Artificially manipulated immune cells

Abstract

The present invention relates to compositions for artificially manipulating immune cells. More particularly, the present invention relates to compositions for artificially manipulating immune cells, as well as methods for producing manipulated immune cells using the compositions and their uses, wherein the manipulated immune cells comprise artificially modified immunomodulatory genes and artificial receptors.

Description

[0001] This application is a divisional application of the patent application with application number 201880045774.2, filed on May 8, 2018, and with the invention name “Artificially manipulated immune cells”. Technical Field

[0002] The present invention relates to artificial manipulation or modification of immunomodulatory genes, and more particularly to a gene manipulation composition for artificially manipulating immunomodulatory genes and immune cells comprising the artificially manipulated immunomodulatory genes. Background Art

[0003] Cell therapy agents are drugs that use living cells to induce regeneration to repair damaged or diseased cells / tissues / entities. They are drugs produced by physical, chemical, or biological manipulation (e.g., in vitro culture, proliferation, selection, etc. of autologous, allogeneic, or xenogeneic cells).

[0004] Among them, immunomodulatory cell therapy agents are drugs that achieve the purpose of disease treatment by regulating the immune response in the body using immune cells (such as dendritic cells, natural killer cells, T cells, etc.).

[0005] Currently, immunomodulatory cell therapies are being developed primarily for cancer treatment. Unlike conventional cancer treatments such as surgery, anticancer drugs, and radiation, immunomodulatory cell therapies utilize immune cells directly administered to the patient to activate immune function, achieving therapeutic effects. Immunomodulatory cell therapies are expected to play a significant role in the emerging biology of the future.

[0006] The physical and chemical properties of the antigens introduced into cells vary depending on the type of immunomodulatory cell therapy. When foreign genes are introduced into immune cells in the form of viral vectors, these cells can have the characteristics of both cell therapy and gene therapy.

[0007] The administration of immunomodulatory cell therapy can be implemented as follows: by activating a variety of immune cells (such as peripheral blood mononuclear cells (PBMCs), T cells, NK cells, etc. isolated from the patient by apheresis) using a variety of antibodies and cytokines, followed by ex vivo proliferation and reinjection into the patient; or by reinjecting immune cells into which genes (such as T cell receptors (TCRs) or chimeric antigen receptors (CARs)) are introduced.

[0008] Adoptive immunotherapy involves the delivery of autologous antigen-specific immune cells (e.g., T cells) generated ex vivo and may be a promising strategy for treating a variety of immune diseases as well as cancer.

[0009] Recently, it has been reported that immune cell therapeutics can be used in a variety of ways, such as as autoimmune suppressors and exhibiting anti-cancer properties. Therefore, immune cell therapeutics can be used for a variety of indications by modulating immune responses. Consequently, there is a significant need to develop and improve the therapeutic efficacy of manipulated immune cells for adoptive immunotherapy. Summary of the Invention

[0010] Technical issues

[0011] As an exemplary embodiment, the present invention provides a composition for manipulating immune cells, wherein the composition is used for artificially manipulating immune cells.

[0012] As an exemplary embodiment, the present invention provides a manipulated immune cell comprising at least one artificially modified immunomodulatory gene and at least one artificial receptor.

[0013] As an exemplary embodiment, the present invention provides a method for producing an artificial immune cell comprising at least one artificially modified immunomodulatory gene and at least one artificial receptor.

[0014] As an exemplary embodiment, the present invention provides a method for treating an immune disease, the method comprising an artificial immune cell comprising at least one artificially modified immunomodulatory gene and at least one artificial receptor as active ingredients.

[0015] Technical Solution

[0016] To address these issues, the present invention relates to a composition for manipulating immune cells. More specifically, the present invention relates to a composition for manipulating immune cells, the composition being used to artificially manipulate immune cells; and manipulated immune cells comprising artificially modified immunomodulatory genes and artificial receptors produced using the composition, as well as uses thereof.

[0017] The present invention provides compositions for manipulating immune cells for specific purposes.

[0018] The term "composition for manipulating immune cells" refers to one or more substances selected from DNA, RNA, nucleic acids, proteins, viruses, chemical compounds, etc., which are used to artificially manipulate or modify immune cells.

[0019] In certain embodiments, a composition for manipulating immune cells may comprise:

[0020] A guide nucleic acid capable of forming a complementary bond with a target sequence in a nucleic acid sequence of at least one immunomodulatory gene selected from the group consisting of: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene, and KDM6A gene; and

[0021] Artificial receptors, wherein the artificial receptors are artificially prepared receptors and are not wild-type receptors.

[0022] The term "immunomodulatory gene" is intended to include any gene that is directly expected to or indirectly affects the formation and performance of an immune function or response. In the present invention, immunomodulatory genes include any gene that is directly expected to or indirectly affects the functional regulation of immune cells, and also directly expected to or indirectly affects the functional regulation of cells (e.g., phagocytes) that interact with immune cells. Here, immunomodulatory genes can implement functions related to the formation and performance of an immune function or response in the form of the immunomodulatory gene itself or a protein expressed by the immunomodulatory gene.

[0023] The term "artificial receptor" refers to an artificially prepared functional entity that is not a wild-type receptor and has a specific ability to recognize an antigen and perform a specific function.

[0024] The composition for manipulating immune cells may optionally further comprise at least one editing protein selected from the group consisting of a Cas9 protein derived from Streptococcus pyogenes, a Cas9 protein derived from Campylobacter jejuni, a Cas9 protein derived from Streptococcus thermophilus, a Cas9 protein derived from Staphylococcus aureus, a Cas9 protein derived from Neisseria meningitidis, and a Cpf1 protein.

[0025] The target sequence can be a continuous 10 bp-25 bp nucleotide sequence located in the promoter region of an immunomodulatory gene.

[0026] The target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the intron region of the immunomodulatory gene.

[0027] The target sequence can be a continuous 10 bp-25 bp nucleotide sequence located in the exon region of the immunomodulatory gene.

[0028] The target sequence can be a continuous 10 bp-25 bp nucleotide sequence located in the enhancer region of the immunomodulatory gene.

[0029] The target sequence may be a continuous 10 bp to 25 bp nucleotide sequence located in the 3'-UTR (untranslated region) or 5'-UTR of an immunomodulatory gene.

[0030] The target sequence may be a continuous 10 bp to 25 bp nucleotide sequence adjacent to the 5' end and / or 3' end of a PAM (protospacer adjacent motif) sequence in the nucleic acid sequence of the immunomodulatory gene.

[0031] Here, the PAM sequence may be at least one sequence selected from the following sequences:

[0032] 5'-NGG-3' (N is A, T, C, or G);

[0033] 5'-NNNNRYAC-3' (N is independently A, T, C or G; R is A or G; Y is C or T);

[0034] 5'-NNAGAAW-3' (N is independently A, T, C or G; W is A or T);

[0035] 5'-NNNNGATT-3' (each N is independently A, T, C or G);

[0036] 5'-NNGRR(T)-3' (each N is independently A, T, C or G; R is A or G; Y is C or T); and

[0037] 5'-TTN-3' (N is A, T, C or G).

[0038] In certain embodiments, the target sequence may be one or more selected from SEQ ID NO: 1-SEQ ID NO: 289.

[0039] The guide nucleic acid may comprise a guide domain capable of forming complementary binding with a target sequence on an immune modulatory gene, wherein the complementary binding may comprise 0-5 mismatches.

[0040] Here, the guide domain may comprise a nucleotide sequence complementary to the target sequence on the immunomodulatory gene, wherein the complementary nucleotide sequence may contain 0-5 mismatches.

[0041] The guide nucleic acid may comprise at least one domain selected from the group consisting of: a first complementary domain, a linker domain, a second complementary domain, a proximal domain, and a tail domain.

[0042] The artificial receptor can have binding specificity for at least one antigen.

[0043] Here, the at least one antigen may be an antigen specifically expressed by cancer cells and / or viruses.

[0044] Here, at least one antigen may be a tumor-associated antigen.

[0045] Here, the at least one antigen may be one or more selected from the group consisting of: A33, ALK, alpha-fetoprotein (AFP), adrenergic receptor β3 (ADRB3), α-folate receptor, AD034, AKT1, BCMA, β-human chorionic gonadotropin, B7H3 (CD276), BST2, BRAP, CD5, CD13, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD40, CD44v6, CD52, CD72, CD79a, C D79b, CD89, CD97, CD123, CD138, CD160, CD171, CD179a, carbonic anhydrase IX (CAIX), CA-125, carcinoembryonic antigen (CEA), CCR4, C-type lectin-like molecule (CLL-1 or CLECL1), claudin6 (CLDN6), CXORF61, CAGE, CDX2, CLP, CT-7, CT8 / HOM-TES-85, cTAGE-1, ERBB2, epidermal growth factor receptor (EGFR), EGFR Type III variant (EGFRvIII), epithelial cell adhesion molecule (EPCAM), E74-like factor 2 mutant (ELF2M), ephrin type A receptor 2 (EphA2), EMR2, Fms-like tyrosine kinase 3 (FLT3), FCRL5, Fibulin-1, G250, GD2, glycoprotein 36 (gp36), glycoprotein 100 (gp100), glucocorticoid-induced tumor necrosis factor receptor (GITR), GPRC5D, GloboH, G protein-coupled receptor 20 (GPR20), GPC3, hsp70-2, human high molecular weight melanoma-associated antigen (HMWMAA), hepatitis A virus cellular receptor 1 (HAVCR1), human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), HAGE, HCA587 / MAGE-C2, hCAP-G, HCE661, HER2 / neu, HLA-Cw, HOM-HD-21 / Galectin-9, HOM-MEEL-40 / SSX2, and HOM-RCC-3.1.3 / CAXII, HOXA7, HOXB6, Hu, HUB 1, insulin growth factor (IGF1)-I, IGF-II, IGF1 receptor, interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), interleukin-11 receptor alpha (IL-11Ra), IGLL1, KIT (CD117), KM-HN-3, KM-KN-1, KOC1, KOC2, KOC3, KOC3, LAGA-1a, LAGE-1, LAIR1, LILRA2, LY75, Lewis Y antigen, MUC1, MN-CA IX, M-CSF, MAGE-1, MAGE-4a, mesothelin, MAGE-A1, MAD-CT-1, MAD-CT-2, MART1, MPP1 1. MSLN, neural cell adhesion molecule (NCAM), NY-ESO-1, NY-ESO-5, Nkp30, NKG2D, NY-BR-1, NY-BR-62, NY-BR-85, NY-CO-37, NY-CO-38, NNP-1, NY-LU-12, NY-REN-10, NY-REN-19 / LKB / STK1 1. NY-REN-21, NY-REN-26 / BCR, NY-REN-3 / NY-CO-38, NY-REN-33 / SNC6, NY-REN-43, NY-REN-65, NY-REN-9, NY-SAR-35, o-acetyl-GD2 ganglioside (OAcGD2), OGFr, PSMA, prostatic acid phosphatase (PAP), p53, prostate cancer tumor antigen 1 (PCTA-1), prostate stem cell antigen (PSCA), serine protease 21 (testisin or PRSS21), platelet-derived growth factor receptor β (PDGFR-β), PLAC1, pan-linked protein 3 (PANX3), PLU-1, ROR-1, RAGE-1, RU1, RU2, Rab38, RBPJκ, RHAMM, stage-specific embryonic antigen 4 (SSEA-4), SCP1, SSX3, SSX4, SSX5, Tyrp-1, TAG72, thyroglobulin, human telomerase reverse transcriptase (hTERT), 5T4, tumor-associated glycoprotein (TAG72), tyrosinase, transglutaminase 5 (TGS5), TEM1, TEM7R, thyroid-stimulating hormone receptor (TSHR), Tie 2, TRP-2, TOP2A, TOP2B, uroplakin 2 (UPK2), vimentin, vascular endothelial growth factor receptor 2 (VEGFR2), Wilms tumor protein 1 (WT1), and Lewis (Y) antigen.

[0046] The artificial receptor can be a chimeric antigen receptor (CAR).

[0047] The artificial receptor can be an artificially manipulated or modified T cell receptor (TCR).

[0048] The guide nucleic acid, artificial receptor, and editing protein can be in the form of nucleic acid sequences encoding their respective ones.

[0049] The nucleic acid sequence may be contained in a plasmid or viral vector.

[0050] Here, the viral vector may be one or more selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus or herpes simplex virus.

[0051] The artificial receptor and editing protein can be in the form of mRNA encoding their respective mRNAs.

[0052] The artificial receptors and editing proteins can be in the form of polypeptides or proteins.

[0053] When the composition for manipulating immune cells optionally further comprises an editing protein, the composition may be in the form of a guide nucleic acid-editing protein complex.

[0054] The present invention provides manipulated immune cells for specific purposes.

[0055] "Manipulated immune cells" refer to immune cells that have been artificially manipulated rather than wild-type.

[0056] In certain embodiments, the manipulated immune cells may comprise at least one artificially engineered immunomodulatory gene and / or a product expressed by the artificially engineered immunomodulatory gene, wherein the immunomodulatory gene is selected from the group consisting of the following genes: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and KDM6A gene; and at least one artificial receptor protein and / or a nucleic acid encoding the artificial receptor protein.

[0057] At least one artificially engineered immunomodulatory gene may comprise an artificial modification within the nucleotide sequence of the immunomodulatory gene.

[0058] At least one artificially engineered immunomodulatory gene may comprise a deletion and / or insertion of at least one nucleotide in the target sequence of the immunomodulatory gene or in the 1 bp to 50 bp nucleotide sequence region adjacent to the 5' end and / or 3' end of the target sequence.

[0059] At least one artificially engineered immunomodulatory gene may comprise a deletion and / or insertion of at least one nucleotide in a continuous 1 bp-50 bp nucleotide sequence region adjacent to the 5' end and / or 3' end of the PAM sequence in the nucleic acid sequence of the immunomodulatory gene.

[0060] Here, the deletion of at least one nucleotide may be a continuous deletion of 1 bp to 50 bp, a discontinuous deletion of 1 bp to 50 bp, or a deletion of 1 bp to 50 bp in which continuous and discontinuous forms are mixed.

[0061] Here, the deletion of at least one nucleotide may be a continuous deletion of 2 bp to 50 bp.

[0062] Here, the insertion of at least one nucleotide may be a continuous insertion of 1 bp to 50 bp, a discontinuous insertion of 1 bp to 50 bp, or an insertion of 1 bp to 50 bp in which the continuous form and the discontinuous form are mixed.

[0063] Here, the insertion of at least one nucleotide may be the insertion of a continuous 5 bp to 1000 bp nucleotide fragment.

[0064] Here, the insertion of at least one nucleotide may be the insertion of a portion or the entire nucleotide sequence of a specific gene.

[0065] The specific gene may be an exogenous gene introduced from an external region that is not contained in immune cells containing immunomodulatory genes.

[0066] The specific gene may be an endogenous gene present in the genome of an immune cell comprising an immunomodulatory gene.

[0067] Here, deletion and insertion of at least one nucleotide may occur in the same nucleotide sequence region.

[0068] Here, deletion and insertion of at least one nucleotide may occur in different nucleotide sequence regions.

[0069] The at least one product expressed by the artificially engineered immunomodulatory gene may be in the form of mRNA and / or protein.

[0070] The product expressed by the artificially engineered immunomodulatory gene may have a reduced or suppressed expression level compared to the amount of the product expressed by the immunomodulatory gene in wild-type immune cells that have not been artificially manipulated.

[0071] Here, the wild-type immune cells that have not been artificially manipulated may be immune cells isolated from humans.

[0072] Here, the wild-type immune cells that have not been artificially manipulated may be immune cells before artificial manipulation.

[0073] The nucleic acid encoding the artificial receptor protein is present in the cell but may not be inserted into the genome of the manipulated immune cell.

[0074] Nucleic acids encoding artificial receptor proteins can be inserted into the 3'-UTR, 5'-UTR, introns, exons, promoters and / or enhancer regions of immune modulatory genes in the genome of manipulated immune cells.

[0075] The nucleic acid encoding the artificial receptor protein can be inserted into at least one intron selected from introns present in the genome of the manipulated immune cell.

[0076] The nucleic acid encoding the artificial receptor protein can be inserted into at least one exon selected from the exons present in the genome of the manipulated immune cell.

[0077] The nucleic acid encoding the artificial receptor protein can be inserted into at least one promoter selected from promoters present in the genome of the manipulated immune cell.

[0078] The nucleic acid encoding the artificial receptor protein can be inserted into at least one enhancer selected from enhancers present in the genome of the manipulated immune cell.

[0079] The nucleic acid encoding the artificial receptor protein can be inserted into one or more regions other than introns, exons, promoters, and enhancers present in the genome of the manipulated immune cell.

[0080] The manipulated immune cells may be artificially manipulated immune cells selected from the group consisting of dendritic cells, T cells, NK cells, NKT cells, and CIK cells.

[0081] The present invention provides manipulated immune cells for specific purposes, wherein the immune cells display at least one characteristic.

[0082] In certain embodiments, the at least one characteristic may be one or more selected from the group consisting of:

[0083] increased production and / or secretion of cytokines;

[0084] Cell proliferation, and

[0085] Increased cytotoxicity.

[0086] Here, the cytokine may be one or more selected from the group consisting of IL-2, TNFα, and IFN-γ.

[0087] The explanation regarding the manipulated immune cells is as described above.

[0088] The present invention provides methods for producing manipulated immune cells for specific purposes.

[0089] In certain embodiments, a method for producing manipulated immune cells may comprise contacting:

[0090] (a) Immune cells;

[0091] (b) an artificial receptor protein or a composition for expressing an artificial receptor protein; and

[0092] (c) A composition for gene manipulation, wherein the composition is capable of artificially manipulating at least one immunomodulatory gene selected from the group consisting of: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d, TET2 gene, PSGL-1 gene, A20 gene and KDM6A gene.

[0093] (a) The immune cells may be immune cells isolated from the human body or immune cells differentiated from stem cells.

[0094] (b) The composition for expressing the artificial receptor protein may comprise a nucleic acid sequence encoding the artificial receptor protein.

[0095] (c) The composition for genetic manipulation may comprise:

[0096] A guide nucleic acid or a nucleic acid encoding the guide nucleic acid, wherein the guide nucleic acid has homology with or is capable of forming a complementary binding with a target sequence SEQ ID NO: 1 to SEQ ID NO: 289 in the nucleic acid sequence of at least one immunomodulatory gene selected from the group consisting of: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene, and KDM6A gene; and

[0097] At least one editing protein or a nucleic acid encoding the editing protein, wherein the at least one editing protein is selected from the group consisting of the following editing proteins: Cas9 protein derived from Streptococcus pyogenes, Cas9 protein derived from Campylobacter jejuni, Cas9 protein derived from Streptococcus thermophilus, Cas9 protein derived from Staphylococcus aureus, Cas9 protein derived from Neisseria meningitidis, and Cpf1 protein.

[0098] Here, the guide nucleic acid and the editing protein may each be in the form of a nucleic acid sequence in at least one vector, or may be in the form of a guide nucleic acid-editing protein complex in which the guide nucleic acid and the editing protein are bound.

[0099] The contacting can be performed ex vivo.

[0100] The contacting may be sequential or simultaneous contacting of (a) the immune cell with (b) the composition for expressing the artificial receptor protein and (c) the composition for gene manipulation.

[0101] The contacting can be performed by at least one method selected from the group consisting of electroporation, liposome, plasmid, viral vector, nanoparticle, and protein translocation domain (PTD) fusion protein.

[0102] The present invention provides methods for treating immune diseases using manipulated immune cells for specific purposes.

[0103] In certain embodiments, a method for treating an immune disease comprises administering to a subject a pharmaceutical composition comprising manipulated immune cells as an active ingredient.

[0104] The explanation regarding the manipulated immune cells is as described above.

[0105] The pharmaceutical composition may further comprise additional components.

[0106] Here, the additional component may be an immune checkpoint inhibitor.

[0107] Immune checkpoint inhibitors can be inhibitors of PD-1, PD-L1, LAG-3, TIM-3, CTLA-4, TIGIT, BTLA, IDO, VISTA, ICOS, KIR, CD160, CD244, or CD39.

[0108] Here, the additional component may be an antigen binding agent, a cytokine, a cytokine secretagogue, or a cytokine inhibitor.

[0109] Here, the additional component may be a suitable vehicle for delivering the manipulated immune cells into the body.

[0110] The manipulated immune cells contained in the pharmaceutical composition can be autologous cells of the subject, or allogeneic cells.

[0111] The immune disease may be an autoimmune disease.

[0112] Here, the autoimmune disease may be graft-versus-host disease (GVHD), systemic lupus erythematosus, celiac disease, type 1 diabetes, Graves' disease, inflammatory bowel disease, psoriasis, rheumatoid arthritis, or multiple sclerosis.

[0113] The disease may be a difficult-to-treat disease in which the causative agent is known but the treatment is unknown.

[0114] Here, the intractable disease may be a viral infection disease, a disease caused by a prion pathogen, or cancer.

[0115] Administration of the pharmaceutical composition to a subject suffering from an immune disease can be performed by a method selected from injection, infusion, implantation or transplantation.

[0116] The subjects are mammals, including humans, monkeys, mice and rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0117] Figures 1 to 27 Examples of artificially modified or manipulated target genes are illustrated.

[0118] Figure 28 Figure 2 is a graph showing the expression levels of CAR in 139CAR-T cells treated with CRISPR / Cas9.

[0119] Figure 29 is a graph showing T cell growth after electroporation of the CRISPR / Cas9 complex.

[0120] Figure 30 Shown is selective knockout of DGK by CRISPR / Cas9 in T cells, with graphs confirming DGK indels (%) (A) and DGK protein expression (B).

[0121] Figure 31 Figure 2 is a graph illustrating off-target sites of gRNAs for each DGK identified using Digenome-Seq.

[0122] Figure 32 is a graph showing the cytotoxic effect of 139CAR-T cells in which AAVS1 was knocked out by CRISPR / Cas9.

[0123] Figure 33 The figure compares the cytotoxic effect (A) and cytokine secretion level (B) of 139CAR-T cells, wherein the 139CAR-T cells are AAVS1 knockout 139CAR-T cells or DGK knockout 139CAR-T cells generated by CRISPR / Cas9.

[0124] Figure 34 Graph comparing the PDL-1 expression level of U87vIII (A) and the PD-1 expression level of T cells (B) after co-culture of U87 cells or U87vIII cells with 139CAR-T cells.

[0125] Figure 35 Graphs showing changes in calcium influx (A) and pERK protein expression (B) in DGK-knockout 139CAR-T cells.

[0126] Figure 36The graphs compare the cytotoxic effects and cytokine secretion levels of 139CAR-T cells with AAVS1 knockout or 139CAR-T cells with DGK knockout in the presence of immunosuppressive factors, showing the presence of TGF-β (A) and PEG2 (B).

[0127] Figure 37 Graph showing the effector function of AAVS1-knockout 139CAR-T cells or DGK-knockout 139CAR-T cells in the presence of immunosuppressive factors.

[0128] Figure 38 Graphs showing the effector function of AAVS1-knockout c259 TCR T cells or DGK-knockout c259 TCR T cells in the presence of immunosuppressive factors.

[0129] Figure 39 Described is the experimental design used to identify the effector activity of DGK knockout T cells under repeated antigen exposure.

[0130] Figure 40 Illustrated are cell proliferation of DGK knockout T cells upon repeated antigen exposure, where the graphs compare (A) the number of surviving cells and (B) the number of proliferating cells (%).

[0131] Figure 41 The Fas-mediated activity inducing cell death in DGK knockout T cells is illustrated, wherein the graphs show (A) activation-induced cell death (AICD, %) and (B) Fas expression level (%).

[0132] Figure 42 Graph showing cytokine secretion levels of AAVS1-knockout 139CAR-T cells or DGK-knockout 139CAR-T cells, followed by repeated tumor inoculation.

[0133] Figure 43 Graphs showing the accumulation of (A) naive T cells and (B) effector memory T cells in AAVS1-knockout 139CAR-T cells or DGK-knockout 139CAR-T cells.

[0134] Figure 44 Graphs showing the expression levels of (A) effector memory regulatory factors, (B) type 1 cytokines, and (C) type 2 cytokines in AAVS1-knockout 139CAR-T cells or DGK-knockout 139CAR-T cells.

[0135] Figure 45Graphs showing the expression levels of markers associated with T cell exhaustion in AAVS1-knockout 139CAR-T cells or DGK-knockout 139CAR-T cells.

[0136] Figure 46 The antitumor effects of AAVS1-knockout 139CAR-T cells or DGK-knockout 139CAR-T cells are illustrated, with graphs showing the antitumor effects upon intravenous injection (A) and the antitumor effects upon intratumoral injection (B), and images comparing tumor sizes in each case (C).

[0137] Figure 47 Shown are graphs comparing the maintenance status of AAVS1 139CAR-T cells, αKO 139CAR-T cells, ζKO 139CAR-T cells, and dKO 139CAR-T cells injected in vivo (A, B) and the tumor size in each case (C); and a chart illustrating the number of tumor-infiltrating T cells in each case (D).

[0138] Figure 48 Graphs comparing IFN-γ, TNFα-positive cells (%) (A), Ki-67-positive cells (%) (B), and T-bet-positive cells (%) (C) of AAVS1 139CAR-T cells, αKO 139CAR-T cells, ζKO 139CAR-T cells, and dKO 139CAR-T cells injected in vivo. DETAILED DESCRIPTION

[0139] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention pertains. Although methods and materials similar or identical to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0140] One aspect disclosed herein relates to guide nucleic acids.

[0141] The term "guide nucleic acid" refers to a nucleotide sequence that can recognize a target nucleic acid, target gene, or target chromosome and interact with an editing protein. A guide nucleic acid can complementarily bind to a portion of the nucleotide sequence in the target nucleic acid, target gene, or target chromosome. Furthermore, a portion of the nucleotide sequence in the guide nucleic acid can interact with a portion of the amino acids in the editing protein, forming a guide nucleic acid-editing protein complex.

[0142] The guide nucleic acid can play a role in inducing the guide nucleic acid-editing protein complex to localize to the target region of the target nucleic acid, target gene or target chromosome.

[0143] The guide nucleic acid can be in the form of DNA, RNA, or a DNA / RNA mixture and have a sequence of 5-150 nucleic acids.

[0144] The guide nucleic acid can be a continuous nucleic acid sequence.

[0145] For example, the continuous nucleic acid sequence may be (N)m, wherein N is A, T, C or G, or is A, U, C or G; and m is an integer of 1-150.

[0146] The guide nucleic acid can be two or more consecutive nucleic acid sequences.

[0147] For example, the two or more consecutive nucleic acid sequences may be (N)m and (N)o, wherein N represents A, T, C or G, or represents A, U, C or G; m and o are integers of 1-150, and may be the same or different from each other.

[0148] The guide nucleic acid comprises one or more domains.

[0149] The domain may be a functional domain, such as a leader domain, a first complementary domain, a linker domain, a second complementary domain, a proximal domain or a tail domain, but is not limited thereto.

[0150] Here, a guide nucleic acid may have two or more functional domains. In addition, the two or more functional domains may be different from each other. Alternatively, the two or more functional domains contained in the guide nucleic acid may be identical to each other. For example, a guide nucleic acid may have two or more proximal domains, and in another example, a guide nucleic acid may have two or more tail domains. However, the fact that the functional domains contained in the guide nucleic acid are two identical domains does not mean that the two functional domains have the same sequence; although the sequences of the domains are different, as long as they perform the same function, they can be considered to be the same.

[0151] Details regarding the functional domains are described below.

[0152] i) Guide domain

[0153] The term "guide domain" refers to a domain that has a complementary guide sequence capable of forming a complementary bond with a portion of a target gene or nucleic acid, and functions to specifically interact with the target gene or nucleic acid. For example, a guide domain can be used to direct the guide nucleic acid-editing protein complex to a location with a specific nucleotide sequence in the target gene or nucleic acid.

[0154] The guide domain can be a nucleotide sequence of 10 bp to 35 bp.

[0155] In one example, the guide domain may be a nucleotide sequence of 10 bp-35 bp, 15 bp-35 bp, 20 bp-35 bp, 25 bp-35 bp, or 30 bp-35 bp.

[0156] In another example, the guide domain may be a nucleotide sequence of 10 bp-15 bp, 15 bp-20 bp, 20 bp-25 bp, 25 bp-30 bp, or 30 bp-35 bp.

[0157] The leader domain may comprise a leader sequence.

[0158] The term "guide sequence" is a nucleotide sequence that is complementary to a portion of a sequence in one strand of the double-stranded chain of a target gene or nucleic acid, wherein the guide sequence can be a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.

[0159] The guide sequence can be a nucleotide sequence of 10 bp to 25 bp.

[0160] In one example, the guide sequence may be a nucleotide sequence of 10 bp-25 bp, 15 bp-25 bp, or 20 bp-25 bp.

[0161] In another example, the guide sequence may be a nucleotide sequence of 10 bp-15 bp, 15 bp-20 bp, or 20 bp-25 bp.

[0162] Furthermore, the leader domain may have additional nucleotide sequences.

[0163] The additional nucleotide sequence may be a sequence that promotes or inhibits the function of the leader domain.

[0164] The additional nucleotide sequence may be a sequence that promotes or inhibits the function of the leader sequence.

[0165] The additional nucleotide sequence may be a nucleotide sequence of 1 bp to 10 bp.

[0166] In one example, the additional nucleotide sequence may be a nucleotide sequence of 2 bp-10 bp, 4 bp-10 bp, 6 bp-10 bp, or 8 bp-10 bp.

[0167] In another example, the additional nucleotide sequence may be a nucleotide sequence of 1 bp-3 bp, 3 bp-6 bp, or 7 bp-10 bp.

[0168] In an embodiment, the additional nucleotide sequence may be a nucleotide sequence of 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp or 10 bp.

[0169] For example, the additional nucleotide sequence may be a 1-base nucleotide sequence G (guanine) or a 2-base nucleotide sequence GG.

