Base editing system for optimizing engineered t-cells, and use thereof

Abstract

A base editing system for optimizing engineered T-cells, and a use thereof. Provided is a base editing system for modifying engineered T-cells, comprising: i) a base editing polypeptide, and / or an expression construct encoding the base editing polypeptide, the base editing polypeptide comprising a DNA binding protein and at least one deaminase domain; and ii) a guide RNA, and / or an expression construct encoding the guide RNA, the guide RNA targeting any one or any combination of the TRAC, TGFBR2, PD -1, CISH and / or FAS genome regions. The base editing system has efficient gene editing tool efficiency and low indel frequency, can implement precise editing of genes relating to T-cell function enhancement, and has important value for expanding application scenarios of base editing technology in fields such as the preparation of safe and efficient engineered T-cells.

Description

Optimizing the base editing system for engineered T cells and its applications

[0001] Priority and related applications

[0002] This application claims priority to Chinese patent application No. 202311662136.4, filed on December 6, 2023, entitled “Optimized base editing system and its application”, Chinese patent application No. 202410044584.6, filed on January 11, 2024, entitled “Optimized base editing system and its application”, and Chinese patent application No. 202410718712.0, filed on June 4, 2024, entitled “Optimized base editing system and its application”. The entire contents of the above applications, including the appendices, are incorporated into this application by reference. Technical Field

[0003] The present invention belongs to the field of genetic engineering. Specifically, the present invention relates to a base editing system for optimizing engineered T cells and its application, and more specifically, to a base editing system and method for modifying or editing engineered T cells. Background Art

[0004] Chimeric Antigen Receptor T-Cell (CAR-T cell or CAR-T) is a type of T cell modified by genetic engineering technology. It integrates recognition receptors for a target antigen epitope (such as some tumor cell-specific antigen molecules) into the T lymphocyte genome and stimulates the activation of T cells, thereby enhancing the T lymphocyte's ability to recognize and kill specific tumor cells.

[0005] CAR-T cells can be divided into two categories based on the source of the engineered T cells: autologous CAR-T cells and allogeneic (or off-the-shelf) CAR-T cells. The former utilizes T cells extracted from the patient's own body for engineering, while the latter utilizes T cells isolated from a healthy, non-human individual. Each type of CAR-T cell has its own advantages and disadvantages in practical application. For example, a key advantage of autologous CAR-T cells is that, since they are derived from the patient's own body, the CAR gene is integrated and engineered ex vivo. When infused back into the patient, they often exhibit a strong immune response and are immune-resistant. However, in patients with tumors, especially in the later stages of the disease, the number of healthy T cells is relatively limited. Therefore, it is sometimes difficult to isolate sufficient T cells from the patient and complete the production of autologous CAR-T cells in a timely manner. Off-the-shelf CAR-T cells, on the other hand, can be isolated from healthy donors, ensuring a guaranteed quantity and a faster supply cycle, significantly reducing the cost of treatment for patients. Therefore, they represent a highly promising new type of CAR-T cell therapy. However, ordinary off-the-shelf CAR-T cells have the problem of poor clinical efficacy, which is mainly caused by two reasons. First, because they come from different individuals, off-the-shelf CAR-T cells may encounter immune rejection reactions after being injected into the patient's body, and thus will be quickly cleared by the patient's immune system; secondly, the occurrence of graft-versus-host disease (GvHD) causes CAR-T cells from heterologous individuals to identify and mistakenly kill the patient's original immune cells, resulting in the weakening of the patient's immune system. The superposition of these two factors has led to the poor clinical effect of previous off-the-shelf CAR-T cells.

[0006] However, recent studies have found that during the functional modification and preparation of off-the-shelf CAR-T cells, knocking out some genes through gene editing can inhibit immune rejection reactions and the occurrence of GvHD. It can also help improve the proliferation ability, tumor killing ability and maintenance time of off-the-shelf CAR-T cells after they are injected into the patient's body, thereby improving their clinical effects.

[0007] For example, the CISH gene encodes an immune negative regulatory protein that can inhibit the IL-15 signaling pathway in NK cells and T cells to suppress immune responses. Studies have found that downregulating the expression of the CISH gene helps T cell activation, survival, and expansion (Zhu et al., 2022. Metabolic Reprogramming via Deletion of CISH in Human iPSC-Derived NK Cells Promotes In Vivo Persistence and Enhances Anti-tumor Activity.).

[0008] FAS, encoded by the FAS gene, is a receptor protein that interacts with the FASL protein to promote T cell apoptosis. Studies have found that by expressing a dominant-negative effector protein of FAS in CAR-T cells, disrupting the interaction between FAS and FASL, the persistence of CAR-T cells can be prolonged and their tumor-killing efficacy can be enhanced (Yamamoto et al., 2019. T cells genetically engineered to overcome death signaling and enhance adoptive cancer immunotherapy).

[0009] TGF-β is an anti-inflammatory factor expressed in the immune microenvironment of the blood and many tumors. Studies have found that binding of TGF-β to the receptor protein TBR2, encoded by the TGFBR2 gene, triggers activation of downstream signaling pathways, inhibiting T cell activation and survival, thereby suppressing T cell immunity (Rouce et al., 2015. The TGF-β / SMAD pathway is an important mechanism for NK cell immune evasion in childhood B-acute lymphoblastic leukemia). Disrupting the binding and interaction between TGF-β and the receptor TBR2 can help improve the survival of activated T cells (Bollard et al., 2018. Tumor-Specific T-Cells Engineered to Overcome Tumor Immune Evasion Induce Clinical Responses in Patients With Relapsed Hodgkin Lymphoma). Therefore, knocking out the TGFBR2 gene in appropriate regions may improve the efficacy of CAR-T cells.

[0010] In addition, knocking out the immune checkpoint protein PD-1 has been reported to promote T cell activation, expansion, and tumor killing (McGowan et al., 2020. PD-1 disrupted CAR-T cells in the treatment of solid tumors: Promises and challenges). Knockout of TRAC can help eliminate GvHD reactions and improve the viability and tumor killing efficacy of CAR-T cells (Eyquem et al., 2017. Targeting a CAR to the TRAC locus with CRISPR / Cas9 enhances tumor rejection).

[0011] Although there have been reports of knocking out one or several of these genes to improve the effectiveness of CAR-T cells, there have been no reports of using base editing to knock out all five genes at the same time.

[0012] Base editing, a type of genome editing technology, refers to the process of replacing nucleotides at specific DNA sites through genetic engineering. In humans and animals, many genetic diseases are caused by point mutations in functional genes. Base editing technology can be used to modify and alter the genomic DNA of animals, plants, and microorganisms to create new genotypes and obtain desired traits that are beneficial for production applications. It can also be used to correct severe congenital genetic variations in the treatment of genetic diseases. Therefore, base editing holds important application prospects in the creation of superior germplasm and the treatment of diseases.

[0013] Base editing is achieved with the help of a base editor system. Based on the type of bases affected by the base editor system, base editing systems can be divided into two categories: 1) cytosine base editor system (CBE) and 2) adenine base editor system (ABE). Among them, CBE can act on the cytidine base on the cytosine deoxyribonucleotide (abbreviated as cytidine, C), while ABE can act on the adenine base on the adenine deoxyribonucleotide (abbreviated as adenosine, A). CBE editing can convert CG base pairs on double-stranded DNA to TA base pairs; ABE editing can convert AT base pairs on double-stranded DNA to GC base pairs.

[0014] At present, accurate, effective, stable and safe base editing systems are still needed to further optimize CAR-T cells, improve the survival, expansion, maintenance, activation and tumor killing performance of CAR-T cells, and effectively inhibit or even eliminate immune rejection reactions or GvHD reactions. Summary of the Invention

[0015] Problems to be solved by the invention

[0016] Given that there is still a need for accurate, effective, stable and safe base editing systems to further optimize cells, the purpose of the present invention is to provide a base editing system that can be used to edit T cell-related enhancement genes to meet the actual needs of optimizing engineered T cells (such as CAR-T cells).

[0017] Solutions for solving problems

[0018] In a first aspect of the present invention, a method for obtaining modified T cells is provided, wherein the method comprises knocking out or down-regulating endogenous genes expressing T cells by gene editing technology, wherein the endogenous genes include T cell receptor α chain constant region (TRAC) gene, transforming growth factor β receptor 2 (TGFBR2) gene, programmed death receptor 1 (PD-1) gene, cytokine-inducible SH2 protein (CISH) gene and cell suicide-associated factor (FAS) gene.

[0019] In some embodiments, the gene editing technology includes DNA insertion, deletion or replacement of the gene target site. Preferably, the gene editing technology is DNA replacement of the gene target site.

[0020] In some embodiments, the gene editing technology comprises single base editing.

[0021] In some embodiments, the method comprises:

[0022] a) providing a purified T cell population;

[0023] b) introducing a single-base editing system into the T cell population in a);

[0024] c) expanding and harvesting the modified T cells;

[0025] Wherein, the single-base editing system in step b) includes: i) a base editing polypeptide, and / or an expression construct encoding a base editing polypeptide; ii) a guide RNA (gRNA) containing a spacer corresponding to the original spacer on the TRAC, TGFBR2, PD-1, CISH, FAS gene, and / or an expression construct encoding the guide RNA.

