed without altering the target gene's DNA sequence, successfully constructing low-immunogenic CTL cells capable of evading immune rejection. Simultaneously, the resulting universal mesenchymal stem cells, compared to unedited mesenchymal stem cells and those obtained through other transfection methods, exhibit stable survival in the inflammatory pathological environment of the lungs, continuously exerting therapeutic effects. Furthermore, they show more significant improvement in the pathological phenotype and multiple biochemical indicators of acute pneumonia. This method solves the problem of immune rejection of allogeneic stem cells by the human immune system in stem cell transplantation-based regenerative therapies, providing an effective new cell therapy for acute pneumonia and offering technical support and new ideas for its clinical treatment, demonstrating promising application prospects.

[0013] To achieve the above objectives, the present invention employs the following solution:

[0014] On the one hand, the present invention provides a method for constructing a universal cell system, comprising the following steps:

[0015] (1) Construction of recombinant plasmids;

[0016] (2) Prepare the transfection complex;

[0017] (3) Transfect the plasmid into the cells;

[0018] In step (3), the plasmid mixture is transfected into the cells using electrotransfection.

[0019] Furthermore, the cells are mesenchymal stem cells or CTL cells.

[0020] In the invention patents published under application numbers CN113846063A and CN113801881A, the inventors had previously constructed and obtained "a universal human stem cell suitable for allogeneic transplantation," but it was not applied to specific indications such as acute lung injury. This is related to the use of lentiviral transduction in the early stages: on the one hand, the lentiviral transduction method is complex, highly selective for cell types, and requires highly skilled operators, making it difficult to popularize in general laboratories; on the other hand, although mesenchymal stem cells are generally considered to have low immunogenicity, lentiviral vectors and their transgenic products may trigger an immune response, leading to rejection of the modified cells by the body; simultaneously, the random insertion of lentiviruses into the cell genome may damage important gene functions within the cell, or even lead to malignant transformation; all of these may pose potential safety hazards. Therefore, in this invention, the construction method of universal mesenchymal stem cells was further optimized to construct a safer and more stable universal mesenchymal stem cell, which has good therapeutic effects in various indications.

[0021] Therefore, this invention employs electroporation combined with CRISPRoff to construct a novel universal cell system, including universal mesenchymal stem cells and universal CTL cells. The universal mesenchymal stem cells maintain low immunogenicity in an inflammatory environment, enabling them to avoid immune rejection and stably exert their therapeutic effects after transplantation, significantly improving ALI symptoms and exhibiting better regenerative and repair effects on damaged lung tissue. Compared to methods using lentiviral transduction and liposome transfection, the universal mesenchymal stem cells constructed in this invention have the best transfection efficiency and cell viability. This is likely because electroporation induces changes in cell membrane potential through a high-intensity electric field, instantaneously increasing cell membrane permeability and creating reversible pores on the cell membrane to facilitate the entry of exogenous nucleic acids, making it suitable for almost all cell types.

[0022] In the construction of the universal CTL cells, based on the super enhancer (B2M-SE) on the B2M gene discovered in the aforementioned patent, the epigenetic editor CRISPRoff and sgRNA targeting B2M-SE are co-transfected into human CTL cells using electroporation. This allows for epigenetic editing of the super enhancer at specific sites within the CTL cells, resulting in the first construction of universal CTL cells without any alteration to the DNA sequence. Furthermore, compared to the aforementioned strategy of constructing universal human stem cells using a lentiviral system, this invention modifies and upgrades the editing strategy to address the issue of natural resistance of CTL and other lymphocytes to lentiviral system modification. It employs a virus-free transient transfection method, where a single transient epigenetic editing can permanently reduce the immunogenicity of CTL cells. Moreover, the constructed universal CTL cells can circumvent allogeneic immune rejection.

[0023] Meanwhile, the construction method provided by this invention avoids both the technical difficulties of editing CTLs in viral systems and the safety issues such as off-target editing caused by the continuous and stable expression of viral systems. This construction strategy does not require multiple gene editing of CTL cells, nor does it require the introduction of exogenous viral genes into the CTL genome, and it does not cause permanent changes to the CTL genome sequence like ordinary gene editing strategies. Therefore, it combines simplicity and safety.

[0024] Further, in step (2), the transfection complex includes a plasmid mixture and either mesenchymal stem cells or CTL cells; the plasmid mixture includes CRISPRoff plasmid and sgRNA plasmid.

[0025] Furthermore, in step (2), the amount of plasmid used is 100-200 ng, or the amount of plasmid used is 400-800 ng.

[0026] Furthermore, in step (2), when the cells are mesenchymal stem cells, the amount of plasmid used is 400-800 ng.

[0027] Furthermore, in step (2), when the cells are CTL cells, the amount of plasmid used is 100-200 ng.

[0028] In some embodiments, to further improve the transfection efficiency and cell viability in the universal mesenchymal stem cell construction method of the present invention, different plasmid dosages were screened. Experimental results showed that as the plasmid dosage increased, the proportion of double-positive cells gradually increased from 3.7% to 15.9%. However, when the plasmid dosage exceeded 800 ng, cell viability decreased significantly, possibly due to a saturation effect or unintended effects caused by excessive plasmid-cell interaction. Therefore, the preferred plasmid dosage in this invention is 800 ng.

[0029] Similarly, in some embodiments, to further improve the transfection efficiency and cell viability in the universal CTL cell construction method of the present invention, different plasmid dosages were screened. Experimental results showed that the proportion of GFP fluorescently positive cells was highest when the plasmid dosage was 150 ng, while the proportion of GFP fluorescently positive cells decreased significantly when the plasmid dosage was reduced or increased. This is because, on the one hand, when the plasmid dosage is too low, the number of expressed CRISPRoff editors and sgRNAs is insufficient, failing to achieve the optimal epigenetic editing effect; on the other hand, when the plasmid dosage is too high, a saturation effect occurs or some unintended effects arise due to excessive plasmid interaction with cells. Therefore, in this embodiment, the preferred plasmid dosage is 150 ng.

[0030] Furthermore, in step (2), the mass ratio of CRISPRoff plasmid to sgRNA plasmid is 1:1.

[0031] Furthermore, in the electrotransfection parameters of step (3), the voltage is 990V-1400V, the duration of the electrical pulse is 10ms-40ms, and the number of pulses is 1-3; or the voltage is 1200V-1600V, the duration of the electrical pulse is 10ms-20ms, and the number of pulses is 2-3.

[0032] Furthermore, in the electrotransfection parameters of step (3), when the cells are mesenchymal stem cells, the voltage is 990V-1400V, the duration of the electrical pulse is 10ms-40ms, and the number of pulses is 1-3.

[0033] Furthermore, in the electrotransfection parameters of step (3), when the cells are CTL cells, the voltage is 1200V-1600V, the duration of the electrical pulse is 10ms-20ms, and the number of pulses is 2-3.

[0034] In some implementations, different electroporation parameters were screened to minimize cell damage and improve cell survival while achieving good transfection efficiency. Experimental results showed that a high electroporation efficiency of 93.58% was achieved under the electroporation conditions of 1400V / 10ms / 3pulse. Therefore, the preferred electroporation parameters of this invention are 1400V / 10ms / 3pulse for preparing universal mesenchymal stem cells.

[0035] Similarly, in some embodiments, in order to achieve good transfection efficiency while minimizing cell damage and improving cell survival, different electroporation parameters were screened. Experimental results showed that a high electroporation efficiency of 95% was achieved under the electroporation conditions of 1600V / 10ms / 3pulses. Therefore, the preferred electroporation parameters of this invention are 1600V / 10ms / 3pulses for preparing universal CTL cells.

[0036] On the other hand, the present invention provides a universal mesenchymal stem cell, which is constructed using any one of the above technical solutions.

[0037] In another aspect, the present invention provides the use of a universal mesenchymal stem cell for preparing a formulation that enhances the lung injury repair and regeneration capacity, wherein the universal mesenchymal stem cell used in the application is constructed using any one of the above technical solutions.

[0038] In some embodiments, to verify that the universal mesenchymal stem cells prepared by the present invention have a more significant therapeutic effect on acute lung injury, an LPS-induced acute pneumonia model was constructed and an in vivo bioluminescence imaging tracking experiment was performed. At the same time, HE staining experiments and biochemical index detection experiments of bronchoalveolar lavage fluid were also performed.

[0039] Among them, the results of the in vitro observation experiment of lung tissue showed that, compared with the PBS treatment group, the ntMSCs treatment group showed improvement in lung tissue inflammation and congestion, but compared with... The group still had severe pneumonia symptoms, indicating that ntMSCs had limited therapeutic effect on pneumonia; while the GLOBES treatment group showed a much better improvement in pneumonia compared to the PBS treatment group, and... The results showed that the GLOBES group closely resembled normal lung tissue, indicating that GLOBES had a better therapeutic effect on pneumonia. Subsequent experiments compared the GLOBES-1 and GLOBES-2 treatment groups. The results showed that, compared to the GLOBES-1 and GLOBES-2 treatment groups, the GLOBES treatment group exhibited significantly reduced inflammation and congestion in its lung tissue, suggesting that the GLOBES treatment group constructed via electroporation had a more significant effect on improving pneumonia.

[0040] HE staining results showed that: compared to In the normal lung tissue of the control group, the lung tissue of the LPS-induced PBS group mice showed typical acute pneumonia injury. Although ntMSCs treatment alleviated the above-mentioned acute lung injury to some extent, compared with... In the normal lung tissue of the group, numerous areas of lung injury were still clearly visible. However, the lung tissue sections of the GLOBES treatment group differed significantly from those of the ntMSCs group, and were more similar to those of the ntMSCs group. The normal lung tissue section morphology of the group indicates that lung injury was significantly improved and repaired after GLOBES treatment. Subsequent experiments compared the group with the GLOBES-1 and GLOBES-2 treatment groups. The results showed that, compared to the GLOBES-1 and GLOBES-2 treatment groups, the GLOBES treatment group showed significant improvement and repair of lung injury after treatment. This further demonstrates that the GLOBES treatment group constructed via electroporation has a more significant lung injury repair effect.

[0041] The results of bronchoalveolar lavage fluid (BAL) content detection showed that the LDH content in the BAL of the PBS, ntMSCs, GLOBES-1, and GLOBES-2 groups was significantly higher than that in the GLOBES treatment group. The study included two groups of patients. The ntMSCs treatment group had an LDH content approximately 2.05 times higher than the GLOBES treatment group. Under normal circumstances, LDH exists only in the cytoplasm of body tissue cells. Higher LDH content in bronchoalveolar lavage fluid indicates more severe tissue damage, thus demonstrating that GLOBES treatment significantly improved and repaired lung injury. Subsequent experiments also measured LDH content in bronchoalveolar lavage fluid from the GLOBES-1 and GLOBES-2 treatment groups. Results showed that the LDH content in the GLOBES-1 treatment group was approximately 1.72 times that of the GLOBES treatment group, and the LDH content in the GLOBES-2 treatment group was approximately 1.85 times that of the GLOBES treatment group. While the LDH content in the GLOBES-1 and GLOBES-2 treatment groups decreased slightly compared to the ntMSCs treatment group, it was still significantly higher than that in the GLOBES treatment group. This further indicates that the GLOBES treatment group constructed via electroporation has a more significant lung injury repair effect. Therefore, the detection results of LDH content in bronchoalveolar lavage fluid further prove that the GLOBES treatment group constructed by electroporation has a more significant effect on repairing acute pneumonia damage.

[0042] The results of bronchoalveolar lavage fluid albumin content detection showed that although the ntMSCs treatment group could reduce the albumin content in bronchoalveolar lavage fluid to some extent, the corresponding albumin content was still significantly higher than that in the ntMSCs treatment group. The albumin content in the bronchoalveolar lavage fluid of the ntMSCs treatment group was approximately 1.43 times that of the GLOBES treatment group. Subsequent experiments also measured the LDH content in the bronchoalveolar lavage fluid of the GLOBES-1 and GLOBES-2 treatment groups. The results showed that the albumin content in the bronchoalveolar lavage fluid of the GLOBES-1 treatment group was approximately 1.28 times that of the GLOBES treatment group, and approximately 1.32 times that of the GLOBES treatment group. Compared to the ntMSCs treatment group, the albumin content in the bronchoalveolar lavage fluid of the GLOBES-1 and GLOBES-2 treatment groups decreased slightly, but still increased compared to the GLOBES treatment group. The albumin content in the GLOBES treatment group was close to that of the ntMSCs treatment group. The albumin content in the bronchoalveolar lavage fluid was normal. Therefore, the results of albumin content detection further demonstrate that the GLOBES treatment group constructed by electroporation has a more significant effect on repairing acute pneumonia damage.

