Base editing system for optimizing engineered T cells and application thereof

Abstract

The invention belongs to the field of gene engineering. Specifically, the invention relates to a base editing system for optimizing engineered T cells and application of the base editing system. The present invention provides a base editing system for modifying engineered T cells, comprising: i) a base editing polypeptide, and / or an expression construct encoding a base editing polypeptide, the base editing polypeptide comprising a DNA binding protein and at least one deaminase domain; ii) a guide RNA, and / or an expression construct encoding a guide RNA, the guide RNA targeting any one of TRAC, TGFBR2, PD-1, CISH, and / or FAS genomic regions, or any combination thereof. The base editing system disclosed by the invention has high efficiency of a gene editing tool and low Indel frequency, can realize accurate editing of T cell function enhancement related genes, and has important value for expanding application scenes of a base editing technology in the fields of preparation of safe and efficient engineered T cells and the like.

Description

[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”. All contents of the above applications, including 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 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 according to the different sources of modified T cells, namely autologous CAR-T cells and allogeneic (or off-the-shelf) CAR-T cells; the former extracts the patient's own T cells for modification, while the latter separates T cells from healthy heterologous individuals for modification. The two types of CAR-T cells have their own advantages and disadvantages in practical application. For example, an important advantage of autologous CAR-T cells is that since they are derived from the patient themselves, the integration and modification of the CAR gene is completed in vitro, and when they are infused back into the patient's body, they often have a good immune effect and will not cause immune rejection; however, due to the relatively limited number of healthy T cells in patients with tumor cells, especially in the later stages of the disease, it is sometimes difficult to actually isolate a sufficient number of T cells from the patient's body and complete the preparation of autologous CAR-T cells in a timely manner. Off-the-shelf CAR-T cells can be isolated from healthy donors, so that the quantity can be guaranteed, and the supply cycle will be faster, and the cost of patient application will be greatly reduced, so it is a new type of CAR-T cell therapy with great potential. However, ordinary off-the-shelf CAR-T cells have the problem of poor clinical effects, 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 original immune cells in the patient's body, resulting in a weakening of the patient's immune system. The combination of these two factors has led to the poor clinical effects of previous off-the-shelf CAR-T cells.

[0006] However, recent studies have found that in the process of functional transformation 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 being injected into the patient's body, thereby improving its clinical effect.

[0007] For example, the CISH gene encodes an immune negative regulator 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 the activation, survival, and expansion of T cells (Zhu et al., 2022. Metabolic Reprograming 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 FASL protein to promote T cell apoptosis. Studies have found that by expressing a dominant negative effector protein of FAS protein in CAR-T cells and disrupting the interaction between FAS and FASL, the maintenance time of CAR-T cells can be increased and their tumor killing effect can be enhanced (Yamamoto et al., 2019. T cells genetically engineered to overcome death signaling enhance adoptive cancer immunotherapy).

[0009] TGF-β is an anti-inflammatory factor expressed in the immune microenvironment of blood and many tumors. Studies have found that the binding of TGF-β to the receptor protein TBR2 encoded by the TGFBR2 gene can trigger the activation of downstream signaling pathways, inhibit the activation and survival of T cells, and thus inhibit 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 ability 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 the appropriate area may contribute to the application effect of CAR-T cells.

[0010] In addition, knocking out the immune checkpoint protein PD-1 has also been reported to promote T cell activation, proliferation, and tumor killing effects (McGowan et al., 2020. PD-1 disrupted CAR-T cells in the treatment of solid tumors: Promises and challenges). Knockout of TRAC helps eliminate GvHD reactions and improve the viability and tumor killing effects of CAR-T cells (Eyquem et al., 2017. Targeting a CAR to the TRAClocus with CRISPR / Cas9 enhances tumour 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 refers to the process of replacing nucleotides at specific DNA sites through genetic engineering. Base editing is a type of genome editing technology. In humans and animals, many genetic diseases are caused by point mutations in functional genes. Base editing technology can be used to modify and transform the genomic DNA of animals, plants, and microorganisms to create new genotypes and obtain target traits that are beneficial to production applications. It can also be used to correct some serious congenital genetic variations in the treatment of genetic diseases. Therefore, base editing has important application prospects in the creation of excellent germplasm and disease treatment.

[0013] Base editing is achieved with the help of a base editor system. According to the type of bases acted on by the base editor system, the base editor system 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 cytosine deoxyribonucleotide (abbreviated as cytidine, C), while ABE can act on the adenine base on adenine deoxyribonucleotide (abbreviated as adenosine, A). CBE editing can convert CG base pairs on double-stranded DNA into TA base pairs; ABE editing can convert AT base pairs on double-stranded DNA into GC base pairs.

[0014] At present, an accurate, effective, stable and safe base editing system is still needed to further optimize CAR-T cells, improve the survival, proliferation, 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] Problem that the invention aims to solve

[0016] In view of the fact 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 comprise 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 a gene target site. Preferably, the gene editing technology is DNA replacement of a gene target site.

[0020] In some embodiments, the gene editing technique 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, and FAS genes, and / or an expression construct encoding the guide RNA.

[0026] In some 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.

[0027] In some 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.

[0028] In some embodiments, the base editing polypeptide includes 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 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.

[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%, 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 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 an APOBEC family deaminase, a 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 their variants.

[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%, 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, FAS gene;

[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, SEQID NO:18, or SEQ ID NO:20.

[0055] In some 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.

[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 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.

[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 the fourth aspect of the present invention, a composition is provided, wherein the composition comprises the modified T cells as described in 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 the editing efficiency results of CISH gene preferred targets.

[0064] Figure 2 , Schematic diagram of the editing efficiency results of the preferred target of 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 site of TGFBR2 gene.

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

[0068] Figure 6Schematic diagram of the editing efficiency results of 5 genes by mini-Sdd9-QBEmax and mini-Sdd9-BE4max.

[0069] Figure 7 Schematic diagram of the indel (insertion / deletion) efficiency results of mini-Sdd9-QBEmax and mini-Sdd9-BE4max for 5 genes.

[0070] Figure 8 Schematic diagram of the editing efficiency / indel results of mini-Sdd9-QBEmax and mini-Sdd9-BE4max for 5 genes.

[0071] Fig. 9 Schematic diagram of the editing efficiency results of Sdd7-QBEmax and Sdd7-BE4max on 5 genes.

[0072] Fig.10 Schematic diagram of the indel efficiency results of Sdd7-QBEmax and Sdd7-BE4max for 5 genes.

[0073] Fig.11 Schematic diagram of the editing efficiency / indel results of Sdd7-QBEmax and Sdd7-BE4max for 5 genes.

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

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

[0076] Fig.14 , schematic diagram of the editing efficiency results of mini-Sdd9-BE4max for the combined knockout of five genes.

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

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

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

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

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

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

[0083] Fig.21 , Schematic diagram of the relative positions of protospacers / target sites in five genes.

[0084] Fig. 22 , BE4max, QBEmax base editing system polypeptide structure schematic diagram, the promoter, NLS, and UGI are selected according to the known techniques in the art. DETAILED DESCRIPTION

[0085] I. Definition

[0086] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology related terms and laboratory operation procedures 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 documents: 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, the definitions and explanations of the relevant terms are provided below.

[0087] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and each combination should be considered to have been listed separately 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 term "comprising" is used herein to describe a protein or nucleic acid sequence, the protein or nucleic acid may consist 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 at this time, and accordingly, its encoding nucleotide sequence may also contain a start codon; and vice versa.