[0026] In some embodiments, the protospacer sequence of the TRAC gene comprises SEQ ID NO:51; the protospacer sequence of the TGFBR2 gene comprises SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:28; the protospacer sequence of the PD-1 gene comprises SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, or SEQ ID NO:47; the protospacer sequence of the CISH gene comprises SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:10; the protospacer sequence of the FAS gene comprises SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:20.

[0027] In some embodiments, the protospacer sequence of the TRAC gene comprises SEQ ID NO:51; the protospacer sequence of the TGFBR2 gene comprises SEQ ID NO:24, or SEQ ID NO:28; the protospacer sequence of the PD-1 gene comprises SEQ ID NO:41, or SEQ ID NO:47; the protospacer sequence of the CISH gene comprises SEQ ID NO:1; and the protospacer sequence of the FAS gene comprises SEQ ID NO:14.

[0028] In some embodiments, the base editing polypeptide comprises a DNA binding protein and at least one deaminase domain.

[0029] In some embodiments, the base editing polypeptide further comprises at least one uracil DNA glycosylase inhibitor (UGI) domain.

[0030] In some embodiments, the base editing polypeptide further comprises a nuclear localization sequence.

[0031] In some embodiments, the deaminase is selected from a cytidine deaminase, optionally, the cytidine deaminase is selected from an SCP1.201 family deaminase, optionally, the SCP1.201 family deaminase is selected from an Sdd2, Sdd3, Sdd4 deaminase, Sdd6 deaminase, Sdd7 deaminase, mini-Sdd7 deaminase, mini-Sdd9 deaminase, Sdd10 deaminase, Sdd59 deaminase, mini-Sdd3 deaminase or mini-Sdd6 deaminase.

[0032] In some embodiments, the deaminase is Sdd7 and / or mini-Sdd9 cytidine deaminase.

[0033] In some embodiments, the DNA binding protein is selected from TALE, ZFP or CRISPR effector protein or variants thereof; optionally, the CRISPR effector protein is selected from Cas9 nickase, inactivated Cas9 or variants thereof.

[0034] In some embodiments, the base editing polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 74-80.

[0035] In some embodiments, the method further comprises introducing a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor (CAR) comprises an antigen binding domain and a transmembrane domain.

[0036] In some embodiments, the T cell population is derived from human T cells, optionally from T cells enriched from human blood cells, optionally from T cells enriched from human blood cells and cryopreserved.

[0037] In some embodiments, the introduction method includes electroporation, liposome transfection, microinjection, viral infection (such as adenovirus, adeno-associated virus, lentivirus or other virus), N-acetylgalactosamine (GalNAc)-mediated, gene gun-mediated, ribonucleoprotein (RNP) complex-mediated, and nanoparticle-mediated transformation.

[0038] In a second aspect of the present invention, a base editing system for modifying T cells is provided, comprising:

[0039] i) a base editing polypeptide, and / or an expression construct encoding a base editing polypeptide, wherein the base editing polypeptide comprises a DNA binding protein and at least one deaminase domain; and

[0040] ii) a guide RNA, and / or an expression construct encoding the guide RNA, wherein the guide RNA targets any one or any combination of the T cell receptor alpha chain constant region (TRAC) gene, the transforming growth factor beta receptor 2 (TGFBR2) gene, the programmed death receptor 1 (PD-1) gene, the cytokine-inducible SH2 protein (CISH) gene, and / or the cell suicide-associated factor (FAS) genomic region.

[0041] In some embodiments, the deaminase domain is a cytidine deaminase.

[0042] In some embodiments, the cytidine deaminase is selected from the group consisting of an APOBEC family deaminase, an SCP1.201 family deaminase, or a homolog thereof;

[0043] Optionally, the APOBEC family deaminase is selected from AID deaminase, APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase or APOBEC3H deaminase;

[0044] Optionally, the SCP1.201 family deaminase is selected from Sdd2, Sdd3, Sdd4 deaminase, Sdd6 deaminase, Sdd7 deaminase, mini-Sdd7 deaminase, mini-Sdd9 deaminase, Sdd10 deaminase, Sdd59 deaminase, mini-Sdd3 deaminase or mini-Sdd6 deaminase.

[0045] In some embodiments, the cytidine deaminase is Sdd7 and / or mini-Sdd9 cytidine deaminase.

[0046] In some embodiments, the DNA binding protein is selected from a TALE, a ZFP, or a CRISPR effector protein.

[0047] In some embodiments, the CRISPR effector protein is selected from Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cast10d, Cas12, Cas13, Cas14, CasX, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse 3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn1, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, Cu1966, TraC or variants thereof.

[0048] In some embodiments, the DNA binding protein is a Cas9 nickase, an inactivated Cas9, or a variant thereof.

[0049] In some embodiments, the base editing system further contains at least one uracil DNA glycosylase inhibitor (UGI).

[0050] In some embodiments, the base editing system further comprises a nuclear localization sequence.

[0051] In some embodiments, the base editing polypeptide comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 74-80.

[0052] In some embodiments, the base editing polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 77-80.

[0053] In some embodiments, the guide RNA contains a spacer corresponding to the protospacer of the TRAC, TGFBR2, PD-1, CISH, or FAS genes;

[0054] Optionally, the protospacer sequence of the TRAC gene includes SEQ ID NO:51; the protospacer sequence of the TGFBR2 gene includes SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:28; the protospacer sequence of the PD-1 gene includes SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, or SEQ ID NO:47; the protospacer sequence of the CISH gene includes SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:10; the protospacer sequence of the FAS gene includes SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:20.

[0055] In some embodiments, the protospacer sequence of the TRAC gene comprises SEQ ID NO:51; the protospacer sequence of the TGFBR2 gene comprises SEQ ID NO:24, or SEQ ID NO:28; the protospacer sequence of the PD-1 gene comprises SEQ ID NO:41, or SEQ ID NO:47; the protospacer sequence of the CISH gene comprises SEQ ID NO:1; and the protospacer sequence of the FAS gene comprises SEQ ID NO:14.

[0056] In the third aspect of the present invention, a modified T cell is provided, wherein the T cell is modified by the method described in the first aspect of the present invention, or the base editing system described in the second aspect of the present invention is introduced, and the T cell knocks out or downregulates the expression of endogenous genes, wherein the endogenous genes include T cell receptor α chain constant region (TRAC) gene, transforming growth factor β receptor 2 (TGFBR2) gene, programmed death receptor 1 (PD-1) gene, cytokine-inducible SH2 protein (CISH) gene and cell suicide-associated factor (FAS) gene.

[0057] In some embodiments, the T cells are CAR-T cells.

[0058] In some embodiments, the CAR-T cells recognize one or more target antigens.

[0059] In some embodiments, the T cells are used to prevent or treat autoimmune diseases, tumors, viral infectious diseases, bacterial infectious diseases, genetic diseases, infectious diseases, or cardiovascular diseases.

[0060] In a fourth aspect of the present invention, a composition is provided, wherein the composition comprises the modified T cells according to the third aspect of the present invention, and, optionally, a pharmaceutically acceptable carrier or excipient.

[0061] Effects of the Invention

[0062] The base editing system provided by the present invention can simultaneously knock out TRAC, TGFBR2, PD-1, CISH and FAS genes, that is, edit T cell-related enhancement genes, thereby meeting the needs of optimizing engineered T cells, such as CAR-T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1. Schematic diagram of editing efficiency results of CISH gene preferred targets.

[0064] Figure 2. Schematic diagram of the editing efficiency results of the preferred target sites of the FAS gene.

[0065] Figure 3. Schematic diagram of the editing efficiency results of the preferred targets of the PD-1 gene.

[0066] Figure 4. Schematic diagram of the editing efficiency results of the preferred target sites of the TGFBR2 gene.

[0067] Figure 5. Schematic diagram of the editing efficiency results of the TRAC gene's preferred target sites.

[0068] Figure 6. Schematic diagram of the editing efficiency of mini-Sdd9-QBEmax and mini-Sdd9-BE4max on five genes.

[0069] Figure 7. Schematic diagram of the indel (insertion / deletion) efficiency results of mini-Sdd9-QBEmax and mini-Sdd9-BE4max for five genes.

[0070] Figure 8. Schematic diagram of the editing efficiency / indel results of mini-Sdd9-QBEmax and mini-Sdd9-BE4max for five genes.

[0071] Figure 9. Schematic diagram of the editing efficiency of Sdd7-QBEmax and Sdd7-BE4max on five genes.

[0072] Figure 10. Schematic diagram of the indel efficiency results of Sdd7-QBEmax and Sdd7-BE4max for five genes.

[0073] Figure 11. Schematic diagram of the editing efficiency / indel results of Sdd7-QBEmax and Sdd7-BE4max for five genes.

[0074] Figure 12. Schematic diagram of the editing efficiency of Sdd7-BE4max for the combined knockout of five genes.

[0075] Figure 13. Schematic diagram of the editing efficiency of Sdd7-QBEmax for the combined knockout of five genes.

[0076] Figure 14. Schematic diagram of the editing efficiency of mini-Sdd9-BE4max for the combined knockout of five genes.

[0077] Figure 15. Schematic diagram of the editing efficiency of mini-Sdd9-QBEmax for the combined knockout of five genes.

