KR20180018457A - Engineered-immune regulatory factor and Immunity Activation thereby - Google Patents

Abstract

The present invention relates to an immune system which is artificially manipulated and shows improved immune functions. More specifically, the present invention relates to an artificially manipulated immunoregulatory factor, and an immune system whose function is artificially modified while having cells including the same. Particularly, the present invention relates to an artificially manipulated immunoregulatory gene such as PD-1, CTLA-4, A20, DGKα, DGKζ, FAS, EGR2, PPP2R2D, PSGL-1, KDM6A, and TET2, and/or an immune system by expressed products thereof.

Description

Engineered immune regulatory elements and immune activity thereby resulting in a < RTI ID = 0.0 >

The present invention relates to an artificially engineered immune system with improved immune effects. More specifically, it relates to an artificially engineered immune system comprising an artificially engineered immune modulatory element and cells comprising it.

Cell Therapeutics are drugs that induce therapeutic effects such as regeneration using living cells to repair damaged or diseased cells, tissues, and individuals. They are used for culturing, propagating, or screening autologous, allogeneic, xenogeneic cells, Refers to pharmaceuticals manufactured by chemical or biological manipulation.

Among them, the immune modulating cell therapeutic agent is a drug used for the purpose of treating diseases by controlling the immune response in the body using immune cells such as dendritic cells, natural killer cells and T cells.

Currently, immunotherapeutic cell therapy is mainly developed as an indication for cancer treatment. Since the immune function is activated by administering immune cells directly to the patient, the therapeutic effect is obtained. Therefore, And is expected to be a major part of future biopharmaceuticals.

Immunomodulatory cell therapy agents have both physical and chemical characteristics of the substances to be introduced into the cell depending on the type of cell, and a cell therapy agent and a gene therapy agent when the foreign gene is introduced into the immune cell as a viral vector.

The immune regulatory cell therapeutic agent can be prepared by activating various immune cells such as peripheral blood mononuclear cells (PBMC), T cells and NK cells isolated from a patient through apheresis with various antibodies and cytokines, Or by injecting the patient with an immune cell into which a gene such as TCR (T-Cell Receptor) or CAR (Chimeric Antigen Receptor) has been introduced.

Adoptive immunotherapy involving the delivery of autologous antigen-specific immune cells (e. G., T cells) produced in ex vivo can be used to treat cancer as well as various immune diseases It could be a promising strategy.

Recently, it has been reported that an immune cell therapeutic agent can be used variously as an autoimmune suppressant as well as an anticancer function. Immune cell therapeutic agents can be used for various indications by controlling the immune response. Therefore, there is a great demand for improvement and development of therapeutic efficacy of manipulated immune cells used for adoptive immunotherapy.

The present invention, in one embodiment, provides an artificially engineered immune system with improved immune effect.

In one embodiment, the present invention provides an artificially manipulated immunomodulatory element and a cell comprising the same.

In one embodiment, the present invention provides a method for modifying (e.g., enhancing or inhibiting) the function of immune cells.

In one embodiment, the present invention provides a therapeutic and / or prophylactic use of a disease accompanied by an immunological abnormality, which comprises an immune modulating factor and / or an immune cell as an active ingredient.

The present invention provides an anticancer function by enhancing the proliferation, survival, cytotoxicity, infiltration, and cytokine-release of immune cells. do.

In one embodiment, the present invention provides an immunomodulatory gene such as artificially engineered PD-1, CTLA-4, A20, DGKa, DGKζ, FAS, EGR2, PPP2R2D, PSGL-1, KDM6A, TET2 and / .

In one embodiment, the present invention provides a composition for immune cell-based genetic modification comprising a guide nucleic acid-editor protein complex applicable to modulation of the activity of an immunomodulatory gene, and a method of using the same.

In one embodiment, the present invention relates to a method of manipulating an immunoregulatory gene such as artificially manipulated PD-1, CTLA-4, A20, DGKα, DGKζ, FAS, EGR2, PPP2R2D, PSGL-1, KDM6A and TET2 Guide nucleic acids and editor proteins.

In order to solve the above problems, the present invention relates to an artificially manipulated immune system with improved immune effect. More specifically, it relates to an artificially engineered immune system comprising an artificially engineered immune modulatory element and cells comprising it.

The present invention provides a genetically engineered or modified immunomodulatory element for a particular purpose.

"Immunomodulatory element" is a substance that functions in connection with the formation and performance of an immune response. It includes all of a variety of substances that may have an unnatural, artificially manipulated, immune response modulating function. For example, it may be a genetically engineered or modified gene or protein expressed in an immune cell.

The immunomodulatory element may function in the activation or inactivation of immune cells. It can function to suppress the immune response. It can function to promote the immune response. For example, an immune cell growth regulatory element, an immune cell death regulatory element, an immune cell function loss element, or a cytokine secretion element.

In one embodiment of the present invention, for example, genetically engineered or modified PD-1 gene, CTLA-4 gene, TNFAIP3 (A20) gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene , PPP2R2D gene, TET2 gene, PSGL-1 gene, and KDM6A gene.

In one embodiment of the invention, the immunomodulatory element may comprise two or more genes genetically engineered or modified. For example, in the group consisting of the PD-1 gene, CTLA-4 gene, TNFAIP3 (A20) gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2R2D gene, TET2 gene, PSGL-1 gene and KDM6A gene Two or more genes selected can be manipulated or modified.

As a preferred example of the present invention, genetically engineered or modified TNFAIP3, DGKA, DGKZ, FAS, EGR2, PSGL-1, and KDM6A genes can be used.

Therefore, in one embodiment of the present invention, one selected from the group consisting of PD-1, CTLA-4, A20, DGK ?, DGK ?, FAS, EGR2, PPP2R2D, PSGL-1, KDM6A and TET2, RTI ID = 0.0 > artificially engineered < / RTI > immunoregulatory genes.

Such modifications in the nucleic acid sequence can be artificially manipulated by the guide nucleic acid-editor protein complex, without limitation.

The "guide nucleic acid-editor protein complex" refers to a complex formed through the interaction of a guide nucleic acid and an editor protein, and the nucleic acid-protein complex includes a guide nucleic acid and an editor protein.

The guide nucleic acid-editor protein complex can modify the target. The subject may be a target nucleic acid, gene, chromosome or protein.

For example, the gene is an immunoregulatory gene engineered by a guide nucleic acid-editor protein complex,

In a sequence sequence of 1 bp to 50 bp adjacent to the 5 'end and / or the 3' end of the PAM (proto-spacer-adjacent motif) sequence in the nucleic acid sequence constituting the immunomodulatory gene

Deletion or insertion of one or more nucleotides;

Substitution with one or more nucleotides that is different from the wild type gene;

Insert one or more nucleotides from outside

A variant of one or more of the nucleic acids, or

The chemical modification of one or more nucleotides in the nucleic acid sequence constituting said immunomodulatory gene

Lt; RTI ID = 0.0 > an < / RTI > artificially engineered immunomodulatory gene.

Modifications of the nucleic acid can occur in the promoter region of the gene.

Modifications of the nucleic acid can occur in the exon region of the gene. In one embodiment, deformation may occur in the region within the top 50% of the coding region of the gene.

Modifications of the nucleic acid can occur in the intron region of the gene.

The modification of the nucleic acid may occur in the enhancer region of the gene.

The PAM sequence may be, for example, one or more of the following sequences (described in the 5 'to 3' direction).

NGG (wherein N is A, T, C or G);

NNNNRYAC wherein each N is independently A, T, C or G, R is A or G, and Y is C or T;

NNAGAAW wherein N is each independently A, T, C or G and W is A or T;

NNNNGATT, where each N is independently A, T, C or G;

NNGRR (T) wherein each N is independently A, T, C or G, R is A or G, and Y is C or T; And

TTN (where N is A, T, C or G).

In another embodiment, the present invention provides a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: To 289 in the target sequence, respectively.

For example, one or more guide nucleic acids selected from the following group can be provided:

A guide nucleic acid capable of forming a complementary bond to each of the target sequences of SEQ ID NOS: 6 and 11 in the A20 gene nucleic acid sequence;

A guide nucleic acid capable of forming a complementary bond to each of the target sequences of SEQ ID NOS: 19, 20, 21, and 23 in the DGKa gene nucleic acid sequence;

A guide nucleic acid capable of forming a complementary bond to the target sequence of SEQ ID NO: 25 in the EGR2 gene nucleic acid sequence;

A guide nucleic acid capable of forming a complementary bond to the target sequence of SEQ ID NO: 64, respectively, of the PPP2R2D gene nucleic acid sequence;

A guide nucleic acid capable of forming a complementary bond to the target sequence of SEQ ID NO: 87 and 89, respectively, in the PD-1 gene nucleic acid sequence;

A guide nucleic acid capable of forming a complementary bond to a target sequence of SEQ ID NO: 109, 110, 111, 112 and 113, respectively, in the DGKζ gene nucleic acid sequence;

A guide nucleic acid capable of forming a complementary bond to the target sequence of SEQ ID NO: 126, 128 and 129, respectively, in the TET-2 gene nucleic acid sequence;

A guide nucleic acid capable of forming a complementary bond to the target sequence of SEQ ID NO: 182 in the PSGL-1 gene nucleic acid sequence;

A guide nucleic acid capable of forming a complementary bond to each of the target sequences of SEQ ID NOS: 252, 254, 257 and 264 in the FAS gene nucleic acid sequence; And

A guide nucleic acid capable of forming a complementary bond, respectively, in the target sequence of SEQ ID NO: 285 of the KDM6A gene nucleic acid sequence.

The guide nucleic acid may be, without limitation, a nucleotide of 18-25 bp, 18-24 bp, 18-23 bp, 19-23 bp, 19-23 bp, or 20-23 bp.

Also, in an embodiment, the invention provides an artificially engineered immune cell comprising said artificially engineered immunomodulatory gene and at least one of the products expressed therefrom.

Such cells are, but are not limited to, immune cells and stem cells. Immune cell "is a cell involved in the immune response, including all cells directly or indirectly involved in the immune response and their pre-differentiation cells.

The stem cells may be derived from embryonic stem cells, adult stem cells, induced pluripotent stem cells (iPS cells) or induced pluripotent stem cells having autologous replication and differentiation ability (Eg, an iPS cell generated from a subject, manipulated to alter (eg, induce a mutation in).

The immune cell may be a CD3 positive cell. For example, a T cell or a CAR-T cell.

The immune cell may be a CD56 positive cell. For example, NK cells, such as NK92, primary NK cells.

In an embodiment, the immune cell may be a CD3 and a CD56 double positive cell (CD3 / CD56 double positive cell). For example, it may be an NKT (Natural Killer T) cell or a CIK (Cytokine Induced Killer cell).

Specifically, for example, the cell may be a T cell such as CD8 + T cells (eg, 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, stem cell memory T cells, lymphoid progenitor cells, hematopoietic stem cells, natural killer cells (NK cells), dendritic cells, cytokine induced cells (CIK: Cytokine Induced Killer cell, PBMC (peripheral blood mononuclear cell), monocyte, macrophage, NKT (natural killer T) cell, and the like. Preferably, the immune cell may be a T cell, a CAR-T cell, an NK cell, or an NKT cell.

Immune cells can be artificially manipulated to inhibit or inactivate the activity of the immunomodulatory gene.

The immune cell may further comprise a chimeric antigen receptor (CAR).

In one example, the T cell may further comprise a chimeric antigen receptor (CAR) or engineered TCR (T-cell receptor).

The immune cells may further comprise a guide nucleic acid-editor protein complex or a nucleic acid sequence encoding them.

The editor protein includes Cas9 protein derived from Streptococcus pyogenes, Cas9 protein derived from Campylobacter jejuni, Cas9 protein derived from Streptococcus thermophilus, Streptococcus aureus A Cas9 protein derived from Streptococcus aureus, a Cas9 protein derived from Neisseria meningitidis, and a Cpf1 protein. In one example, it may be a Cas9 protein derived from Streptococcus pyogenes or a Cas9 protein derived from Campylobacter jejuni.

Wherein the guide nucleic acid is a complementary binding to a part of a nucleic acid sequence of at least one gene selected from the group consisting of PD-1, CTLA-4, A20, DGK ?, DGK ?, FAS, EGR2, PPP2R2D, PSGL-1, KDM6A, Can be formed. 0 to 5, 0 to 4, 0 to 3, 0 to 2 mismatching. As a preferred example, the guide nucleic acid may be a nucleotide which forms a complementary bond to each of at least one of the target sequences of SEQ ID NOS: 1 to 289 of Table 1. [

SEQ ID NOS: 6 and 11 (A20), SEQ ID NOS: 19, 20, 21 and 23 (DGKa), SEQ ID NO: 25 (EGR2), SEQ ID NO: 64 (PPP2R2D), SEQ ID NOS: 87 and 89 SEQ ID NOS: 109, 110, 111, 112 and 113 (DGKζ), SEQ ID NOS 126,128 and 129 (TET-2), SEQ ID NO: 182 (PSGL-1), SEQ ID NOS 252, 254, 257 and 264 ), And a nucleotide that respectively forms a complementary bond to at least one of the target sequences of SEQ ID NO: 285 (KDM6A).

In one embodiment, the immune cell may comprise one or more of the artificially engineered DGKa and DGKζ genes that have undergone modifications in the nucleic acid sequence.

In another embodiment, the immune cell may comprise an artificially engineered DGKa and DGK < z > gene, wherein a modification has occurred in the nucleic acid sequence.

In an embodiment, a composition is provided that causes the desired immune response. May be referred to as a pharmaceutical composition or a therapeutic composition.

In one embodiment, the present invention provides a nucleic acid sequence comprising at least one of SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: A guide nucleic acid capable of forming a complementary bond to the target sequence of SEQ ID NO: 289; And an editor protein or a nucleic acid encoding the same

≪ / RTI >

The description of the relevant configuration is as described above.

In an embodiment, the present invention provides a method of treating immune cells

(a) a target of SEQ ID NOS: 1 to 289 of the nucleic acid sequence of one or more genes selected from the group consisting of PD-1, CTLA-4, A20, DGK ?, DGK ?, FAS, EGR2, PPP2R2D, PSGL-1, KDM6A and TET2 A guide nucleic acid capable of forming a complementary bond to each of the sequences; And

(b) Cas9 protein derived from Streptococcus pyogenes, Cas9 protein derived from Campylobacter jejuni, Cas9 protein derived from Streptococcus thermophilus, Streptococcus aureus A Cas9 protein derived from Streptococcus aureus, a Cas9 protein derived from Neisseria meningitidis, and an edible protein selected from the group consisting of Cpf1 protein

And contacting the immune cells with an antigen.

The guide nucleic acid and the editor protein may be present in one or more vectors each in the form of a nucleic acid sequence or may be present by forming a complex by binding of a guide nucleic acid and an editor protein.

The contacting step may be carried out in vivo or ex vivo.

The step of contacting may be carried out by one or more methods selected from electroporation, liposomes, plasmids, viral vectors, nanoparticles and protein translocation domain (PTD) fusion protein methods.

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

In an embodiment, the sequence of one or more of PD-1, CTLA-4, A20, DGK ?, DGK ?, FAS, EGR2, PPP2R2D, PSGL-1, KDM6A, and TET2 is sequenced to the sequence of the immune cell target location And provides a method for providing information on the information.

The present invention also provides a method of constructing a library using the information provided by such a method.

In an embodiment, a gene manipulation kit is provided comprising:

(a) a target of SEQ ID NOS: 1 to 289 of the nucleic acid sequence of one or more genes selected from the group consisting of PD-1, CTLA-4, A20, DGK ?, DGK ?, FAS, EGR2, PPP2R2D, PSGL-1, KDM6A and TET2 A guide nucleic acid capable of forming a complementary bond to each of the sequences;

(b) Cas9 protein derived from Streptococcus pyogenes, Cas9 protein derived from Campylobacter jejuni, Cas9 protein derived from Streptococcus thermophilus, Streptococcus aureus A Cas9 protein derived from Streptococcus aureus, a Cas9 protein derived from Neisseria meningitidis, and a Cpf1 protein, or a nucleic acid encoding the same.

These kits can be used to artificially manipulate the desired gene.

In an embodiment, the invention provides all aspects of the use of an immune therapy approach, including administration of an artificially engineered cell, such as genetically engineered immune cells or stem cells, to a subject. It is particularly useful for adoptive immunotherapy.

The subject to be treated may be a mammal including humans, primates such as monkeys, rodents such as mice and rats, and the like.

An effective immune cell therapeutic agent can be obtained by an artificially manipulated immune modulating factor and an immune system that is artificially modified by cells containing it.

For example, when an artificial immune modulating factor is modulated by the methods and compositions of the present invention, the survival, proliferation, persistency, cytotoxicity, cytokine secretion -release) and / or infiltration may be improved.

1 is a graph showing the median fluorescence intensity (MFI) of CD25 in cells knocked out of the DGK-alpha gene using sgRNA (# 11; denoted as DGK-alpha # 11) for DGK-alpha.
Figure 2 is a graph showing CD25 MFI in cells knocked out of the A20 gene using sgRNA (denoted # 11; A20 # 11) for A20.
Figure 3 is a graph showing CD25 MFI in cells where the EGR2 gene was knocked out using sgRNA (# 1; denoted EGR2 # 1) for EGR2.
Figure 4 is a graph showing the CD25 MFI in cells knocked out of the PPP2R2D gene using sgRNA (# 10; designated PPP2R2D # 10) for PPP2R2D.
Figure 5 shows the expression of A20 (# 11; A20 # 11) using the sgRNA (# 11; A20 # 11) for DGKalpha gene knockout cells, A20 using sgRNA (# 11; denoted DGK-alpha # Gamma level in the culture medium of cells knocked out of the EGR2 gene using gene knockout cells, sgRNA (# 1; denoted as EGR2 # 1) for EGR2 (IFN-gamma level unit : pg / ml).
FIG. 6 is a graph showing the effect of sgRNA (# 8 and # 11 used together; DGKalpha # 8 +) on DGKalpha gene knockout cell, DGK-alpha, using sgRNA (# 11; denoted DGK- 11), cells knocked out of the DGK-zeta gene using sgRNA (indicated by # 5; DGK-zeta # 5) against DGK-zeta, Cells in which the A20 gene was knocked out using sgRNA (# 11; denoted as A20 # 11)
(IFN-gamma level units: pg / ml), respectively.
FIG. 7 is a graph showing the results of experiments on DGK-alpha knockout cells using DGK-alpha # 11, DGK-alpha knockout cells using DGKalpha # 8 + 11 and DGK-zeta # (IL-2 level unit: pg / ml) in the culture medium of the cells knocked out of the A20 gene and knocked out the A20 gene using A20 # 11.
FIG. 8A relates to the knockout result of CRISPR / Cas9 mediated DGK gene in human primary T cells, showing (A) the gene knockout timeline in human primary T cells (cell activation by CD3 / CD28 beads, (B) Knockout of DGK gene using lentiviral transfer and electroporation method d. (B) Indel efficiency test for DGKα and DGKζ using Mi-seq system. FIG. 8b shows the result of off-target analysis Fig.
FIG. 9A shows effect and proliferation of CAR-T cells by knockout of DGK gene. (A) Killing activity of 139 CAR-T cells according to 7-AAD positive U87vIII cell measurement using flow cytometry (B) ELISA (IFN-γ, IL-2 kit, Biolegend) and FIG. 9b is a graph showing the results of evaluation of the proliferative activity of 139 CAR T-cells using flow cytometry.
Figure 10 relates to the results of DGKs knockout enhancement of 139 CAR expression following antigen exposure and amplification of CD3 terminal signaling, with (A) stimulation of CD3 / CD28 beads on the phosphorylated ERK signal of 139 CAR-T cells Western blot analysis. (B) 139 CAR expression using flow cytometry Results: CAR expression according to the presence of the antigen on the left, and CAR on the right after 3 days of antigen exposure.
Figure 11 shows the results of DGKs knockout not inducing tonic activation and T-cell exhaustion. (A) Evaluation of IFN-y secretion by ELISA, (B) 1 (left) and TIM-3 (right).
12 shows the results of avoiding the immunosuppressive effects of TGF-β and PGE2 on DGK-knockout T cells, and (A) 139 CAR-T and 139 with TGF-β (10 ng / (B) Killing activity of 139 CAR-T and 139 DGKαζ CAR-T cells, with or without PGE2 (0.5 ug / mL) (Killing activity of DGKαζ CAR-T cells, Killing activity, IFN-γ secretion ability and IL- activity, IFN-y secretion ability and IL-2 secretion ability.
FIG. 13 is a graph showing the effect of CRISPR / Cas9-mediated DKKα on knockout efficiency and effector function in human NK cells, (A) and (B) showing the effect of NK-92 cells and human primary (C) is a graph showing the killing activity of NK-92 by measuring 7-AAD-positive Raji cells.
FIG. 14 shows knockout efficiencies of DGKα and DGKζ mediated by CRISPR / Cas9 in human NKT cells. (A) Indel efficiency, (B) Cell growth (C) The results of Western blotting experiments for confirming expression are shown.
FIG. 15 shows the effect of knockout of DGKα and DGKζ on effector function in human NKT cells, (A) killing activity of each of DGKα and DGKζ and simultaneous knockout and (B) ELISA (IFN-kit, Biolegend ) ≪ / RTI >
FIG. 16 shows the results of knockout of PA-1 in NKT cells for evaluation of knockout of DGKα and DGKζ in human NKT cells, and then (A) improvement of indel efficiency and (B) cytotoxicity, Fig.
FIGS. 17a to 17c show the results of analysis for hPSGL-1 sgRNA screening in Jurkat cells. The results are shown in FIG. 17. The results are shown in FIG. 17a and FIG. 17b. (17b, 17c) showing the degree of expression of PSGL-1.
18 shows results of hPSGL-1 knockout (KO) experiments on human primary T cells, showing (A) the indel efficiency and (B) the degree of T cell not expressing PSGL-1 after knockout And (C) the degree of expression of PSGL-1 expressed on the T cell surface after knockout.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. 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 embodiments are illustrative only and not intended to be limiting

The present invention relates to an artificially engineered immune system with improved immune effects. More specifically, it relates to an artificially engineered immune system comprising an artificially engineered immune modulatory element and cells comprising it.