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

[0090] "Genetically modified organism" or "genetically modified cell" means an organism or cell that contains an exogenous polynucleotide or a modified gene or expression control sequence in its genome. For example, the exogenous polynucleotide can be stably integrated into the genome of the organism or cell and inherited for consecutive generations. The exogenous polynucleotide can be integrated into the genome alone or as part of a recombinant DNA construct. The modified gene or expression control sequence is a sequence in the genome of the organism or cell that contains single or multiple deoxynucleotide substitutions, deletions and additions.

[0091] "Polynucleotide", "nucleic acid sequence", "nucleotide sequence" or "nucleic acid fragment" are used interchangeably and are single-stranded or double-stranded RNA or DNA polymers that optionally may 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" represents cytidine or deoxycytidine, "G" represents guanosine or deoxyguanosine, "U" represents uridine, "T" represents deoxythymidine, "R" represents purine (A or G), "Y" represents pyrimidine (C or T), "K" represents G or T, "H" represents A or C or T, "I" represents inosine, and "N" represents any nucleotide.

[0092] "Polypeptide", "peptide", and "protein" are used interchangeably herein to refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids, 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 full 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). Although there are many methods to measure the identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill (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 generally can be carried out without changing 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 change 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 a DNA chain using RNA as a template. It can recognize the base sequence on the RNA chain and synthesize a complementary DNA sequence on the DNA chain.

[0096] "Domain" refers to the part of a protein that has a relatively stable three-dimensional structure. Proteins are usually composed of one or more domains, each of which has its own spatial folding form. Domains can be continuous or discontinuous, and they are usually composed of one or more segments in the amino acid sequence. The existence of domains enables proteins to have functional diversity, 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, the 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 can be translated. An "expression construct" may contain 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 is normally naturally present.

[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 in the present invention 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 to perform its function without the stable inheritance of the exogenous nucleotide sequence. In transient transformation, the exogenous nucleic acid sequence is not integrated into the genome.

[0101] "T cell" refers 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 a T cell receptor on the cell surface.

[0102] "Engineered T cells" refer to any T cells containing 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 different organs, with specificity for epithelial cell apoptosis and crypt shedding. GvHD is often associated with stem cell transplants, such as those occurring with bone marrow transplants, and can also be induced by CAR therapies based on donor leukocyte infusions, virus-specific T cells, T cell receptor-deficient T cells, lymphoid progenitor cells, and regulatory T cells.

[0106] "HEK293T" refers to a variant of human embryonic kidney 293 cells (HEK293) containing the SV40 large T-antigen. This antigen allows episomal replication of transfected plasmids containing the SV40 replication origin, which results 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 system 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" (Nickingenzyme or Nickase) is a special enzyme, a kind 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 off 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, the 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 polypeptides 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 variant thereof. Inactivated nucleases can target nucleic acids without producing chain breaks. For example, residues such as Asp10, His840, Asn854, and Asn856 in a 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] Herein, "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: APOBEC family deaminase, SCP1.201 family deaminase or their homologs; 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 and APOBEC3H deaminase; 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, mini-Sdd6 deaminase or their respective functional mutants, and a non-limiting example of the SCP1.201 family deaminase comprises the amino acid sequence SEQ ID 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 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, usually a short amino acid sequence, which 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 may 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) comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at or near the N-terminus. 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 at or near the C-terminus. In some embodiments, the polypeptide comprises a combination of these, such as one or more NLSs at the N-terminus and one or more NLSs at the C-terminus. When there is more than one NLS, each can be selected to be independent of the other NLS. Generally speaking, NLS consists of one or more short sequences of positively charged lysine or arginine exposed on the protein surface, but other types of NLS are also known. Non-limiting examples of NLS include amino acid sequences SEQ ID NO: 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 bacteriophageMu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity[J]. Science advances, 2017, 3(8):eaao4774.;

[0121] BE4max is the BE4 in the document, and its structure is as shown in the document Figure 5 A) or QBEmax.

[0122] In this specification, the base editor (BE) system includes BE1, BE2, BE3, BE4, etc., wherein BE1 is composed of a fusion protein of dCas9 and APOBEC1. BE2 is a BE system obtained by introducing UGI (Uracil DNA glycosylase inhibitor) into BE1. BE3 refers to a BE system obtained by replacing dCas9 in BE2 with nCas9 (D10A). BE4 refers to a BE system obtained by optimizing the length of the linker region between APOBEC1 and nCas9 (D10A), the length of the linker region between nCas9 (D10A) and UGI, and adding a copy of UGI based on BE3.

[0123] In some embodiments, QBEmax comprises a deaminase domain and a pair of DNA binding protein domains having a DNA single-strand cleavage function.

[0124] In some embodiments of the present invention, the DNA binding protein having single-stranded DNA cleavage activity is two paired DNA binding protein domains, and the paired DNA binding protein domains are respectively connected to the N-terminus and the C-terminus of the deaminase domain through a linker. In some embodiments of the present invention, the paired DNA binding protein domains can be obtained by the following method: by performing a circular transformation on the amino acid sequence of the DNA binding protein having a single-stranded cleavage function (the linker of the circular transformation can be selected from GGSGGSGGSGGSGGSGGSGG; SEQ ID NO: 90), a circular transformation sequence with a length of 1388 amino acids is obtained (the circular transformation sequence is as follows

[0125] 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 DNA binding protein are split to obtain a pair of separated polypeptides, which are paired DNA binding protein domains with DNA single-strand cleavage function. The above-mentioned paired DNA binding protein domains with DNA single-strand cleavage function are fused or linked to the N-terminus and C-terminus of the deaminase domain, respectively. According to the fusion position with the deaminase, the domains connected to the N-terminus or C-terminus of the deaminase are respectively defined as the deaminase N-terminal connecting domain (QBE-N) and the deaminase C-terminal connecting domain (QBE-C).

[0126] For example:

[0127]

[0128] Among them, the first split site is defined as the deaminase attachment site, in which 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, in which 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.

[0129] 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.

[0130] In some embodiments, the N-terminus or C-terminus of the deaminase domain comprises one or two single domains, and the single domains are connected by a linker. Herein, a "single domain" is a part of a DNA binding protein domain, and all single domains are combined and spliced ​​into a complete DNA binding protein domain. The single domain does not contain a linker inside, and has linkers at both ends to connect to other single domains or functional regions (such as deaminase, NLS or UGI, etc.).

[0131] 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.

[0132] 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.

[0133] The deaminase is as described above under "deaminase domain".

[0134] Herein, "joint" refers to connecting two molecules or parts, such as two domains of a fusion protein. Typically, a joint is located between or flanking two groups, molecules or other parts and is connected to each by a covalent bond, thereby connecting the two. In some embodiments, the joint is an organic molecule, group, polymer or chemical part.

[0135] 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.

[0136] “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.

[0137] In the present invention, "target nucleic acid" is a polynucleotide (e.g., DNA such as genomic DNA) including a site ("target site" or "target sequence") targeted by a DNA binding protein (e.g., Cas protein, etc.) guided by a guide RNA. "Target sequence" is a sequence to which the guide sequence of the guide RNA will hybridize.

[0138] In this specification, "protospacer", "protospacer" and "editing window" are used interchangeably and refer to nucleic acid sequences in the genome that are 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.

[0139] 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.