[0170] Additional nucleotide sequences may be located 5' to the leader sequence.

[0171] Additional nucleotide sequences may be located 3' to the leader sequence.

[0172] ii) First complementary domain

[0173] The term "first complementary domain" is a domain comprising a nucleotide sequence complementary to a second complementary domain, as described below, which has sufficient complementarity to form a double chain with the second complementary domain. For example, the first complementary domain can be a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity with the second complementary domain.

[0174] The first complementary domain can form a double chain with the second complementary domain through complementary binding. The double chain can be used to interact with some amino acids in the editing protein, thereby forming a guide nucleic acid-editing protein complex.

[0175] The first complementary domain may be a sequence of 5-35 nucleotides.

[0176] In one example, the first complementarity domain can be a sequence of 5-35 nucleotides, 10-35 nucleotides, 15-35 nucleotides, 20-35 nucleotides, 25-35 nucleotides, or 30-35 nucleotides.

[0177] In another example, the first complementarity domain can be a sequence of 1-5 nucleotides, 5-10 nucleotides, 10-15 nucleotides, 15-20 nucleotides, 20-25 nucleotides, 25-30 nucleotides, or 30-35 nucleotides.

[0178] iii) Linker domain

[0179] The term "linker domain" is a nucleic acid sequence that connects two or more domains (two or more identical or different domains). A linker domain can be connected to two or more domains by means of a covalent bond or a non-covalent bond, or can be connected to two or more domains by means of a covalent bond or a non-covalent bond.

[0180] The linker domain may be a sequence of 1-30 nucleotides.

[0181] In one example, the linker domain can be a sequence of 1-5 nucleotides, 5-10 nucleotides, 10-15 nucleotides, 15-20 nucleotides, 20-25 nucleotides, or 25-30 nucleotides.

[0182] In another example, the linker domain can be a sequence of 1-30 nucleotides, 5-30 nucleotides, 10-30 nucleotides, 15-30 nucleotides, 20-30 nucleotides, or 25-30 nucleotides.

[0183] iv) Second complementary domain

[0184] The term "second complementary domain" refers to a domain comprising a nucleotide sequence that comprises a nucleic acid sequence complementary to the first complementary domain described above and that has sufficient complementarity to form a double strand with the first complementary domain. For example, the second complementary domain can be a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity with the first complementary domain.

[0185] The second complementary domain can form a double chain with the first complementary domain through complementary binding. The formed double chain can be used to interact with some amino acids in the editing protein, thereby forming a guide nucleic acid-editing protein complex.

[0186] The second complementary domain may have a nucleotide sequence complementary to the first complementary domain and a nucleotide sequence that has no complementarity with the first complementary domain (e.g., a nucleotide sequence that does not form a double strand with the first complementary domain), and may have a longer nucleotide sequence than the first complementary domain.

[0187] The second complementarity domain may have a sequence of 5-35 nucleotides.

[0188] In examples, the second complementarity domain can be a sequence of 1-35 nucleotides, 5-35 nucleotides, 10-35 nucleotides, 15-35 nucleotides, 20-35 nucleotides, 25-35 nucleotides, or 30-35 nucleotides.

[0189] In another example, the second complementarity domain can be a sequence of 1-5 nucleotides, 5-10 nucleotides, 10-15 nucleotides, 15-20 nucleotides, 20-25 nucleotides, 25-30 nucleotides, or 30-35 nucleotides.

[0190] v) Proximal domain

[0191] The term "proximal domain" is a nucleotide sequence that is located proximal to the second complementarity domain.

[0192] The proximal domain may have a complementary nucleotide sequence and may form a double strand based on the complementary nucleotide sequence.

[0193] The proximal domain may be a sequence of 1-20 nucleotides.

[0194] In one example, the proximal domain can be a sequence of 1-20 nucleotides, 5-20 nucleotides, 10-20 nucleotides, or 15-20 nucleotides.

[0195] In another example, the proximal domain can be a sequence of 1-20 bases, 5-20 bases, 10-20 bases, or 15-20 bases. The proximal domain can be a sequence of 1-5 nucleotides, 5-10 nucleotides, 10-15 nucleotides, or 15-20 nucleotides.

[0196] vi) Tail domain

[0197] The term "tail domain" is a nucleotide sequence located at one or more of the two ends of a guide nucleic acid.

[0198] The tail domain may have a complementary nucleotide sequence and may form a double strand based on the complementary nucleotide sequence.

[0199] The tail domain may be a sequence of 1-50 nucleotides.

[0200] In examples, the tail domain can be a sequence of 5-50 nucleotides, 10-50 nucleotides, 15-50 nucleotides, 20-50 nucleotides, 25-50 nucleotides, 30-50 nucleotides, 35-50 nucleotides, 40-50 nucleotides, or 45-50 nucleotides.

[0201] In another example, the tail domain can be a sequence of 1-5 nucleotides, 5-10 nucleotides, 10-15 nucleotides, 15-20 nucleotides, 20-25 nucleotides, 25-30 nucleotides, 30-35 nucleotides, 35-40 nucleotides, 40-45 nucleotides, or 45-50 nucleotides.

[0202] Meanwhile, part or all of the nucleic acid sequences contained in the domains (ie, the leader domain, the first complementary domain, the linker domain, the second complementary domain, the proximal domain and the tail domain) may optionally or additionally contain chemical modifications.

[0203] Chemical modifications may be, but are not limited to, methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl 3' phosphorothioate (MS), or 2'-O-methyl 3' thioPACE (MSP).

[0204] The guide nucleic acid comprises one or more domains.

[0205] The guide nucleic acid may comprise a guide domain.

[0206] The guide nucleic acid may comprise a first complementarity domain.

[0207] The guide nucleic acid may comprise a linker domain.

[0208] The guide nucleic acid may comprise a second complementarity domain.

[0209] The guide nucleic acid may comprise a proximal domain.

[0210] The guide nucleic acid may comprise a tail domain.

[0211] Here, 1, 2, 3, 4, 5, 6 or more domains may be present.

[0212] The guide nucleic acid may comprise 1, 2, 3, 4, 5, 6 or more guide domains.

[0213] The guide nucleic acid may comprise 1, 2, 3, 4, 5, 6 or more first complementary domains.

[0214] The guide nucleic acid may comprise 1, 2, 3, 4, 5, 6 or more linker domains.

[0215] The guide nucleic acid may comprise 1, 2, 3, 4, 5, 6 or more second complementary domains.

[0216] The guide nucleic acid may comprise 1, 2, 3, 4, 5, 6 or more proximal domains.

[0217] The guide nucleic acid may comprise 1, 2, 3, 4, 5, 6 or more tail domains.

[0218] Here, in the guide nucleic acid, one type of domain may be repeated.

[0219] A guide nucleic acid may comprise several domains with or without repeats.

[0220] The guide nucleic acid may comprise the same type of domains. Here, the same type of domains may have the same nucleic acid sequence or different nucleic acid sequences.

[0221] The guide nucleic acid may comprise two types of domains. Here, the two different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.

[0222] The guide nucleic acid may comprise three types of domains. Here, the three different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.

[0223] The guide nucleic acid may comprise four types of domains. Here, the four different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.

[0224] The guide nucleic acid may comprise five types of domains. Here, the five different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.

[0225] The guide nucleic acid may comprise six types of domains. Here, the six different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.

[0226] For example, a guide nucleic acid may consist of a [guide domain]-[first complementary domain]-[linker domain]-[second complementary domain]-[linker domain]-[guide domain]-[first complementary domain]-[linker domain]-[second complementary domain]. Here, the two guide domains may contain guide sequences targeting different or the same targets; the two first complementary domains and the two second complementary domains may have the same or different nucleic acid sequences. When the guide domains contain guide sequences targeting different targets, the guide nucleic acid can specifically bind to two different targets; here, this specific binding can occur simultaneously or sequentially. Furthermore, the linker domain can be cleaved by a specific enzyme, and in the presence of the specific enzyme, the guide nucleic acid can be separated into two or three parts.

[0227] As an embodiment of the content disclosed in this specification, the guide nucleic acid can be gRNA.

[0228] gRNA

[0229] The term "gRNA" refers to a nucleic acid that can specifically guide a gRNA-CRISPR enzyme complex (i.e., a CRISPR complex) to a target gene or nucleic acid. In addition, a gRNA is a nucleic acid-specific RNA that can bind to a CRISPR enzyme and guide the CRISPR enzyme to a target gene or nucleic acid.

[0230] gRNA can comprise multiple domains.Based on each domain, interactions can occur within the three-dimensional structure or the chains of the active form of the gRNA or between these chains.

[0231] gRNA can refer to single-stranded gRNA (a single RNA molecule) or double-stranded gRNA (comprising more than one RNA molecule, typically two separate RNA molecules).

[0232] In an exemplary embodiment, the single-stranded gRNA may comprise, from 5' to 3' direction, a guide domain (i.e., a domain comprising a guide sequence capable of forming complementary binding with a target gene or nucleic acid), a first complementary domain, a linker domain, a second complementary domain (which has a sequence complementary to the first complementary domain sequence and thus forms a double-stranded nucleic acid with the first complementary domain), a proximal domain, and an optional tail domain.

[0233] In another embodiment, the double-stranded gRNA may comprise a first chain and a second chain, wherein the first chain comprises a guide domain (i.e., a domain comprising a guide sequence capable of forming complementary binding with a target gene or nucleic acid) and a first complementary domain; the second chain comprises, from the 5' to the 3' direction, a second complementary domain (the domain having a sequence complementary to the first complementary domain sequence, thereby forming a double-stranded nucleic acid with the first complementary domain), a proximal domain, and an optional tail domain.

[0234] Herein, the first strand may be referred to as crRNA, and the second strand may be referred to as tracrRNA. The crRNA may comprise a guide domain and a first complementary domain; the tracrRNA may comprise a second complementary domain, a proximal domain, and an optional tail domain.

[0235] In another embodiment, the single-stranded gRNA may comprise, from 3' to 5' direction, a guide domain (i.e., a domain comprising a guide sequence capable of forming complementary binding with a target gene or nucleic acid), a first complementary domain, and a second complementary domain (the domain having a sequence complementary to the first complementary domain sequence, thereby forming a double-stranded nucleic acid with the first complementary domain).

[0236] The first complementary domain may be homologous to a naturally occurring first complementary domain or may be derived from a naturally occurring first complementary domain. Furthermore, the first complementary domain may differ in its base sequence depending on the naturally occurring species, may be derived from a first complementary domain contained in a naturally occurring species, or may be partially or completely homologous to a first complementary domain contained in a naturally occurring species.

[0237] In an exemplary embodiment, the first complementarity domain may be partially (ie, at least 50% or more) or completely homologous to the first complementarity domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus, or Neisseria meningitidis, or a first complementarity domain derived therefrom.

[0238] For example, when the first complementary domain is the first complementary domain of Streptococcus pyogenes or a first complementary domain derived therefrom, the first complementary domain may be 5'-GUUUUAGAGCUA-3' or a base sequence having partial (i.e., at least 50% or more) or complete homology to 5'-GUUUUAGAGCUA-3'. Here, the first complementary domain may further comprise (X) n , such that it is 5'-GUUUUAGAGCUA(X) n -3'. X can be selected from the group consisting of bases A, T, U and G; n can represent the number of bases, which is an integer from 5 to 15. Here, (X) n It can be n repeats of the same base, or a mixture of n bases A, T, U and G.

[0239] In another embodiment, when the first complementarity domain is the first complementarity domain of Campylobacter jejuni or a first complementarity domain derived therefrom, the first complementarity domain may be 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3' or 5'-GUUUUAGUCCCUU-3', or a base sequence having partial (i.e., at least 50% or more) or complete homology to 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3' or 5'-GUUUUAGUCCCUU-3'. Here, the first complementarity domain may further comprise (X) n , such that it is 5'-GUUUUAGUCCCUUUUUAAAUUUCUU(X) n -3' or 5'-GUUUUAGUCCCUU(X) n -3'. X can be selected from the group consisting of bases A, T, U and G; n can represent the number of bases, which is an integer from 5 to 15. Here, (X) n It can represent n repeats of the same base, or a mixture of n bases A, T, U, and G.

[0240] In another embodiment, the first complementarity domain may have partial (i.e., at least 50% or more) or complete homology with the first complementarity domain of the following bacteria or the first complementarity domain derived therefrom: Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp. (SC_KO8D17), Leptospira inadai, Lachnospira (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum, or Eubacterium eligens.

[0241] For example, when the first complementary domain is the first complementary domain of Thriparia or the first complementary domain derived therefrom, the first complementary domain may be 5'-UUUGUAGAU-3' or a base sequence having partial (i.e., at least 50% or more) homology to 5'-UUUGUAGAU-3'. Here, the first complementary domain may further comprise (X) n , such that it is 5'-(X) n UUUGUAGAU-3'. X can be selected from the group consisting of bases A, T, U and G; n can represent the number of bases, which is an integer from 1 to 5. Here, (X) n It can represent n repeats of the same base, or a mixture of n bases A, T, U, and G.

[0242] Here, the linker domain may be a nucleotide sequence that connects the first complementary domain to the second complementary domain.

[0243] The linker domain can be linked to the first complementarity domain and the second complementarity domain, respectively, by covalent or non-covalent bonds.

[0244] The linker domain can connect the first complementarity domain and the second complementarity domain through a covalent or non-covalent bond.

[0245] The linker domain is suitable for use in a single-stranded gRNA molecule and can be used to connect to the first and second strands of a double-stranded gRNA via covalent or non-covalent bonds or to connect the first and second strands to produce a single-stranded gRNA.

[0246] The linker domain can be used to connect the crRNA and tracrRNA of a double-stranded gRNA via covalent or non-covalent bonds, or to connect the crRNA and tracrRNA to generate a single-stranded gRNA.

[0247] Here, the second complementary domain may have homology with a naturally occurring second complementary domain or may be derived from a naturally occurring second complementary domain. In addition, the second complementary domain may differ in its base sequence depending on the naturally occurring species, may be derived from a second complementary domain contained in a naturally occurring species, or may have partial or complete homology with a second complementary domain contained in a naturally occurring species.

[0248] In exemplary embodiments, the second complementarity domain may be partially (ie, at least 50% or more) or completely homologous to the second complementarity domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus, or Neisseria meningitidis, or a second complementarity domain derived therefrom.

[0249] For example, when the second complementarity domain is the second complementarity domain of Streptococcus pyogenes or a second complementarity domain derived therefrom, the second complementarity domain may be 5'- UAGC AAGU UAAAA U-3' or with 5'- UAGC AAGU UAAAA U-3' has a base sequence with partial (i.e., at least 50% or more) homology (the base sequence forming a double strand with the first complementary domain is underlined). Here, the second complementary domain may further comprise (X) n and / or (X) m , such that it is 5'-(X) n UAGC AAGU UAAAA U(X) m -3'. X can be selected from the group consisting of bases A, T, U and G; n and m can each represent the number of bases, wherein n can be an integer from 1 to 15, and m can be an integer from 1 to 6. Here, (X) n It can represent n repeats of the same base, or a mixture of n bases A, T, U and G. In addition, (X) m It can represent m repeats of the same base, or a mixture of m bases A, T, U, and G.

[0250] In another example, when the second complementarity domain is the second complementarity domain of Campylobacter jejuni or a second complementarity domain derived therefrom, the second complementarity domain may be 5'- AAGAAAUUUAAAAAGGGACUAAAA U-3' or 5'- AAGGGACUAAAA U-3, or with 5'- AAGAAAUUUAAAAAGGGACUAAAA U-3' or 5'- AAGGGACUAAAA U-3' has a base sequence with partial (i.e., at least 50% or more) homology (the base sequence forming a double strand with the first complementary domain is underlined). Here, the second complementary domain may further comprise (X) n and / or (X) m , such that it is 5'-(X) n AAGAAAUUUAAAAAGGGACUAAAA U(X) m -3' or 5'-(X)n AAGAAAUUUAAAAA U(X)m-3'. X can be selected from the group consisting of bases A, T, U and G; n and m can each represent the number of bases, wherein n can be an integer from 1 to 15, and m can be an integer from 1 to 6. Here, (X) n It can represent n repeats of the same base, or a mixture of n bases A, T, U and G. In addition, (X) m It can represent m repeats of the same base, or a mixture of m bases A, T, U, and G.

[0251] In another embodiment, the second complementarity domain may have partial (i.e., at least 50% or more) or complete homology with the first complementarity domain of the following bacteria or the second complementarity domain derived therefrom: Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp. (SC_KO8D17), Leptospira inadai, Lachnospira (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum, or Eubacterium eligens.

[0252] For example, when the second complementary domain is the second complementary domain of Bacteria or a second complementary domain derived therefrom, the second complementary domain may be 5'-AAAUU UCUAC U-3' or with 5'-AAAUU UCUAC U-3' has a base sequence with partial (i.e., at least 50% or more) homology (the base sequence forming a double strand with the first complementary domain is underlined). Here, the second complementary domain may further comprise (X) n and / or (X) m , making it 5'-(X)nAAAUU UCUAC U(X)m-3'. X can be selected from the group consisting of bases A, T, U and G, and n and m can each represent the number of bases, wherein n can be an integer from 1 to 10, and m can be an integer from 1 to 6. Here, (X) n It can represent n repeats of the same base, or a mixture of n bases A, T, U and G. In addition, (X) m It can represent m repeats of the same base, or a mixture of m bases A, T, U, and G.

[0253] Here, the first complementary domain and the second complementary domain can form a complementary binding.

[0254] The first complementary domain and the second complementary domain can form a double chain through complementary binding.

[0255] The resulting duplex can interact with the CRISPR enzyme.

[0256] Optionally, the first complementary domain may comprise an additional nucleotide sequence that does not form complementary binding with the second complementary domain of the second chain.

[0257] Here, the additional nucleotide sequence may be a nucleotide sequence of 1 bp to 15 bp. For example, the additional nucleotide sequence may be a nucleotide sequence of 1 bp to 5 bp, 5 bp to 10 bp, or 10 bp to 15 bp.

[0258] Here, the proximal domain may be a domain in the 5' to 3' direction of the second complementary domain.

[0259] The proximal domain may have homology with a naturally occurring proximal domain or may be derived from a naturally occurring proximal domain. Furthermore, the proximal domain may differ in base sequence depending on the naturally occurring species, may be derived from a proximal domain contained in a naturally occurring species, or may have partial or complete homology with a proximal domain contained in a naturally occurring species.

[0260] In exemplary embodiments, the proximal domain may be partially (ie, at least 50% or more) or completely homologous to the proximal domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus, or Neisseria meningitidis, or a proximal domain derived therefrom.

[0261] For example, when the proximal domain is the proximal domain of Streptococcus pyogenes or a proximal domain derived therefrom, the proximal domain may be 5'-AAGGCUAGUCCG-3' or a base sequence having partial (i.e., at least 50% or more) homology to 5'-AAGGCUAGUCCG-3'. Here, the proximal domain may further comprise (X) n , making it 5'-AAGGCUAGUCCG(X) n -3'. X can be selected from the group consisting of bases A, T, U and G; n can represent the number of bases, which can be an integer from 1 to 15. Here, (X) n It can represent n repeats of the same base, or a mixture of n bases A, T, U, and G.

[0262] In another embodiment, when the proximal domain is the proximal domain of Campylobacter jejuni or a proximal domain derived therefrom, the proximal domain may be 5'-AAAGAGUUUGC-3' or a base sequence having at least 50% or higher homology to 5'-AAAGAGUUUGC-3'. Here, the proximal domain may further comprise (X) n , making it 5'-AAAGAGUUUGC(X) n -3'. X can be selected from the group consisting of bases A, T, U and G; n can represent the number of bases, which can be an integer from 1 to 40. Here, (X) n It can represent n repeats of the same base, or a mixture of n bases A, T, U, and G.

[0263] Here, a tail domain can be optionally added to the 3' end of the first strand or the 3' end of the second strand of the single-stranded gRNA or double-stranded gRNA.

[0264] In addition, the tail domain may have homology with a natural tail domain, or may be derived from a natural tail domain. In addition, the tail domain may differ in base sequence depending on the naturally occurring species, may be derived from a tail domain contained in a naturally occurring species, or may have partial or complete homology with a tail domain contained in a naturally occurring species.

[0265] In an exemplary embodiment, the tail domain may be partially (ie, at least 50% or more) or completely homologous to the tail domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus, or Neisseria meningitidis, or a tail domain derived therefrom.

[0266] For example, when the tail domain is the tail domain of Streptococcus pyogenes or a tail domain derived therefrom, the tail domain may be 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' or a base sequence having partial (i.e., at least 50% or more) homology to 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3'. Here, the tail domain may further comprise (X) n , making it 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(X) n -3'. X can be selected from the group consisting of bases A, T, U and G; n can represent the number of bases, which can be an integer from 1 to 15. Here, (X) n It can represent n repeats of the same base, or a mixture of n bases (such as A, T, U, and G).

[0267] In another example, when the tail domain is the tail domain of Campylobacter jejuni or a tail domain derived therefrom, the tail domain may be 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3' or a base sequence having partial (i.e., at least 50% or more) homology to 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3'. Here, the tail domain may further comprise (X) n , making it 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU(X) n -3'. X can be selected from the group consisting of bases A, T, U and G; n can represent the number of bases, which can be an integer from 1 to 15. Here, (X) n It can represent n repeats of the same base, or a mixture of n bases A, T, U, and G.

[0268] In another embodiment, the tail domain may comprise a 1-10 base sequence at the 3' end that participates in in vitro or in vivo transcription methods.

[0269] For example, when the T7 promoter is used for in vitro transcription of the gRNA, the tail domain can be any base sequence present at the 3' end of the DNA template. Furthermore, when the U6 promoter is used for in vivo transcription, the tail domain can be UUUUUU; when the H1 promoter is used for transcription, the tail domain can be UUUU; and when the Pol III promoter is used, the tail domain can contain several uracil bases or alternative bases.

[0270] The gRNA may comprise multiple domains as described above, and thus the length of the nucleic acid sequence may be adjusted depending on the domains contained in the gRNA; based on each domain, interactions may occur within or between chains of the three-dimensional structure or active form of the gRNA.

[0271] gRNA can refer to single-stranded gRNA (a single RNA molecule) or double-stranded gRNA (comprising more than one RNA molecule, typically two separate RNA molecules).

[0272] double-stranded gRNA

[0273] Double-stranded gRNA consists of a first strand and a second strand.

[0274] Here, the first chain can be

[0275] 5'-[guide domain]-[first complementary domain]-3'; and

[0276] The second chain can be

[0277] 5'-[second complementary domain]-[proximal domain]-3' or

[0278] 5'-[second complementary domain]-[proximal domain]-[tail domain]-3'.

[0279] Here, the first strand may refer to crRNA, and the second strand may refer to tracrRNA.

[0280] Here, the first strand and the second strand may optionally contain additional nucleotide sequences.

[0281] In one example, the first chain may be

[0282] 5'-(N 靶标 )-(Q) m -3'; or

[0283] 5'-(X) a -(N 靶标 )-(X) b -(Q) m -(X) c -3'.

[0284] Here, N 靶标 It is a nucleotide sequence that is complementary to a partial sequence in one of the double-stranded strands of a target gene or nucleic acid, and is a nucleotide sequence region that can be altered according to the target sequence on the target gene or nucleic acid.

[0285] Here, (Q) m It is a base sequence comprising a first complementary domain, which is capable of forming a complementary bond with the second complementary domain of the second chain. (Q) m The first complementary domain may be a sequence that is partially or completely homologous to a naturally occurring species' first complementary domain. The base sequence of the first complementary domain may be altered depending on the species of origin. Q may be independently selected from the group consisting of A, U, C, and G. m may be the number of bases, which is an integer from 5 to 35.

[0286] For example, when the first complementarity domain has partial or complete homology to the first complementarity domain of Streptococcus pyogenes or a first complementarity domain derived from Streptococcus pyogenes, (Q) m It may be 5'-GUUUUAGAGCUA-3' or a base sequence having at least 50% or higher homology to 5'-GUUUUAGAGCUA-3'.

[0287] In another embodiment, when the first complementarity domain is partially or completely homologous to the first complementarity domain of Campylobacter jejuni or a first complementarity domain derived from Campylobacter jejuni, (Q) mIt may be 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3' or 5'-GUUUUAGUCCCUU-3', or a base sequence having at least 50% or higher homology with 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3' or 5'-GUUUUAGUCCCUU-3'.

[0288] In another example, when the first complementarity domain has partial or complete homology to the first complementarity domain of Streptococcus thermophilus or the first complementarity domain derived from Streptococcus thermophilus, (Q) m It may be 5'-GUUUUAGAGCUGUGUUGUUUCG-3' or a base sequence having at least 50% or higher homology to 5'-GUUUUAGAGCUGUGUUGUUUCG-3'.

[0289] In addition, (X) a 、(X) b 、(X) c Each is an optional additional base sequence, wherein X can be independently selected from the group consisting of A, U, C and G; a, b, c can be a base number, which is 0 or an integer of 1-20.

[0290] In an exemplary embodiment, the second strand may be 5'-(Z) h -(P) k -3'; or 5'-(X) d -(Z) h -(X) e -(P) k -(X) f -3'.

[0291] In another embodiment, the second strand may be 5'-(Z) h -(P) k -(F) i -3'; or 5'-(X) d -(Z) h -(X) e -(P) k -(X) f -(F) i -3'.

[0292] Here, (Z) h It is a base sequence containing a second complementary domain, which can form a complementary bond with the first complementary domain of the first chain. (Z) hThe second complementary domain may be a sequence that is partially or completely homologous to a naturally occurring species' second complementary domain. The base sequence of the second complementary domain may be modified depending on the species of origin. Z may be independently selected from the group consisting of A, U, C, and G. h may be the number of bases, which may be an integer from 5 to 50.

[0293] For example, when the second complementarity domain has partial or complete homology to the second complementarity domain of Streptococcus pyogenes or a second complementarity domain derived from Streptococcus pyogenes, (Z) h It may be 5'-UAGCAAGUUAAAAU-3' or a base sequence having at least 50% or higher homology to 5'-UAGCAAGUUAAAAU-3'.

[0294] In another embodiment, when the second complementarity domain has partial or complete homology to the second complementarity domain of Campylobacter jejuni or a second complementarity domain derived from Campylobacter jejuni, (Z) h It may be 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3' or 5'-AAGGGACUAAAAU-3', or a base sequence having at least 50% or higher homology to 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3' or 5'-AAGGGACUAAAAU-3'.

[0295] In another embodiment, when the second complementarity domain has partial or complete homology to the second complementarity domain of Streptococcus thermophilus or a second complementarity domain derived from Streptococcus thermophilus, (Z) h It may be 5'-CGAAACAACACAGCGAGUUAAAAU-3' or a base sequence having at least 50% or higher homology to 5'-CGAAACAACACAGCGAGUUAAAAU-3'.

[0296] (P) k The base sequence comprising the proximal domain may be partially or completely homologous to the proximal domain of a naturally occurring species. The base sequence of the proximal domain may be modified depending on the species of origin. P may be independently selected from the group consisting of A, U, C, and G. k may be the number of bases, which is an integer from 1 to 20.

[0297] For example, when the proximal domain has partial or complete homology to the proximal domain of Streptococcus pyogenes or a proximal domain derived from Streptococcus pyogenes, (P) k It may be 5'-AAGGCUAGUCCG-3' or a base sequence having at least 50% or higher homology to 5'-AAGGCUAGUCCG-3'.

[0298] In another embodiment, when the proximal domain has partial or complete homology to the proximal domain of Campylobacter jejuni or a proximal domain derived from Campylobacter jejuni, (P) k It may be 5'-AAAGAGUUUGC-3' or a base sequence having at least 50% or higher homology to 5'-AAAGAGUUUGC-3'.

[0299] In another embodiment, when the proximal domain has partial or complete homology to the proximal domain of Streptococcus thermophilus or the proximal domain derived from Streptococcus thermophilus, (P) k It may be 5'-AAGGCUUAGUCCG-3' or a base sequence having at least 50% or higher homology to 5'-AAGGCUUAGUCCG-3'.

[0300] (F) i The base sequence of the tail domain may be partially or completely homologous to the tail domain of a naturally occurring species; the base sequence of the tail domain may be modified depending on the species of origin. F may be independently selected from the group consisting of A, U, C, and G; and i may be a base number, which is an integer from 1 to 50.

[0301] For example, when the tail domain has partial or complete homology to the tail domain of Streptococcus pyogenes or a tail domain derived from Streptococcus pyogenes, (F) i It may be 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' or a base sequence having at least 50% or higher homology to 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3'.

[0302] In another embodiment, when the tail domain has partial or complete homology to the tail domain of Campylobacter jejuni or a tail domain derived from Campylobacter intestinalis, (F) i It may be 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3' or a base sequence having at least 50% or higher homology to 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3'.

[0303] In another embodiment, when the tail domain has partial or complete homology to the tail domain of Streptococcus thermophilus or a tail domain derived from Streptococcus thermophilus, (F) iIt may be 5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3' or a base sequence having at least 50% or higher homology to 5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3'.

[0304] In addition, (F) i A sequence of 1-10 bases that participates in in vitro or in vivo transcription methods may be included at the 3' end.

[0305] For example, when the T7 promoter is used for in vitro transcription of the gRNA, the tail domain can be any base sequence present at the 3' end of the DNA template. Furthermore, when the U6 promoter is used for in vivo transcription, the tail domain can be UUUUUU; when the H1 promoter is used for in vivo transcription, the tail domain can be UUUU; and when the Pol III promoter is used, the tail domain can contain several uracil bases or alternative bases.

[0306] In addition, (X) d 、(X) e and (X) f It can be an optionally added base sequence, wherein X can be independently selected from the group consisting of A, U, C and G; d, e, and f can each be a base number, which is 0 or an integer of 1-20.

[0307] single-stranded gRNA

[0308] Single-stranded gRNA can be divided into first single-stranded gRNA and second single-stranded gRNA.