[Immune Regulation Factor]

Immune modulating factors ( immune regulatory factor )

"Immunomodulatory element" is a substance that functions in connection with the formation and performance of an immune response. It includes all of a variety of substances that may have an unnatural, artificially manipulated, immune response modulating function. For example, it may be a genetically engineered or modified gene or protein expressed in an immune cell.

The term "artificially manipulated" means an artificially deformed state, not a state of being as it exists in nature.

The term "genetically engineered" refers to the case where an artificial genetic modification is applied to a biological or non-biological material referred to in the present invention. For example, the genome may be artificially modified And / or gene products (polypeptides, proteins, etc.).

As a preferred example, the present invention provides a genetically engineered or modified immunomodulatory element for a particular purpose.

The following listed elements are only examples of immunoregulatory factors and thus do not limit the types of immunoregulatory elements encompassed by the present invention. The genes or proteins listed below may not have only one type of immunoregulatory function, but may have a plurality of functions. In addition, two or more immunoregulatory elements can be provided as needed.

[Immune cell activity regulating elements]

"Factor regulating immune cell activity" is an element that functions to regulate the degree or activity of an immune response, for example, a genetically engineered or modified gene or protein that functions to regulate the degree or activity of an immune response Lt; / RTI >

The immune cell activity regulatory element may function related to the activation or deactivation of immune cells.

The immune cell activity regulatory element may function to promote or enhance the immune response.

The immune cell activity regulatory element may function to suppress the immune response.

The immune cell activity regulator can function in the cell membrane to function as a channel protein, a receptor, signal transduction that regulates the immune response, and synthesis and degradation of proteins.

E.g,

The immune cell activity regulatory factor may be PD-1.

The PD-1 gene (also referred to as the PDCD1 gene; the PD-1 gene and the PDCD1 gene are hereinafter used to mean the same gene) is a protein PD-1 (also referred to as cluster of differentiation 279) (Full-length DNA, cDNA, or mRNA). In one example, the PD-1 gene may be but is not limited to one or more selected from the group consisting of: a gene encoding human PD-1 (eg, NCBI Accession No. NP - 005009.2) Accession No. PD-1 gene represented by NM_005018.2, NG_012110.1, and the like.

The immune cell activity regulatory factor may be CTLA-4.

The CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding the protein CTLA-4, also referred to as CD152 (cluster of differentiation 152). In one example, the CTLA-4 gene may be but is not limited to one or more selected from the group consisting of: a gene encoding human CTLA-4 (eg, NCBI Accession No. NP_001032720.1, NP_005205.2, etc.) For example, the CTLA-4 gene represented by NCBI Accession No. NM_001037631.2, NM_005214.4, NG_011502.1, and the like.

The immune cell activity regulatory element may be CBLB.

The immune cell activity regulatory element may be PSGL-1.

The immune cell activity regulatory element may be ILT2.

The immune cell activity regulatory element may be KIR2DL4.

The immune cell activity regulatory element may be SHP-1.

The genes may be derived from mammals including humans, primates such as monkeys, rodents such as rats and mice, and the like.

In one embodiment, the immune cell activity regulatory element may function to promote an immune response.

The immune cell activity regulatory element may be an immune cell growth regulating element.

The term "immune cell growth regulatory element" means an element that regulates the growth of immune cells by regulating protein synthesis in immune cells, and may be, for example, a gene or protein expressed in immune cells.

The immune cell growth regulatory element may function related to transcription of DNA, detoxification of RNA, and cell differentiation.

Examples of immune cell growth regulatory elements may be genes or proteins involved in the NFAT, IKB / NF-KB, AP-1, 4E-BP1, eIF4E, S6 expression pathways.

E.g,

The immune cell growth regulator may be DGK-alpha.

The DGKA (Dgk-alpDha) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding DGKA (Diacylglycerol kinase alpha). In one example, the DGKA gene may be but is not limited to one or more selected from the group consisting of human DGKA (eg, NCBI Accession No. NP_001336.2, NP_958852.1, NP_958853.1, NP_963848.1, etc.) For example, NCBI Accession No. < / RTI > DGKA gene represented by NM_001345.4, NM_201444.2, NM_201445.1, NM_201554.1, NC_000012.12, and the like.

The immune cell growth regulator may be DGK-zeta.

The DGKZ (Dgk-zeta) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding DGKZ (Diacylglycerol kinase zeta). In one example, the DGKZ gene may be but is not limited to one or more selected from the group consisting of: human DGKZ (eg, NCBI Accession No. NP_001099010.1, NP_001186195.1, NP_001186196.1, NP_001186197.1, NP_003637 .2, NP_963290.1, NP_963291.2, etc.), such as NCBI Accession No. DGKZ gene represented by 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.

The immune cell growth regulator may be EGR2.

The EGR2 gene refers to a gene (full-length DNA, cDNA or mRNA) encoding EGR2 (Early growth response protein 2). In one example, the EGR2 gene may be, but is not limited to, one or more selected from the group consisting of:

The immune cell growth regulator may be EGR3.

The immune cell growth regulator may be PPP2R2D.

The immune cell growth regulator may be A20 (TNFAIP3).

The immune cell growth regulator may be PSGL-1.

The genes may be derived from mammals including humans, primates such as monkeys, rodents such as rats and mice, and the like.

The immune cell activity regulatory element may be an immune cell death regulating element.

The "immune cell death regulatory element" may be a gene or a protein expressed in an immune cell, which functions as an element involved in the death of an immune cell.

The immune cell death regulatory element may function in apoptosis or necrosis of immune cells.

In one embodiment, the immune cell death regulatory element may be a caspase cascade-associated protein or gene.

Immune cell death control factor may be Fas. When referring to the following protein or gene, it is natural for a person skilled in the art to manipulate the receptor or the binding portion on which the protein or gene functions.

In another embodiment, the immune cell death regulatory element may be a Death domain-associated protein or gene. At this time, the immune cell death control factor may be Daxx.

The immune cell death regulatory element may be a Bcl-2 family protein.

The immune cell death regulatory element may be a BH3-only family protein.

The immune cell death control factor may be Bim.

The immune cell death control factor may be Bid.

The immune cell death control factor may be BAD.

The immune cell death regulatory element may be a ligand or receptor located in the immune extracellular membrane.

At this time, the immune cell death control factor may be PD-1.

In addition, the immune cell death control factor may be CTLA-4.

The immune cell activity regulatory element may be an immune cell exhaustion regulating element.

An " immune cell function loss factor "is a gene or protein expressed in an immune cell, which functions as a factor involved in the disappearance of the gradual function of immune cells.

The immune cell dysfunction factor may function to help transcription or translation of genes involved in immune cell deactivation.

At this time, the function of assisting transcription may be a function of demethylating the gene.

In addition, the gene involved in inactivation of immune cells includes the gene of the immune cell activity regulatory element.

At this time, the immune cell function loss factor may be TET2.

A gene encoding human TET2 (e.g., NCBI Accession No. NP_001120680.1, NP_060098.3, etc.), such as NCBI Accession NM001127208.2. TET2 gene represented by NM_017628.4, NG_028191.1, etc.

Immune cell function loss factor can function in overgrowth of immune cells. Immune cells that over-grow and do not regenerate lose function.

At this time, the immune cell function loss factor may be Wnt. When referring to the following protein or gene, it is natural for a person skilled in the art to manipulate the gene in the signal transduction pathway containing the protein or its protein, and the receptor, binding part, on which the gene functions.

In addition, the immune cell function loss factor may be Akt. When referring to the following protein or gene, it is natural for a person skilled in the art to manipulate the gene in the signal transduction pathway containing the protein or its protein, and the receptor, binding part, on which the gene functions.

The immune cell activity regulatory element may be a cytokine production regulating element

The "cytokine secretory element" is an element involved in the secretion of cytokines of immune cells, and may be a gene or protein expressed in immune cells that performs this function.

Cytokine is a signal protein that plays an important role in living organisms. Infection, immune, inflammation, trauma, corruption, cancer, and the like. Cytokines can be secreted from cells and then affect other cells or secreted cells themselves. For example, it induces the proliferation of macrophages or promotes the differentiation of the secretory cells themselves. However, when it is secreted in too much amount, it causes problems such as attack of normal cells, so proper secretion of cytokine is also important in immune response.

IL-12, IL-13, IL-1, IL-6, IL-12, IL-7, IL-15, IL-17, IFN-a.

Alternatively, the cytokine may be capable of inducing or killing the antigen-bearing cells recognized by the immune cells by transmitting a signal to the other immune cells. Wherein the cytokine secretion component is preferably a gene or protein in the gene pathway associated with IL-2 secretion.

In an embodiment, the immunomodulatory element may refer to a group of molecules expressing in an immune cell. These molecules can effectively work to down-regulate or inhibit / promote the immune response.

For example, as a group of molecules that express T cells, an "immune checkpoint" is defined as a Programmed Death1 (PD-1. PDCD1 or CD279, accession number: NM_005018) cytotoxic T lymphocyte antigen 4 (CD272, Accession No .: NM_002286.5), Tim3 (HAVCR2, GenBank accession No .: JX049979.1), BTLA (CD272, Accession No .: NM_181780.3), GenBank Accession No. AF414120.1), LAG3 , BY55 (CD160, GenBank accession No .: CR541888.1), TIGIT (IVSTM3, Accession No .: NM_173799), LAIR1 (CD305, GenBank accession No .: CR542051.1), SIGLEC10 (GeneBank accession No .: AY358337.1), 2B4 TGFBRII, TGFRBRI, SMAD2, and TGFRB1, accession number: NM_001166664.1), PPP2CA, PPP2CB, PTPN6, PTPN22, CD96, CRTAM, SIGLEC7, SIGLEC9, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3 However, it is not limited to this.

In one embodiment of the present invention, for example, genetically engineered or modified PD-1 gene, CTLA-4 gene, TNFAIP3 (A20) gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene , The PPP2R2D gene, the TET2 gene, the PSGL-1 gene, and the KDM6A gene.

In one embodiment of the invention, the immunomodulatory element may comprise two or more genes genetically engineered or modified. For example, in the group consisting of the PD-1 gene, CTLA-4 gene, TNFAIP3 (A20) gene, DGKA gene, DGKZ gene, FAS gene, EGR2 gene, PPP2R2D gene, TET2 gene, PSGL-1 gene and KDM6A gene Two or more genes selected can be manipulated or modified.

As a preferred example of the present invention, genetically engineered or modified TNFAIP3, DGKA, DGKZ, FAS, EGR2, PSGL-1, and KDM6A genes can be used.

The gene manipulation or modification can be obtained by artificially inserting, deleting, substituting, and inverting the mutation in a part or all of the genome sequence of the wild type gene. In addition, the genetic manipulation or modification may be obtained by fusing manipulation or modification of two or more genes

For example, such a gene manipulation or modification may inactivate the gene so that a protein encoded from the gene is not expressed in a protein form having an original function.

For example, by further activating the gene by such genetic manipulation or modification, the protein encoded from the gene can be expressed in a protein form having an improved function than the original function. For example, when the function of a protein encoded by a specific gene is A, the function of the protein expressed by the manipulated gene is completely different from that of A, or it may have an additional function (A + B) have.

For example, two or more genes having functions that are different from each other or complementary to each other by such genetic manipulation or modification may be used so that two or more proteins are expressed in a fused form.

For example, by using two or more genes having functions that are different from each other or complementary to each other by such genetic manipulation or modification, two or more proteins can be expressed in separate independent forms in cells.

The genetic information can be obtained from a known database such as GenBank of National Center for Biotechnology Information (NCBI).

In one embodiment, manipulation or modification of the gene may be one or more of the following:

1 to 30, 1 to 27, 1 to 25, 1 to 23, 1 to 23, and 1 to 24 nucleotides of the target gene, for example, all or part of the target gene (hereinafter, 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 nucleotide,

1 to 30, 1 to 27, 1 to 25, 1 to 23, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1, 2 or 3 nucleotides 1 to 30, 1 to 27, 1 to 25, 1 to 23, 1 to 20, or 3 to 5 nucleotides, and substitution of one nucleotide with a nucleotide different from the original (wild type) Insertion of any of the nucleotides (selected independently from A, T, C, and G) into any position of the target gene of SEQ ID NO: 1, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 nucleotide .

A portion of the target gene that is modified (the 'target site') may be at least 1 bp, 3 bp, 5 bp, 7 bp, 10 bp, 12 bp, 15 bp, 17 bp, 20 bp, 3bp to 30bp, 5bp to 30bp, 10bp to 30bp, 12bp to 30bp, 15bp to 30bp, 17bp to 30bp, 20bp to 30bp, 1bp to 27bp, 3bp to 27bp, 5bp to 27bp, 7bp to 27bp, 27bp, 15bp to 27bp, 17bp to 27bp, 20bp to 27bp, 1bp to 25bp, 3bp to 25bp, 5bp to 25bp, 7bp to 25bp, 10bp to 25bp, 12bp to 25bp, 15bp to 25bp, 17bp to 25bp, 20 bp to 23 bp, 3 bp to 23 bp, 5 bp to 23 bp, 7 bp to 23 bp, 10 bp to 23 bp, 12 bp to 23 bp, 15 bp to 23 bp, 17 bp to 23 bp, 20 bp to 23 bp, 1 bp to 20 bp, 3 bp to 20 bp, 20bp, 7bp to 20bp, 10bp to 20bp, 12bp to 20bp, 15bp to 20bp, 17bp to 20bp, 21bp to 25 bp, 18 bp to 22 bp, or 21 bp to 23 bp.

[Cells Containing Immune Regulatory Elements]

One aspect of the invention is a cell comprising said artificially engineered immune modulatory element.

Such cells are, but are not limited to, immune cells and stem cells.

The term "immune cell" of the present invention is a cell involved in the immune response, and includes all the cells directly or indirectly involved in the immune response and their pre-differentiation cells.

Immune cells may have the function of cytokine secretion, differentiation into other immune cells, and cytotoxicity. Immune cells include cells that have undergone mutations from the natural state.

Immune cells differentiate from hematopoietic stem cells in the bone marrow and include lymhoid progenitor cells and myeloid progenitor cells. T cells and B cells in which lymphoid precursor cells differentiate and are responsible for immunity; Eosinophil, basophil, basophil, granule megakaryocyte, and erythrocyte, which are differentiated from myeloid lineage progenitor cells and the like.

Specifically, for example, the cell may be a T cell such as CD8 + T cells (eg, 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, stem cell memory T cells, lymphoid progenitor cells, hematopoietic stem cells, natural killer cells (NK cells), dendritic cells, cytokine induced cells (CIK: Cytokine Induced Killer cell, PBMC (peripheral blood mononuclear cell), monocyte, macrophage, NKT (natural killer T) cell, and the like. Macrophages and dendritic cells are referred to as "antigen presenting cells" or "APCs ", which are specialized cells capable of activating T cells when their major histocompatibility complex (MHC) receptors on their cell surface interact with TCRs on the T- . Alternatively, any hematopoietic stem or immune system cells can be converted to APC by introducing a nucleic acid molecule that expresses an antigen recognized by a TCR or other antigen binding protein (e. G., CAR).

In an embodiment, the immune cell can be a cell used for immunotherapy by inactivating or replacing a gene that synthesizes a protein (e.g., an immunocompromised protein) associated with MHC recognition and / or immune function.

In an embodiment, the immune cell may further comprise a polynucleotide encoding a short chain and multi-subunit receptor (e.g., CAR, TCR) for specific cell recognition.

In an embodiment, the immune cells of the invention may be administered to a healthy donor and / or patient's blood (e.g., peripheral blood), stem cells (e.g., embryonic stem cells, induced pluripotent stem cells, etc.) ), Cord blood, bone marrow, and the like, and may have been manipulated in vitro.

In an embodiment, the immune cell may be a CD3 positive cell. For example, a T cell or a CAR-T cell. CD3 is a receptor in which TCR and various proteins are present as a complex on the T cell surface. Five proteins called γ, δ, ε, ζ, and η chain form CD3, and they exist as TCR / CD3 complex with αβ: γδεζζ or αβ: γδεζη state together with TCR. They are known to have the function of signal transduction into the cell when T cells recognize the antigen.

In an embodiment, the immune cell may be a CD56 positive cell. For example, NK cells, such as NK92, primary NK cells.

NK cells have the third largest number of immune cells, and about 10% of peripheral blood immunocytes are NK cells. NK cells have CD56 and CD16 and mature in the liver or bone marrow. Viruses infect cells or tumor cells. When abnormal cells are recognized, perforin is sprayed on the cell membrane to dissolve the cell membrane, to pierce it, to spray granzyme into the cell membrane, to disrupt the cytoplasm, to cause apotosis, It injects water and salt to cause necrosis. It has the ability to kill many cancer cells. In particular, NK cells are well known as cells in which introduction of external genetic material is not easy.

In an embodiment, the immune cell can be CD3 and CD56 double positive cells. For example, NKT (natural killer T) or CIK (cytokine-induced killer cell) cells.

Natural killer T (NKT) or cytokine-induced killer cell (CIK) cells are generally immune cells expressing CD3, a T cell marker, and CD56, a natural killer cell (NK cell) marker. T cells and has both NK cell characteristics and functions, thus killing tumor cells independent of the main histocompatibility complex (MHC). In particular, NKT (Natural Killer T) cells are cells expressing T cell receptor (TCR) and NK1.1 or NKR-P1A (CD161), an NK cell specific surface marker.

As an example, NKT cells recognize a glycolipid presented by CD1d, a monomorphic protein with a structure similar to MHC class I. NKT cells secrete various types of cytokines such as IL-4, IL-13, IL-10 and IFN-γ when activated by ligands such as α-GalCer. In addition, the NKT cells have anti-tumor activity.

In another example, CIK cells are cells that are proliferated when cultured for 2-3 weeks by treating the cells with an interleukin 2 and CD3 antibody in vitro, and are positive for CD3 and CD56. CIK cells produce large amounts of IFN-y and TNF-a.

In an embodiment, the cell is selected from the group consisting of embryonic stem cells, adult stem cells, induced pluripotent stem cells (iPS cells) or induced pluripotent stem cells (Eg, an iPS cell derived cell).