[0140] In some embodiments, the guide RNA is present as a single RNA molecule. In some embodiments, the guide RNA is a sgRNA, which comprises two parts: (1) a guide sequence complementary to a target sequence of a target nucleic acid; and (2) a part that binds to a DNA binding protein.

[0141] 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 genomes, and the spacer sequence is complementary to the target sequence.

[0142] Herein, "site1, site2..." refers to a protospacer sequence. In some specific embodiments, the protospacer sequence is shown in SEQ ID NO: 1-51.

[0143] In some specific embodiments, the target sequence is a DNA sequence complementary to the original spacer sequence shown in SEQ ID NO: 1-51 (it can be understood that it is located in the non-editing chain). As used herein, "sg1, sg2..." represents an RNA sequence (guide sequence) complementary to the target sequence, that is, an RNA sequence complementary to the complementary DNA sequence of SEQ ID NO: 1-51.

[0144] 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 NO:97-99.

[0145] In some embodiments, the guide RNA includes a spacer sequence corresponding to the original spacer sequence of the following target site, wherein the target site is selected from the TRAC, TGFBR2, PD-1, CISH or FAS genome, and the genome sequence refers to GRCh38.p14.

[0146] In some optional embodiments, the protospacer sequence comprises:

[0147] (a) at least one of the nucleotide sequences shown in SEQ ID NOs: 1-10; and / or

[0148] (b) at least one of the nucleotide sequences shown in SEQ ID NOs: 11-20; and / or

[0149] (c) at least one of the nucleotide sequences shown in SEQ ID NOs: 21-30; and / or

[0150] (d) at least one of the nucleotide sequences shown in SEQ ID NOs: 31-47; and / or

[0151] (e) at least one of the nucleotide sequences shown in SEQ ID NOs: 48-51.

[0152] In some specific embodiments, the guide RNA contains spacers corresponding to the following protospacers of TRAC, TGFBR2, PD-1, CISH, FAS genes:

[0153] 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 SEQID 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.

[0154] 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.

[0155] 3. Methods for obtaining modified T cells

[0156] On the other hand, the present invention provides a method for obtaining modified T cells, the method comprising 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.

[0157] In some embodiments, the gene editing technology includes DNA insertion, deletion or replacement of a gene target site. Preferably, the gene editing technology is DNA replacement of a gene target site.

[0158] In some specific embodiments, the gene editing technology includes single base editing.

[0159] In some specific embodiments, the method comprises:

[0160] a) providing a purified T cell population;

[0161] b) introducing a single base editing system into the T cell population in a);

[0162] c) Expanding and harvesting the modified T cells.

[0163] 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 genes, and / or an expression construct encoding the guide RNA.

[0164] 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 includes at least one of the nucleotide sequences shown in SEQ ID NOs:21-30; the protospacer sequence of the PD-1 gene includes at least one of the nucleotide sequences shown in SEQ ID NOs:31-47; the protospacer sequence of the CISH gene includes at least one of the nucleotide sequences shown in SEQ ID NOs:1-10; the protospacer sequence of the FAS gene includes at least one of the nucleotide sequences shown in SEQ ID NOs:11-20.

[0165] 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.

[0166] 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 SEQID NO:1; and the protospacer sequence of the FAS gene includes SEQ ID NO:14.

[0167] In some embodiments, the base editing polypeptide includes a DNA binding protein and at least one deaminase domain.

[0168] In some embodiments, the base editing polypeptide further comprises at least one uracil DNA glycosylase inhibitor (UGI) domain.

[0169] In some embodiments, the base editing polypeptide further comprises a nuclear localization sequence.

[0170] In some embodiments, 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.

[0171] In some embodiments, the deaminase is Sdd7 and / or mini-Sdd9 cytidine deaminase.

[0172] 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.

[0173] In some embodiments, the base editing polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of SEQ ID NOs:74-80.

[0174] 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.

[0175] In some embodiments, the T cell population is from human T cells, optionally from T cells enriched from human blood cells, optionally from T cells enriched from human blood cells and cryopreserved.

[0176] 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.

[0177] In some embodiments, the single base editing system is a base editing system for modifying T cells as 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 (eg, CAR-T cells) by a method selected from the following: electroporation, liposome transfection, microinjection, viral infection (eg, adenovirus, adeno-associated virus, lentivirus, or other virus), N-acetylgalactosamine (GalNAc)-mediated, gene gun-mediated, ribonucleoprotein (RNP) complex-mediated, nanoparticle-mediated transformation.

[0178] 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.

[0179] 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 the delivery of "naked" polynucleotides (such as electroporation, "gene gun" delivery and various other technologies for the introduction of polynucleotides). The introduced polynucleotides can be stably or transiently maintained in the host cell. Stable maintenance generally requires that the introduced polynucleotides 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 a nuclear or mitochondrial chromosome. Many "vectors" are known to be able to mediate the transfer of genes to mammalian cells, as known in the art and described herein.

[0180] 4. A modified T cell

[0181] 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 the base editing system described in the above “2. Base editing system for modifying T cells” is introduced.

[0182] 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.

[0183] In some embodiments, the T cells are CAR-T cells.

[0184] In some specific embodiments, the CAR-T cells recognize one or more target antigens.

[0185] 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 (eg, an autologous human CAR-T cell).

[0186] 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.

[0187] 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;

[0188] The tumor is selected from the group consisting of lymphoma, hematologic tumor 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 tumor, 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, brain low-grade glioma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, One or more of: cancer, mesothelial cell cancer, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, colorectal 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.

[0189] The virus is selected from one or more of influenza virus, parainfluenza virus, measles virus, mumps virus, herpes virus, adenovirus, respiratory syncytial virus, polio virus, coxsackie virus or echovirus;

[0190] The bacteria are selected from one or more of Escherichia coli, Lactobacillus casei, Bacteroides fragilis, Acinetobacter lwoffii, Fusobacterium nucleatum, Bacteroides johnsonii, Bacteroides arabidopsis, Lactobacillus rhamnosus, Bacteroides massiliense, Bacteroides ovale, Campylobacter jejuni, Staphylococcus saprophyticus, Enterococcus faecalis, Bacteroides thetaiotaomicron, Bacteroides vulgaris, Bacteroides monomorpha, Bacteroides faecalis, Fusobacterium mortis and Bifidobacterium breve.

[0191] 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.

[0192] The cardiovascular disease is selected from the group consisting of coronary heart disease, cerebrovascular disease, peripheral arterial vascular disease, rheumatic heart disease, congenital heart disease, deep vein thrombosis and pulmonary embolism.

[0193] 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.

[0194] 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.

[0195] Example

[0196] References for experimental methods in 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 baseeditors 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.

[0197] Example 1. Identification of sgRNA

[0198] In the preparation of engineered T cells, especially CAR-T cells, knocking out five genes: PD-1, CISH, FAS, TGFBR2, and TRAC, helps the survival, expansion, maintenance, activation, and tumor killing performance of T cells, while effectively inhibiting or even eliminating immune rejection or GvHD reactions. In addition to being restricted by PAM, protospacer, and off-target sites, it is also subject to codon restrictions. The cytidine base editing system (CBE) converts the DNA of the codons encoding arginine (CGA) or glutamine (CAA, CAG) or tryptophan (UGG) in the editing window into a stop codon (UAG, UGA, UAA), or by editing important splicing sites, in order to achieve the effect of gene knockout. For this reason, taking the above factors into consideration, a group of possible editing targets were finally selected from these five genes, with 10 targets selected for CISH, FAS, and TGFBR2, 4 targets selected for TRAC, and 17 targets selected for PD-1, for a total of 51 targets. The original spacer sequence of the above 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 Fig.21 As shown. sgRNA is designed for the above target, wherein the guide sequence of sgRNA is complementary to the target sequence of the non-editing chain (i.e., the complementary sequence of the original spacer sequence), so that 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 gene sequences in the cell genome, HEK293T cells were selected as verification cells in this example.

