Increasing post-implantation viability and therapeutic effects of cells by controlling glycogen accumulation

Abstract

The invention relates to a method for improving survival ability and treatment effect after cell implantation by controlling glycogen accumulation. The invention discloses a cell. According to the embodiment of the invention, glycogen synthesis key enzymes in the cells are up-regulated, and the cells are selected from at least one of stem cells and T cells. According to the cell provided by the embodiment of the invention, the glycogen accumulation is improved, the hunger resistance and the cell activity after implantation are enhanced, the killing effect on tumors is enhanced, the treatment effect on stem cell indications is improved, and particularly, the treatment effect on pulmonary fibrosis is improved.

Description

Technical Field

[0001] The present invention relates to the field of cell genetic engineering. Specifically, the present invention relates to improving the survival ability and therapeutic effect after cell implantation by controlling glycogen accumulation. More specifically, the present invention relates to a cell, a method for increasing glycogen accumulation in cells, a pharmaceutical composition, and related uses thereof. Background Art

[0002] Stem cells are a class of undifferentiated or incompletely differentiated cells with self-renewal ability and multi-lineage differentiation potential. They are the basis for constructing and regenerating various tissues and organs in the body. Stem cell therapy has been used to treat certain blood and immune system diseases, such as leukemia and lymphoma, through hematopoietic stem cell transplantation (bone marrow transplantation). In addition, stem cells have the potential to treat neurodegenerative diseases, heart diseases, diabetes, and tissue injuries. However, due to the poor survival rate and residence time of implanted stem cells in the pathological environment, their therapeutic effect is still limited.

[0003] T cells, also known as T lymphocytes, are a core component of the adaptive immune system. They originate from hematopoietic stem cells in the bone marrow and mature in the thymus. They recognize antigens through unique T cell receptors (TCRs) and differentiate into multiple subsets, including helper T cells, cytotoxic T cells, regulatory T cells, memory T cells, natural killer T cells, and γδ T cells, etc. T cells play a key role in immune responses, including activating B cells to produce antibodies, directly killing infected or cancerous cells, regulating immune responses, and forming immune memory. Their activation depends on dual signals: the binding of TCR to the major histocompatibility complex (pMHC), and the interaction of co-stimulatory molecules. T cells have a wide range of applications in clinical treatment, especially in vaccine development and adoptive cell therapies such as CAR-T cell therapy. However, due to the metabolic inhibition of implanted T cells in the pathological environment, resulting in poor survival and cell killing ability, their therapeutic effect is still limited.

[0004] Therefore, there is an urgent need to improve the survival ability and therapeutic effect of stem cells and T cells after implantation. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to some extent. For this purpose, the present invention provides a method for improving the survival ability and therapeutic effect after cell implantation by controlling glycogen accumulation.

[0006] The present invention is completed based on the following discoveries of the inventors: The depletion of intracellular energy storage substances such as glucose is the main cause of apoptosis of cells after being implanted into the in - vivo pathological environment. In the existing technologies for improving the survival ability of cells after implantation, there are mainly problems such as high cost, low R & D efficiency, and potential safety issues. To overcome this problem, the present invention has genetically engineered the glycogen metabolism of cells (the present invention exemplarily demonstrates with mesenchymal stem cells, induced pluripotent stem cells, and T lymphocytes), promoted the accumulation of glycogen, enhanced the anti - starvation ability of stem cells and T cells, thereby enhancing the killing effect on tumors and the therapeutic effect on stem cell indications, especially the therapeutic effect on pulmonary fibrosis.

[0007] In the first aspect of the present invention, the present invention provides a cell. According to an embodiment of the present invention, the key glycogen - synthesizing enzyme in the cell is up - regulated, and the cell is selected from at least one of stem cells and T cells. For the cell according to the embodiment of the present invention, the glycogen accumulation is increased, the anti - starvation ability is enhanced, the cell activity after implantation is enhanced, the killing effect on tumors is enhanced, the therapeutic effect on stem cell indications is improved, especially the therapeutic effect on pulmonary fibrosis is improved.

[0008] In the second aspect of the present invention, the present invention provides a method for increasing glycogen accumulation in cells. According to an embodiment of the present invention, the method includes: up - regulating the key glycogen - synthesizing enzyme in the cell, and the cell is selected from at least one of stem cells and T cells. For the method according to the embodiment of the present invention, the glycogen accumulation, anti - starvation ability, and cell activity after implantation in stem cells and T cells are increased, thereby improving the killing effect on tumors and the therapeutic effect on stem cell indications, especially improving the therapeutic effect on pulmonary fibrosis.

[0009] In the third aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition includes: the stem cells described in the first aspect of the present invention. For the pharmaceutical composition according to the embodiment of the present invention, the killing effect on tumors is enhanced, the therapeutic effect on stem cell indications is improved, especially the therapeutic effect on pulmonary fibrosis is improved.

[0010] In the fourth aspect of the present invention, the present invention provides the use of the stem cells described in the first aspect of the present invention or the pharmaceutical composition described in the third aspect of the present invention in the preparation of a drug, and the drug is used for treating or preventing diseases applicable to the stem cells and / or T cells or for anti - aging health care.

[0011] In the fifth aspect of the present invention, the present invention provides the use of a key glycogen - synthesizing enzyme or a reagent for up - regulating a key glycogen - synthesizing enzyme in enhancing the stress resistance of stem cells.

[0012] Additional aspects and advantages of the present invention will be given in part in the following description, become apparent in part from the following description, or be learned by practice of the present invention. Brief Description of the Drawings

[0013] The above and / or additional aspects and advantages of the present invention will become apparent and be readily understood from the description of embodiments in conjunction with the following drawings, in which:

[0014] Figure 1 Graph showing the effect of the key enzyme combination expression scheme in Example 1 on the glycogen accumulation in cells.

[0015] Figure 2 、 3 、4 is a graph showing the effect of expressing dephosphorylation-inhibited glycogen synthase on the glycogen accumulation in mesenchymal stem cells in Example 2.

[0016] Figure 5 Graph showing the effect of expressing dephosphorylation-inhibited glycogen synthase on the glycogen accumulation in induced pluripotent stem cells in Example 3.

[0017] Figure 6 Graph showing the effect of expressing dephosphorylation-inhibited glycogen synthase on the glycogen accumulation in chimeric antigen receptor T cells in Example 4.

[0018] Figure 7 Graph showing the survival of glycogen-accumulating mesenchymal stem cells under in vitro starvation conditions in Example 5.

[0019] Figure 8 Graph of in vivo imaging of the lungs of mice in Example 6.

[0020] Figure 9 Graph of fluorescence intensity quantification in Example 6.

[0021] Figure 10 Graph showing the survival and body weight change curves of mesenchymal stem cell-treated mice in Example 7.

[0022] Figure 11 Schematic diagram of the dynamic control circuit for glycogen synthesis in Example 8.

[0023] Figure 12 Graph showing doxycycline (DOX)-induced glycogen accumulation achieved by the circuit in Example 8.

[0024] Figure 13 Graph showing that doxycycline (DOX)-induced glycogen accumulation in TET-GYS mesenchymal stem cells in Example 8 improves the survival ability of cells under starvation conditions.

[0025] Figure 14 Graph showing the survival of pluripotent stem cells (iPSCs) under starvation conditions in Example 9.

[0026] Figure 15 It is a diagram showing the survival of induced pluripotent stem cells (iPSCs) after tracheal implantation in Example 9.