Cells as preferred examples of the present invention include manipulated or modified genes, which are immunomodulatory elements.

The cell may be part or all of the engineered or modified gene; Or an expression product thereof.

For example, a protein encoded from the gene by inactivation of the gene by genetic manipulation or modification may not be expressed in the protein form having the original function.

For example, the gene can be further activated by such manipulation or modification, whereby the protein encoded from the gene can be expressed in the form of a protein having an improved function than the original function.

For example, such a gene manipulation or modification may be such that a protein encoded from the gene is expressed in a protein form that has an original function and / or a function other than that

For example, the cell may be a cell in which two or more proteins are expressed in a modified form using two or more genes having mutually different or mutually complementary functions due to such gene manipulation or modification.

For example, such genetic modification or modification may be one of three types of cytokine producing or secretory ability of IL-2, TNF [alpha] and IFN [gamma]

As an example, the cell of the present invention may further comprise the following constitution.

- receptor

A cell of the invention may comprise an "immune receptor ".

"Immune receptor" means a receptor that exists on the surface of an artificially manipulated or modified immune cell, and refers to a substance that recognizes a substance involved in an immune response, for example, an antigen and performs a specific function.

The receptor may be in a wild state or an artificially engineered state

The receptor may have affinity for the antigen.

Receptors may have the ability to recognize the structure of MHC structural proteins and antigens disclosed in structural proteins.

The receptor may produce an immune response signal.

"Immune response signal" means any signal that arises in the immune response process.

The immune response signal may be a signal related to the growth and differentiation of immune cells.

The immune response signal may be a signal related to the death of immune cells.

The immune response signal may be a signal related to the activity of the immune cell.

The immune response signal may be a signal associated with the assistance of the immune response.

The immune response signal may be a signal that regulates the expression of the gene of interest.

The immune response signal may be to stimulate or inhibit the synthesis of cytokines.

The immune response signal may be to stimulate or inhibit the secretion of cytokines

The immune response signal may be a signal that aids in the growth or differentiation of other immune cells.

The immune response signal may be a signal that regulates the activity of other immune cells.

The immune response signal may be a signal that attracts other immune cells to a position where the signal occurs.

In one embodiment,

The receptor may be a TCR (T cell receptor).

In one example, the cell may be modified to include a specific T cell receptor (TCR) gene (e.g., a TRAC or TRBC gene). In another example, the TCR has been shown to be associated with tumor associated antigen (eg, MART1 (melanoma antigen recognized by T cells 1, MAGEA3, NY-ESOl), NYESOl, CEA (carcinoembryonic antigen) Binding specificity.

The receptor may be a TLR (Toll like receptor).

The receptor may be CD4 and CD8 which are airway receptors involved in MHC-restricted T cell activation.

The receptor may be CTLA-4 (CD152).

The receptor may be CD28.

The receptor may be CD137, 4-1BB, which is a receptor that amplifies the response of T cells

Lt; RTI ID = 0.0 > CD3 < / RTI > that is the signaling component of the T cell antigen receptor

The receptor may be a CAR (Chimeric Antigen Receptor).

In one embodiment of the present invention, the receptor may be an artificially engineered artificial receptor

"Artificial receptor" means an artificially created functional entity that recognizes an antigen and does a specific function, rather than a wild-type receptor.

Such artificial receptors may contribute to an improved immune response by improving the cognitive ability to specific antigens or by producing an enhanced immune response signal.

The artificial receptor may have the following constitution as an example.

(i) an antigen recognition unit

Artificial receptors include antigen recognition sites.

"Antigen recognition part" means a part recognizing an antigen as part of an artificial receptor.

The antigen recognition portion may be an improved recognition of the specific antigen over the wild receptor. At this time, the specific antigen may be an antigen of a cancer cell. In addition, certain antigens may be antigens of normal body cells.

The antigen recognizing part may have affinity with the antigen.

The antigen recognition unit can generate signals while binding to the antigen. The signal may be an electrical signal. The signal may be a chemical signal.

The antigen recognizing part may comprise a signal sequence.

The signal sequence refers to a peptide sequence that allows a protein to be transferred to a specific position in the process of synthesizing the protein.

The signal sequence may be located near the N-terminus of the antigen recognition site. At this time, the distance from the N-terminus may be around 100 amino acids. The signal sequence may be located close to the C-terminus of the antigen recognition site. At this time, the distance from the C terminus may be around 100 amino acids.

The antigen recognizing unit may have an organic functional relationship with the first signal generating unit.

The antigen recognition site may be homologous to the Fab (fragment antigen binding) domain of the antibody.

The antigen recognition portion may be a single-chain variable fragment (scFv).

The antigen recognition unit may recognize an antigen by itself or by recognizing an antigen or by forming an antigen recognition structure.

 The antigen recognizing structure can recognize an antigen by having a specific structure, and the unit and the combination of the units having such a specific structure can be easily understood by those having ordinary skill in the art. The antigen recognizing structure can also be composed of one or more than two units.

The antigen recognizing structure may be a structure in which the units are connected in a line, or may be a structure in which the units are connected in parallel.

A structure in which two or more units are connected in series in one direction and a structure in which two or more units are connected in parallel can be formed on the distal end of one unit at the same time, Means a combined form.

E.g,

The unit may be an inorganic material.

The unit may be a biochemical ligand.

The unit may be homologous to the antigen recognition portion of the wild-type receptor.

The monomers may be homologous to the antibody protein.

The unit may be a heavy chain or homologous to the immunoglobulin.

The unit may be a light chain or homologous to the immunoglobulin.

The unit may include a signal sequence.

On the other hand, the unit may be bonded by a chemical bond or may be bonded through a specific bond.

An " antigen recognition unit combining part "is a site where antigen recognition structural units are bonded to each other, and may be an optional configuration existing when there is an antigen recognition structure composed of two or more antigen recognition structural units .

The antigen recognizing structural unit binding moiety may be a peptide. At this time, the binding portion may have a high ratio of serine to threonine.

The antigen recognizing structural unit binding moiety may be a chemical bond.

The antigen recognizing structural unit binding moiety may have a specific length to aid in the expression of the stereostructure of the antigen recognizing structural unit.

The antigen recognizing structural unit binding site can help the function of the antigen recognizing structure by having a specific positional relationship between the antigen recognizing structural units.

(ii)

The artificial receptor includes a receptor body portion.

The "receptor body part" may be a part that mediates the connection between the antigen recognition part and the signal generating part, and physically connects the antigen recognition part and the signal generating part.

The function of the receptor body part may be to transmit a signal generated by the antigen recognition part or the signal generation part.

The structure of the receptor body part may also have the function of the signal generating part at the same time.

The function of the receptor body portion may be to allow the artificial receptor gas to be immobilized on the immune cells.

The receptor body portion may comprise an amino acid helical structure.

The structure of the receptor body portion may include a portion that is homologous to a portion of a common receptor protein present in the body. The range of homology may be 50 to 100%.

The structure of the receptor body may include a portion that is homologous to the protein on the immune cell. The range of homology may be 50 to 100%.

E.g,

The receptor body may be the CD8 transmembrane domain.

The receptor body portion may be the CD28 transmembrane region. At this time, when the second signal generator is CD28, the CD28 can function as the second signal generator and the receiver body at the same time.

(iii)

The artificial receptor may comprise a signal generator.

The "first signal generating part" refers to a part of the artificial receptor that generates an immune response signal.

The "second signal generating part" refers to a part of the artificial receptor that interacts with the first signal generating part or independently generates an immune response signal.

The artificial receptor may include a first signal generator and / or a second signal generator.

Or two or more first and / or second signal generators.

The first and / or second signal generators may comprise specific sequence motifs.

Sequence motifs may be homologous to motifs of CD (cluster of designation) proteins.

At this time, the CD protein may be CD3, CD247, CD79.

The sequence motif may be the amino acid sequence of YxxL / I.

The sequence motifs may be included in the first and / or second signal generators in multiple ways.

At this time, the first sequence motif may be located at a distance of 1 to 200 amino acids from the start position of the first signal generator. The second sequence motif may be located at a distance of 1 to 200 amino acids from the start position of the second signal generator.

In addition, the spacing between each sequence motif may be from 1 to 15 amino acids.

At this time, the preferred spacing between each sequence motif is 6 to 8 amino acids.

E.g,

The first and / or second signal generators may be CD3z.

The first and / or second signal generators may be FcεRIγ.

The first and / or second signal generators may generate an immune response signal only when certain conditions are satisfied.

Certain conditions may be one that recognizes an antigen recognizing additive antigen.

Certain conditions may be that the antigen recognizing portion forms a bond with the antigen.

Certain conditions may be that a signal generated when an antigen recognition moiety and an antigen form a bond is received.

Certain conditions may be that the antigen recognizing portion recognizes or binds to the antigen and then separates.

The immune response signal may be a signal related to the growth and differentiation of immune cells.

The immune response signal may be a signal related to the death of immune cells.

The immune response signal may be a signal related to the activity of the immune cell.

The immune response signal may be a signal associated with the assistance of the immune response.

The immune response signal can be activated specifically for the signal generated by the antigen recognition unit.

The immune response signal may be a signal that regulates the expression of the gene of interest.

The immune response signal may be a signal that suppresses the immune response.

In one example, the signal generator may include an additional signal generator.

The "additional signal generating part" means a part generating an additional immune response signal with respect to the immune reaction signal generated by the first and / or second signal generating part as part of the artificial receptor.

Hereinafter, the additional signal generating unit is referred to as an n-th signal generating unit (n? 1) according to the order.

The artificial receptor may include an additional signal generator in addition to the first signal generator.

Two or more additional signal generators may be included in the artificial receptors.

The additional signal generating part may be a structure generating 4-1BB, CD27, CD28, ICOS, OX40 or other immune response signal.

The condition that the additional signal generating unit generates the immune response signal and the characteristic of the generated immune reaction signal include the content corresponding to the immune response signal of the first and / or second signal generating unit.

The immune response signal may be one that promotes the synthesis of cytokines. The immune response signal may be to stimulate or inhibit the secretion of the cytokine. Wherein the cytokine is preferably IL-2, TNF [alpha] or IFN- [gamma].

The immune response signal may be a signal that aids in the growth or differentiation of other immune cells.

The immune response signal may be a signal that regulates the activity of other immune cells

The immune response signal may be a signal that attracts other immune cells to a position where the signal occurs.

The present invention encompasses all possible binding relationships of artificial receptors. Thus, embodiments of the artificial receptors of the present invention are not limited to those mentioned herein.

The artificial receptor may consist of an antigen recognition-receptor body-first signal generator. The receiver body portion may optionally be included.

The artificial receptor may be composed of an antigen recognizing part-receptor body part-second signal generating part-first signal generating part. The receiver body portion may optionally be included. At this time, the positions of the first signal generator and the second signal generator may be changed.

The artificial receptor may be composed of an antigen recognizing part-receptor body part-second signal generating part-third signal generating part-first signal generating part. The receiver body portion may optionally be included. At this time, the positions of the first to third signal generators may be changed.

The number of signal generating units in the artificial receptor is not limited to one to three, but may be more than three.

In addition to the above examples, the artificial receptor may have the structure of the antigen recognizing part-signal generating part-receptor body part. Such a structure may be advantageous when it is necessary to produce an extracellularly acting immune response signal with an artificial receptor.

Artificial receptors can function corresponding to wild receptors.

An artificial receptor can function to form a specific positional relationship by forming a bond with a specific antigen.

Artificial receptors can function to recognize specific antigens and generate immune response signals that promote immune responses to specific antigens.

The artificial receptor recognizes the antigen of a general body cell and can suppress the immune response to the body cell.

(iv) signal sequence

In one example, the artificial receptor may optionally comprise a signal sequence.

If an artificial receptor contains a signal sequence for a particular protein, it can help facilitate the placement of artificial receptors on the immune cell membrane. Preferably, if the artificial receptor in the immune cell comprises a signal sequence of a transmembrane protein, it may help the artificial receptor to pass through the immune cell membrane and be located in the outer membrane of the immune cell.

An artificial receptor may comprise one or more signal sequences.

The signal sequence may contain many positively charged amino acids.

The signal sequence may comprise a positively charged amino acid at a position near the N or C terminus.

The signal sequence may be the signal sequence of the transmembrane protein.

The signal sequence may be a signal sequence of a protein located in the outer membrane of the immune cell.

The signal sequence may be included in the structure of the artificial receptor, i.e., the antigen recognition unit, the receptor body, the first signal generator, and the additional signal generator.

At this time, the signal sequence may be located near the N or C terminal of each structure.

At this time, the distance from the N or C terminal may be around 100 amino acids.

In one example, the cell may be modified to include a specific T cell receptor (TCR) gene.

In another example, the TCR may be a tumor-associated antigen (eg, MALL1 (melanoma antigen recognized by T cells 1), MAGEA3 (melanoma-associated antigen 3), NY- ESOl, carcinoembryonic antigen (CEA) melanoma). < / RTI >

In another example, the cell may be modified to include a specific chimeric antigen receptor (CAR). In one example, the CAR is a tumor associated antigen (eg, CD 19, CD20, carbonic anhydrase IX (CAIX), CD 171, CEA, ERBB2, GD2, alpha-folate receptor, Lewis Y antigen, prostate specific membrane antigen Or tumor associated glycoprotein 72 (TAG72)).

In another example, the cell may be modified to bind to one or more of the following tumor antigens, e.g., by TCR or CAR.

Tumor antigens include, but are not limited to, AD034, AKT1, BRAP, CAGE, CDX2, CLP, CT-7, CT8 / HOM-TES-85, cTAGE-1, EGFR, EGFRvIII, Fibulin- , HOM-MEEL-40 / SSX2, HOM-RCC-3.1.3 / CAXII, HOXA7, HOXB6 , Hu, HUB 1, KM-HN-3, KM-KN- 1, KOCl, KOC2, KOC3, KOC3, LAGE-1, MAGE-1, MAGE-4a, MPPl, MSLN, NNP- 1, NY-BR-62, NY-BR-85, NY-CO-37, NY-CO-38, NY-ESO-1, NY-ESO-5, NY- NY-REN-33 / SNC6, NY-REN-26 / BCR, NY-REN-19 / LKB / STK1 1, NY- 43, NY-REN-65, NY-REN-9, NY-SAR-35, OGFr, PSMA, PSCA PLU-1, Rab38, RBPJkappa, RHAMM, SCP1, SCP-1, SSX3, SSX4, SSX5, TOP2A, TOP2B , Or Tyrosinase, and the like.

- antigen binding modulator ( antigen binding regulating element )

The cells of the present invention may further comprise an "antigen binding modulator ".

An " antigen binding regulatory element "is a gene or protein that functions as a factor that enables receptor-antigen binding.

These antigen binding regulatory elements can be used to regulate the immune response. For example, when an externally manipulated cell is put into a living body, HVGD (host HVGD (graft disease), host graft disease (HVGD), in which an immune response to an externally manipulated cell is activated, ) Is a problem, it can be solved by suppressing the antigen binding force of the immune cell receptor

The antigen binding regulatory element may be a protein or gene related to the structure of the receptor.

The antigen binding control element may be a protein or a gene that is homologous to the structure of the receptor.

E.g,

The antigen binding modulator may be dCK.

The antigen binding modulator may be CD52.

The antigen binding control element may be B2M.

The antigen binding modulator may be a protein or a gene related to the construct recognized by the receptor.

For example, the antigen binding control element may be an MHC protein.

One embodiment of the present invention is an immune cell comprising said artificially engineered immunomodulatory genes or proteins thereof.

Another embodiment of the present invention relates to the above artificially engineered immunomodulatory genes or proteins thereof; And a receptor.

Yet another embodiment of the present invention relates to the above artificially engineered immunomodulatory genes or proteins thereof; Receptor; And an antigen binding regulatory element.

A representative example of the cell of the present invention is an immune cell.

In some embodiments of the invention, the immune cells are selected from the group consisting of PBMC (Peripheral blood mononuclear cell), NK cells (Natural killer cell), monocytes, T cells, CAR-T cells, macrophages, NKT cells Natural killer T cell), and the like. Preferably, it may be a T cell, a CAR-T cell, a NK cell (natural killer cell), or a NKT cell (natural killer T cell).

Factors that limit the efficacy of genetically engineered immune cells, such as T cells, NK cells, NKT cells,

(1) immune cell proliferation, for example, limited propagation of immune cells after adoptive transfer;

(2) induction of immune cell survival, e. G., Apoptosis of immune cells by factors in the tumor environment; And

(3) immune cell function, such as inhibition of cytotoxic immune cell function by inhibitory factors secreted by host immune cells and cancer cells.

One or more genes selected from the group consisting of one or more immune cell expressing genes such as PD-1, CTLA-4, TNFAIP3, DGKA, DGKZ, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A and TET2 Lt; RTI ID = 0.0 > inactivated, < / RTI > immune cells.

For example, one or more immune cell expressing genes such as PD-1, CTLA-4, TNFAIP3, DGKA, DGKZ, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A and / , Knockout, knockdown, or knock-in, respectively, independently of each other to affect the function, survival, and function of the target.

For example, in one immune cell, two or more of the expression genes, for example, PD-1, CTLA-4, TNFAIP3, DGKA, DGKZ, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A and / Knock-out, knock-down or knock-in at the same time. In one embodiment, DGKA and DGKZ were knocked out simultaneously.

For example, one or more immune cell-expressing genes such as PD-1, CTLA-4, TNFAIP3, DGKA, DGKZ, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A and / Survival, or function of one or more immune cells, for example, by targeting a promoter region, an enhancer, a 3'UTR, and / or a polyadenylation signal sequence, or a transcription sequence such as an intron or exon sequence Knock-out, knock-down or knock-in, respectively.

For example, one or more immune cell expression genes, such as PD-1, CTLA-4, TNFAIP3, DGKA, DGKZ, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A and / or TET2 genes, Knockdown, or knock-in, respectively, to affect the proliferation, survival, and function of one or more immune cells, by induction of a change involving deletion, substitution, insertion, or mutation.

At this time, it is obvious that the immunomodulatory genes not disclosed in this specification can be combined and targeted.

[Immune system]

Further, another aspect of the present invention relates to said artificially engineered immunomodulating element; ≪ / RTI > and / or cells comprising it.

An "immune system " of the present invention is a term that includes all phenomena that affect an immune response in the body by altering the function of an artificially manipulated immune modulating factor, i.e., a mechanism exhibiting a new immune effect, Compositions, methods, and uses that directly or indirectly involve such immune systems. For example, genes involved in innate immunity, adaptive immunity, cellular immunity, humoral immunity, active immunity, passive immune response, immune cells and immune organ / tissue.

Each component of this immune system is collectively referred to as the "immune system factor".

The immune system of the present invention comprises manipulated immune cells.

A manipulated immune cell refers to an immune cell that has undergone an artificial manipulation rather than a natural state. Recently, techniques for enhancing immunity by extracting immune cells from the body and applying artificial manipulation have been actively studied. Immune cells that have undergone this manipulation are now being illuminated as new therapeutic methods because of their excellent immune efficacy against certain diseases. In particular, studies on manipulated immune cells have been actively conducted in relation to the treatment of cancer.

The manipulated immune cell may be a function-regulated immune cell or an artificial-structured immune cell.

Functional immune cells ( functionally manipulated immune cell )

"Functionally-functioning immune cell" of the present invention means an immune cell in which a wild receptor or an immune regulatory element has been manipulated in its natural state.

Hereinafter, the term "manipulation" refers to all manipulations that ordinary technicians can utilize to manipulate proteins and genes, such as cutting, connecting, removing, inserting, modifying, or removing, adding, or modifying proteins. Hereinafter, immune cells include not only already differentiated immune cells but also differentiated cells (e.g., stem cells).

Functionally engineered immune cells may be immune cells engineered with wild-type receptors. At this time, the wild receptor may be a TCR.

Functional immune cells may be those where the wild-type receptor is absent or present at a lower rate.

Functional immune cells may be those where the wild receptor is present at a greater rate on the surface.

Functional immune cells may be those with enhanced recognition of wild-type receptors for specific antigens.

Functional immune cells may be immune cells that have been manipulated with an immunomodulatory element.

Functional immune cells may be engineered immune cell activity regulatory elements.