[0043] Results of bronchoalveolar lavage fluid (BAL) analysis showed that the total protein content was similar to that of albumin, with the total protein content in the ntMSCs treatment group being approximately 1.22 times that of the GLOBES treatment group. Subsequent experiments also revealed that the total protein content in BAL of the GLOBES-1 and GLOBES-2 treatment groups was approximately 1.12 times and 1.15 times that of the GLOBES treatment group, respectively. While the total protein content in the BAL of the GLOBES-1 and GLOBES-2 treatment groups was slightly lower than that in the ntMSCs treatment group, the results were still significant. The number of cells decreased, but still increased compared to the GLOBES treatment group; similarly, the total number of cells in the bronchoalveolar lavage fluid of the ntMSCs treatment group was 1.77 times that of the GLOBES treatment group; and in subsequent experiments, it was also found that the total number of cells in the bronchoalveolar lavage fluid of the GLOBES-1 treatment group and the GLOBES-2 treatment group were 1.31 times and 1.42 times that of the GLOBES treatment group, respectively. The total number of cells in the bronchoalveolar lavage fluid of the GLOBES-1 treatment group and the GLOBES-2 treatment group also increased significantly compared to the GLOBES treatment group. At the same time, the total number of cells in the bronchoalveolar lavage fluid of the GLOBES treatment group and the total number of cells in the bronchoalveolar lavage fluid of the GLOBES treatment group were also significantly increased. There were no statistically significant differences between the groups.

[0044] Therefore, the above results all confirm that endowing allogeneic MSCs with immunocompatibility characteristics helps MSCs survive for a long time in pathological environments, thereby stably exerting anti-inflammatory and therapeutic effects, and achieving regeneration and repair of damaged tissues. Moreover, compared with universal MSCs constructed by lentiviral transduction and liposome transfection, the universal MSCs constructed by the optimal electrotransfection method in this invention have the most significant therapeutic effect on lung injury.

[0045] In another aspect, the present invention provides the use of universal mesenchymal stem cells for preparing agents that reduce allogeneic immune rejection, wherein the universal mesenchymal stem cells used in the present invention are constructed using any one of the above technical solutions.

[0046] In some implementations, to verify that GLOBES can maintain its low immunogenicity under inflammatory pathological conditions in the lungs, a GLOBES in vivo transplantation and recovery experiment was conducted. The results showed that the HLA-I expression level of control ntMSCs in vivo was 8.5 times higher than that of in vitro cultured ntMSCs in the in vivo pneumonia environment; while GLOBES in vivo maintained low HLA-I expression levels, with an increase of only about 2 times compared to in vitro cultured GLOBES, and this HLA-I expression level was only 20.7% of the HLA-I expression level in ntMSCs in vivo. Furthermore, subsequent experiments also showed a significant increase in HLA-I expression levels in both the GLOBES-1 and GLOBES-2 groups compared to the in vitro culture groups. Therefore, it can be seen that, compared with unedited MSCs, lentivirus-transduced GLOBES-1 and liposome-transfected GLOBES-2, the GLOBES constructed by the optimal electrotransfection method in this invention can still maintain its low immunogenicity in the inflammatory pathological environment of the lungs.

[0047] In another aspect, the present invention provides the use of a universal mesenchymal stem cell for preparing an agent that reduces the content of lactate dehydrogenase, albumin and / or total protein in bronchoalveolar lavage fluid, wherein the universal mesenchymal stem cell used in the application is constructed using the construction method described in any one of the above technical solutions.

[0048] In some implementations, the survival rate of transplanted cells in mice was tested in different groups of luc-GLOBES and luc-ntMSCs. The experimental results showed that, compared with unedited MSCs, GLOBES constructed by the optimal electroporation method in this invention did not induce an immune response in allogeneic MSCs and could survive for a longer time in mice with reconstituted human immune systems.

[0049] In subsequent experiments, the same procedure was performed on mice injected with equal amounts of luc-GLOBES-1 or luc-GLOBES-2 cells, and the cell survival rates were compared. The results showed that although no obvious immune rejection-induced cell death was observed in the GLOBES-1 and GLOBES-2 groups, the cell survival rate was significantly reduced.

[0050] Therefore, it is further demonstrated that, compared with unedited MSCs, lentivirally transduced GLOBES-1, and liposome-transfected GLOBES-2, the GLOBES constructed by the optimal electrotransfection method in this invention does not induce an immune response in allogeneic MSCs and can survive for a longer time in mice with reconstituted human immune systems.

[0051] In another aspect, the present invention provides the use of a universal mesenchymal stem cell for preparing a formulation that improves the survival time of mesenchymal stem cells in an in vivo inflammatory environment or maintains low immunogenicity of mesenchymal stem cells in vivo, wherein the universal mesenchymal stem cells used in the present invention are constructed using the construction method described in any one of the above technical solutions.

[0052] In another aspect, the present invention provides a universal CTL cell, which is constructed using any one of the above technical solutions.

[0053] In another aspect, the present invention provides the use of a universal CTL cell for preparing a formulation that inhibits NK cell activation or reduces the CD107a positivity rate, wherein the universal CTL cell used in the application is constructed using the construction method described in any one of the above technical solutions.

[0054] In another aspect, the present invention provides the use of a universal CTL cell for preparing an agent that reduces LDH content, wherein the universal CTL cell used in the application is constructed using the construction method described in any one of the above technical solutions.

[0055] In some embodiments, to further demonstrate that the universal CTL of the present invention can evade the killing effect of allogeneic NK cells, an LDH content detection experiment was conducted. The results showed that compared with the negative control group, the killing effect of NK cells on K562 cells in the positive control K562 group was significantly enhanced, reaching 21.3%; while in the CTL group and the universal CTL group, the killing effect of NK cells on CTL (4.84%) and universal CTL (4.4%) was not significantly different from that in the negative control group. This further demonstrates that the universal CTL can evade the killing effect of allogeneic NK cells.

[0056] In subsequent experiments, the NK cell killing ability of two groups of universal CTL cells constructed using lentivirus transduction and three groups of universal CTL cells constructed using liposome transfection was tested. The results showed that the NK cell killing ability of the universal CTL cells constructed using lentivirus transduction reached 6.85%, while that of the universal CTL cells constructed using liposome transfection reached 7.06%. Although this was significantly lower than the killing ability of NK cells in the positive control group, it was still higher than that in the universal CTL group. This may be because lentivirus introduces viral DNA, and liposome transfection also introduces liposomes. Since both viral DNA and liposomes have certain toxicity, they may cause mutations in the sequence of the super-enhancer in the B2M gene that responds to IFN-γ stimulation. This mutation may affect the interaction between the super-enhancer and surrounding regulatory elements, thereby affecting the expression of the B2M gene. The β2-microglobulin encoded by the B2M gene is an important component of major histocompatibility complex (MHC) class I molecules and plays an important role in the immune response. Therefore, alterations in B2M gene expression may affect NK cell recognition and killing, ultimately leading to enhanced NK cell killing ability against lentivirally transduced universal CTL cells 2 and liposome-transfected universal CTL cells 3. Conversely, lentiviral and liposome transfection may also alter the antigens or ligands expressed on the CTL cell surface, affecting NK cell recognition and killing, thus further enhancing NK cell killing ability against lentivirally transduced universal CTL cells 2 and liposome-transfected universal CTL cells 3. This further demonstrates that the universal CTL cells constructed using the optimal electrotransfection method in this invention exhibit the best performance and are the most reliable in evading allogeneic NK cell killing.

[0057] In another aspect, the present invention provides the use of a universal CTL cell for preparing a formulation that permanently reduces the immunogenicity of CTL cells or evades allogeneic lymphocyte killing, wherein the universal CTL cell used in the application is constructed using the construction method described in any one of the above technical solutions.

[0058] In some embodiments, to verify that the optimal universal CTL cells constructed in this invention do not induce allogeneic immune responses, allogeneic PBMCs (peripheral blood mononuclear cells) were labeled with CFSE dye, and co-cultured with CTL cells, universal CTL cells, K562 cells, and human PBMCs, respectively. Flow cytometry was used to detect PBMC cell proliferation, and the effects of different cell types on human PBMCs were compared. The results showed that PBMCs in the CTL group exhibited significant proliferation (47.5%), while the proliferation rate of PBMCs in the universal CTL group (17.1%) was not significantly different from that in the single-cell culture group (12.0%). This indicates that universal CTLs did not induce a strong immune response. This may be because when CTL cells stimulate PBMCs, PBMCs undergo a strong immune response, resulting in a large proliferation of PBMCs. Therefore, when there is no significant difference between the proliferation rate of PBMCs in the universal CTL group and the proliferation rate of PBMCs in the culture-only group (12.0%), it indicates that the optimal universal CTL cells constructed in this invention do not induce allogeneic immune responses, have low immunogenicity, and can maintain low immunogenicity in an inflammatory environment.

[0059] In some embodiments, to verify that the optimal universal CTL cells constructed in this invention can evade the killing effect of allogeneic NK cells, co-culture experiments were conducted using CTL, universal CTL, K562 cells, and NK cells, and the activation marker CD107a of NK cells was detected. The results showed that compared to the negative control group (CD107a positive rate of 3.05%), the positive rate of CD107a in the positive control K562 group was significantly increased (CD107a positive rate of 18.1%), while there was no significant difference between the CTL group (CD107a positive rate of 5.52%) and the universal CTL group (CD107a positive rate of 4.54%) and the negative control group; this indicates that universal CTL does not activate allogeneic NK cells.

[0060] To further demonstrate that the universal CTLs constructed using the method of this invention have the best performance, subsequent experiments further compared universal CTL cells 2 constructed using lentiviral transduction and universal CTL cells 3 constructed using liposome transfection. Under the same culture conditions, co-culture experiments were conducted with NK cells, and the activation marker CD107a of NK cells was detected. The results showed that the CD107a positivity rate was 7.12% in the universal CTL cell 2 group constructed using lentiviral transduction, and 7.95% in the universal CTL cell 3 group constructed using liposome transfection. Although the CD107a positivity rate was significantly lower than that in the positive control group, it was still higher than that in the universal CTL group. Therefore, it can be concluded that the universal CTLs constructed using the optimal electrotransfection method in this invention have the best performance and are the most reliable in terms of performance without activating allogeneic NK cells.

[0061] In another aspect, the present invention provides the use of a universal CTL cell for preparing a formulation that enhances the killing ability of CTL cells against tumor cells, wherein the universal CTL cell used in the present invention is constructed using any one of the above technical solutions.

[0062] The beneficial effects of this invention are as follows:

[0063] 1. This invention utilizes electroporation combined with CRISPR-based epigenetic editing to construct a universal mesenchymal stem cell and universal CTL cell that maintain low immunogenicity in an inflammatory environment: 1) The universal mesenchymal stem cells, when injected intravenously, do not induce an immune rejection response; and compared to untreated mesenchymal stem cells, universal mesenchymal stem cells constructed through lentiviral transduction or liposome transfection, they exhibit better therapeutic effects on pneumonia and regenerative repair effects on damaged lung tissue, and show more significant improvement in the pathological phenotype and multiple biochemical indicators of acute pneumonia; furthermore, they can be used in the lungs... Maintaining low immunogenicity in the pathological environment of disease solves the problem of immune rejection of allogeneic stem cells by the human immune system in regenerative therapies based on stem cell transplantation; at the same time, it can survive for a longer time in the pathological environment of lung diseases, thereby exerting a better anti-inflammatory effect; 2) The universal TCL cells described can exert significant therapeutic effects in the treatment of tumors and autoimmune diseases, effectively improve the targeted killing ability of tumor cells, and better balance the immune system. This provides a solid theoretical basis for its wide application in clinical practice, and also opens up new avenues and possibilities for the treatment of various related diseases, with broad application prospects.

[0064] 2. This invention utilizes electroporation combined with CRISPR-based epigenetic editing to construct universal mesenchymal stem cells. In terms of safety, this epigenetic editing method does not affect the genomic sequence of the cells, and the construction method is safe and simple. Furthermore, the universal mesenchymal stem cells gene-edited using this method do not possess the ability to form tumors in vivo. Using the universal mesenchymal stem cells constructed through epigenetic editing in an inflammatory environment, maintaining low immunogenicity, to treat acute lung injury provides a novel, safe, and effective treatment strategy for acute pneumonia. It also provides a new cell source and effective treatment method for mesenchymal stem cell therapy of acute lung injury. After transplantation, these universal mesenchymal stem cells can stably exert their therapeutic effect without immune rejection, significantly improving the symptoms of acute lung injury. This provides technical support and new ideas for the clinical treatment of acute pneumonia and has promising application prospects.