[0027] Figure 16 It is a diagram showing the survival of glycogen-accumulating T cells cultured under in vitro starvation conditions in Example 10.

[0028] Figure 17 It is a diagram showing the effect of expressing dephosphorylation-inhibited glycogen synthase on the tumor cell killing ability of chimeric antigen receptor T cells in Example 10. Detailed implementation manners

[0029] The embodiments of the present invention will be described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as a limitation of the present invention.

[0030] It should be noted that the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present invention, unless otherwise stated, the meaning of "a plurality" is two or more.

[0031] In the present invention, the term "comprising" or "including" is an open expression, that is, it includes the content specified by the present invention, but does not exclude other aspects of the content.

[0032] In the present invention, the terms "optionally", "optional" or "option" generally mean that the subsequent events or conditions may or may not occur, and this description includes the cases where such events or conditions occur, as well as the cases where such events or conditions do not occur.

[0033] In the present invention, the term "key glycogen synthase enzyme" refers to a class of enzymes that play a key role in the process of glycogen synthesis. Glycogen is a polysaccharide formed by the connection of glucose molecules through α-1,4 and α-1,6 glycosidic bonds and is the main form of storing glucose in animals. Glycogen is mainly stored in the liver and muscles and can be quickly decomposed into glucose when needed to provide energy for the body. In the present invention, the "key glycogen synthase enzyme" specifically refers to glycogen synthase (GYS), UDP-glucose pyrophosphorylase (UGP), glycogen branching enzyme (GBE) and glycogenin (GYG).

[0034] In the present invention, the term "mesenchymal stem cells (MSCs)" refers to a type of adult stem cells that exist in various tissues including adipose tissue, bone marrow, etc., and have the potential for self-renewal and multi-directional differentiation. These cells can differentiate into various types of cells, such as osteocytes, chondrocytes, and adipocytes, etc., under appropriate conditions, and have the characteristics of immune regulation and promoting tissue repair.

[0035] In the present invention, the term "induced pluripotent stem cells (iPSCs)" refers to the cells generated by the process of reprogramming mature somatic cells into pluripotent stem cells through the overexpression of transcription factors (such as Oct4, Sox2, Klf4, and c-Myc). These iPSCs have pluripotency similar to that of embryonic stem cells and can differentiate into any cell type in the body.

[0036] In the present invention, the term "chimeric antigen receptor T-cells (CAR-T cells)" refers to genetically engineered T lymphocytes that express a specific chimeric antigen receptor on their surface. This receptor is usually composed of a single-chain antibody fragment (scFv) that can recognize the antigen on the surface of tumor cells and one or more signal transduction domains for T cell activation, enabling CAR-T cells to specifically recognize and kill tumor cells.

[0037] In the present invention, the term "glycogen-engineered mesenchymal stem cells" refers to the modification of mesenchymal stem cells by genetic engineering means. According to the embodiments of the present invention, key glycogen synthase genes, such as GYS and UGP, are overexpressed in mesenchymal stem cells through lentiviral vectors, and similar effects can also be achieved by techniques such as mRNA transfection.

[0038] In the present invention, the term "glycogen-engineered induced pluripotent stem cells" refers to the modification of induced pluripotent stem cells by genetic engineering means. According to the embodiments of the present invention, key glycogen synthase genes, such as GYS and UGP, are overexpressed in induced pluripotent stem cells through lentiviral vectors, and similar effects can also be achieved by techniques such as mRNA transfection.

[0039] In the present invention, the term "glycogen-engineered chimeric antigen receptor T-cells" refers to the modification of chimeric antigen receptor T-cells by genetic engineering means. According to the embodiments of the present invention, key glycogen synthase genes, such as GYS and UGP, are overexpressed in chimeric antigen receptor T-cells through lentiviral vectors, and similar effects can also be achieved by techniques such as mRNA transfection.

[0040] The present invention provides a cell, a method for increasing glycogen accumulation in cells, a pharmaceutical composition and related uses, which will be described in detail below.

[0041] Cell

[0042] In the first aspect of the present invention, the present invention provides a cell. According to an embodiment of the present invention, the key enzymes for glycogen synthesis in the cell are upregulated, and the cell is selected from at least one of stem cells and T cells. According to the cell of the embodiment of the present invention, the glycogen accumulation is increased, the anti-starvation ability is enhanced, the cell activity after implantation is enhanced, the killing effect on tumors is enhanced, the therapeutic effect on stem cell indications is improved, especially the therapeutic effect on pulmonary fibrosis is improved.

[0043] According to an embodiment of the present invention, the upregulation of the key enzymes for glycogen synthesis or the upregulation of the key enzymes for glycogen synthesis includes at least one of overexpressing the key enzymes for glycogen synthesis and increasing the activity of the key enzymes for glycogen synthesis.

[0044] According to an embodiment of the present invention, the upregulation of the key enzymes for glycogen synthesis or the upregulation of the key enzymes for glycogen synthesis includes overexpressing the key enzymes for glycogen synthesis. In the embodiment of the present invention, for example, overexpressing at least one of GYS, UGP, GBE, GYG, overexpressing GYS, co-expressing GYS and UGP, co-expressing GYS and GBE, co-expressing GYS and GYG.

[0045] According to an embodiment of the present invention, the upregulation of the key enzymes for glycogen synthesis or the upregulation of the key enzymes for glycogen synthesis includes increasing the activity of the key enzymes for glycogen synthesis. In the embodiment of the present invention, for example, overexpressing GYS with phosphorylated site mutations (GYSMUT group), overexpressing GYS with phosphorylated sequence truncated (GYSDELC group), overexpressing GYS with phosphorylated site mutations (GYS(698,709) group), overexpressing GYS with phosphorylated site mutations (GYS(709,711) group), overexpressing GYS with phosphorylated site mutations (GYS(10,11) group).

[0046] According to an embodiment of the present invention, the upregulation of the key enzymes for glycogen synthesis or the upregulation of the key enzymes for glycogen synthesis includes overexpressing the key enzymes for glycogen synthesis and increasing the activity of the key enzymes for glycogen synthesis. In the embodiment of the present invention, for example, co-expressing dephosphorylation-inhibited (phosphorylated sequence truncated) GYS and UGP (GYSDELC-UGP group).

[0047] According to an embodiment of the present invention, the key enzymes for glycogen synthesis include at least one of GYS and UGP.

[0048] According to an embodiment of the present invention, the key enzymes for glycogen synthesis include GYS and UGP.

[0049] According to an embodiment of the present invention, the overexpression of the key enzyme for glycogen synthesis is achieved by at least one of the following methods:

[0050] Virus / non-virus vector-mediated gene / mRNA transfection technology, transposase or recombinase-mediated gene integration, and CRISPR / CAS system-based gene editing or transcriptional activation.

[0051] Among them, it should be explained that "virus vector-mediated gene / mRNA transfection" refers to using a virus vector (such as adenovirus, lentivirus, etc.) to deliver the target gene or mRNA into host cells to achieve gene expression or silencing.

[0052] "Non-virus vector-mediated gene / mRNA transfection" refers to using non-virus methods (such as liposomes, electroporation, etc.) to deliver genes or mRNA into cells, avoiding the immune response and safety issues that may be caused by virus vectors.

[0053] "Transposase or recombinase-mediated gene integration" refers to using a transposase (such as PiggyBac transposase, TALE protein, zinc finger protein, etc.) or a recombinase (such as Bxb1, Cre / LoxP system) to integrate foreign genes into the genome of host cells to achieve long-term and stable gene expression.