At this time, the functional immune cells may be immune cells in which at least one selected from among SHP-1, PD-1, CTLA-4, CBLB, ILT-2, KIR2DL4 and PSGL-1 is inactivated.

Functional immune cells may be engineered immune cell growth regulators.

At this time, the functional immune cells may be immune cells in which at least one selected from DGK-alpha, DGK-zeta, Fas, EGR2, Egr3, PPP2R2D and A20 is inactivated. A preferred embodiment is to inactivate at least one selected from DGK-alpha, DGK-zeta, EGR2, PPP2R2D, A20.

Functional immune cells may be engineered immune cell death control elements.

At this time, the functional immune cells may be immune cells in which at least one selected from Daxx, Bim, Bid, BAD, PD-1, and CTLA-4 is inactivated.

In addition, the functionally-functioning immune cell may be an immune cell into which an element inducing self-destruction has been inserted.

Functional immune cells may be engineered immune cell function loss factors.

At this time, the functional immune cells may be immune cells in which at least one selected from TET2 and Wnt Akt is inactivated.

Functional immune cells may be engineered with a cytokine secretory element.

Functional immune cells may be engineered with an antigen binding regulatory element.

At this time, the functional immune cells may be immune cells in which at least one selected from dCK, CD52, B2M, and MHC is inactivated.

Functionally engineered immune cells may be engineered with immune modulatory elements that differ from those mentioned above.

Functional immune cells may be those in which one or more immunoregulatory elements have been engineered simultaneously. At this time, one or more kinds of immunoregulatory factors can be manipulated.

By the manipulation of the wild-type receptor and the immunoregulatory component, the functionally-functioning immune cells may have new immunological efficacy.

At this time, manipulating an immune modulator does not necessarily mean that one kind of new immunomodulatory effect has to occur. The manipulation of one immunomodulatory element may cause or inhibit a variety of new immunomodulatory effects.

The new immunomodulatory effect may be the ability to regulate the recognition of specific antigens.

The new immunomodulatory effect may be an improved recognition of specific antigens.

At this time, the specific antigen may be an antigen of the disease. For example, it may be an antigen of a cancer cell.

The new immunomodulatory effect may be a reduced ability to recognize specific antigens.

New immune efficacy may be improved immunity.

The new immunomodulatory effect may be a regulated immune cell growth. At this time, the immunological effect may be promoted or alleviated by the growth and differentiation of immune cells.

The new immunomodulatory effect may be controlled immune cell death. At this time, the immunological effect may be to prevent the death of immune cells. In addition, immunological efficacy may be such that the immune cells kill themselves when appropriate.

The new immunomodulatory effect may be mitigation of immune cell dysfunction.

The new immunomodulatory effect may be the modulation of the cytokine secretion of immune cells. At this time, the immunological effect may be to promote or suppress the secretion of the cytokine.

The new immunomodulatory effect may be to modulate the antigen binding capacity of wild-type receptors in immune cells. At this time, the immunological effect may be to improve the specificity of the wild-type receptor for a specific antigen.

Artificial structure addition type  Immune cells ( artificial structure supplemented immune cell )

"Artificial structural appendicular immune cells" means that an artificial structure is added to the immune cells.

For example, an artificial structural appendicular immune cell may be an immune cell to which an artificial receptor has been added.

An artificial receptor may be one that is capable of recognizing an antigen of a particular disease. For example, an artificial extra-cellular immune cell may be a CAR-T cell.

It may also be the addition of artificial receptors that are capable of recognizing each of two or more antigens caused by a particular disease. At this time, each artificial receptor may be expressed in a time-dependent manner depending on conditions.

For example, in the case of a manipulated immune cell for the treatment of cancer, the first artificial receptor may generate an immune response signal that initiates the expression of the second artificial receptor gene, and then the second artificial receptor may be expressed. The second artificial receptor can produce an immune response signal that induces an immune response to cancer cells. In this case, the ability of the manipulated immune cells to attack cancer cells can be improved.

Artificial receptors may be capable of recognizing manipulated immune cells.

Artificial receptors may be capable of recognizing normal body cells. As an example, an artificial structural appendicular immune cell may be an iCAR-T cell.

The artificial receptor may be one that is cognitive to the third substance. At this time, the third substance may be capable of binding to an antigen of a specific disease.

At this time, the third substance can bind to an artificial receptor and at the same time be capable of binding an antigen of a specific disease. For example, it may have the ability to bind cancer cell antigens concurrently with an artificial receptor.

As another example, the artificial structural addition type immune cells may be immune cells to which an artificial structure having a specific function is added in addition to artificial receptors.

If an artificial structure is added to the immune cells different from the natural state, the artificial structural appendicular immune cells may have a new immunological effect.

E.g,

The new immunomodulatory effect may be that it binds to a specific antigen so that the immune cell has a specific positional relationship with the antigen.

The new immunomodulatory effect may be a function that recognizes a particular antigen and promotes an immune response to a particular antigen.

New immune efficacy may be a function that inhibits excessive immune response.

The new immunomodulatory effect may be a function of regulating the signaling pathway of the immune response.

The new immunomodulatory effect may be a function to identify a specific disease by forming a bond with a third substance. At this time, the third substance may be a biomarker of a specific disease.

A preferred example of the above-mentioned specific antigens may be an antigen of a cancer cell.

The antigens of cancer cells include but are not limited to AD034, AKT1, BRAP, CAGE, CDX2, CLP, CT-7, CT8 / HOM-TES-85, cTAGE-1, EGFR, EGFRvIII, Fibulin- HOM-MEEL-40 / SSX2, HOM-RCC-3.1.3 / CAXII, HOXA7, HOXB6, Hu, HEK-C2, hCAP-G, HCE661, HER2 / neu, HLA- 1, MAGE-4a, MPP1, MSLN, NNP-1, NY-BR-1, KM-HN-3, KM-KN-1, KOC1, KOC2, KOC3, KOC3, LAGE- NY-BR-62, NY-BR-85, NY-CO-37, NY-CO-38, NY-ESO-1, NY- NY-REN-33 / SNC6, NY-REN-43, NY, REN-19 / LKB / STK1 1, NY-REN-21, NY-REN-26 / -REN-65, NY-REN-9, NY-SAR-35, OGFr, PLU-1, PSMA, PSCA, Rab38, RBPJkappa, RHAMM, SCP1, SCP-1, SSX3, SSX4, SSX5, TOP2A, TOP2B, ROR -1 or Tyrosinase, and the like.

Complex manipulated immune cells ( hybrid manipulated immune cell )

"Complex manipulated immune cells" means immune cells, cells that have both an immune modulatory element and an artificial structure added.

The manipulation of the immunoregulatory factor in the combined manipulated immune cells is as described for the functionally-functioning immune cells. In addition, the addition of the artificial structure is as described in the artificial structural addition type immune cells.

Where the functional function of the immune cell is a genetic manipulation, the location of the added artificial structure may be the same as the position of the gene where the functional manipulation occurred.

The new immunological efficacy of the combined manipulated immune cells may include new immunological efficacy of the functionally-functioning immune cells and the artificial addition-type immune cells, and may further exhibit improved immune efficacy by the interaction therebetween.

Improved immune efficacy may be due to improved specificity and immune response to certain diseases. In one preferred example, the combined immunodeficient cells may be those with improved specificity and immune response to cancer.

The immune system of the present invention comprises a desired immune response and a disease treatment mechanism therefor, which is made by a manipulated immunomodulatory component and / or a manipulated immune cell.

In an embodiment, it may be used to affect immune cell proliferation using immune regulatory elements and / or manipulated immune cells inactivating genes that inhibit immune cell proliferation.

In an embodiment, an immune regulatory element inactivating a gene that mediates immune cell apoptosis and / or manipulated immune cells may be used to affect immune cell survival.

In an embodiment, an immune modulating factor that inactivates a gene encoding an immunosuppressive and inhibitory signaling factor and / or manipulated immune cells may be used to affect immune cell function.

The methods and compositions discussed herein may be used to treat one or more of the factors that limit the efficacy of genetically modified immune cells, such as immune cell proliferation, immune cell survival, immune cell function, or any combination thereof, And may be used individually or in combination.

Immune-regulating therapy, on the other hand, refers to the treatment of diseases by manipulating the immune response in the body using engineered immune regulatory elements and / or manipulated immune cells.

For example, immune cells such as dendritic cells, natural killer cells, and T cells can be used to treat diseases by activating or inactivating immune responses in the body.

These immunomodulatory therapies are mainly developed for the indication of cancer therapy, and since the immune function is activated by administering the immune cells directly to the patient, the therapeutic effect is obtained. Therefore, the surgery, chemotherapy or radiation therapy It is a differentiated treatment mechanism.

One example of the immunomodulatory therapy is a treatment with a dendritic cell regulator, a lymphokine activated killer (LAK), a tumor invading T cell including T-lymphocytes (TIL), T-receptor-modified T cells (TCR-T), and chimeric antigen-receptor T-cells do.

[Genetic manipulation or modification]

The manipulation or modification of the substances involved in the immunomodulatory elements, immune cells, and immune system of the present invention can preferably be through genetic manipulation.

In one aspect, compositions and methods for targeting and genetically engineering some or all of the non-coding or coding regions of immunomodulatory genes that affect the proliferation, survival and / or function of immune cells can be provided.

The composition and method

In embodiments, for the formation of the desired immune system, one or more of the immunoregulatory genes involved may be engineered or modified. This can be done through modification of the nucleic acid that constitutes the gene. As a result of the operation, it includes knock down, knock out, and knock in forms.

In embodiments, a promoter region, or a transcription sequence, such as an intron or exon sequence, may be targeted. A cipher sequence, for example a cipher region, an initial cipher region, can be targeted for alteration of expression and knockout.

In an embodiment, the nucleic acid modification is carried out in the presence of one or more nucleotides, such as 1 to 30 bp, 1 to 27 bp, 1 to 25 bp, 1 to 23 bp, 1 to 20 bp, 1 to 15 bp, 1 to 10 bp, 1 to 5 bp, Deletion, and / or insertion of 1 bp nucleotides.

In embodiments, deletions in one or more of the immunomodulatory genes may be used to knockout one or more of the immunomodulatory genes, or to eliminate one or more expressions, or to knock out one or more one or two alleles Or < / RTI > mutations.

In an embodiment, genetic knockdown can be used to reduce the expression of unwanted alleles or transcripts.

In an embodiment, it can be used to alter an immunomodulatory gene that affects immune cell function by targeting a non-coding sequence of a promoter, enhancer, intron, 3'UTR, and / or polyadenylation signal.

In an embodiment, alteration of the gene nucleic acid may result in modulation of the activity of the immunomodulatory gene, e.g., activation or inactivation.

In embodiments, the genetic nucleic acid modification may be a single stranded or double stranded cleavage of a specific site in a gene targeted by the guide nucleic acid-editor protein complex, i.e., deactivating the targeted gene by catalyzing the nucleic acid strand damage have.

In an embodiment, nucleic acid strand breaks can be repaired via mechanisms such as homologous recombination or nonhomologous end joining (NHEJ).

In this case, when the NHEJ mechanism occurs, a change is made in the DNA sequence at the cleavage site, whereby the gene can be inactivated. Repair via NHEJ results in short gene fragment substitutions, insertions or deletions and can be used to induce corresponding gene knockouts.

In another aspect, the present invention can provide said genetic manipulation position.

Refers to a position in the gene that, in embodiments, results in a decrease or elimination of the expression of an immunomodulatory gene product if altered by an NHEJ-mediated alteration.

E.g,

May be in the initial cipher domain.

It can be in the top 50% cipher area.

Lt; / RTI > promoter sequence.

May be in a particular intron sequence

It may be in a certain exon sequence.

May be at a particular position in the gene that affects the proliferation, survival and / or function of the immune cell.

It may be at a specific position in the gene that affects the function of the protein involved in the immune response.

It may be at a particular position in the gene that affects cognitive abilities for a particular antigen.

It may be at a specific position in the gene that affects the function of regulating the secretion of cytokines in immune cells.

May be at a particular position in the gene that affects the ability of the receptor to control antigen binding capacity in immune cells.

The gene manipulation may be performed in consideration of the gene expression regulatory process.

In embodiments, selection may be made of suitable manipulation means for each step in transcription regulation, RNA processing regulation, RNA transport regulation, RNA degradation regulation, translational regulation or protein modification regulation.

In embodiments, RNAi (RNA interference or RNA silencing) can be used to prevent small RNAs (sRNAs) from interfering with mRNA, degrading stability and, in some cases, destroying protein synthesis information, Expression can be controlled.

In embodiments, wild-type or variant enzymes that can catalyze the hydrolysis (cleavage) of DNA or RNA molecules, preferably bonds between nucleic acids in DNA molecules, can be used. A guide nucleic acid-editor protein complex can be used.

For example, one or more selected from the group consisting of meganuclease, Zinc finger nuclease, CRISPR / Cas9 (Cas9 protein), CRISPR-Cpf1 (Cpf1 protein) and TALE- The expression of genetic information can be controlled by manipulating the gene using a cleavage agent.

Preferred examples include, but are not limited to, non-homologous end joining (NHEJ) or homologous recombination repair using the guide nucleic acid-editor protein complex, for example, using the CRISPR / Cas system homology-directed repair (HDR).

In one embodiment, the PD-1 gene, the CTLA-4 gene, the TNFAIP3 gene, the DGKA gene, the DGKZ gene, the Fas gene, the EGR2 gene, and the PPP2R2D gene are used as immunomodulatory genes that affect the proliferation, survival and / , The TET2 gene, or the PSGL-1 gene and the KDM6A gene.

Table 1 summarizes the target sequence regions of the above genes, that is, sites at which the nucleic acid modification can occur (the target sequence regions shown in Table 1 include PAM sequence 5'-NGG-3 'at the 3' end Lt; / RTI &

The target sequence may be two or more targets simultaneously.

Two or more of the genes may be targeted simultaneously.

Two or more target sequences in a homologous gene or two or more target sequences in a heterologous gene can be simultaneously targeted.

In one embodiment, DGKa or DGKz may be targeted, respectively.

In one embodiment, DGKa and DGKz can be targeted simultaneously.

[Table 1] Target sequence

[Gene scissors system]

Genetic manipulation or modification of a substance involved in the immunomodulatory elements, immune cells and immune system of the present invention can be carried out using a "guide nucleic acid-editor protein complex ".

Guide nucleic acid - Editor protein  Complex

The "guide nucleic acid-editor protein complex" refers to a complex formed through the interaction of a guide nucleic acid and an editor protein, and the nucleic acid-protein complex includes a guide nucleic acid and an editor protein.

The " guide nucleic acid " means a nucleic acid capable of recognizing a target nucleic acid, gene, chromosome or protein.

The guide nucleic acid may be in the form of a DNA, RNA or DNA / RNA blend, and may have 5 to 150 nucleic acid sequences.

The guide nucleic acid may comprise one or more domains.

The domain may be, but is not limited to, a guiding domain, a first complementary domain, a connecting domain, a second complementary domain, a proximal domain, a tail domain, and the like.

The guide nucleic acid may include two or more domains, and may include the same domain repeatedly or include different domains.

The guide nucleic acid may be a single contiguous nucleic acid sequence.

For example, one contiguous nucleic acid sequence may be (N) m, where N is A, T, C or G, or A, U, C or G, and m is an integer from 1 to 150 .

The guide nucleic acid may have two or more consecutive nucleic acid sequences.

For example, two or more consecutive nucleic acid sequences may be (N) m and (N) o, wherein N is A, T, C or G, or A, U, C or G, Means an integer of 1 to 150, and m and o may be the same or different from each other.

The " editor protein " means a peptide, polypeptide, or protein that is capable of directly binding to, or not interacting directly with, a nucleic acid. The editor protein may also be conceptually referred to as "gene scissors" or RGEN (RNA-Guided Endonuclease).

The editor protein may be an enzyme.

The above-mentioned editor protein may be a fusion protein.

Herein, the term " fusion protein " refers to a protein produced by fusion of an enzyme and additional domains, peptides, polypeptides or proteins.

The term " enzyme " means a protein comprising a domain capable of cleaving a nucleic acid, gene, chromosome or protein.

The additional domains, peptides, polypeptides or proteins may be functional domains, peptides, polypeptides or proteins having the same or different functions as the enzymes.

Wherein the fusion protein is at or near the amino terminus of the enzyme; Carboxy terminus or vicinal thereof; The middle part of the enzyme; Or an additional domain, peptide, polypeptide or protein in one or more of these combinations.

Wherein the fusion protein is at or near the amino terminus of the enzyme; Carboxy terminus or vicinal thereof; The middle part of the enzyme; Or a functional domain, peptide, polypeptide or protein in one or more of these combinations.

The guide nucleic acid-editor protein complex can modify the target.

The subject may be a target nucleic acid, gene, chromosome or protein.

For example, the guide nucleic acid-editor protein complex can be used to control (e.g., inhibit, inhibit, decrease, increase, or promote) the expression of the protein of interest, or to remove the protein or to express a new protein .

At this time, the guide nucleic acid-editor protein complex can act at DNA, RNA, gene or chromosome level.

The guide nucleic acid-editor protein complex can act at the transcription and translation step of the gene.

The guide nucleic acid-editor protein complex can act at the protein level.

One. Guide nucleic acid

The guide nucleic acid is a nucleic acid capable of recognizing a target nucleic acid, gene, chromosome or protein, and forms a guide nucleic acid-protein complex.

At this time, the guide nucleic acid serves to recognize or target the nucleic acid, gene, chromosome or protein targeted by the guide nucleic acid-protein complex.

The guide nucleic acid may be in the form of a DNA, RNA or DNA / RNA blend, and may have 5 to 150 nucleic acid sequences.

The guide nucleic acid may be linear or circular.

The guide nucleic acid may be a single contiguous nucleic acid sequence.

For example, one contiguous nucleic acid sequence may be (N) m, where N is A, T, C or G, or A, U, C or G, and m is an integer from 1 to 150 .

The guide nucleic acid may have two or more consecutive nucleic acid sequences.

For example, two or more consecutive nucleic acid sequences may be (N) m and (N) o, wherein N is A, T, C or G, or A, U, C or G, Means an integer of 1 to 150, and m and o may be the same or different from each other.

The guide nucleic acid may comprise one or more domains.

Here, the domain may be a guide domain, a first complementary domain, a connection domain, a second complementary domain, a proximal domain, a tail domain, and the like, but is not limited thereto.

The guide nucleic acid may include two or more domains, and may include the same domain repeatedly or include different domains.

The description of the domain is described below.

i) Guide domain

A " guiding domain " is a domain containing a complementary guide sequence capable of binding complementary to a target sequence on a target gene or nucleic acid, and serves for specific interaction with a target gene or nucleic acid.

The guide sequence is a nucleic acid sequence complementary to a target sequence on a target gene or nucleic acid, for example, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% % Or more complementary or completely complementary nucleic acid sequence.

The guide domain may be from 5 to 50 nucleotides in length.

For example, the guiding domain may comprise 5 to 50 nucleotides, 10 to 50 nucleotides, 15 to 50 nucleotides, 20 to 50 nucleotides, 25 to 50 nucleotides, 30 to 50 nucleotides, 35 To 50 nucleotides, 40 to 50 nucleotides, or 45 to 50 nucleotides.

In another embodiment, the guiding domain comprises one to five nucleotide sequences, 5 to 10 nucleotides, 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, 25 to 30 nucleotides, From 30 to 35 nucleotides, from 35 to 40 nucleotides, from 40 to 45 nucleotides, or from 45 to 50 nucleotides.

The guide domain may comprise a guide sequence.

The guide sequence may be a complementary base sequence capable of binding complementary to the target sequence on the target gene or nucleic acid.

The guiding sequence may be a nucleic acid sequence complementary to the target sequence on the target gene or nucleic acid, and may be, for example, at least 70%, 75%, 80%, 85%, 90% or 95% complementary or completely complementary Nucleic acid sequence.

The guide sequence may be from 5 to 50 nucleotide sequences.

For example, the guiding domain may comprise 5 to 50 nucleotides, 10 to 50 nucleotides, 15 to 50 nucleotides, 20 to 50 nucleotides, 25 to 50 nucleotides, 30 to 50 nucleotides, 35 To 50 nucleotides, 40 to 50 nucleotides, or 45 to 50 nucleotides.