[0078] Figure 16. Schematic diagram of the editing efficiency / editing purity results of CISH gene knockout by mini-Sdd9-QBEmax and mini-Sdd9-BE4max, where arrows indicate knockout editing sites.

[0079] Figure 17. Schematic diagram of the editing efficiency / editing purity results of FAS gene knockout by mini-Sdd9-QBEmax and mini-Sdd9-BE4max, where arrows indicate knockout editing sites.

[0080] Figure 18. Schematic diagram of the editing efficiency / editing purity results of PD-1 gene knockout by mini-Sdd9-QBEmax and mini-Sdd9-BE4max, where arrows indicate knockout editing sites.

[0081] Figure 19. Schematic diagram of the editing efficiency / editing purity results of TGFBR2 gene knockout by mini-Sdd9-QBEmax and mini-Sdd9-BE4max, where arrows indicate knockout editing sites.

[0082] Figure 20. Schematic diagram of the editing efficiency / editing purity results of TRAC gene knockout by mini-Sdd9-QBEmax and mini-Sdd9-BE4max, where arrows indicate knockout editing sites.

[0083] Figure 21. Schematic diagram of the relative positions of protospacers / targets in five genes.

[0084] Figure 22. Schematic diagram of the polypeptide structure of the BE4max and QBEmax base editing systems. The promoter, NLS, and UGI are selected according to the techniques known in the art. DETAILED DESCRIPTION

[0085] 1. Definition

[0086] In the present invention, unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are terms and routine procedures widely used in the corresponding fields. For example, the standard recombinant DNA and molecular cloning techniques used in the present invention are well known to those skilled in the art and are more fully described in the following literature: Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter referred to as "Sambrook"). At the same time, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0087] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and should be treated as if each combination had been individually listed herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0088] When the word "comprising" is used herein to describe a sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention. In addition, it is clear to those skilled in the art that the methionine encoded by the start codon at the N-terminus of the polypeptide may be retained in certain practical situations (for example, when expressed in a specific expression system), but it does not substantially affect the function of the polypeptide. Therefore, when describing a specific polypeptide amino acid sequence in the specification and claims of this application, although it may not contain a methionine encoded by a start codon at the N-terminus, a sequence containing the methionine is also covered, and accordingly, its encoding nucleotide sequence may also contain a start codon; and vice versa.

[0089] "Gene" and "genome" as used herein encompass not only the chromosomal DNA present in the cell nucleus, but also the organelle DNA present in subcellular components of the cell (eg, mitochondria, plastids).

[0090] "Genetically modified organism" or "genetically modified cell" refers to an organism or cell that contains an exogenous polynucleotide or a modified gene or expression control sequence within its genome. For example, the exogenous polynucleotide is capable of stably integrating into the genome of the organism or cell and being inherited through successive generations. The exogenous polynucleotide can be integrated into the genome alone or as part of a recombinant DNA construct. A modified gene or expression control sequence is one that contains single or multiple deoxynucleotide substitutions, deletions, and additions within the genome of the organism or cell.

[0091] "Polynucleotide," "nucleic acid sequence," "nucleotide sequence," or "nucleic acid fragment" are used interchangeably and are single-stranded or double-stranded polymers of RNA or DNA that optionally contain synthetic, non-natural, or altered nucleotide bases. Nucleotides are referred to by their single-letter names as follows: "A" is adenosine or deoxyadenosine (RNA or DNA, respectively), "C" is cytidine or deoxycytidine, "G" is guanosine or deoxyguanosine, "U" is uridine, "T" is deoxythymidine, "R" is a purine (A or G), "Y" is a pyrimidine (C or T), "K" is G or T, "H" is A or C or T, "I" is inosine, and "N" is any nucleotide.

[0092] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical analog of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms "polypeptide," "peptide," "amino acid sequence," and "protein" may also include modified forms including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation.

[0093] Sequence "identity" has a meaning recognized in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule. (See, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). While there are many methods to measure the identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48: 1073 (1988)).

[0094] In peptides or proteins, suitable conservative amino acid substitutions are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Generally, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224).

[0095] "RNA-dependent DNA polymerase" refers to an enzyme that can synthesize DNA using RNA as a template. It recognizes the base sequence on the RNA chain and synthesizes a complementary DNA sequence on the DNA chain.

[0096] A "domain" refers to a portion of a protein with a relatively stable three-dimensional structure. Proteins are typically composed of one or more domains, each with its own spatial folding configuration. Domains can be continuous or discontinuous, typically consisting of one or more segments of an amino acid sequence. The presence of domains enables functional diversity in proteins, and new functions can be achieved through the recombination and combination of domains.

[0097] "Expression construct" or "construct" refers to a vector, such as a recombinant vector, suitable for expressing a nucleotide sequence of interest in an organism. "Expression" refers to the production of a functional product. For example, expression of a nucleotide sequence may refer to the transcription of the nucleotide sequence (such as transcription to generate mRNA or functional RNA) and / or the translation of RNA into a precursor or mature protein. An "expression construct" may be a linear nucleic acid fragment, a circular plasmid, a viral vector, or, in some embodiments, an RNA (such as mRNA) that is capable of translation. An "expression construct" may comprise regulatory sequences and a nucleotide sequence of interest from different sources, or regulatory sequences and a nucleotide sequence of interest from the same source but arranged in a manner different from that which normally occurs in nature.

[0098] "Introducing" a nucleic acid molecule (e.g., a plasmid, a linear nucleic acid fragment, RNA, etc.) or a protein into an organism refers to transforming an organism cell with the nucleic acid or protein so that the nucleic acid or protein can function in the cell. "Transformation" as used herein includes stable transformation and transient transformation.

[0099] "Stable transformation" refers to the introduction of an exogenous nucleotide sequence into a genome, resulting in the stable inheritance of the exogenous nucleotide sequence. Once stably transformed, the exogenous nucleic acid sequence is stably integrated into the genome of the organism and any successive generations thereof.

[0100] "Transient transformation" refers to the introduction of a nucleic acid molecule or protein into a cell where it functions without the exogenous nucleotide sequence being stably inherited. In transient transformation, the exogenous nucleic acid sequence does not integrate into the genome.

[0101] "T cells" refer to a type of lymphocyte that develops in the thymus and plays a central role in immune responses. T cells can be distinguished from other lymphocytes by the presence of T cell receptors on their cell surface.

[0102] "Engineered T cells" refer to any T cells that contain a cell receptor that is heterologous to the T cell. For example, T cells containing a chimeric antigen receptor (CAR); or T cells containing a heterologous TCR that binds to an antigen, such as MART1, NY-ESO-1, p53, and the like.

[0103] "Chimeric antigen receptor," "CAR," or "CARs" are terms understood by those skilled in the art to refer to a class of antigen binding proteins comprising a fusion protein of a selected single-chain fragment variable domain from a specific monoclonal antibody and one or more T-cell receptor intracellular signaling domains.

[0104] "CAR-T cell therapy" refers to a type of treatment that involves genetically modifying a patient's autologous T cells to express a CAR specific for a tumor antigen, followed by ex vivo cell expansion and re-infusion back into the patient. This T cell genetic modification can occur via viral-based gene transfer methods or non-viral methods such as DNA-based transposons, CRISPR / Cas9 technology, or direct transfer of in vitro transcribed mRNA by electroporation.

[0105] "GvHD" refers to a syndrome characterized by inflammation in various organs, with specificity for epithelial cell apoptosis and crypt shedding. GvHD is commonly associated with stem cell transplants, such as those occurring with bone marrow transplants, and can also be induced by CAR therapies based on infusions of donor leukocytes, virus-specific T cells, T cell receptor-deficient T cells, lymphocyte progenitor cells, and regulatory T cells.

[0106] "HEK293T" refers to a variant of human embryonic kidney 293 cells (HEK293) that contains the SV40 large T-antigen. This antigen allows episomal replication of transfected plasmids containing the SV40 origin of replication, resulting in amplification of the transfected plasmid and prolonged expression of the desired gene product.

[0107] "In vitro" refers to procedures performed outside of a living organism, typically using biological material isolated from its normal biological host or usual biological environment.

[0108] "Orthologs" or "orthologous sequences" refer to homologous nucleic acid or amino acid sequences that are derived from the same ancestral sequence separated by a speciation event.

[0109] "Homologs" or "homologous sequences" refer to nucleic acid or amino acid sequences that are derived from a common ancestral sequence.

[0110] 2. Base editing systems for modifying T cells

[0111] In one aspect, the present invention provides a base editing system for modifying T cells, comprising:

[0112] i) a base editing polypeptide, and / or an expression construct encoding a base editing polypeptide, wherein the base editing polypeptide comprises a DNA binding protein and at least one deaminase domain; and

[0113] ii) a guide RNA, and / or an expression construct encoding the guide RNA, wherein the guide RNA can target any one of the T cell receptor alpha chain constant region (TRAC) gene, the transforming growth factor beta receptor 2 (TGFBR2) gene, the programmed death receptor 1 (PD-1) gene, the cytokine-inducible SH2 protein (CISH) gene, and / or the cell suicide-associated factor (FAS) genomic region or any combination thereof.