[0054] "CRISPR / CAS system-based gene editing" refers to the CRISPR / CAS system (especially Cas9 protein) being used to precisely cut the genomic target DNA sequence to achieve gene knockout, repair, or insertion.

[0055] "CRISPR / CAS system-based transcriptional activation" refers to the CRISPR / CAS system, especially the dCas9 protein, which achieves transcriptional activation of specific genes by binding to specific transcriptional activators, thereby increasing the expression of specific genes.

[0056] According to an embodiment of the present invention, the overexpression of the key enzyme for glycogen synthesis is achieved by virus / non-virus vector-mediated gene / mRNA transfection technology.

[0057] According to an embodiment of the present invention, the virus / non-virus vector-mediated gene / mRNA transfection technology is achieved by using at least one of the following methods:

[0058] Liposome or lipid nanoparticle (LNP)-mediated transfection technology, calcium phosphate-mediated transfection technology, cationic transfection reagent-mediated transfection technology, virus-like particle (VLP)-mediated transfection technology, electroporation transfection technology, gene delivery using lentiviral vectors, gene delivery using adeno-associated virus vectors, gene delivery using adenoviral vectors, and gene delivery using Sendai virus vectors.

[0059] According to an embodiment of the present invention, the gene / mRNA transfection technology is achieved by gene delivery using a lentiviral vector.

[0060] According to an embodiment of the present invention, the increase in the activity of the key glycogen synthase enzyme is achieved by at least one of the following methods: activator activation treatment, inducible expression of the key glycogen synthase enzyme element controlled by a transcriptional activation element, and reduction or removal of the phosphorylation inhibition of the key glycogen synthase enzyme.

[0061] According to an embodiment of the present invention, the activator includes insulin.

[0062] According to an embodiment of the present invention, the transcriptional activation element includes at least one of TetR, estrogen receptor, and vitamin D receptor.

[0063] According to an embodiment of the present invention, the transcriptional activation element includes TetR.

[0064] According to an embodiment of the present invention, the reduction or removal of the phosphorylation inhibition of the key glycogen synthase enzyme is carried out by at least one of the following methods: mutating the phosphorylation inhibition site on the cell genome and truncating the region where the phosphorylation inhibition site is located.

[0065] According to an embodiment of the present invention, the phosphorylation inhibition site includes at least one of Ser-8, Ser-641, Ser-645, Ser-649, Ser-653, Ser-654, and Ser-657.

[0066] According to an embodiment of the present invention, the phosphorylation inhibition site includes Ser-8, Ser-641, Ser-645, Ser-649, Ser-653, Ser-654, and Ser-657.

[0067] According to an embodiment of the present invention, the region where the phosphorylation inhibition site is truncated includes 100 amino acids at the C-terminus of the key glycogen synthase enzyme.

[0068] According to an embodiment of the present invention, the region where the phosphorylation inhibition site is truncated includes 100 amino acids at the C-terminus of GYS. Taking GYS from mice as an example, the truncated sequence is

[0069] PASVPPSPSLSRHSSPHQSEDEEEPRDGPLGEDSERYDEEEEAAKDRRNIRAPEWPRRASCSSSTGGSKRSNSVDTGPSSSLSTPTEPLSPTSSLGEERN (SEQ ID NO:1).

[0070] According to an embodiment of the present invention, the mutated phosphorylation inhibitory site is achieved by at least one of the following methods: lentiviral vector-mediated gene editing, genome editing using the CRISPR / CAS system, oligonucleotide-mediated homology-directed repair technology to introduce mutations, and mRNA base editing technology to introduce mutations at the RNA level.

[0071] According to an embodiment of the present invention, the mutated phosphorylation inhibitory site is achieved by lentiviral vector-mediated gene editing.

[0072] According to an embodiment of the present invention, truncating the region where the phosphorylation inhibitory site is located is achieved by at least one of the following methods: lentiviral vector-mediated gene editing and genome editing using the CRISPR / CAS system.

[0073] According to an embodiment of the present invention, truncating the phosphorylation inhibitory site is achieved by lentiviral vector-mediated gene editing.

[0074] According to an embodiment of the present invention, it further includes: overexpressing the key glycogen synthase enzyme whose phosphorylation inhibition is reduced or removed.

[0075] According to an embodiment of the present invention, overexpressing the key glycogen synthase enzyme whose phosphorylation inhibition is reduced or removed is achieved by at least one of the following methods: virus / non-virus vector-mediated gene / mRNA transfection technology, transposase or recombinase-mediated gene integration, and CRISPR / CAS system-based gene editing or transcriptional activation.

[0076] According to an embodiment of the present invention, overexpressing the key glycogen synthase enzyme whose phosphorylation inhibition is reduced or removed is achieved by virus / non-virus vector-mediated gene / mRNA transfection technology.

[0077] According to an embodiment of the present invention, the gene / mRNA transfection technology is achieved by at least one of the following methods: liposome- or LNP-mediated transfection technology, calcium phosphate-mediated transfection technology, cationic transfection reagent-mediated transfection technology, virus-like particle VLP-mediated transfection technology, electroporation transfection technology, lentiviral vector for gene delivery, adeno-associated virus vector for gene delivery, adenovirus vector for gene delivery, and Sendai virus vector for gene delivery.

[0078] According to an embodiment of the present invention, the gene / mRNA transfection technology is achieved by gene delivery through a lentiviral vector.

[0079] According to an embodiment of the present invention, the stem cells are selected from at least one of adult stem cells, pluripotent stem cells, unipotent stem cells, and totipotent stem cells.

[0080] According to an embodiment of the present invention, the cells are preferably pluripotent stem cells and T lymphocytes, and more preferably mesenchymal stem cells, induced pluripotent stem cells, and chimeric antigen receptor T cells.

[0081] Method for increasing glycogen accumulation in cells

[0082] In a second aspect of the present invention, the present invention provides a method for increasing glycogen accumulation in cells. According to an embodiment of the present invention, the method includes: upregulating key glycogen synthase enzymes in cells selected from at least one of stem cells and T cells. According to the method of the embodiment of the present invention, the glycogen accumulation, anti-starvation ability, and cell viability after implantation in stem cells and T cells are increased, thereby improving the killing effect on tumors and the therapeutic effect on stem cell indications, especially improving the therapeutic effect on pulmonary fibrosis.

[0083] According to an embodiment of the present invention, the upregulation of the key glycogen synthase enzyme or the upregulation of the key glycogen synthase enzyme includes overexpressing the key glycogen synthase enzyme or increasing the activity of the key glycogen synthase enzyme.

[0084] According to an embodiment of the present invention, the key glycogen synthase enzyme includes at least one of GYS and UGP.

[0085] According to an embodiment of the present invention, the key glycogen synthase enzyme includes GYS and UGP.

[0086] According to an embodiment of the present invention, the overexpression of the key glycogen synthase enzyme is achieved by at least one of the following methods:

[0087] Virus / non-virus vector-mediated gene / mRNA transfection technology, transposase or recombinase-mediated gene integration, and CRISPR / CAS system-based gene editing or transcriptional activation.

[0088] According to an embodiment of the present invention, the overexpression of the key glycogen synthase enzyme is achieved by virus / non-virus vector-mediated gene / mRNA transfection technology.