In another example, the guiding sequence may comprise one to five nucleotide sequences, 5 to 10 nucleotides, 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, 25 to 30 nucleotides, From 30 to 35 nucleotides, from 35 to 40 nucleotides, from 40 to 45 nucleotides, or from 45 to 50 nucleotides.

Also, the guiding domain may comprise a guiding sequence and an additional base sequence.

The additional base sequence may be one for enhancing or reducing the function of the guide domain.

The additional base sequence may be one for enhancing or reducing the function of the guide sequence.

The additional base sequence may be from 1 to 35 base sequences.

As an example, the additional base sequence may comprise 5 to 35 nucleotides, 10 to 35 nucleotides, 15 to 35 nucleotides, 20 to 35 nucleotides, 25 to 35 nucleotides, or 30 to 35 nucleotides .

In another example, the additional base sequence may be selected from the group consisting of 1 to 5 nucleotides, 5 to 10 nucleotides, 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, 25 to 30 nucleotides Or 30 to 35 nucleotide sequences.

The additional base sequence may be located at the 5 ' end of the guide sequence.

The additional base sequence may be located at the 3 ' end of the guide sequence.

ii ) 1st  Complementary domain

A " first complementary domain " is a nucleic acid sequence comprising a second complementary domain and a complementary nucleic acid sequence, and is complementary enough to form a double strand with a second complementary domain.

The first complementary domain may be from 5 to 35 nucleotide sequences.

In one example, the first complementary domain comprises a sequence of 5 to 35 nucleotides, 10 to 35 nucleotides, 15 to 35 nucleotides, 20 to 35 nucleotides, 25 to 35 nucleotides, or 30 to 35 nucleotides Lt; / RTI >

In another embodiment, the first complementary domain comprises a sequence selected from the group consisting of 1 to 5 nucleotides, 5 to 10 nucleotides, 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, 25 to 30 nucleotides, Or 30 to 35 nucleotide sequences.

iii ) Connection domain

A " connection domain " is a nucleic acid sequence linking two or more domains. A connection domain connects two or more identical or different domains. A connection domain may have a covalent or non-covalent association with two or more domains, or two or more domains may be covalently or non-covalently linked.

The linking domain may be from 1 to 30 nucleotide sequences.

In one embodiment, the linking domain comprises one to five nucleotide sequences, 5 to 10 nucleotides, 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, or 25 to 30 nucleotides .

In another embodiment, the linking domain comprises a nucleotide sequence selected from the group consisting of 1 to 30 nucleotides, 5 to 30 nucleotides, 10 to 30 nucleotides, 15 to 30 nucleotides, 20 to 30 nucleotides, or 25 to 30 nucleotides Lt; / RTI >

iv ) Second  Complementary domain

A " second complementary domain " is a nucleic acid sequence comprising a first complementary domain and a complementary nucleic acid sequence, and is complementary enough to form a double strand with the first complementary domain.

The second complementary domain includes a base sequence that is not complementary to the first complementary domain and the complementary base sequence and the first complementary domain, for example, a base sequence that does not form a double strand with the first complementary domain And the length of the base sequence may be longer than that of the first complementary domain.

The second complementary domain may be from 5 to 35 nucleotide sequences.

In one embodiment, the second complementary domain comprises a sequence selected from the group consisting of 1 to 35 nucleotides, 5 to 35 nucleotides, 10 to 35 nucleotides, 15 to 35 nucleotides, 20 to 35 nucleotides, 25 to 35 nucleotides, Sequence or a 30 to 35 base sequence.

In another embodiment, the second complementary domain comprises one to five nucleotide sequences, 5 to 10 nucleotides, 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, 25 to 30 nucleotides, Base sequence or 30 to 35 base sequences.

v) Proximal  domain( proximal domain )

A " proximal domain " is a nucleic acid sequence located close to a second complementary domain.

The proximal domain may comprise a complementary base sequence in the proximal domain and may form a double strand by a complementary base sequence.

The proximal domain may be from 1 to 20 nucleotide sequences.

As an example, the proximal domain may be from 1 to 20 nucleotides, from 5 to 20 nucleotides, from 10 to 20 nucleotides, or from 15 to 20 nucleotides.

In another example, the proximal domain may be from 1 to 5 nucleotides, from 5 to 10 nucleotides, from 10 to 15 nucleotides, or from 15 to 20 nucleotides.

vi ) Tail domain

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

The tail domain may comprise a complementary base sequence in the tail domain and may form a double strand by a complementary base sequence.

The tail domain may be from 1 to 50 nucleotide sequences.

For example, the tail domain may comprise 5 to 50 nucleotides, 10 to 50 nucleotides, 15 to 50 nucleotides, 20 to 50 nucleotides, 25 to 50 nucleotides, 30 to 50 nucleotides, 35 To 50 nucleotides, 40 to 50 nucleotides, or 45 to 50 nucleotides.

In another embodiment, the tail domain may comprise one to five nucleotide sequences, five to ten nucleotides, 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, 25 to 30 nucleotides, From 30 to 35 nucleotides, from 35 to 40 nucleotides, from 40 to 45 nucleotides, or from 45 to 50 nucleotides.

On the other hand, some or all of the nucleic acid sequences including the above-mentioned domains, i.e., the guide domain, the first complementary domain, the connecting domain, the second complementary domain, the proximal domain and the tail domain may optionally or additionally include a chemical modification have.

The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl 3'phosphorothioate (MS), or 2'-O-methyl 3'thioPACE It is not limited.

The guide nucleic acid comprises one or more domains.

The guide nucleic acid may comprise a guide domain.

The guide nucleic acid may comprise a first complementary domain.

The guide nucleic acid may comprise a linking domain.

The guide nucleic acid may comprise a second complementary domain.

The guide nucleic acid may comprise a proximal domain.

The guide nucleic acid may comprise a tail domain.

At this time, the number of the domains may be 1, 2, 3, 4, 5, 6, or more.

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

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

The guide nucleic acid may comprise one, two, three, four, five, six or more linking domains.

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

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

The guide nucleic acid may comprise one, two, three, four, five, six or more tail domains.

At this time, the guide nucleic acid may include one domain in duplicate.

The guide nucleic acid may comprise multiple domains without overlapping or overlapping.

The guide nucleic acid may comprise the same kind of domain, wherein the same kind of domains may have the same nucleic acid sequence or may have different nucleic acid sequences.

The guide nucleic acid may comprise two kinds of domains, wherein the two other domains may have different nucleic acid sequences or may have the same nucleic acid sequence.

The guide nucleic acid may comprise three kinds of domains, wherein the three other domains may have different nucleic acid sequences or may have the same nucleic acid sequence.

The guide nucleic acid may comprise four kinds of domains, wherein the other four kinds of domains may have different nucleic acid sequences or may have the same nucleic acid sequences.

The guide nucleic acid may include five kinds of domains, wherein the other five kinds of domains may have different nucleic acid sequences or may have the same nucleic acid sequences.

The guide nucleic acid may comprise six kinds of domains, wherein the other six kinds of domains may have different nucleic acid sequences or may have the same nucleic acid sequence.

For example, the guide nucleic acid may be selected from [guide domain] - [first complementary domain] - [link domain] - [second complementary domain] - [link domain] - [guide domain] - [first complementary domain] - [Linked domain] - [Second complementary domain], where the two guide domains may contain a guide sequence for different or identical targets, and the two first complementary domains Two second complementary domains may have the same nucleic acid sequence or have different nucleic acid sequences. Where the guiding domain comprises a guiding sequence for a different target, the guiding nucleic acid may specifically bind to two targets, wherein specific binding may occur simultaneously or sequentially. Also, the linking domain can be cleaved by a specific enzyme, and in the presence of a specific enzyme, the guide nucleic acid can be divided into two parts or three parts.

As one example of the guide nucleic acid of the present invention, the gRNA is described below.

gRNA

&Quot; gRNA " refers to the gRNA-CRISPR enzyme complex for the target gene or nucleic acid, i. E., The specifically targeting nucleic acid of the CRISPR complex. Also, the gRNA means a target gene or a nucleic acid-specific RNA. The gRNA can be coupled with a CRISPR enzyme to direct a CRISPR enzyme to a target gene or a nucleic acid.

The gRNA may comprise a plurality of domains. Each domain can interact within the strand or between strands of the three-dimensional behavior or the active form of the gRNA.

gRNA is a single stranded gRNA (single RNA molecule); Or a double gRNA (including more than one and typically two separate RNA molecules).

In one embodiment, the single stranded gRNA comprises a guide domain in the 5 'to 3' direction, a domain comprising a guide sequence capable of binding complementary to a target gene or nucleic acid; A first complementary domain; Connection domain; A second complementary domain, a domain capable of forming a double-stranded nucleic acid with a first complementary domain since the first complementary domain has a sequence complementary to the first complementary domain sequence; Proximal domain; And optionally a tail domain.

In another embodiment, the double gRNA comprises a guide domain in the 5 'to 3' direction, a domain comprising a guide sequence capable of a complementary binding to a target gene or nucleic acid, and a first complementary domain ≪ / RTI > And a second complementary domain; a domain capable of forming a double-stranded nucleic acid with a first complementary domain having a sequence complementary to the first complementary domain sequence; a proximal domain; And optionally a second strand comprising a tail domain.

Wherein the first strand may be referred to as a crRNA and the second strand may be referred to as a tracrRNA. The crRNA may comprise a guiding domain and a first complementary domain, wherein the tracrRNA may comprise a second complementary domain, a proximal domain and optionally a tail domain.

In yet another embodiment, the single stranded gRNA comprises a guide domain in the 3 'to 5' direction, a domain comprising a guide sequence capable of binding complementary to the target gene or nucleic acid; A first complementary domain; And a second complementary domain, a domain complementary to the first complementary domain sequence, and a domain capable of forming a double-stranded nucleic acid with the first complementary domain.

Guide domain

The guiding domain comprises a complementary guide sequence capable of binding complementary to the target sequence on the target gene or nucleic acid. The guide sequence is a nucleic acid sequence complementary to the target sequence on the target gene or nucleic acid, for example, at least 70%, 75%, 80%, 85%, 90% or 95% complementary or completely complementary nucleic acid sequence Lt; / RTI > The guiding domain is believed to play a role in allowing specific interaction with the target gene or nucleic acid of the gRNA-Cas complex, i.e. the CRISPR complex.

The guide domain may be from 5 to 50 nucleotides in length.

In one embodiment, the guide domain comprises 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, Lt; / RTI > nucleotides or 25 nucleotides in length.

In another embodiment, the guiding domain comprises 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 base sequences or 25 base sequences.

At this time, the guide domain may include a guide sequence.

The guide sequence may be a complementary base sequence capable of binding complementary to the target sequence on the target gene or nucleic acid.

The guiding sequence may be a nucleic acid sequence complementary to the target sequence on the target gene or nucleic acid, and may be, for example, at least 70%, 75%, 80%, 85%, 90% or 95% complementary or completely complementary Nucleic acid sequence.

The guide sequence may be from 5 to 50 nucleotide sequences.

In one embodiment, the guide sequence comprises 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, Lt; / RTI > nucleotides or 25 nucleotides in length.

In another embodiment, the guide sequence comprises 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 base sequences or 25 base sequences.

At this time, the guide domain may include a guide sequence and an additional base sequence.

The additional base sequence may be from 1 to 35 base sequences.

In one embodiment, the additional base sequence comprises one nucleotide sequence, two nucleotide sequences, three nucleotide sequences, four nucleotide sequences, five nucleotide sequences, six nucleotide sequences, seven nucleotide sequences, eight nucleotide sequences, 9 base sequences or 10 base sequences.

For example, the additional base sequence may be one base sequence G (guanine) or two base sequences GG.

The additional base sequence may be located at the 5 ' end of the guide sequence.

The additional base sequence may be located at the 3 ' end of the guide sequence.

Optionally, some or all of the nucleotide sequence of the guiding domain may comprise a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl 3'phosphorothioate (MS), or 2'-O-methyl 3'thioPACE It is not limited.

The first complementary domain

The first complementary domain comprises a second complementary domain and a complementary nucleic acid sequence, and is complementary enough to form a double strand with the second complementary domain.

At this time, the first complementary domain may be 5 to 35 nucleotide sequences. The first complementary domain may comprise 5 to 35 nucleotide sequences.

In one embodiment, the first complementary domain comprises 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 bases, A nucleotide sequence of 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotide sequences, 24 nucleotide sequences, or 25 nucleotide sequences.

In another embodiment, the first complementary domain comprises 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, A base sequence, 13 nucleotide sequences, 14 nucleotide sequences, 15 nucleotide sequences, 16 nucleotide sequences, 17 nucleotide sequences, 18 nucleotide sequences, 19 nucleotide sequences, 20 nucleotide sequences, 21 nucleotide sequences, 22 nucleotide sequences A nucleotide sequence, 23 nucleotide sequences, 24 nucleotide sequences, or 25 nucleotide sequences.

The first complementary domain may have homology with the first complementary domain of nature, or may be derived from a first complementary domain of nature. In addition, the first complementary domain may have a difference in the base sequence of the first complementary domain depending on the species present in nature, and may be derived from a first complementary domain comprising the species present in nature, or And may have some or complete homology with the first complementary domain comprising the species present in nature.

In one embodiment, the first complementary domain is selected from the group consisting of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Streptococcus aureus, ), Or at least 50% or more, or complete homology with the first complementary domain or the derived first complementary domain of Neisseria meningitides.

For example, when the first complementary domain is a first complementary domain of Streptococcus fyogenes or a first complementary domain derived from Streptococcus fyogenses, the first complementary domain is 5'-GUUUUAGAGCUA-3 Or 5'-GUUUUAGAGCUA-3 ', and at least 50% or more homology with 5'-GUUUUAGAGCUA-3'. At this time, the first complementary domain may further include (X) n, i.e., 5'-GUUUUAGAGCUA (X) n-3 '. X may be selected from the group consisting of bases A, T, U and G, and n is the number of base sequences and may be an integer of 5 to 15. Here, (X) n may be an integer of n repeats of the same base sequence, or may be an integer n base sequence in which bases A, T, U and G are mixed.

In another example, when the first complementary domain is a first complementary domain of Campylobacter jejuni or a first complementary domain of Campylobacter herbaceae, the first complementary domain is 5'-GUUUUAGUCCCUUUUUAAAUUUCUU -3 ', or may be a base sequence having at least 50% or more homology with 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3'. At this time, the first complementary domain may further include (X) n, i.e., 5'-GUUUUAGUCCCUUUUUAAAUUUCUU (X) n-3 '. X may be selected from the group consisting of bases A, T, U and G, and n is the number of base sequences and may be an integer of 5 to 15. Here, (X) n may be an integer of n repeats of the same base sequence, or may be an integer n base sequence in which bases A, T, U and G are mixed.

In another embodiment, the first complementary domain is selected from the group consisting of Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasicus, , Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Pseudomonas spp. ), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp. (SC_KO8D17), Reppose Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112)), At least 50% or more complete homology with the first complementary domain or the derived first complementary domain of Candidatus Methanoplasma termitum or Eubacterium eligens, Lt; / RTI >

For example, when the first complementary domain is a first complementary domain of P. bacterium or a first complementary domain of P. bacterium, the first complementary domain is 5'-UUUGUAGAU-3 ' Or may be a base sequence having at least 50% or more homology with 5'-UUUGUAGAU-3 '. At this time, the first complementary domain may further include (X) n, that is, 5 '- (X) nUUUGUAGAU-3'. X may be selected from the group consisting of bases A, T, U and G, and n is the number of base sequences and may be an integer of 1 to 5. Here, (X) n may be an integer of n repeats of the same base sequence, or may be an integer n base sequence in which bases A, T, U and G are mixed.

Optionally, some or all of the base sequence of the first complementary domain may comprise a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl 3'phosphorothioate (MS), or 2'-O-methyl 3'thioPACE It is not limited.

Connected Domains

A linking domain is a nucleic acid sequence linking two or more domains, and a linking domain links two or more identical or different domains. A connection domain may have a covalent or non-covalent association with two or more domains, or two or more domains may be covalently or non-covalently linked.

The linking domain may be a nucleic acid sequence that links a first complementary domain and a second complementary domain to produce single stranded gRNA.

The connecting domain may be covalently or non-covalently bound to the first complementary domain and the second complementary domain.

The connection domain may connect the first complementary domain and the second complementary domain in a covalent or non-covalent manner.

The linking domain may be from 1 to 30 nucleotide sequences. The linking domain may comprise from 1 to 30 nucleotide sequences.

In one embodiment, the linking domain comprises one to five nucleotide sequences, 5 to 10 nucleotides, 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, or 25 to 30 nucleotides Lt; / RTI >

In another embodiment, the linking domain comprises one to five nucleotide sequences, 5 to 10 nucleotides, 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, or 25 to 30 nucleotides Sequence.

The linking domain is suitable for use in single-stranded gRNA molecules, and can be covalently or non-covalently linked to the first and second strands of the double gRNA, or covalently or non-covalently linked the first and second strands Can be used to generate single stranded gRNA. The linking domain can be used for covalent or noncovalent binding with the crRNA and tracrRNA of the double gRNA, or covalently or noncovalently linking the crRNA and the tracrRNA to produce single stranded gRNA.

The linking domain may be homologous to, or derived from, a naturally occurring sequence, e. G., A partial sequence of a tracrRNA.

Optionally, some or all of the nucleotide sequence of the linking domain may comprise a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl 3'phosphorothioate (MS), or 2'-O-methyl 3'thioPACE It is not limited.

Second complementary domain

The second complementary domain comprises a first complementary domain and a complementary nucleic acid sequence and is complementary enough to form a double strand with the first complementary domain. The second complementary domain includes a base sequence that is not complementary to the first complementary domain and the complementary base sequence and the first complementary domain, for example, a base sequence that does not form a double strand with the first complementary domain And the length of the base sequence may be longer than that of the first complementary domain.

At this time, the second complementary domain may be 5 to 35 nucleotide sequences. The first complementary domain may comprise 5 to 35 nucleotide sequences.

In one embodiment, the second complementary domain comprises 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 bases, A nucleotide sequence of 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotide sequences, 24 nucleotide sequences, or 25 nucleotide sequences.

In another embodiment, the second complementary domain comprises 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, A base sequence, 13 nucleotide sequences, 14 nucleotide sequences, 15 nucleotide sequences, 16 nucleotide sequences, 17 nucleotide sequences, 18 nucleotide sequences, 19 nucleotide sequences, 20 nucleotide sequences, 21 nucleotide sequences, 22 nucleotide sequences A nucleotide sequence, 23 nucleotide sequences, 24 nucleotide sequences, or 25 nucleotide sequences.

The second complementary domain may have a homology with a second complementary domain derived from nature, or may be derived from a second complementary domain derived from nature. The second complementary domain may have a difference in the base sequence of the second complementary domain depending on the species present in nature and may be derived from a second complementary domain comprising the species present in nature, May have some or complete homology with a second complementary domain comprising the species present in nature.

In one embodiment, the second complementary domain is selected from the group consisting of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Streptococcus aureus, ), Or at least 50% or more, or complete homology with the second complementary domain or the derived second complementary domain of Neisseria meningitides.

For example, if the second complementary domain is a second complementary domain of Streptococcus fyogenes or a second complementary domain of Streptococcus fyogenses, the second complementary domain is 5'- UAGC AAGU UAAAA U-3 ', or may be a nucleotide sequence having at least 50% or more homology with the 5'- UAGC AAGU UAAAA U-3' (underlined indicates that the first complementary domain forms a double strand Base sequence). In this case, the second complementary domain may further include (X) n or / and (X) m, i.e., 5 '- (X) n UAGC AAGU UAAAA U (X) m-3'. X may be selected from the group consisting of bases A, T, U, and G, wherein n and m are numbers of base sequences, n may be an integer of 1 to 15, and m is an integer of 1 to 6 have. Here, (X) n may be an integer of n repeats of the same base sequence, or may be an integer n base sequence in which bases A, T, U and G are mixed. Also, (X) m may be an integer number of m repeats of the same base sequence, or may be an integer m base sequence in which bases A, T, U and G are mixed.