[0309] First single-stranded gRNA

[0310] The first single-stranded gRNA is a single-stranded gRNA in which the first strand and the second strand of the double-stranded gRNA are connected via a linker domain.

[0311] Specifically, single-stranded gRNA can be

[0312] 5'-[leader domain]-[first complementary domain]-[linker domain]-[second complementary domain]-3',

[0313] 5'-[leader domain]-[first complementary domain]-[linker domain]-[second complementary domain]-[proximal domain]-3'; or

[0314] 5'-[leader domain]-[first complementary domain]-[linker domain]-[second complementary domain]-[proximal domain]-[tail domain]-3'.

[0315] The first single-stranded gRNA may optionally comprise additional nucleotide sequences.

[0316] In an exemplary embodiment, the first single-stranded gRNA may be

[0317] 5'-(N 靶标 )-(Q) m -(L) j -(Z) h -3';

[0318] 5'-(N 靶标 )-(Q) m -(L) j -(Z) h -(P) k -3'; or

[0319] 5'-(N 靶标 )-(Q) m -(L) j -(Z) h -(P) k -(F) i -3'.

[0320] In another exemplary embodiment, the single-stranded gRNA may be

[0321] 5'-(X) a -(N 靶标 )-(X) b -(Q) m -(X) c -(L) j -(X) d -(Z) h -(X) e -3';

[0322] 5'-(X) a -(N 靶标 )-(X) b -(Q) m -(X) c -(L) j -(X) d -(Z) h -(X) e -(P) k -(X) f -3'; or

[0323] 5'-(X) a -(N 靶标 )-(X) b -(Q) m -(X) c -(L)j -(X) d -(Z) h -(X) e -(P) k -(X) f -(F) i -3'.

[0324] Here, N 靶标 It is a base sequence that can form a complementary bond with a target sequence on a target gene or nucleic acid, and is a base sequence region that can be changed according to the target sequence on a target gene or nucleic acid.

[0325] (Q) m Contains a base sequence comprising a first complementary domain, which is capable of forming a complementary bond with a second complementary domain. (Q) m The first complementary domain may be a sequence that is partially or completely homologous to a naturally occurring species' first complementary domain. The base sequence of the first complementary domain may be altered depending on the species of origin. Q may be independently selected from the group consisting of A, U, C, and G. m may be the number of bases, which may be an integer from 5 to 35.

[0326] For example, when the first complementarity domain has partial or complete homology to the first complementarity domain of Streptococcus pyogenes or a first complementarity domain derived from Streptococcus pyogenes, (Q) m It may be 5'-GUUUUAGAGCUA-3' or a base sequence having at least 50% or higher homology to 5'-GUUUUAGAGCUA-3'.

[0327] In another embodiment, when the first complementarity domain is partially or completely homologous to the first complementarity domain of Campylobacter jejuni or a first complementarity domain derived from Campylobacter jejuni, (Q) m It may be 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3' or 5'-GUUUUAGUCCCUU-3', or a base sequence having at least 50% or higher homology with 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3' or 5'-GUUUUAGUCCCUU-3'.

[0328] In another example, when the first complementarity domain has partial or complete homology to the first complementarity domain of Streptococcus thermophilus or the first complementarity domain derived from Streptococcus thermophilus, (Q) m It may be 5'-GUUUUAGAGCUGUGUUGUUUCG-3' or a base sequence having at least 50% or higher homology to 5'-GUUUUAGAGCUGUGUUGUUUCG-3'.

[0329] In addition, (L) j is a base sequence comprising a linker domain that connects the first complementary domain and the second complementary domain, thereby generating a single-stranded gRNA. Here, L can be independently selected from the group consisting of A, U, C, and G; j can be a base number, which is an integer of 1-30.

[0330] (Z) h It is a base sequence comprising a second complementary domain, which is capable of forming a complementary bond with the first complementary domain. (Z) h The second complementary domain may be a sequence that is partially or completely homologous to a naturally occurring species; the base sequence of the second complementary domain may be altered depending on the species of origin. Z may be independently selected from the group consisting of A, U, C, and G; and h is the number of bases, which may be an integer from 5 to 50.

[0331] For example, when the second complementarity domain has partial or complete homology to the second complementarity domain of Streptococcus pyogenes or a second complementarity domain derived from Streptococcus pyogenes, (Z) h It may be 5'-UAGCAAGUUAAAAU-3' or a base sequence having at least 50% or higher homology to 5'-UAGCAAGUUAAAAU-3'.

[0332] In another embodiment, when the second complementarity domain has partial or complete homology to the second complementarity domain of Campylobacter jejuni or a second complementarity domain derived from Campylobacter jejuni, (Z) h It may be 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3' or 5'-AAGGGACUAAAAU-3', or a base sequence having at least 50% or higher homology to 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3' or 5'-AAGGGACUAAAAU-3'.

[0333] In another embodiment, when the second complementarity domain has partial or complete homology to the second complementarity domain of Streptococcus thermophilus or a second complementarity domain derived from Streptococcus thermophilus, (Z) h It may be 5'-CGAAACAACACAGCGAGUUAAAAU-3' or a base sequence having at least 50% or higher homology to 5'-CGAAACAACACAGCGAGUUAAAAU-3'.

[0334] (P) kThe base sequence comprising the proximal domain may be partially or completely homologous to the proximal domain of a naturally occurring species. The base sequence of the proximal domain may be modified depending on the species of origin. P may be independently selected from the group consisting of A, U, C, and G. k may be the number of bases, which is an integer from 1 to 20.

[0335] For example, when the proximal domain has partial or complete homology to the proximal domain of Streptococcus pyogenes or a proximal domain derived from Streptococcus pyogenes, (P) k It may be 5'-AAGGCUAGUCCG-3' or a base sequence having at least 50% or higher homology to 5'-AAGGCUAGUCCG-3'.

[0336] In another embodiment, when the proximal domain has partial or complete homology to the proximal domain of Campylobacter jejuni or a proximal domain derived from Campylobacter jejuni, (P) k It may be 5'-AAAGAGUUUGC-3' or a base sequence having at least 50% or higher homology to 5'-AAAGAGUUUGC-3'.

[0337] In another embodiment, when the proximal domain has partial or complete homology to the proximal domain of Streptococcus thermophilus or the proximal domain derived from Streptococcus thermophilus, (P) k It may be 5'-AAGGCUUAGUCCG-3' or a base sequence having at least 50% or higher homology to 5'-AAGGCUUAGUCCG-3'.

[0338] (F) i The base sequence may include a tail domain, which may be partially or completely homologous to the tail domain of a naturally occurring species; the base sequence of the tail domain may be modified depending on the species of origin. F may be independently selected from the group consisting of A, U, C, and G; and i may be a base number, which is an integer from 1 to 50.

[0339] For example, when the tail domain has partial or complete homology to the tail domain of Streptococcus pyogenes or a tail domain derived from Streptococcus pyogenes, (F) i It may be 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' or a base sequence having at least 50% or higher homology to 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3'.

[0340] In another embodiment, when the tail domain has partial or complete homology to the tail domain of Campylobacter jejuni or a tail domain derived from Campylobacter jejuni, (F)i It may be 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3' or a base sequence having at least 50% or higher homology to 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3'.

[0341] In another embodiment, when the tail domain has partial or complete homology to the tail domain of Streptococcus thermophilus or a tail domain derived from Streptococcus thermophilus, (F) i It may be 5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3' or a base sequence having at least 50% or higher homology to 5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3'.

[0342] In addition, (F) i A sequence of 1-10 bases that participates in in vitro or in vivo transcription methods may be included at the 3' end.

[0343] For example, when the T7 promoter is used for in vitro transcription of the gRNA, the tail domain can be any base sequence present at the 3' end of the DNA template. Furthermore, when the U6 promoter is used for in vivo transcription, the tail domain can be UUUUUU; when the H1 promoter is used for transcription, the tail domain can be UUUU; and when the Pol III promoter is used, the tail domain can contain several uracil bases or alternative bases.

[0344] In addition, (X) a 、(X) b 、(X) c 、(X) d 、(X) e and (X) f It can be an optionally added base sequence, wherein X can be independently selected from the group consisting of A, U, C and G; a, b, c, d, e and f can each be a base number, which is 0 or an integer of 1-20.

[0345] Second single-stranded gRNA

[0346] The second single-stranded gRNA may be a single-stranded gRNA consisting of a guide domain, a first complementary domain, and a second complementary domain.

[0347] Here, the second single-stranded gRNA can be

[0348] 5'-[second complementary domain]-[first complementary domain]-[leader domain]-3'; or

[0349] 5'-[second complementary domain]-[linker domain]-[first complementary domain]-[guide domain]-3'.

[0350] The second single-stranded gRNA may optionally comprise additional nucleotide sequences.

[0351] In an exemplary embodiment, the second single-stranded gRNA may be

[0352] 5'-(Z) h -(Q) m -(N 靶标 )-3'; or

[0353] 5'-(X) a -(Z) h -(X) b -(Q) m -(X) c -(N 靶标 )-3'.

[0354] In another embodiment, the single-stranded gRNA may be

[0355] 5'-(Z) h -(L) j -(Q) m -(N 靶标 )-3'; or

[0356] 5'-(X) a -(Z) h -(L) j -(Q) m -(X) c -(N 靶标 )-3'.

[0357] Here, N 靶标 It is a base sequence that can form a complementary bond with a target sequence on a target gene or nucleic acid, and is a base sequence region that can be changed according to the target sequence on a target gene or nucleic acid.

[0358] (Q) m It is a base sequence comprising a first complementary domain, which is capable of forming a complementary bond with the second complementary domain of the second chain. (Q) m The first complementary domain may be a sequence that is partially or completely homologous to a naturally occurring species' first complementary domain. The base sequence of the first complementary domain may be altered depending on the species of origin. Q may be independently selected from the group consisting of A, U, C, and G. m may be the number of bases, which may be an integer from 5 to 35.

[0359] For example, when the first complementarity domain has partial or complete homology with the first complementarity domain of Bacteria or a first complementarity domain derived therefrom, (Q) m It may be 5'-UUUGUAGAU-3' or a base sequence having at least 50% or higher homology to 5'-UUUGUAGAU-3'.

[0360] (Z) h It is a base sequence containing a second complementary domain, which can form a complementary bond with the first complementary domain of the first chain. (Z) h The second complementary domain may be a sequence that is partially or completely homologous to a naturally occurring species' second complementary domain. The base sequence of the second complementary domain may be modified depending on the species of origin. Z may be independently selected from the group consisting of A, U, C, and G. h may be the number of bases, which is an integer between 5 and 50.

[0361] For example, when the second complementarity domain has partial or complete homology with the second complementarity domain of Threptomyces or a second complementarity domain derived from Threptomyces, (Z) h It may be 5'-AAAUUUCUACU-3' or a base sequence having at least 50% or higher homology to 5'-AAAUUUCUACU-3'.

[0362] In addition, (L) j is a base sequence comprising a linker domain, which connects the first complementary domain and the second complementary domain. Here, L can be independently selected from the group consisting of A, U, C and G; j can be a base number, which is an integer of 1-30.

[0363] In addition, (X) a 、(X) b and (X) c Each is an optional additional base sequence, wherein X can be independently selected from the group consisting of A, U, C and G; a, b and c can be the number of bases, which is 0 or an integer of 1-20.

[0364] As an aspect disclosed in the present invention, the guide nucleic acid may be a gRNA that can form a complementary bond with the target sequence of the immunomodulatory gene.

[0365] The term "immunomodulatory gene" refers to all genes that directly participate in or indirectly affect the regulation of immune function or the regulation of functions related to the formation and performance of an immune response. In the present invention, immunomodulatory genes include all genes that directly participate in or indirectly affect the regulation of the functions of immune cells and phagocytes that can interact with immune cells. In particular, immunomodulatory genes can perform immune functions or functions related to the formation and performance of an immune response due to the immunomodulatory gene itself or the protein expressed by the immunomodulatory gene.

[0366] Can classify immunomodulatory gene according to the function of the protein of immunomodulatory gene expression.The immunomodulatory gene listed below is only the example of the immunomodulatory gene based on function, therefore is not the type of immunomodulatory gene that the present invention is contained in is restricted.The gene listed below may not only have a type of immunomodulatory function, but may have polytype function.In addition, two or more immunomodulatory genes (if needed) can be provided.

[0367] In one example, the immune regulatory gene can be an immune cell activity regulatory gene.

[0368] The term "immune cell activity regulating gene" refers to a gene that regulates the extent or activity of an immune response. For example, it can be a gene that stimulates or suppresses the extent or activity of an immune response. Here, the immune cell activity regulating gene can control the extent or activity of an immune response through the immune cell activity regulating gene or the protein expressed by the immune cell activity regulating gene.

[0369] Immune cell activity regulatory genes can perform functions related to the activation or inactivation of immune cells.

[0370] Immune cell activity regulatory genes can perform functions related to the activation or inactivation of immune cells.

[0371] Immune cell activity regulatory genes can function to suppress immune responses.

[0372] Immune cell activity regulatory genes can bind to channel proteins and receptors on the cell membrane, thereby executing functions related to protein synthesis that regulates immune responses.

[0373] For example, the immune cell activity regulator gene can be programmed cell death protein (PD-1)

[0374] The PD-1 gene (also referred to as the PDCD1 gene; hereinafter, the PD-1 gene and the PDCD1 gene are used to refer to the same gene) refers to a gene (full-length DNA, cDNA, or mRNA) encoding the PD-1 protein (also referred to as cluster of differentiation 279 (CD279)). In an embodiment, the PD-1 gene may be one or more selected from the group consisting of, but not limited to, genes encoding human PD-1 (e.g., NCBI accession number NP_005009.2, etc.), such as PD-1 genes represented by NCBI accession numbers NM_005018.2, NG_012110.1, etc.

[0375] The immune cell activity regulatory gene may be cytotoxic T lymphocyte-associated protein 4 (CTLA-4).

[0376] The CTLA-4 gene refers to a gene (full-length DNA, cDNA, or mRNA) encoding the CTLA-4 protein (also known as cluster of differentiation 152 (CD152)). In an embodiment, the CTLA-4 gene may be one or more selected from the group consisting of, but not limited to, genes encoding human CTLA-4 (e.g., NCBI accession numbers NP_001032720.1, NP_005205.2, etc.), such as CTLA-4 genes represented by NCBI accession numbers NM_001037631.2, NM_005214.4, NG_011502.1, etc.

[0377] The immune cell activity regulating gene may be CBLB.

[0378] The immune cell activity regulating gene may be PSGL-1.

[0379] The immune cell activity regulating gene may be ILT2.

[0380] The immune cell activity regulating gene may be KIR2DL4.

[0381] The immune cell activity regulating gene may be SHP-1.

[0382] The above genes may be derived from mammals including primates (eg, humans, monkeys, etc.) and rodents (eg, mice, rats, etc.).

[0383] Genetic information can be obtained from known databases such as GenBank at the National Center for Biotechnology Information (NCBI).

[0384] In one embodiment, the immune cell activity regulating gene may function to stimulate an immune response.

[0385] The immune cell activity regulating gene may be an immune cell growth regulating gene.

[0386] The term "immune cell growth regulatory gene" refers to a gene that regulates immune cell growth by regulating protein synthesis in immune cells, for example, a gene that stimulates or inhibits immune cell growth. In this case, the immune cell growth regulatory gene can control immune cell growth by controlling protein synthesis in immune cells that have the immune cell growth regulatory gene itself or the protein expressed by the immune cell growth regulatory gene.

[0387] Immune cell growth regulatory genes can function in DNA transcription, RNA translation and cell differentiation.

[0388] Examples of immune cell growth regulatory genes may include genes involved in expression pathways of NFAT, IκB / NF-κB, AP-1, 4E-BP1, eIF4E, and S6.

[0389] For example, the immune cell growth regulatory gene may be DGK-α.

[0390] The DGKA (Dgk-alpha, DGKα) gene refers to a gene (full-length DNA, cDNA, or mRNA) encoding the diacylglycerol kinase alpha protein (DGKA). In an embodiment, the DGKA gene may be one or more selected from the group consisting of, but not limited to, genes encoding human DGKA (e.g., NCBI accession numbers NP_001336.2, NP_958852.1, NP_958853.1, NP_963848.1, etc.), such as DGKA genes represented by NCBI accession numbers NM_001345.4, NM_201444.2, NM_201445.1, NM_201554.1, NC_000012.12, etc.

[0391] The immune cell growth regulatory gene may be DGK-ζ.

[0392] The DGKZ (Dgk-zeta, DGKζ) gene refers to a gene (full-length DNA, cDNA, or mRNA) encoding the diacylglycerol kinase zeta protein (DGKZ). In an embodiment, the DGKZ gene may be one or more selected from the group consisting of, but not limited to, genes encoding human DGKZ (e.g., NCBI Accession Nos. NP_001099010.1, NP_001186195.1, NP_001186196.1, NP_001186197.1, NP_003637.2, NP_963290. 1, NP_963291.2, etc.), for example, DGKZ genes represented by NCBI accession numbers NM_001105540.1, NM_001199266.1, NM_001199267.1, NM_001199268.1, NM_003646.3, NM_201532.2, NM_201533.3, NG_047092.1, etc.

[0393] The immune cell growth regulatory gene may be EGR2.

[0394] The EGR2 gene refers to a gene (full-length DNA, cDNA, or mRNA) encoding early growth response protein 2 (EGR2). In an embodiment, the EGR2 gene may be one or more selected from the group consisting of the following genes, but is not limited thereto: genes encoding human EGR2 (e.g., NCBI accession numbers NP_000390, NP_001129649, NP_001129650, NP_001129651, NP_001307966, etc.). For example, the EGR2 gene represented by NCBI accession numbers NM_000399, NM_001136177, NM_001136178, NM_001136179, NM_001321037, etc.

[0395] The immune cell growth regulatory gene may be EGR3.

[0396] The immune cell growth regulatory gene may be PPP2r2d.

[0397] The immune cell growth regulatory gene may be A20 (TNFAIP3).

[0398] The above genes can be derived from mammals including primates (eg, humans, monkeys, etc.) and rodents (eg, mice, rats, etc.).

[0399] Genetic information can be obtained from known databases such as GenBank at the National Center for Biotechnology Information (NCBI).

[0400] In an embodiment, the immune cell activity regulating gene may be an immune cell death regulating gene.

[0401] The term "immune cell death regulating gene" refers to a gene whose function involves immune cell death, for example, stimulating or inhibiting immune cell death. Here, the immune cell death regulating gene can perform the function of controlling immune cell death through the immune cell death regulating gene itself or the protein expressed by the immune cell death regulating gene.

[0402] Immune cell death regulatory genes can perform functions related to immune cell apoptosis or necrosis.

[0403] For example, the immune cell death regulatory gene may be a caspase cascade-associated gene.

[0404] In this case, the immune cell death regulatory element may be Fas. Where reference is made to a gene below, it will be apparent to one of ordinary skill in the art that manipulation may be performed on the receptor or binding region on which the gene acts.

[0405] The immune cell death regulatory gene may be a death domain-related gene.

[0406] Here, the immune cell death regulatory gene may be Daxx.

[0407] The immune cell death regulatory gene may be a Bcl-2 family gene.

[0408] Immune cell death regulatory genes may be BH3-only family genes.

[0409] The immune cell death regulatory gene may be Bim.

[0410] The immune cell death regulatory gene may be Bid.

[0411] The immune cell death regulatory gene may be BAD.

[0412] The immune cell death regulating gene may be a gene encoding a ligand or receptor located on the outer membrane of an immune cell.

[0413] Here, the immune cell death regulatory gene may be PD-1.

[0414] In addition, the immune cell death regulatory gene may be CTLA-4.

[0415] The above genes can be derived from mammals including primates (eg, humans, monkeys, etc.) and rodents (eg, mice, rats, etc.).

[0416] Genetic information can be obtained from known databases such as GenBank at the National Center for Biotechnology Information (NCBI).

[0417] In an embodiment, the immune cell activity regulating gene may be an immune cell exhaustion regulating gene.

[0418] The term "immune cell depletion regulating gene" is a gene that performs a function associated with the gradual loss of immune cell function, and here, the immune cell depletion regulating gene can perform the function of controlling the gradual loss of immune cell function through the immune cell depletion regulating gene itself or a protein expressed by the immune cell depletion regulating gene.

[0419] Immune cell exhaustion regulatory genes may function to assist the transcription or translation of genes involved in immune cell inactivation.

[0420] Here, the function of assisting transcription may be a function of demethylating the corresponding gene.

[0421] In addition, genes involved in immune cell inactivation include immune cell activity regulatory genes.

[0422] For example, the immune cell exhaustion regulatory gene may be TET2.

[0423] The TET2 gene refers to a gene (full-length DNA, cDNA, or mRNA) encoding TET2 (Tet methylcytosine dioxygenase 2). In an embodiment, the TET2 gene may be one or more genes encoding human TET2 (e.g., NCBI accession numbers NP_001120680.1, NP_060098.3, etc.) selected from the group consisting of, but not limited to, TET2 genes represented by NCBI accession numbers NM_001127208.2, NM_017628.4, NG_028191.1, etc.).

[0424] Immune cell exhaustion regulatory elements may function in immune cell overgrowth, where immune cells that undergo overgrowth and fail to regenerate lose their function.

[0425] Here, the immune cell exhaustion regulatory gene may be Wnt.

[0426] In addition, the immune cell exhaustion regulatory gene may be Akt.

[0427] The above genes can be derived from mammals including primates (eg, humans, monkeys, etc.) and rodents (eg, mice, rats, etc.).

[0428] Genetic information can be obtained from known databases such as GenBank at the National Center for Biotechnology Information (NCBI).

[0429] In another embodiment, the immune cell activity regulatory element may be a cytokine production regulatory gene.

[0430] The term "cytokine production regulatory gene" is an element involved in cytokine secretion by immune cells, which is expressed by immune cells that perform such functions, and here, the cytokine production regulatory gene can function to control cytokine production by immune cells through the cytokine production regulatory gene itself or the protein expressed by the cytokine production regulatory gene.

[0431] Cytokines are a general term for proteins secreted by immune cells and are signal proteins that play an important role in the body. Cytokines are related to infection, immunity, inflammation, wounds, ulcers, cancer, etc. Cytokines can be secreted by cells and then affect other cells, or affect the cells that secrete them themselves. For example, cytokines can induce macrophage proliferation or promote the differentiation of secretory cells themselves. However, when cytokine secretion is excessive, it can cause problems such as attacking normal cells. Therefore, the appropriate secretion of cytokines is also important in the immune response.

[0432] The cytokine production regulating gene may preferably be, for example, a gene in the secretory pathway of TNFα, IFN-γ, TGF-β, IL-2, IL-4, IL-10, IL-13, IL-1, IL-6, IL-12, and IFN-α.

[0433] Alternatively, cytokines may function to deliver signals to other immune cells to induce immune cells to kill recognized antigen-bearing cells or to help differentiation. In this case, the cytokine production regulatory gene may preferably be a gene in a gene pathway involved in IL-2 secretion.

[0434] The above genes can be derived from mammals including primates (eg, humans, monkeys, etc.) and rodents (eg, mice, rats, etc.).

[0435] Genetic information can be obtained from known databases such as GenBank at the National Center for Biotechnology Information (NCBI).

[0436] In an embodiment, the immunomodulatory gene disclosed in this specification may be an immune cell activity regulating gene.

[0437] The immunomodulatory gene may be PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene.

[0438] In one embodiment of the contents disclosed in this specification, the guide nucleic acid may be a gRNA that complementary binds to the target sequence of PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene.

[0439] The term "target sequence" refers to a nucleotide sequence in a target gene or nucleic acid, particularly a partial nucleotide sequence in a target region in a target gene or nucleic acid, wherein the "target region" is a region in a target gene or nucleic acid that can be modified by a guided nucleic acid-editing protein.

[0440] The target gene disclosed in this specification may be an immune regulatory gene.

[0441] The target gene disclosed in the present specification may be PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene.

[0442] Hereinafter, the term "target sequence" may refer to two types of nucleotide sequence information. For example, for a target gene, the target sequence may refer to the transcribed strand sequence information of the target gene DNA, or the nucleotide sequence information of the non-transcribed strand.

[0443] For example, the target sequence may refer to a partial nucleotide sequence (transcribed chain) 5'-ATCATTGGCAGACTAGTTCG-3' in the target region of target gene A or a complementary nucleotide sequence (non-transcribed chain) 5'-CGAACTAGTCTGCCAATGAT-3'.

[0444] The target sequence may be a sequence of 5-50 nucleotides.

[0445] In an embodiment, the target sequence may be a nucleotide sequence of 16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp or 25 bp.

[0446] The target sequence comprises a guide nucleic acid binding sequence or a guide nucleic acid non-binding sequence.

[0447] The term "guide nucleic acid binding sequence" refers to a nucleotide sequence that is partially or fully complementary to a guide sequence contained in the guide domain of a guide nucleic acid, and can form a complementary bond with the guide sequence contained in the guide domain of the guide nucleic acid. Target sequences and guide nucleic acid binding sequences are nucleotide sequences that can be modified depending on the target gene or nucleic acid (i.e., the subject of genetic manipulation or modification) and can be designed in various forms depending on the target gene or nucleic acid.

[0448] The term "guide nucleic acid non-binding sequence" refers to a nucleotide sequence that has partial or complete homology to a guide sequence contained in a guide domain of a guide nucleic acid and that is unable to form a complementary bond with the guide sequence contained in the guide domain of the guide nucleic acid. Furthermore, a guide nucleic acid non-binding sequence is a nucleotide sequence that is complementary to a guide nucleic acid binding sequence and can form a complementary bond with the guide nucleic acid binding sequence.

[0449] The guide nucleic acid binding sequence is a partial nucleotide sequence in the target sequence and can be either of two nucleotide sequences having sequences in different order from the target sequence, i.e., two nucleotide sequences that form complementary binding. Here, the guide nucleic acid non-binding sequence can be the nucleotide sequence of the target sequence excluding the guide nucleic acid binding sequence.

[0450] For example, when the partial nucleotide sequence 5'-ATCATTGGCAGACTAGTTCG-3' and the complementary nucleotide sequence 5'-CGAACTAGTCTGCCAATGAT-3' in the target region of target gene A are the target sequences, the guide nucleic acid binding sequence can be either of the two target sequences, namely 5'-ATCATTGGCAGACTAGTTCG-3' or 5'-CGAACTAGTCTGCCAATGAT-3'. Here, when the guide nucleic acid binding sequence is 5'-ATCATTGGCAGACTAGTTCG-3', the guide nucleic acid non-binding sequence can be 5'-CGAACTAGTCTGCCAATGAT-3'; or when the guide nucleic acid binding sequence is 5'-CGAACTAGTCTGCCAATGAT-3', the guide nucleic acid non-binding sequence can be 5'-ATCATTGGCAGACTAGTTCG-3'.

[0451] The guide nucleic acid binding sequence may be a nucleotide sequence selected from a nucleotide sequence homologous to the target sequence (i.e., the transcribed strand) and a nucleotide sequence homologous to the non-transcribed strand. Here, the guide nucleic acid non-binding sequence may be a nucleotide sequence other than a nucleotide sequence homologous to the guide nucleic acid binding sequence (i.e., the transcribed strand) in the target sequence and a nucleotide sequence homologous to the non-transcribed strand.

[0452] The guide nucleic acid binding sequence can be the same length as the target sequence.

[0453] The guide nucleic acid non-binding sequence can have the same length as the target sequence or the guide nucleic acid binding sequence.

[0454] The guide nucleic acid binding sequence can be a sequence of 5-50 nucleotides.

[0455] In an embodiment, the guide nucleic acid binding sequence may be a nucleotide sequence of 16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp or 25 bp.

[0456] The non-binding sequence of the guide nucleic acid can be a nucleotide sequence of 5 bp to 50 bp.

[0457] In an embodiment, the guide nucleic acid non-binding sequence may be a nucleotide sequence of 16 bp, 17 bp, 18 bp, 19 bp, 20 bp, 21 bp, 22 bp, 23 bp, 24 bp or 25 bp.

[0458] The guide nucleic acid binding sequence can form partial or complete complementary binding with the guide sequence contained in the guide domain of the guide nucleic acid, and the length of the guide nucleic acid binding sequence can be the same as the length of the guide sequence.

[0459] The guide nucleic acid binding sequence can be a nucleotide sequence that is complementary to the guide sequence contained in the guide domain of the guide nucleic acid, having, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.

[0460] In one example, the guide nucleic acid binding sequence may have or include a nucleotide sequence of 1 bp to 8 bp that is not complementary to the guide sequence included in the guide domain of the guide nucleic acid.

[0461] The guide nucleic acid non-binding sequence may have partial or complete homology to the guide sequence contained in the guide domain of the guide nucleic acid, and the length of the guide nucleic acid non-binding sequence may be the same as the length of the guide sequence.

[0462] The non-binding sequence of the guide nucleic acid can be a nucleotide sequence having homology to the guide sequence contained in the guide domain of the guide nucleic acid, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more homology or complete homology.

[0463] In one example, the guide nucleic acid non-binding sequence may have or include a 1 bp-8 bp nucleotide sequence that is not complementary to the guide sequence included in the guide domain of the guide nucleic acid.

[0464] The non-binding sequence of the guide nucleic acid can form a complementary bond with the binding sequence of the guide nucleic acid, and the length of the non-binding sequence of the guide nucleic acid can be the same as the length of the binding sequence of the guide nucleic acid.

[0465] The guide nucleic acid non-binding sequence can be a nucleotide sequence that is complementary to the guide nucleic acid binding sequence, having, for example, at least 90% or 95% or higher complementarity or complete complementarity.

[0466] In one example, the guide nucleic acid non-binding sequence may have or include a 1 bp-2 bp nucleotide sequence that is not complementary to the guide nucleic acid binding sequence.

[0467] In addition, the guide nucleic acid binding sequence can be a nucleotide sequence located near a nucleotide sequence that can be recognized by the editing protein.