[0114] "DNA binding protein" refers to a protein associated with a nucleic acid (e.g., DNA or RNA), for example, a DNA binding protein can be guided to a specific nucleic acid sequence by a guide RNA. In some embodiments, the DNA binding protein is selected from a transcription activator-like effector (TALE), a zinc finger protein (ZFP), or a CRISPR effector protein. In some embodiments, the CRISPR effector protein is selected from Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cast10d, Cas12, Cas13, Cas14, CasX, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn1, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, Cu1966, TraC, or variants thereof.

[0115] In some embodiments, the DNA binding protein is a nickase or a variant thereof. "Nickase" (Nicking enzyme or Nickase) is a special enzyme, a type of endonuclease, which can recognize a specific sequence on double-stranded DNA and only make a nick on one of the chains without completely cutting the double-stranded DNA. This nick is usually at the phosphodiester bond, resulting in an open end of the DNA, but keeping the other chain intact. For example, in the gene editing technology CRISPR / Cas9, a nickase version of Cas9 (called nCas9 or Cas9n) is used to create a single-stranded nick, rather than a double-stranded break. For example, residues such as Asp10, His840, Asn854, and Asn856 in the wild-type exemplary Streptococcus pyogenes Cas9 polypeptide are mutated to inactivate one of the multiple nucleic acid cleavage domains (e.g., nuclease domains). In some embodiments, the DNA binding protein is selected from the cyclic conversion polypeptide other than the deaminase used in Cas9 nickase or QBEmax.

[0116] In some embodiments, the DNA binding protein is an inactivated and / or conditionally enzymatically inactivated nuclease or a variant thereof. The inactivated nuclease can target nucleic acids without producing chain breaks. For example, residues such as Asp10, His840, Asn854, and Asn856 in the wild-type exemplary Streptococcus pyogenes Cas9 polypeptide are mutated to inactivate the nucleic acid cleavage domain of the multiple nucleic acid cleavage domains (e.g., nuclease domains). In some embodiments, the DNA binding protein is selected from inactivated Cas9 (dCas9).

[0117] As used herein, a "deaminase domain" refers to an enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is a cytidine deaminase or an adenosine deaminase, ie, an enzyme that can remove the amino group of a cytidine molecule or an adenosine molecule. In some embodiments, the deaminase is selected from: an APOBEC family deaminase, an SCP1.201 family deaminase, or a homolog thereof; the APOBEC family deaminase is selected from: an AID deaminase, an APOBEC1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an APOBEC3F deaminase, an APOBEC3G deaminase, and an APOBEC3H deaminase; the SCP1.201 family deaminase is selected from: an Sdd2, an Sdd3, an Sdd4 deaminase, an Sdd6 deaminase, an Sdd7 deaminase, a mini-Sdd7 deaminase, a mini-Sdd9 deaminase, an Sdd10 deaminase, an Sdd59 deaminase, a mini-Sdd3 deaminase, a mini-Sdd6 deaminase, or a functional mutant thereof, a non-limiting example of an SCP1.201 family deaminase comprises the amino acid sequence SEQ ID NO: NO:70-73 and SEQ ID NO:81-88.

[0118] In some embodiments, the base editing system of the present invention further contains a uracil DNA glycosylase inhibitor (UGI), a non-limiting example of which comprises the amino acid sequence of SEQ ID NO:89.

[0119] In some embodiments, the base editing system of the present invention further includes a nuclear localization sequence. Herein, “nuclear localization sequence (NLS)” or “nuclear localization signal” are used interchangeably and refer to a domain of a protein, typically a short amino acid sequence, that can interact with a nuclear import carrier so that the protein can be transported into the cell nucleus. In general, one or more NLSs in the base editing system (e.g., a base editing polypeptide) should have sufficient strength to drive the base editing system (e.g., a base editing polypeptide) to accumulate in an amount that can achieve its base editing function in the nucleus of the cell. In general, the intensity of nuclear localization activity is determined by the number, position, one or more specific NLSs used, or a combination of these factors in the base editing system (e.g., a base editing polypeptide). In some embodiments of the present invention, the NLS of the base editing system (e.g., a base editing polypeptide) of the present invention may be located at the N-terminus and / or the C-terminus. In some embodiments of the present invention, the NLS of the base editing system (e.g., base editing polypeptide) of the present invention can be located between the deaminase domain (e.g., adenosine deaminase domain and / or cytidine deaminase domain), the DNA binding protein, and / or the UGI. In some embodiments, the base editing system (e.g., base editing polypeptide) comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs. In some embodiments, the base editing system (e.g., base editing polypeptide) is contained at or near the N-terminus about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs. In some embodiments, the base editing system (e.g., base editing polypeptide) is contained at or near the C-terminus about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs. In some embodiments, the polypeptide comprises a combination of these, such as one or more NLSs contained at the N-terminus and one or more NLSs at the C-terminus. When more than one NLS is present, each can be selected independently of the other NLSs. Generally speaking, an NLS consists of one or more short sequences of positively charged lysine or arginine residues exposed on the surface of the protein, but other types of NLSs are also known. Non-limiting examples of NLSs include the amino acid sequences SEQ ID NOs: 52-68.

[0120] "Base editing polypeptide" refers to a polypeptide that binds to a polynucleotide and has base modification activity. In some embodiments, the base editing polypeptide includes a DNA binding protein and at least one deaminase domain. In some embodiments, the framework of the base editing polypeptide is selected from the BE system, such as BE1, BE2, BE3 or BE4max (reference: Komor AC, Zhao KT, Packer MS, et al. Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity[J]. Science advances, 2017, 3(8): eaao4774.; BE4max is BE4 in the document, and its structure is shown in Figure 5A of the document) or QBEmax.

[0121] In this specification, base editor (BE) systems include BE1, BE2, BE3, and BE4. BE1 consists of a fusion protein of dCas9 and APOBEC1. BE2 is a BE system obtained by introducing the UGI (Uracil DNA glycosylase inhibitor) into BE1. BE3 refers to a BE system obtained by replacing the dCas9 in BE2 with nCas9 (D10A). BE4 is a BE system obtained by optimizing the linker region length between APOBEC1 and nCas9 (D10A), the linker region length between nCas9 (D10A) and UGI, and adding a copy of UGI based on BE3.

[0122] In some embodiments, QBEmax comprises a deaminase domain and a pair of DNA binding protein domains capable of single-strand DNA cleavage.

[0123] In some embodiments of the present invention, the DNA binding protein having single-stranded DNA cleavage activity is a pair of DNA binding protein domains, wherein the paired DNA binding protein domains are connected to the N-terminus and C-terminus of the deaminase domain via a linker, respectively. In some embodiments of the present invention, the paired DNA binding protein domains can be obtained by the following method: by circularly transforming the amino acid sequence of the DNA binding protein having single-stranded DNA cleavage function (the linker of the circular transformation can be selected from GGSGGSGGSGGSGGSGGSGG; SEQ ID NO: 90), obtaining a circular transformation sequence with a length of 1388 amino acids (the circular transformation sequence is such as SEQ ID NO: 91, wherein the amino acid site of the circular transformation sequence is defined as the first amino acid by the methionine M encoded by the start codon, and the 1388th amino acid by the glycine G directly connected to the first amino acid in the linker). The circular transformation DNA binding protein is split at two sites to obtain a pair of separated polypeptides, i.e., the paired DNA binding protein domains having DNA single-stranded DNA cleavage function. The paired DNA-binding protein domains with single-strand DNA cleavage function are fused or linked to the N-terminus and C-terminus of the deaminase domain, respectively. Depending on the position of their fusion with the deaminase, the domains linked to the N-terminus or C-terminus of the deaminase are defined as the deaminase N-terminal linking domain (QBE-N) and the deaminase C-terminal linking domain (QBE-C), respectively.

[0124] For example:

[0125] Among them, the first split site is defined as the deaminase attachment site, where the lower-coded amino acid is attached to the deaminase N-terminus and the higher-coded amino acid is attached to the amino acid C-terminus; the second split site is defined as the non-deaminase attachment site, where the lower-coded amino acid is the C-terminus of the QBE-C domain and the higher-coded amino acid is the N-terminus of the QBE-N domain.

[0126] Therefore, in some embodiments, the paired DNA-binding protein domains having DNA single-strand cleavage function include a deaminase N-terminal connecting domain and a deaminase C-terminal connecting domain, and the deaminase N-terminal connecting domain and the deaminase C-terminal connecting domain are respectively connected to the N-terminus and C-terminus of the deaminase domain through a linker.

[0127] In some embodiments, the N-terminus or C-terminus of the deaminase domain comprises one or two single domains, which are connected by a linker. Herein, a "single domain" is a portion of a DNA-binding protein domain, and all single domains are combined to form a complete DNA-binding protein domain. A single domain does not contain a linker internally, but has linkers at both ends to connect to other single domains or functional regions (such as deaminases, NLSs, or UGIs, etc.).

[0128] In some embodiments, QBEmax comprises the following structure: [X2]-[Deaminase]-[X3]-[X4], wherein "-" represents a linker or a ring-transforming linker, wherein the linker is 0-100 amino acids in length.

[0129] In some embodiments, X 2-4 In some preferred embodiments, X2 is SEQ ID NO: 94, X3 is SEQ ID NO: 95, and X4 is SEQ ID NO: 96.

[0130] The deaminase is as described above under "deaminase domain".