[0199] HEK293T cells were transfected with the above-mentioned target sequence complementary sgRNA and BE4max backbone editing system plasmids fused to APOBEC1, mini-Sdd3, mini-Sdd6, Sdd7 and mini-Sdd9 deaminases (SEQ ID NO: 74-78). The transfection conditions were 60,000 cells per well in a 48-well cell plate, and transfection was performed 18-24 hours after plating; transfection was performed in the form of plasmids, with 375ng of editing system plasmid and 125ng of sgRNA plasmid added to each well, and then mixed and transfected with 1μL of jetPRIME transfection reagent (Cat. No.: 101000046, Polyplus Inc.). After 72 hours of transfection, cell genomic DNA was extracted, the target fragment was amplified by two rounds of PCR and the product was purified, and the base editing effect at the target site was detected by the second-generation detection technology (NGS).

[0200] The results are shown in Table 1 (where "\" indicates that no editing efficiency was detected). The inventors verified the sgRNA screened by bioinformatics in actual HEK293T cells. The editing efficiency of different protospacers varied greatly, with the highest reaching 71.26% and the lowest undetectable editing efficiency. At the same time, base editing polypeptides composed of different deaminases also had obvious preferences for editing different targets.

[0201] Table 1

[0202]

[0203]

[0204]

[0205] Table 1 shows the editing efficiency of the editing system adapted to each high editing efficiency target, and the editing targets with high editing efficiency are obtained. For the CISH gene, the targets corresponding to site1, site5, site8 and site10 have better effects, with editing efficiencies of 47.46%-71.26%; for the PD-1 gene, the targets corresponding to site14, site15, site16 and site17 have the best effects, with editing efficiencies of 57.45%-67.68%; for the FAS gene, the targets corresponding to site4, site7, site8 and site10 have better effects, with editing efficiencies of 49.05%-61.68%; for the TGFBR2 gene, the targets corresponding to site1, site4, site7 and site8 have better effects, with editing efficiencies of 46.91%-64.48%; for the TRAC gene, the target corresponding to site4 has better effects, with editing efficiencies of 69.06%.

[0206] Example 2: Screening of deaminase

[0207] In order to construct a single-base editing system with universal applicability to multiple genes, this example further verified and screened out deaminases with editing effects on multiple targets based on the preferred targets in Example 1. The experimental results are as follows: Figure 1-Figure 5 As shown. Figure 1-Figure 5The editing efficiency of the base editing system using different deaminases at the preferred target sites was verified in the experiment, where the editing efficiency was defined as efficient editing when it was greater than or equal to 40%, marked as "+"; less than 40% was defined as non-efficient editing, marked as "-", and the statistical results are shown in Table 2. The data show that the base editors with APOBEC1, miniSdd3, and mini-Sdd6 as deaminases did not produce efficient editing in the preferred target sites of some target genes. Only the base editing system with Sdd7 and miniSdd9 as deaminases had efficient editing targets in all five genes, indicating that they are ideal deaminases for the base editing system in the present invention.

[0208] Table 2

[0209]

[0210]

[0211] Example 3. Validation of different base editing systems

[0212] To verify the effect of different base editing polypeptides on the editing effect, this example selects a circular cleavage variant of a CRISPR protein as the QBEmax system of a DNA binding protein as a verification system. In this system, the amino acid sequence of a DNA binding protein with a single-stranded cleavage function is subjected to a circular transformation (the linker of the circular transformation is GGSGGSGGSGGSGGSGGSGG; SEQ ID NO: 90), and a circular transformation sequence with a length of 1388 amino acids is 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 circular transformation sequence is split at two sites to obtain a pair of separated polypeptides (SEQ ID NO: 92 and SEQ ID NO: 93), and the separated polypeptides are paired DNA binding protein domains. According to the 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 connection domain (QBE-N) and the deaminase C-terminal connection domain QBE-C, respectively. The polypeptide of the QBEmax system comprises the following structure: [QBE-N]-[deaminase]-[QBE-C], wherein "-" is a linker. Fig. 22 Schematic diagram of the structure of the base editing polypeptide of this example.

[0213] The two preferred deaminases Sdd7 and mini-Sdd9 screened in Example 2 were used as the deaminase domains of the base editing polypeptides, and four base editing polypeptides were obtained: 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).

[0214] On the one hand, HEK293T cells were transfected with plasmids expressing the above-mentioned editing peptides and the corresponding sgRNAs. The editing effects of these targets were statistically analyzed, and their performance at these CAR-T related sites was compared in parallel.

[0215] Figure 6 It showed that the average editing efficiency of mini-Sdd9-QBEmax in all five target genes was comparable to the overall average editing efficiency of mini-Sdd9-BE4max; Figure 7 The results showed that the average indel of mini-Sdd9-QBEmax in all five target genes was significantly lower than that of mini-Sdd9-BE4max; Figure 8 The editing efficiency / indel ratios were statistically analyzed, and it was found that the editing efficiency / indel ratios of mini-Sdd9-QBEmax were significantly higher than those of mini-Sdd9-BE4max in all five genes, ranging from 1.99 to 9.91 times higher.

[0216] Fig. 9 The average editing efficiency of Sdd7-QBEmax on the five genes was slightly lower than that of Sdd7-BE4max. Fig.10 It showed that the average indel of Sdd7-QBEmax was lower than that of Sdd7-BE4max; Fig.11 The editing efficiency / indel ratio was calculated and the editing effect of Sdd7-QBEmax was better than that of Sdd7-BE4max in CISH, PD-1, and TRAC, while the editing effect of Sdd7-BE4max in FAS and TGFBR2 was better than that of Sdd7-QBEmax.

[0217] 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 the editing of gene sites related to T cell function enhancement, and each has its own advantages. When selecting different deaminases, different editing systems can be adapted as appropriate.

[0218] Example 4: Verification of target editing purity

[0219] In this example, the 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 efficiency while also having a high editing purity to ensure safety. Figure 16-20 The editing effect and purity of the above five genes (51 target sites) knocked out by mini-Sdd9-QBEmax and mini-Sdd9-BE4max are shown in the figure above: a shows the editing efficiency and indel of different sites (the position indicated by the blue arrow is the expected mutant base); b shows the ratio of editing efficiency / indel of 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 indicates the purity of editing. Figure 16-20 The analysis and statistics are shown in Table 4. Editing efficiency greater than or equal to 40% is marked as "+", less than 40% is marked as "-"; editing purity greater than or equal to 95% is marked as "+", less than 95% is marked as "-". Efficiency and purity are both "+" indicating that the editing system has a good comprehensive effect at a certain site.