[0089] According to an embodiment of the present invention, the virus / non-virus vector-mediated gene / mRNA transfection technology is achieved by using at least one of the following methods: liposome or lipid nanoparticle (LNP)-mediated transfection technology, calcium phosphate-mediated transfection technology, cationic transfection reagent-mediated transfection technology, virus-like particle (VLP)-mediated transfection technology, electroporation transfection technology, lentiviral vector for gene delivery, adeno-associated virus vector for gene delivery, adenovirus vector for gene delivery, and Sendai virus vector for gene delivery.

[0090] According to an embodiment of the present invention, the gene transfection technology is achieved by gene delivery using a lentiviral vector.

[0091] According to an embodiment of the present invention, the increase in the activity of the key glycogen synthase enzyme is achieved by at least one of the following methods: activator activation treatment, inducible expression of the key glycogen synthase enzyme element controlled by a transcriptional activation element, and reduction or removal of the phosphorylation inhibition of the key glycogen synthase enzyme.

[0092] According to an embodiment of the present invention, the activator includes insulin.

[0093] According to an embodiment of the present invention, the transcriptional activation element includes at least one of TetR, estrogen receptor, and vitamin D receptor.

[0094] According to an embodiment of the present invention, the transcriptional activation element includes TetR.

[0095] According to an embodiment of the present invention, the reduction or removal of the phosphorylation inhibition of the key glycogen synthase enzyme is carried out by at least one of the following methods: mutating the phosphorylation inhibition site on the cell genome and truncating the region where the phosphorylation inhibition site is located.

[0096] According to an embodiment of the present invention, the phosphorylation inhibition site includes at least one of Ser-8, Ser-641, Ser-645, Ser-649, Ser-653, Ser-654, and Ser-657.

[0097] According to an embodiment of the present invention, the phosphorylation inhibition site includes at least one of Ser-8, Ser-641, Ser-645, Ser-649, Ser-653, Ser-654, and Ser-657 on GYS.

[0098] According to an embodiment of the present invention, the phosphorylation inhibition site includes Ser-8, Ser-641, Ser-645, Ser-649, Ser-653, Ser-654, and Ser-657.

[0099] According to an embodiment of the present invention, the phosphorylation inhibitory sites include Ser-8, Ser-641, Ser-645, Ser-649, Ser-653, Ser-654, and Ser-657 on GYS.

[0100] According to an embodiment of the present invention, the region where the phosphorylation inhibitory site is truncated includes 100 amino acids at the C-terminus of the key glycogen synthase enzyme.

[0101] According to an embodiment of the present invention, the region where the phosphorylation inhibitory site is truncated includes 100 amino acids at the C-terminus of GYS.

[0102] According to an embodiment of the present invention, the mutation of the phosphorylation inhibitory site is achieved by using at least one of the following methods: lentiviral vector-mediated gene editing, genome editing using the CRISPR / CAS system, oligonucleotide-mediated homology-directed repair technology to introduce mutations, and mRNA base editing technology to introduce mutations at the RNA level.

[0103] According to an embodiment of the present invention, the mutation of the phosphorylation inhibitory site is achieved by lentiviral vector-mediated gene editing.

[0104] According to an embodiment of the present invention, the truncation of the region where the phosphorylation inhibitory site is located is achieved by using at least one of the following methods: lentiviral vector-mediated gene editing and genome editing using the CRISPR / CAS system.

[0105] According to an embodiment of the present invention, the truncation of the region where the phosphorylation inhibitory site is located is achieved by lentiviral vector-mediated gene editing.

[0106] According to an embodiment of the present invention, it further includes: overexpressing the key glycogen synthase enzyme whose phosphorylation inhibition is reduced or removed.

[0107] According to an embodiment of the present invention, the overexpression of the key glycogen synthase enzyme whose phosphorylation inhibition is reduced or removed is achieved by at least one of the following methods: virus / non-virus vector-mediated gene / mRNA transfection technology, transposase or recombinase-mediated gene integration, and CRISPR / CAS system-based gene editing or transcriptional activation.

[0108] According to an embodiment of the present invention, the overexpression of the key glycogen synthase enzyme whose phosphorylation inhibition is reduced or removed is achieved by virus / non-virus vector-mediated gene / mRNA transfection technology.

[0109] According to an embodiment of the present invention, the gene / mRNA transfection technology is achieved by using at least one of the following methods: liposome- or LNP-mediated transfection technology, calcium phosphate-mediated transfection technology, cationic transfection reagent-mediated transfection technology, virus-like particle (VLP)-mediated transfection technology, electroporation transfection technology, gene delivery using a lentiviral vector, gene delivery using an adeno-associated virus vector, gene delivery using an adenovirus vector, and gene delivery using a Sendai virus vector.

[0110] According to an embodiment of the present invention, the gene / mRNA transfection technology is achieved by gene delivery using a lentiviral vector.

[0111] According to an embodiment of the present invention, the stem cells are selected from at least one of adult stem cells, pluripotent stem cells, unipotent stem cells, and totipotent stem cells.

[0112] According to an embodiment of the present invention, the cells are preferably pluripotent stem cells and T lymphocytes, and more preferably mesenchymal stem cells, induced pluripotent stem cells, and chimeric antigen receptor T cells.

[0113] Pharmaceutical composition

[0114] In a third aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the stem cells described in the first aspect of the present invention. The pharmaceutical composition according to the embodiment of the present invention has enhanced killing effect on tumors and improved therapeutic effect on stem cell indications, especially improved therapeutic effect on pulmonary fibrosis.

[0115] Use

[0116] In a fourth aspect of the present invention, the present invention provides the use of the stem cells described in the first aspect of the present invention or the pharmaceutical composition described in the third aspect of the present invention in the preparation of a drug for treating or preventing diseases applicable to the stem cells and / or T cells or for anti-aging health care.

[0117] According to an embodiment of the present invention, the diseases include at least one selected from the following: emphysema, bronchopulmonary dysplasia, pulmonary fibrosis, rheumatoid arthritis, pulmonary embolism, pneumothorax, skin trauma, skin scar, cartilage injury, myocardial injury, diabetes, autoimmune cirrhosis, and cancer.

[0118] According to an embodiment of the present invention, the cancer is selected from at least one of the following: liver cancer, gastric cancer, lung cancer, pancreatic cancer, breast cancer, cervical cancer, skin cancer, prostate cancer, melanoma, thyroid cancer, uterine fibroids, lymphoma, esophageal cancer, intestinal cancer, myeloma, nasal cancer, bone cancer, head and neck cancer, oral cancer, kidney cancer, and Kaposi's sarcoma.

[0119] Use

[0120] In the fifth aspect of the present invention, the present invention provides the use of key glycogen synthase enzymes or reagents that upregulate key glycogen synthase enzymes in enhancing the stress resistance of stem cells.

[0121] According to an embodiment of the present invention, the key glycogen synthase enzyme includes at least one of GYS and UGP.

[0122] According to an embodiment of the present invention, the key glycogen synthase enzyme includes GYS and UGP.

[0123] According to an embodiment of the present invention, the stem cells are selected from at least one of adult stem cells, pluripotent stem cells, unipotent stem cells, and totipotent stem cells.

[0124] According to an embodiment of the present invention, the cells are preferably pluripotent stem cells and T lymphocytes, and more preferably mesenchymal stem cells, induced pluripotent stem cells, and chimeric antigen receptor T cells.