[0131] As used herein, a "linker" refers to a linker that connects two molecules or moieties, such as two domains of a fusion protein. Typically, a linker is located between or flanking two groups, molecules, or other moieties and is connected to each by a covalent bond, thereby connecting the two. In some embodiments, a linker is an organic molecule, group, polymer, or chemical moiety.

[0132] In some embodiments, the base editing polypeptide comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 74-80. In some embodiments, the base editing polypeptide comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 77-80.

[0133] "Guide RNA" and "gRNA" are used interchangeably and refer to a polynucleotide that can be specific to a target sequence of a target nucleic acid and can form a complex with a DNA-binding protein.

[0134] In the present invention, "target nucleic acid" is a polynucleotide (e.g., DNA such as genomic DNA) comprising a site ("target site" or "target sequence") targeted by a DNA binding protein (e.g., Cas protein, etc.) guided by a guide RNA. A "target sequence" is a sequence to which the guide sequence of the guide RNA will hybridize.

[0135] In this specification, "protospacer", "protospacer" and "editing window" are used interchangeably and refer to the nucleic acid sequence in the genome that is complementary to the target sequence. In the natural state, the protospacer sequence is adjacent to the protospacer sequence adjacent motif (PAM). During the base editing process, the nucleotides in this sequence undergo deamination and nucleotide replacement under the action of the base editing system.

[0136] In this specification, a "spacer" is an RNA sequence within the guide sequence of the gRNA that complementarily hybridizes with the target sequence, that is, an RNA version corresponding to the original spacer sequence.

[0137] In some embodiments, the guide RNA is present as a single RNA molecule. In some embodiments, the guide RNA is an sgRNA, which comprises two parts: (1) a guide sequence that is complementary to a target sequence of a target nucleic acid; and (2) a portion that binds to a DNA binding protein.

[0138] In some embodiments, the guide sequence of portion (1) comprises a spacer sequence corresponding to a protospacer on the TRAC, TGFBR2, PD-1, CISH, and FAS genome, and the spacer sequence is complementary to the target sequence.

[0139] Herein, "site 1, site 2..." refers to a protospacer sequence. In some specific embodiments, the protospacer sequence is as shown in SEQ ID NO: 1-51.

[0140] In some specific embodiments, the target sequence is a DNA sequence complementary to the protospacer sequence shown in SEQ ID NOs: 1-51 (which is understood to be located on the non-editing strand). As used herein, "sg1, sg2..." represents an RNA sequence (guide sequence) complementary to the target sequence, i.e., an RNA sequence complementary to the complementary DNA sequence of SEQ ID NOs: 1-51.

[0141] In some embodiments, portion (2) corresponds to a sequence called a backbone sequence, which is responsible for specific binding to a DNA binding protein. In some embodiments, portion (2) is identical or homologous to the backbone sequence provided in Jinek et al., Science 337:816-821 (2012) or Nguyen et al., Science 386, eado9336 (2024). In some embodiments, the backbone sequence is as shown in SEQ ID NOs:97-99.

[0142] In some embodiments, the guide RNA comprises a spacer sequence corresponding to the following target site protospacer sequence, wherein the target site is selected from the TRAC, TGFBR2, PD-1, CISH or FAS genome, and the genomic sequence refers to GRCh38.p14.

[0143] In some optional embodiments, the protospacer sequence comprises:

[0144] (a) at least one of the nucleotide sequences shown in SEQ ID NOs: 1-10; and / or

[0145] (b) at least one of the nucleotide sequences shown in SEQ ID NOs: 11-20; and / or

[0146] (c) at least one of the nucleotide sequences shown in SEQ ID NOs: 21-30; and / or

[0147] (d) at least one of the nucleotide sequences shown in SEQ ID NOs: 31-47; and / or

[0148] (e) at least one of the nucleotide sequences shown in SEQ ID NOs: 48-51.

[0149] In some specific embodiments, the guide RNA contains spacers corresponding to the following protospacers of the TRAC, TGFBR2, PD-1, CISH, and FAS genes:

[0150] The protospacer sequence of the TRAC gene includes SEQ ID NO:51; the protospacer sequence of the TGFBR2 gene includes SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:28; the protospacer sequence of the PD-1 gene includes SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, or SEQ ID NO:47; the protospacer sequence of the CISH gene includes SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:10; the protospacer sequence of the FAS gene includes SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:20.

[0151] In some preferred embodiments, the protospacer sequence of the TRAC gene includes SEQ ID NO:51; the protospacer sequence of the TGFBR2 gene includes SEQ ID NO:24, or SEQ ID NO:28; the protospacer sequence of the PD-1 gene includes SEQ ID NO:41, or SEQ ID NO:47; the protospacer sequence of the CISH gene includes SEQ ID NO:1; and the protospacer sequence of the FAS gene includes SEQ ID NO:14.

[0152] 3. Methods for Obtaining Modified T Cells

[0153] On the other hand, the present invention provides a method for obtaining modified T cells, which comprises knocking out or down-regulating endogenous genes expressing T cells through gene editing technology, wherein the endogenous genes include T cell receptor α chain constant region (TRAC) gene, transforming growth factor β receptor 2 (TGFBR2) gene, programmed death receptor 1 (PD-1) gene, cytokine-inducible SH2 protein (CISH) gene and cell suicide-associated factor (FAS) gene.

[0154] In some embodiments, the gene editing technology includes DNA insertion, deletion or replacement of the gene target site. Preferably, the gene editing technology is DNA replacement of the gene target site.

[0155] In some specific embodiments, the gene editing technology includes single base editing.

[0156] In some specific embodiments, the method comprises:

[0157] a) providing a purified T cell population;

[0158] b) introducing a single-base editing system into the T cell population in a);

[0159] c) Expanding and harvesting the modified T cells.

[0160] In some embodiments, the single-base editing system in step b) includes: i) a base editing polypeptide, and / or an expression construct encoding a base editing polypeptide; ii) a guide RNA (gRNA) containing a spacer corresponding to the original spacer on the TRAC, TGFBR2, PD-1, CISH, FAS gene, and / or an expression construct encoding the guide RNA.

[0161] In some embodiments, the protospacer sequence of the TRAC gene comprises at least one of the nucleotide sequences shown in SEQ ID NOs:48-51; the protospacer sequence of the TGFBR2 gene comprises at least one of the nucleotide sequences shown in SEQ ID NOs:21-30; the protospacer sequence of the PD-1 gene comprises at least one of the nucleotide sequences shown in SEQ ID NOs:31-47; the protospacer sequence of the CISH gene comprises at least one of the nucleotide sequences shown in SEQ ID NOs:1-10; the protospacer sequence of the FAS gene comprises at least one of the nucleotide sequences shown in SEQ ID NOs:11-20.

[0162] In some preferred embodiments, the protospacer sequence of the TRAC gene includes SEQ ID NO:51; the protospacer sequence of the TGFBR2 gene includes SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:28; the protospacer sequence of the PD-1 gene includes SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, or SEQ ID NO:47; the protospacer sequence of the CISH gene includes SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:10; the protospacer sequence of the FAS gene includes SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:20.

[0163] In some more preferred embodiments, the protospacer sequence of the TRAC gene includes SEQ ID NO:51; the protospacer sequence of the TGFBR2 gene includes SEQ ID NO:24, or SEQ ID NO:28; the protospacer sequence of the PD-1 gene includes SEQ ID NO:41, or SEQ ID NO:47; the protospacer sequence of the CISH gene includes SEQ ID NO:1; and the protospacer sequence of the FAS gene includes SEQ ID NO:14.

[0164] In some embodiments, the base editing polypeptide comprises a DNA binding protein and at least one deaminase domain.

[0165] In some embodiments, the base editing polypeptide further comprises at least one uracil DNA glycosylase inhibitor (UGI) domain.

[0166] In some embodiments, the base editing polypeptide further comprises a nuclear localization sequence.

[0167] In some embodiments, the deaminase is selected from a cytidine deaminase, optionally, the cytidine deaminase is selected from an SCP1.201 family deaminase, optionally, the SCP1.201 family deaminase is selected from an Sdd2, Sdd3, Sdd4 deaminase, Sdd6 deaminase, Sdd7 deaminase, mini-Sdd7 deaminase, mini-Sdd9 deaminase, Sdd10 deaminase, Sdd59 deaminase, mini-Sdd3 deaminase or mini-Sdd6 deaminase.

[0168] In some embodiments, the deaminase is Sdd7 and / or mini-Sdd9 cytidine deaminase.

[0169] In some embodiments, the DNA binding protein is selected from TALE, ZFP or CRISPR effector protein or variants thereof; optionally, the CRISPR effector protein is selected from Cas9 nickase, inactivated Cas9 or variants thereof.

[0170] In some embodiments, the base editing polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 74-80.

[0171] In some embodiments, the method further comprises introducing a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor (CAR) comprises an antigen binding domain and a transmembrane domain.

[0172] In some embodiments, the T cell population is derived from human T cells, optionally from T cells enriched from human blood cells, optionally from T cells enriched from human blood cells and cryopreserved.

[0173] In some embodiments, the introduction method includes electroporation, liposome transfection, microinjection, viral infection (such as adenovirus, adeno-associated virus, lentivirus or other virus), N-acetylgalactosamine (GalNAc)-mediated, gene gun-mediated, ribonucleoprotein (RNP) complex-mediated, and nanoparticle-mediated transformation.