[0220] Depend on Figure 16-20The comprehensive editing efficiency and editing purity analysis in Table 3 showed that for the CISH gene, mini-Sdd9-BE4max had the best effect at site 1, and mini-Sdd9-QBEmax had the best effect at site 1 and site 9; for the FAS gene, mini-Sdd9-BE4max had the best effect at site 7, and mini-Sdd9-QBEmax had the best effect at site 4 and site 8; for the PD-1 gene, mini-Sdd9-BE4max had the highest editing efficiency at site 17, and had a good comprehensive effect at site 5 and site 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. The editing purity of mini-Sdd9-QBEmax was high in all sites of the gene, and the editing efficiency was average; for the TRAC gene, both mini-Sdd9-BE4max and mini-Sdd9-QBEmax showed good effects at site 4.

[0221] In summary, the editing purity was included in the screening scope and 51 targets were screened for the second time. It was found that there were some differences in the preferred editing sites of QBEmax and BE4max in the five targeted genes. Different editing peptides can be used as appropriate when actually knocking out genes. In addition, some of the preferred targets of QBEmax obtained in this example (for example, CISH gene site9; PD-1 gene site2, site3, site7, site11, site12) were used as candidate targets for subsequent co-knockout experiments as a supplement.

[0222] Table 3

[0223]

[0224]

[0225] Example 5: Joint knockout of five genes

[0226] In Examples 1-4, the inventors gradually screened preferred targets for targeting five genes, preferably deaminases, and preferred base editing polypeptides. In this example, the inventors permuted and combined the above parameters to obtain the combination in Table 4, which verified the experimental effect of multi-gene joint knockout. The base editing polypeptide and five different sgRNAs were co-transfected into HEK293T cells to knock out the five genes.

[0227] Table 4

[0228]

[0229]

[0230] Fig.12 The editing efficiency of combination 1 is shown to be relatively stable and relatively high, ranging from 42.9% to 54.88%; Fig.13 The editing efficiency of combination 2 is shown as 54.21%, which is relatively high for CISH gene. The comprehensive editing efficiency of combination 2 is 30.35%-54.21%; Fig.14 The editing efficiency of combination 3 is shown. The editing ability of this system for PD-1 and TRAC is relatively weak, but the editing ability for CISH, FAS and TGFBR2 is still good, ranging from 37.66% to 39.86%; Fig.15 The editing efficiency of combination 4 is shown, and the overall editing ability is also at a high level, with the editing efficiency between 32.65% and 56.78%. Therefore, the technical solution of the present application can achieve the modification of genes related to T cell function enhancement.

[0231] 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. Nucleotide or amino acid sequences involved in the present invention: >SEQ ID NO:1CISH-site1TCTGCGTTCAGGGGTAAGCG

[0232] >SEQ ID NO:2CISH-site2GGACTGGGCAGCGGCCCCTG

[0233] >SEQ ID NO:3CISH-site3GACTGGGCAGCGGCCCCTGT

[0234] >SEQ ID NO:4CISH-site4GTTCCAGGGACGGGGCCCAC

[0235] >SEQ ID NO:5CISH-site5GTCATGCAGCCCTTGCCTGC

[0236] >SEQ ID NO:6CISH-site6TGGAACCCCAATACCAGCCT

[0237] >SEQ ID NO:7CISH-site7TCATGCAGCCCTTGCCTGCT

[0238] >SEQ ID NO:8CISH-site8CACCTGCAGAAGATGCCAGA

[0239] >SEQ ID NO:9CISH-site9CAGCCTTGTGCAGCACTATG

[0240] >SEQ ID NO:10CISH-site10CTCACCAGATTCCCGAAGGT

[0241] >SEQ ID NO:11FAS-site1GACTGCGTGCCCTGCCAAGA

[0242] >SEQ ID NO:12FAS-site2ACTGCGTGCCCTGCCAAGAA>SEQ ID NO:13 FAS-site3CGTGCCCTGCCAAGAAGGGA>SEQ ID NO:14 FAS-site4GAGGGTCCAGATGCCCAGCA>SEQ IDNO:15 FAS-site5TACAGTTGAGACTCAGAACT>SEQ ID NO:16 FAS-site6GTTGAGACTCAGAACTTGGA>SEQ ID NO:17 FAS-site7AAGCCACCCCAAGTTAGATC>SEQ IDNO:18 FAS-site8CCCCAAACAATTAGTGGAAT>SEQ ID NO:19 FAS-site9AACTTACCCCAAACAATTAG>SEQ ID NO:20 FAS-site10AAGTTCAACTGCTTCGTAAT>SEQ IDNO:21 TGFBR2-site1TTCAGAGCAGTTTGAGACAG>SEQ ID NO:22 TGFBR2-site2GAACATACTCCAGTTCCTGA>SEQ ID NO:23 TGFBR2-site3CGGAGTTGGGGAAACAATAC>SEQID NO:24 TGFBR2-site4ACCTACAGGAGTACCTGACG>SEQ ID NO:25 TGFBR2-site5CCGTCTCACAGCCCAGTGTG>SEQ ID NO:26 TGFBR2-site6GTCCTTCTCTGTCTTCCAAG>SEQID NO:27 TGFBR2-site7ACTCCAGTTCCTGACGGCTG>SEQ ID NO:28 TGFBR2-site8TTCCCAGAGCACCAGAGCCA>SEQ ID NO:29 TGFBR2-site9CTGGTGGTTGAGCCAGAAGC>SEQID NO:30 GFbR2-site10TGGTGGTTGAGCCAGAAGCT>SEQ ID NO:31 PD-1-site1ACGACTGGCCAGGGCGCCTG>SEQ ID NO:32 PD-1-site2CGACTGGCCAGGGCGCCTGT>SEQ IDNO:33 PD-1-site3TGTAGCACCGCCCAGACGAC>SEQ ID NO:34PD-1-site4CACCGCCCAGACGACTGGCC>SEQ ID NO:35 PD-1-site5ACCGCCCAGACGACTGGCCA>SEQ IDNO:36 PD-1-site6GTCTGGGCGGTGCTACAACT>SEQ ID NO:37 PD-1-site7GGGCGGTGCTACAACTGGGC>SEQ ID NO:38 PD-1-site8CGGTGCTACAACTGGGCTGG>SEQ IDNO:39PD-1-site9CTACAACTGGGCTGGCGGCC>SEQ ID NO:40 PD-1-site10CAGCAACCAGACGGACAAGC>SEQ ID NO:41 PD-1-site11CGTGTCACACAACTGCCCAA>SEQID NO:42 PD-1-site12GTGTCACACAACTGCCCAAC>SEQ ID NO:43 PD-1-site13CACCTACCTAAGAACCATCC>SEQ ID NO:44 PD-1-site14CGCAGATCAAAGAGAGCCTG>SEQID NO:45 PD-1-site15CGGCCAGTTCCAAACCCTGG>SEQ ID NO:46 PD-1-site16AGCCGGCCAGTTCCAAACCC>SEQ ID NO:47 PD-1-site17CAGTTCCAAACCCTGGTGGT>SEQID NO:48 TRAC-site1AACAAATGTGTCACAAAGTA