[0125] The solutions of the present invention will be explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. For those not specifically noted in the examples regarding technical or conditions, they shall be in accordance with the techniques or conditions described in the literature in this field or in accordance with the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0126] Example 1: A large amount of glycogen accumulation was achieved in mesenchymal stem cells through overexpression and co-expression of key enzymes

[0127] In this example, GYS was overexpressed (GYS1 group, also known as GYS1WT group), GYS and UGP were co-expressed (GYS-UGP group), GYS was co-expressed with glycogen branching enzyme GBE (GYS-GBE group), and GYS was co-expressed with GYG responsible for glycogen initiation (GYS-GYG group) in mouse mesenchymal stem cells (MSCs) (product number: CP-M138).

[0128] The specific experimental steps of this example are as follows:

[0129] (1) The gene sequences of GYS, UGP, GBE, and GYG were obtained through gene synthesis.

[0130] (2) Preparation of lentiviral vectors: The above sequences were respectively integrated into the pCDH lentiviral vector backbone through Gibson (NEB#E5510S) to generate replication-defective lentiviral vectors expressing GYS, UGP, GBE, and GYG, respectively.

[0131] Among them, the preparation process of the lentiviral vector with replication defect is as follows: Seed 293T cells in a cell culture dish with a diameter of 10 cm, and transduce the 293T cells with viral plasmids using Lipo8000 (Beyotime #C0533). Add 4.5 μg of lentiviral vector transgenic plasmids (correspondingly expressing GYS, UGP, GBE, GYG or GFP, luciferase Akaluc, puromycin resistance gene), 1.5 μg of pVSV-G (VSV glycoprotein expression plasmid), and 3 μg of psPAX2 plasmid to each dish of cells. Mix the plasmids in 450 μL of opti-MEM with 12 μL of lipo8000 and add them to the medium of 293T cells. Collect the supernatants at 24 hours and 48 hours respectively. Concentrate the lentiviral vector particles in the supernatant using the lentiviral vector concentrate (5X) purchased from Wuhan Sevier Biotechnology Co., Ltd. After concentration, resuspend the precipitate with 200 μL of PBS, and centrifuge to collect the lentiviral vector GYS, UGP, GBE, and GYG suspensions respectively.

[0132] (3) Transduce mouse mesenchymal stem cells: Mouse mesenchymal stem cells are purchased from Procell (product number: CP-M138) and cultured using DMEM F-12 medium (10% FBS, 1% PS). Add the lentiviral vector GYS (corresponding to the GYS1 group, also known as the GYS1 WT group), lentiviral vector UGP (also known as the UGP group), lentiviral vector GYS + UGP (corresponding to the GYS-UGP group), lentiviral vector GYS + GBE (corresponding to the GYS-GBE group), and lentiviral vector GYS + GYG (corresponding to the GYS-GYG group) suspensions obtained in step (2) into the mesenchymal stem cell medium respectively. Passage the mesenchymal stem cells 48 hours later, add puromycin (final concentration 1 μg / mL) to the medium for drug screening, and passage the mesenchymal stem cells for more than one week respectively.

[0133] (4) Glycogen content determination: Determine the glycogen content of the above-mentioned mesenchymal stem cells respectively, and perform quantitative detection using a glycogen content determination kit (Solarbio, BC0345-100T / 96S).

[0134] The experimental results of glycogen content determination are as Figure 1 shown. Compared with the mesenchymal stem cells not transduced with any lentiviral vector, overexpression of GYS in mesenchymal stem cells can promote the increase of glycogen content in mesenchymal stem cells; co-expression of GYS and UGP in mesenchymal stem cells can significantly promote the increase of glycogen content in mesenchymal stem cells; co-expression of GYS and GBE, and co-expression of GYS and GYG cannot promote the increase of glycogen content in mesenchymal stem cells.

[0135] Example 2: A large amount of glycogen accumulation in mesenchymal stem cells was achieved through functional optimization of key enzymes

[0136] The implementation method was referred to Example 1, with the difference that in this example, GYS with phosphorylated site mutations (GYSMUT group), GYS with phosphorylated sequence truncated (GYSDELC group), co-expression of GYS with dephosphorylation inhibition (truncated phosphorylated sequence) and UGP (GYSDELC-UGP group), GYS with phosphorylated site mutations (GYS(698,709) group), GYS with phosphorylated site mutations (GYS(709,711) group), and GYS with phosphorylated site mutations (GYS(10,11) group) were overexpressed in mouse mesenchymal stem cells.

[0137] (1) GYSMUT (GYS with phosphorylated site mutations), GYSDELC (GYS with phosphorylated sequence truncated), GYS(698,709), GYS(709,711), and GYS(10,11) were obtained by gene synthesis. Among them, GYSMUT is a mutant of wild-type GYS that mutated its phosphorylation inhibitory sites (Ser-8, Ser-641, Ser-645, Ser-649, Ser-653, Ser-654, and Ser-657 were all mutated); GYSDELC is a mutant of wild-type GYS with the C-terminal 100 amino acids truncated; GYS(698,709) is a mutant of wild-type GYS that mutated its phosphorylation inhibitory sites (Ser-698 and Ser-709); GYS(709,711) is a mutant of wild-type GYS that mutated its phosphorylation inhibitory sites (Ser-709 and Ser-711); GYS(10,11) is a mutant of wild-type GYS that mutated its phosphorylation inhibitory sites (Ser-10 and Ser-11).

[0138] (2) Preparation of lentiviral vectors: The above sequences were respectively integrated into the pCDH lentiviral vector backbone by Gibson to generate replication-defective lentiviral vectors expressing GYSMUT, GYSDELC, GYS(698,709), GYS(709,711), and GYS(10,11).

[0139] Among them, the preparation process of the replication-defective lentiviral vector was as shown in Example 1. Lentiviral vector suspensions of GYSMUT, GYSDELC, GYS(698,709), GYS(709,711), and GYS(10,11) were collected by centrifugation respectively.

[0140] (3) Preparation of mRNA: Construct in vitro transcription linear DNA templates for the expression of GYSMUT and UGP+GYSDELC driven by the T7 promoter (sequence: TAATACGACTCACTATAGG (SEQ ID NO:2)) through PCR, and obtain mRNA for the expression of GYSMUT and UGP+GYSDELC through in vitro transcription using the T7 ARCA mRNA kit (NEB, E2060S).

[0141] (4) Transduction of mouse mesenchymal stem cells (MSCs): Culture using DMEM F-12 medium (10% FBS, 1% PS). Respectively add the lentiviral vector suspensions of GYSMUT, GYSDELC, GYS(698,709), GYS(709,711), and GYS(10,11) obtained in step (2) into the mesenchymal stem cell medium, and jointly add the lentiviral vector UGP obtained in Example 1 and the above GYSDELC suspension into the mesenchymal stem cell medium. After 48 hours, passage the mesenchymal stem cells, add puromycin (final concentration 1 μg / mL) to the medium for drug screening, and passage and culture the mesenchymal stem cells for more than one week.

[0142] For mRNA transduction, seed mesenchymal stem cells into a 24-well plate at a density of 50,000 cells per well. After 24 hours, use Lipofectamine MessengerMAX liposome (ThermoFisher, LmRNA001) to transfect the mRNA expressing GYSMUT into mesenchymal stem cells according to the instructions, and perform subsequent tests after 48 hours.