[0065] 3. This invention provides a method for constructing universal CTL cells. Utilizing transient CRISPRoff technology combined with electroporation, specific sites of the super-enhancer are edited in the epigenetics of CTL cells, resulting in the first universal CTL cells constructed without any alteration to the DNA sequence. Furthermore, addressing issues such as modifying the natural resistance of CTL and other lymphocytes to lentiviral systems, the editing strategy has been modified and upgraded. A virus-independent transient transfection method is employed, achieving a permanent reduction in CTL cell immunogenicity with a single transient epigenetic edit. The constructed universal CTLs can avoid allogeneic immune rejection. Simultaneously, this construction method avoids the technical difficulties of editing CTLs with viral systems and the safety issues such as off-target editing caused by the continuous stable expression of viral systems. It eliminates the need for multiple gene edits of CTL cells, the introduction of exogenous viral genes into the CTL genome, the need for persistent expression of the CRISPRi editing system, and the need for gene knockout or overexpression. Unlike conventional gene editing strategies, it does not cause permanent changes to the CTL genome sequence, thus combining simplicity and safety. Attached Figure Description

[0066] Figure 1 shows the transfection efficiency and cell viability results under different electroporation parameters.

[0067] Figure 2 shows the transfection efficiency and cell viability results under different plasmid dosages.

[0068] Figure 3A is a schematic diagram of mouse lung tissue after different treatment groups in vitro.

[0069] Figure 3B shows the HE staining results of mouse lung tissue after different treatment groups.

[0070] Figure 4A shows the LDH content in bronchoalveolar lavage fluid of different groups.

[0071] Figure 4B shows the albumin content in bronchoalveolar lavage fluid of different groups.

[0072] Figure 4C shows the results of the determination of total protein content in bronchoalveolar lavage fluid of different groups.

[0073] Figure 4D shows the results of the determination of total cell count in bronchoalveolar lavage fluid of different groups.

[0074] Figure 5A shows the qualitative imaging fluorescence results of BLI in mice from different groups.

[0075] Figure 5B shows the survival rate of transplanted cells in mice in different groups.

[0076] Figure 6 shows the flow cytometry results of HLA-I expression levels in mice in different groups, both in vivo and in vitro.

[0077] Figure 7A shows the fluorescence results of co-transfecting human CTL cells with CRISPRoff and sgRNA targeting B2M-SE.

[0078] Figure 7B shows the flow cytometry results of HLA-I protein expression in CTL cells.

[0079] Figure 8 shows the transfection efficiency results under different electrotransfer parameters.

[0080] Figure 9 shows the cell viability results under different plasmid dosages.

[0081] Figure 10A shows the flow cytometry results of PBMCs co-cultured with CTLs or general-purpose CTLs, and the proliferation of the cells detected by flow cytometry.

[0082] Figure 10B is a bar chart showing the proliferation of PBMCs after co-culturing CTL or universal CTL with allogeneic PBMCs.

[0083] Figure 11A shows the results of the CD107a positivity rate in different groups.

[0084] Figure 11B is a bar chart showing the proportion of CD107a positive cases in different groups.

[0085] Figure 12 shows the results of NK cell killing ability in different groups. Detailed Implementation

[0086] The present invention will be further described in detail below with reference to the embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0087] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0088] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0089] In this invention, CTL cells, PBMCs, and NK cells were all obtained from the Zhejiang Provincial Blood Center, with the ethics approval number being ZJBC / YW / 05-01.

[0090] CTL cells: cytotoxic T lymphocytes;

[0091] Universal CTL cells: CTL cells of allogeneic origin that can evade immune rejection;

[0092] PBMC: Peripheral blood mononuclear cell;

[0093] NK cells: Natural killer cells.

[0094] Example 1: Optimal Construction Method of Universal Mesenchymal Stem Cells (Universal MSCs) of the Present Invention

[0095] This invention proposes a novel method for constructing a universal cell system, aiming to provide an efficient, stable, and widely applicable construction strategy. This method plays a crucial role in enhancing cell therapy efficacy, expanding the scope of cell applications, and permanently reducing cellular immunogenicity. In the following embodiments, the construction of universal mesenchymal stem cells and universal CTL cells are used as examples to describe the specific construction process and the functions of the resulting universal mesenchymal stem cells and universal CTL cells in detail.

[0096] In this embodiment, the optimal method for constructing universal mesenchymal stem cells is first described in detail.

[0097] Given the recognized immunomodulatory and anti-inflammatory capabilities of MSCs, MSC stem cell therapy has emerged as a potential treatment for ALI. However, autologous MSCs face limitations in clinical application due to insufficient cell sources, batch instability, high cost, and long preparation cycles. While allogeneic MSCs can address these limitations to some extent, they are subject to allogeneic immune rejection, preventing them from sustaining their therapeutic effects in the recipient and resulting in poor efficacy.

[0098] In the invention patents with publication numbers CN113846063A and CN113801881A, the inventors had previously constructed and obtained "a universal human stem cell suitable for allogeneic transplantation," but it was not applied to specific indications such as acute lung injury. This may be related to the use of lentiviral transduction: on the one hand, the lentiviral transduction procedure is relatively complex, highly selective for cell types, and requires high operational skills from researchers, making it difficult to popularize in general laboratories; on the other hand, although mesenchymal stem cells are generally considered to have low immunogenicity, lentiviral vectors and their transgenic products may trigger an immune response, leading to rejection of the modified cells by the body; at the same time, the random insertion of lentiviruses into the cell genome may damage important gene functions within the cell, or even lead to cell malignancy; all of these may pose potential safety hazards. Therefore, in subsequent research, the construction method of universal mesenchymal stem cells was further optimized in order to construct a safer and more stable universal mesenchymal stem cell, which has good therapeutic effects in various indications.

[0099] Therefore, in this embodiment, a novel universal mesenchymal stem cell was constructed using electroporation combined with CRISPRoff. This universal mesenchymal stem cell can maintain low immunogenicity in an inflammatory environment, enabling it to avoid immune rejection and stably exert its therapeutic effect after transplantation, significantly improving ALI symptoms and exhibiting better regenerative and repair effects on damaged lung tissue. The specific construction method is as follows:

[0100] 1. sgRNA design and recombinant plasmid construction (the specific construction process of the recombinant plasmid, the sequence of the sgRNA, and the sequence of the enhancer are all based on the construction process in the invention patent with publication number CN113846063A):

[0101] ① Design sgRNAs targeting the enhancer sequence (SEQ ID NO.1) (Benchling website: https: / / www.benchling.com / crispr / ). The sgRNA sequences are shown in the table below:

[0102] Table 1. sgRNA Sequences

[0103] Restriction sites were added to both ends of the sgRNA. A CACC was added to the 5' end of the sense strand and an AAAC was added to the 5' end of the antisense strand, forming sticky ends complementary to those of the pLV-U6-gRNA-UbC-eGFP-P2A-Bsr plasmid (Addgene:#83925) after Fast Digest BBSI digestion. If the first base at the 5' end of the sense strand was not G, a G was added after the CACC at the 5' end, and a C was added to the 3' end of the corresponding antisense strand. pLV-U6-gRNA-UbC-eGFP-P2A-Bsr is an sgRNA backbone expression vector containing the U6 promoter, carrying the GFP (green fluorescent protein) gene and ampicillin resistance.

[0104] ① pLV-U6-gRNA-UbC-eGFP-P2A-Bsr was digested with Fast Digest Bbs I, and the linearized vector was recovered after DNA gel electrophoresis.

[0105] ② The sgRNA sequence was phosphorylated and annealed using T4 PNK; the linear pLV-U6-gRNA-UbC-eGFP-P2A-Bsr plasmid vector was ligated with the annealed sgRNA double-stranded sequence using T4 ligase at room temperature for 1 h. The ligation product was transformed into competent bacteria Trans 109, incubated on ice for 30 min, at 42℃ for 45 s, and on ice for 2 min. Clones were screened on ampicillin-resistant LB plates. Positive clones were picked, cultured, and sequenced. The sequencing primer was the forward primer sequence of the U6 promoter, 5'-GAGGGCCTATTTCCCATGATTCC-3' (SEQ ID NO.18). The clone with correct sequencing was the recombinant plasmid.

[0106] 2. Collect mesenchymal stem cells in centrifuge tubes and centrifuge at 400×g for 5 minutes. Discard the supernatant, resuspend in PBS, and count the cells. The method for obtaining the mesenchymal stem cells is specifically based on the method in Chinese invention patent application CN111849885A, which involves inducing hESCs to differentiate into MSCs to obtain a large number of mature MSCs (wherein, the hESC line is WA09, purchased from the WiCell Research Center, and relevant terms and conditions were signed).

[0107] 3. Preparation of the transfection complex: Electroporation was performed using a Neon transfection system (Invitrogen, MPK5000). For a 10 μL electroporation system, take 2 × 10⁻⁶ ppm of the solution. 5Each mesenchymal stem cell was resuspended in 9 μL of electroporation buffer and 1 μL of electroporation buffer containing 300 ng of plasmid mixture (including 150 ng of CRISPRoff and 150 ng of sgRNA) was added (the CRISPRoff is a commercially available plasmid).

[0108] 4. Electroporation: Transfer the resulting mixture containing cells and plasmids to 10 μL tip (Neon) and perform electroporation using the electroporation conditions of "1400V / 10ms / 3pulses" (tip here refers to a laboratory pipette / absorption tool);

[0109] 5. Immediately after electroporation, transfer the cells to wells containing preheated fresh culture medium and incubate at 37°C with 5% CO2. For 24-well plates, seed each well with 5 reactants (total 1×10⁻⁶). 6 (1 cell), inoculated with 0.5 mL of culture medium.

[0110] Based on the invention patent with publication number CN113846063A, which describes how knocking down or silencing a portion of the enhancer gene sequence of the B2M gene in stem cells can maintain low immunogenicity in inflammatory environments, this embodiment utilizes the super enhancer SE on the B2M gene that responds to IFN-γ stimulation, as described in the aforementioned patent, and further employs electroporation combined with CRISPRoff epigenetic editing to construct a universal mesenchymal stem cell.

[0111] In this embodiment, a universal mesenchymal stem cell (MSC) was constructed using electroporation combined with epigenetic editing CRISPRoff: (1) it maintains low immunogenicity in an inflammatory environment and does not cause immune rejection after intravenous injection; (2) compared to unedited MSCs, it can survive in vivo for a longer time, thus exerting a better anti-inflammatory effect; (3) in terms of safety, the epigenetic editing CRISPRoff method does not affect the genomic sequence of the cells, and the construction method is safe and simple. Experimental data in the following embodiments also demonstrate that MSCs gene-edited using the method of this invention do not have the ability to form tumors in vivo.

[0112] This invention provides a novel, safe, and effective treatment strategy for acute lung injury by constructing universal MSCs with low immunogenicity under inflammatory conditions through epigenetic editing.

[0113] Example 2: Further exploration and optimization of a universal mesenchymal stem cell construction method

[0114] I. Screening of Transfection Methods

[0115] In this embodiment, to construct a safer, more stable, and less immunogenic universal mesenchymal stem cell, different transfection methods were further screened based on the original method for constructing universal mesenchymal stem cells, including: lentiviral transduction, electroporation, and liposome transfection. The specific operations are as follows:

[0116] 1. A universal MSC was constructed using the electroporation method described in Example 1;

[0117] 2. Construction of universal MSCs using lentivirus transduction 2: The day before, 2×10 5 MSCs were seeded into 6-well plates, aiming for 70% confluence at infection the following day. Before infection, the medium was replaced with fresh complete medium, and the virus particles were diluted with an appropriate amount of fresh complete medium at a multiplicity of infection (MOI) of 0.3. The diluted virus particles were then added dropwise through a 0.2 μm filter to the wells containing MSCs. 2 μl of 10 mg / ml polybrene (final concentration 10 μg / ml) was added. After mixing by cross-rotation, the cells were centrifuged at 1300 rpm at 37°C for 45 min (both acceleration and deceleration were set to "0") to facilitate better binding of the virus to the target cells. After cell expansion culture, flow cytometry was used to sort cells that were double-positive for both mCherry (red fluorescence) and GFP (green fluorescence). The surviving MSCs were classified as universal MSCs 2.

[0118] 3. Construct universal MSCs using liposome transfection: 3: One day in advance, prepare 2×10 6 Seed MSCs in 10cm cell culture dishes, ensuring 60% cell confluence by the second day. The optimal transfection time is 12-18 hours after seeding. Following the Lipofectamine 3000 reagent (Invitrogene, catalog number: L3000015) instructions, incubate 2.4μg CRISPRoff, 1.2μg sgRNA, and 20μl Lipofectamine 3000 at room temperature for 10 minutes, then co-transfect MSCs. After 48 hours, perform flow cytometry sorting to separate cells that are double-positive for BFP (blue fluorescence) and GFP (green fluorescence). The surviving MSCs are considered universal MSCs.

[0119] The transfection efficiency and cell viability of the different universal MSCs constructed were then tested.