[0243] >SEQ ID NO:49 TRAC-site2TTTCAAAACCTGTCAGTGAT

[0244] >SEQ ID NO:50 TRAC-site3

[0245] TTCAAAACCTGTCAGTGATT

[0246] >SEQ ID NO:51 TRAC-site4TTCGTATCTGTAAAACCAAG

[0247] >SEQ ID NO:52 NLS

[0248] KKRKV

[0249] >SEQ ID NO:53 NLS

[0250] PKKKRKV

[0251] >SEQ ID NO:54 NLS

[0252] KRPAATKKAGQAKKKK

[0253] >SEQ ID NO:55 NLS

[0254] MDSLLMNRRKFLYQFKNVRWAKGRRETYLC

[0255] >SEQ ID NO:56 NLS

[0256] KRTADGSEFEPKKKRKV

[0257] >SEQ ID NO:57 NLS

[0258] PAAKRVKLD

[0259] >SEQ ID NO:58 NLS

[0260] RQRRNELKRSP

[0261] >SEQ ID NO:59 NLS

[0262] NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY

[0263] >SEQ ID NO:60 NLS

[0264] RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV

[0265] >SEQ ID NO:61NLS

[0266] VSRKRPRP

[0267] >SEQ ID NO:62NLS

[0268] PPKKARED

[0269] >SEQ ID NO:63NLS

[0270] POPKKKPL

[0271] >SEQ ID NO:64NLS

[0272] SALIKKKKKMAP

[0273] >SEQ ID NO:65 NLS

[0274] RKLKKKIKKL

[0275] >SEQ ID NO:66 NLS

[0276] REKKKFLKRR

[0277] >SEQ ID NO:67 NLS

[0278] KRKGDEVDGVDEVAKKKSKK

[0279] >SEQ ID NO:68 NLS

[0280] RKCLQAGMNLEARKTKK

[0281] >SEQ ID NO:69 Deaminase rAPOBEC1:

[0282] SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIE

[0283] KFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLIS

[0284] SGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQ

[0285] LTFFTIALQSCHYQRLPPHILWATGLK

[0286] >SEQ ID NO:70 mini - Sdd3

[0287] PRRFNPACWQGLKPYDQAGTAEGNLFIAPGKRWNTRPMQASKLEVGPQSDLHPQWRSRKAPWHI

[0288] EGKIAAYMRQKGFTDGCVYLNARPCSGPDGCARNLPDLLPVGSTLHVHARYIDRTGETRFYYREY

[0289] RGTGKALT

[0290] >SEQ ID NO:71 mini-Sdd6

[0291] PASKPSRFKPMRTDPAKADEIRPHVGKDRAVATLWDADGNRVLGLHSADDDGPAATAAWKPPW

[0292] RDYVRLRRHVEAHAAARMHQDGHKTMVMYINLPPCKYFDGCKLNLEDILPKGSTLWMHRVFQN

[0293] GGTKIYQFNGTGRAYV

[0294] >SEQ ID NO:72 mini-Sdd7

[0295] EGGGPGAVPEGGDGPPAVPAEEVERLRGELPPPVVPGTGQKTHGRWIGPDGRVRAIVSGRDEDAA

[0296] LVHAQLAAKGIPDEPTRNSDVEQKLAAHMVANGIRHVTLVINHRPCRGFDDSCDTLVPIILPEGCT

[0297] LTVHGQTDKGMRVRVRYTGGARPWWS

[0298] >SEQ ID NO:73 mini-Sdd9

[0299] CPIWVKNALQELVGRKETSGKVFDVDGKPIGPDIQSGYKDRELRRGVYETLRKSPFFQKHFPSTAT

[0300] WYVSLHVEAQYAVWMLRNRIKHATVVINNTYVCSDMNRLHDNCMTAVPHILPEGYTMTVWKA

[0301] DRTEVTLRGKAPKE

[0302] >SEQ ID NO:74 rAPOBEC1-BE4max

[0303] MKRTADGSEFESPKKKRKVSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRH

[0304] SIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLSRYPHVTLFIYI

[0305] ARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNEAHWPRYPHLWVRLYVLE

[0306] LYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLKSGGSSSGGSSGSETPGTSESAT

[0307] PESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGE

[0308] TAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD

[0309] EVAYHEKYPTIYHLRKKLVDSTDKADLRIYLALAMHIKFRGHFLIEGDLNPDNSDVDKLFIQLVQ

[0310] TYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFD

[0311] LAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIK

[0312] RYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEEL

[0313] LVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLAR

[0314] GNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYN

[0315] ELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFN

[0316] ASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRIEMIEERLKTYAHLFDDKVMKQLKRR

[0317] RYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL

[0318] HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIE

[0319] EGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDD

[0320] SIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKA

[0321] GFIKRQLVETRQITKHVAQILDSRMNTKYENDKLIREVKVITLSKLVSDFRKDFQFYKVREINN

[0322] YHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMN

[0323] FFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIL

[0324] PKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEK

[0325] NPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY

[0326] EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI

[0327] IHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSGGSG

[0328] GSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKP

[0329] WALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDIL

[0330] VHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEFEPKKKRKV>SEQID NO:75 mini-Sdd9-BE4max

[0331] MKRTADGSEFESPKKKRKVPIWVKNALQELVGRKETSGKVFDVDGKPIGPDIQSGYKDRELRRG

[0332] VYETLRKSPFFQKHFPSTATWYVSLHVEAQYAVWMLRNRIKHATVVINNTYVCSDMNRLHDNC

[0333] MTAVPHILPEGYTMTVWKADRTEVTLRGKAPKESGGSSGGSSGSETPGTSESATPESSGGSSGGSD

[0334] KKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTA

[0335] RRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTI

[0336] YHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPI

[0337] NASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSK

[0338] DTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL

[0339] LKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR

[0340] KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYVGPLARGNSRFAWMTR

[0341] KSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEG

[0342] MRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLK

[0343] IIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRK

[0344] LINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPA

[0345] IKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKE

[0346] HPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKN

[0347] RGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAEGGGLSELDKAGFIKRQLVETRQIT

[0348] KHVAQILDSRMNTKYDENDKLIREVKIVTLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAV

[0349] VGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFKTEITLANGEIR

[0350] KRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKK

[0351] DWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE

[0352] VKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQ

[0353] KQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPA

[0354] AFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSGGSTNLSDIIEKETG

[0355] KQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGEN

[0356] KIKMLSGGSGGSGNLSDIIEKETGKQLVIQESILMLPEEVEVIGNKPESDILVHTAYDESTDEN

[0357] VMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEFEPKKKRKV

[0358] >SEQ ID NO:76 mini-Sdd6-BE4max

[0359] MKRTADGSEFESPKKKRKVPASKPSRFKPMRTDPAKADEIRPHVGKDRAVATLWDADGNRVLGL

[0360] HSADDDGPAATAAWKPPWRDYVRLRRHVEAHAAARMHQDGHKTMVMYINLPPCKYFDGCKLN

[0361] LEDILPKGSTLWMHRVFQNGGTKIYQFNGTGRAYVSGGSSGGSSGSETPGTSESATPESSGGSSGG

[0362] SDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR

[0363] TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY

[0364] PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE

[0365] NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ

[0366] LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD

[0367] LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED

[0368] LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW

[0369] MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY

[0370] VTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYH

[0371] DLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRIEMIEERLKTYAHLFDDKVMKQLKRRRYTGWGR

[0372] LSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA

[0373] GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQ

[0374] ILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTR

[0375] SDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAEGGGLSELDKAGFIKRQLVE

[0376] TRQITKHVAQILDSRMNTKYDENDKLIVEKVITLSKLVSDFRKDFQFYKVREINNYHHAHDAY

[0377] LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFKTEITLAN

[0378] GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA

[0379] RKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKG

[0380] YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED

[0381] NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLG

[0382] APAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSGGSGGSTNLSDIIEK

[0383] ETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNG

[0384] ENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTD

[0385] ENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEFEPKKKRKV

[0386] >SEQ ID NO:77 Sdd7-BE4max

[0387] MKRTADGSEFESPKKKRKVLEAVRARLIGGGPGAVPEGGDGPPAVPAEEVERLRGELPPPVVP

[0388] GTGQKTHGRWIGPDGRVRAIVSGRDEDAALVHAQLAAKGIPDEPTRNSDVEQKLAAHMVANGIR

[0389] HVTLVINHRPCRGFDDSCDTLVPIILPEGCTLTVHGQTDKGMRVRVRYTGGARPWWSSGGSSGGS

[0390] SGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIK

[0391] KNLIGALLFDSGETAEATRLKRTARRRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEED

[0392] KKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRIYLALAMHIKFRGHFLIEGDLNP

[0393] DNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIA

[0394] LSLGLTPNFKSNFDLAEDAKLQLSKDTYDDLDLNLLAQIGDQYADLFLAAKNLSDAILLSDILRVN

[0395] TEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFI

[0396] KPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKIL

[0397] TFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPK

[0398] HSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECF

[0399] DSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLF

[0400] DDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDI

[0401] QKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKG

[0402] QKNSRERMKRIEEGIGELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDV

[0403] DHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTK

[0404] AERGGGLSELDKAGFIKRKQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRK

[0405] DFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKA

[0406] TAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTE

[0407] VQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELL

[0408] GITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSK

[0409] YVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKH

[0410] RDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSKEVLDATLIHQSITGLYETRIDLSQLG

[0411] GDSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVM

[0412] LLTSDAPEYKPWALVIQDSNGENKIKMLGSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVE

[0413] EVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSE

[0414] FEPKKKRKV

[0415] >SEQ ID NO:78 mini-Sdd3-BE4max

[0416] MKRTADGSEFESPKKKKRKVPRRFNPACWQGLKPYDQAGTAEGNLFIAPGKRWNTRPMQASKLEV

[0417] GPQSDLHPQWRSRKAPWHIEGKIAAYMRQKGFTDGCVYLNARPCSGPDGCARNLPDLLPVGSTL

[0418] HVHARYIDRTGETRFYYREYRGTGKALTSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIG

[0419] LAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTR

[0420] RKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKK

[0421] LVDSTDKADLRIYLALAMHIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVD

[0422] AKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDD

[0423] DLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALV

[0424] RQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTF

[0425] DNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETI

[0426] TPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKP

[0427] AFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDK

[0428] DFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGI

[0429] RDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGI

[0430] LQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEGIGELGSQILKEHPVE

[0431] NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGK

[0432] SDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV

[0433] AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTA

[0434] LIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFKTEITLANGEIRKRPLI

[0435] ETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP

[0436] KKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKD

[0437] LIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFV

[0438] EQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF

[0439] DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSGGSTGNSTNLSDIIEKETGKQLVI

[0440] QESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML

[0441] SGGSGGSGGSTNLSIIEKETGKQLVIQESILMLPEEVEVIGNKPESDILVHTAYDESTDENVMLLT

[0442] SDAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEFEPKKKRKV

[0443] >SEQ ID NO:79 Sdd7-QBEmax

[0444] MKRTADGSFESPKKKRKVKATAKYFFYSNIMNFKTEITLANGEIRKRPLIETNGETGEIVWDKG

[0445] RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYS

[0446] VLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENG

[0447] RKRMLASAGELQKGNELALPSKYVNFLYLASHYEKSGGSSGGSSGSETPGTSESATPESSGGSSGG

[0448] SLEEAVARLIEGGGPGAVPEGGDGPPAVPAEEVERLRGELPPPVVPGTGQKTHGRWIGPDGRVR

[0449] AIVSGRDEDAALVHAQLAAKGIPDEPTRNSDVEQKLAAHMVANGIRHVTLVINHRPCRGFDDSCD

[0450] TLVPIILPEGCTLTVHGQTDKGMRVRVRYTGGARPWWSSGGSSGGSSGSETPGTSESATPESSGGS

[0451] SGGSLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAE

[0452] NIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGSGGSG

[0453] GSGGSGGSGGSGGMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALL

[0454] FDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIF

[0455] GNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLF

[0456] IQLVQTYNQLFEEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFK

[0457] SNFDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSA

[0458] SMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDG

[0459] TEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVG

[0460] PLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF

[0461] TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGV

[0462] EDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMK

[0463] QLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSG

[0464] QGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRER

[0465] MKRIEEGIGELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSF

[0466] LKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSE

[0467] LDKAGFIKRQLVETRQITKHVAQILDSRMNTKYENDKLIREVKVITLSKLVSDFRKDFQFYKVR

[0468] EINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGSGGSGGSGGST

[0469] NLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWA

[0470] LVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVH

[0471] TAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEFEPKKKRKV

[0472] >SEQ ID NO:80 mini-Sdd9-QBEmax

[0473] MKRTADGSFESPKKKRKVKATAKYFFYSNIMNFKTEITLANGEIRKRPLIETNGETGEIVWDKG

[0474] RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYS

[0475] VLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENG

[0476] RKRMLASAGELQKGNELALPSKYVNFLYLASHYEKSGGSSGGSSGSETPGTSESATPESSGGSSGG

[0477] SCPIWVKNALQELVGRKETSGKVFDVDGKPIGPDIQSGYKDRELRRGVYETLRKSPFFQKHFPSTA

[0478] TWYVSLHVEAQYAVWMLRNRIKHATVVINNTYVCSDMNRLHDNCMTAVPHILPEGYTMTVWK

[0479] ADRTEVTLRGKAPKESGGSSGGSSGSETPGTSESATPESSGGSSGGSLKGSPEDNEQKQLFVEQHK

[0480] HYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTI

[0481] DRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGSGGSGGSGGSGGSGGSGGMDKKYSIGL

[0482] AIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRR

[0483] KNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKL

[0484] VDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDA

[0485] KAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDL

[0486] DNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ

[0487] QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDN

[0488] GSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITP

[0489] WNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAF

[0490] LSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDF

[0491] LDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD

[0492] KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQ

[0493] TVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEGIKELGSQILKEHPVENT

[0494] QLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSD

[0495] NVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVA

[0496] QILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTAL

[0497] IKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGSGGSGGSGGSTNLSDIIEKETGKQLVIQESILML

[0498] PEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSSGGSGGS

[0499] GGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYK

[0500] PWALVIQDSNGENKIKMLSGGSKRTADGSEFEPKKKRKV

[0501] >SEQ ID NO:81 Sdd2

[0502] APDSLVWFDPLGLIVLQQVPYNDHPLFGAVSEFIQGKSRSDLRGRNVAAVLLDDGTVIVRASEGG

[0503] GNHAERVLMGLSEVDPAKVVAVYTERSPCTGRINCHDLLDSSLGADVPVYYTHEMIRGQEGKTA

[0504] QQIEADRNQFCRGG

[0505] >SEQ ID NO:82 Sdd3

[0506] SASAQLNTYLAAIGNSTTTVEAQPEAAPPPAAAESLDSTPRLPDGGIDFHALAKRLGLLEARPTEQP

[0507] PFDPRRFNPACWQGLKPYDQAGTAEGNLFIAPGKRWNTRPMQASKLEVGPQSDLHPQWRSRKAP

[0508] WHIEGKIAAYMRQKGFTDGCVYLNARPCSGPDGCARNLPDLLPVGSTLHVHARYIDRTGETRFYY

[0509] REYRGTGKALT

[0510] >SEQ ID NO:83 Sdd4

[0511] LDAMDAYLSEIAGGNAPARAGPKAPEPKQPGGSSSPRARDGRIDFRALLERLKAQGVVGLEGRSD

[0512] DPIPDFDPKKQNPACYQGLAPRQKGKPVRGNLFFPDGRRWNDVALESSRGEPAFDLNIIKPEYRSL

[0513] SPARGHLEGNVAAWMRSTFHQEMVLYINESPCRKHGKGCLYTLEHFLPRGYVLHVWSRNDRGE

[0514] WRGNTFRGSGEAFTEGA

[0515] >SEQ ID NO:84 Sdd6

[0516] VETRDKIIAAKSRSDAGLLAFQQATNGSIDSRPAEAIANLQRAKTHLDEAQRLVANSDAAVDNYIN

[0517] AILGGASAATAQPSAVIPASKPSRFKPMRTDPAKADEIRPHVGKDRAVATLWDADGNRVLGLHSA

[0518] DDDGPAATAAWKPPWRDYVRLRRHVEAHAAARMHQDGHKTMVMYINLPPCKYFDGCKLNLED

[0519] ILPKGSTLWMHRVFQNGGTKIYQFNGTGRAYV

[0520] >SEQ ID NO:85 Sdd7

[0521] LEAVRARLIGEGGGPGAVPEGGDGPPAVPAEEVERLRGELPPPVVPGTGQKTHGRWIGPDGRVRA

[0522] IVSGRDEDAALVHAQLAAKGIPDEPTRNSDVEQKLAAHMVANGIRHVTLVINHRPCRGFDDSCDT

[0523] LVPIILPEGCTLTVHGQTDKGMRVRVRYTGGARPWWS

[0524] >SEQ ID NO:86 Sdd9

[0525] LDVDDPHTFYVLAGKTPVLVHNSECPIWVKNALQELVGRKETSGKVFDVDGKPIGPDIQSGYKDR

[0526] ELRRGVYETLRKSPFFQKHFPSTATWYVSLHVEAQYAVWMLRNRIKHATVVINNTYVCSDMNRL

[0527] HDNCMTAVPHILPEGYTMTVWKADRTEVTLRGKAPKE

[0528] >SEQ ID NO:87 Sdd10

[0529] LDAALGAVRRIIAALGTSGAERASPGANGSERVDELAERLPPTVVPNTSAKTHGWWFTGQGAAQE

[0530] LISGEGPDARAAYEALREEGYPRPGMPFVAMHVEIKLAAHMRRNDIEHATVVINNIPCPLVWGCE

[0531] NLIGVVLPEGSSLTVHGSNGYERTFTGGRKPPWPR

[0532] >SEQ ID NO:88 Sdd59

[0533] LLTPPPRPAAPPTTRPKPLVARTGDAYPPGTEWALPLIVQPHPPVGGTVPVEGHVRALRPESQISHV

[0534] FHPGGGHWTEQARARLRVLPGFGWAVNLGHHVELQIAAWMTACGIHHAELVLNRPPCGERYGL

[0535] GCHQALPVLLPRGYRLTVSSTRGGPQPYQHHYEGKA

[0536] >SEQ ID NO:89 UGI

[0537] TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPW

[0538] ALVIQDSNGENKIKML

[0539] >SEQ ID NO:90 Linker

[0540] GGSGGSGGSGGSGGSGGSGG

[0541] >SEQ ID NO:91 Circular permutation sequence

[0542] MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR

[0543] TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY

[0544] PTIYHLRKKLVDSTDKADLRIYLALAMHIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE

[0545] NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ

[0546] LSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD

[0547] LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED

[0548] LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW

[0549] MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY

[0550] VTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYH

[0551] DLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRIEMIEERLKTYAHLFDDKVMKQLKRRRYTGWGR

[0552] LSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA

[0553] GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQ

[0554] ILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTR

[0555] SDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAEGGGLSELDKAGFIKRQLVE

[0556] TRQITKHVAQILDSRMNTKYDENDKLIVEKVITLSKLVSDFRKDFQFYKVREINNYHHAHDAY

[0557] LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFKTEITLAN

[0558] GEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIA

[0559] RKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKG

[0560] YKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPED

[0561] NEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLG

[0562] APAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGSGGSGGSGGSGGSGGS

[0563] GG

[0564] >SEQ ID NO:92 QBE3-N

[0565] KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVK

[0566] KTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVK

[0567] ELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELAL

[0568] PSKIDFLASHYEK

[0569] >SEQ ID NO:93 QBE3-C

[0570] LKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH

[0571] LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGSGGSGGSG

[0572] GSGGSGGSGGMDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDS

[0573] GETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNI

[0574] VDEVAYHEKYPTIYHLRKKLVDSTDKADLRIYLALAMHIKFRGHFLIEGDLNPDNSDVDKLFIQL

[0575] VQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSN

[0576] FDLAEDAKLQLSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASM

[0577] IKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTE <h2 style=";text-align:left;direction:ltr">

[0578] <h2 style=";text-align:left;direction:ltr"> ELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0579] <h2 style=";text-align:left;direction:ltr"> ARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTV<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0580] <h2 style=";text-align:left;direction:ltr"> YNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVED<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0581] <h2 style=";text-align:left;direction:ltr"> RFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQL<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0582] <h2 style=";text-align:left;direction:ltr"> KRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0583] <h2 style=";text-align:left;direction:ltr"> DSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0584] <h2 style=";text-align:left;direction:ltr"> RIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLK<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0585] <h2 style=";text-align:left;direction:ltr"> DDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0586] <h2 style=";text-align:left;direction:ltr"> KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0587] <h2 style=";text-align:left;direction:ltr"> NNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0588] <h2 style=";text-align:left;direction:ltr"> >SEQ ID No.94 QBE3-X2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0589] KATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVK

[0590] KTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVK

[0591] ELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELAL

[0592] PSKYVNFLYLASHYEK

[0593] >SEQ ID No.95QBE3-X3

[0594] LKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIH

[0595] LFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

[0596] >SEQ ID No.96QBE3-X4

[0597] MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR

[0598] TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY

[0599] PTIYHLRKKLVDSTDKADLRLIYLALAMHIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE

[0600] NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ

[0601] LSKDTYDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQD

[0602] LTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNRED

[0603] LLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW

[0604] MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY

[0605] VTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYH

[0606] DLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRIEMIEERLKTYAHLFDDKVMKQLKRRRYTGWGR

[0607] LSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA

[0608] GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIGELGSQ

[0609] ILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTR

[0610] SDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAEGGGLSELDKAGFIKRQLVE

[0611] TRQITKHVAQILDSRMNTKYDENDKLIVEKVITLSKLVSDFRKDFQFYKVREINNYHHAHDAY

[0612] LNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIG

[0613] > SEQ ID NO: 97 sgRNA backbone sequence

[0614] guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugc

[0615] > SEQ ID NO: 98 gRNA backbone sequence

[0616] GUUUGAGAGCUAUGCUGGAAACAGCAUAGCAAGUUCAAAUAAGGCUAGUCCGUUAUCAAC

[0617] UUGAAAAAGUGGCACCGAGUCGGUGC

[0618] > SEQ ID NO: 99 gRNA backbone sequence

[0619] GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUAAUCAACUUUUAAAGAG

[0620] UGAGCACUGAUUCGGUGC

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 SEQID 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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