In another example, when the first complementary domain is a second complementary domain of Campylobacter jejuni or a second complementary domain of Campylobacter sp. Njuni , the second complementary domain is 5'- AAGAAAUUUAAAAAGGGACUAAAAU -3 ', or may be a base sequence having a part, at least 50% or more homology with 5'- AAGAAAUUUAAAAAGGGACUAAAAAGGGACUAAAAU- 3' (underlined indicates base sequence forming double strand with first complementary domain). In this case, the second complementary domain may further include (X) n or / and (X) m, that is, 5 '- (X) _n AAGAAAUUUAAAAAGGGACUAAAAU (X) _m -3'. The X may be selected from the group consisting of bases A, T, U, and G, wherein n may be an integer of 1 to 15, and m may be 1 to 6. Here, (X) n may be an integer of n repeats of the same base sequence, or may be an integer n base sequence in which bases A, T, U and G are mixed. Also, (X) m may be an integer number of m repeats of the same base sequence, or may be an integer m base sequence in which bases A, T, U and G are mixed.

In another embodiment, the first complementary domain is selected from the group consisting of Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasicus, , Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Pseudomonas spp. ), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp. (SC_KO8D17), Reppose Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112)), At least 50% or more complete homology with the first complementary domain or the derived first complementary domain of Candidatus Methanoplasma termitum or Eubacterium eligens, Lt; / RTI >

For example, if the second complementary domain is a second complementary domain of P. bacterium or a second complementary domain of P. bacterium, the second complementary domain is 5'-AAAUU UCUAC U-3 Or 5'-AAAUU UCUAC U-3 '(underlined is a nucleotide sequence forming a double strand with the first complementary domain). The second complementary domain may further include (X) n or / and (X) m, that is, 5 '- (X) _n AAAUU UCUAC U (X) _m -3'. X may be selected from the group consisting of bases A, T, U and G, wherein n and m are the number of base sequences, n may be an integer of 1 to 10, and m is an integer of 1 to 6 have. Here, (X) n may be an integer of n repeats of the same base sequence, or may be an integer n base sequence in which bases A, T, U and G are mixed. Also, (X) m may be an integer number of m repeats of the same base sequence, or may be an integer m base sequence in which bases A, T, U and G are mixed.

Optionally, some or all of the base sequence of the second complementary domain may comprise a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl 3'phosphorothioate (MS), or 2'-O-methyl 3'thioPACE It is not limited.

The proximal domain

The proximal domain is a 1 to 20 nucleotide sequence located close to the second complementary domain, and is a domain located in the 3 'direction of the second complementary domain. At this time, the proximal domain can form double stranded bonds between complementary base sequences in the proximal domain.

In one embodiment, the proximal domain comprises 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotide sequences, 14 nucleotide sequences, or 15 nucleotide sequences.

In another embodiment, the proximal domain comprises 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotide sequences, 14 nucleotide sequences, or 15 nucleotide sequences.

In addition, the proximal domain may have homology with a naturally occurring proximal domain or may be derived from a naturally occurring proximal domain. In addition, the proximal domain may have a difference in the base sequence of the proximal domain depending on the species present in nature, and may be derived from a proximal domain comprising the species present in nature, or a proximal domain Lt; RTI ID = 0.0 > and / or < / RTI >

In one embodiment, the proximal domain is selected from the group consisting of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Streptococcus aureus, At least 50% or more, or complete homology with the proximal or derived proximal domain of Neisseria meningitides.

For example, when the proximal domain is a proximal domain of Streptococcus fjigogenes or a proximal domain derived from Streptococcus fjigenses, the proximal domain may be 5'-AAGGCUAGUCCG-3 ', or 5'-AAGGCUAGUCCG-3' And at least 50% or more homology with the nucleotide sequence shown in SEQ ID NO. At this time, the proximal domain may further include (X) n, i.e., 5'-AAGGCUAGUCCGG (X) n-3 '. X may be selected from the group consisting of bases A, T, U and G, and n is the number of base sequences and may be an integer of 1 to 15. Here, (X) n may be an integer of n repeats of the same base sequence, or may be an integer n base sequence in which bases A, T, U and G are mixed.

For example, if the proximal domain is a proximal domain of Campylobacter jejuni or a proximal domain derived from Campylobacter jejuni, the proximal domain may be 5'-AAAGAGUUUGC-3 ', or 5'-AAAGAGUUUGC -3 'and at least 50% or more homology with the nucleotide sequence of SEQ ID NO: 3. At this time, the proximal domain may further include (X) n, i.e., 5'-AAAGAGUUUGC (X) n-3 '. X may be selected from the group consisting of bases A, T, U, and G, wherein n is the number of base sequences and may be an integer of 1 to 40. Here, (X) n may be an integer of n repeats of the same base sequence, or may be an integer n base sequence in which bases A, T, U and G are mixed.

Optionally, some or all of the base sequence of the proximal domain may comprise a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl 3'phosphorothioate (MS), or 2'-O-methyl 3'thioPACE It is not limited.

Tail domain

The tail domain can be a single strand gRNA or a domain that can be selectively added to the 3 'end of the double gRNA, the tail domain can be from 1 to 50 nucleotide sequences, or the tail domain comprises from 1 to 50 nucleotide sequences can do. At this time, the tail domain can form double stranded bonds between complementary base sequences in the tail domain.

In one embodiment, the tail domain comprises one to five nucleotide sequences, 5 to 10 nucleotides, 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, 25 to 30 nucleotides , 30 to 35 nucleotides, 35 to 40 nucleotides, 40 to 45 nucleotides, or 45 to 50 nucleotides.

In another embodiment, the tail domain comprises a nucleotide sequence selected from the group consisting of 1 to 5 nucleotides, 5 to 10 nucleotides, 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, 25 to 30 nucleotides , 30 to 35 nucleotides, 35 to 40 nucleotides, 40 to 45 nucleotides, or 45 to 50 nucleotides.

In addition, the tail domain may have homology with a naturally occurring tail domain, or may be derived from a naturally occurring tail domain. In addition, the tail domain may have a difference in the base sequence of the tail domain depending on the species present in nature, and may be derived from a tail domain including a species present in nature, or a tail domain including a species present in nature Lt; RTI ID = 0.0 > and / or < / RTI >

In one embodiment, the tail domain is selected from the group consisting of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Streptococcus aureus, At least 50% or more, or complete homology with the tail domain or the derived tail domain of Neisseria meningitides.

For example, if the tail domain is the tail domain of Streptococcus fjigogenes or the tail domain of Streptococcus fjogneses, the tail domain may be 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3 ', or 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3 And at least 50% or more homology with the nucleotide sequence shown in SEQ ID NO. At this time, the tail domain may further include (X) n, i.e., 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC (X) n-3 '. X may be selected from the group consisting of bases A, T, U and G, and n is the number of base sequences and may be an integer of 1 to 15. Here, (X) n may be an integer of n repeats of the same base sequence, or may be an integer n base sequence in which bases A, T, U and G are mixed.

In another example, if the tail domain is the tail domain of Campylobacter jejuni or the tail domain of Campylobacter zoojuni, the tail domain may be 5'-GGGACUCUGCGGGUUACAAUCCCCUAAAACCGCUUUU-3 ', or 5'-GGGACUCUGCGGGUUACAAUCCCCUAAAACCGCUUUU -3 'and at least 50% or more homology with the nucleotide sequence of SEQ ID NO: 3. At this time, the tail domain may further include (X) n, i.e., 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU (X) n-3 '. X may be selected from the group consisting of bases A, T, U and G, and n is the number of base sequences and may be an integer of 1 to 15. Here, (X) n may be an integer of n repeats of the same base sequence, or may be an integer n base sequence in which bases A, T, U and G are mixed.

In another embodiment, the tail domain may comprise from 1 to 10 nucleotide sequences at the 3'end associated with in vitro or in vivo transcription methods.

For example, when a T7 promoter is used for in vitro transcription of a gRNA, the tail domain may be any base sequence present at the 3 ' end of the DNA template. In addition, when the U6 promoter is used for in vivo transcription, the tail domain may be UUUUUU, and when the H1 promoter is used for transcription, the tail domain may be UUUU, and when the pol-III promoter is used , The tail domain may comprise several uracil bases or bases that can be substituted.

Optionally, some or all of the base sequence of the tail domain may comprise a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl 3'phosphorothioate (MS), or 2'-O-methyl 3'thioPACE It is not limited.

The gRNA may comprise multiple domains as described above and may control the length of the nucleic acid sequence along the domain that the gRNA comprises and may be modified by each domain to include strands or strands of the three- Can interact with each other.

gRNA is a single stranded gRNA (single RNA molecule); Or a double gRNA (including more than one and typically two separate RNA molecules).

double gRNA

The double gRNA consists of a first strand and a second strand.

At this time, the first strand

5 '- [guide domain] - [first complementary domain] -3'

The second strand

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

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

Wherein the first strand may be referred to as a crRNA and the second strand may be referred to as a tracrRNA.

First strand

[Guide domain]

The guide domain in the first strand comprises a complementary guide sequence capable of binding complementary to the target sequence on the target gene or nucleic acid. The guide sequence is a nucleic acid sequence complementary to the target sequence on the target gene or nucleic acid, for example, at least 70%, 75%, 80%, 85%, 90% or 95% complementary or completely complementary nucleic acid sequence Lt; / RTI > The guiding domain is believed to play a role in allowing specific interaction with the target gene or nucleic acid of the gRNA-Cas complex, i.e. the CRISPR complex.

At this time, the guiding domain may have 5 to 50 nucleotide sequences or 5 to 50 nucleotide sequences. For example, the guiding domain may have 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, Or 25 base sequences, or may comprise the same.

In addition, the guide domain may include a guide sequence.

The guide sequence may be a complementary base sequence or complementary base sequence capable of binding complementary to the target sequence on the target gene or nucleic acid, and may be, for example, at least 70%, 75%, 80%, 85% , 90%, or 95% or more complementary or completely complementary base sequence.

At this time, the guide sequence may be 5 to 50 nucleotide sequences, or may include 5 to 50 nucleotide sequences. For example, the guide sequence may comprise 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, Base sequence or 25 base sequences, or may comprise the same.

Optionally, the guiding domain may comprise a guiding sequence and additional base sequences.

At this time, the additional base sequence may be 1 to 35 base sequences. For example, the additional base sequence may include one base sequence, two base sequences, three base sequences, four base sequences, five base sequences, six base sequences, seven base sequences, eight base sequences, nine base sequences, Lt; RTI ID = 0.0 > 10 < / RTI > nucleotide sequences.

In one embodiment, the additional base sequence may be one base sequence G (guanine) or two base sequences GG.

At this time, the additional base sequence may be located at the 5 'end of the guide domain, or may be located at the 5' end of the guide sequence.

The additional base sequence may be located at the 3 'end of the guide domain or at the 3' end of the guide sequence.

[ 1st  Complementary domain]

The first complementary domain is a domain containing a complementary nucleic acid sequence with a second complementary domain of the second strand and having a degree of complementarity enough to form a double strand with the second complementary domain.

The first complementary domain may have 5 to 35 nucleotide sequences or 5 to 35 nucleotide sequences. For example, the first complementary domain may comprise 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, A nucleotide sequence having a nucleotide sequence of 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 base sequences, 24 base sequences, or 25 base sequences.

The first complementary domain may have homology with the first complementary domain of nature, or may be derived from a first complementary domain of nature. In addition, the first complementary domain may have a difference in the base sequence of the first complementary domain depending on the species present in nature, and may be derived from a first complementary domain comprising the species present in nature, or And may have some or complete homology with the first complementary domain comprising the species present in nature.

In one embodiment, the first complementary domain is selected from the group consisting of Streptococcus pyogenes , Campylobacter jejuni ), Streptococcus thermophilus ( Streptococcus thermophilus , Streptococcus aureus , or Neisseria < RTI ID = 0.0 > at least 50%, or complete homology with the first complementary domain or the derived first complementary domain of the first and second complementary domains.

Optionally, the first complementary domain may comprise additional base sequences that do not undergo a complementary binding with the second complementary domain of the second strand.

Herein, the additional base sequence may be 1 to 15 base sequences. For example, the additional base sequence may be from 1 to 5 nucleotides, from 5 to 10 nucleotides, or from 10 to 15 nucleotides.

Optionally, some or all of the nucleotide sequences of the guide domain and / or the first complementary domain may comprise chemical modifications. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl 3'phosphorothioate (MS), or 2'-O-methyl 3'thioPACE It is not limited.

Thus, the first strand may consist of 5 '- [guiding domain] - [first complementary domain] -3' as described above.

In addition, the first strand may optionally comprise additional base sequences.

In one embodiment, the first strand comprises

5 '- (N _target ) - (Q) _m -3'; or

5 '- (X) _a - (N _target ) - (X) _b - (Q) _m - (X) _c -3'.

Here, the N _target is a base sequence capable of binding complementary to a target sequence on a target gene or a nucleic acid, and the N _target is a base sequence region that can be changed according to a target sequence on a target gene or nucleic acid.

Herein, (Q) _m is a base sequence comprising a first complementary domain, and includes a base sequence capable of complementary binding with a second complementary domain of a second strand. The (Q) _m may be a sequence having partial or complete homology with the first complementary domain of the species existing in nature, and the nucleotide sequence of the first complementary domain may be changed depending on the derived species. The Q may be independently selected from the group consisting of A, U, C and G, and m is the number of base sequences and may be an integer of 5 to 35.

For example, when the first complementary domain has partial or complete homology with the first complementary domain of Streptococcus fyogenes or the first complementary domain derived from Streptococcus fjogneses, the (Q) _m May be 5'-GUUUUAGAGCUA-3 ', or may be a base sequence having at least 50% homology with 5'-GUUUUAGAGCUA-3'.

In another example, when the first complementary domain has partial or complete homology with the first complementary domain of Campylobacter zeaxanth or the first complementary domain derived from Campylobacter zein, the (Q) _m is 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3 ', or may be a nucleotide sequence having at least 50% homology with 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3'.

As another example, when the first complementary domain has partial or complete homology with the first complementary domain of Streptococcus thermophilus or the first complementary domain derived from Streptococcus thermophilus, the (Q) _m May be 5'-GUUUUAGAGCUGUGUUGUUUCG-3 ', or may be a base sequence having at least 50% or more homology with 5'-GUUUUAGAGCUGUGUUGUUUCG-3'.

(X) _a , (X) _b and (X) _c are optionally added base sequences, wherein X may be independently selected from the group consisting of A, U, C and G, A, b, and c are numbers of base sequences and may be 0 or an integer of 1 to 20.

Second strand

The second strand is comprised of a second complementary domain and a proximal domain, and optionally may further comprise a tail domain.

[ Second  Complementary domain]

The second complementary domain in the second strand comprises a complementary nucleic acid sequence with the first complementary domain of the first strand and is complementary enough to form a double strand with the first complementary domain. The second complementary domain includes a base sequence that is not complementary to the first complementary domain and the complementary base sequence and the first complementary domain, for example, a base sequence that does not form a double strand with the first complementary domain And the length of the base sequence may be longer than that of the first complementary domain.

The second complementary domain may have 5 to 35 nucleotide sequences or 5 to 35 nucleotide sequences. For example, the second complementary domain may comprise 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, , 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides , 23 nucleotides, 24 nucleotides, or 25 nucleotides.

The second complementary domain may have a homology with the second complementary domain derived from nature, or may be derived from a second complementary domain derived from nature. The second complementary domain may have a difference in the base sequence of the second complementary domain depending on the species present in nature and may be derived from a second complementary domain comprising the species present in nature, May have some or complete homology with a second complementary domain comprising the species present in nature.

In one embodiment, the second complementary domain is selected from the group consisting of Streptococcus pyogenes , Campylobacter jejuni ), Streptococcus thermophilus ( Streptococcus thermophilus , Streptococcus aureus , or Neisseria < RTI ID = 0.0 > at least 50% or more, or complete homology with the second complementary domain or the derived second complementary domain of the first and second complementary domains.

Optionally, the second complementary domain may comprise additional base sequences that do not undergo a complementary binding with the first complementary domain of the first strand.

Herein, the additional base sequence may be 1 to 25 base sequences. For example, the additional base sequence may be from 1 to 5 nucleotides, from 5 to 10 nucleotides, from 10 to 15 nucleotides, from 15 to 20 nucleotides, or from 20 to 25 nucleotides.

[ Proximal  domain]

The proximal domain in the second strand is a 1 to 20 nucleotide sequence and is located in the 3 'direction of the second complementary domain. For example, the proximal domain may comprise 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, A base sequence, 13 base sequences, 14 base sequences, or 15 base sequences.

At this time, the proximal domain can form double stranded bonds between complementary base sequences in the proximal domain.

In addition, the proximal domain may have homology with a naturally occurring proximal domain or may be derived from a naturally occurring proximal domain. In addition, the proximal domain may have a difference in the base sequence of the proximal domain depending on the species present in nature, and may be derived from a proximal domain comprising the species present in nature, or a proximal domain Lt; RTI ID = 0.0 > and / or < / RTI >

In one embodiment, the proximal domain is selected from the group consisting of Streptococcus pyogenes , Campylobacter jejuni ), Streptococcus thermophilus ( Streptococcus thermophilus , Streptococcus aureus , or Neisseria < RTI ID = 0.0 > proximal domain or domains derived from the proximal portion of the meningitides), may have at least 50% or more, or complete homology.

[Tail domain]

Alternatively, the tail domain in the second strand may be selectively added to the 3 ' end of the second strand, wherein the tail domain may be from 1 to 50 nucleotides or from 1 to 50 nucleotides in length . For example, the tail domain may comprise one to five nucleotide sequences, 5 to 10 nucleotides, 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, 25 to 30 nucleotides, 30 to 35 base sequences, 35 to 40 base sequences, 40 to 45 base sequences, or 45 to 50 base sequences.

At this time, the tail domain can form double stranded bonds between complementary base sequences in the tail domain.

In addition, the tail domain may have homology with a naturally occurring tail domain, or may be derived from a naturally occurring tail domain. In addition, the tail domain may have a difference in the base sequence of the tail domain depending on the species present in nature, and may be derived from a tail domain including a species present in nature, or a tail domain including a species present in nature Lt; RTI ID = 0.0 > and / or < / RTI >

In one embodiment, the tail domain is selected from the group consisting of Streptococcus pyogenes , Campylobacter jejuni ), Streptococcus thermophilus ( Streptococcus thermophilus , Streptococcus aureus , or Neisseria < RTI ID = 0.0 > at least 50%, or complete homology with the tail domain or the derived tail domain of the human immunoglobulins ( e.g., meningitides ).

In another embodiment, the tail domain may comprise from 1 to 10 nucleotide sequences at the 3'end associated with in vitro or in vivo transcription methods.

For example, when a T7 promoter is used for in vitro transcription of a gRNA, the tail domain may be any base sequence present at the 3 ' end of the DNA template. In addition, when the U6 promoter is used for in vivo transcription, the tail domain may be UUUUUU, and when the H1 promoter is used for transcription, the tail domain may be UUUU, and when the pol-III promoter is used , The tail domain may comprise several uracil bases or bases that can be substituted.

Optionally, some or all of the base sequences of the second complementary domain, the proximal domain, and / or the tail domain may comprise a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl 3'phosphorothioate (MS), or 2'-O-methyl 3'thioPACE It is not limited.

Thus, the second strand may be 5 '- [second complementary domain] - [proximal domain] -3' or 5 '- [second complementary domain] - [proximal domain] - [tail domain] 3 '.

In addition, the second strand may optionally comprise additional base sequences.

In one embodiment, the second strand comprises

5 '- (Z) _h - (P) _k- 3'; or

5 '- (X) _d - (Z) _h- (X) _e - (P) _k- (X) _f -3'.

In another embodiment, the second strand comprises

5 '- (Z) _h- (P) _k - (F) _i -3'; or

5 '- (X) _d- (Z) _h- (X) _e- (P) _k- (X) _f- (F) _i -3'.

Herein, (Z) _h is a base sequence comprising a second complementary domain, and includes a base sequence capable of complementary binding with a first complementary domain of the first strand. The (Z) _h may be a sequence having partial or complete homology with the second complementary domain of the species present in nature, and the nucleotide sequence of the second complementary domain may be changed depending on the derived species. Z may be independently selected from the group consisting of A, U, C and G, and h is the number of nucleotide sequences and may be an integer of 5 to 50.