[0120] The steps for detecting the transfection efficiency are as follows: cells are transfected according to different transfection methods, and the fluorescence value is detected on a flow cytometer (BD Biosciences, FACSAria Fusion SORP). Successfully transfected cells will have red (mCherry) and green (GFP) fluorescence. The position of the control group is the position where no fluorescence is expressed. A cross gate is drawn to determine the percentage of cells in the experimental group that have shifted, i.e., the transfection rate.

[0121] The cell viability assay procedure is as follows: Take 0.5 mL of cell suspension (1 × 10⁻⁶) 5 Approximately 10 μL of 7-aminoactinomycin D (7-AAD) staining agent was added to each cell, and the cells were incubated at room temperature for 2 minutes. The fluorescence intensity was then detected by flow cytometry (FL3 channel) based on the excitation / emission wavelengths (Ex / Em = 545 nm / 650 nm). 7-AAD is a non-permeable fluorescent dye; it cannot pass through the cell membrane of living cells but can penetrate the cell membrane of dead cells and bind to their DNA, thus facilitating the detection of the ratio of living to dead cells using flow cytometry.

[0122] The specific results are shown in Table 2:

[0123] Table 2. Transfection efficiency and cell viability of universal MSCs constructed using different transfection methods

[0124] As shown in Table 1, the universal mesenchymal stem cells constructed by electroporation have the best transfection efficiency and cell viability. This may be because: (1) Lentiviral transduction has some unavoidable drawbacks. Its viral DNA can be permanently integrated into the MSC genome, causing the potential risk of insertional mutations, which may lead to abnormal cell function or even cancer. At the same time, the preparation process of lentivirus is relatively complicated, the operation is relatively complex, and the requirements for experimental environment and operators are relatively strict. (2) The transfection efficiency of liposome transfection is often affected by a variety of factors, such as the ratio of liposomes to nucleic acids and the state of cells, which has a certain degree of instability. In addition, liposomes themselves may produce a certain degree of toxicity to cells, affecting the normal physiological function of cells. (3) However, electroporation exhibits unique characteristics. Electroporation causes changes in cell membrane potential through a high-intensity electric field, which instantaneously increases cell membrane permeability and creates reversible pores on the cell membrane to facilitate the entry of exogenous nucleic acids. It is applicable to almost all types of cells. Compared to the former two, electroporation has the following advantages: it can control the transfection process more precisely, and the transfection efficiency is relatively high and more stable; it causes less damage to cells because the electric field is applied for a short time; and it is applicable to a wider range of cell types, making it more versatile.

[0125] Therefore, in this embodiment, electrotransfection is preferred for constructing universal mesenchymal stem cells.

[0126] II. Screening of Electroporation Parameters

[0127] Electroporation parameters have a significant impact on transfection efficiency, cell viability, and subsequent growth status. Therefore, in this embodiment, different electroporation parameters were further screened in order to minimize cell damage and improve cell viability while achieving good transfection efficiency.

[0128] Specifically, a negative control (i.e., no electroporation) and three experimental groups were set up. The electroporation parameters for the three experimental groups were as follows: (1) 990V voltage, 40ms pulse duration, one pulse; (2) 1200V voltage, 20ms pulse duration, two pulses; (3) 1400V voltage, 10ms pulse duration, three pulses. The transfection efficiency was determined using a flow cytometer (BD Biosciences, USA, FACSAria Fusion SORP).

[0129] The specific results are shown in Figure 1. As can be seen from Figure 1, a high electroporation efficiency was achieved under the electroporation conditions of 1400V / 10ms / 3pulse (voltage 1400V, pulse duration 10ms, 3 pulses). The electroporation efficiency was 93.58%. Therefore, in this embodiment, the preferred electroporation parameters are 1400V / 10ms / 3pulse to prepare general-purpose MSCs.

[0130] III. Screening of Plasmid Amount

[0131] In electroporation, the amount of plasmid used has a significant impact on transfection efficiency and cell viability. Therefore, in this embodiment, different amounts of plasmid were further screened: the amount of plasmid was set to three dosage groups of 400ng, 600ng and 800ng, and different universal mesenchymal stem cells were constructed. The optimal amount of plasmid was determined by comparing the proportion of BFP and GFP fluorescent double positive cells. The specific results are shown in Figure 2.

[0132] As shown in Figure 2, the proportion of double-positive cells gradually increased from 3.7% to 15.9% with increasing plasmid dosage. However, when the plasmid dosage exceeded 800 ng, cell viability decreased significantly. This may be due to a saturation effect or unintended effects caused by excessive plasmid interaction with cells. Therefore, in this embodiment, the preferred plasmid dosage is 800 ng.

[0133] Example 3: Construction of a universal mesenchymal stem cell line stably expressing luciferase

[0134] In this embodiment, to better track universal mesenchymal stem cells in subsequent verification experiments and thus verify the experimental results, a step of "constructing universal mesenchymal stem cells stably expressing luciferase" was added. Specifically, lentiviral transduction was used to construct universal mesenchymal stem cells stably expressing luciferase. The specific construction steps are as follows (wherein, GLOBES is the universal mesenchymal stem cell constructed by optimal electrotransfection according to this invention, ntMSCs is untreated mesenchymal stem cells, GLOBES-1 is the universal mesenchymal stem cell constructed by lentiviral transduction in Example 2, and GLOBES-2 is the universal mesenchymal stem cell constructed by liposome transfection in Example 2):

[0135] 1. Preparation of luciferase lentivirus: Take 5 μg of core plasmid luciferase-neo (Addgene, catalog number: 105621), 6 μg of packaging plasmid psPAX2 (Addgene, catalog number: 12260) and 6 μg of envelope plasmid pMD2.G (Addgene, catalog number: 12259) to prepare luciferase lentivirus;

[0136] 2. Cell infection: The prepared luciferase lentivirus was used to infect GLOBES, ntMSCs, GLOBES-1 and GLOBES-2 respectively;

[0137] 3. Neomycin drug screening: Neomycin drug screening was performed at a concentration of 300 μg / μL;

[0138] 4. Obtaining stable expression cells: GLOBES, ntMSCs, GLOBES-1 and GLOBES-2 cells that stably express luciferase were obtained through drug screening and named luc-GLOBES, luc-ntMSCs, luc-GLOBES-1 and luc-GLOBES-2, respectively.

[0139] In subsequent embodiments, the therapeutic effects of luc-GLOBES, luc-ntMSCs, luc-GLOBES-1, and luc-GLOBES-2 constructed using three different transfection methods in Example 2 on acute lung injury were compared and tested. Subsequent verification experiments showed that luc-GLOBES constructed using the optimal electrotransfection method of this invention exhibited the best therapeutic effect on lung injury. Therefore, this does not conflict with the use of lentiviral transduction to construct stable luciferase-expressing universal mesenchymal stem cells for better tracking of universal mesenchymal stem cells. This indicates that in subsequent clinical applications, since the effectiveness of the optimal universal mesenchymal stem cells constructed using electrotransfection in this invention has been verified, it is unnecessary to construct "stable luciferase-expressing universal mesenchymal stem cells" again, and therefore, the lentiviral transduction method is unnecessary. Thus, by omitting the step of "constructing stable luciferase-expressing universal mesenchymal stem cells," the efficacy of the optimal universal mesenchymal stem cells constructed using electrotransfection in this invention in the treatment of lung injury can be further improved.

[0140] Example 4: Testing of GLOBES' more significant therapeutic effect on acute pneumonia

[0141] I. Construction of an LPS-induced acute pneumonia model and in vivo bioluminescence imaging tracking experiment

[0142] In this embodiment, to better compare the therapeutic effects of luc-GLOBES, luc-ntMSCs, and luc-GLOBES-1 and luc-GLOBES-2 on acute lung injury, an LPS-induced acute pneumonia model was constructed, and BLI (bioluminescence imaging) detection was performed. Bronchoalveolar lavage and HE staining experiments were then conducted in subsequent experiments. The specific operational steps are as follows:

[0143] (1) Three days in advance, allogeneic PBMCs were co-cultured with wild-type MSCs to sensitize PBMCs;

[0144] (2) Purchase B-NDG hIL15 mice from Biocytogen. After one week of acclimatization, anesthetize the mice with pentobarbital at a dose of 60 mg / kg and fix the mice vertically to expose the throat.

[0145] (3) Insert the indwelling tube accurately into the trachea of ​​the mouse, and use a micropipette to drip the LPS solution into the lungs of the mouse through the indwelling tube at a dose of 2 mg / kg.

[0146] (4) After the LPS infusion is completed, continue to maintain an upright posture for 2 minutes to ensure that the LPS is fully inhaled into the lungs;

[0147] (5) Five hours after LPS stimulation, administer 1×10 7 / mouse, inject the sensitized PBMCs from step 1 into mice via the tail vein to construct a mouse model of human immune system reconstruction by PBMC reinfusion.

[0148] (6) One hour after re-injecting the PBMC, press 1×10 6 Cell therapy was performed by intravenous injection of luc-GLOBES, luc-ntMSCs, luc-GLOBES-1 or luc-GLOBES-2 into the tail vein, with a PBS group serving as a negative control.

[0149] (7) Two hours after injection of luc-GLOBES, luc-ntMSCs, luc-GLOBES-1 or luc-GLOBES-2, BLI was measured and used as the initial BLI signal value on day 0.

[0150] (8) Three days later, administer the same amount of LPS via tracheal infusion again as in step (3) to maintain pneumonia symptoms; and test BLI again as in step (7);

[0151] (9) Two days after the second LPS infusion, the sample was collected; after taking a BLI image, the bronchoalveolar lavage experiment was performed or lung tissue was removed for HE staining.

[0152] II. HE staining

[0153] (1) Remove the lung tissue from the mouse and fix it with 4% paraformaldehyde for 24 hours;

[0154] (2) After dehydration and paraffin embedding, the sections were cut into 7μm thick sections;

[0155] (3) The sections were dewaxed with xylene in a fume hood and then hydrated with a gradient of ethanol. The time for each step was as follows: xylene (I) 1 hour, xylene (II) 1 hour, a mixture of xylene and ethanol of equal volume for 30 min, anhydrous ethanol (I) 2 min, anhydrous ethanol (II) 2 min, 95% ethanol 2 min, 80% ethanol 2 min, 70% ethanol 2 min, and distilled water for 10 min.

[0156] (4) Then stain the kernels with hematoxylin for 9 minutes, and soak and wash them in tap water 3 times, 5 minutes each time;

[0157] (5) Use an equal volume of hydrochloric acid and alcohol mixture to separate the colors for 1 second, then soak and rinse in tap water 3 times, 5 minutes each time;

[0158] (6) Stain with eosin for 1 second, then soak and rinse in tap water 3 times, 5 minutes each time;

[0159] (7) Dehydration: The slices were placed in 50% alcohol for 5 seconds, 70% alcohol for 5 seconds, 80% alcohol for 5 seconds, 90% alcohol for 5 seconds, anhydrous ethanol (I) for 5 seconds, and anhydrous ethanol (II) for 5 seconds in sequence for dehydration treatment.

[0160] (8) Transparency: The dehydrated slices were placed in a solution of equal volumes of xylene and ethanol for 1.5 min; xylene (I) for 2 min; xylene (II) for >2 min;

[0161] (9) Cover with a glass slide and seal with neutral resin; after the resin has dried completely, wipe off the excess resin with 95% alcohol, blow dry, and then sweep the slide with a slide sweeper.

[0162] III. Detection of Biochemical Indicators in Bronchoalveolar Lavage Fluid

[0163] (1) Alveolar lavage: After anesthetizing the mice, they were fixed to the operating table in a supine position, and the trachea was dissected and exposed using surgical instruments; a small incision was made in the trachea with surgical scissors, and an indwelling tube was inserted through the small incision and fixed to the trachea with surgical sutures; 0.5 mL of PBS was instilled into the lungs through the indwelling tube using a 1 mL syringe, and after repeated blowing and aspiration several times, it was aspirated and transferred to a 1.5 mL centrifuge tube and stored on ice; then the lungs were lavaged once more with 0.5 mL of PBS.

[0164] (2) Cell count: Centrifuge at 400×g at 4℃ for 10 min, resuspend the pellet in 1 mL PBS, and count cells using a CountStar instrument;

[0165] (3) The supernatant is used to determine various biochemical indicators, including: LDH content detection, albumin content detection, and total protein content detection. Specific procedures and results are as follows:

[0166] ①LDH content detection

[0167] Following the instructions of the LDH Cytotoxicity Assay Kit, control and detection media supernatants were prepared to measure lactate dehydrogenase (LDH) levels. Five replicates were set up for each experimental group, including a "spontaneous LDH group" and a "maximum LDH group." After co-culture, 10 μL of the lysis buffer from the kit was added to the wells of the "maximum LDH group," and the mixture was incubated at 37°C for 45 min. After lysis, the culture media from all groups were collected and centrifuged at 400 × g for 5 min. 50 μL of the centrifuged supernatant was transferred to a new 96-well plate, and 50 μL of the pre-prepared reaction mixture was added. After mixing, the plate was incubated at room temperature in the dark for 0.5 h. The reaction was terminated by adding 50 μL of Stop Solution, and the absorbance at 490 nm and 680 nm was measured using a microplate reader. LDH cytotoxicity was calculated using the formula provided in the kit.