[0174] In some embodiments, the single-base editing system is the base editing system for modifying T cells described in "II. Base editing system for modifying T cells" above. The method comprises introducing the base editing system into T cells in vitro or ex vivo. In some embodiments, the method comprises knocking out or performing dominant negative mutations on the TRAC, TGFBR2, PD-1, CISH, and FAS genes. In some embodiments, the base editing system is introduced into T cells (e.g., CAR-T cells) by a method selected from the following: electroporation, liposome transfection, microinjection, viral infection (such as adenovirus, adeno-associated virus, lentivirus or other virus), N-acetylgalactosamine (GalNAc)-mediated, gene gun-mediated, ribonucleoprotein (RNP) complex-mediated, and nanoparticle-mediated transformation.

[0175] The base editing system described in the present invention can be transfected or injected into cells, tissues or organs in the form of DNA, RNA, protein or virus.

[0176] In some embodiments, gene delivery vehicles can be used to deliver polynucleotides (e.g., polynucleotides encoding base editing systems described herein) to cells or tissues. "Gene delivery," "gene transfer," "transduction," etc., as used herein, refer to the introduction of exogenous polynucleotides into host cells, such as vector-mediated gene transfer (by, for example, viral infection / transfection, or various other protein-based or lipid-based gene delivery complexes) and assisting in the delivery of "naked" polynucleotides (e.g., electroporation, "gene gun" delivery, and various other technologies for the introduction of polynucleotides). The polynucleotides introduced can be stably or transiently maintained in the host cell. Stable maintenance generally requires that the polynucleotides introduced include a replication origin compatible with the host cell or a replicon incorporated into the host cell, such as an extrachromosomal replicon (e.g., plasmid) or nuclear or mitochondrial chromosome. Many "vectors" are known to mediate the transfer of genes to mammalian cells, as known in the art and described herein.

[0177] 4. A modified T cell

[0178] On the other hand, the present invention provides a modified T cell, which is modified by the method described in the above "3. Method for obtaining modified T cells" or introduced with the base editing system described in the above "2. Base editing system for modifying T cells".

[0179] In some embodiments, the T cells knock out or downregulate the expression of endogenous genes, wherein the endogenous genes include the T cell receptor α chain constant region (TRAC) gene, the transforming growth factor β receptor 2 (TGFBR2) gene, the programmed death receptor 1 (PD-1) gene, the cytokine-inducible SH2 protein (CISH) gene, and the cell suicide-associated factor (FAS) gene.

[0180] In some embodiments, the T cells are CAR-T cells.

[0181] In some specific embodiments, the CAR-T cells recognize one or more target antigens.

[0182] In some specific embodiments, the CAR-T cell includes a chimeric antigen receptor, and the preparation method or composition of the chimeric antigen receptor is a prior art, and the present invention is not specifically limited. For example, the chimeric antigen receptor includes a transmembrane domain, an intracellular domain, and an extracellular domain, and the extracellular domain includes an antibody targeting an antigen, and the specific antibody type can be selected according to the different antigens. In some embodiments, the CAR-T cell is an allogeneic CAR-T cell or an autologous CAR-T cell (e.g., an autologous human CAR-T cell).

[0183] The CAR-T cells provided by the present invention can be used to prevent or treat autoimmune diseases, tumors, viral infectious diseases, bacterial infectious diseases, genetic diseases, infectious diseases or cardiovascular diseases.

[0184] The autoimmune disease is selected from one or more of systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, lupus nephritis, neuromyelitis optica, systemic sclerosis, Sjögren's syndrome, and polymyositis;

[0185] The tumor is selected from the group consisting of lymphoma, hematologic malignancy or solid tumor; preferably, selected from the group consisting of adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, endocervical adenocarcinoma, bile duct carcinoma, colon adenocarcinoma, lymphoid tumors, diffuse large B-cell lymphoma, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe cell carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, acute myeloid leukemia, low-grade glioma of the brain, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, One or more of: carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic cancer, endometrial cancer, uterine sarcoma, uveal melanoma, multiple myeloma, acute lymphoid leukemia, chronic lymphoid leukemia, chronic myeloid leukemia, T-cell lymphoma, B-cell lymphoma, lung cancer, anal cancer, intraocular melanoma, retinoblastoma.

[0186] The virus is selected from one or more of influenza virus, parainfluenza virus, measles virus, mumps virus, herpes virus, adenovirus, respiratory syncytial virus, poliovirus, coxsackie virus or echovirus;

[0187] The bacteria are selected from one or more of Escherichia coli, Lactobacillus casei, Bacteroides fragilis, Acinetobacter lwoffii, Fusobacterium nucleatum, Bacteroides johnsonii, Bacteroides thaliana, Lactobacillus rhamnosus, Bacteroides massiliense, Bacteroides ovatus, Campylobacter jejuni, Staphylococcus saprophyticus, Enterococcus faecalis, Bacteroides thetaiotaomicron, Bacteroides vulgaris, Bacteroides monomorpha, Bacteroides faecalis, Fusobacterium mortis and Bifidobacterium breve.

[0188] The genetic disease is selected from chromosomal disease or chromosomal syndrome, single gene disease, and polygenic disease. The polygenic disease is, for example, asthma, cleft lip, schizophrenia, anencephaly, hypertension, congenital heart disease, and epilepsy.

[0189] The cardiovascular disease is selected from the group consisting of coronary heart disease, cerebrovascular disease, peripheral arterial disease, rheumatic heart disease, congenital heart disease, deep vein thrombosis and pulmonary embolism.

[0190] The present invention also provides a method for preventing or treating a disease, comprising administering a therapeutically effective amount of the T cells or CAR-T cells to a subject in need thereof.

[0191] The present invention also provides a composition comprising the modified T cells as described in "IV. A modified T cell" and, optionally, a pharmaceutically acceptable carrier or excipient.

[0192] Example

[0193] References for the experimental methods in the subsequent examples: Shan Q, et al. Genome editing in rice and wheat using the CRISPR / Cas system. Nat Protoc. 2014; 9(10): 2395-2410. doi: 10.1038 / nprot.2014.157; Liang Z, et al. Genome editing of bread wheat using biolistic delivery of CRISPR / Cas9 in vitro transcripts or ribonucleoproteins. Nat Protoc. 2018; 13(3): 413-430. doi: 10.1038 / nprot.2017.145; Jin S,. An unbiased method for evaluating the genome-wide specificity of base editors in rice. Nat Protoc.2021;16(1):431-457.doi:10.1038 / s41596-020-00423-y;Huang J,et al.Discovery of deaminase functions by structure-based protein clustering[published correction appears in Cell.2024Aug8;187(16):4426-4428.doi:10.1016 / j.cell.2024.07.003].Cell.2023;186(15):3182-3195.e14.doi:10.1016 / j.cell.2023.05.041, the above documents are incorporated into this article by reference.

[0194] Example 1. Identification of sgRNA

[0195] In engineered T cells, particularly CAR-T cell development, knockout of five genes—PD-1, CISH, FAS, TGFBR2, and TRAC—can enhance T cell survival, expansion, maintenance, activation, and tumorigenicity, while effectively suppressing or even eliminating immune rejection or GvHD. In addition to restrictions imposed by PAMs, protospacers, and off-target sites, codons are also required. Gene knockout can be achieved by converting DNA codons encoding arginine (CGA), glutamine (CAA, CAG), or tryptophan (UGG) within the editing window to stop codons (UAG, UGA, UAA) using the cytidine base editing system (CBE), or by editing key splice sites. Taking these factors into consideration, a group of potential editing targets within these five genes was selected: 10 targets each for CISH, FAS, and TGFBR2, 4 for TRAC, and 17 for PD-1, for a total of 51 targets. The original spacer sequence of the above-mentioned target site for the target nucleic acid is shown in SEQ ID NO: 1-51, and the relative position of the target site in the gene is shown in Figure 21. sgRNA is designed for the above-mentioned target site, wherein the guide sequence of the sgRNA is complementary to the target sequence of the non-editing chain (i.e., the complementary sequence of the original spacer sequence), so that the sgRNA binds to the target sequence of the non-editing chain (the complementary sequence of the original spacer). The backbone sequence of the sgRNA is shown in SEQ ID NO: 97. Based on the consistency of the above-mentioned gene sequences in the cell genome, HEK293T cells were selected as verification cells in this example.

[0196] HEK293T cells were transfected with sgRNAs complementary to the aforementioned target sequences and plasmids containing the BE4max backbone editing system fused to APOBEC1, mini-Sdd3, mini-Sdd6, Sdd7, and mini-Sdd9 deaminases (SEQ ID NOs: 74-78). Transfections were performed 18-24 hours after plating 60,000 cells per well in a 48-well plate. The cells were transfected with 375 ng of the editing system plasmid and 125 ng of the sgRNA plasmid per well. The cells were mixed and transfected with 1 μL of jetPRIME transfection reagent (Cat. No. 101000046, Polyplus Inc.). 72 hours after transfection, genomic DNA was extracted, the target fragments were amplified by two rounds of PCR, and the products were purified. The base editing efficacy at the target site was analyzed using next-generation sequencing (NGS).