[0468] In one example, the guide nucleic acid binding sequence may be a continuous 5 bp-50 bp nucleotide sequence adjacent to the 5' end and / or 3' end of the nucleotide sequence capable of being recognized by the editing protein.

[0469] In an embodiment, the target sequence disclosed in the present specification may be a continuous 10 bp-35 bp nucleotide sequence located in the promoter region of an immunomodulatory gene.

[0470] Here, the target sequence may be a 10 bp-35 bp, 15 bp-35 bp, 20 bp-35 bp, 25 bp-35 bp, or 30 bp-35 bp nucleotide sequence.

[0471] Alternatively, the target sequence may be a 10 bp-15 bp, 15 bp-20 bp, 20 bp-25 bp, 25 bp-30 bp, or 30 bp-35 bp nucleotide sequence.

[0472] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the promoter region of the PD-1 gene.

[0473] In another example, the target sequence can be a continuous 10 bp-25 bp nucleotide sequence located in the promoter region of the CTLA-4 gene.

[0474] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the promoter region of the A20 gene.

[0475] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the promoter region of the DGKA gene.

[0476] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the promoter region of the DGKZ gene.

[0477] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the promoter region of the FAS gene.

[0478] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the promoter region of the EGR2 gene.

[0479] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the promoter region of the PPP2r2d gene.

[0480] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the promoter region of the TET2 gene.

[0481] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the promoter region of the PSGL-1 gene.

[0482] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the promoter region of the KDM6A gene.

[0483] The target sequence disclosed in the present specification may be a continuous 10 bp-35 bp nucleotide sequence located in the intron region of the immunomodulatory gene.

[0484] Here, the target sequence may be a 10 bp-35 bp, 15 bp-35 bp, 20 bp-35 bp, 25 bp-35 bp, or 30 bp-35 bp nucleotide sequence.

[0485] Alternatively, the target sequence may be a 10 bp-15 bp, 15 bp-20 bp, 20 bp-25 bp, 25 bp-30 bp, or 30 bp-35 bp nucleotide sequence.

[0486] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the intron region of the PD-1 gene.

[0487] In another example, the target sequence can be a continuous 10 bp-25 bp nucleotide sequence located in the intron region of the CTLA-4 gene.

[0488] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the intron region of the A20 gene.

[0489] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the intron region of the DGKA gene.

[0490] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the intron region of the DGKZ gene.

[0491] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the intron region of the FAS gene.

[0492] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the intron region of the EGR2 gene.

[0493] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the intron region of the PPP2r2d gene.

[0494] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the intron region of the TET2 gene.

[0495] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the intron region of the PSGL-1 gene.

[0496] In yet another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the intron region of the KDM6A gene.

[0497] The target sequence disclosed in the present specification may be a continuous 10 bp-35 bp nucleotide sequence located in the exon region of an immunomodulatory gene.

[0498] Here, the target sequence may be a 10 bp-35 bp, 15 bp-35 bp, 20 bp-35 bp, 25 bp-35 bp, or 30 bp-35 bp nucleotide sequence.

[0499] Alternatively, the target sequence may be a 10 bp-15 bp, 15 bp-20 bp, 20 bp-25 bp, 25 bp-30 bp, or 30 bp-35 bp nucleotide sequence.

[0500] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the exon region of the PD-1 gene.

[0501] In another example, the target sequence can be a continuous 10 bp-25 bp nucleotide sequence located in the exon region of the CTLA-4 gene.

[0502] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the exon region of the A20 gene.

[0503] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the exon region of the DGKA gene.

[0504] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the exon region of the DGKZ gene.

[0505] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the exon region of the FAS gene.

[0506] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the exon region of the EGR2 gene.

[0507] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the exon region of the PPP2r2d gene.

[0508] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the exon region of the TET2 gene.

[0509] In another example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence located in the exon region of the PSGL-1 gene.

[0510] In yet another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the exon region of the KDM6A gene.

[0511] The target sequence disclosed in the present specification may be a continuous 10 bp-35 bp nucleotide sequence located in the enhancer region of an immunomodulatory gene.

[0512] Here, the target sequence may be a 10 bp-35 bp, 15 bp-35 bp, 20 bp-35 bp, 25 bp-35 bp, or 30 bp-35 bp nucleotide sequence.

[0513] Alternatively, the target sequence may be a 10 bp-15 bp, 15 bp-20 bp, 20 bp-25 bp, 25 bp-30 bp, or 30 bp-35 bp nucleotide sequence.

[0514] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the enhancer region of the PD-1 gene.

[0515] In another example, the target sequence can be a continuous 10 bp-25 bp nucleotide sequence located in the enhancer region of the CTLA-4 gene.

[0516] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the enhancer region of the A20 gene.

[0517] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the enhancer region of the DGKA gene.

[0518] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the enhancer region of the DGKZ gene.

[0519] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the enhancer region of the FAS gene.

[0520] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the enhancer region of the EGR2 gene.

[0521] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the enhancer region of the PPP2r2d gene.

[0522] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the enhancer region of the TET2 gene.

[0523] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the enhancer region of the PSGL-1 gene.

[0524] In yet another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the enhancer region of the KDM6A gene.

[0525] The target sequence disclosed in the present specification may be a continuous 10 bp-35 bp nucleotide sequence located in the coding region, non-coding region, or a combination thereof of an immunomodulatory gene.

[0526] Here, the target sequence may be a 10 bp-35 bp, 15 bp-35 bp, 20 bp-35 bp, 25 bp-35 bp, or 30 bp-35 bp nucleotide sequence.

[0527] Alternatively, the target sequence may be a 10 bp-15 bp, 15 bp-20 bp, 20 bp-25 bp, 25 bp-30 bp, or 30 bp-35 bp nucleotide sequence.

[0528] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the coding region, non-coding region, or a combination thereof of the PD-1 gene.

[0529] In another example, the target sequence can be a continuous 10 bp to 25 bp nucleotide sequence located in the coding region, the non-coding region, or a combination thereof of the CTLA-4 gene.

[0530] In one example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence located in the coding region, the non-coding region, or a combination thereof of the A20 gene.

[0531] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the coding region, the non-coding region, or a combination thereof of the DGKA gene.

[0532] In one example, the target sequence can be a continuous 10 bp-25 bp nucleotide sequence located in the coding region, the non-coding region, or a combination thereof of the DGKZ gene.

[0533] In another example, the target sequence can be a continuous 10 bp-25 bp nucleotide sequence located in the coding region, the non-coding region, or a combination thereof of the FAS gene.

[0534] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the coding region, the non-coding region, or a combination thereof of the EGR2 gene.

[0535] In another example, the target sequence can be a continuous 10 bp-25 bp nucleotide sequence located in the coding region, non-coding region, or a combination thereof of the PPP2r2d gene.

[0536] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the coding region, the non-coding region, or a combination thereof of the TET2 gene.

[0537] In another example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence located in the coding region, the non-coding region, or a combination thereof of the PSGL-1 gene.

[0538] In yet another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the coding region, the non-coding region, or a combination thereof of the KDM6A gene.

[0539] The target sequence disclosed in the present specification may be a continuous 10 bp-35 bp nucleotide sequence located in the promoter region, enhancer region, 3'-UTR region, 5'-UTR region, polyA region or a combination thereof of an immunomodulatory gene.

[0540] Here, the target sequence may be a 10 bp-35 bp, 15 bp-35 bp, 20 bp-35 bp, 25 bp-35 bp, or 30 bp-35 bp nucleotide sequence.

[0541] Alternatively, the target sequence may be a 10 bp-15 bp, 15 bp-20 bp, 20 bp-25 bp, 25 bp-30 bp, or 30 bp-35 bp nucleotide sequence.

[0542] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the promoter region, enhancer region, 3'-UTR region, 5'-UTR region, poly A region, or a combination thereof of the PD-1 gene.

[0543] In another example, the target sequence can be a continuous 10 bp to 25 bp nucleotide sequence located in the promoter region, enhancer region, 3'-UTR region, 5'-UTR region, poly A region, or a combination thereof of the CTLA-4 gene.

[0544] In one example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence located in the promoter region, enhancer region, 3'-UTR region, 5'-UTR region, polyA region, or a combination thereof of the A20 gene.

[0545] In another example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence located in the promoter region, enhancer region, 3'-UTR region, 5'-UTR region, polyA region, or a combination thereof of the DGKA gene.

[0546] In one example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence located in the promoter region, enhancer region, 3'-UTR region, 5'-UTR region, polyA region, or a combination thereof of the DGKZ gene.

[0547] In another example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence located in the promoter region, enhancer region, 3'-UTR region, 5'-UTR region, polyA region, or a combination thereof of the FAS gene.

[0548] In one example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence located in the promoter region, enhancer region, 3'-UTR region, 5'-UTR region, polyA region, or a combination thereof of the EGR2 gene.

[0549] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the promoter region, enhancer region, 3'-UTR region, 5'-UTR region, polyA region, or a combination thereof of the PPP2r2d gene.

[0550] In one example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence located in the promoter region, enhancer region, 3'-UTR region, 5'-UTR region, polyA region, or a combination thereof of the TET2 gene.

[0551] In another example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence located in the promoter region, enhancer region, 3'-UTR region, 5'-UTR region, polyA region, or a combination thereof of the PSGL-1 gene.

[0552] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the promoter region, enhancer region, 3'-UTR region, 5'-UTR region, polyA region, or a combination thereof of the KDM6A gene.

[0553] The target sequence disclosed in the present specification may be a continuous 10 bp-35 bp nucleotide sequence located in the exon region, intron region, or a combination thereof of an immunomodulatory gene.

[0554] Here, the target sequence may be a 10 bp-35 bp, 15 bp-35 bp, 20 bp-35 bp, 25 bp-35 bp, or 30 bp-35 bp nucleotide sequence.

[0555] Alternatively, the target sequence may be a 10 bp-15 bp, 15 bp-20 bp, 20 bp-25 bp, 25 bp-30 bp, or 30 bp-35 bp nucleotide sequence.

[0556] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the exon region, intron region, or a combination thereof of the PD-1 gene.

[0557] In another example, the target sequence can be a continuous 10 bp to 25 bp nucleotide sequence located in the exon region, intron region, or a combination thereof of the CTLA-4 gene.

[0558] In one example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence located in the exon region, intron region, or a combination thereof of the A20 gene.

[0559] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the exon region, intron region, or a combination thereof of the DGKA gene.

[0560] In one example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence located in the exon region, intron region, or a combination thereof of the DGKZ gene.

[0561] In another example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence located in the exon region, intron region, or a combination thereof of the FAS gene.

[0562] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the exon region, intron region, or a combination thereof of the EGR2 gene.

[0563] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the exon region, intron region, or a combination thereof of the PPP2r2d gene.

[0564] In one example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the exon region, intron region, or a combination thereof of the TET2 gene.

[0565] In another example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence located in the exon region, intron region, or a combination thereof of the PSGL-1 gene.

[0566] In yet another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the exon region, intron region, or a combination thereof of the KDM6A gene.

[0567] The target sequence disclosed in the present specification may be a continuous 10 bp-35 bp nucleotide sequence that includes or is adjacent to a mutation region (eg, a region that is different from the wild-type gene) of an immunomodulatory gene.

[0568] Here, the target sequence may be a 10 bp-35 bp, 15 bp-35 bp, 20 bp-35 bp, 25 bp-35 bp, or 30 bp-35 bp nucleotide sequence.

[0569] Alternatively, the target sequence may be a 10 bp-15 bp, 15 bp-20 bp, 20 bp-25 bp, 25 bp-30 bp, or 30 bp-35 bp nucleotide sequence.

[0570] In one example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence comprising a mutated region (eg, a region different from the wild-type gene) of the PD-1 gene or adjacent to the PD-1 gene.

[0571] In another example, the target sequence can be a continuous 10 bp to 25 bp nucleotide sequence that includes or is adjacent to a mutant region of the CTLA-4 gene (eg, a region that differs from the wild-type gene).

[0572] In one example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence that includes or is adjacent to a mutation region (eg, a region that is different from the wild-type gene) of the A20 gene.

[0573] In another example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence that includes or is adjacent to a mutant region of the DGKA gene (eg, a region that is different from the wild-type gene).

[0574] In one example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence that includes or is adjacent to a mutant region of the DGKZ gene (eg, a region that is different from the wild-type gene).

[0575] In another example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence that includes or is adjacent to a mutant region of the FAS gene (eg, a region that is different from the wild-type gene).

[0576] In one example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence that includes or is adjacent to a mutation region of the EGR2 gene (eg, a region that is different from the wild-type gene).

[0577] In another example, the target sequence may be a continuous 10 bp-25 bp nucleotide sequence that includes or is adjacent to a mutant region (eg, a region that is different from the wild-type gene) of the PPP2r2d gene.

[0578] In one example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence that includes or is adjacent to a mutant region of the TET2 gene (eg, a region that is different from the wild-type gene).

[0579] In another example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence that includes or is adjacent to a mutant region of the PSGL-1 gene (eg, a region that is different from the wild-type gene).

[0580] In another example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence that includes or is adjacent to a mutant region of the KDM6A gene (eg, a region that is different from the wild-type gene).

[0581] The target sequence disclosed in the present specification may be a continuous 10 bp to 35 bp nucleotide sequence at the 5' end and / or 3' end of the protospacer adjacent motif (PAM) sequence in the nucleic acid sequence of the immunomodulatory gene.

[0582] The term "protospacer adjacent motif (PAM) sequence" is a nucleotide sequence that can be recognized by an editing protein. Here, the PAM sequence may have a nucleotide sequence that varies depending on the type and source species of the editing protein.

[0583] Here, the PAM sequence can be, for example, one or more of the following sequences (described in the 5' to 3' direction):

[0584] NGG (N is A, T, C or G);

[0585] NNNNRYAC (each N is independently A, T, C or G; R is A or G; Y is C or T);

[0586] NNAGAAW (each N is independently A, T, C or G; W is A or T);

[0587] NNNNGATT (each N is independently A, T, C or G);

[0588] NNGRR(T) (each N is independently A, T, C or G; R is A or G; Y is C or T); and

[0589] TTN (N is A, T, C or G).

[0590] Here, the target sequence may be a 10 bp-35 bp, 15 bp-35 bp, 20 bp-35 bp, 25 bp-35 bp, or 30 bp-35 bp nucleotide sequence.

[0591] Alternatively, the target sequence may be a 10 bp-15 bp, 15 bp-20 bp, 20 bp-25 bp, 25 bp-30 bp, or 30 bp-35 bp nucleotide sequence.

[0592] In one example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence adjacent to the 5' end and / or 3' end of the PAM sequence in the nucleic acid sequence of the PD-1 gene.

[0593] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the PD-1 gene.

[0594] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' or / and 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NGGNG-3' or / and 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the PD-1 gene.

[0595] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the PD-1 gene.

[0596] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the PD-1 gene.

[0597] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10 bp-25 bp nucleotide sequence at the 5' end and / or 3' end of 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the PD-1 gene.

[0598] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or 3' end of 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the PD-1 gene.

[0599] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N = A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10-25 nucleotide sequence at the 5' end or / and 3' end of 5'-TTN-3' (N = A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the PD-1 gene.

[0600] In another example, the target sequence can be a continuous 10 bp to 25 bp nucleotide sequence adjacent to the 5' end and / or 3' end of the PAM sequence in the nucleic acid sequence of the CTLA-4 gene.

[0601] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the CTLA-4 gene.

[0602] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' or / and 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10 bp-25 bp nucleotide sequence at the 5' end or / and 3' end of 5'-NGGNG-3' or / and 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the CTLA-4 gene.

[0603] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10 bp-25 bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the CTLA-4 gene.

[0604] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10 bp-25 bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the CTLA-4 gene.

[0605] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C), the target sequence can be a continuous 10 bp-25 bp nucleotide sequence at the 5' end and / or 3' end of 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the CTLA-4 gene.

[0606] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the CTLA-4 gene.

[0607] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N = A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10 bp-25 bp nucleotide sequence at the 5' end and / or 3' end of 5'-TTN-3' (N = A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the CTLA-4 gene.

[0608] In one example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence adjacent to the 5' end and / or 3' end of the PAM sequence in the nucleic acid sequence of the A20 gene.

[0609] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the A20 gene.

[0610] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' or / and 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NGGNG-3' or / and 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the A20 gene.

[0611] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the A20 gene.

[0612] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the A20 gene.

[0613] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10 bp-25 bp nucleotide sequence at the 5' end and / or 3' end of 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the A20 gene.

[0614] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the A20 gene.

[0615] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N = A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end and / or 3' end of 5'-TTN-3' (N = A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the A20 gene.

[0616] In another example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence adjacent to the 5' end and / or 3' end of the PAM sequence in the nucleic acid sequence of the DGKA gene.

[0617] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the DGKA gene.

[0618] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' or / and 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NGGNG-3' or / and 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the DGKA gene.

[0619] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the DGKA gene.

[0620] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the DGKA gene.

[0621] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10 bp-25 bp nucleotide sequence at the 5' end and / or 3' end of 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the DGKA gene.

[0622] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the DGKA gene.

[0623] In one embodiment, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N = A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end and / or 3' end of 5'-TTN-3' (N = A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the DGKA gene.

[0624] In one example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence adjacent to the 5' end and / or the 3' end of the PAM sequence in the nucleic acid sequence of the DGKZ gene.

[0625] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the DGKZ gene.

[0626] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' or / and 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NGGNG-3' or / and 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the DGKZ gene.

[0627] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the DGKZ gene.

[0628] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the DGKZ gene.

[0629] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10 bp-25 bp nucleotide sequence at the 5' end and / or 3' end of 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the DGKZ gene.

[0630] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the DGKZ gene.

[0631] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N = A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end and / or 3' end of 5'-TTN-3' (N = A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the DGKZ gene.

[0632] In another example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence adjacent to the 5' end and / or 3' end of the PAM sequence in the nucleotide sequence of the FAS gene.

[0633] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the FAS gene.

[0634] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' or / and 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NGGNG-3' or / and 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the FAS gene.

[0635] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the FAS gene.

[0636] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the FAS gene.

[0637] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10 bp-25 bp nucleotide sequence at the 5' end and / or 3' end of 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the FAS gene.

[0638] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the FAS gene.

[0639] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N = A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end and / or 3' end of 5'-TTN-3' (N = A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the FAS gene.

[0640] In one example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence adjacent to the 5' end and / or 3' end of the protospacer adjacent motif (PAM) in the nucleic acid sequence of the EGR2 gene.

[0641] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the EGR2 gene.

[0642] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' or / and 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NGGNG-3' or / and 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the EGR2 gene.

[0643] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the EGR2 gene.

[0644] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the EGR2 gene.

[0645] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end and / or 3' end of 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the EGR2 gene.

[0646] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the EGR2 gene.

[0647] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N = A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end and / or 3' end of 5'-TTN-3' (N = A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the EGR2 gene.

[0648] In another example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence adjacent to the 5' end and / or the 3' end of the PAM sequence in the nucleotide sequence of the PPP2r2d gene.

[0649] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the PPP2r2d gene.

[0650] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' or / and 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NGGNG-3' or / and 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the PPP2r2d gene.

[0651] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the PPP2r2d gene.

[0652] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the PPP2r2d gene.

[0653] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the PPP2r2d gene.

[0654] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the PPP2r2d gene.

[0655] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N = A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-TTN-3' (N = A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the PPP2r2d gene.

[0656] In one example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence adjacent to the 5' end and / or the 3' end of the PAM sequence in the nucleic acid sequence of the TET2 gene.

[0657] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the TET2 gene.

[0658] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' or / and 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NGGNG-3' or / and 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the TET2 gene.

[0659] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the TET2 gene.

[0660] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the TET2 gene.

[0661] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end and / or 3' end of 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the TET2 gene.

[0662] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the TET2 gene.

[0663] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N = A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end and / or 3' end of 5'-TTN-3' (N = A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the TET2 gene.

[0664] In another example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence adjacent to the 5' end and / or the 3' end of the PAM sequence in the nucleic acid sequence of the PSGL-1 gene.

[0665] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the PSGL-1 gene.

[0666] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3', 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end and / or 3' end of 5'-NGGNG-3', 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the PSGL-1 gene.

[0667] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the PSGL-1 gene.

[0668] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the PSGL-1 gene.

[0669] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10 bp-25 bp nucleotide sequence at the 5' end and / or 3' end of 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the PSGL-1 gene.

[0670] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the PSGL-1 gene.

[0671] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N = A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end and / or 3' end of 5'-TTN-3' (N = A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the PSGL-1 gene.

[0672] In another example, the target sequence may be a continuous 10 bp to 25 bp nucleotide sequence at the 5' end and / or the 3' end of the protospacer adjacent motif (PAM) in the nucleic acid sequence of the KDM6A gene.

[0673] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NGG-3', 5'-NAG-3' or / and 5'-NGA-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the KDM6A gene.

[0674] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' or / and 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NGGNG-3' or / and 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the KDM6A gene.

[0675] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNNGATT-3' or / and 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the KDM6A gene.

[0676] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the KDM6A gene.

[0677] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C), the target sequence can be a continuous 10 bp-25 bp nucleotide sequence at the 5' end and / or 3' end of 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the KDM6A gene.

[0678] In another embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end or / and 3' end of 5'-NNGRR-3', 5'-NNGRRT-3' or / and 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C) in the nucleic acid sequence adjacent to the KDM6A gene.

[0679] In an embodiment, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N = A, T, G or C; or A, U, G or C), the target sequence may be a continuous 10bp-25bp nucleotide sequence at the 5' end and / or 3' end of 5'-TTN-3' (N = A, T, G or C; or A, U, G or C) in the nucleic acid sequence adjacent to the KDM6A gene.

[0680] The following table lists examples of target sequences that can be used in one embodiment of the present invention. The target sequences described in the table are guide nucleic acid non-binding sequences; complementary sequences, that is, guide nucleic acid binding sequences can be predicted by the sequences described.

[0681] [Table 1] Target sequences of immune regulatory genes

[0682]

[0683]

[0684]

[0685]

[0686]

[0687]

[0688]

[0689]

[0690]

[0691]

[0692] Aspects of the disclosure herein relate to gene manipulation compositions for artificially manipulating immune regulatory genes.

[0693] The composition for gene manipulation can be used to produce artificially modified immunomodulatory genes. In addition, the immunomodulatory genes artificially modified by the composition for gene manipulation can regulate the immune system.

[0694] The term "artificially modified or engineered or artificially engineered" refers to a state in which artificial modifications are applied, rather than the original state existing in the natural state. The artificially modified or engineered non-natural immune regulatory genes hereinafter can be used interchangeably with artificial immune regulatory genes.

[0695] The "immune system" of the present invention is a term that includes all phenomena that affect the immune response in the body (i.e., participate in mechanisms that manifest new immune effects) through functional changes of manipulated immunoregulatory factors, and includes all substances, compositions, methods and uses that directly or indirectly participate in such an immune system. For example, the immune system includes all genes, immune cells and immune organs / tissues that participate in innate immunity, adaptive immunity, cellular immunity, humoral immunity, active immunity and passive immune responses.

[0696] The composition for gene manipulation disclosed in this specification may include a guide nucleic acid and an editing protein.

[0697] A composition for genetic manipulation may comprise:

[0698] (a) a guide nucleic acid that can form a complementary bond with a target sequence of an immune-regulating gene or a nucleic acid sequence encoding the target sequence; and

[0699] (b) one or more editing proteins or nucleic acid sequences encoding the same.

[0700] The explanations about the above-mentioned immunoregulatory genes are as described above.

[0701] The explanation about the above target sequence is as described above.

[0702] Compositions for genetic manipulation may comprise a guide nucleic acid-editing protein complex.

[0703] The term "guide nucleic acid-editing protein complex" refers to a complex formed by the interaction between a guide nucleic acid and an editing protein.

[0704] The explanation about the guide nucleic acid is as described above.

[0705] An "editing protein" refers to a peptide, polypeptide, or protein that is capable of directly binding to a nucleic acid or interacting with a nucleic acid without direct binding.

[0706] In this case, the nucleic acid may be a target nucleic acid, a gene, or a nucleic acid contained in a chromosome. Here, the nucleic acid may be a guide nucleic acid.

[0707] The editing protein may be an enzyme.

[0708] Herein, the term "enzyme" refers to a polypeptide or protein containing a domain capable of cleaving nucleic acids, genes or chromosomes.

[0709] The enzyme may be a nuclease or a restriction enzyme.

[0710] The editing protein may comprise a fully active enzyme.

[0711] Here, a "fully active enzyme" refers to an enzyme that has the same function as the original function of a wild-type enzyme that cleaves nucleic acids, genes, or chromosomes. For example, a wild-type enzyme that cleaves double-stranded DNA may be a fully active enzyme that cleaves all double-stranded DNA. In another example, when a wild-type enzyme that cleaves double-stranded DNA is artificially modified to delete or replace part of its amino acid sequence, if the artificially modified enzyme mutant cleaves double-stranded DNA equally well as the wild-type enzyme, then the artificially modified enzyme mutant may be a fully active enzyme.

[0712] In addition, an enzyme with full activity can include an enzyme with improved function compared to the function of the wild-type enzyme. For example, a specifically modified or engineered form of a wild-type enzyme that cleaves double-stranded DNA can have improved full enzymatic activity compared to the wild-type enzyme, i.e., improved activity in cleaving double-stranded DNA.

[0713] Editing proteins may include enzymes with incomplete or partial activity.

[0714] Herein, the term "enzyme with incomplete or partial activity" refers to an enzyme that has only a portion of the original function of a wild-type enzyme that cleaves nucleic acids, genes, or chromosomes. For example, a specifically modified or engineered form of a wild-type enzyme that cleaves double-stranded DNA may have a first function or a second function. Herein, the first function may be the function of cleaving the first strand of double-stranded DNA, and the second function may be the function of cleaving the second strand of double-stranded DNA. Herein, the enzyme with the first function or the enzyme with the second function may be an enzyme with incomplete or partial activity.

[0715] The editing protein may include an inactivated enzyme.

[0716] Herein, the term "inactivated enzyme" refers to an enzyme in which the original function of a wild-type enzyme for cleaving nucleic acids, genes, or chromosomes is completely inactivated. For example, a specifically modified or engineered form of a wild-type enzyme may lose both its primary function and its secondary function, i.e., both its primary function of cleaving the first strand of double-stranded DNA and its secondary function of cleaving the second strand of double-stranded DNA are lost. Herein, an enzyme that loses both its primary function and its secondary function may be considered an inactivated enzyme.

[0717] The editing protein can be a fusion protein.

[0718] Here, fusion protein refers to a protein produced by fusing an enzyme with an additional domain, peptide, polypeptide or protein.

[0719] The additional domain, peptide, polypeptide or protein may be a functional domain, peptide, polypeptide or protein having the same or different function as that of the functional domain, peptide, polypeptide or protein contained in the enzyme.

[0720] The fusion protein can comprise additional functional domains, peptides, polypeptides or proteins at one or more regions located at or near the amino terminus of the enzyme, at or near the carboxyl terminus of the enzyme, in the middle of the enzyme, and combinations thereof.

[0721] Here, the functional domain, peptide, polypeptide or protein may be a domain, peptide, polypeptide or protein having methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity or nucleic acid binding activity, or a tag or reporter gene for purifying and isolating proteins (including peptides), but the present invention is not limited thereto.

[0722] The functional domain, peptide, polypeptide or protein may be a deaminase.

[0723] Tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags and thioredoxin (Trx) tags; reporter genes include glutathione S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, autofluorescent proteins (including green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and blue fluorescent protein (BFP)), but the present invention is not limited thereto.

[0724] Furthermore, the functional domain, peptide, polypeptide or protein may be a nuclear localization sequence or signal (NLS) or a nuclear export sequence or signal (NES).

[0725] The NLS can be: the NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV; an NLS derived from a nucleoplasmin (e.g., the nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK); the c-myc NLS having the amino acid sequence PAAKRVKLD or RQRRNELKRSP; the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the IBB domain sequence derived from importin-α, RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV; the myoma T protein sequences VSRKRPRP and PPKKARED; the human p53 sequence POPKKKPL; the mouse c-abl IV sequence SALIKKKKKMAP; influenza virus NS1 sequences DRLRR and PKQKKRK; hepatitis virus δ antigen sequence RKLKKKIKKL; mouse Mx1 protein sequence REKKKFLKRR; human poly (ADP-ribose) polymerase sequence KRKGDEVDGVDEVAKKKSKK; or steroid hormone receptor (human) glucocorticoid sequence RKCLQAGMNLEARKTKK, but the present invention is not limited thereto.

[0726] The additional domain, peptide, polypeptide or protein may be a dysfunctional domain, peptide, polypeptide or protein that does not perform a specific function. Here, the dysfunctional domain, peptide, polypeptide or protein may be a domain, peptide, polypeptide or protein that does not affect the function of the enzyme.

[0727] The fusion protein can comprise an additional dysfunctional domain, peptide, polypeptide or protein at one or more regions located at or near the amino terminus of the enzyme, at or near the carboxyl terminus of the enzyme, in the middle of the enzyme, and combinations thereof.

[0728] The editing protein can be a natural enzyme or a fusion protein.

[0729] The editing protein can exist in the form of a partially modified native enzyme or a fusion protein.

[0730] The editing protein can be an artificially produced enzyme or fusion protein that does not exist in nature.

[0731] The editing protein can exist in the form of a partially modified artificial enzyme or a fusion protein that does not exist in the natural state.

[0732] Here, the modification may be substitution, deletion, addition, or a combination of the above modifications of the amino acids contained in the editing protein.

[0733] Furthermore, the modification may be a substitution, deletion, addition, or a combination of the above modifications to some bases in the base sequence encoding the editing protein.

[0734] In addition, the composition for gene manipulation may optionally further comprise a donor or a nucleic acid sequence encoding the donor, wherein the donor comprises a specific nucleotide sequence desired to be inserted.