[0168] ② Albumin content detection

[0169] 0.5 hours before the experiment, remove the ELISA strips and reagents from the plate and allow them to reach room temperature. Add 100 μL of the sample and standard working solution to each well, with 5 replicates per group. Seal the plate and incubate at 37°C for 90 min. Discard the liquid in the plate and shake off any remaining liquid. Add 100 μL of biotin-labeled albumin antibody working solution to each well, seal the plate, and incubate at 37°C for 60 min. Discard the liquid in the plate and shake off any remaining liquid. Add 350 μL of washing buffer to each well, soak for 2 min, and then shake off the washing buffer. Repeat this washing process 4 times. Add 100 μL of... HRP-labeled streptavidin working solution was sealed and incubated at 37°C for 30 min. 300 μL of washing buffer was added to each well, and the plate was soaked for 2 min before being spun dry. This washing process was repeated 4 times. Under light-protected conditions, 90 μL of chromogenic reagent was added to each well, and the plate was sealed and incubated at 37°C for 15 min. 50 μL of stop solution was added to each well, and the OD value at 450 nm was measured using a microplate reader within 5 min. The albumin concentration was calculated by plotting the standard concentration on the x-axis and the absorbance OD value on the y-axis.

[0170] ③ Total protein content detection

[0171] A. Preparation of Protein Standards: a. Add 1.2 mL of protein standard preparation solution to one tube of protein standard (30 mg BSA), dissolve thoroughly to prepare a 25 mg / mL protein standard solution. This solution can be used immediately or stored long-term at -20°C. b. Take an appropriate amount of 25 mg / mL protein standard and dilute to a final concentration of 0.5 mg / mL. For example, take 20 μL of 25 mg / mL protein standard and add 980 μL of diluent to prepare a 0.5 mg / mL protein standard. The standard should be diluted with the same solution as the protein sample. However, for simplicity, the standard can also be diluted with 0.9% NaCl or PBS. The diluted 0.5 mg / mL protein standard can be stored long-term at -20°C.

[0172] B. Preparation of BCA working solution: Based on the number of samples, prepare an appropriate amount of BCA working solution by adding 1 volume of BCA reagent B to 50 volumes of BCA reagent A (50:1), and mix thoroughly. For example, add 100 μL of BCA reagent B to 5 mL of BCA reagent A, mix well, and prepare 5.1 mL of BCA working solution. The BCA working solution is stable at room temperature for 24 hours.

[0173] C. Protein Concentration Detection: a. Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of standard solution to the standard wells of a 96-well plate, and then add standard diluent to bring the total volume to 20 μL. This corresponds to standard concentrations of 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg / mL, respectively. b. Add an appropriate volume of sample to the sample wells of the 96-well plate. If the sample volume is less than 20 μL, add standard diluent to bring the total volume to 20 μL. Please record the sample volume. c. Add 200 μL of BCA working solution to each well and incubate at 37°C for 20-30 minutes. d. Measure the absorbance at A562 or a wavelength between 540-595 nm using a microplate reader. e. Calculate the protein concentration of the sample based on the standard curve and the sample volume used.

[0174] IV. Experimental Results

[0175] 1. Results of in vitro observation and HE staining of lung tissue

[0176] In this embodiment, the lung tissue was first subjected to gross observation and HE staining after ex vivo to identify the improvement of lung inflammation and the integrity of tissue structure. The specific results are shown in Figures 3A and 3B.

[0177] The macroscopic view in Figure 3A shows the lung tissue of the PBS-treated group compared to that of normal mice. In the ntMSCs treatment group, significant lung inflammation and congestion were observed, indicating the successful establishment of the LPS-induced pneumonia model. Compared to the PBS treatment group, the ntMSCs treatment group showed some improvement in lung inflammation and congestion, but compared to the PBS treatment group, the ntMSCs treatment group showed improvement in lung inflammation and congestion. The group still had severe pneumonia symptoms, indicating that ntMSCs had limited therapeutic effect on pneumonia; while the GLOBES treatment group showed a much better improvement in pneumonia compared to the PBS treatment group, and... The normal lung tissue in the group is closer to that in the study, indicating that GLOBES has a better therapeutic effect on pneumonia.

[0178] Subsequent experiments compared the GLOBES-1 and GLOBES-2 treatment groups. Results showed that the GLOBES-1 treatment group exhibited significantly reduced inflammation and congestion in lung tissue compared to the GLOBES-1 and GLOBES-2 groups. This indicates that the GLOBES treatment group, constructed via electroporation, had a more significant effect on improving pneumonia. This may be because lentiviruses introduce viral DNA, and liposome transfection also introduces liposomes. Since both viral DNA and liposomes possess some toxicity, they may affect cell activity or biological function, thus impacting the therapeutic effect on lung injury and ultimately leading to a significant reduction in the overall therapeutic efficacy.

[0179] The HE staining in Figure 3B further confirms the above conclusions. As can be seen from Figure 3B, compared to... In the normal lung tissue of the control group, the lung tissue of mice in the LPS-induced PBS group showed typical acute pneumonia injury, namely, thickening of the alveolar septa, alveolar hemorrhage, and increased neutrophil infiltration. Although ntMSCs treatment alleviated the above-mentioned acute lung injury to some extent, compared with... In the normal lung tissue of the group, numerous areas of lung injury were still clearly visible. However, the lung tissue sections of the GLOBES treatment group differed significantly from those of the ntMSCs group, and were more similar to those of the ntMSCs group. The normal lung tissue section morphology of the group indicates that lung injury was significantly improved and repaired after GLOBES treatment. Subsequent experiments compared the group with the GLOBES-1 and GLOBES-2 treatment groups. The results showed that, compared to the GLOBES-1 and GLOBES-2 treatment groups, the GLOBES treatment group showed significant improvement and repair of lung injury after treatment. This further demonstrates that the GLOBES treatment group constructed via electroporation has a more significant lung injury repair effect.

[0180] Therefore, it can be concluded that, compared with the ntMSCs, GLOBES-1 and GLOBES-2 groups, the GLOBES treatment group constructed by electroporation has a more significant therapeutic effect on acute pneumonia.

[0181] 2. LDH content detection results

[0182] Figure 4A shows the LDH content in bronchoalveolar lavage fluid of different groups. As can be seen from Figure 4A, the LDH content in the bronchoalveolar lavage fluid of the PBS and ntMSCs groups was significantly higher than that of the GLOBES treatment group. The study found that the LDH content in the ntMSCs treatment group was approximately 2.05 times that of the GLOBES treatment group. LDH normally exists only in the cytoplasm of body tissue cells; a higher LDH content in bronchoalveolar lavage fluid indicates more severe tissue damage. This suggests that GLOBES treatment significantly improved and repaired lung injury. Subsequent experiments also measured the LDH content in the bronchoalveolar lavage fluid of the GLOBES-1 and GLOBES-2 treatment groups. The results showed that the LDH content in the GLOBES-1 treatment group was approximately 1.72 times that of the GLOBES treatment group, and the LDH content in the GLOBES-2 treatment group was approximately 1.85 times that of the GLOBES treatment group. While the LDH content in the GLOBES-1 and GLOBES-2 treatment groups decreased slightly compared to the ntMSCs treatment group, it was still significantly higher than that in the GLOBES treatment group. This further indicates that the GLOBES treatment group constructed via electroporation has a more significant lung injury repair effect.

[0183] Therefore, the detection results of LDH content in bronchoalveolar lavage fluid further prove that the GLOBES treatment group constructed by electroporation has a more significant effect on repairing acute pneumonia damage.

[0184] 3. Albumin content test results

[0185] Figure 4B shows the albumin content in bronchoalveolar lavage fluid of different groups. As can be seen from Figure 4B, although the ntMSCs treatment group can reduce the albumin content in bronchoalveolar lavage fluid to some extent, the corresponding albumin content is still significantly higher than that in the ntMSCs treatment group. The albumin content in the bronchoalveolar lavage fluid of the ntMSCs treatment group was approximately 1.43 times that of the GLOBES treatment group. Subsequent experiments also measured the albumin content in the bronchoalveolar lavage fluid of the GLOBES-1 and GLOBES-2 treatment groups. The results showed that the albumin content in the bronchoalveolar lavage fluid of the GLOBES-1 treatment group was approximately 1.28 times that of the GLOBES treatment group, and approximately 1.32 times that of the GLOBES treatment group. Compared to the ntMSCs treatment group, the albumin content in the bronchoalveolar lavage fluid of the GLOBES-1 and GLOBES-2 treatment groups decreased slightly, but still increased compared to the GLOBES treatment group. The albumin content in the GLOBES treatment group was close to that of the ntMSCs treatment group. The group's normal albumin content.

[0186] Therefore, the results of albumin content detection in bronchoalveolar lavage fluid further demonstrate that the GLOBES treatment group constructed by electroporation has a more significant effect on repairing acute pneumonia damage.

[0187] 4. Total protein content test results

[0188] The albumin and total protein content in bronchoalveolar lavage fluid (BALF) can reflect the permeability of lung tissue; higher protein permeability indicates more severe lung tissue damage. As shown in Figure 4C, the results of total protein content determination are similar to those of albumin determination. The total protein content in the ntMSCs treatment group was approximately 1.22 times that of the GLOBES treatment group. Subsequent experiments also found that the total protein content in the BALF of the GLOBES-1 and GLOBES-2 treatment groups was approximately 1.12 times and 1.15 times that of the GLOBES treatment group, respectively. Although the total protein content in the BALF of the GLOBES-1 and GLOBES-2 treatment groups was slightly lower than that of the ntMSCs treatment group, it was still higher than that of the GLOBES treatment group.

[0189] The total cell count in the bronchoalveolar lavage fluid can reflect the degree of inflammatory cell infiltration and permeation in lung tissue. As shown in Figure 4D, although the total cell count in the bronchoalveolar lavage fluid of the ntMSCs treatment group was significantly lower than that of the PBS treatment group, it was still significantly higher than that of the GLOBES treatment group. The total cell count in the bronchoalveolar lavage fluid of the ntMSCs treatment group was 1.77 times that of the GLOBES treatment group. Subsequent experiments also showed that the total cell count in the bronchoalveolar lavage fluid of the GLOBES-1 and GLOBES-2 treatment groups was 1.31 times and 1.42 times that of the GLOBES treatment group, respectively. The total cell count in the bronchoalveolar lavage fluid of the GLOBES-1 and GLOBES-2 treatment groups was also significantly increased compared to the GLOBES treatment group. The total cell count in the bronchoalveolar lavage fluid of the GLOBES treatment group and... There were no statistically significant differences between the groups.

[0190] Therefore, the above results all confirm that endowing allogeneic MSCs with immunocompatibility characteristics helps MSCs survive for a long time in pathological environments, thereby stably exerting anti-inflammatory and therapeutic effects, and achieving regeneration and repair of damaged tissues. Moreover, compared with universal MSCs constructed by lentiviral transduction and liposome transfection, the universal MSCs constructed by the optimal electrotransfection method in Example 1 have the most significant therapeutic effect on lung injury.

[0191] Example 5: GLOBES did not induce an immune response in allogeneic MSCs.

[0192] In this embodiment, mice were injected with equal amounts of luc-GLOBES and luc-ntMSCs cells. Two hours later, BLI imaging was performed using an IVIS imaging system of a small animal imaging device, which served as the initial BLI signal value on day 0. Subsequently, BLI detection was performed at the same time points on days 3 and 5, and the BLI signal values ​​on days 3 and 5 were recorded. Qualitative BLI fluorescence images were recorded. At the same time, the cell viability in the mice was recorded on days 0, 3, and 5, and the fluorescence signal was quantitatively analyzed using the built-in software of the IVIS imaging system of the small animal imaging device. The specific results are shown in Figures 5A and 5B.

[0193] Figure 5A shows the qualitative fluorescence imaging results of BLI in mice of different groups, and Figure 5B shows the survival rate of transplanted cells in mice of different groups. As shown in Figures 5A and 5B, after injecting equal amounts of GLOBES and control ntMSCs, unedited ntMSCs exhibited significant immune rejection and cell death in the inflammatory pathological environment of the lungs. On day 3, the survival rate of ntMSCs was only 45.15% of the initial transplanted amount, and by day 5, only 12.17% of ntMSCs survived. In contrast, GLOBES showed stable survival in the inflammatory pathological environment of the lungs, with a cell survival rate of 95.82% on day 3 and 85.48% of GLOBES still surviving by day 5.