[0197] The results are shown in Table 1 (where "\" indicates no editing efficiency was detected). The inventors verified the sgRNAs screened by bioinformatics in actual HEK293T cells. The editing efficiencies of different protospacers varied greatly, with the highest reaching 71.26% and the lowest undetectable editing efficiency. At the same time, base editing peptides composed of different deaminases also showed obvious preferences for editing different targets.

[0198] Table 1

[0199] Table 1 shows the editing efficiency of each high-editing-efficiency target site. The target sites with the highest editing efficiency are shown in Table 1. For the CISH gene, sites 1, 5, 8, and 10 showed the best results, with editing efficiencies ranging from 47.46% to 71.26%. For the PD-1 gene, sites 14, 15, 16, and 17 showed the best results, with editing efficiencies ranging from 57.45% to 67.68%. For the FAS gene, sites 4, 7, 8, and 10 showed the best results, with editing efficiencies ranging from 49.05% to 61.68%. For the TGFBR2 gene, sites 1, 4, 7, and 8 showed the best results, with editing efficiencies ranging from 46.91% to 64.48%. For the TRAC gene, site 4 showed the best results, with an editing efficiency of 69.06%.

[0200] Example 2: Screening of deaminase

[0201] To construct a single-base editing system with universal applicability across multiple genes, this example further validated and screened deaminases with editing effects on multiple targets based on the preferred targets identified in Example 1. The experimental results are shown in Figures 1-5. The editing efficiency of the base editing system using different deaminases at the preferred targets in Figures 1-5 was verified. High-efficiency editing was defined as 40% or higher, marked as "+," while low-efficiency editing was defined as "-." The statistical results are shown in Table 2. The data show that base editors using APOBEC1, miniSdd3, and mini-Sdd6 as deaminases failed to achieve high-efficiency editing at the preferred target sites in some target genes. Only base editing systems using Sdd7 and miniSdd9 as deaminases achieved high-efficiency editing targets across all five genes, demonstrating that these deaminases are ideal for use in base editing systems in this invention.

[0202] Table 2

[0203] Example 3. Validation of different base editing systems

[0204] To verify the effect of different base editing polypeptides on the editing effect, this example selected a circular cleavage variant of a CRISPR protein as the QBEmax system for DNA binding proteins as a verification system. In this system, the amino acid sequence of a DNA binding protein with a single-stranded cleavage function was circularly transformed (the circular transformation linker was GGSGGSGGSGGSGGSGGSGG; SEQ ID NO: 90), and a circular transformation sequence with a length of 1388 amino acids was obtained (the circular transformation sequence is such as SEQ ID NO: 91, wherein the amino acid site of the circular transformation sequence is defined as the first amino acid by the methionine M encoded by the start codon, and the glycine G directly connected to the first amino acid in the linker is defined as the 1388th amino acid). The two sites of the circular transformation sequence were split to obtain a pair of separated polypeptides (SEQ ID NO: 92 and SEQ ID NO: 93), which are the paired DNA binding protein domains. Based on their fusion position with the deaminase, the domains connected to the N-terminus or C-terminus of the deaminase are defined as the deaminase N-terminal linking domain (QBE-N) and the deaminase C-terminal linking domain (QBE-C), respectively. The polypeptides of the QBEmax system comprise the following structure: [QBE-N]-[deaminase]-[QBE-C], where "-" represents a linker. Figure 22 shows a schematic diagram of the structure of the base editing polypeptide of this example.

[0205] The two preferred deaminases Sdd7 and mini-Sdd9 screened in Example 2 were used as the deaminase domains of the base editing polypeptides to obtain four base editing polypeptides: Sdd7-QBEmax (SEQ ID NO: 79), Sdd7-BE4max (SEQ ID NO: 77), mini-Sdd9-QBEmax (SEQ ID NO: 80), and mini-Sdd9-BE4max (SEQ ID NO: 75).

[0206] On the one hand, HEK293T cells were transfected with plasmids expressing the aforementioned editing peptides and the corresponding sgRNAs. The editing effects of these targets were statistically analyzed and compared with their performance at these CAR-T related sites.

[0207] Figure 6 shows that the average editing efficiency of mini-Sdd9-QBEmax in all five target genes is comparable to the overall average editing efficiency of mini-Sdd9-BE4max; Figure 7 shows that the average indels of mini-Sdd9-QBEmax in all five target genes are significantly lower than the average indels of mini-Sdd9-BE4max; Figure 8 summarizes the editing efficiency / indel ratio and finds that the editing efficiency / indel ratio of mini-Sdd9-QBEmax is significantly higher than that of mini-Sdd9-BE4max in all five genes, ranging from 1.99 to 9.91 times higher.

[0208] Figure 9 shows that the average editing efficiency of Sdd7-QBEmax on the five genes is slightly lower than that of Sdd7-BE4max; at the same time, Figure 10 shows that the average indel of Sdd7-QBEmax is lower than that of Sdd7-BE4max; Figure 11 statistics the editing efficiency / indel ratio. The editing effect of Sdd7-QBEmax on CISH, PD-1, and TRAC is better than that of Sdd7-BE4max, while the editing effect of Sdd7-BE4max on FAS and TGFBR2 is better than that of Sdd7-QBEmax.

[0209] In summary, the QBEmax and BE4max base editing peptides obtained by fusing mini-Sdd9 and Sdd7 deaminases with the circular cleavage variant proteins of nCas9 or CRISPR, respectively, have similar editing efficiencies in editing gene sites related to T cell function enhancement. Each has its own advantages. When selecting different deaminases, different editing systems can be adapted as appropriate.

[0210] Example 4: Verification of target editing purity

[0211] In this example, mini-Sdd9 deaminase was connected to different base editing polypeptide backbones to verify the editing purity of each site of the above five genes. When using common BE tools for base editing (C-to-T), unexpected insertions and deletions and impure base editing byproducts (such as C-to-G and C-to-A) are still often observed. The formation of impure editing may appear as a new missense mutation in the target gene that was originally knocked out. Therefore, the ideal BE tool should have a high editing purity while having a high editing efficiency to ensure safety. Figures 16-20 show the editing effect and purity of mini-Sdd9-QBEmax and mini-Sdd9-BE4max knockout of the above five genes (51 target sites), wherein the above figures: a shows the editing efficiency and indel at different sites (the position indicated by the blue arrow is the expected mutation base); b shows the ratio of editing efficiency / indel at different sites; c shows the proportion of C-to-T in each edited site (C-to-T / C-to-R, R is A or G), which represents the purity of the editing. The analysis and statistics of Figures 16-20 are shown in Table 4. The editing efficiency is greater than or equal to 40% and is marked as "+", and less than 40% is marked as "-"; the editing purity is greater than or equal to 95% and is marked as "+", and less than 95% is marked as "-". Both efficiency and purity are "+", indicating that the editing system has a good overall effect at a certain site.

[0212] From the comprehensive editing efficiency and editing purity analysis in Figures 16-20 and Table 3, it can be seen that for the CISH gene, mini-Sdd9-BE4max has the best effect at site 1, and mini-Sdd9-QBEmax has the best effect at sites 1 and 9; for the FAS gene, mini-Sdd9-BE4max has the best effect at site 7, and mini-Sdd9-QBEmax has the best effect at sites 4 and 8; for the PD-1 gene, mini-Sdd9-BE4max has the highest editing efficiency at site 17, and has a good comprehensive effect at sites 5 and 8, and mini- Sdd9-QBEmax showed good effects at site 2, site 3, site 7, site 11, site 12, site 15, site 16, and site 17. For the TGFBR2 gene, mini-Sdd9-BE4max had the best editing efficiency at site 8 and the best overall effect at site 4. Mini-Sdd9-QBEmax had high editing purity and average editing efficiency at all sites of the gene. For the TRAC gene, both mini-Sdd9-BE4max and mini-Sdd9-QBEmax showed good effects at site 4.

[0213] In summary, a secondary screening of 51 targets, including editing purity as a screening factor, revealed slight differences in the preferred editing sites of QBEmax and BE4max in the five targeted genes. This suggests that different editing peptides can be used as appropriate for actual gene knockout. Furthermore, some of the preferred targets of QBEmax identified in this example (e.g., CISH gene site 9; PD-1 gene sites 2, 3, 7, 11, and 12) were selected as candidate targets for subsequent co-knockout experiments.

[0214] Table 3

[0215] Example 5: Joint knockout of five genes

[0216] In Examples 1-4, the inventors gradually screened for preferred targets for five genes, preferably deaminases, and preferred base editing peptides. In this example, the inventors permuted and combined the above parameters to obtain the combinations in Table 4, which verified the experimental effect of multi-gene knockout. The base editing peptides were co-transfected with five different sgRNAs into HEK293T cells to knock out the five genes.

[0217] Table 4

[0218] Figure 12 shows the editing efficiency of combination 1, which is relatively stable and relatively high, ranging from 42.9% to 54.88%; Figure 13 shows the editing efficiency of combination 2, which has a relatively high editing efficiency of 54.21% for the CISH gene, and the comprehensive editing efficiency of combination 2 is 30.35% to 54.21%; Figure 14 shows the editing efficiency of combination 3. The system has relatively weak editing ability for PD-1 and TRAC, but the editing ability for CISH, FAS and TGFBR2 is still good, ranging from 37.66% to 39.86%; Figure 15 shows the editing efficiency of combination 4. The overall editing ability is also at a high level, with an editing efficiency between 32.65% and 56.78%. Thus, the technical solution of the present application can achieve the modification of genes related to T cell function enhancement.