For example, when the second complementary domain has partial or complete homology with the second complementary domain of Streptococcus fjigogenes or the second complementary domain derived from Streptococcus fyiogenses, the (Z) _h May be 5'-UAGCAAGUUAAAAU-3 ', or may be a base sequence having at least 50% or more homology with 5'-UAGCAAGUUAAAAU-3'.

In another example, when the second complementary domain has partial or complete homology with the second complementary domain of Campylobacter zeaxanth or the second complementary domain from Campylobacter zephyran, the (Z) _h is 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3 ', or may be a base sequence having at least 50% or more homology with 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3'.

As another example, when the second complementary domain has partial or complete homology with the second complementary domain of Streptococcus thermophilus or the second complementary domain derived from Streptococcus thermophilus, the (Z) _h May be 5'-CGAAACAACACAGCGAGUUAAAAU-3 ', or may be a base sequence having at least 50% or more homology with 5'-CGAAACAACACAGCGAGUUAAAAU-3'.

(P) _k is a base sequence comprising a proximal domain, and may be a sequence having partial or complete homology with a proximal domain of a species present in nature, and the base sequence of the proximal domain may be altered depending on the species derived . P may be independently selected from the group consisting of A, U, C and G, and k is the number of base sequences and may be an integer of 1 to 20.

For example, when the proximal domain has some or complete homology with the proximal domain of Streptococcus fjigogenes or the proximal domain derived from Streptococcus fyogeensis, (P) _k is 5'-AAGGCUAGUCCG-3 ' Or may be a base sequence having at least 50% or more homology with 5'-AAGGCUAGUCCG-3 '.

In another example, (P) _k may be 5'-AAAGAGUUUGC-3 'when the proximal domain has some or complete homology with the proximal domain of Campylobacter jejuni or Campylobacter sp. , Or a base sequence having at least 50% or more homology with 5'-AAAGAGUUUGC-3 '.

As another example, when the proximal domain has some or complete homology with the proximal domain of Streptococcus thermophilus or the proximal domain derived from Streptococcus thermophilus, (P) _k is 5'-AAGGCUUAGUCCG-3 ' Or may be a base sequence having at least 50% or more homology with 5'-AAGGCUUAGUCCG-3 '.

(F) _i is a base sequence comprising a tail domain, and may be a sequence having partial or complete homology with the tail domain of a species present in nature, and the base sequence of the tail domain may be changed depending on the species derived . The above F may be independently selected from the group consisting of A, U, C and G, wherein i is the number of base sequences and may be an integer of 1 to 50.

For example, when the tail domain has partial or complete homology with the tail domain of Streptococcus fjigogenes or the tail domain from Streptococcus fjogneses, (F) _i is 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3 ' Or may be a base sequence having at least 50% homology with 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3 '.

In another example, when the tail domain has partial or complete homology with the tail domain of Campylobacter zeaxanthus or the tail domain from Campylobacter zephyran, the (F) _i may be 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3 ' Or 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3 ', having at least 50% homology.

As another example, when the tail domain has partial or complete homology with the tail domain of Streptococcus thermophilus or the tail domain from Streptococcus thermophilus, (F) _i is 5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3 ' Or may be a base sequence having at least 50% homology with 5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3 '.

In addition, (F) _i above may contain 1 to 10 nucleotide sequences at the 3 'terminus associated with in vitro or in vivo transcription methods.

For example, when a T7 promoter is used for in vitro transcription of a gRNA, the tail domain may be any base sequence present at the 3 ' end of the DNA template. In addition, when the U6 promoter is used for in vivo transcription, the tail domain may be UUUUUU, and when the H1 promoter is used for transcription, the tail domain may be UUUU, and when the pol-III promoter is used , The tail domain may comprise several uracil bases or bases that can be substituted.

(X) _d , (X) _e and (X) _f are optionally added base sequences, and X may be independently selected from the group consisting of A, U, C and G, D, e and f are the number of nucleotide sequences and may be 0 or an integer of 1 to 20.

Single strand gRNA

Single-stranded gRNA can be divided into two types.

i) Single strand gRNA

First, there is a single-stranded gRNA in which the first strand and the second strand of the double gRNA are connected to each other through a linking domain, wherein the single-stranded gRNA comprises 5 '- [first strand] - [ ] -3 '.

Specifically, the single stranded gRNA comprises

5 '- [guide domain] - [first complementary domain] - [connection domain] - [second complementary domain] - [proximal domain] - 3' or

5 '- [guide domain] - [first complementary domain] - [connection domain] - [second complementary domain] - [proximal domain] - [tail domain] -3' .

Each domain except for the connecting domain is identical to the description of each domain of the first strand and the second strand of the double gRNA.

- Connection domain

In the single-stranded gRNA, the linking domain is a domain connecting the first strand and the second strand, specifically, a nucleic acid sequence capable of forming a single-stranded gRNA by connecting a first complementary domain and a second complementary domain to be. At this time, the connection domain may have a covalent bond or a non-covalent bond with the first complementary domain and the second complementary domain, or both the first complementary domain and the second complementary domain may be covalently or noncovalently linked .

The linking domain may be from 1 to 30 nucleotides in length, or from 1 to 30 nucleotides in length. For example, the linking domain may comprise one to five nucleotides, five to ten nucleotides, 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, or 25 to 30 nucleotides Or may include, a < / RTI >

The linking domain is suitable for use in single-stranded gRNA molecules, and can be covalently or non-covalently linked to the first and second strands of the double gRNA, or covalently or non-covalently linked the first and second strands Can be used to generate single stranded gRNA. The linking domain can be used for covalent or noncovalent binding with the crRNA and tracrRNA of the double gRNA, or covalently or noncovalently linking the crRNA and the tracrRNA to produce single stranded gRNA.

The linking domain may be homologous to, or derived from, a naturally occurring sequence, e. G., A partial sequence of a tracrRNA.

Optionally, some or all of the nucleotide sequence of the linking domain may comprise a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl 3'phosphorothioate (MS), or 2'-O-methyl 3'thioPACE It is not limited.

Thus, a single stranded gRNA may be a 5 '- [guiding domain] - [first complementary domain] - [ligated domain] - [second complementary domain] - [proximal domain] Or 5 '- [guide domain] - [first complementary domain] - [connection domain] - [second complementary domain] - [proximal domain] - [tail domain] -3' have.

In addition, the single-stranded gRNA may optionally comprise additional base sequences.

In one embodiment, the single stranded gRNA comprises

5 '- (N _target ) - (Q) _m - (L) _j- (Z) _h- (P) _k- 3'; or

5 '- (N _target ) - (Q) _m - (L) _j- (Z) _h- (P) _k- (F) _i -3'.

In another embodiment, the single stranded gRNA comprises

_{5 '- (X) a -} (N target) - (X) b - (Q) m - (X) c - (L) j - (X) d - (Z) h - (X) e - (P ) _k - (X) _f- 3 '; or

_{5 '- (X) a -} (N target) - (X) b - (Q) m - (X) c - (L) j - (X) d - (Z) h - (X) e - (P ) _k - (X) _f - (F) _i -3 '.

Here, the N _target is a base sequence capable of binding complementary to a target sequence on a target gene or a nucleic acid, and the N _target is a base sequence region that can be changed according to a target sequence on a target gene or nucleic acid.

(Q) _m is a base sequence including a first complementary domain, and includes a base sequence capable of complementary binding with a second complementary domain. The (Q) _m may be a sequence having partial or complete homology with the first complementary domain of the species existing in nature, and the nucleotide sequence of the first complementary domain may be changed depending on the derived species. The Q may be independently selected from the group consisting of A, U, C and G, and m is the number of base sequences and may be an integer of 5 to 35.

For example, when the first complementary domain has partial or complete homology with the first complementary domain of Streptococcus fyogenes or the first complementary domain derived from Streptococcus fjogneses, the (Q) _m May be 5'-GUUUUAGAGCUA-3 ', or may be a base sequence having at least 50% homology with 5'-GUUUUAGAGCUA-3'.

In another example, when the first complementary domain has partial or complete homology with the first complementary domain of Campylobacter zeaxanth or the first complementary domain derived from Campylobacter zein, the (Q) _m is 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3 ', or may be a nucleotide sequence having at least 50% homology with 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3'.

As another example, when the first complementary domain has partial or complete homology with the first complementary domain of Streptococcus thermophilus or the first complementary domain derived from Streptococcus thermophilus, the (Q) _m May be 5'-GUUUUAGAGCUGUGUUGUUUCG-3 ', or may be a base sequence having at least 50% or more homology with 5'-GUUUUAGAGCUGUGUUGUUUCG-3'.

In addition, (L) _j is a nucleotide sequence comprising a linking domain, and is a nucleotide sequence that allows a first complementary domain and a second complementary domain to be joined to produce single stranded gRNA. Herein, L may be independently selected from the group consisting of A, U, C and G, and j is the number of base sequences and may be an integer of 1 to 30.

(Z) _h is a base sequence comprising a second complementary domain, and includes a base sequence capable of a complementary binding with a first complementary domain. The (Z) _h may be a sequence having partial or complete homology with the second complementary domain of the species present in nature, and the nucleotide sequence of the second complementary domain may be changed depending on the derived species. Z may be independently selected from the group consisting of A, U, C and G, and h is the number of nucleotide sequences and may be an integer of 5 to 50.

For example, when the second complementary domain has partial or complete homology with the second complementary domain of Streptococcus fjigogenes or the second complementary domain derived from Streptococcus fyiogenses, the (Z) _h May be 5'-UAGCAAGUUAAAAU-3 ', or may be a base sequence having at least 50% or more homology with 5'-UAGCAAGUUAAAAU-3'.

In another example, when the second complementary domain has partial or complete homology with the second complementary domain of Campylobacter zeaxanth or the second complementary domain from Campylobacter zephyran, the (Z) _h is 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3 ', or may be a base sequence having at least 50% or more homology with 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3'.

As another example, when the second complementary domain has partial or complete homology with the second complementary domain of Streptococcus thermophilus or the second complementary domain derived from Streptococcus thermophilus, the (Z) _h May be 5'-CGAAACAACACAGCGAGUUAAAAU-3 ', or may be a base sequence having at least 50% or more homology with 5'-CGAAACAACACAGCGAGUUAAAAU-3'.

(P) _k is a base sequence comprising a proximal domain, and may be a sequence having partial or complete homology with a proximal domain of a species present in nature, and the base sequence of the proximal domain may be altered depending on the species derived . P may be independently selected from the group consisting of A, U, C and G, and k is the number of base sequences and may be an integer of 1 to 20.

For example, when the proximal domain has some or complete homology with the proximal domain of Streptococcus fjigogenes or the proximal domain derived from Streptococcus fyogeensis, (P) _k is 5'-AAGGCUAGUCCG-3 ' Or may be a base sequence having at least 50% or more homology with 5'-AAGGCUAGUCCG-3 '.

In another example, (P) _k may be 5'-AAAGAGUUUGC-3 'when the proximal domain has some or complete homology with the proximal domain of Campylobacter jejuni or Campylobacter sp. , Or a base sequence having at least 50% or more homology with 5'-AAAGAGUUUGC-3 '.

As another example, when the proximal domain has some or complete homology with the proximal domain of Streptococcus thermophilus or the proximal domain derived from Streptococcus thermophilus, (P) _k is 5'-AAGGCUUAGUCCG-3 ' Or may be a base sequence having at least 50% or more homology with 5'-AAGGCUUAGUCCG-3 '.

(F) _i is a base sequence comprising a tail domain, and may be a sequence having partial or complete homology with the tail domain of a species present in nature, and the base sequence of the tail domain may be changed depending on the species derived . The above F may be independently selected from the group consisting of A, U, C and G, wherein i is the number of base sequences and may be an integer of 1 to 50.

For example, when the tail domain has partial or complete homology with the tail domain of Streptococcus fjigogenes or the tail domain from Streptococcus fjogneses, (F) _i is 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3 ' Or may be a base sequence having at least 50% homology with 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3 '.

In another example, when the tail domain has partial or complete homology with the tail domain of Campylobacter zeaxanthus or the tail domain from Campylobacter zephyran, the (F) _i may be 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3 ' Or 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3 ', having at least 50% homology.

As another example, when the tail domain has partial or complete homology with the tail domain of Streptococcus thermophilus or the tail domain from Streptococcus thermophilus, (F) _i is 5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3 ' Or may be a base sequence having at least 50% homology with 5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3 '.

In addition, (F) _i above may contain 1 to 10 nucleotide sequences at the 3 'terminus associated with in vitro or in vivo transcription methods.

For example, when a T7 promoter is used for in vitro transcription of a gRNA, the tail domain may be any base sequence present at the 3 ' end of the DNA template. In addition, when the U6 promoter is used for in vivo transcription, the tail domain may be UUUUUU, and when the H1 promoter is used for transcription, the tail domain may be UUUU, and when the pol-III promoter is used , The tail domain may comprise several uracil bases or bases that can be substituted.

In addition, the _{(X) a, (X)} b, (X) c, (X) d, (X) e and (X) _f is the nucleotide sequence that can be optionally added, wherein X is A, U, C, and G, and a, b, c, d, e, and f are numbers of nucleotide sequences and may be 0 or an integer of 1 to 20.

ii ) Single strand gRNA

The single-stranded gRNA may then be a single-stranded gRNA consisting of a guiding domain, a first complementary domain and a second complementary domain,

At this time, the single stranded gRNA

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

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

- Guide domain

In the single-stranded gRNA, the guiding domain comprises a complementary guide sequence capable of binding complementary to the target sequence on the target gene or nucleic acid. The guide sequence is a nucleic acid sequence complementary to the target sequence on the target gene or nucleic acid, for example, at least 70%, 75%, 80%, 85%, 90% or 95% complementary or completely complementary nucleic acid sequence Lt; / RTI > The guiding domain is believed to play a role in allowing specific interaction with the target gene or nucleic acid of the gRNA-Cas complex, i.e. the CRISPR complex.

At this time, the guiding domain may have 5 to 50 nucleotide sequences or 5 to 50 nucleotide sequences. For example, the guiding domain may have 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, Or 25 base sequences, or may comprise the same.

In addition, the guide domain may include a guide sequence.

The guide sequence may be a complementary base sequence or complementary base sequence capable of binding complementary to the target sequence on the target gene or nucleic acid, and may be, for example, at least 70%, 75%, 80%, 85% , 90%, or 95% or more complementary or completely complementary base sequence.

At this time, the guide sequence may be 5 to 50 nucleotide sequences, or may include 5 to 50 nucleotide sequences. For example, the guide sequence may comprise 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, Base sequence or 25 base sequences, or may comprise the same.

Optionally, the guiding domain may comprise a guiding sequence and additional base sequences.

At this time, the additional base sequence may be 1 to 35 base sequences. For example, the additional base sequence may include one base sequence, two base sequences, three base sequences, four base sequences, five base sequences, six base sequences, seven base sequences, eight base sequences, nine base sequences, Lt; RTI ID = 0.0 > 10 < / RTI > nucleotide sequences.

In one embodiment, the additional base sequence may be one base sequence G (guanine) or two base sequences GG.

At this time, the additional base sequence may be located at the 5 'end of the guide domain, or may be located at the 5' end of the guide sequence.

The additional base sequence may be located at the 3 'end of the guide domain or at the 3' end of the guide sequence.

- First complementary domain

The first complementary domain includes a second complementary domain and a complementary nucleic acid sequence, and is a domain having a degree of complementarity enough to form a double strand with the second complementary domain.

The first complementary domain may have 5 to 35 nucleotide sequences or 5 to 35 nucleotide sequences. For example, the first complementary domain may comprise 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, A nucleotide sequence having a nucleotide sequence of 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 base sequences, 24 base sequences, or 25 base sequences.

The first complementary domain may have homology with the first complementary domain of nature, or may be derived from a first complementary domain of nature. In addition, the first complementary domain may have a difference in the base sequence of the first complementary domain depending on the species present in nature, and may be derived from a first complementary domain comprising the species present in nature, or And may have some or complete homology with the first complementary domain comprising the species present in nature.

In one embodiment, the first complementary domain is selected from the group consisting of Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasicus , Pseudomonas aeruginosa, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp . (BV3L6), Porphyromonas macacae , Lachnospiraceae bacterium (ND2006)), Porphyromonas crevioricanis , Prevotella disiens , Moraxella bovoculi (237)), Smiihella sp . (SC_KO8D17)), rep force pyrazolyl or die (Leptospira inadai , Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum ) or a part of the first complementary domain of Eubacterium eligens , or a part of the first complementary domain derived from Eubacterium eligens , at least 50% or more, or complete homology.

Optionally, the first complementary domain may comprise additional base sequences that do not undergo a complementary binding with the second complementary domain.

Herein, the additional base sequence may be 1 to 15 base sequences. For example, the additional base sequence may be from 1 to 5 nucleotides, from 5 to 10 nucleotides, or from 10 to 15 nucleotides.

[ Second  Complementary domain]

The second complementary domain comprises a complementary nucleic acid sequence with the first complementary domain of the first strand and is complementary enough to form a double strand with the first complementary domain. The second complementary domain includes a base sequence that is not complementary to the first complementary domain and the complementary base sequence and the first complementary domain, for example, a base sequence that does not form a double strand with the first complementary domain And the length of the base sequence may be longer than that of the first complementary domain.

The second complementary domain may have 5 to 35 nucleotide sequences or 5 to 35 nucleotide sequences. For example, the second complementary domain may comprise 5 nucleotides, 6 nucleotides, 7 nucleotides, 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, , 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides , 23 nucleotides, 24 nucleotides, or 25 nucleotides.

The second complementary domain may have a homology with the second complementary domain derived from nature, or may be derived from a second complementary domain derived from nature. The second complementary domain may have a difference in the base sequence of the second complementary domain depending on the species present in nature and may be derived from a second complementary domain comprising the species present in nature, May have some or complete homology with a second complementary domain comprising the species present in nature.

In one embodiment, the second complementary domain is selected from the group consisting of Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasicus , Pseudomonas aeruginosa, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp . (BV3L6), Porphyromonas macacae , Lachnospiraceae bacterium (ND2006)), Porphyromonas crevioricanis , Prevotella disiens , Moraxella bovoculi (237)), Smiihella sp . (SC_KO8D17)), rep force pyrazolyl or die (Leptospira inadai , Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum ) or a partial, at least 50% or more complete homology with the second complementary domain or the derived second complementary domain of Eubacterium eligens .

Optionally, the second complementary domain may comprise additional base sequences that do not undergo a complementary binding with the first complementary domain.

Herein, the additional base sequence may be 1 to 15 base sequences. For example, the additional base sequence may be from 1 to 5 nucleotides, from 5 to 10 nucleotides, or from 10 to 15 nucleotides.

[Linked domain]

Alternatively, the linkage domain is a nucleic acid sequence that links a first complementary domain and a second complementary domain to produce single stranded gRNA. At this time, the connection domain may have a covalent bond or a non-covalent bond with the first complementary domain and the second complementary domain, or both the first complementary domain and the second complementary domain may be covalently or noncovalently linked .

The linking domain may be from 1 to 30 nucleotides in length, or from 1 to 30 nucleotides in length. For example, the linking domain may comprise one to five nucleotides, five to ten nucleotides, 10 to 15 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, or 25 to 30 nucleotides Or may include, a < / RTI >

Optionally, some or all of the nucleotide sequences of the guiding domain, the first complementary domain, the second complementary domain, and the connecting domain may comprise a chemical modification. The chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl 3'phosphorothioate (MS), or 2'-O-methyl 3'thioPACE It is not limited.

Thus, the single-stranded gRNA may be a 5 '- [second complementary domain] - [first complementary domain] - [guiding domain] -3' or 5 '- [second complementary domain] Connection domain] - [first complementary domain] - [guide domain] -3 '.

In addition, the single-stranded gRNA may optionally comprise additional base sequences.

In one embodiment, the single stranded gRNA comprises

5 '- (Z) _h- (Q) _m - (N _target ) -3'; or

5 '- (X) _a- (Z) _h- (X) _b- (Q) _m- (X) _c- (N _target ) -3'.