[0194] In subsequent experiments, the same procedure was performed on mice injected with equal amounts of luc-GLOBES-1 or luc-GLOBES-2 cells, and the cell survival rates were compared. The results showed that although no obvious immune rejection-induced cell death was observed in the GLOBES-1 and GLOBES-2 groups, the cell survival rate was significantly reduced. By day 5, only 56.48% and 55.65% of GLOBES-1 and GLOBES-2 cells survived, respectively.

[0195] Therefore, it can be seen that, compared with unedited MSCs, lentivirally transduced GLOBES-1 and liposome-transfected GLOBES-2, the GLOBES constructed by the optimal electrotransfection method in Example 1 does not induce an immune response in allogeneic MSCs and can survive for a longer time in mice with reconstituted human immune systems.

[0196] Example 6: Tests demonstrating that GLOBES maintains low immunogenicity in an inflammatory pathological environment of the lungs.

[0197] To verify that GLOBES can maintain its low immunogenicity under the inflammatory pathological environment of the lungs, this example conducts a GLOBES in vivo transplantation and recovery experiment, the specific operation of which is as follows:

[0198] I. Preparation of single-cell suspension from lung tissue

[0199] (1) Humanized mice with LPS-induced acute pneumonia (mice with reconstituted human immune system) were treated with GLOBES, ntMSCs, GLOBES-1 or GLOBES-2 for 48 hours. The mice were then euthanized by cervical dislocation, and the lungs were removed from the mice in a clean bench and soaked in PBS.

[0200] (2) Place 3 mL of mouse lung cell separation medium (DMEM / F12+15mM HEPES+1% (v / v) penicillin-streptomycin+1×Glutamax) in a 35 mm culture dish. This means that the mouse lung cell separation medium consists of DMEM / F12 cell culture medium with 15 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (also called HEPES) buffer, 1% penicillin-streptomycin mixture, and glutamine solution.

[0201] (3) Transfer the mouse lungs to the above-mentioned 35mm culture dish and cut the lung tissue into 1mm pieces using surgical scissors. 2 Fragments of various sizes;

[0202] (4) Transfer all lung fragments to a 15mL centrifuge tube, add 4mL of mouse lung digestion solution (lung cell separation solution + 2mg / mL collagenase + 0.1mg / mL DNase I, indicating that 2mg / mL collagenase and 0.1mg / mL deoxyribonuclease I were added to the lung cell separation solution), digest in a 37℃ water bath for 2 hours, shaking and mixing once every half hour;

[0203] (6) After digestion, the digestion solution was passed through a 40μm sieve and centrifuged at 460rpm for 5min.

[0204] (7) After centrifugation, discard the supernatant, add 3 mL of red blood cell lysis buffer, and lyse at room temperature for 10 min;

[0205] (8) After the red blood cell lysis was completed, the red blood cell lysis buffer was diluted with 9 mL of lung cell separation solution, centrifuged at 460 rpm for 5 min, the supernatant was discarded after centrifugation and resuspended in PBS to obtain a single cell suspension of lung tissue.

[0206] (9) The expression levels of HLA-I in mice in vivo and in vitro were detected by flow cytometry (Beckman Coulter, DxFLEX), including: blank group (no treatment), GLOBES group, ntMSCs group, GLOBES-1 group and GLOBES-2 group.

[0207] II. Experimental Results

[0208] Figure 6 shows the flow cytometry results of HLA-I expression levels in mice in different groups, both in vivo and in vitro. As can be seen from Figure 6, the flow cytometry results, obtained through the GLOBES in vivo transplantation and recovery experiment, indicate that the HLA-I expression level in control ntMSCs in vivo was 8.5 times higher than that in vitro cultured ntMSCs in the in vivo pneumonia environment. In contrast, GLOBES in vivo maintained low HLA-I expression levels, with an increase of only about 2 times compared to in vitro cultured GLOBES, and this HLA-I expression level was only 20.7% of the HLA-I expression level in ntMSCs.

[0209] In subsequent experiments, it was also found that the HLA-I expression levels in the GLOBES-1 group (GLOBES-1 in vivo) and the GLOBES-2 group (GLOBES-2 in vivo) were significantly higher than those in the in vitro culture group.

[0210] Therefore, it can be seen that, compared with unedited MSCs, lentivirus-transduced GLOBES-1 and liposome-transfected GLOBES-2, the GLOBES constructed by the optimal electrotransfection method in Example 1 can still maintain its low immunogenicity in the inflammatory pathological environment of the lungs.

[0211] Example 7: Optimal Construction Method of Universal CTL Cells Provided by the Present Invention

[0212] In this embodiment, the method for constructing universal CTL cells was further optimized in order to construct a safer and more stable universal CTL cell, which has good therapeutic effects in various indications.

[0213] Therefore, in this embodiment, based on the super enhancer (B2M-SE) on the B2M gene discovered in the aforementioned patent, the epigenetic editor CRISPRoff and sgRNA targeting B2M-SE are co-transfected into human CTL cells using electroporation. This allows for epigenetic editing of the super enhancer at specific sites within the CTL cells, thus constructing, for the first time, universal CTL cells without any DNA sequence alterations. Furthermore, compared to the aforementioned strategy of constructing universal human stem cells using a lentiviral system, this invention modifies and upgrades the editing strategy to address the issues of natural resistance of CTL and other lymphocytes to lentiviral system modification. It employs a virus-independent transient transfection method, enabling a permanent reduction in CTL cell immunogenicity with a single transient epigenetic edit. Moreover, the constructed universal CTL cells can circumvent allogeneic immune rejection.

[0214] Meanwhile, the construction method provided by this invention avoids both the technical difficulties of editing CTLs in viral systems and the safety issues such as off-target editing caused by the continuous and stable expression of viral systems. This construction strategy does not require multiple gene editing of CTL cells, nor does it require the introduction of exogenous viral genes into the CTL genome, and it does not cause permanent changes to the CTL genome sequence like ordinary gene editing strategies. Therefore, it combines simplicity and safety.

[0215] This invention provides a method for constructing universal CTL cells, the specific steps of which are as follows:

[0216] 1. sgRNA design and recombinant plasmid construction (the specific construction process of the recombinant plasmid, the sequence of the sgRNA, and the sequence of the enhancer are all based on the construction process in the invention patent with publication number CN113846063A):

[0217] ① Design sgRNAs targeting the enhancer sequence (SEQ ID NO.1) (Benchling website: https: / / www.benchling.com / crispr / ). The sgRNA sequences are shown in the table below:

[0218] Table 3. sgRNA Sequences

[0219] Restriction sites were added to both ends of the sgRNA. A CACC was added to the 5' end of the sense strand and an AAAC was added to the 5' end of the antisense strand, forming sticky ends complementary to those of the pLV-U6-gRNA-UbC-eGFP-P2A-Bsr plasmid (Addgene:#83925) after Fast Digest BBSI digestion. If the first base at the 5' end of the sense strand was not G, a G was added after the CACC at the 5' end, and a C was added to the 3' end of the corresponding antisense strand. pLV-U6-gRNA-UbC-eGFP-P2A-Bsr is an sgRNA backbone expression vector containing the U6 promoter, carrying the GFP (green fluorescent protein) gene and ampicillin resistance.

[0220] ① pLV-U6-gRNA-UbC-eGFP-P2A-Bsr was digested with Fast Digest Bbs I, and the linearized vector was recovered after DNA gel electrophoresis.

[0221] ② The sgRNA sequence was phosphorylated and annealed using T4 PNK; the linear pLV-U6-gRNA-UbC-eGFP-P2A-Bsr plasmid vector was ligated with the annealed sgRNA double-stranded sequence using T4 ligase at room temperature for 1 h. The ligation product was transformed into competent bacteria Trans 109, incubated on ice for 30 min, at 42℃ for 45 s, and on ice for 2 min. Clones were screened on ampicillin-resistant LB plates. Positive clones were picked, cultured, and sequenced. The sequencing primer was the forward primer sequence of the U6 promoter, 5'-GAGGGCCTATTTCCCATGATTCC-3' (SEQ ID NO: 18). The clone with correct sequencing was the recombinant plasmid.

[0222] 2. Cell sorting: using "human CD8" + T cell enrichment kit (STEMCELL, catalog number: 19053) will add CD8 + Cytotoxic T lymphocytes (CTL cells) were sorted out;

[0223] 3. Activation of CTL cells: Before electroporation, CTL cells were activated using a CD3 / CD28 kit (BioLegend, catalog number: 422603) for 3 days.

[0224] 4. Collect the activated CTL cells in centrifuge tubes and centrifuge at 600×g for 8 minutes. Discard the supernatant, resuspend in PBS, and count the cells.

[0225] 5. Preparation of the transfection complex: Electroporation was performed using a Neon transfection system (Invitrogen, MPK5000). For a 10 μL electroporation system, take 2 × 10⁻⁶ ppm of the solution. 5 One CTL cell was resuspended in 9 μL of electroporation buffer and 1 μL of electroporation buffer containing 300 ng of plasmid mixture (including 150 ng of CRISPRoff plasmid and 150 ng of sgRNA) was added (CRISPRoff is a commercially available plasmid).

[0226] 6. Transfer the resulting mixture containing cells and plasmids to 10 μL tip (Neon) and perform electroporation using the electroporation conditions of "1600V / 10ms / 3pulses" (here, tip refers to the pipetting / absorption tool specifically for the Neon transfection system);

[0227] 7. Immediately after electroporation, transfer the cells to wells containing preheated fresh culture medium and incubate at 37°C with 5% CO2. For 24-well plates, seed each well with 5 reactants (total 1×10⁻⁶). 6 (1 cell), inoculated with 0.5 ml of culture medium.

[0228] Based on the invention patent with publication number CN113846063A, which describes how knocking down or silencing a portion of the enhancer gene sequence of the B2M gene in cells can maintain low immunogenicity in inflammatory environments, this embodiment utilizes the super enhancer SE on the B2M gene that responds to IFN-γ stimulation, as described in the aforementioned patent, and further employs electroporation combined with CRISPRoff epigenetic editing to construct universal intercellular CTL cells.

[0229] In this embodiment, an optimal universal CTL cell was constructed, as shown in Figure 7A. The fluorescence results of BF (bright field) and GFP (green fluorescence) in Figure 7A show that the epigenetic editor CRISPRoff was successfully delivered into the CTLs. Forty-eight hours later, the expression level of HLA-I (human leukocyte antigen class I) protein in the cells was detected by flow cytometry (BD Biosciences, FACSAria Fusion SORP). HLA-I is known to be positively correlated with cellular immunogenicity. The results are shown in Figure 7B. In Figure 7B, the horizontal axis represents the expression level of HLA-ABC on the cell surface, expressed as average fluorescence intensity. The results indicate that, compared to unedited CTL cells, the epigenetically edited CTL cells showed significant clustering, with a subpopulation of cells expressing low HLA-I (the small peak to the left of the dashed line). This subpopulation of low-immunogenic CTL cells was termed the universal CTL.

[0230] Therefore, in this embodiment, a universal CTL cell was successfully constructed using the optimal construction method. The universal CTL cell has permanent low immunogenicity, does not cause immune rejection, is suitable for allogeneic transplantation, and can be used for the treatment of diseases such as tumors.

[0231] Example 8: Further exploration and optimization of a universal CTL cell construction method

[0232] I. Screening of Transfection Methods

[0233] In this embodiment, to construct a safer, more stable, and less immunogenic universal CTL cell line, different transfection methods were further screened based on the original method for constructing universal CTL cells, including: lentiviral transduction, electroporation, and liposome transfection. The specific operations are as follows:

[0234] 1. Universal CTL cells were constructed using the electrotransfection method described in Example 7;

[0235] 2. Constructing universal CTL cells using lentivirus transduction: The day before, administer 1×10⁻⁶ cells. 6 CTLs were seeded into 6-well plates. Before infection, the medium was replaced with fresh complete medium, and the virus particles were diluted with an appropriate amount of fresh complete medium at a multiplicity of infection (MOI) of 0.3. The diluted virus particles were then added dropwise through a 0.2 μm filter membrane to the wells containing CTLs. 2 μL of 10 mg / ml polybrene (final concentration 10 μg / ml) was added. After mixing by cross-rotation, the cells were centrifuged at 1300 rpm at 37°C for 45 min (both acceleration and deceleration were set to "0") to facilitate better binding of the virus to the target cells. After cell expansion culture, flow cytometry was performed to sort out cells that were double-positive for both mCherry (red fluorescence) and GFP (green fluorescence). The surviving CTLs were identified as universal CTL cells.