[0219] Although the present invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt specific circumstances, materials, compositions of matter, processes, process steps or steps to the purpose, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

[0220] The nucleotide or amino acid sequence involved in the present invention is:

Claims

1. A method for obtaining modified T cells, characterized in that: The method comprises knocking out or down-regulating endogenous genes expressing T cells by gene editing technology, wherein the endogenous genes include T cell receptor α chain constant region (TRAC) gene, transforming growth factor β receptor 2 (TGFBR2) gene, programmed death receptor 1 (PD-1) gene, cytokine-inducible SH2 protein (CISH) gene and cell suicide-associated factor (FAS) gene.

2. The method according to claim 1, characterized in that: The gene editing technology includes DNA insertion, deletion or replacement of the gene target site. Preferably, the gene editing technology is DNA replacement of the gene target site.

3. The method according to claim 1, characterized in that The gene editing technology includes single base editing.

4. The method according to any one of claims 1 to 3, characterized in that The method comprises: a) providing a purified T cell population; b) introducing a single base editing system into the T cell population in a); c) expanding and harvesting the modified T cells; Wherein, the single-base editing system in step b) includes: i) a base editing polypeptide, and / or an expression construct encoding a base editing polypeptide; ii) a guide RNA (gRNA) containing a spacer corresponding to the original spacer on the TRAC, TGFBR2, PD-1, CISH, and FAS genes, and / or an expression construct encoding the guide RNA.

5. The method according to claim 4, characterized in that The protospacer sequence of the TRAC gene includes SEQ ID NO:51; the protospacer sequence of the TGFBR2 gene includes SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:28; the protospacer sequence of the PD-1 gene includes SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, or SEQ ID NO:47; the protospacer sequence of the CISH gene includes SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:10; the protospacer sequence of the FAS gene includes SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:

20.

6. The method according to claim 4, characterized in that The protospacer sequence of the TRAC gene includes SEQ ID NO:51; the protospacer sequence of the TGFBR2 gene includes SEQ ID NO:24, or SEQ ID NO:28; the protospacer sequence of the PD-1 gene includes SEQ ID NO:41, or SEQ ID NO:47; the protospacer sequence of the CISH gene includes SEQ ID NO:1; the protospacer sequence of the FAS gene includes SEQ ID NO:

14.

7. The method according to any one of claims 4 to 6, characterized in that: The base editing polypeptide includes a DNA binding protein and at least one deaminase domain.

8. The method according to any one of claims 4 to 7, characterized in that: The base editing polypeptide also includes at least one uracil DNA glycosylase inhibitor (UGI) domain.

9. The method according to any one of claims 4 to 8, characterized in that: The base editing polypeptide also includes a nuclear localization sequence.

10. The method according to any one of claims 4 to 9, characterized in that: The deaminase is selected from cytidine deaminase, optionally, the cytidine deaminase is selected from SCP1.201 family deaminase, optionally, the SCP1.201 family deaminase is selected from Sdd2, Sdd3, Sdd4 deaminase, Sdd6 deaminase, Sdd7 deaminase, mini-Sdd7 deaminase, mini-Sdd9 deaminase, Sdd10 deaminase, Sdd59 deaminase, mini-Sdd3 deaminase or mini-Sdd6 deaminase.

11. The method according to any one of claims 4 to 10, characterized in that: The deaminase is Sdd7 and / or mini-Sdd9 cytidine deaminase.

12. The method according to any one of claims 4 to 11, characterized in that: The DNA binding protein is selected from TALE, ZFP or CRISPR effector protein or its variants; optionally, the CRISPR effector protein is selected from Cas9 nickase, inactivated Cas9 or its variants.

13. The method according to any one of claims 4 to 12, characterized in that: The base editing polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity with any one of SEQ ID NOs: 74-80.

14. The method according to any one of claims 4 to 13, characterized in that: The method also includes introducing a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor (CAR) includes an antigen binding domain and a transmembrane domain.

15. The method according to any one of claims 4 to 14, characterized in that: The T cell population is derived from human T cells, optionally from T cells enriched from human blood cells, or optionally from T cells enriched from human blood cells and frozen.

16. The method according to any one of claims 4 to 15, characterized in that The introduction method includes electroporation, liposome transfection, microinjection, viral infection (such as adenovirus, adeno-associated virus, lentivirus or other viruses), N-acetylgalactosamine (GalNAc)-mediated, gene gun-mediated, ribonucleoprotein (RNP) complex-mediated, and nanoparticle-mediated transformation.

17. A base editing system for modifying T cells, comprising: i) a base editing polypeptide, and / or an expression construct encoding a base editing polypeptide, wherein the base editing polypeptide comprises a DNA binding protein and at least one deaminase domain; and ii) a guide RNA, and / or an expression construct encoding the guide RNA, wherein the guide RNA targets any one or any combination of the T cell receptor alpha chain constant region (TRAC) gene, the transforming growth factor beta receptor 2 (TGFBR2) gene, the programmed death receptor 1 (PD-1) gene, the cytokine-inducible SH2 protein (CISH) gene, and / or the cell suicide-associated factor (FAS) genomic region.

18. The base editing system according to claim 17, characterized in that The deaminase domain is a cytidine deaminase.

19. The base editing system according to claim 18, characterized in that The cytidine deaminase is selected from the group consisting of APOBEC family deaminase, SCP1.201 family deaminase or homologs thereof; Optionally, the APOBEC family deaminase is selected from AID deaminase, APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase or APOBEC3H deaminase; Optionally, the SCP1.201 family deaminase is selected from Sdd2, Sdd3, Sdd4 deaminase, Sdd6 deaminase, Sdd7 deaminase, mini-Sdd7 deaminase, mini-Sdd9 deaminase, Sdd10 deaminase, Sdd59 deaminase, mini-Sdd3 deaminase or mini-Sdd6 deaminase.

20. The base editing system according to claim 19, characterized in that The cytidine deaminase is Sdd7 and / or mini-Sdd9 cytidine deaminase.

21. The base editing system according to any one of claims 17 to 20, characterized in that The DNA binding protein is selected from TALE, ZFP or CRISPR effector protein.

22. The base editing system according to claim 21, characterized in that The CRISPR effector protein is selected from Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cast10d, Cas12, Cas13, Cas14, CasX, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or Ca sE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn1, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpf1, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, Cu1966, TraC or variants thereof.

23. The base editing system according to claim 22, characterized in that The DNA binding protein is Cas9 nickase, inactivated Cas9 or a variant thereof.

24. The base editing system of any one of claims 17 to 23, characterized in that The base editing system also contains at least one uracil DNA glycosylase inhibitor (UGI).

25. The base editing system of any one of claims 17-24, characterized in that The base editing system also includes a nuclear localization sequence.

26. The base editing system according to any one of claims 17 to 25, characterized in that The base editing polypeptide comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 74-80.

27. The base editing system according to claim 26, characterized in that The base editing polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 77-80.

28. The base editing system according to any one of claims 17 to 27, characterized in that The guide RNA contains a spacer corresponding to the protospacer of the TRAC, TGFBR2, PD-1, CISH, and FAS genes; Optionally, the protospacer sequence of the TRAC gene includes SEQ ID NO:51; the protospacer sequence of the TGFBR2 gene includes SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:28; the protospacer sequence of the PD-1 gene includes SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, or SEQ ID NO:47; the protospacer sequence of the CISH gene includes SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:10; the protospacer sequence of the FAS gene includes SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:

20.

29. The base editing system according to claim 28, characterized in that The protospacer sequence of the TRAC gene includes SEQ ID NO:51; the protospacer sequence of the TGFBR2 gene includes SEQ ID NO:24, or SEQ ID NO:28; the protospacer sequence of the PD-1 gene includes SEQ ID NO:41, or SEQ ID NO:47; the protospacer sequence of the CISH gene includes SEQ ID NO:1; the protospacer sequence of the FAS gene includes SEQ ID NO:

14.

30. A modified T cell, wherein the T cell is modified by the method according to any one of claims 1-16, or the base editing system according to any one of claims 17-29 is introduced, and the T cell knocks out or down-regulates the expression of endogenous genes, wherein the endogenous genes include T cell receptor α chain constant region (TRAC) gene, transforming growth factor β receptor 2 (TGFBR2) gene, programmed death receptor 1 (PD-1) gene, cytokine-inducible SH2 protein (CISH) gene and cell suicide-associated factor (FAS) gene.

31. The T cell according to claim 30, characterized in that The T cells are CAR-T cells.

32. The T cell according to claim 31, characterized in that The CAR-T cells recognize one or more target antigens.

33. The T cell according to any one of claims 30 to 32, characterized in that The T cells are used to prevent or treat autoimmune diseases, tumors, viral infectious diseases, bacterial infectious diseases, genetic diseases, infectious diseases or cardiovascular diseases.

34. A composition, wherein the composition comprises the modified T cell of any one of claims 30-33, and, optionally, a pharmaceutically acceptable carrier or excipient.

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