[0143] (5) Periodic acid-Schiff staining experiment: Perform periodic acid-Schiff staining experiments on the above mesenchymal stem cells transfected with lentiviral vectors GYSMUT, GYSDELC, UGP+GYSDELC, GYS(698,709), GYS(709,711), and GYS(10,11). Use the periodic acid-Schiff staining kit (product number C0142M) to perform staining according to the standard procedure in the instructions. This example exemplarily shows the results of the periodic acid-Schiff staining experiment of mesenchymal stem cells transfected with the lentiviral vector GYSMUT, as Figure 2 shown, a large amount of glycogen accumulation is achieved in GYSMUT mesenchymal stem cells.

[0144] Perform periodic acid-Schiff staining on mesenchymal stem cells transfected with GYSMUT and UGP+GYSDELC mRNA, as Figure 3 shown, compared with the wild-type mesenchymal stem cell control group, a significant increase in glycogen is achieved in both the GYSMUT and UGP+GYSDELC groups of cells.

[0145] (6) Glycogen content determination: The glycogen content of the above-mentioned mesenchymal stem cells was determined respectively, and a glycogen content determination kit was used for quantitative detection (Solarbio, BC0345-100T / 96S).

[0146] The experimental results of glycogen content determination are as Figure 4 shown. Compared with mesenchymal stem cells not transduced with any lentiviral vector, overexpression of wild-type GYS1 and UGP in mesenchymal stem cells can increase the glycogen content of mesenchymal stem cells. Overexpression of GYSMUT in mesenchymal stem cells can promote the accumulation of a large amount of glycogen in mesenchymal stem cells; overexpression of GYSDELC in mesenchymal stem cells can promote the accumulation of a large amount of glycogen in mesenchymal stem cells; co-expression of UGP and GYSDELC in mesenchymal stem cells can further increase the glycogen content in mesenchymal stem cells. However, GYS(698, 709), GYS(709, 711) and GYS(10, 11) did not achieve a large accumulation of glycogen, and there was no significant difference from the effect of wild-type GYS1.

[0147] Example 3: A large amount of glycogen accumulation in induced pluripotent stem cells was achieved through the functional optimization of key enzymes

[0148] The implementation method refers to Example 2, with the difference that in this example, phosphorylated site-mutated GYS (GYSMUT group) was overexpressed in induced pluripotent stem cells.

[0149] (1) Transduction of induced pluripotent stem cells (iPSCs): iPSCs derived from healthy humans were purchased from Zhejiang Meisen Cell Technology (product number: CTCC-003-0381), cultured in complete medium (product number: CTCC-003-0381-CM), transfected with the lentiviral vector expressing GYSMUT in step (2) of Example 2, and the iPSCs were passaged and cultured with puromycin drug screening (final concentration 1 μg / mL) for more than one week to obtain GYSMUT iPSCs.

[0150] (2) Glycogen content determination: The glycogen content of the above-mentioned induced pluripotent stem cells was determined respectively, and a glycogen content determination kit was used for quantitative detection (Solarbio, BC0345-100T / 96S).

[0151] The experimental results of glycogen content determination are as Figure 5 shown. Compared with induced pluripotent stem cells not transduced with any lentiviral vector, overexpression of GYSMUT in induced pluripotent stem cells can achieve a large accumulation of glycogen.

[0152] Example 4: A large amount of glycogen accumulation in chimeric antigen receptor T cells was achieved through the functional optimization of key enzymes

[0153] The implementation method refers to Example 2, with the difference that in this example, GYS with phosphorylated site mutation (GYSMUT) is overexpressed in chimeric antigen receptor T cells.

[0154] (1) Two chimeric antigen receptors HA-28ζ targeting GD2 were obtained by gene synthesis, and their amino acid sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively.

[0155] MELGLSWVFLVAILEGVQCEVQLLQSGPELEKPGASVMISCKASGSSFTGYNMNWVRQNIGKSLE

[0156] WIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMKYWGQGTSVT

[0157] VSSGGGGSGGGGSGGGGSDVVMTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPG

[0158] QSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELE

[0159] PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG

[0160] VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE

[0161] PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT

[0162] VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKFWVLVVVGGVLACYSLLVTVAFIIFWVR

[0163] SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNEL

[0164] NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:3)

[0165] MPHSSDSSDSSFSRSPPPGKQDSSDDVRRVQRREKNRIAAQKSRQRQTQKADTLHLESEDLEKQNAALRKEIKQLTEELKYFTSVLNSHEPLCSVLAASTPSPPEVVYSAHAFHQPHVSSPRFQPRAKRGSGSGGGSGGGTGAGSGEGRGSLLTCGDVEENPGPMEFGLSWLFLVAILKGVQCSRDILLTQTPLSLPVSLGDQASISCRSSQSLVHRNGNTYLHWYLQKPGQSPKLLIHKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPPLTFGAGTKLELKGGGGSGGGGSGGGGSEVKLQQSGPSLVEPGASVMISCKASGSSFTGYNMNWVRQNIGKSLEWIGAIDPYYGGTSYNQKFKGRATLTVDKSSSTAYMHLKSLTSEDSAVYYCVSGMKYWGQGTSVTVSSAKTTPPSVYGRVTVSSAEPKSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:4)

[0166] (2) Preparation of lentiviral vector: Integrate GYSMUT and chimeric antigen receptor HA-28ζ sequences in Example 2 into the pCDH lentiviral vector backbone by Gibson to generate a lentiviral vector co-expressing GYSMUT and HA-H28ζ.

[0167] Among them, the preparation process of the replication-defective lentiviral vector is as shown in Example 1. Centrifuge and collect to obtain lentiviral vectors co-expressing GYSMUT and HA-H28ζ respectively.

[0168] (3) Transduction of chimeric antigen receptor T cells (CAR-T cells): Lymphatic T cells are purchased from Milestone Biological Science & Technology Co., Ltd. The cells are cultured in RPMI1640 medium (HyClone, SH30027), which is supplemented with 10% fetal bovine serum (FBS) (Gibco, 2168090RP), 55 μM β-mercaptoethanol (Thermo, 21985023), 300 U / mL interleukin-2 (IL-2) (PeproTech, 200-02-50UG), and 100 mM L-glutamine; to promote cell activation, the cells are co-cultured with anti-CD3 / CD28 nanobeads (Miltenyi Biotec, 5220609381) for stimulation.

[0169] Transfect the above-mentioned lymphatic T cells with the lentiviral vector expressing GYSMUT and chimeric antigen receptor HA-28ζ in step (2), passage and culture with puromycin drug screening (final concentration 1 μg / mL) for more than one week to obtain GYSMUT CAR-T cells.

[0170] (4) Glycogen content determination: Determine the glycogen content of the above-mentioned chimeric antigen receptor T cells respectively, and use a glycogen content determination kit for quantitative detection (Solarbio, BC0345-100T / 96S).

[0171] As Figure 6 , the experimental results of glycogen content determination show that overexpression of GYSMUT in chimeric antigen receptor T cells can achieve a large amount of glycogen accumulation compared with chimeric antigen receptor T cells not transduced with any lentiviral vector.

[0172] Example 5: By increasing the glycogen accumulation of mesenchymal stem cells, the anti-starvation ability of mesenchymal stem cells is greatly improved.

[0173] In this example, DPBS buffer was used as the starvation-conditioned medium to culture the glycogen-engineered mesenchymal stem cells prepared in Example 1 and Example 2 and the control group of wild-type mesenchymal stem cells under starvation conditions, and CCK8 was used to monitor the cell survival. The results are as Figure 7 shown. The cells in the control group apoptosed within 8 hours, while the cells in the experimental group (GYSMUT mesenchymal stem cells) survived for more than 72 hours. This example confirmed the anti-starvation stress ability of glycogen-engineered mesenchymal stem cells through in vitro starvation treatment.