[0236] 3. Construct universal CTL cells using liposome transfection: 3. One day in advance, administer 2×10⁻⁶ cells. 6 CTLs were seeded in 10cm cell culture dishes. The optimal transfection time is 12-18 hours after seeding. Following the Lipofectamine 3000 reagent (Invitrogene, catalog number: L3000015) instructions, 2.4μg CRISPRoff, 1.2μg sgRNA, and 20μL Lipofectamine 3000 were incubated at room temperature for 10 minutes before co-transfecting CTLs. Forty-eight hours later, flow cytometry sorting was performed to separate cells that were double-positive for BFP (blue fluorescence) and GFP (green fluorescence). The surviving CTLs were designated as universal CTL cells.

[0237] The transfection efficiency and cell viability of the different universal CTL cells constructed were then tested.

[0238] The steps for detecting the transfection efficiency are as follows: Cells are transfected according to different transfection methods, and the fluorescence value is detected on a flow cytometer (BD Biosciences, FACSAria Fusion SORP). Successfully transfected cells will have red (mCherry) or BFP (blue fluorescence) or green (GFP) fluorescence. The position of the control group is the position where no fluorescence is expressed. A cross gate is drawn, and the percentage of cells in the experimental group that are shifted is determined, which is the transfection rate.

[0239] The cell viability assay procedure is as follows: Take 0.5 mL of cell suspension (1 × 10⁻⁶) 5 Approximately 10 μL of 7-aminoactinomycin D (7-AAD) staining agent was added to each cell, and the cells were incubated at room temperature for 2 minutes. The fluorescence intensity was then detected by flow cytometry (FL3 channel) based on the excitation / emission wavelengths (Ex / Em = 545 nm / 650 nm). 7-AAD is a non-permeable fluorescent dye; it cannot pass through the cell membrane of living cells but can penetrate the cell membrane of dead cells and bind to their DNA, thus facilitating the detection of the ratio of living to dead cells using flow cytometry.

[0240] The specific results are shown in Table 4:

[0241] Table 4. Transfection efficiency and cell viability of universal CTL cells constructed using different transfection methods

[0242] As shown in Table 4, the universal CTL cells constructed by electroporation have the best transfection efficiency and cell viability. This may be because: (1) Lentiviral transduction has some unavoidable drawbacks. Its viral DNA can be permanently integrated into the CTL cell genome, causing the potential risk of insertion mutations, which may lead to abnormal cell function or even cancer. At the same time, the preparation process of lentivirus is relatively complicated, the operation is relatively complex, and the requirements for experimental environment and operators are relatively strict. (2) The transfection efficiency of liposome transfection is often affected by a variety of factors, such as the ratio of liposomes to nucleic acids and the state of cells, which has a certain degree of instability. In addition, liposomes themselves may produce a certain degree of toxicity to cells, affecting the normal physiological function of cells. (3) However, electroporation exhibits unique characteristics. Electroporation causes changes in cell membrane potential through a high-intensity electric field, which instantaneously increases cell membrane permeability and creates reversible pores on the cell membrane to facilitate the entry of exogenous nucleic acids. It is applicable to almost all types of cells. Compared to the former two, electroporation has the following advantages: it can control the transfection process more precisely, and the transfection efficiency is relatively high and more stable; it causes less damage to cells because the electric field is applied for a short time; and it is applicable to a wider range of cell types, making it more versatile.

[0243] Therefore, in this embodiment, electrotransfection is preferred for constructing universal CTL cells.

[0244] II. Screening of Electroporation Parameters

[0245] Electroporation parameters have a significant impact on transfection efficiency, cell viability, and subsequent growth status. Therefore, in this embodiment, different electroporation parameters were further screened in order to minimize cell damage and improve cell viability while achieving good transfection efficiency.

[0246] Specifically, a negative control (i.e., no electroporation) and three experimental groups were set up. The electroporation parameters for the three experimental groups were as follows: (1) 1200V voltage, 20ms pulse duration, 2 pulses; (2) 1400V voltage, 10ms pulse duration, 3 pulses; (3) 1600V voltage, 10ms pulse duration, 3 pulses. The transfection efficiency was determined by flow cytometry.

[0247] The specific results are shown in Figure 8. As can be seen from Figure 8, a high electrotransfer efficiency was achieved under the electrotransfer conditions of 1600V / 10ms / 3pulse (voltage 1600V, pulse duration 10ms, 3 pulses). The electrotransfer efficiency was 95.3%. Therefore, in this embodiment, the preferred electrotransfer parameters are 1600V / 10ms / 3pulse to prepare a general-purpose CTL.

[0248] III. Screening of Plasmid Amount

[0249] In electroporation, the amount of plasmid used has a significant impact on transfection efficiency and cell viability. Therefore, in this embodiment, different amounts of plasmid were further screened: the amount of plasmid was set to three dosage groups of 100ng, 150ng and 200ng, and different universal CTL cells were constructed using an electroporator (Invitrogen, MPK5000). The optimal amount of plasmid was determined by comparing the proportion of GFP fluorescent positive cells. The specific results are shown in Figure 9.

[0250] As shown in Figure 9, the proportion of GFP-positive cells was highest when the plasmid dosage was 150 ng. However, the proportion of GFP-positive cells decreased significantly when the plasmid dosage was reduced or increased. This is because, on the one hand, when the plasmid dosage is low, the number of expressed CRISPRoff editors and sgRNAs is insufficient, failing to achieve the optimal epigenetic editing effect; on the other hand, when the plasmid dosage is excessive, a saturation effect occurs, or unintended effects arise due to excessive plasmid interaction with cells. Therefore, in this embodiment, a plasmid dosage of 150 ng is preferred.

[0251] Example 9: Verification experiment to confirm that universal CTL cells do not induce allogeneic immune responses

[0252] To verify that the optimal universal CTL cells constructed in this invention do not induce allogeneic immune responses, in this embodiment, allogeneic PBMCs were labeled with CFSE dye, and co-cultured with CTL cells, the optimal universal CTL cells from Example 7, K562 cells (human chronic myeloid leukemia cell line, ATCC website, catalog number CCL-243; the K562 cells can specifically activate NK cells and are used only as a positive control for NK cell activation experiments) and human PBMCs (peripheral blood mononuclear cells). Flow cytometry was used to detect PBMC cell proliferation (PBMC proliferation reflects the intensity of immune rejection), and the effects of different cell types on human PBMCs were compared. The specific steps of the co-culture experiment are as follows:

[0253] 1. Collect human peripheral blood (source: human peripheral blood leukocytes obtained from Zhejiang Provincial Blood Center; ethics approval number: ZJBC / YW / 05-01) and mix and dilute with an equal volume of PBS;

[0254] 2. Take 3 mL of Ficoll (manufacturer: GE Life, product number: 17144003-1) and place it in a 15 mL centrifuge tube. Slowly add 6 mL of diluted blood to the top layer of Ficoll (do not disturb the Ficoll layer).

[0255] 3. Centrifuge at 300×g for 30 minutes (with acceleration and deceleration both at 0).

[0256] 4. Take the middle flocculent layer into a new centrifuge tube and add 10 mL of PBS to wash the cells;

[0257] 5. Centrifuge at 300×g for 10 minutes;

[0258] 6. Discard the supernatant, add 10 mL of PBS to resuspend and wash the cells; repeat the washing process twice more.

[0259] 7. Resuspend the cells in 1 mL of PBS and count them;

[0260] 8. According to the instructions of the CFDA-SE Cell Proliferation and Tracing Detection Kit (Beyotime, catalog number: C0051), the isolated PBMCs were stained. The staining conditions were: incubation at 37°C for 10 minutes, with gentle mixing every 5 minutes. Then, the cells were washed twice with complete cell culture medium, centrifuged, resuspended in complete cell culture medium, and counted to obtain the reaction cells.

[0261] 9. Using 1×10 4 The stimulated cells, namely CTL, universal CTL and K562, were seeded in a U-shaped 96-well plate at a cell density of 100 μL / cells.

[0262] 10. Using 1×10 5 The reaction cells PBMCs were seeded in U-shaped 96-well plates at a cell density of cells / 100μL.

[0263] 11. After culturing the U-shaped 96-well plates in a cell culture incubator for 3 days, the proliferation of PBMCs was detected by flow cytometry, and the effects of different stimulating cell CTLs and universal CTLs on PBMC culture were recorded.

[0264] The specific results are shown in Figures 10A and 10B. Among them, PBMC cultured alone was used as the negative control group, CTL co-cultured with PBMC was used as the positive control group, and general CTL co-cultured with PBMC was used as experimental group 1.

[0265] Figures 10A and 10B are schematic diagrams of the proliferation results of PBMCs co-cultured with CTLs or general-purpose CTLs and collected by flow cytometry. In Figure 10B, the average value is the final value obtained after four repeated experiments. The lowest value is 47.5%, and the highest value is higher than 60%. Therefore, the average value is higher than 47.5%, which is slightly different from that in Figure 4A.

[0266] As shown in Figures 10A and 10B, the results indicate that CTLs can induce a strong immune response. PBMCs in the CTL group showed significant proliferation (47.5%), while the proliferation rate of PBMCs in the universal CTL group (17.1%) was not significantly different from that in the culture-only group (12.0%). This suggests that the universal CTLs did not induce a strong immune response. This may be because when CTL cells stimulate PBMCs, the PBMCs undergo a strong immune response, resulting in massive proliferation. Therefore, when the proliferation rate of PBMCs in the universal CTL group was not significantly different from that in the culture-only group (12.0%), it indicates that the optimal universal CTL cells constructed in this invention do not induce allogeneic immune responses, have low immunogenicity, and can maintain low immunogenicity in an inflammatory environment.

[0267] Example 10: A universal CTL with low HLA-I expression can evade allogeneic NK cell killing.

[0268] I. NK cell activation experiment

[0269] To verify that the optimal universal CTL cells constructed in this invention can evade the killing effect of allogeneic NK cells, this embodiment uses CTL cells, the optimal universal CTL cells from Example 7, K562 cells, and NK cells for co-culture experiments to detect the rejection of different cell types by NK cells and to detect the NK cell activation marker CD107a. The specific steps of the co-culture experiment are as follows:

[0270] 1. Obtain human PBMCs according to the method in Example 9;

[0271] 2. NK cells were sorted using a human NK cell isolation kit (STEMCELL, catalog number: 17955);

[0272] 3. Allogeneic NK cells were co-cultured with CTLs or universal CTLs at a 1:1 ratio for 1 day.

[0273] 4. At the same time, an allogeneic NK cell co-culture group with K562 was set up as a positive control, and an NK cell culture group alone was set up as a negative control.

[0274] 5. One day later, the NK cells with suspension properties were pipetted from the bottom of the culture dish and collected; the activation status of NK cells was detected by flow cytometry using CD107a-APC antibody (1:100 dilution).

[0275] The specific results are shown in Figures 11A and 11B. In Figure 11A, "Isotype" represents the isotype control, used to set the background control in flow cytometry analysis to help accurately distinguish between specific and non-specific signals; "Native NK" represents untreated NK cells in their natural state, i.e., NK cells cultured alone in the negative control group; MFI represents the mean fluorescence intensity. Combining Figures 11A and 11B, it can be seen that compared to the negative control group (CD107a positivity rate of 3.05%), the CD107a positivity rate in the positive control K562 group was significantly increased (CD107a positivity rate of 18.1%), while the CTL group (CD107a positivity rate of 5.52%) and the universal CTL group (CD107a positivity rate of 4.54%) showed no significant difference from the negative control group; this indicates that universal CTL does not activate allogeneic NK cells.

[0276] To further demonstrate that the universal CTLs constructed using the method of this invention have the best performance, subsequent experiments further compared the universal CTL cells 2 constructed using lentiviral transduction and the universal CTL cells 3 constructed using liposome transfection, as described in Example 8. Under the same culture conditions, co-culture experiments were conducted with NK cells, and the activation marker CD107a of NK cells was detected. The results showed that the CD107a positivity rate was 7.12% in the universal CTL cell 2 group constructed using lentiviral transduction, and 7.95% in the universal CTL cell 3 group constructed using liposome transfection. Although the CD107a positivity rate was significantly lower than that in the positive control group, it was still higher than that in the universal CTL group. Therefore, it can be concluded that the universal CTL cells constructed using the optimal electrotransfection method in Example 7 have the best performance and are the most reliable in terms of performance without activating allogeneic NK cells.