[0735] Here, the nucleotide sequence desired to be inserted may be a partial nucleotide sequence in a gene involved in immunity.

[0736] Here, the nucleotide sequence desired to be inserted may be a nucleotide sequence that corrects or introduces a mutation in the immunomodulatory gene to be manipulated.

[0737] The term "donor" refers to a nucleic acid sequence that helps repair damaged genes or nucleic acids through HDR.

[0738] The donor can be a double-stranded nucleic acid or a single-stranded nucleic acid.

[0739] The donor can be linear or circular.

[0740] The donor can comprise a nucleic acid sequence that has homology to the target gene or nucleic acid.

[0741] For example, the donor may contain nucleotide sequences that share homology with nucleotide sequences at locations where the specific nucleic acid is to be inserted (e.g., upstream and downstream of the damaged nucleic acid). Specifically, the specific nucleic acid to be inserted may be located between a nucleotide sequence that shares homology with a nucleotide sequence downstream of the damaged nucleic acid and a nucleotide sequence that shares homology with a nucleotide sequence upstream of the damaged nucleic acid. Specifically, the nucleotide sequences sharing such homology may share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or greater homology, or may be completely homologous.

[0742] The donor may optionally contain additional nucleotide sequences. Specifically, the additional nucleotide sequences may play a role in enhancing the HDR efficiency, stability or knock-in efficiency of the donor.

[0743] For example, the additional nucleotide sequence may be a nucleotide sequence rich in A and T bases (ie, an AT-rich domain). Alternatively, the additional nucleotide sequence may be a scaffold / matrix attachment region (S / MAR).

[0744] The guide nucleic acid, editing protein, or guide nucleic acid-editing protein complex disclosed herein can be delivered or introduced into a subject in various forms.

[0745] The term "subject" refers to an organism into which a guide nucleic acid, editing protein, or guide nucleic acid-editing protein complex is introduced; an organism in which a guide nucleic acid, editing protein, or guide nucleic acid-editing protein complex operates; or a specimen or sample obtained from such an organism.

[0746] The subject can be an organism comprising a target gene or chromosome for the guide nucleic acid-editing protein complex.

[0747] The organism can be an animal, animal tissue, or animal cell.

[0748] The organism may be a human, a human tissue, or a human cell.

[0749] The tissue can be eye, skin, liver, kidney, heart, lung, brain, muscle, or blood.

[0750] The cell may be an immune cell, such as a natural killer cell (NK cell), a T cell, a B cell, a dendritic cell, and a macrophage, or a stem cell.

[0751] A test specimen or sample can be obtained from an organism containing the target gene or chromosome (e.g., saliva, blood, liver tissue, brain tissue, hepatocytes, neurons, phagocytes, T cells, B cells, astrocytes, cancer cells, or stem cells).

[0752] Preferably, the subject may be an organism comprising an immunomodulatory gene.

[0753] The guide nucleic acid, editing protein, or guide nucleic acid-editing protein complex can be delivered or introduced into a subject in the form of DNA, RNA, or a mixed form.

[0754] Here, DNA, RNA, or a mixture thereof encoding the guide nucleic acid and / or editing protein can be delivered or introduced into a subject using methods known in the art.

[0755] Alternatively, DNA, RNA, or a mixture thereof encoding the guide nucleic acid and / or editing protein can be delivered or introduced into a subject via a vector, non-vector, or a combination thereof.

[0756] The vector can be a viral vector or a non-viral vector (eg, a plasmid).

[0757] The non-vector can be naked DNA, DNA complex or mRNA.

[0758] Nucleic acid sequences encoding guide nucleic acids and / or editing proteins can be delivered or introduced into a subject with the aid of a vector.

[0759] The vector may comprise a nucleic acid sequence encoding a guide nucleic acid and / or an editing protein.

[0760] For example, a vector may contain nucleic acid sequences encoding both a guide nucleic acid and an editing protein, respectively.

[0761] For example, a vector can comprise a nucleic acid sequence encoding a guide nucleic acid.

[0762] As an example, the domains included in the guide nucleic acid may all be included in one vector, or may be divided and then included in different vectors.

[0763] For example, a vector can comprise a nucleic acid sequence encoding an editing protein.

[0764] In one example, in the case of an editing protein, the nucleic acid sequence encoding the editing protein may be contained in one vector, or may be divided and then contained in several vectors.

[0765] A vector may contain one or more regulatory / control components.

[0766] Here, the regulatory / control components may include: a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, an internal ribosome entry site (IRES), a splice acceptor and / or a 2A sequence.

[0767] The promoter may be a promoter recognized by RNA polymerase II.

[0768] The promoter may be a promoter recognized by RNA polymerase III.

[0769] The promoter may be an inducible promoter.

[0770] The promoter may be a subject-specific promoter.

[0771] The promoter can be a viral or non-viral promoter.

[0772] As for the promoter, an appropriate promoter can be used depending on the control region (ie, the nucleic acid sequence encoding the guide nucleic acid or the editing protein).

[0773] For example, a promoter that can be used to guide nucleic acids can be an H1, EF-1a, tRNA, or U6 promoter. For example, a promoter that can be used to edit proteins can be a CMV, EF-1a, EFS, MSCV, PGK, or CAG promoter.

[0774] The vector may be a viral vector or a recombinant viral vector.

[0775] The virus can be a DNA virus or an RNA virus.

[0776] Here, the DNA virus may be a double-stranded DNA (dsDNA) virus or a single-stranded DNA (ssDNA) virus.

[0777] Here, the RNA virus may be a single-stranded RNA (ssRNA) virus.

[0778] The virus may be a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus (AAV), a vaccinia virus, a poxvirus, or a herpes simplex virus, but the present invention is not limited thereto.

[0779] In general, viruses can infect a host (e.g., a cell), thereby introducing a nucleic acid encoding viral genetic information into the host or inserting a nucleic acid encoding genetic information into the host genome. Viruses with such characteristics can be used to introduce guide nucleic acids and / or editing proteins into a subject. The guide nucleic acids and / or editing proteins introduced using viruses can be transiently expressed in a subject (e.g., a cell). Alternatively, the guide nucleic acids and / or editing proteins introduced using viruses can be continuously expressed in a subject (e.g., a cell) for a long time (e.g., 1, 2, 3, 6, or 9 months, 1 or 2 years, or permanently).

[0780] Depending on the type of virus, the packaging capacity of the virus can vary from at least 2 kb to 50 kb. Depending on such packaging capacity, a viral vector comprising either a guide nucleic acid or an editing protein or a viral vector comprising both a guide nucleic acid and an editing protein can be designed. Alternatively, a viral vector comprising a guide nucleic acid, an editing protein, and additional components can be designed.

[0781] In one example, recombinant lentiviruses can be used to deliver or introduce nucleic acid sequences encoding guide nucleic acids and / or editing proteins.

[0782] In another example, recombinant adenovirus can be used to deliver or introduce nucleic acid sequences encoding guide nucleic acids and / or editing proteins.

[0783] In yet another embodiment, recombinant AAV can be used to deliver or introduce nucleic acid sequences encoding guide nucleic acids and / or editing proteins.

[0784] In yet another example, hybrid viruses (e.g., a mixture of one or more of the viruses listed herein) can be used to deliver or introduce nucleic acid sequences encoding guide nucleic acids and / or editing proteins.

[0785] Nucleic acid sequences encoding guide nucleic acids and / or editing proteins can be delivered or introduced into a subject using non-vectors.

[0786] The non-vector may comprise a nucleic acid sequence encoding a guide nucleic acid and / or an editing protein.

[0787] The non-vector can be naked DNA, DNA complex, mRNA or a mixture thereof.

[0788] The non-vector can be delivered into or introduced into a subject by means of electroporation, particle bombardment, sonoporation, magnetic transfection, transient cell compression or squeezing (as described in, for example, Lee et al. (2012) NanoLett., 12, 6322-6327), lipid-mediated transfection, dendrimers, nanoparticles, calcium phosphate, silica, ormosil, or a combination thereof.

[0789] As an example, delivery via electroporation can be performed by mixing cells with nucleic acid sequences encoding guide nucleic acids and / or editing proteins in a cartridge, chamber, or cuvette and applying an electrical stimulus to the cells at a predetermined duration and amplitude.

[0790] In another example, nanoparticles can be used to deliver non-carriers. The nanoparticles can be inorganic nanoparticles (e.g., magnetic nanoparticles, silica, etc.) or organic nanoparticles (e.g., lipids coated with polyethylene glycol (PEG), etc.). The outer surface of the nanoparticles can be conjugated with a positively charged polymer (e.g., polyethyleneimine, polylysine, polyserine, etc.) that can be attached.

[0791] In certain embodiments, non-vectors may be delivered using a lipid shell.

[0792] In certain embodiments, non-vector delivery can be performed using exosomes, which are endogenous nanovesicles that transfer proteins and RNA and can deliver RNA to the brain and other target organs.

[0793] In certain embodiments, non-carriers can be delivered using liposomes. Liposomes are spherical vesicle structures composed of a single or multiple lamellar lipid bilayer surrounding an internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Although liposomes can be made from several different types of lipids, phospholipids are most commonly used to produce liposomes as drug carriers.

[0794] Additionally, compositions for non-vector delivery may contain other additives.

[0795] The editing protein can be delivered or introduced into a subject in the form of a peptide, polypeptide, or protein.

[0796] The editing protein can be delivered or introduced into a subject in the form of a peptide, polypeptide, or protein by methods known in the art.

[0797] The peptide, polypeptide or protein form can be delivered into or introduced into a subject by means of electroporation, microinjection, transient cell compression or squeezing (such as described in the document "Lee et al. (2012) NanoLett., 12, 6322-6327"), lipid-mediated transfection, nanoparticles, liposomes, peptide-mediated delivery or a combination thereof.

[0798] The peptide, polypeptide or protein can be delivered together with a nucleic acid sequence encoding a guide nucleic acid.

[0799] In one example, delivery via electroporation can be performed by mixing cells into which the editing protein is to be introduced with or without a guide nucleic acid in a cartridge, chamber, or cuvette and applying an electrical stimulus to the cells at a predetermined duration and amplitude.

[0800] The guide nucleic acid and editing protein can be delivered or introduced into a subject as a nucleic acid-protein mixture.

[0801] The guide nucleic acid and editing protein can be delivered or introduced into a subject in the form of a guide nucleic acid-editing protein complex.

[0802] For example, the guide nucleic acid can be DNA, RNA, or a mixture thereof. The editing protein can be a peptide, polypeptide, or protein.

[0803] In one example, the guide nucleic acid and editing protein can be delivered or introduced into a subject in the form of a guide nucleic acid-editing protein complex (i.e., ribonucleoprotein (RNP)) comprising an RNA-type guide nucleic acid and a protein-type editing protein.

[0804] The guide nucleic acid-editing protein complex disclosed in this specification can modify target nucleic acids, genes or chromosomes.

[0805] For example, the guide nucleic acid-editing protein complex induces modification of the sequence of the target nucleic acid, gene or chromosome. Thus, the protein expressed by the target nucleic acid, gene or chromosome can have its modified structure and / or function, its controlled expression or its deleted expression.

[0806] The guide nucleic acid-editing protein complex can act at the DNA, RNA, gene, or chromosome level.

[0807] In one example, the guide nucleic acid-editing protein complex can be used to modulate (e.g., inhibit, repress, reduce, increase, or promote) the expression of a protein encoded by a target gene, or to modulate (e.g., inhibit, repress, reduce, increase, or promote) the activity of a protein, or to express a modified protein by engineering or modifying the target gene.

[0808] The guide nucleic acid-editing protein complex can play a role in both gene transcription and translation.

[0809] In one example, the guide nucleic acid-editing protein complex can promote or repress the transcription of the target gene, thereby regulating (e.g., inhibiting, repressing, reducing, increasing, or promoting) the expression of the protein encoded by the target gene.

[0810] In another example, the guide nucleic acid-editing protein complex can promote or repress the translation of the target gene, thereby regulating (e.g., inhibiting, repressing, reducing, increasing, or promoting) the expression of the protein encoded by the target gene.

[0811] In the embodiments disclosed herein, the composition for gene manipulation may include gRNA and CRISPR enzyme.

[0812] A composition for genetic manipulation may comprise:

[0813] (a) gRNA, which is capable of forming a complementary bond with a target sequence of an immunomodulatory gene or a nucleic acid sequence encoding the target sequence of the immunomodulatory gene; and

[0814] (b) one or more CRISPR enzymes or nucleic acid sequences encoding said CRISPR enzymes.

[0815] The explanations about the above-mentioned immunoregulatory genes are as described above.

[0816] The explanation about the above target sequence is as described above.

[0817] The genetic manipulation composition may comprise a gRNA-CRISPR enzyme complex.

[0818] The term "gRNA-CRISPR enzyme complex" refers to the complex formed by the interaction between gRNA and CRISPR enzyme.

[0819] The explanation about the above gRNA is as described above.

[0820] "CRISPR enzyme" is the main protein component of the CRISPR-Cas system, which forms a complex with gRNA to form the CRISPR-Cas system.

[0821] The CRISPR enzyme may be a nucleic acid or a polypeptide (or protein) having a sequence encoding a CRISPR enzyme.

[0822] The CRISPR enzyme may be a type II CRISPR enzyme.

[0823] The crystal structure of the type II CRISPR enzyme was determined based on studies of more than two types of naturally occurring microbial type II CRISPR enzyme molecules (Jinek et al., Science, 343(6176):1247997, 2014) and studies of Streptococcus pyogenes Cas9 (SpCas9) in complex with gRNA (Nishimasu et al., Cell, 156:935-949, 2014; and Anders et al., Nature, 2014, doi:10.1038 / nature13579).

[0824] Type II CRISPR enzymes comprise two lobes, the recognition (REC) lobe and the nuclease (NUC) lobe, each containing several domains.

[0825] The REC lobe contains an arginine-rich helical bridge (BH) domain, the REC1 domain, and the REC2 domain.

[0826] Here, the BH domain is a long α-helix and an arginine-rich region, while the REC1 domain and REC2 domain play an important role in recognizing the duplex formed in gRNA (e.g., single-stranded gRNA, double-stranded gRNA, or tracrRNA).

[0827] The NUC lobe comprises a RuvC domain, an HNH domain and a PAM interaction (PI) domain. Here, the RuvC domain includes a RuvC-like domain, or the HNH domain is used to include an HNH-like domain.

[0828] Here, the RuvC domain shares structural similarity with members of the naturally occurring microbial family with type II CRISPR enzymes and cuts a single strand (e.g., a non-complementary strand of a target gene or nucleic acid, i.e., a strand that does not form a complementary bond with the gRNA). In the art, the RuvC domain is sometimes referred to as the RuvCI domain, the RuvCII domain, or the RuvCIII domain, generally referred to as RuvCI, RuvCII, or RuvCIII.

[0829] The HNH domain shares structural similarity with the HNH endonuclease and cuts a single strand (e.g., the complementary strand of the target nucleic acid molecule, i.e., the strand that forms a complementary bond with the gRNA). The HNH domain is located between the RuvCII and III motifs.

[0830] The PI domain recognizes a specific nucleotide sequence (i.e., a protospacer adjacent motif (PAM)) in a target gene or nucleic acid or interacts with a PAM. Here, the PAM may vary depending on the source of the type II CRISPR enzyme. For example, when the CRISPR enzyme is SpCas9, the PAM may be 5'-NGG-3'; when the CRISPR enzyme is Streptococcus thermophilus Cas9 (StCas9), the PAM may be 5'-NNAGAAW-3' (W = A or T); when the CRISPR enzyme is Neisseria meningitidis Cas9 (NmCas9), the PAM may be 5'-NNNNGATT-3'; when the CRISPR enzyme is Campylobacter jejuni Cas9 (CjCas9), the PAM may be 5'-NNNVRYAC-3' (V = G or C or A; R = A or G; Y = C or T), where N may be A, T, G or C, or A, U, G or C. However, although it is generally understood that the PAM is determined according to the origin of the enzyme as described above, the PAM may change as research progresses on mutants of the enzyme from that origin.

[0831] The type II CRISPR enzyme can be Cas9.

[0832] Cas9 can be derived from various microorganisms, such as Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp.), Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor bescii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicu, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsonii, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngby asp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, and Acaryochloris marina.

[0833] Cas9 is an enzyme that binds to a gRNA to cleave or modify a target sequence or location on a target gene or nucleic acid. It is composed of an HNH domain (capable of cleaving a nucleic acid strand complementary to the gRNA), a RuvC domain (capable of cleaving a nucleic acid strand non-complementarily bound to the gRNA), a REC domain (target recognition), and a PI domain (PAM recognition). For detailed structural features of Cas9, see Hiroshi Nishimasu et al. (2014) Cell 156:935-949.

[0834] Cas9 can be isolated from naturally occurring microorganisms or produced non-naturally by recombinant or synthetic methods.

[0835] Furthermore, the CRISPR enzyme may be a type V CRISPR enzyme.

[0836] Type V CRISPR enzymes contain a similar RuvC domain (corresponding to the RuvC domain of type II CRISPR enzymes) and may be composed of a Nuc domain (instead of the HNH domain of type II CRISPR enzymes), a REC domain and a WED domain (interacting with the target), and a PI domain (recognizing PAM). For the specific structural features of type V CRISPR enzymes, see Takashi Yamano et al. (2016) Cell 165: 949-962.

[0837] Type V CRISPR enzymes can interact with gRNAs to form a gRNA-CRISPR enzyme complex, or CRISPR complex, and can, in collaboration with the gRNA, allow the guide sequence to access a target sequence containing a PAM sequence. Here, the ability of type V CRISPR enzymes to interact with target genes or nucleic acids depends on the PAM sequence.

[0838] The PAM sequence is a sequence present in the target gene or nucleic acid that is recognized by the PI domain of the V-type CRISPR enzyme. The PAM sequence may vary depending on the source of the V-type CRISPR enzyme. That is, depending on the species, there are different PAM sequences that can be specifically recognized. For example, the PAM sequence recognized by Cpf1 may be 5'-TTN-3' (N is A, T, C or G). However, although it is generally understood that the PAM is determined based on the source of the enzyme as described above, the PAM may change as research progresses on mutants of the enzyme from that source.

[0839] The type V CRISPR enzyme may be Cpf1.

[0840] Cpf1 may be a Cpf1 derived from Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridium, diaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium, or Acidaminococcus.

[0841] Cpf1 contains a RuvC domain (similar to and corresponding to the RuvC domain of type II CRISPR enzymes) and may be composed of a Nuc domain (instead of the HNH domain of Cas9), a REC domain and a WED domain (interacting with the target), and a PI domain (recognizing PAM). For the specific structural features of Cpf1, see Takashi Yamano et al. (2016) Cell 165: 949-962.

[0842] Cpf1 can be isolated from naturally occurring microorganisms or produced non-naturally by recombinant or synthetic methods.

[0843] The CRISPR enzyme can be a nuclease or restriction enzyme that has the function of cleaving both strands of the target gene or nucleic acid.

[0844] The CRISPR enzyme may be a fully active CRISPR enzyme.

[0845] Here, "fully active" refers to a state in which the enzyme has the same function as a wild-type CRISPR enzyme, and a CRISPR enzyme in this state is referred to as a "fully active CRISPR enzyme." Here, "function of a wild-type CRISPR enzyme" refers to a state in which the enzyme has the function of cleaving double-stranded DNA, i.e., a state in which the enzyme has the first function of cleaving the first strand of double-stranded DNA and the second function of cleaving the second strand of double-stranded DNA.

[0846] A fully active CRISPR enzyme may be a wild-type CRISPR enzyme that cleaves double-stranded DNA.

[0847] A fully active CRISPR enzyme may be a CRISPR enzyme mutant in which the wild-type CRISPR enzyme that cleaves double-stranded DNA is modified or manipulated.

[0848] A CRISPR enzyme mutant may be an enzyme in which one or more amino acids in the amino acid sequence of a wild-type CRISPR enzyme are substituted with another amino acid or one or more amino acids are deleted.

[0849] A CRISPR enzyme mutant can be an enzyme in which one or more amino acids are added to the amino acid sequence of a wild-type CRISPR enzyme. Here, the position of the added amino acid can be the N-terminus, the C-terminus, or within the amino acid sequence of the wild-type enzyme.

[0850] The CRISPR enzyme mutant can be a fully active enzyme with improved function compared to the wild-type CRISPR enzyme.

[0851] For example, a particular modified or manipulated form of a wild-type CRISPR enzyme (i.e., a CRISPR enzyme mutant) can cleave double-stranded DNA without binding to or remaining at a distance from the double-stranded DNA to be cleaved. In this case, the modified or manipulated form can be a fully active CRISPR enzyme with improved function compared to the wild-type CRISPR enzyme.

[0852] A CRISPR enzyme mutant can be a fully active enzyme with reduced function compared to a wild-type CRISPR enzyme.

[0853] For example, a specific modified or manipulated form of a wild-type CRISPR enzyme (i.e., a CRISPR enzyme mutant) can cleave double-stranded DNA at a specific distance from or closer to the double-stranded DNA to be cleaved, or in the presence of a certain binding. Here, the certain binding can be, for example, binding between an amino acid at a specific position of the enzyme and a DNA nucleotide sequence in the cleavage position. In this case, the modified or manipulated form can be a fully active CRISPR enzyme with reduced function compared to the wild-type CRISPR enzyme.

[0854] The CRISPR enzyme may be a CRISPR enzyme with incomplete or partial activity.

[0855] The term "having incomplete or partial activity" refers to a state in which a function is selected from the function of a wild-type CRISPR enzyme (i.e., a first function of cleaving the first strand of double-stranded DNA, and a second function of cleaving the second strand of double-stranded DNA). In addition, a CRISPR enzyme having incomplete or partial activity may be referred to as a nickase.

[0856] The term "nickase" refers to a CRISPR enzyme that has been manipulated or modified to cleave only one strand of a double-stranded target gene or nucleic acid. The nickase has nuclease activity that cleaves a single strand (e.g., a strand that is not complementary to the gRNA of the target gene or nucleic acid or a strand that is complementary to it). Therefore, the nuclease activity of two nickases is required to cleave a double strand.

[0857] The nickase may have the nuclease activity of the RuvC domain of the CRISPR enzyme. That is, the nickase may not comprise the nuclease activity of the HNH domain of the CRISPR enzyme, for which the HNH domain may be manipulated or modified.

[0858] In one example, when the CRISPR enzyme is a type II CRISPR enzyme, the nickase may be a type II CRISPR enzyme comprising a modified HNH domain.

[0859] For example, when the type II CRISPR enzyme is wild-type SpCas9, the nickase can be a SpCas9 mutant in which residue 840 in the amino acid sequence of the wild-type SpCas9 is mutated from histidine to alanine and the nuclease activity of the HNH domain is inactivated. Here, the resulting nickase (i.e., the SpCas9 mutant) has the nuclease activity of the RuvC domain and is therefore able to cleave the non-complementary strand of the target gene or nucleic acid, i.e., the strand that does not form a complementary bond with the gRNA.

[0860] In another example, when the type II CRISPR enzyme is wild-type CjCas9, the nickase may be a CjCas9 mutant in which residue 559 in the wild-type CjCas9 amino acid sequence is mutated from histidine to alanine and the nuclease activity of the HNH domain is inactivated. Here, the resulting nickase (i.e., CjCas9 mutant) has the nuclease activity of the RuvC domain and is therefore able to cut the non-complementary strand of the target gene or nucleic acid, i.e., the strand that does not form a complementary bond with the gRNA.

[0861] Furthermore, the nickase may possess the nuclease activity of the HNH domain of the CRISPR enzyme. That is, the nickase may not comprise the nuclease activity of the RuvC domain of the CRISPR enzyme, for which purpose the RuvC domain may be manipulated or modified.

[0862] In one example, when the CRISPR enzyme is a type II CRISPR enzyme, the nickase may be a type II CRISPR enzyme comprising a modified RuvC domain.

[0863] For example, when the type II CRISPR enzyme is wild-type SpCas9, the nickase can be a SpCas9 mutant in which the 10th residue in the amino acid sequence of the wild-type SpCas9 is mutated from aspartic acid to alanine and the nuclease activity of the RuvC domain is inactivated. Here, the resulting nickase (i.e., the SpCas9 mutant) has the nuclease activity of the HNH domain and is therefore able to cut the complementary strand of the target gene or nucleic acid, i.e., the strand that forms a complementary bond with the gRNA.

[0864] In another example, when the type II CRISPR enzyme is wild-type CjCas9, the nickase may be a CjCas9 mutant in which residue 8 in the amino acid sequence of the wild-type CjCas9 is mutated from aspartic acid to alanine and the nuclease activity of the RuvC domain is inactivated. Here, the resulting nickase (i.e., the CjCas9 mutant) has the nuclease activity of the HNH domain and is therefore able to cut the complementary strand of the target gene or nucleic acid, i.e., the strand that forms a complementary bond with the gRNA.

[0865] The CRISPR enzyme may be an inactivated CRISPR enzyme.

[0866] The term "inactivated" refers to a state in which the functions of a wild-type CRISPR enzyme (i.e., the first function of cleaving the first strand of double-stranded DNA and the second function of cleaving the second strand of double-stranded DNA) are completely lost. The CRISPR enzyme in this state is called an inactivated CRISPR enzyme.

[0867] By mutation in a domain of a wild-type CRISPR enzyme that has nuclease activity, an inactivated CRISPR enzyme can be made to have an inactivated nuclease.

[0868] The inactivated CRISPR enzyme may be one in which the nuclease activity of the RuvC domain and the HNH domain is inactivated due to a mutation. That is, the inactivated CRISPR enzyme may not comprise the nuclease activity of the RuvC domain and the HNH domain of the CRISPR enzyme, for which purpose the RuvC domain and the HNH domain may be manipulated or modified.

[0869] In one example, when the CRISPR enzyme is a type II CRISPR enzyme, the inactivated CRISPR enzyme may be a type II CRISPR enzyme comprising a modified RuvC domain and an HNH domain.

[0870] For example, when the type II CRISPR enzyme is wild-type SpCas9, the inactivated CRISPR enzyme may be a SpCas9 mutant in which the nuclease activity of the RuvC domain and the HNH domain is inactivated by mutating residue 10 and residue 840 in the amino acid sequence of the wild-type SpCas9 from aspartic acid and histidine to alanine, respectively. Here, the resulting inactivated CRISPR enzyme (i.e., the SpCas9 mutant) has the nuclease activity of the inactivated RuvC domain and the HNH domain, and thus may not cleave the double strand of the target gene or nucleic acid at all.

[0871] In another example, when the type II CRISPR enzyme is wild-type CjCas9, the inactivated CRISPR enzyme may be a CjCas9 mutant in which the nuclease activity of the RuvC domain and the HNH domain is inactivated by mutating residues 8 and 559 in the amino acid sequence of the wild-type CjCas9 from aspartic acid and histidine to alanine, respectively. Here, the resulting inactivated CRISPR enzyme (i.e., SpCas9 mutant) has the nuclease activity of the inactivated RuvC domain and HNH domain, and thus may not cleave the double strand of the target gene or nucleic acid at all.

[0872] In addition to the nuclease activity described above, CRISPR enzymes may have helicase activity, i.e., the ability to unwind the helical structure of double-stranded nucleic acids.

[0873] Furthermore, the CRISPR enzyme can be modified so that the CRISPR enzyme has full, incomplete, or partial helicase activity.

[0874] The CRISPR enzyme may be a CRISPR enzyme mutant in which the wild-type CRISPR enzyme has been artificially manipulated or modified.

[0875] A CRISPR enzyme mutant may be a CRISPR enzyme mutant that has been artificially manipulated or modified to modify the function of a wild-type CRISPR enzyme (i.e., a first function of cleaving a first strand of double-stranded DNA, and / or a second function of cleaving a second strand of double-stranded DNA).

[0876] For example, a CRISPR enzyme mutant can be a form that has lost the first function of the wild-type CRISPR enzyme.

[0877] Alternatively, the CRISPR enzyme mutant may be a form that has lost a second function of the wild-type CRISPR enzyme.

[0878] For example, a CRISPR enzyme mutant may be a form that has lost the functions (i.e., the first function and the second function) of the wild-type CRISPR enzyme.

[0879] CRISPR enzyme mutants can form a gRNA-CRISPR enzyme complex by interacting with gRNA.

[0880] The CRISPR enzyme mutant can be a CRISPR enzyme mutant that has been artificially manipulated or modified to modify the function of the wild-type CRISPR enzyme in interacting with the gRNA.

[0881] For example, a CRISPR enzyme mutant can be a form that has reduced interaction with the gRNA compared to the wild-type CRISPR enzyme.

[0882] Alternatively, the CRISPR enzyme mutant may be a form that has increased interaction with the gRNA compared to the wild-type CRISPR enzyme.

[0883] For example, a CRISPR enzyme mutant can be a form that has the primary function of a wild-type CRISPR enzyme and has reduced interaction with the gRNA.

[0884] Alternatively, the CRISPR enzyme mutant may be a form that has the primary function of the wild-type CRISPR enzyme and has increased interaction with the gRNA.

[0885] For example, a CRISPR enzyme mutant can be a form that has a secondary function of the wild-type CRISPR enzyme and reduced interaction with the gRNA.

[0886] Alternatively, the CRISPR enzyme mutant may be a form that has a secondary function of the wild-type CRISPR enzyme and increased interaction with the gRNA.

[0887] For example, a CRISPR enzyme mutant may be a form that lacks the first and second functions of the wild-type CRISPR enzyme but has reduced interaction with the gRNA.

[0888] Alternatively, the CRISPR enzyme mutant may be a form that lacks the primary and secondary functions of the wild-type CRISPR enzyme but has increased interaction with the gRNA.

[0889] Here, depending on the strength of the interaction between gRNA and CRISPR enzyme mutants, various gRNA-CRISPR enzyme complexes can be formed, and the function of accessing or cleaving the target sequence can be changed depending on the CRISPR enzyme mutant.