[0174] Example 6: By increasing the glycogen accumulation of mesenchymal stem cells, the survival ability after implantation into mice was greatly improved

[0175] To test the cell survival ability of glycogen-accumulating mesenchymal stem cells after implantation in vivo, a bleomycin-induced pulmonary fibrosis mouse model was used for testing. A pulmonary fibrosis mouse model was constructed by perfusing bleomycin (3 mg / kg) into the trachea. The soft tissue above the trachea of the mouse was cut open with scissors, and a 1 mL syringe was used to aspirate the PBS suspension of the glycogen-engineered mesenchymal stem cells prepared in Example 1 and Example 2 (10,000 cells / μL), and then injected into the pulmonary fibrosis mice through the trachea, and the wound of the mouse was sutured with surgical sutures.

[0176] The GYSMUT mesenchymal stem cells co-expressing Akaluc luciferase were implanted into the lungs of mice through the trachea. The control group was implanted with mesenchymal stem cells expressing only Akaluc. The substrate of Akaluc, Akalumine-HCL (product number: V41343), was dissolved in PBS (5 mg / mL), and the mice were intraperitoneally injected (1 mg / mouse) on the 3rd and 7th days and imaged in vivo using a Lumina III instrument. The results are as Figure 8 and Figure 9 shown, showing that the survival rate of GYSMUT mesenchymal stem cells on the 7th day was significantly higher than that of the control group, proving that glycogen-engineered mesenchymal stem cells have stronger in vivo survival ability.

[0177] This example confirmed through in vivo imaging that glycogen-engineered mesenchymal stem cells can significantly improve the survival ability after implantation.

[0178] Example 7: Glycogen-engineered mesenchymal stem cells significantly improve the therapeutic effect

[0179] Taking pulmonary fibrosis treatment as an application scenario, the therapeutic ability of glycogen-engineered mesenchymal stem cells was verified. According to the method described in Example 4, the glycogen-engineered mesenchymal stem cells prepared in Example 1 and Example 2 and the control group of mesenchymal stem cells expressing GFP were perfused into the trachea of the pulmonary fibrosis mouse model for treatment, and the body weight and survival of the mice were continuously observed. The results are as Figure 10As shown, glycogen-engineered mesenchymal stem cells have better long-term therapeutic effects. This example confirms that the therapeutic effects of glycogen-engineered mesenchymal stem cells are significantly improved compared to ordinary mesenchymal stem cells.

[0180] Example 8: Achieving control of glycogen synthesis in mesenchymal stem cells through the TetR-doxycycline induction system

[0181] In this example, a tetR-controlled inducible dynamic regulation system for glycogen accumulation was established, allowing it to induce glycogen accumulation in vitro and stop the overexpression of GYS in vivo to further improve the survival ability of mesenchymal stem cells. The schematic diagram is as Figure 11 shown.

[0182] According to the above circuit scheme, the TET-GYS lentiviral vector was constructed with reference to the method of Example 1, and the TET-GYS mesenchymal stem cells were constructed. The effect of achieving glycogen accumulation control using doxycycline (DOX) is as follows (periodic acid-Schiff staining), and the experimental results are as Figure 12 shown.

[0183] The mesenchymal stem cells with induced glycogen accumulation were cultured under starvation conditions. The results show that the glycogen accumulation induced by doxycycline promotes the survival of TET-GYS mesenchymal stem cells under starvation conditions. The uninduced TET-GYS mesenchymal stem cells and wild-type mesenchymal stem cells were used as control groups, as Figure 13 shown.

[0184] This example confirms that by introducing the TetR transcription factor to control key glycogen synthesis enzymes such as GYSMUT, dynamic glycogen accumulation in mesenchymal stem cells can be achieved under the induction of inducers such as doxycycline, and the anti-starvation ability of mesenchymal stem cells can be improved.

[0185] Example 9: Glycogen engineering enhances the anti-starvation and post-implantation survival ability of induced pluripotent stem cells (iPSCs)

[0186] The iPSCs in Example 3 were cultured under starvation conditions using DPBS. After 24 hours, CCK8 was used to detect the cell survival situation. The results show that GYSMUT iPSCs have higher cell activity, as Figure 14 shown, indicating that glycogen modification enhances the anti-starvation ability of iPSCs.

[0187] Co-express GYSMUT and Gluc luciferase in iPSCs by lentiviral vector transfection, implant them into the lungs of pulmonary fibrosis mice through the trachea, implant iPSCs expressing only Gluc in the control group, collect the lung tissues of mice on day 0 and day 7 in 1.5 mL centrifuge tubes, add 200 μL of PBS and 10 1-mm grinding beads, then use a grinder to grind thoroughly at low temperature, centrifuge at 1000 g for 10 minutes at 4°C, and take the supernatant to detect the Gluc enzyme activity using a Gluc detection kit (product number: LF061). Evaluate the survival of implanted cells based on the ratio of Gluc signal intensity between the samples on day 7 and day 0. As Figure 15 shown, the survival ratio of GYSMUT iPSCs was significantly higher than that of the control group (P < 0.05). This example shows that glycogen engineering can enhance the anti-starvation ability and in vivo survival ability of pluripotent stem cells represented by iPSCs.

[0188] Example 10: Glycogen engineering enhances the tumor cell killing effect of chimeric antigen receptor T cells

[0189] Culture the GYSMUT CAR-T cells in Example 4 under DPBS starvation conditions using DPBS, and use CAR-T cells expressing EGFP as the control group. After 24 hours, use CCK8 (Beijing Bairuiji Biotechnology, BN15201-500T) to detect the cell survival and calculate its survival ratio. As Figure 16 shown, the results show that GYSMUT chimeric antigen receptor T cells have higher cell activity, indicating that glycogen modification enhances the anti-starvation ability of chimeric antigen receptor T cells.

[0190] Construct the GFP-2A-GD2 fusion protein sequence through gene synthesis. MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKEGRGSLLTCGDVEENPGPMRLDRRALYALVLLLACASLGLLYSSTRNAPSLPNPLALWSPPQGPPRLDLLDLAPEPRYAHIPVRIKEQVVGLLAQNNCSCESKGGSLPLPFLRQVRAVDLTKAFDAEELRAVSVAREQEYQAFLARSRSLADQLLIAPANSPLQYPLQGVEVQPLRSILVPGLSLQEASVQEIYQVNLSASLGTWDVAGEVTGVTLTGEGQPDLTLASPVLDKLNRQLQLVTYSSRSYQANTADTVRFSTKGHEVAFTILVRHPPNPRLYPPSSLPQGAEYNISALVTIATKTFLRYDRLRTLIASIRRFYPTVTIVIADDSDKPERISDPHVEHYFMPFGKGWFAGRNLAVSQVTTKYVLWVDDDFVFTARTRLEKLVDVLEKTPLDLVGGAVREISGYATTYRQLLSVEPGAPGLGNCFRQKQGFHHELVGFPSCVVTDGVVNFFLARTDKVRQVGFDPRLNRVAHLEFFLDGLGFLRVGSCSDVVVDHASKVKLPWTAKDPGAETYARYRYPGSLDQSQVAKHRLLFFKHRLQCMTAE*(SEQ ID NO:5). And construct the GFP-2A-GD2 lentivirus, and transfect Nalm6 cells (IMMOCELL, IML-012) with the lentivirus to construct GFP-2A-GD2 Nalm6 cells.