[0277] II. LDH Content Detection Experiment

[0278] Following the instructions of the LDH cytotoxicity assay kit (Thermo Fisher, C20300), a control group and a test culture medium supernatant were set up to measure lactate dehydrogenase (LDH) levels. Five replicates were set up for each experimental group, including a "spontaneous LDH group" and a "maximum LDH group." After co-culture, 10 μL of the lysis buffer from the kit was added to the wells of the "maximum LDH group," and the mixture was incubated at 37°C for 45 min. After lysis, the culture medium from all groups was collected and centrifuged at 400 × g for 5 min. 50 μL of the supernatant was transferred to a new 96-well plate, and 50 μL of pre-prepared reaction mixture was added. After mixing, the plate was incubated at room temperature in the dark for 0.5 h. The reaction was terminated by adding 50 μL of Stop Solution, and the plate was then readjusted using a microplate reader (Thermo Fisher, Varioskan). LUX) was used to measure the absorbance at 490 nm and 680 nm; LDH cytotoxicity was calculated according to the formula provided in the kit, which is: cytotoxicity% = (experimental group LDH activity - spontaneous group LDH activity) / (maximum lysis group LDH activity - spontaneous group LDH activity) × 100%.

[0279] LDH is an enzyme found inside cells. When cells are damaged or die, LDH is released extracellularly. Further detection of LDH content in the co-culture supernatant indirectly reflects the cytotoxicity of NK cells against universal CTLs. Higher LDH content in the co-culture supernatant indicates a greater cytotoxic ability of NK cells against the cells in this group.

[0280] The results in Figure 12 show that, compared to the negative control group, the killing effect of NK cells on K562 cells was significantly enhanced in the positive control K562 group, reaching 21.3%; while in the CTL group and the universal CTL group, the killing effect of NK cells on CTLs (4.84%) and universal CTLs (4.4%) was not significantly different from that in the negative control group. This further indicates that universal CTLs can evade the killing effect of allogeneic NK cells.

[0281] In subsequent experiments, the NK cell killing ability of lentiviral transduced universal CTL cells (Group 2) and liposome-transfected universal CTL cells (Group 3) was tested. The results showed that the NK cell killing ability of lentiviral transduced universal CTL cells (Group 2) reached 6.85%, while that of liposome-transfected universal CTL cells (Group 3) reached 7.06%. Although this was significantly lower than the killing ability of NK cells in the positive control group, it was still higher than that in the universal CTL group. This may be because lentivirus introduces viral DNA, and liposome transfection also introduces liposomes. Since both viral DNA and liposomes have certain toxicity, they may alter the cell's activity or biological characteristics. On the other hand, lentiviral and liposome transfection may also cause changes in the antigens or ligands expressed on the surface of CTL cells, thus affecting NK cell recognition and killing, ultimately leading to enhanced NK cell killing ability against lentiviral transduced universal CTL cells (Group 2) and liposome-transfected universal CTL cells (Group 3). In this embodiment, the content regarding "the discovered super enhancer SE on the B2M gene that responds to IFN-γ stimulation" is specifically referred to in the description in the invention patent application publication number CN113846063A.

[0282] Therefore, it can be further demonstrated that the universal CTL cells constructed by the optimal electrotransfection method in Example 7 have the best performance and are the most reliable in evading allogeneic NK cell killing.

[0283] Example 11: Application of the universal CTL cells constructed in this invention in diseases such as tumors.

[0284] The verification experiments in the above embodiments demonstrate that the universal CTL cells constructed using the optimal electrotransfection method of this invention exhibit the best performance: they not only do not induce allogeneic immune responses and have low immunogenicity, but also maintain low immunogenicity even under inflammatory conditions, and are the most reliable in evading allogeneic NK cell killing. Therefore, given the excellent characteristics exhibited by the universal CTL cells constructed in this invention, further in-depth and comprehensive research will be conducted on their application in the treatment of various diseases in future studies, aiming to achieve precise application and high efficacy in the treatment of multiple diseases, bringing new breakthroughs and progress to the medical field.

[0285] In this embodiment, the role of the universal CTL cells in tumor diseases, autoimmune diseases, and other fields was preliminarily explored. Preliminary experimental results show that:

[0286] (1) In the treatment of tumor diseases, CTLs that specifically recognize tumor-associated antigens are isolated from PBMCs co-cultured with tumor cells, and universal CTLs are constructed using the above method. The universal CTL cells can activate a targeted immune response by specifically recognizing tumor-associated antigens. This may be because the universal CTL cells constructed in this invention can accurately locate and act on tumor cells, and because they have permanent low immunogenicity, they can also stimulate immune effects through specific mechanisms without triggering a widespread immune response, induce tumor cell apoptosis, and achieve effective killing of tumor cells, ultimately achieving significant therapeutic effects and effectively curbing the development and spread of tumors. Meanwhile, in this embodiment, comparative tests were also conducted on the universal CTL cells constructed by different construction methods: humanized mice (Biocytogen (Beijing) Pharmaceutical Technology Co., Ltd., catalog number: 112478, a humanized mouse based on the CD34+ immune system of B-NDG hIL15 mice; humanized mice refer to mice with reconstructed human immune systems that have or partially have human immune systems and can better mimic human immune functions and pathology) were injected with tumor cells via the tail vein for 6 hours, and then different universal CTL cells were injected for treatment. The changes in tumor volume and survival time of mice in different groups were observed. The results showed that, compared with universal CTL cells 2 constructed by lentiviral transduction and universal CTL cells 3 constructed by liposome transfection, the universal CTL cells constructed by electrotransfection significantly enhanced the killing effect on tumor cells and showed a good inhibitory effect on tumor growth, with the slowest tumor growth. Furthermore, in mice injected with universal CTL cells constructed by different methods, nearly 62% of the mice treated with universal CTL cells constructed by electrotransfection survived for more than 30 days, while in the other two groups of mice injected with universal CTL cells 2 and universal CTL cells 3, only approximately 36% and 32% of the mice, respectively, survived for more than 20 days. This further demonstrates that the universal CTL cells constructed by electrotransfection in this invention can significantly inhibit tumor growth and have good anti-tumor ability. This may be because the universal CTL cells constructed by electroporation, while maintaining low immunogenicity and evading allogeneic NK cell killing, can more precisely activate specific immune responses against tumor cells, thereby enhancing the targeted killing ability of tumor cells. Furthermore, the unique electroporation mechanism of this invention may induce specific changes in the expression and function of cell surface receptors, making their interaction with tumor cells more efficient and accurate. At the same time, the universal CTL cells can better maintain their own stability and activity, exerting a killing effect on tumor cells while having a smaller impact on normal cells, thus prolonging the survival time of normal cells. This is also due to their ability to maintain low immunogenicity even in inflammatory environments, reducing unnecessary immune attacks and damage to normal cells.Therefore, future research will delve deeper into its specific molecular mechanisms and signaling pathways, which will help to more comprehensively understand the source of these advantages and provide a more solid theoretical basis for its widespread clinical application.

[0287] (2) In the treatment of autoimmune diseases, CTLs that specifically recognize antigens associated with activated immune cells are isolated from PBMCs co-cultured with abnormally activated immune cells, and universal CTLs are constructed using the above method. These universal CTLs can play a crucial regulatory role, effectively regulating the balance of the immune system and significantly reducing inflammatory responses. This may be because universal CTLs possess unique low immunogenicity and the ability to evade allogeneic NK cell killing, enabling them to exist stably in complex immune environments and accurately recognize and eliminate abnormally activated immune cells. This ability is not contradictory but rather synergistic. While maintaining its own characteristics, the universal CTLs, through their precise immune recognition mechanism, can accurately locate these abnormally activated immune cells and trigger specific immune responses to eliminate them, thereby restoring the normal functional state of the immune system and alleviating the symptoms of autoimmune diseases. This regulation of the immune system balance requires both effective intervention against abnormal immune cells and avoidance of excessive immune responses that could cause new damage to the body. The universal CTLs constructed in this invention possess precisely this ability, demonstrating unique advantages and value in the treatment of autoimmune diseases. In this embodiment, comparative tests were also conducted on universal CTL cells constructed using different methods. The results showed that, compared to universal CTL cells 2 constructed via lentiviral transduction and universal CTL cells 3 constructed via liposome transfection, universal CTL cells constructed using electroporation exhibited better efficacy in the treatment of autoimmune diseases. This may be because electroporated universal CTL cells retain their advantages of more reliable low immunogenicity and evasion of allogeneic NK cell killing, allowing them to function more stably in vivo. They can precisely identify and specifically eliminate abnormally activated immune cells in autoimmune diseases, while avoiding triggering excessive immune responses that could lead to new immune damage. Furthermore, the use of transient CRISPRoff technology combined with electroporation may endow universal CTL cells with a more efficient immunomodulatory mechanism, enabling them to better balance the function of the immune system, promote the establishment of immune tolerance, and thus more effectively alleviate the inflammatory response and related symptoms of autoimmune diseases. Their unique cellular characteristics and functional performance allow them to demonstrate superior regulatory capacity and therapeutic effects in addressing the complex immune imbalances of autoimmune diseases. Therefore, future research will delve deeper into the mechanism of action of transient CRISPRoff technology combined with electroporation in immune regulation. This will help to more comprehensively understand the source of the above advantages, providing a more solid theoretical basis for its widespread clinical application and opening up new avenues and technical support for the treatment of autoimmune diseases.

[0288] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

[0289]

Claims

1. A method for constructing a universal cell system, characterized by, The method comprises the following steps: (1) construction of a recombinant plasmid; (2) preparation of a transfection complex; (3) transfection of the plasmid into cells; In the step (3), the plasmid mixture is transfected into the cells by using an electroporation method.

2. The construction method of claim 1, wherein, The cells are mesenchymal stem cells or CTL cells.

3. The construction method of claim 2, wherein, In the step (2), the transfection complex comprises the plasmid mixture and any one of the mesenchymal stem cells or the CTL cells; and the plasmid mixture comprises a CRISPRoff plasmid and an sgRNA plasmid.

4. The construction method of claim 3, wherein, In the step (2), the amount of the plasmid is 100-200 ng, or the amount of the plasmid is 400-800 ng.

5. The construction method of claim 4, wherein, In the step (2), when the cells are mesenchymal stem cells, the amount of the plasmid is 400-800 ng.

6. The construction method of claim 5, wherein, In the step (2), when the cells are CTL cells, the amount of the plasmid is 100-200 ng.

7. The construction method of claim 6, wherein, In the step (2), the mass ratio of the CRISPRoff plasmid to the sgRNA plasmid is 1:

1.

8. The construction method of claim 7, wherein, In the step (3), the voltage is 990-1400 V, the duration of the electric pulse is 10-40 ms, and the number of pulses is 1-3; or the voltage is 1200-1600 V, the duration of the electric pulse is 10-20 ms, and the number of pulses is 2-3.

9. The construction method of claim 8, wherein, In the step (3), when the cells are mesenchymal stem cells, the voltage is 990-1400 V, the duration of the electric pulse is 10-40 ms, and the number of pulses is 1-3.

10. The construction of claim 9, wherein, In the step (3), when the cells are CTL cells, the voltage is 1200-1600 V, the duration of the electric pulse is 10-20 ms, and the number of pulses is 2-3.

11. A universal mesenchymal stem cell, characterized by, The construction method is constructed by using any one of the construction methods in claims 1-10.

12. Use of a universal mesenchymal stem cell for the preparation of a preparation for improving the regenerative capacity of lung injury repair, characterized in that, The construction method is constructed by using any one of the construction methods in claims 1-10.

13. Use of a universal mesenchymal stem cell for the preparation of a preparation for reducing allogeneic immune rejection, characterized in that, The construction method is constructed by using any one of the construction methods in claims 1-10.

14. Use of a universal mesenchymal stem cell for the manufacture of a preparation for reducing the lactate dehydrogenase, albumin and / or total protein content in the alveolar lavage fluid, characterized in that, The construction method is constructed by using any one of the construction methods in claims 1-10.

15. Use of a universal mesenchymal stem cell for the preparation of a preparation for increasing the survival time of mesenchymal stem cells in an inflammatory environment in vivo or for maintaining mesenchymal stem cells in a low immunogenicity in vivo, characterized in that, The universal mesenchymal stem cells are constructed by using any one of the construction methods in claims 1-10.

16. A universal CTL cell, characterized by, The construction method is constructed by using any one of the construction methods in claims 1-10.

17. Use of universal CTL cells for the preparation of a preparation for inhibiting the activation of NK cells or reducing the proportion of CD 107a positive cells, characterized in that, The universal CTL cells are constructed by using any one of the construction methods in claims 1-10.

18. Use of universal CTL cells for the preparation of a formulation for reducing the LDH content, characterized in that, The universal CTL cells are constructed by using any one of the construction methods in claims 1-10.

19. Use of a universal CTL cell for the preparation of a formulation for permanently reducing the immunogenicity of CTL cells or for evading killing by allogeneic lymphocytes, characterized in that, The universal CTL cells are constructed by using any one of the construction methods in claims 1-10.

20. Use of universal CTL cells for the preparation of a preparation for increasing the killing ability of CTL cells against tumor cells, characterized in that, The universal CTL cells are constructed by using any one of the construction methods in claims 1-10.

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