[0890] For example, a gRNA-CRISPR enzyme complex formed by a CRISPR enzyme mutant with reduced interaction with the gRNA is able to cleave the double-stranded or single-stranded target sequence only when it is close to or positioned at the target sequence that forms a fully complementary binding with the gRNA.

[0891] A CRISPR enzyme mutant may comprise a wild-type CRISPR enzyme with at least one amino acid modification.

[0892] In one example, a CRISPR enzyme mutant may comprise at least one substitution of an amino acid in a wild-type CRISPR enzyme.

[0893] In another example, a CRISPR enzyme mutant may have at least one amino acid deleted from a wild-type CRISPR enzyme.

[0894] In yet another example, the CRISPR enzyme mutant may have at least one addition of an amino acid to the wild-type CRISPR enzyme.

[0895] In one example, a CRISPR enzyme mutant may comprise a substitution, deletion, and / or addition of at least one of the amino acids of a wild-type CRISPR enzyme.

[0896] Furthermore, in addition to the original function of the wild-type CRISPR enzyme (i.e., a first function of cleaving the first strand of double-stranded DNA and a second function of cleaving the second strand of double-stranded DNA), the CRISPR enzyme mutant may further comprise an optional functional domain. Here, the CRISPR enzyme mutant may have additional functions in addition to the original function of the wild-type CRISPR enzyme.

[0897] The functional domain may be a domain having methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity or nucleic acid binding activity, or a tag or reporter gene for separating and purifying proteins (including peptides), but the present invention is not limited thereto.

[0898] Tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags and thioredoxin (Trx) tags; reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, autofluorescent proteins (including green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and blue fluorescent protein (BFP)), but the present invention is not limited thereto.

[0899] The functional domain may be a deaminase.

[0900] For example, an incomplete or partial CRISPR enzyme can additionally contain a cytidine deaminase as a functional domain. In one exemplary embodiment, a cytidine deaminase (e.g., apolipoprotein B editing complex 1 (APOBEC1)) can be added to the SpCas9 nickase to generate a fusion protein. The resulting [SpCas9 nickase]-[APOBEC1] can be used for editing from nucleotides C to T or U, or from nucleotides G to A, or for nucleotide repair.

[0901] In another example, an incomplete or partial CRISPR enzyme may further comprise a cytidine deaminase as a functional domain. In one embodiment, an adenine deaminase (e.g., TadA variant, ADAR2 variant, ADAT2 variant, etc.) can be added to the SpCas9 nickase to produce a fusion protein. The [SpCas9 nickase]-[TadA variant], [SpCas9 nickase]-[ADAR2 variant], or [SpCas9 nickase]-[ADAT2 variant] thus formed modifies nucleotide A to inosine, which is recognized by the polymerase as nucleotide G and essentially exhibits a repair or editing effect from nucleotide A to G, and can therefore be used in editing or nucleotide repair from nucleotide A to G, or from nucleotide T to C.

[0902] The functional domain can be a nuclear localization sequence or signal (NLS) or a nuclear export sequence or signal (NES).

[0903] In one example, the CRISPR enzyme may comprise one or more NLSs. Here, the one or more NLSs may be contained at or near the N-terminus of the CRISPR enzyme, at or near the C-terminus of the enzyme, or a combination thereof. The NLS may be an NLS sequence derived from the following NLSs, but the present invention is not limited thereto: the NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV; an NLS from a nucleoplasmic protein (e.g., a bipartite nucleoplasmic protein NLS having the sequence KRPAATKKAGQAKKKK); a c-myc NLS having the amino acid sequence PAAKRVKLD or RQRRNELKRSP; an hRNPA1M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV from the IBB domain of importin α; the sequences VSRKRPRP and PPKKARED of myoma T protein; the sequence POPKKKPL of human p53; and the sequence of mouse c-abl The sequence of IV is SALIKKKKKMAP; the sequences of influenza virus NS1 are DRLRR and PKQKKRK; the sequence of hepatitis virus delta antigen is RKLKKKIKKL; the sequence of mouse Mx1 protein is REKKKFLKRR; the sequence of human poly (ADP-ribose) polymerase is KRKGDEVDGVDEVAKKKSKK; or the NLS sequence derived from the sequence of steroid hormone receptor (human) glucocorticoid is RKCLQAGMNLEARKTKK.

[0904] In addition, CRISPR enzyme mutants may include split-type CRISPR enzymes prepared by dividing the CRISPR enzyme into two or more parts. The term "split" refers to the functional or structural division of a protein, or the random division of a protein into two or more parts.

[0905] The split CRISPR enzyme can be a fully active enzyme, an enzyme with incomplete or partial activity, or an inactivated enzyme.

[0906] For example, when the CRISPR enzyme is SpCas9, SpCas9 can be divided into two parts between residue 656 (tyrosine) and residue 657 (threonine) to produce a split SpCas9.

[0907] Split CRISPR enzymes may optionally comprise additional domains, peptides, polypeptides or proteins for reconstitution.

[0908] Additional domains, peptides, polypeptides or proteins for remodeling can be assembled such that the split CRISPR enzyme is identical or similar in structure to the wild-type CRISPR enzyme.

[0909] The additional domains, peptides, polypeptides or proteins used for remodeling can be FRB and FKBP dimerization domains; inteins; ERT and VPR domains; or domains that form heterodimers under specific conditions.

[0910] For example, SpCas9 can be divided into two parts between residues 713 (serine) and 714 (glycine), thereby generating a split-type SpCas9. The FRB domain can be attached to one of the two parts, and the FKBP domain can be attached to the other part. In the resulting split-type SpCas9, the FRB domain and the FKBP domain can form a dimer in the presence of rapamycin, thereby generating a reconstituted CRISPR enzyme.

[0911] The CRISPR enzyme or CRISPR enzyme mutant described in the present invention can be a polypeptide, a protein, or a nucleic acid having a sequence encoding the polypeptide or protein, and codon optimization can be performed for the subject to be introduced with the CRISPR enzyme or CRISPR enzyme mutant.

[0912] The term "codon optimization" refers to a modification process to a nucleic acid sequence that improves expression in a host cell by replacing at least one codon in the native sequence with a codon that is more common or most frequently used in the host cell while maintaining the native amino acid sequence. Various species have specific preferences for specific codons for specific amino acids, and this codon preference (difference in codon usage between different organisms) is generally related to the translation efficiency of mRNA, believing that this depends on the characteristics of the codons translated and the availability of specific tRNA molecules. The dominant tRNA selected in a cell generally reflects the most frequently used codons in peptide synthesis. Therefore, genes can be customized in a given organism by optimizing gene expression based on codon optimization.

[0913] The gRNA, CRISPR enzyme, or gRNA-CRISPR enzyme complex disclosed herein can be delivered or introduced into a subject in various forms.

[0914] The explanation about the above subjects is as described above.

[0915] In embodiments, the gRNA and / or CRISPR enzyme may be delivered or introduced into a subject via a vector comprising nucleic acid sequences encoding the gRNA and / or CRISPR enzyme, respectively.

[0916] The vector may comprise a nucleic acid sequence encoding a gRNA and / or a CRISPR enzyme.

[0917] In one example, a vector can contain nucleic acid sequences encoding both a gRNA and a CRISPR enzyme.

[0918] In another example, the vector may comprise a nucleic acid sequence encoding a gRNA.

[0919] For example, the domains contained in the gRNA may all be contained in the vector, or the domains may be separated and contained individually in the vector.

[0920] In another example, a vector may comprise a nucleic acid sequence encoding a CRISPR enzyme.

[0921] For example, with respect to a CRISPR enzyme, the nucleic acid sequence encoding the CRISPR enzyme may be contained entirely within the vector, or it may be split and contained individually within the vector.

[0922] A vector may comprise one or more regulatory / control components.

[0923] Here, the regulatory / control components may include: a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, an internal ribosome entry site (IRES), a splice acceptor and / or a 2A sequence.

[0924] The promoter may be a promoter recognized by RNA polymerase II.

[0925] The promoter may be a promoter recognized by RNA polymerase III.

[0926] The promoter may be an inducible promoter.

[0927] The promoter may be a subject-specific promoter.

[0928] The promoter can be a viral promoter or a non-viral promoter.

[0929] With respect to the promoter, an appropriate promoter can be used depending on the control region (i.e., the nucleic acid sequence encoding the gRNA and / or CRISPR enzyme).

[0930] For example, a promoter useful for gRNA may be an H1, EF-1a, tRNA, or U6 promoter. For example, a promoter useful for CRISPR enzyme may be a CMV, EF-1a, EFS, MSCV, PGK, or CAG promoter.

[0931] The vector may be a viral vector or a recombinant viral vector.

[0932] The virus can be a DNA virus or an RNA virus.

[0933] Here, the DNA virus may be a double-stranded DNA (dsDNA) virus or a single-stranded DNA (ssDNA) virus.

[0934] Here, the RNA virus may be a single-stranded RNA (ssRNA) virus.

[0935] The virus can be, but is not limited to, a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus (AAV), a vaccinia virus, a poxvirus, or a herpes simplex virus.

[0936] In one example, the nucleic acid sequence encoding the gRNA and / or CRISPR enzyme can be delivered or introduced via a recombinant lentivirus.

[0937] In another example, the nucleic acid sequence encoding the gRNA and / or CRISPR enzyme can be delivered or introduced via a recombinant adenovirus.

[0938] In another example, nucleic acid sequences encoding gRNA and / or CRISPR enzymes can be delivered or introduced via recombinant AAV.

[0939] In yet another example, nucleic acid sequences encoding gRNA and / or CRISPR enzymes can be delivered or introduced via a hybrid virus (e.g., a hybrid of one or more of the viruses listed herein).

[0940] In embodiments, a form of a gRNA-CRISPR enzyme complex may be delivered or introduced into a subject.

[0941] For example, gRNA can be DNA, RNA, or a mixture thereof. CRISPR enzymes can be peptides, polypeptides, or proteins.

[0942] In one example, the gRNA and CRISPR enzyme can be delivered or introduced into a subject in the form of a gRNA-CRISPR enzyme complex (i.e., ribonucleoprotein (RNP)) comprising an RNA-type gRNA and a protein-type CRISPR.

[0943] The gRNA-CRISPR enzyme complex can be delivered into or introduced into a subject by means of electroporation, microinjection, transient cell compression or squeezing (such as described in the literature [Lee et al. (2012) NanoLett., 12, 6322-6327]), lipid-mediated transfection, nanoparticles, liposomes, peptide-mediated delivery, or a combination thereof.

[0944] The gRNA-CRISPR enzyme complex disclosed in this specification can be used to artificially manipulate or modify target genes (i.e., immune regulatory genes).

[0945] The gRNA-CRISPR enzyme complex (i.e., CRISPR complex) described above can be used to manipulate or modify a target gene. Here, manipulation or modification of a target gene includes all of the following stages: i) cutting or damaging the target gene; and ii) repairing or restoring the damaged target gene.

[0946] i) Cutting or damaging the target gene The CRISPR complex can be used to cut or damage the target gene, in particular to cut or damage the target sequence in the target gene.

[0947] The target sequence may be the target of the gRNA-CRISPR enzyme complex, and the target sequence may or may not contain a PAM sequence recognized by the CRISPR enzyme. Such a target sequence can provide the practitioner with important criteria for designing gRNA.

[0948] The target sequence can be specifically recognized by the gRNA of the gRNA-CRISPR enzyme complex, thereby positioning the gRNA-CRISPR enzyme complex in proximity to the recognized target sequence.

[0949] "Cleavage" at the target site refers to the breakage of the covalent backbone of the polynucleotide. Cleavage can include, but is not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds and can be performed by a variety of other methods. Both single-strand cleavage and double-strand cleavage are possible, and double-strand cleavage can occur as a result of two different single-strand cleavages. Double-strand cleavage can produce blunt ends or staggered ends.

[0950] In one example, using the CRISPR complex to cut or damage the target gene can be to completely cut or damage both strands of the target sequence.

[0951] In an embodiment, when the CRISPR enzyme is wild-type SpCas9, the CRISPR complex can completely cut the double strand of the target sequence that forms a complementary binding with the gRNA.

[0952] In another embodiment, when the CRISPR enzymes are SpCas9 Nickase (D10A) and SpCas9 Nickase (H840A), each CRISPR complex can independently cleave both single strands of the target sequence that forms a complementary binding with the gRNA. That is, SpCas9 Nickase (D10A) can cleave the complementary single strand of the target sequence that forms a complementary binding with the gRNA, while SpCas9 Nickase (H840A) can cleave the non-complementary single strand of the target sequence that forms a complementary binding with the gRNA. The cleavage can be performed sequentially or simultaneously.

[0953] In another example, using the CRISPR complex to cut or damage the target gene or nucleic acid can be to cut or damage only a single strand of the double strand of the target sequence. Here, the single strand can be a guide nucleic acid binding sequence in the target sequence that forms a complementary bond with the gRNA (i.e., a complementary single strand), or a guide nucleic acid non-binding sequence that does not form a complementary bond with the gRNA (i.e., a single strand that is non-complementary to the gRNA).

[0954] In one embodiment, when the CRISPR enzyme is SpCas9 nickase (D10A), the CRISPR complex can cut the guide nucleic acid binding sequence in the target sequence that forms a complementary binding with the gRNA, that is, SpCas9 nickase (D10A) can cut the complementary single strand, while the guide nucleic acid non-binding sequence that does not form a complementary binding with the gRNA (that is, a single strand that is non-complementary to the gRNA) may not be cut.

[0955] In another embodiment, when the CRISPR enzyme is SpCas9 nickase (H840A), the CRISPR complex can cut the non-binding sequence of the guide nucleic acid in the target sequence that does not form a complementary binding with the gRNA, that is, SpCas9 nickase (H840A) can cut the single strand that is non-complementary to the gRNA, while the guide nucleic acid binding sequence (i.e., the complementary single strand) in the target sequence that forms a complementary binding with the gRNA may not be cut.

[0956] In yet another example, the cleavage or damage of a target gene or nucleic acid using a CRISPR complex can be the partial removal of a nucleic acid fragment.

[0957] In an embodiment, when a CRISPR complex is formed by two gRNAs that form complementary binding with respective different target sequences and a wild-type SpCas9, the double-stranded target sequence that forms complementary binding with the first gRNA can be cut, and the double-stranded target sequence that forms complementary binding with the second gRNA can be cut, thereby deleting the nucleic acid fragment with the help of the first gRNA, the second gRNA and SpCas9.

[0958] ii) Repair or restoration of damaged target genes can be performed by non-homologous end joining (NHEJ) or homology-directed repair (HDR).

[0959] Non-homologous end joining (NHEJ) is a method for repairing double-strand breaks in DNA by ligating the ends of the cleaved double-stranded or single-stranded DNA. Generally speaking, the damaged double strand is repaired when the two compatible ends formed by the double-strand break (e.g., cleavage) are brought into contact with each other, completely joining the ends. NHEJ is a repair method that can be used throughout the cell cycle and typically occurs when the cell lacks a homologous genome template (e.g., during the G1 phase).

[0960] During the process of repairing damaged genes or nucleic acids using NHEJ, some insertions and / or deletions (indels) occur in the nucleic acid sequence of the NHEJ repair region. Such insertions and / or deletions cause frame shifts, resulting in frameshifted transcriptome mRNA. As a result, due to nonsense-mediated decay or the inability to synthesize normal proteins, inherent function is lost. In addition, even if the reading frame remains unchanged, mutations caused by a considerable number of insertions or deletions in the sequence can also lead to disruption of protein function. Because mutations in important functional domains may be less tolerated than mutations in non-critical regions of the protein, mutations are locus-dependent.

[0961] Since it is not possible to predict the indel mutations generated by NHEJ in nature, the specific indel sequence is preferably located in the designated damaged region and can be derived from a small region of microhomology. Conventionally, the length of the deletion ranges from 1 bp to 50 bp, while the insertion tends to be shorter and usually contains short repeat sequences directly surrounding the damaged region.

[0962] Furthermore, NHEJ is a mutagenic process that can be used to delete short sequence motifs when a specific final sequence is not necessary.

[0963] This NHEJ can be used to perform specific knockout of the gene targeted by the CRISPR complex. A CRISPR enzyme (e.g., Cas9 or Cpf1) can be used to cut the double strand or two single strands of the target gene or nucleic acid, and the damaged double strand or two single strands in the target gene or nucleic acid can be made to have insertions and deletions by means of NHEJ, thereby inducing specific knockout of the target gene or nucleic acid. Here, the site of the target gene or nucleic acid cut by the CRISPR enzyme can be in a non-coding region or a coding region; in addition, the site of the target gene or nucleic acid restored by NHEJ can be in a non-coding region or a coding region.

[0964] In one example, due to the process of cleavage of both strands of the target gene using the CRISPR complex and restoration by NHEJ, various insertions and deletions (indels) may occur in the restoration region.

[0965] The term "indel" collectively refers to mutations in which nucleotides are inserted or deleted from the nucleotide sequence of DNA. As described above, when directing the nucleic acid-editing protein complex to cleave the nucleic acid (DNA, RNA) of an immunomodulatory gene, indels can be insertions or deletions introduced into the target sequence during repair via homologous recombination (HDR) or non-homologous end joining (NHEJ) mechanisms.

[0966] Homology-directed repair (HDR) is an error-free correction method that uses homologous sequences as templates to repair or restore damaged genes or nucleic acids. Generally speaking, to repair or restore damaged DNA (i.e., to restore the cell's intrinsic information), the damaged DNA is repaired or restored using the information of unmodified complementary nucleotide sequences or the information of sister chromatids. The most common type of HDR is homologous recombination (HR). HDR is a repair or recovery method that usually occurs during the S phase or G2 / M phase of actively dividing cells.

[0967] In order to repair or restore damaged DNA by means of HDR without using the sister chromatids or complementary nucleotide sequences of the cell, a DNA template (i.e., a nucleic acid template comprising a complementary nucleotide sequence or a homologous nucleotide sequence) artificially synthesized using the information of the complementary nucleotide sequence or the homologous nucleotide sequence can be provided to the cell to repair or restore the damaged DNA. Here, when a nucleic acid sequence or a nucleic acid fragment is further added to the nucleic acid template to repair the damaged DNA, the nucleic acid sequence or nucleic acid fragment further added to the damaged DNA can be knocked in. The nucleic acid sequence or nucleic acid fragment further added can be a nucleic acid sequence or nucleic acid fragment that corrects a target gene or nucleic acid modified by a mutation of a normal gene or nucleic acid, or a gene or nucleic acid desired to be expressed in a cell, but is not limited thereto.

[0968] In one example, a CRISPR complex can be used to cut a double-stranded or single-stranded portion of a target gene or nucleic acid, and a nucleic acid template (which comprises a nucleotide sequence complementary to a nucleotide sequence adjacent to the cleavage site) can be provided to a cell to repair or restore the cut nucleotide sequence in the target gene or nucleic acid through an HDR method.

[0969] Here, the nucleic acid template comprising a complementary nucleotide sequence may have damaged DNA (i.e., a double-stranded or single-stranded sequence cut in the complementary nucleotide sequence), and further comprises a nucleic acid sequence or nucleic acid fragment desired to be inserted into the damaged DNA. A nucleic acid template comprising a complementary base sequence and a nucleic acid sequence or nucleic acid fragment to be inserted may be used to insert an additional nucleic acid sequence or nucleic acid fragment into the damaged DNA (i.e., the cleavage site of the target gene or nucleic acid). Here, the nucleic acid sequence or nucleic acid fragment to be inserted and the additional nucleic acid sequence or nucleic acid fragment may be a nucleic acid sequence or nucleic acid fragment that corrects a modified target gene or nucleic acid caused by a mutation of a normal gene or nucleic acid, or a gene or nucleic acid to be expressed in a cell. The complementary nucleotide sequence may be a nucleotide sequence that forms a complementary bond with the damaged DNA (i.e., a nucleotide sequence on the left or right side of the double-stranded or single-stranded sequence that the target gene or nucleic acid is cut). Alternatively, the complementary nucleotide sequence may be a nucleotide sequence that forms a complementary bond with the damaged DNA (i.e., the 3' and 5' ends of the double-stranded or single-stranded sequence that the target gene or nucleic acid is cut). The complementary nucleotide sequence may be a nucleotide sequence of 15 bp to 3000 bp, and the length or size of the complementary nucleotide sequence may be appropriately designed according to the size of the nucleic acid template or the target gene or nucleic acid. Here, as the nucleic acid template, a double-stranded or single-stranded nucleic acid may be used, or it may be linear or circular, but the present invention is not limited thereto.

[0970] In another example, a CRISPR complex can be used to cut a double-stranded or single-stranded target gene or nucleic acid, a nucleic acid template (which contains a homologous nucleotide sequence adjacent to the nucleotide sequence of the cleavage site) can be provided to the cell, and the cleaved nucleotide sequence in the target gene or nucleic acid can be repaired or restored by HDR methods.

[0971] Here, the nucleic acid template comprising a homologous nucleotide sequence may have damaged DNA (i.e., a double-stranded or single-stranded homologous nucleotide sequence that is cut), and further comprises a nucleic acid sequence or nucleic acid fragment that is desired to be inserted into the damaged DNA. A nucleic acid template comprising a homologous base sequence and a nucleic acid sequence or nucleic acid fragment to be inserted can be used to insert an additional nucleic acid sequence or nucleic acid fragment into the damaged DNA (i.e., the cleavage site of the target gene or nucleic acid). Here, the nucleic acid sequence or nucleic acid fragment to be inserted and the additional nucleic acid sequence or nucleic acid fragment can be a nucleic acid sequence or nucleic acid fragment that corrects a modified target gene or nucleic acid caused by a mutation of a normal gene or nucleic acid, or a gene or nucleic acid to be expressed in a cell. The homologous nucleotide sequence can be a nucleotide sequence with homology to the damaged DNA, i.e., a nucleotide sequence with homology to the left and right nucleotide sequences of the double-stranded or single-stranded cut in the target gene or nucleic acid. Alternatively, the homologous nucleotide sequence can be a nucleotide sequence with homology to the damaged DNA, i.e., a base sequence with homology to the 3' and 5' ends of the double-stranded or single-stranded cut in the target gene or nucleic acid. The homologous nucleotide sequence can be a nucleotide sequence of 15 bp to 3000 bp, and the length or size of the homologous nucleotide sequence can be appropriately designed according to the size of the nucleic acid template or the target gene or nucleic acid. Here, as the nucleic acid template, a double-stranded or single-stranded nucleic acid can be used, or it can be linear or circular, but the present invention is not limited thereto.

[0972] In addition to NHEJ and HDR, there are methods for repairing or restoring damaged target genes. For example, the method for repairing or restoring damaged target genes can be single-strand annealing, single-strand break repair, mismatch repair, or nucleotide damage repair or a method using nucleotide damage repair.

[0973] Single-strand annealing (SSA) is a method for repairing double-strand breaks between two repetitive sequences present in a target nucleic acid, generally using repetitive sequences of more than 30bp of nucleotide sequence. The repetitive sequence can be cut (to produce sticky ends) to produce single strands at each broken end of the double strand of the target nucleic acid; and, after cutting, the single-stranded overhang containing the repetitive sequence is coated with RPA protein to prevent inappropriate annealing of the repetitive sequences to each other. RAD52 binds to each repetitive sequence on the overhang and arranges sequences that can anneal to complementary repetitive sequences. After annealing, the single-stranded overhang (flap) of the overhang is cut, and new DNA is synthesized to fill the specific gap, thereby restoring the DNA double strand. The result of this repair is that the DNA sequence between the two repeats is deleted, and the length of the deletion can depend on a variety of factors (including the position of the two repeats used here and the path or degree of cutting).

[0974] Similar to HDR, SSA uses complementary sequences (i.e., complementary repeats) to modify or correct a target nucleic acid sequence; however, unlike HDR, SSA does not require a nucleic acid template.

[0975] Single-strand break repair (SSBR) can repair single-strand breaks in the genome using mechanisms different from the above-mentioned repair mechanisms. In the case of single-strand DNA breaks, PARP1 and / or PARP2 recognize the break and mobilize the repair machinery. The binding and activity of PARP1 on DNA breaks is temporary and promotes SSBR by promoting the stability of the SSBR protein complex in the damaged area. The most important protein in the SSBR complex is XRCC1, which interacts with proteins that promote the 3' and 5' end processing of DNA to stabilize DNA. End processing generally involves repairing the damaged 3' end to a hydroxylated state and / or repairing the damaged 5' end to have a phosphate moiety, and DNA gap filling occurs after end processing. There are two methods of DNA gap filling, namely short patch repair and long patch repair, and short patch repair involves the insertion of a translocated single nucleotide. After DNA gap filling, DNA ligase promotes end joining.

[0976] Mismatch repair (MMR) can act on mismatched DNA nucleotides. The MSH2 / 6 or MSH2 / 3 complex each has ATPase activity and therefore plays an important role in recognizing mismatches and initiating repair. MSH2 / 6 mainly recognizes nucleotide-nucleotide mismatches and recognizes mismatches of one or two nucleotides, while MSH2 / 3 mainly recognizes longer mismatches.

[0977] Base excision repair (BER) is a repair process active throughout the cell cycle that is used to remove small, non-helical distorted nucleotide damage regions from the genome. In damaged DNA, the damaged nucleotide is removed by cleaving the N-glycosidic bond connecting the base to the deoxyribose-phosphate backbone, followed by cleavage of the phosphodiester bond backbone, thereby generating single-stranded DNA breaks. The damaged single-stranded ends thus formed are removed, and the gaps created by the removal of the single strand are filled with new complementary bases. DNA ligase is then used to connect the ends of the newly filled complementary bases to the backbone, thereby achieving repair or recovery of the damaged DNA.

[0978] Nucleotide excision repair (NER) is an important excision mechanism for removing large helical lesions from DNA. When damage is detected, short single-stranded DNA fragments containing the damaged region are removed, creating a single-stranded gap of 22-30 bp nucleotide sequence. The gap is then filled with new complementary bases, and DNA ligase is used to connect the ends of the newly filled complementary bases to the backbone, thereby repairing or restoring the damaged DNA.

[0979] The effects of artificial manipulation of target genes (i.e., immune regulatory genes) using gRNA-CRISPR complexes can largely be knockout, knockdown, and knockin.

[0980] The term "knockout" refers to the inactivation of a target gene or nucleic acid, while "target gene or nucleic acid inactivation" refers to a state in which transcription and / or translation of the target gene or nucleic acid is absent. Knockout can inhibit the transcription and translation of disease-causing genes or genes with abnormal functions, thereby preventing protein expression.

[0981] For example, when a gRNA-CRISPR enzyme complex (i.e., a CRISPR complex) is used to edit or correct a target gene or chromosome, the CRISPR complex can be used to cut the target gene or chromosome. The CRISPR complex can be used to repair the damaged target gene or chromosome through NHEJ. Due to NHEJ, the damaged target gene or chromosome may have insertions and deletions, thereby inducing specific knockout of the target gene or chromosome.

[0982] In another example, when a gRNA-CRISPR enzyme complex (i.e., a CRISPR complex) and a donor are used to edit or correct a target gene or chromosome, the CRISPR complex can be used to cut the target gene or nucleic acid. The target gene or nucleic acid damaged by the CRISPR complex can be restored with the aid of HDR using a donor. Here, the donor comprises a complementary nucleotide sequence and a nucleotide sequence desired to be inserted. Here, the number of nucleotide sequences desired to be inserted can be adjusted according to the position or purpose of insertion. When the damaged gene or chromosome is repaired by using a donor, the nucleotide sequence desired to be inserted is inserted into the damaged nucleotide sequence region, thereby inducing specific knockout of the target gene or chromosome.

[0983] The term "knockdown" refers to a decrease in the transcription and / or translation of a target gene or nucleic acid or the expression of a target protein. Knockdown is used to regulate the overexpression of a gene or protein to prevent or treat a disease.

[0984] For example, when a target gene or chromosome is edited or corrected using a gRNA-CRISPR inactivation enzyme-transcription repression activity domain complex (i.e., a CRISPR inactivation complex comprising a transcription repression activity domain), the CRISPR inactivation complex can specifically bind to the target gene or chromosome, and the transcription of the target gene or chromosome can be inhibited by the transcription repression activity domain contained in the CRISPR inactivation complex, thereby inducing knockdown (wherein the expression of the corresponding gene or chromosome is inhibited).

[0985] In another example, when a gRNA-CRISPR enzyme complex (i.e., a CRISPR complex) is used to edit or correct a target gene or chromosome, the CRISPR complex can cut the promoter and / or enhancer region of the target gene or chromosome. Here, the gRNA can recognize a partial nucleotide sequence as a target sequence in the promoter and / or enhancer region of the target gene or chromosome. The target gene or chromosome damaged by the CRISPR complex can be restored by NHEJ. Due to NHEJ, the damaged target gene or chromosome may have an insertion or deletion, thereby inducing specific knockout for the target gene or chromosome. Alternatively, when a donor is selectively used, the target gene or chromosome damaged by the CRISPR complex can be restored by HDR. When a donor is used to restore the damaged gene or chromosome, the nucleotide sequence to be inserted is inserted into the damaged nucleotide sequence region, thereby inducing specific knockout for the target gene or chromosome.

[0986] The term "knock-in" refers to the insertion of a specific nucleic acid or gene into a target gene or nucleic acid. Specifically, the term "specific nucleic acid or gene" refers to a nucleic acid or gene that is intended to be inserted or desired to be expressed. Knock-in can be used to treat disease by precisely correcting a disease-causing mutant gene or inducing normal gene expression by inserting a normal gene.

[0987] Furthermore, knock-in may require additional donors.

[0988] For example, when a gRNA-CRISPR enzyme complex (i.e., CRISPR complex) and a donor are used to edit or correct a target gene or nucleic acid, the CRISPR complex can be used to cut the target gene or nucleic acid. The CRISPR complex can be used to restore the damaged target gene or nucleic acid through HDR. Here, the donor comprises a specific nucleic acid or gene, and the donor can be used to insert the specific nucleic acid or gene into the damaged gene or chromosome. Here, the inserted specific nucleic acid or gene can induce protein expression.