[0191] Fifty thousand GFP +Nalm6-GD2 cells were seeded into transparent 96-well flat-bottom plates and co-cultured with an equal amount of GYSMUT HA-28ζ CAR T cells in a medium with a total volume of 200 μL. The total GFP fluorescence intensity per well was used as an indicator to quantify the GFP + of Nalm6-GD2 viable cells. As a control, HA-28ζ CAR T cells that did not express GYSMUT were used. After 24 hours of co-culture, fluorescent cells were counted by flow cytometry, and the proportion of surviving cancer cells to the initial number of seeded cells was calculated. As Figure 17 shown, compared with the control group, the proportion of surviving tumor cells in the experimental group was significantly lower, indicating that glycoengineering significantly enhanced the anti-tumor function of CAR-T cells.

[0192] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0193] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limitations on the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A cell, characterized in that, The key enzymes for glycogen synthesis in the cells are upregulated, and the cells are selected from at least one of stem cells and T cells.

2. A method for increasing glycogen accumulation in cells, characterized in that, Including: Upregulating the key enzymes for glycogen synthesis in cells, where the cells are selected from at least one of stem cells and T cells.

3. The cell according to claim 1 or the method according to claim 2, characterized in that, The upregulation of the key enzymes for glycogen synthesis or the upregulation of the key enzymes for glycogen synthesis includes at least one of overexpressing the key enzymes for glycogen synthesis and enhancing the activity of the key enzymes for glycogen synthesis.

4. The cell according to claim 1 or the method according to claim 2, characterized in that, The key enzymes for glycogen synthesis include at least one of GYS and UGP; Preferably, the key enzymes for glycogen synthesis include GYS and UGP.

5. The cell or method according to claim 3, characterized in that, The overexpression of the key enzymes for glycogen synthesis is achieved by at least one of the following methods: Virus / non-virus vector-mediated gene / mRNA transfection technology, transposase or recombinase-mediated gene integration, and CRISPR / CAS system-based gene editing or transcriptional activation.

6. The cell or method according to claim 5, wherein The gene / mRNA transfection technology is achieved by at least one of the following methods: liposome- or LNP-mediated transfection, calcium phosphate-mediated transfection, cationic transfection reagent-mediated transfection, virus-like particle VLP-mediated transfection, electroporation transfection, lentiviral vector-mediated gene delivery, adeno-associated virus vector-mediated gene delivery, adenovirus vector-mediated gene delivery, and Sendai virus vector-mediated gene delivery.

7. The cell or method according to claim 3, wherein The enhancement of the activity of the key enzymes for glycogen synthesis is achieved by at least one of the following methods: Activator activation treatment, inducible expression of the key enzyme element for glycogen synthesis controlled by a transcriptional activation element, and reduction or removal of the phosphorylation inhibition of the key enzymes for glycogen synthesis; Optionally, the activator includes insulin; Optionally, the transcriptional activation element includes at least one of TetR, estrogen receptor, and vitamin D receptor.

8. The cell or method according to claim 7, characterized in that, The reduction or removal of the phosphorylation inhibition of the key enzymes for glycogen synthesis is carried out by the following method: Mutating the phosphorylation inhibition site and / or truncating the region where the phosphorylation inhibition site is located on the cell genome.

9. The cell or method according to claim 8, characterized in that, The phosphorylation inhibition site includes at least one of Ser-8, Ser-641, Ser-645, Ser-649, Ser-653, Ser-654, and Ser-657; Optionally, the phosphorylation inhibition site includes Ser-8, Ser-641, Ser-645, Ser-649, Ser-653, Ser-654, and Ser-657; Optionally, the truncation of the region where the phosphorylation inhibition site is located includes 100 amino acids at the C-terminus of the key enzymes for glycogen synthesis.

10. The cell or method according to claim 8, wherein The mutation of the phosphorylation inhibition site is achieved by at least one of the following methods: lentiviral vector-mediated gene editing, genome editing by the CRISPR / CAS system, introduction of mutations by oligonucleotide-mediated homologous directed repair technology, and introduction of mutations at the RNA level by mRNA base editing technology.

11. The cell or method according to claim 8, characterized in that, The truncation of the region where the phosphorylation inhibition site is located is achieved by at least one of the following methods: lentiviral vector-mediated gene editing and genome editing by the CRISPR / CAS system.

12. The cell or method according to claim 7, characterized in that, Further including: Overexpress the key glycogen synthase enzyme with reduced or removed phosphorylation inhibition.

13. The cell or method according to claim 12, characterized in that, The overexpression of the key glycogen synthase enzyme with reduced or removed phosphorylation inhibition is achieved by at least one of the following methods: Virus / non-virus vector-mediated gene / mRNA transfection technology, transposase or recombinase-mediated gene integration, and CRISPR / CAS system-based gene editing or transcriptional activation.

14. The cell or method according to claim 13, characterized in that, The gene / mRNA transfection technology is achieved by using at least one of the following methods: liposome- or LNP-mediated transfection technology, calcium phosphate-mediated transfection technology, cationic transfection reagent-mediated transfection technology, virus-like particle VLP-mediated transfection technology, electroporation transfection technology, lentiviral vector for gene delivery, adeno-associated virus vector for gene delivery, adenovirus vector for gene delivery, and Sendai virus vector for gene delivery.

15. The cell according to claim 1 or the method according to claim 2, characterized in that, The cells are preferably pluripotent stem cells and T lymphocytes, more preferably mesenchymal stem cells, induced pluripotent stem cells, and chimeric antigen receptor T cells.

16. A pharmaceutical composition, characterized in that, Including: The cells according to any one of claims 1, 3 to 15.

17. Use of the cells according to any one of claims 1, 3 to 15 or the pharmaceutical composition according to claim 16 in the preparation of a drug for treating or preventing diseases applicable to the stem cells and / or T cells or for anti-aging health care.

18. The use according to claim 17, characterized in that, The diseases include at least one selected from the following: emphysema, bronchopulmonary dysplasia, pulmonary fibrosis, rheumatoid arthritis, pulmonary embolism, pneumothorax, skin trauma, skin scar, cartilage injury, myocardial injury, diabetes, autoimmune cirrhosis, and cancer.

19. The use according to claim 18, characterized in that, The cancer is selected from at least one of the following: liver cancer, gastric cancer, lung cancer, pancreatic cancer, breast cancer, cervical cancer, skin cancer, prostate cancer, melanoma, thyroid cancer, uterine fibroids, lymphoma, esophageal cancer, intestinal cancer, myeloma, nasal cancer, bone cancer, head and neck cancer, oral cancer, kidney cancer, Kaposi's sarcoma.

20. Use of a key glycogen synthase enzyme or a reagent that upregulates a key glycogen synthase enzyme in enhancing the stress resistance of stem cells.

21. The use according to claim 20, characterized in that, The key glycogen synthase enzyme includes at least one of GYS and UGP; Preferably, the key glycogen synthase enzyme includes GYS and UGP.

22. The use according to claim 20, characterized in that, The cells are selected from at least one of stem cells and T cells, preferably pluripotent stem cells and T lymphocytes, more preferably mesenchymal stem cells, induced pluripotent stem cells, and chimeric antigen receptor T cells.

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