[0989] As an embodiment disclosed in this specification, the gRNA-CRISPR enzyme complex can artificially manipulate or modify the following genes: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene.

[0990] The gRNA-CRISPR enzyme complex can specifically recognize target sequences of the following genes: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene.

[0991] The gRNA of the gRNA-CRISPR enzyme complex can specifically recognize the target sequence, thereby positioning the gRNA-CRISPR enzyme complex near the recognized target sequence.

[0992] The target sequence may be a region or range for artificially modifying the PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene.

[0993] The target sequence can be a continuous 10bp-25bp nucleotide sequence located in the promoter region of the following genes: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene.

[0994] The target sequence can be a continuous 10bp-25bp nucleotide sequence located in the intron region of the following genes: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene.

[0995] The target sequence can be a continuous 10bp-25bp nucleotide sequence located in the exon region of the following genes: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene.

[0996] The target sequence can be a continuous 10bp-25bp nucleotide sequence located in the enhancer region of the following genes: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene.

[0997] The target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the 3'-UTR region of the following genes: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene.

[0998] The target sequence may be a continuous 10 bp-25 bp nucleotide sequence located in the 5'-UTR region of the following genes: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene.

[0999] The target sequence can be a continuous 10bp-25bp nucleotide sequence in the 5' and / or 3' end region of the protospacer adjacent motif (PAM) sequence in the nucleotide sequence adjacent to the following genes: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene.

[1000] Here, the PAM sequence may be, for example, one or more of the following sequences (described in the 5' to 3' direction):

[1001] NGG (N is A, T, C or G);

[1002] NNNNRYAC (each N is independently A, T, C or G; R is A or G; Y is C or T);

[1003] NNAGAAW (each N is independently A, T, C or G; W is A or T);

[1004] NNNNGATT (each N is independently A, T, C or G);

[1005] NNGRR(T) (each N is independently A, T, C or G; R is A or G; Y is C or T); and

[1006] TTN (N is A, T, C or G).

[1007] In an embodiment, the target sequence may be one or more nucleotide sequences selected from the nucleotide sequences described in Table 1.

[1008] A gRNA-CRISPR enzyme complex can be formed by gRNA and CRISPR enzyme.

[1009] The gRNA may comprise a guide domain capable of forming partial or complete complementary binding with a guide nucleic acid binding sequence in the target sequence of the following genes: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene.

[1010] The guide domain can be a nucleotide sequence that is complementary to the guide nucleic acid binding sequence, for example, having at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.

[1011] The guide domain may comprise a nucleotide sequence complementary to the guide nucleic acid binding sequence in the target sequence of the PD-1 gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3, or 0-2 mismatches.

[1012] The guide domain may comprise a nucleotide sequence complementary to a guide nucleic acid binding sequence in a target sequence of the CTLA-4 gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3, or 0-2 mismatches.

[1013] The guide domain may comprise a nucleotide sequence complementary to the guide nucleic acid binding sequence in the target sequence of the A20 gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3 or 0-2 mismatches.

[1014] The guide domain may comprise a nucleotide sequence complementary to the guide nucleic acid binding sequence in the target sequence of the DGKA gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3 or 0-2 mismatches.

[1015] The guide domain may comprise a nucleotide sequence complementary to the guide nucleic acid binding sequence in the target sequence of the DGKZ gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3 or 0-2 mismatches.

[1016] The guide domain may comprise a nucleotide sequence complementary to the guide nucleic acid binding sequence in the target sequence of the FAS gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3 or 0-2 mismatches.

[1017] The guide domain may comprise a nucleotide sequence complementary to the guide nucleic acid binding sequence in the target sequence of the EGR2 gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3 or 0-2 mismatches.

[1018] The guide domain may comprise a nucleotide sequence complementary to the guide nucleic acid binding sequence in the target sequence of the PPP2r2d gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3 or 0-2 mismatches.

[1019] The guide domain may comprise a nucleotide sequence complementary to the guide nucleic acid binding sequence in the target sequence of the TET2 gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3 or 0-2 mismatches.

[1020] The guide domain may comprise a nucleotide sequence complementary to the guide nucleic acid binding sequence in the target sequence of the PSGL-1 gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3 or 0-2 mismatches.

[1021] The guide domain may comprise a nucleotide sequence complementary to the guide nucleic acid binding sequence in the target sequence of the KDM6A gene. Here, the complementary nucleotide sequence may comprise 0-5, 0-4, 0-3 or 0-2 mismatches.

[1022] The gRNA may comprise one or more domains selected from the group consisting of a first complementary domain, a connecting domain, a second complementary domain, a proximal domain, and a tail domain.

[1023] The CRISPR enzyme may be one or more proteins selected from the group consisting of: a Cas9 protein derived from Streptococcus pyogenes, a Cas9 protein derived from Campylobacter jejuni, a Cas9 protein derived from Streptococcus thermophilus, a Cas9 protein derived from Staphylococcus aureus, a Cas9 protein derived from Neisseria meningitidis, and a Cpf1 protein. In one example, the editing protein may be a Cas9 protein derived from Campylobacter jejuni or a Cas9 protein derived from Staphylococcus aureus.

[1024] Depending on the type of gRNA and CRISPR enzyme, the gRNA-CRISPR enzyme complex can artificially manipulate or modify the following genes: PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene.

[1025] In one example, when the CRISPR enzyme is an SpCas9 protein, the continuous 1 bp-50 bp, 1 bp-40 bp, 1 bp-30 bp, preferably 1 bp-25 bp nucleotide sequence region located at the 5' end and / or 3' end of the 5'-NGG-3' (N is A, T, G or C) PAM sequence present in the target region of the artificially manipulated or modified gene may comprise one or more of the following modifications, wherein the artificially manipulated or modified gene is an artificially manipulated or modified PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene:

[1026] i) deletion of one or more nucleotides;

[1027] ii) replacing one or more nucleotides with nucleotides different from those in the wild-type gene;

[1028] iii) insertion of one or more nucleotides; or

[1029] iv) a combination of two or more selected from the above i) to iii).

[1030] In another example, when the CRISPR enzyme is a CjCas9 protein, the continuous 1 bp-50 bp, 1 bp-40 bp, 1 bp-30 bp, preferably 1 bp-25 bp nucleotide sequence region at the 5' and / or 3' end of the 5'-NNNNRYAC-3' (N is independently A, T, G or C; R is A or G; Y is C or T) PAM sequence present in the target region adjacent to the artificially manipulated or modified gene may comprise one or more of the following modifications:

[1031] i) deletion of one or more nucleotides;

[1032] ii) replacing one or more nucleotides with nucleotides different from those in the wild-type gene;

[1033] iii) insertion of one or more nucleotides; or

[1034] iv) a combination of two or more selected from the above i) to iii).

[1035] In another example, when the CRISPR enzyme is an StCas9 protein, the continuous 1 bp-50 bp, 1 bp-40 bp, 1 bp-30 bp, preferably 1 bp-25 bp nucleotide sequence region at the 5' and / or 3' end of the 5'-NNAGAAW-3' (N is independently A, T, G or C; W is A or T) PAM sequence present in the target region adjacent to the artificially manipulated or modified gene may include one or more of the following modifications:

[1036] i) deletion of one or more nucleotides;

[1037] ii) replacing one or more nucleotides with nucleotides different from those in the wild-type gene;

[1038] iii) insertion of one or more nucleotides; or

[1039] iv) a combination of two or more selected from the above i) to iii).

[1040] In one example, when the CRISPR enzyme is an NmCas9 protein, the continuous 1 bp-50 bp, 1 bp-40 bp, 1 bp-30 bp, preferably 1 bp-25 bp nucleotide sequence region at the 5' end and / or 3' end of the 5'-NNNNGATT-3' (N is independently A, T, G or C) PAM sequence present in the target region of the artificially manipulated or modified gene may include one or more of the following modifications: The artificially manipulated or modified gene is an artificially manipulated or modified PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene:

[1041] i) deletion of one or more nucleotides;

[1042] ii) replacing one or more nucleotides with nucleotides different from those in the wild-type gene;

[1043] iii) insertion of one or more nucleotides; or

[1044] iv) a combination of two or more selected from the above i) to iii).

[1045] In another example, when the CRISPR enzyme is a SaCas9 protein, the continuous 1 bp-50 bp, 1 bp-40 bp, 1 bp-30 bp, preferably 1 bp-25 bp nucleotide sequence region at the 5' and / or 3' end of the 5'-NNGRR(T)-3' (N is independently A, T, G or C; R is A or G; and (T) is any sequence that may be optionally included) PAM sequence present in the target region adjacent to the artificially manipulated or modified gene may include one or more of the following modifications:

[1046] i) deletion of one or more nucleotides;

[1047] ii) replacing one or more nucleotides with nucleotides different from those in the wild-type gene;

[1048] iii) insertion of one or more nucleotides; or

[1049] iv) a combination of two or more selected from the above i) to iii).

[1050] In another example, when the CRISPR enzyme is Cpf1 protein, the continuous 1 bp-50 bp, 1 bp-40 bp, 1 bp-30 bp, preferably 1 bp-25 bp nucleotide sequence region located at the 5' end and / or 3' end of the 5'-TTN-3' (N is independently A, T, G or C) PAM sequence present in the target region of the artificially manipulated or modified gene may comprise one or more of the following modifications:

[1051] i) deletion of one or more nucleotides;

[1052] ii) replacing one or more nucleotides with nucleotides different from those in the wild-type gene;

[1053] iii) insertion of one or more nucleotides; or

[1054] iv) a combination of two or more selected from the above i) to iii).

[1055] The effect of artificial manipulation of the PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene with the help of the gRNA-CRISPR enzyme complex can be knockout.

[1056] Artificial manipulation of the PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene with the help of the gRNA-CRISPR enzyme complex can inhibit the expression of proteins encoded by the PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene.

[1057] The effect of artificially manipulating the PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene with the help of the gRNA-CRISPR enzyme complex can be knockdown.

[1058] Artificial manipulation of the PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene with the help of the gRNA-CRISPR enzyme complex can reduce the expression of proteins encoded by the PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene, respectively.

[1059] The effect of artificially manipulating the PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene with the aid of the gRNA-CRISPR enzyme complex can be knock-in.

[1060] Here, the knock-in effect can be induced by the gRNA-CRISPR enzyme complex (and additionally by a donor comprising a foreign nucleotide sequence or gene).

[1061] Artificial manipulation of the PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene with the help of the gRNA-CRISPR enzyme complex can enable the expression of peptides or proteins encoded by foreign nucleotide sequences or genes.

[1062] One aspect of the present disclosure relates to manipulated immune cells.

[1063] "Immune cells" are cells that participate in an immune response, and include all cells that participate directly or indirectly in an immune response as well as their pre-differentiated cells.

[1064] Immune cells can have the functions of cytokine secretion, differentiation into other immune cells and cytotoxicity. Immune cells also include cells that have undergone mutations from their natural state.

[1065] Immune cells differentiate from hematopoietic stem cells in the bone marrow, and mainly include lymphoid progenitor cells and myeloid progenitor cells; they also include all the following cells: T cells and B cells differentiated from lymphoid progenitor cells and responsible for acquired immunity; and macrophages, eosinophils, neutrophils, basophils, megakaryocytes, red blood cells, etc. differentiated from myeloid progenitor cells.

[1066] Specifically, the cell may be at least one selected from the group consisting of the following cells: T cells, such as CD8 + T cells (e.g. CD8 + Naive T cells, CD8 + effector T cells, central memory T cells or effector memory T cells), CD4 + T cells, natural killer T cells (NKT cells), regulatory T cells (Treg), stem cell memory T cells; lymphoid progenitor cells; hematopoietic stem cells; natural killer cells (NK cells); dendritic cells; cytokine-induced killer cells (CIK); peripheral blood mononuclear cells (PBMC); monocytes; macrophages; natural killer T (NKT) cells, etc.

[1067] "Manipulated immune cells" refer to immune cells that have been artificially manipulated and are no longer in their natural state. Recently, research has been actively underway to enhance immunity by extracting and manipulating immune cells from the body. These manipulated immune cells have proven to be a novel therapeutic approach due to their superior immune efficacy against certain diseases. In particular, research into manipulated immune cells has been actively pursued in connection with cancer treatment.

[1068] The manipulated immune cells may be immune cells that have been artificially manipulated or modified using a composition for immune cell manipulation. Herein, the term "composition for immune cell manipulation" refers to one or more substances (e.g., DNA, RNA, nucleic acids, proteins, viruses, compositions) used to artificially modify or manipulate immune cells. For example, a composition for immune cell manipulation may include part or all of a composition for genetic manipulation, and may further include a nucleic acid encoding an exogenous protein for expressing the exogenous protein.

[1069] The manipulated immune cells may be immune cells produced by genetic manipulation.

[1070] Here, gene manipulation can be performed taking into account the regulatory process of gene expression.

[1071] In one example, gene manipulation can be performed in the following steps: transcription regulation, RNA processing regulation, RNA transport regulation, RNA degradation regulation, translation regulation, or protein modification regulation by selecting a manipulation method appropriate for each step.

[1072] For example, genetic manipulation can control the expression of genetic information by blocking mRNA using RNA interference (RNAi) or RNA silencing, and, in some cases, by disrupting the delivery of protein synthesis information during intermediate steps.

[1073] In another example, genetic manipulation can use wild-type enzymes or variant enzymes that can catalyze the hydrolysis (cleavage) of DNA or RNA molecules, preferably can catalyze the hydrolysis (cleavage) of bonds between nucleic acids in DNA molecules. A guide nucleic acid-editing protein complex can be used.

[1074] For example, gene manipulation can control the expression of genetic information by manipulating genes using one or more nucleases selected from the group consisting of: meganucleases, zinc finger nucleases, CRISPR / Cas9 (Cas9 protein), CRISPR-Cpf1 (Cpf1 protein) and TALE nucleases.

[1075] In a preferred embodiment, without limitation, gene manipulation can be performed by a guide nucleic acid-editing protein complex, and the explanation of the guide nucleic acid-editing protein is as described above.

[1076] Furthermore, the manipulated immune cell may be an immune cell with modified function due to loss or damage of a specific protein function.

[1077] Here, the function of a specific protein may be lost or impaired by the compound.

[1078] The compounds can bind to specific proteins and hinder the function of immune regulatory factors.

[1079] In addition, the compound can bind to specific proteins and modify the structure of immune regulatory factors, thereby hindering their normal function.

[1080] Alternatively, the function of a specific protein can be lost or impaired by modifying the protein binding to the specific protein.

[1081] The manipulated immune cells can be functionally manipulated immune cells or hybrid manipulated immune cells.

[1082] As an embodiment disclosed in the present invention, the manipulated immune cells may be functionally manipulated immune cells.

[1083] The term "functionally manipulated immune cell" refers to an immune cell in which the natural expression of a wild-type immunomodulatory factor has been modified or artificially manipulated to impair the function of the immunomodulatory factor.

[1084] The term "immunomodulatory factor" refers to a polypeptide or protein encoded by an immunomodulatory gene, and may also be referred to as an immunomodulatory protein transcribed, translated, and expressed by an immunomodulatory gene.

[1085] A functionally manipulated immune cell may be an immune cell manipulated to suppress or inhibit the expression of an immunomodulatory factor.

[1086] Here, the functionally manipulated immune cell may be an immune cell in which an immunoregulatory gene is manipulated to repress or inhibit the expression of an immunoregulatory factor.

[1087] The functionally manipulated immune cell may be an immune cell in which an immune cell activity regulatory gene is manipulated.

[1088] Here, the functionally manipulated immune cell may be an immune cell in which one or more genes selected from SHP-1, PD-1, CTLA-4, CBLB, ILT-2, KIR2DL4, and PSGL-1 are inactivated.

[1089] The functionally manipulated immune cell may be an immune cell in which an immune cell growth regulatory gene is manipulated.

[1090] Here, the functionally manipulated immune cells may be immune cells in which one or more genes selected from DGK-α, DGK-ζ, FAS, EGR2, EGR3, PPP2r2d, and A20 are inactivated. In a preferred embodiment, one or more genes selected from DGK-α, DGK-ζ, EGR2, PPP2r2d, and A20 are inactivated.

[1091] The functionally manipulated immune cell may be an immune cell in which an immune cell death regulatory gene is manipulated.

[1092] Here, the functionally manipulated immune cell may be an immune cell in which one or more genes selected from DAXX, BIM, BID, BAD, PD-1, and CTLA-4 are inactivated.

[1093] Furthermore, the functionally manipulated immune cell may be an immune cell into which an element that induces its own death is inserted.

[1094] A functionally manipulated immune cell may be an immune cell in which the immune cell depletion regulatory element is manipulated.

[1095] Here, the functionally manipulated immune cell may be an immune cell in which one or more genes selected from TET2, WNT, and AKT are inactivated.

[1096] The functionally manipulated immune cell may be an immune cell in which a cytokine secretion element is manipulated.

[1097] A functionally manipulated immune cell may be an immune cell in which an antigen binding regulatory element is manipulated.

[1098] Here, the functionally manipulated immune cell may be an immune cell in which one or more genes selected from dCK, CD52, B2M, and MHC are inactivated.

[1099] The functionally manipulated immune cell may be an immune cell in which an immunomodulatory gene other than the aforementioned gene is manipulated.

[1100] Functionally manipulated immune cells can be immune cells in which one or more immune regulatory genes are simultaneously manipulated. Here, one or more immune regulatory genes can be manipulated.

[1101] Here, manipulation of one immunomodulatory gene does not necessarily result in the expression of new immune effects. Manipulation of one immunomodulatory gene may result in multiple new immune effects or suppress multiple new immune effects.

[1102] The functionally manipulated immune cell may be an immune cell in which a gene encoding a wild-type receptor other than an immunomodulatory gene is manipulated.

[1103] Here, the wild-type receptor may be a T cell receptor (TCR).

[1104] A functionally manipulated immune cell can be one in which the wild-type receptor is absent or present at a lower rate on the surface.

[1105] A functionally manipulated immune cell can be one in which the wild-type receptor is present on the surface in a large proportion.

[1106] A functionally manipulated immune cell can be one in which a wild-type receptor has enhanced recognition of a particular antigen.

[1107] By manipulating wild-type receptors and immunomodulatory genes, functionally manipulated immune cells can possess novel immunological effects.

[1108] A new immune effect may be an immune effect in which the ability to recognize a specific antigen is modulated.

[1109] A new immune efficacy may be an immune efficacy in which the ability to recognize a specific antigen is improved.

[1110] In particular, the specific antigen may be a disease antigen, such as a cancer cell antigen.

[1111] A new immune efficacy may be an immune efficacy in which the ability to recognize a specific antigen deteriorates.

[1112] The new immune efficacy may be an immune efficacy in which the new immune efficacy is improved.

[1113] The new immune effect may be an immune effect in which the growth of immune cells is regulated. In particular, the immune effect may be an immune effect in which growth and differentiation are promoted or delayed.

[1114] The new immune effect can be an immune effect that regulates the death of immune cells. In particular, the immune effect can be to prevent the death of immune cells. In addition, the immune effect can be to cause immune cells to commit suicide after a suitable period of time.

[1115] The new immune efficacy may be an immune efficacy in which the functional loss of immune cells is alleviated.

[1116] The novel immune effect may be an immune effect in which cytokine secretion of immune cells is regulated. In particular, the immune effect may be the promotion or inhibition of cytokine secretion.

[1117] New immune efficacy can be to regulate the antigen binding ability of wild-type receptors in immune cells. In particular, immune efficacy can be to improve the specificity of wild-type receptors for specific antigens.

[1118] Furthermore, the functionally manipulated immune cell may be an immune cell manipulated such that the function of an immunoregulatory factor is impaired.

[1119] Here, the function of the immunomodulatory factor can be lost or impaired by the compound.

[1120] The compound can bind to the immunomodulatory factor or a specific protein that interacts with the immunomodulatory factor and hinder the function of the immunomodulatory factor.

[1121] In addition, the compounds can bind to immunomodulatory factors and artificially modify the three-dimensional structure of immunomodulatory factors, thereby hindering their normal functions.

[1122] Alternatively, the function of the immunomodulatory factor can be lost or impaired by modifying proteins that interact with the immunomodulatory factor.

[1123] As an embodiment disclosed in the present specification, the manipulated immune cell may be a functionally manipulated immune cell in which an immunomodulatory gene is artificially manipulated.

[1124] Here, the immunomodulatory gene may be PD-1 gene, CTLA-4 gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2r2d gene, TET2 gene, PSGL-1 gene, A20 gene and / or KDM6A gene.

[1125] Functionally manipulated immune cells can be manipulated by a composition for genetic manipulation.

[1126] The explanation about the above-mentioned composition for gene manipulation is as described above.

[1127] Functionally manipulated immune cells may contain one or more artificially manipulated or modified immune regulatory genes.

[1128] Here, the artificially modified immunomodulatory gene may contain one or more of the following modifications in the target sequence or in the 1 bp-50 bp nucleotide sequence region adjacent to the 5' end and / or 3' end of the target sequence:

[1129] i) deletion of one or more nucleotides;

[1130] ii) replacing one or more nucleotides with nucleotides different from those in the wild-type gene;

[1131] iii) insertion of one or more nucleotides; or

[1132] iv) a combination of two or more selected from the above i) to iii).

[1133] In one example, a functionally manipulated immune cell can comprise one or more artificially manipulated or modified immune regulatory genes.

[1134] Here, the artificially manipulated or modified immunomodulatory gene may comprise a deletion of one or more nucleotides in the target sequence or in a 1 bp to 50 bp nucleotide sequence region adjacent to the 5' end and / or 3' end of the target sequence.

[1135] For example, an artificially manipulated or modified immunomodulatory gene may comprise a deletion of one or more nucleotides in a nucleotide sequence region located within the target sequence.

[1136] Here, the deleted nucleotides may be 1 bp to 50 bp nucleotides that are continuous, discontinuous, or a mixture of both (i.e., continuous and discontinuous). For example, the deleted nucleotides may be 1 bp nucleotides located in the target sequence. Alternatively, the deleted nucleotides may be 1 bp nucleotides located in the target sequence. Alternatively, the deleted nucleotides may be continuous 3 bp nucleotides. Alternatively, the deleted nucleotides may be discontinuous 4 bp nucleotides located in the target sequence, wherein the discontinuous 4 bp nucleotides may be 1 bp nucleotides and continuous 3 bp nucleotides, or continuous 2 bp nucleotides and another continuous 2 bp nucleotides ( Figure 1 For example, the deleted nucleotides may be discontinuous 30 bp nucleotides located in the target sequence, wherein the discontinuous 30 bp nucleotides may be continuous 25 bp nucleotides, continuous 4 bp nucleotides, and discontinuous 1 bp nucleotides.

[1137] Alternatively, the deleted nucleotides may be nucleotide fragments comprising 2 bp or more nucleotides. The nucleotide fragments may be 2 bp-5 bp, 6 bp-10 bp, 11 bp-15 bp, 16 bp-20 bp, 21 bp-25 bp, 26 bp-30 bp, 31 bp-35 bp, 36 bp-40 bp, 41 bp-45 bp, or 46 bp-50 bp. For example, the deleted nucleotides may be 2 bp nucleotide fragments located in the target sequence. Alternatively, the deleted nucleotides may be 10 bp nucleotide fragments located in the target sequence. Alternatively, the deleted nucleotides may be 16 bp nucleotide fragments located in the target sequence ( Figure 2 ).

[1138] Alternatively, the deleted nucleotides may be a nucleotide fragment comprising 2 bp or more nucleotides. Here, the nucleotide fragment comprising 2 bp or more nucleotides may be a single nucleotide fragment having a discontinuous nucleotide sequence (i.e., having one or more nucleotide sequence gaps), and two or more deleted nucleotide fragments may be used to generate two or more deleted regions. For example, the deleted nucleotides may be a 2 bp nucleotide fragment and a 6 bp nucleotide fragment located in the target sequence. Alternatively, the deleted nucleotides may be a 12 bp nucleotide fragment and a 6 bp nucleotide fragment located in the target sequence ( Figure 3 ).

[1139] In another example, the artificially manipulated or modified immunomodulatory gene may comprise a deletion of one or more nucleotides in the 1 bp to 50 bp nucleotide sequence region adjacent to the 5' end and / or 3' end of the target sequence.

[1140] Here, the deleted nucleotides may be 1 bp to 50 bp nucleotides which are continuous, discontinuous, or a mixture of both (i.e., continuous and discontinuous). For example, the deleted nucleotides may be 1 bp nucleotides located in the target sequence. Alternatively, the deleted nucleotides may be continuous 4 bp nucleotides located near the 3' end of the target sequence. Alternatively, the deleted nucleotides may be discontinuous 4 bp nucleotides located near the 5' end and / or 3' end of the target sequence, wherein the discontinuous 4 bp nucleotides may be continuous 3 bp nucleotides located near the 5' end of the target sequence, and 1 bp nucleotides located near the 3' end of the target sequence ( Figure 4 For example, the deleted nucleotides may be discontinuous 25 bp nucleotides located in the target sequence, wherein the discontinuous 25 bp nucleotides may be continuous 15 bp nucleotides, continuous 8 bp nucleotides, discontinuous 1 bp nucleotides, and discontinuous 1 bp nucleotides.

[1141] Alternatively, the deleted nucleotides may be a nucleotide fragment comprising continuous 2 bp or more nucleotides. The nucleotide fragment may be 2bp-5bp, 6bp-10bp, 11bp-15bp, 16bp-20bp, 21bp-25bp, 26bp-30bp, 31bp-35bp, 36bp-40bp, 41bp-45bp, or 46bp-50bp. For example, the deleted nucleotides may be a 2bp nucleotide fragment located adjacent to the 3' end of the target sequence. Alternatively, the deleted nucleotides may be a 10bp nucleotide fragment located adjacent to the 5' end of the target sequence. Alternatively, the deleted nucleotides may be a 20bp nucleotide fragment located adjacent to the 3' end of the target sequence ( Figure 5 ).

[1142] Alternatively, the deleted nucleotides may be a nucleotide fragment comprising more than 2 bp of nucleotides. Here, the nucleotide fragment comprising more than 2 bp of nucleotides may be a single nucleotide fragment having a discontinuous nucleotide sequence (i.e., having one or more nucleotide sequence gaps), and two or more deleted nucleotide fragments may be used to generate two or more deleted regions. For example, the deleted nucleotides may be a 3 bp nucleotide fragment located near the 5' end of the target sequence and a 6 bp nucleotide fragment located near the 3' end of the target sequence. Alternatively, the deleted nucleotides may be a 12 bp nucleotide fragment located near the 3' end of the target sequence and a 6 bp nucleotide fragment ( Figure 6 ).

[1143] In another example, the artificially manipulated or modified immunomodulatory gene may comprise a deletion of one or more nucleotides in the target sequence and in the 1 bp to 50 bp nucleotide sequence region adjacent to the 5' end and / or 3' end of the target sequence.

[1144] Here, the deleted nucleotides may be 1 bp to 50 bp nucleotides that are continuous, discontinuous, or a mixture of both (i.e., continuous and discontinuous). For example, the deleted nucleotides may be continuous 4 bp nucleotides located in the target sequence and adjacent to the 3' end of the target sequence. Alternatively, the deleted nucleotides may be discontinuous 3 bp nucleotides located in the target sequence and adjacent to the 3' end of the target sequence, and the discontinuous 3 bp nucleotides may be continuous 2 bp nucleotides located in the target sequence and 1 bp nucleotide located adjacent to the 3' end of the target sequence ( Figure 7 ). For example, the deleted nucleotides may be discontinuous 40 bp nucleotides located in the target sequence, and the discontinuous 25 bp nucleotides may be continuous 10 bp nucleotides, continuous 8 bp nucleotides and discontinuous 5 bp (discontinuous 1 bp, 1 bp, 1 bp, 1 bp and 1 bp) nucleotides.

[1145] Alternatively, the deleted nucleotides may be nucleotide fragments comprising 2 bp or more nucleotides. The nucleotide fragments may be 2 bp-5 bp, 6 bp-10 bp, 11 bp-15 bp, 16 bp-20 bp, 21 bp-25 bp, 26 bp-30 bp, 31 bp-35 bp, 36 bp-40 bp, 41 bp-45 bp, or 46 bp-50 bp. For example, the deleted nucleotides may be 25 bp nucleotide fragments located in the target sequence and adjacent to the 3 ' end of the target sequence. Alternatively, the deleted nucleotides may be 35 bp nucleotide fragments located in the target sequence and adjacent to the 5 ' end and 3 ' end of the target sequence. Figure 8 ).

[1146] Or here, the nucleotide of deletion can be two or more nucleotide fragments.Here, two or more nucleotide fragments can be single nucleotide fragments with discontinuous nucleotide sequence (i.e., having one or more nucleotide sequence gaps), and two or more nucleotide fragments of deletion can be used to produce two or more deletion regions.For example, the nucleotide of deletion can be a 6bp nucleotide fragment located in the target sequence and adjacent to the position of the 5' end of the target sequence, and a 13bp nucleotide fragment located in the target sequence and adjacent to the position of the 3' end of the target sequence. Or, the nucleotide of deletion can be a 17bp nucleotide fragment located in the target sequence and adjacent to the position of the 3' end of the target sequence, and a 4bp nucleotide fragment located at the 3' end of the target sequence ( Figure 9 ).

[1147] In another example, a functionally manipulated immune cell may comprise one or more artificially manipulated or modified immune regulatory genes.

[1148] Here, the artificially manipulated or modified immunomodulatory gene may comprise one or more inserted nucleotides in the target sequence or in a 1 bp to 50 bp nucleotide sequence region located adjacent to the 5' end and / or 3' end of the target sequence.

[1149] For example, an artificially manipulated or modified immunomodulatory gene may include one or more nucleotides inserted into a region of the nucleotide sequence located within the target sequence.

[1150] Here, the inserted nucleotides can be 1bp-50bp nucleotides that are continuous, discontinuous, or mixed in two forms (i.e., continuous and discontinuous). For example, the inserted nucleotides can be continuous 2bp nucleotides inserted in the nucleotide sequence region of the target sequence. Alternatively, the inserted nucleotides can be discontinuous 3bp nucleotides inserted in the nucleotide sequence region in the target sequence, and the discontinuous 3bp nucleotides can be 1bp nucleotides and continuous 2bp nucleotides. Alternatively, the inserted nucleotides can be discontinuous 4bp nucleotides inserted in the nucleotide sequence region in the target sequence, and the discontinuous 4bp nucleotides can be 1bp nucleotides, continuous 2bp nucleotides, and another 1bp nucleotide ( Figure 10 ). For example, the inserted nucleotides may be discontinuous 30 bp nucleotides inserted into the nucleotide sequence region in the target sequence, and the discontinuous 30 bp nucleotides may be continuous 15 bp nucleotides, continuous 12 bp nucleotides and discontinuous 3 bp (discontinuous 1 bp, 1 bp and 1 bp) nucleotides.

[1151] Alternatively, the inserted nucleotides may be nucleotide fragments comprising continuous 5 bp or more nucleotides. The nucleotide fragments may be 5 bp-10 bp, 11 bp-50 bp, 50 bp-100 bp, 100 bp-200 bp, 200 bp-300 bp, 300 bp-400 bp, 400 bp-500 bp, 500 bp-750 bp, or 750 bp-1000 bp. For example, the inserted nucleotides may be 10 bp nucleotide fragments inserted in the nucleotide sequence region in the target sequence. Alternatively, the inserted nucleotides may be 28 bp nucleotide fragments ( Figure 11 ).

[1152] Alternatively, the inserted nucleotides may be part or all of the nucleotide sequences of a specific gene. A specific gene may be a gene derived from an external region not contained by a subject (e.g., human cell) comprising an immunomodulatory gene. Alternatively, a specific gene may be a gene present in a subject (e.g., human cell) comprising an immunomodulatory gene, such as a gene present in a human cell genome. For example, the inserted nucleotides may be a partial nucleotide sequence of an exogenous gene inserted in a nucleotide sequence region in a target sequence. Alternatively, the inserted nucleotides may be the entire nucleotide sequence of an exogenous gene inserted in a nucleotide sequence region in a target sequence. Alternatively, the inserted nucleotides may be a partial nucleotide sequence of an endogenous gene inserted in a nucleotide sequence region in a target sequence, and the endogenous gene may be an allele of a target gene (i.e., an immunomodulatory gene), or other genes outside the target gene. Alternatively, the inserted nucleotides may be the entire nucleotide sequence of an endogenous gene inserted in a nucleotide sequence region in a target sequence, and the endogenous gene may be an allele of a target gene (i.e., an immunomodulatory gene), or other genes outside the target gene ( Figure 12 ).

[1153] In another example, the artificially manipulated or modified immunomodulatory gene may include one or more inserted nucleotides in the target sequence or in a 1 bp to 50 bp nucleotide sequence region located adjacent to the 5' end and / or 3' end of the target sequence.

[1154] Here, the inserted nucleotides may be 1 bp to 50 bp nucleotides which are continuous, discontinuous, or a mixture of both (i.e., continuous and discontinuous). For example, the inserted nucleotides may be continuous 2 bp nucleotides inserted in a nucleotide sequence region adjacent to the 5' end of the target sequence. Alternatively, the inserted nucleotides may be discontinuous 3 bp nucleotides inserted in a nucleotide sequence region adjacent to the 3' end of the target sequence, and the discontinuous 3 bp nucleotides may be a 1 bp nucleotide and a continuous 2 bp nucleotide ( Figure 13 ). For example, the inserted nucleotides may be discontinuous 40 bp nucleotides inserted into the nucleotide region of the target sequence, and the discontinuous 40 bp nucleotides may be continuous 15 bp nucleotides, continuous 20 bp nucleotides and continuous 5 bp nucleotides.

[1155] Alternatively, the inserted nucleotides may be nucleotide fragments comprising continuous 5 bp or more nucleotides. The nucleotide fragments may be 5 bp-10 bp, 11 bp-50 bp, 50 bp-100 bp, 100 bp-200 bp, 200 bp-300 bp, 300 bp-400 bp, 400 bp-500 bp, 500 bp-750 bp, or 750 bp-1000 bp. For example, the inserted nucleotides may be 22 bp nucleotide fragments inserted in the nucleotide sequence region adjacent to the 5 ' end of the target sequence. Alternatively, the inserted nucleotides may be 37 bp nucleotide fragments inserted in the nucleotide sequence region adjacent to the 3 ' end of the target sequence. Figure 14 ).

[1156] Alternatively, the nucleotides of insertion may be part or all of the nucleotide sequences of a specific gene. A specific gene may be a gene derived from an external region not contained by a subject (such as a human cell) comprising an immunomodulatory gene. Alternatively, a specific gene may be a gene contained in a subject (such as a human cell) comprising an immunomodulatory gene, such as a gene present in a human cell genome. For example, the nucleotides of insertion may be a partial nucleotide sequence of an exogenous gene inserted in a nucleotide sequence region adjacent to the 5' end of the target sequence. Alternatively, the nucleotides of insertion may be the entire nucleotide sequence of an exogenous gene inserted in a nucleotide sequence region adjacent to the 3' end of the target sequence. Alternatively, the nucleotides of insertion may be a partial nucleotide sequence of an endogenous gene inserted in a nucleotide sequence region adjacent to the 5' end of the target sequence, and the endogenous gene may be an allele of a target gene (i.e., an immunomodulatory gene), or other genes outside the target gene. Alternatively, the inserted nucleotides may be the entire nucleotide sequence of an endogenous gene inserted into the nucleotide sequence region adjacent to the 3' end of the target sequence, and the endogenous gene may be an allele of the target gene (i.e., an immunomodulatory gene), or another gene other than the target gene ( Figure 15 ).

[1157] In yet another example, the functionally manipulated immune cell may comprise one or more artificially manipulated or modified immune regulatory genes.

[1158] Here, the artificially manipulated or modified immunomodulatory gene may comprise one or more nucleotide deletions and insertions in the target sequence or in the 1 bp to 50 bp nucleotide sequence region located adjacent to the 5' end and / or 3' end of the target sequence.

[1159] For example, an artificially manipulated or modified immunomodulatory gene may contain one or more nucleotide deletions or insertions in a nucleotide sequence region located within the target sequence.

[1160] Here, the deleted nucleotides may be 1 bp to 50 bp nucleotides in which they are continuous, discontinuous, or a mixture of both forms (ie, continuous and discontinuous).

[1161] Here, the inserted nucleotides may be 1 bp to 50 bp nucleotides in which they are continuous, discontinuous, or a mixture of both forms (ie, continuous and discontinuous); nucleotide fragments; or a partial or complete nucleotide sequence of a specific gene, and the deletion and insertion may be performed sequentially or simultaneously.

[1162] The inserted nucleotide fragment may be 5 bp-10 bp, 11 bp-50 bp, 50 bp-100 bp, 100 bp-200 bp, 200 bp-300 bp, 300 bp-400 bp, 400 bp-500 bp, 500 bp-750 bp, or 750 bp-1000 bp.

[1163] The specific gene may be a gene derived from an external region that is not contained in a subject (e.g., a human cell) comprising the immunomodulatory gene. Alternatively, the specific gene may be a gene contained in a subject (e.g., a human cell) comprising the immunomodulatory gene, such as a gene present in the human cell genome.

[1164] For example, the deletion and insertion of nucleotides may occur at similar positions in the target sequence, and the deleted nucleotide may be a 1bp nucleotide located in the target sequence; in this case, the inserted nucleotide may be a continuous 2bp nucleotide inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotide may be a continuous 3bp nucleotide located in the target sequence; in this case, the inserted nucleotide may be a continuous 20bp nucleotide fragment inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotide may be a continuous 2bp nucleotide located in the target sequence; in this case, the inserted nucleotide may be a partial nucleotide sequence of an exogenous gene inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotide may be a continuous 3bp nucleotide located in the target sequence; in this case, the inserted nucleotide may be the entire nucleotide sequence of an endogenous gene inserted in the position of the deleted nucleotide sequence, and the endogenous gene may be an allele of the target gene (i.e., an immunomodulatory gene), or other genes other than the target gene ( Figure 16 ).

[1165] For example, the deletion and insertion of nucleotides may occur at different positions in the target sequence, and the deleted nucleotides may be continuous 4bp nucleotides located in the target sequence. In this case, the inserted nucleotides may be continuous 12bp nucleotide fragments inserted in different positions that are not deleted in the target sequence. Alternatively, the deleted nucleotides may be continuous 5bp nucleotides located in the target sequence; in this case, the inserted nucleotides may be partial nucleotide sequences of endogenous genes inserted in different positions that are not deleted in the target sequence. The endogenous gene may be an allele of the target gene (i.e., an immunomodulatory gene), or other genes other than the target gene ( Figure 17 ).

[1166] For example, the deletion and insertion of nucleotides may occur at similar or different positions in the target sequence, and the deleted nucleotides may be 1 bp nucleotides and consecutive 4 bp nucleotides located in the target sequence; in this case, the inserted nucleotides may be a continuous 10 bp nucleotide fragment inserted in one of the two deletion positions of the target sequence (i.e., the position of the 1 bp nucleotide deletion). Alternatively, the deleted nucleotides may be consecutive 5 bp nucleotides and 1 bp nucleotides located in the target sequence; in this case, the inserted nucleotides may be the entire nucleotide sequence of an endogenous gene inserted in one of the two deletion positions (i.e., the position of the consecutive 5 bp nucleotide deletion), and the endogenous gene may be an allele of the target gene (i.e., an immunomodulatory gene), or other genes other than the target gene ( Figure 18 ).

[1167] Alternatively, the deleted nucleotides may be a nucleotide fragment containing nucleotides of 2 bp or more.

[1168] The deleted nucleotide fragment may be 2bp-5bp, 6bp-10bp, 11bp-15bp, 16bp-20bp, 21bp-25bp, 26bp-30bp, 31bp-35bp, 36bp-40bp, 41bp-45bp or 46bp-50bp.

[1169] Here, the inserted nucleotides may be 1 bp to 50 bp nucleotides which are continuous, discontinuous or a mixture of both forms (ie, continuous and discontinuous); nucleotide fragments; or a partial or complete nucleotide sequence of a specific gene, and deletion and insertion may be performed sequentially or simultaneously.

[1170] For example, the deletion and insertion of nucleotides may occur at similar positions in the target sequence, and the deleted nucleotide may be a 10bp nucleotide fragment located in the target sequence; in this case, the inserted nucleotide may be a continuous 2bp nucleotide inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotide may be a continuous 17bp nucleotide fragment located in the target sequence; in this case, the inserted nucleotide may be a continuous 20bp nucleotide fragment inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotide may be a 15bp nucleotide fragment located in the target sequence; in this case, the inserted nucleotide may be a partial nucleotide sequence of an endogenous gene inserted in the position of the deleted nucleotide sequence, and the endogenous gene may be an allele of the target gene (i.e., an immunomodulatory gene) or other genes other than the target gene. Alternatively, the deleted nucleotide may be a 7bp nucleotide fragment located in the target sequence; in this case, the inserted nucleotide may be the entire nucleotide sequence of an exogenous gene inserted in the position of the deleted nucleotide sequence ( Figure 19 ).

[1171] Alternatively, the deleted nucleotides may be a nucleotide fragment containing nucleotides of 2 bp or more.

[1172] Here, the inserted nucleotides may be 1 bp to 50 bp nucleotides that are continuous, discontinuous, or a mixture of both (i.e., continuous and discontinuous); nucleotide fragments; or partial or complete nucleotide sequences of specific genes, and deletion and insertion may be performed sequentially or simultaneously. In addition, insertion may occur in part or all of two or more deleted regions.

[1173] For example, nucleotide deletions and insertions may occur at similar and / or different positions in the target sequence, and the deleted nucleotides may be a 6bp nucleotide fragment and a 12bp nucleotide fragment located in the target sequence; in this case, the inserted nucleotides may be a 15bp nucleotide fragment at one of the two deleted positions in the target sequence (i.e., the position of the 6bp nucleotide deletion). Alternatively, the deleted nucleotides may be a 12bp nucleotide fragment and an 8bp nucleotide fragment located in the target sequence; in this case, the inserted nucleotides may be a 13bp nucleotide fragment inserted into the two deleted nucleotide sequences, i.e., a 13bp nucleotide fragment inserted into the position of the deleted 12bp nucleotide fragment and a 13bp nucleotide fragment inserted into the position of the deleted 8bp nucleotide. Alternatively, the deleted nucleotides may be a 7bp nucleotide fragment and an 8bp nucleotide fragment located in the target sequence; in this case, the inserted nucleotides may be partial or complete nucleotide sequences of an endogenous gene inserted into the two deleted nucleotide sequences, i.e., the complete nucleotide sequence of the endogenous gene inserted into the position of the deleted 7bp nucleotide fragment and a partial nucleotide sequence of the endogenous gene inserted into the position of the deleted 8bp nucleotide fragment. Alternatively, the deleted nucleotides may be a 9 bp nucleotide fragment and an 8 bp nucleotide fragment located in the target sequence; in this case, the inserted nucleotides may be an 8 bp nucleotide fragment inserted into the two deleted nucleotide sequences, respectively, and all or part of the nucleotide sequence of the exogenous gene, i.e., an 8 bp nucleotide fragment inserted into the position of the deleted 9 bp nucleotide fragment, and a partial nucleotide sequence of the exogenous gene inserted into the position of the deleted 8 bp nucleotide fragment ( Figure 20 ).

[1174] In another example, the artificially manipulated or modified immunomodulatory gene may contain one or more nucleotide deletions and insertions in the 1 bp to 50 bp nucleotide sequence region adjacent to the 5' end and / or 3' end of the target sequence.

[1175] Here, the deleted nucleotides may be 1 bp to 50 bp nucleotides in which they are continuous, discontinuous, or a mixture of both forms (ie, continuous and discontinuous).

[1176] Here, the inserted nucleotides may be 1 bp to 50 bp nucleotides in which they are continuous, discontinuous, or a mixture of both forms (ie, continuous and discontinuous); nucleotide fragments; or a partial or complete nucleotide sequence of a specific gene, and deletion and insertion may be performed sequentially or simultaneously.

[1177] The inserted nucleotide fragment may be 5 bp-10 bp, 11 bp-50 bp, 50 bp-100 bp, 100 bp-200 bp, 200 bp-300 bp, 300 bp-400 bp, 400 bp-500 bp, 500 bp-750 bp, or 750 bp-1000 bp.

[1178] The specific gene may be a gene derived from an external region that is not contained in a subject (e.g., a human cell) comprising the immunomodulatory gene. Alternatively, the specific gene may be a gene contained in a subject (e.g., a human cell) comprising the immunomodulatory gene, such as a gene present in the human cell genome.

[1179] For example, nucleotide deletions and insertions may occur at similar positions near the 5' and / or 3' ends of the target sequence. The deleted nucleotides may be a 1-bp nucleotide located near the 3' end of the target sequence; in this case, the inserted nucleotides may be a continuous 2-bp nucleotide inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotides may be a continuous 3-bp nucleotide located near the 5' end of the target sequence; in this case, the inserted nucleotides may be a 20-bp nucleotide inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotides may be a continuous 3-bp nucleotide located near the 3' end of the target sequence; in this case, the inserted nucleotides may be a partial nucleotide sequence of an endogenous gene inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotides may be a continuous 2-bp nucleotide located near the 5' end of the target sequence; in this case, the inserted nucleotides may be the entire nucleotide sequence of an exogenous gene inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotides may be a 1 bp nucleotide and a continuous 4 bp nucleotide located adjacent to the 3' end of the target sequence; in this case, the inserted nucleotides may be the entire nucleotide sequence of the endogenous gene and a continuous 4 bp nucleotide sequence inserted in the two deleted nucleotide sequences, respectively, i.e., the entire nucleotide sequence of the endogenous gene inserted in the position of the deleted 1 bp nucleotide sequence, and the continuous 4 bp nucleotide sequence inserted in the position of the deleted continuous 4 bp nucleotides ( Figure 21 ).

[1180] For example, nucleotide deletion and insertion may occur at similar or different positions of the nucleotide sequence located near the 5' end and / or 3' end of the target sequence, and the deleted nucleotide may be a 1 bp nucleotide located near the 5' end of the target sequence and a continuous 3 bp nucleotide located near the 3' end of the target sequence; in this case, the inserted nucleotide may be an 8 bp nucleotide fragment inserted into one of the positions of the deleted nucleotide sequence (i.e., the position of the deleted continuous 3 bp nucleotide). Alternatively, the deleted nucleotide may be a continuous 4 bp nucleotide located near the 5' end of the target sequence; in this case, the inserted nucleotide may be a partial nucleotide sequence of an endogenous gene inserted into a different non-deleted position near the 3' end of the target sequence ( Figure 22 ).

[1181] Alternatively, the deleted nucleotides may be a nucleotide fragment containing nucleotides of 2 bp or more.

[1182] The deleted nucleotide fragment may be 2bp-5bp, 6bp-10bp, 11bp-15bp, 16bp-20bp, 21bp-25bp, 26bp-30bp, 31bp-35bp, 36bp-40bp, 41bp-45bp or 46bp-50bp.

[1183] Here, the inserted nucleotides may be 1 bp to 50 bp nucleotides in which they are continuous, discontinuous, or a mixture of both forms (ie, continuous and discontinuous); nucleotide fragments; or a partial or complete nucleotide sequence of a specific gene, and deletion and insertion may be performed sequentially or simultaneously.

[1184] For example, the deletion and insertion of nucleotides may occur at similar positions near the 5' end and / or 3' end of the target sequence, and the deleted nucleotide may be a 17bp nucleotide fragment located near the 3' end of the target sequence; in this case, the inserted nucleotide may be a continuous 2bp nucleotide inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotide may be a 15bp nucleotide fragment located near the 5' end of the target sequence; in this case, the inserted nucleotide may be a 30bp nucleotide fragment inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotide may be a 15bp nucleotide fragment located near the 5' end of the target sequence; in this case, the inserted nucleotide may be a partial nucleotide sequence of an endogenous gene inserted in the position of the deleted nucleotide sequence. Alternatively, the deleted nucleotide may be a 25bp nucleotide fragment located near the 3' end of the target sequence; in this case, the inserted nucleotide may be the entire nucleotide sequence of the endogenous gene inserted in the position of the deleted nucleotide sequence ( Figure 23 ).

[1185] Alternatively, the deleted nucleotides may be a nucleotide fragment containing nucleotides of 2 bp or more.

[1186] Here, the inserted nucleotides can be 1 bp, 2 bp, 3 bp, 4 bp, or 5 bp; a nucleotide fragment; or part or all of the nucleotide sequence of a specific gene, and deletion and insertion can be performed sequentially or simultaneously. Alternatively, insertion can occur in part or all of two or more deleted regions.

[1187] For example, the deletion and insertion of nucleotides may occur at similar positions near the 5' end and / or 3' end of the target sequence, and the deleted nucleotides may be a 7 bp nucleotide fragment located near the 5' end of the target sequence and an 18 bp nucleotide fragment located near the 3' end of the target sequence; in this case, the inserted nucleotides may be a partial nucleotide sequence of the exogenous gene and a 12 bp nucleotide fragment respectively inserted in the two deleted nucleotide sequences, that is, a partial nucleotide sequence of the exogenous gene inserted in the position of the deleted 7 bp nucleotide fragment and a 12 bp nucleotide fragment inserted in the position of the deleted 18 bp nucleotide fragment. Alternatively, the deleted nucleotides may be a 10 bp nucleotide fragment located near the 3' end of the target sequence and a 6 bp nucleotide fragment located near the 5' end of the target sequence; in this case, the inserted nucleotides may be the entire nucleotide sequence of the endogenous gene and a continuous 4 bp nucleotide inserted in the two deleted nucleotide sequences, ...

Claims

1. A method for producing manipulated immune cells, the method comprising: Bring the following substances into contact: (a) Immune cells isolated from living organisms; (b) a composition for expressing an artificial receptor protein, wherein the composition for expressing an artificial receptor protein comprises a nucleic acid encoding the artificial receptor protein; and (c) a composition for gene manipulation, wherein the composition for gene manipulation is capable of artificially manipulating an immunomodulatory gene, wherein the immunomodulatory gene is a DGKZ gene; Wherein, the composition for gene manipulation comprises: at least one guide RNA or a nucleic acid encoding the guide RNA, wherein the guide RNA is capable of targeting a target sequence in the nucleic acid sequence of the DGKZ gene; and An editing protein or a nucleic acid encoding the editing protein, wherein the editing protein is capable of forming a guide RNA-editing protein complex with the guide RNA, wherein the guide RNA-editing protein complex is capable of inducing modification at the target sequence, Wherein, the target sequence is at least one nucleotide sequence selected from the following sequences: SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113 located in the exon region of the DGKZ gene, Wherein, the target sequence comprises a protospacer adjacent motif (PAM) sequence at the 5' end or the 3' end.

2. The method according to claim 1, wherein The editing protein is a Cas9 protein derived from Streptococcus pyogenes.

3. The method according to claim 1, wherein The artificial receptor protein has binding specificity for one or more antigens selected from the group consisting of: A33, ALK, alpha-fetoprotein (AFP), adrenergic receptor β3 (ADRB3), α-folate receptor, AD034, AKT1, BCMA, β-human chorionic gonadotropin, B7H3 (CD276), BST2, BRAP, CD5, CD13, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD40, CD44v6, CD52, CD72, CD7 9a, CD79b, CD89, CD97, CD123, CD138, CD160, CD171, CD179a, carbonic anhydrase IX (CAIX), CA-125, carcinoembryonic antigen (CEA), CCR4, C-type lectin-like molecule-1 (CLL-1 or CLECL1), claudin6 (CLDN6), CXORF61, CAGE, CDX2, CLP, CT-7, CT8 / HOM-TES-85, cTAGE-1, ERBB2, epidermal growth factor receptor (EGFR), EGFR Type III variant (EGFRvIII), epithelial cell adhesion molecule (EPCAM), E74-like factor 2 mutant (ELF2M), ephrin type A receptor 2 (EphA2), EMR2, Fms-like tyrosine kinase 3 (FLT3), FCRL5, fibulin-1, G250, GD2, glycoprotein 36 (gp36), glycoprotein 100 (gp100), glucocorticoid-induced tumor necrosis factor receptor (GITR), GPRC5D, GloboH, G protein-coupled receptor 20 (GPR20), GPC3, hsp70-2, human high molecular weight melanoma-associated antigen (HMWMAA), hepatitis A virus cellular receptor 1 (HAVCR1), human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), HAGE, HCA587 / MAGE-C2, hCAP-G, HCE661, HER2 / neu, HLA-Cw, HOM-HD-21 / Galectin-9, HOM-MEEL-40 / SSX2, and HOM-RCC-3.1.3 / CAXII, HOXA7, HOXB6, Hu, HUB 1, insulin growth factor (IGF1)-I, IGF-II, IGF1 receptor, interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), interleukin-11 receptor alpha (IL-11Ra), IGLL1, KIT (CD117), KM-HN-3, KM-KN-1, KOC1, KOC2, KOC3, LAGA-1a, LAGE-1, LAIR1, LILRA2, LY75, Lewis Y antigen, MUC1, MN-CAIX, M-CSF, MAGE-1, MAGE-4a, mesothelin, MAGE-A1, MAD-CT-1, MAD-CT-2, MART1, MPP1 1. MSLN, neural cell adhesion molecule (NCAM), NY-ESO-1, NY-ESO-5, Nkp30, NKG2D, mammary differentiation antigen (NY-BR-1), NY-BR-62, NY-BR-85, NY-CO-37, NY-CO-38, NNP-1, NY-LU-12, NY-REN-10, NY-REN-19 / LKB / STK1 1. NY-REN-21, NY-REN-26 / BCR, NY-REN-3 / NY-CO-38, NY-REN-33 / SNC6, NY-REN-43, NY-REN-65, NY-REN-9, NY-SAR-35, o-acetyl-GD2 ganglioside (OAcGD2), OGFr, PSMA, prostatic acid phosphatase (PAP), p53, prostate cancer tumor antigen 1 (PCTA-1), prostate stem cell antigen (PSCA), serine protease 21 (testisin or PRSS21), platelet-derived growth factor receptor beta (PDGFR-β), PLAC1, pan-linked protein 3 (PANX3), PLU-1, ROR-1, RAGE-1, RU1, RU2, Rab38, RBPJκ, RHAMM, stage-specific embryonic antigen 4 (SSEA-4), SCP1, SSX3, SSX4, SSX5, Tyrp-1, TAG72, thyroglobulin, human telomerase reverse transcriptase (hTERT), 5T4, tumor-associated glycoprotein 72 (TAG72), tyrosinase, transglutaminase 5 (TGS5), TEM1, TEM7R, thyroid-stimulating hormone receptor (TSHR), Tie 2, TRP-2, TOP2A, TOP2B, uroplakin2 (UPK2), vimentin, vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms tumor protein 1 (WT1).

4. The method according to claim 1, wherein The artificial receptor protein is a chimeric antigen receptor (CAR) or an artificially manipulated T cell receptor (TCR).

5. The method according to claim 1, wherein One or more selected from the guide RNA, the artificial receptor protein, and the editing protein are in the form of nucleic acid sequences encoding their respective ones.

6. The method according to claim 5, wherein: The nucleic acid sequence is contained in a viral vector selected from the group consisting of a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus (AAV), a vaccinia virus, a poxvirus, or a herpes simplex virus.

7. The method according to claim 1, wherein The composition for gene manipulation is in the form of a guide RNA-editing protein complex.

8. The method according to claim 1, wherein The contacting is performed ex vivo.

9. The method according to claim 1, wherein The contacting is carried out in the following order: contacting the immune cell with the composition for expressing the artificial receptor protein; and The immune cells are contacted with the composition for genetic manipulation.

10. The method according to claim 1, wherein The contacting is performed by one or more methods selected from the group consisting of electroporation, liposomes, plasmids, viral vectors, nanoparticles, and protein translocation domain fusion proteins.

11. The method according to claim 1, wherein The target sequence is SEQ ID NO:

111.

12. The method according to claim 1, wherein The target sequence is SEQ ID NO:

113.

13. The method according to claim 1, wherein The immune cells are selected from the group consisting of T cells, NK cells, NKT cells and immune cells differentiated from stem cells.

14. The method according to claim 1, wherein The immune cells are human immune cells.

15. A kit for manipulating immune cells, comprising: at least one guide RNA or a nucleic acid encoding said guide RNA, wherein The guide RNA is capable of targeting a target sequence in the nucleic acid sequence of an immunomodulatory gene, and the immunomodulatory gene is the DGKZ gene; an editing protein or a nucleic acid encoding the editing protein, wherein the editing protein is capable of forming a guide RNA-editing protein complex with the guide RNA, wherein the guide RNA-editing protein complex is capable of inducing modification at the target sequence, and A composition for expressing an artificial receptor protein, wherein the artificial receptor protein is an artificially produced receptor that does not exist in nature, wherein the composition for expressing the artificial receptor protein comprises a nucleic acid encoding the artificial receptor protein, in, The guide RNA forms a complementary bond with a portion of the nucleic acid sequence of the immunomodulatory gene, wherein the complementary bond formed contains 0-5 mismatches, The guide RNA comprises a guide domain, a first complementary domain, a linker domain, and a second complementary domain, and wherein the guide domain consists of the 1st to 20th bases of a nucleotide sequence selected from SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112 and SEQ ID NO: 113, wherein T is changed to U.

16. The kit according to claim 15, wherein The kit is provided in the form of one composition.

17. The kit according to claim 15, wherein The editing protein is a Cas9 protein derived from Streptococcus pyogenes.

18. The kit according to claim 15, wherein The leader domain consists of the 1st to 20th bases of SEQ ID NO: 111, wherein T is changed to U.

19. The kit according to claim 15, wherein The leader domain consists of the 1st to 20th bases of SEQ ID NO: 113, wherein T is changed to U.

20. An artificially manipulated immune cell produced by the method according to claim 1, wherein The artificially manipulated immune cells include: An artificially manipulated immunomodulatory gene and / or an expression product of the artificially manipulated immunomodulatory gene, wherein the artificially manipulated immunomodulatory gene is obtained by artificially manipulating the DGKZ gene; as well as one or more artificial receptor proteins and / or nucleic acids encoding said artificial receptors, wherein the one or more artificially manipulated immunomodulatory genes contain artificial modifications in the nucleotide sequence of the immunomodulatory genes, Wherein, the artificial modification is the deletion and / or insertion of one or more nucleotides in the nucleotide sequence region, Wherein, based on the wild-type immunomodulatory gene, the artificial modification is located in the protospacer adjacent motif (PAM) sequence, or in a continuous 1 bp-50 bp adjacent to the 5' end or 3' end of the PAM sequence, Wherein, the artificially manipulated DGKZ gene does not comprise a nucleotide sequence identical to at least one nucleotide sequence selected from SEQ ID NO: 109 to SEQ ID NO:

113.

21. The artificially manipulated immune cell according to claim 20, wherein The artificially manipulated immune cell is at least one cell selected from the group consisting of the following cells: T cells, NK cells, NKT cells and immune cells differentiated from stem cells.

22. The artificially manipulated immune cell according to claim 20, wherein The artificially manipulated immune cells are derived from humans.

23. The artificially manipulated immune cell according to claim 20, wherein The artificially manipulated DGKZ gene does not contain a nucleotide sequence identical to the nucleotide sequence of SEQ ID NO:

111.

24. The artificially manipulated immune cell according to claim 20, wherein The artificially manipulated DGKZ gene does not contain a nucleotide sequence identical to the nucleotide sequence of SEQ ID NO: 113.

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