In another embodiment, the single stranded gRNA comprises

5 '- (Z) _h- (L) _j- (Q) _m- (N _target ) -3'; or

5 '- (X) _a- (Z) _h- (L) _j- (Q) _m- (X) _c- (N _target ) -3'.

Here, the N _target is a base sequence capable of binding complementary to a target sequence on a target gene or a nucleic acid, and the N _target is a base sequence region that can be changed according to a target sequence on a target gene or nucleic acid.

(Q) _m is a base sequence including a first complementary domain, and includes a base sequence capable of complementary binding with a second complementary domain. The (Q) _m may be a sequence having partial or complete homology with the first complementary domain of the species existing in nature, and the nucleotide sequence of the first complementary domain may be changed depending on the derived species. The Q may be independently selected from the group consisting of A, U, C and G, and m is the number of base sequences and may be an integer of 5 to 35.

For example, when the first complementary domain has partial or complete homology with the first complementary domain of Falco bacterium bacteria or the first complementary domain derived from Falco bacterium bacteria, (Q) _m is 5 '-UUUGUAGAU-3', or may be a base sequence having at least 50% homology with 5'-UUUGUAGAU-3 '.

(Z) _h is a base sequence comprising a second complementary domain, and includes a base sequence capable of a complementary binding with a first complementary domain. The (Z) _h may be a sequence having partial or complete homology with the second complementary domain of the species present in nature, and the nucleotide sequence of the second complementary domain may be changed depending on the derived species. Z may be independently selected from the group consisting of A, U, C and G, and h is the number of nucleotide sequences and may be an integer of 5 to 50.

For example, when the second complementary domain has partial or complete homology with the second complementary domain of Falco bacterium bacteria or the second complementary domain derived from Falco bacterium bacteria, (Z) _h is 5 '-AAAUUUCUACU-3', or may be a base sequence having at least 50% or more homology with 5'-AAAUUUCUACU-3 '.

In addition, (L) _j is a base sequence including a linking domain, and is a base sequence linking a first complementary domain and a second complementary domain. Herein, L may be independently selected from the group consisting of A, U, C and G, and j is the number of base sequences and may be an integer of 1 to 30.

(X) _a , (X) _b and (X) _c are optionally added base sequences, wherein X may be independently selected from the group consisting of A, U, C and G, A, b, and c are numbers of base sequences and may be 0 or an integer of 1 to 20.

2. Editor protein

An editor protein refers to a peptide, polypeptide or protein that is capable of directly binding to, or not interacting directly with, a nucleic acid. The editor protein may also be conceptually referred to as "gene scissors" or RGEN (RNA-Guided Endonuclease).

The nucleic acid may be a target nucleic acid, a gene, or a nucleic acid contained in a chromosome.

The nucleic acid may be a guide nucleic acid.

The editor protein may be an enzyme.

The above-mentioned editor protein may be a fusion protein.

Herein, the fusion protein refers to a protein produced by fusion of an enzyme and an additional domain, peptide, polypeptide or protein.

The enzyme means a protein comprising a domain capable of cleaving a nucleic acid, a gene, a chromosome or a protein.

The enzyme may be a nuclease, a protease or a restriction enzyme.

The additional domains, peptides, polypeptides or proteins may be functional domains, peptides, polypeptides or proteins having the same or different functions as the enzymes.

Wherein the fusion protein is at or near the amino terminus of the enzyme; Carboxy terminus or vicinal thereof; The middle part of the enzyme; Or an additional domain, peptide, polypeptide or protein in one or more of these combinations.

Wherein the fusion protein is at or near the amino terminus of the enzyme; Carboxy terminus or vicinal thereof; The middle part of the enzyme; Or a functional domain, peptide, polypeptide or protein in one or more of these combinations.

The functional domain, the peptide, the polypeptide or the protein may be selected from the group consisting of methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription activity, peptides, polypeptides, or proteins that have a release factor activity, a histone modification activity, a cleavage activity or a nucleic acid binding activity, or a separation of proteins (including peptides) But is not limited to, a tag or reporter gene for purification.

The functional domains, peptides, polypeptides or proteins may be deaminases.

The tag includes a histidine (His) tag, a V5 tag, a FLAG tag, an influenza hemagglutinin (HA) tag, a Myc tag, a VSV-G tag and a thioredoxin (Trx) tag, (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein But are not limited to, autofluorescent proteins, including, for example, GFP), HcRed, DsRed, Cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and blue fluorescent protein (BFP).

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

The NLS is an NLS of the SV40 virus large T-antigen with the amino acid sequence PKKKRKV; NLS from nucleoplasmin (e. G., Nucleoplasmin NLS with sequence KRPAATKKAGQAKKKK); C-myc NLS with amino acid sequence PAAKRVKLD or RQRRNELKRSP; HRNPA1 M9 NLS with sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; The sequence of the IBB domain from IMPOTIN-ALPHA RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV; Sequences of myoma T protein VSRKRPRP and PPKKARED; Sequence of human p53 POPKKKPL; Sequence of mouse c-abl IV SALIKKKKKMAP; The sequences DRLRR and PKQKKRK of influenza virus NS1; The sequence of the hepatitis virus delta antigen RKLKKKIKKL; Sequence of mouse Mx1 protein REKKKFLKRR; Sequence of human poly (ADP-ribose) polymerase KRKGDEVDGVDEVAKKKSKK; Or an NLS sequence derived from the sequence RKCLQAGMNLEARKTKK of the steroid hormone receptor (human) glucocorticoid, but is not limited thereto.

The editor protein may comprise a fully active enzyme.

Here, the " fully active enzyme " means an enzyme having the same function as a function of a wild type enzyme. For example, a wild type enzyme that cleaves a double strand of DNA is an enzyme It has complete enzyme activity.

Further, the fully active enzyme includes an enzyme having a function that is more improved than that of the wild type enzyme. For example, a specific modified or manipulated form of a wild type enzyme that cleaves a double strand of DNA is an enzyme having an increased Enzyme activity, that is, activity to cleave DNA duplexes.

The editor protein may comprise an incomplete or partially active enzyme.

The term " incomplete or partially active enzyme " means an enzyme having only a part of functions of a wild-type enzyme. For example, a specific modified or manipulated form of a wild-type enzyme that cleaves a double strand of DNA is a DNA double strand But has an incomplete or partial enzymatic activity that only partially cleaves a single strand.

The above-mentioned editor protein may contain an inactive enzyme.

The term " inert enzyme " means an enzyme in which all of the functions of the wild type enzyme are inactivated. For example, a specific modified or manipulated form of the wild type enzyme that cleaves double strands of DNA It has inertity to prevent it from doing.

The above-mentioned editor protein may be an enzyme or a fusion protein existing in a natural state.

The above-mentioned editor protein may be a native form of the enzyme or a modified form of a part of the fusion protein.

The above-mentioned editor protein may be an artificially generated enzyme or a fusion protein which is not present in a natural state.

The editor protein may be an artificially generated enzyme or a part of the fusion protein that is not in a natural state.

At this time, the modification may be substitution, deletion, addition, or a mixture of amino acids contained in the editor protein.

Alternatively, the modification may be substitution, deletion, addition, or a mixture of some bases in the nucleotide sequence encoding the editor protein.

As one embodiment of the editor protein of the present invention, the CRISPR enzyme is described below.

CRISPR  enzyme

&Quot; CRISPR enzyme " is a major protein component of the CRISPR-Cas system, which forms a CRISPR-Cas system by complexing with gRNA.

The CRISPR enzyme is a nucleic acid or a polypeptide (or protein) having a sequence encoding a CRISPR enzyme, typically a Type II CRISPR enzyme or a Type V CRISPR enzyme.

The Type II CRISPR enzyme is Cas9, and Cas9 is Actinobacteria (e.g.,Actinomyces naeslundii) Cas9, Aquificae Cas9, Bacteroidetes Cas9, Chlamydiae Cas9, Chloroflexi Cas9, Cyanobacteria Cas9, Elusimicrobia Cas9, Fibrobacteres Cas9, Firmicutes Cas9Streptococcus pyogenes Cas9,Streptococcus thermophilus Cas9,Listeria innocua Cas9,Streptococcus agalactiae Cas9,Streptococcus mutans Cas9 andEnterococcus faecium Cas9), Fusobacteria Cas9, Proteobacteria (e.g.,Neisseria meningitides,Campylobacter jejuni) Cas9, Spirochaetes (e.g.,Treponema denticola) Cas9, and the like.

"Cas9" is an enzyme that cleaves or transforms a target sequence or position on a target gene or nucleic acid in association with a gRNA, and includes an HNH domain in which a gRNA can cleave a nucleic acid strand that makes a complementary binding, A RuvC domain capable of cleaving a binding nucleic acid strand, a target, i.e., a REC domain recognizing target, and a PI domain recognizing PAM. Concrete structural characteristics of Cas9 are described by Hiroshi Nishimasu et al. (2014) Cell 156: 935-949.

The Type V CRISPR enzyme may be Cpf1 and the Cpf1 may be selected from the group consisting of Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Cpf1 from Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium or Acidaminococcus.

The Cpf1 has a similar RuvC domain corresponding to the RuvC domain of Cas9, a lack of the HNH domain of Cas9, a Nuc domain instead, a REC domain recognizing the target and a WED domain and a PI domain recognizing the PAM Lt; / RTI > Concrete structural features of Cpf1 are described by Takashi Yamano et al. (2016) Cell 165: 949-962.

The CRISPR enzyme such as Cas9 or Cpf1 protein may be isolated from a microorganism existing in a natural state or produced naturally by a recombinant method or a synthetic method.

Type  II CRISPR  enzyme

The crystal structure of the Type II CRISPR enzyme is similar to that of two or more naturally occurring microorganisms Type II CRISPR enzyme molecules (Jinek et al., Science, 343 (6176): 1247997, 2014), and a complex of Streptococcus pyogenes (Nishimasu et al., Cell, 156: 935-949, 2014; and Anders et al., Nature, 2014, doi: 10.1038 / nature13579).

Type II CRISPR enzymes include two lobes, Recognition (REC) and Nucleic Acid (NUC) lobes, each lobe containing multiple domains.

The REC lobe comprises an arginine-rich bridge spiral (BH), a REC1 domain and a REC2 domain.

The BH domain is a long? -Helical and arginine rich region, and the REC1 and REC2 domains play an important role in the recognition of the double strand formed in the gRNA, for example, single strand gRNA, double gRNA or tracrRNA.

The NUC lobe comprises the RuvC domain, the HNH domain and the PAM-interacting (PI) domain. At this time, the RuvC domain is used to mean the RuvC-like domain, and the HNH domain is used to mean the HNH-like domain.

At this time, the RuvC domain shares structural similarity with members of a microorganism existing in a natural state including a Type II CRISPR enzyme, and has a single strand, for example, a complementary strand of a target gene or a nucleic acid, The strands that do not bind are cleaved. The RuvC domain is often referred to in the art as the RuvCI domain, the RuvCII domain, and the RuvCIII domain, typically RuvC I, RuvCII, and RuvCIII. For example, in the case of SpCas9, the RuvC domain is assembled from three split RuvC domains (RuvC I, RuvCII and RuvCIII), respectively, located in amino acid sequences 1 to 59, 718 to 769 and 909 to 1098 of SpCas9.

The HNH domain shares structural similarity with the HNH endonuclease and cleaves a single strand, for example, a complementary strand of the target nucleic acid molecule, i. E., A strand that makes a complementary binding to the gRNA. The HNH domain is located between the RuvC II and III motifs. For example, in the case of SpCas9, the HNH domain is located at amino acid sequence 775-908 of SpCas9.

The PI domain recognizes or interacts with the PAM (Protospacer adjacent motif) with a specific base sequence in the target gene or nucleic acid. For example, in the case of SpCas9, the PI domain is located in amino acid sequence 1099 to 1368 of SpCas9.

At this time, the PAM may be different depending on the origin of Type II CRISPR enzyme. For example, PAM may be 5'-NGG-3 'when the CRISPR enzyme is SpCas9, and 5'-NNAGAAW-3' (W = A or T) when PAM is Streptococcus thermophilus Cas9 (StCas9) PAM may be 5'-NNNNGATT-3 'when Naceria meningitidis Cas9 (NmCas9), and PAM may be 5'-NNNVRYAC-3' (V) in case of Campylobacter jejuni Cas9 (CjCas9) = G or C or A, R = A or G, Y = C or T), wherein N is A, T, G or C; Or A, U, G or C.

Type  V CRISPR  enzyme

The Type V CRISPR enzyme has a similar RuvC domain corresponding to the RuvC domain of the Type II CRISPR enzyme and lacks the HNH domain of the Type II CRISPR enzyme and contains the Nuc domain instead of the REC domain and the WED domain And a PI domain that recognizes PAM. The structural characteristics of the specific Type V CRISPR enzyme are described in Takashi Yamano et al. (2016) Cell 165: 949-962.

The Type V CRISPR enzyme can interact with the gRNA and form a gRNA-CRISPR enzyme complex, i.e., a CRISPR complex, and cooperate with the gRNA to approximate the guiding sequence with the target sequence containing the PAM sequence. At this time, the ability of the Type V CRISPR enzyme to interact with the target gene or nucleic acid depends on the PAM sequence.

The PAM sequence can be recognized by the PI domain of Type V CRISPR enzyme, which is a sequence existing in the target gene or nucleic acid. The sequence of the PAM sequence may be different depending on the origin of the Type V CRISPR enzyme. That is, there is a PAM sequence that can be recognized specifically for each species.

As an example, the PAM sequence recognized by Cpf1 may be 5'-TTN-3 '(N is A, T, C or G).

CRISPR  Enzyme activity

The CRISPR enzyme cleaves double or single strands of the target gene or nucleic acid and has a nuclease activity that results in the breakage or deletion of the double strand or single strand. In general, a wild-type Type II CRISPR enzyme or Type V CRISPR enzyme generally cleaves a double strand of a target gene or nucleic acid.

In order to modify or alter such nuclease activity of the CRISPR enzyme, the CRISPR enzyme may be manipulated or modified and such manipulated or modified CRISPR enzyme may be transformed into an incomplete or partially active enzyme or an inactive enzyme.

Incomplete or partially active enzyme

The CRISPR enzyme, which has been modified to have an incomplete or partial activity by altering the enzyme activity, is referred to as a nickase.

&Quot; Nickase " means a CRISPR enzyme that has been manipulated or modified so that only one of the double strand of the target gene or nucleic acid is cleaved, and the nicarase is a single strand, for example, a gRNA of a target gene or nucleic acid, Lt; RTI ID = 0.0 > complementary < / RTI > strand. Therefore, nuclease activity of two niacas is required to cleave double strands.

For example, the nicacase may have nuclease activity by the RuvC domain. That is, the nicarase may not contain nuclease activity by the HNH domain, for which the HNH domain may be manipulated or altered.

For example, when the CRISPR enzyme is a Type II CRISPR enzyme,

In one embodiment, in the case of SpCas9, when the amino acid sequence 840 of histidine of SpCas9 is mutated to alanine, the nuclease activity of the HNH domain is inactivated, so that it can be used as a niacase. Domain, it is possible to cleave the target gene or the non-complementary strand of the nucleic acid, i.e., the strand that does not bind complementary to the gRNA.

In another specific example, in the case of CjCas9, mutation of histidine of amino acid sequence 559 of CjCas9 to alanine inactivates the nuclease activity of the HNH domain, so that it can be used as a niacase, Since it has nuclease activity by the RuvC domain, it can cleave the target gene or the non-complementary strand of the nucleic acid, i.e., the strand that does not bind complementary to the gRNA.

For example, the niacase may have nuclease activity by the HNH domain. That is, the niacase may not contain nuclease activity by the RuvC domain, and for this, the RuvC domain may be manipulated or altered.

For example, when the CRISPR enzyme is a Type II CRISPR enzyme,

In one specific example, in the case of SpCas9, mutation of aspartic acid at position 10 of the amino acid sequence of SpCas9 to alanine inactivates the nuclease activity of the RuvC domain, so that it can be used as a niacase. Since it has nuclease activity by the HNH domain, it can cleave complementary strands of the target gene or nucleic acid, that is, strands that make a complementary binding to the gRNA.

As another specific example, in the case of CjCas9, mutation of aspartic acid at position 8 of amino acid sequence of CjCas9 to alanine inactivates the nuclease activity of the RuvC domain, so that it can be used as a niacase, Has a nuclease activity by the HNH domain, so that a complementary strand of the target gene or nucleic acid, that is, a strand that makes a complementary binding to the gRNA, can be cleaved.

Inert enzyme

The CRISPR enzyme, in which the enzyme activity is completely inactivated by modification of the CRISPR enzyme, is referred to as an inactive CRISPR enzyme.

&Quot; Inactive CRISPR enzyme " means a CRISPR enzyme modified so as not to be able to cleave both the target gene or the double strand of the nucleic acid, and the inactive CRISPR enzyme has a nuclease activity due to a mutation in the nuclease activity domain of the wild type CRISPR enzyme Inactivity. The inactive CRISPR enzyme may be one in which the nuclease activity by the RuvC domain and the HNH domain is mutated.

For example, the inactive CRISPR enzyme can manipulate or alter the RuvC domain and HNH domain to inactivate nuclease activity.

For example, when the CRISPR enzyme is a Type II CRISPR enzyme,

In a specific example, in the case of SpCas9, mutation of the amino acid sequence 10 aspartate of SpCas9 and 840 of histidine to alanine is inactivated by the RuvC domain and the HNH domain, so that the target gene or nucleic acid Can not be cut off.

In another embodiment, mutation of aspartic acid at position 8 and histidine at position 559 in amino acid sequence CjCas9 of CjCas9 to alanine inactivates nuclease activity by RuvC domain and HNH domain, The double strand of the nucleic acid can not be cleaved.

Other activity

In addition to the nuclease activity described above, the CRISPR enzyme may have endonuclease activity, exonuclease activity or helicase activity, i.e., the ability to resolve the helical structure of the double-stranded nucleic acid.

In addition, the CRISPR enzyme can modify the CRISPR enzyme such that the endonuclease activity, exonuclease activity, or helicase activity of the CRISPR enzyme is fully active, incomplete or partially active, or inactive.

CRISPR  Enzymatic Targeting

The CRISPR enzyme may interact with the gRNA and form a gRNA-CRISPR enzyme complex, i.e., a CRISPR complex, and cooperate with the gRNA to approximate the guiding sequence with the target sequence comprising the PAM sequence. At this time, the ability of the CRISPR enzyme to interact with the target gene or nucleic acid is dependent on the PAM sequence.

The PAM sequence can be recognized by the PI domain of the CRISPR enzyme, with the sequence present in the target gene or nucleic acid. The sequence of the PAM sequence may differ depending on the origin of the CRISPR enzyme. That is, there is a PAM sequence that can be recognized specifically for each species.

For example, when the CRISPR enzyme is a Type II CRISPR enzyme,

In the case of SpCas9, the PAM sequence may be 5'-NGG-3 ', 5'-NAG-3' and / or 5'-NGA-

In the case of StCas9, the PAM sequence may be 5'-NGGNG-3 'and / or 5'-NNAGAAW-3' (W = A or T)

In case of NmCas9, the PAM sequence may be 5'-NNNNGATT-3 'and / or 5'-NNNGCTT-3'

For CjCas9, the PAM sequence may be 5'-NNNVRYAC-3 '(V = G, C or A; R = A or G; Y = C or T)

In the case of Streptococcus mutans Cas9 (SmCas9), the PAM sequence may be 5'-NGG-3 'and / or 5'-NAAR-3' (R = A or G)

In the case of Staphylococcus aureus Cas9 (SaCas9), the PAM sequence is 5'-NNGRR-3 ', 5'-NNGRRT-3' and / or 5'-NNGRRV- G, C or A).

In another example, when the CRISPR enzyme is a Type V CRISPR enzyme,

For Cpf1, the PAM sequence may be 5'-TTN-3 '.

Wherein N is A, T, G or C; Or A, U, G or C.

Using a PAM sequence that is specifically recognizable for each of these species, the CRISPR enzyme capable of recognizing a specific PAM sequence can be manipulated or modified. For example, the PI domain of SpCas9 can be replaced with the PI domain of CjCas9 to recognize the CjCas9-specific PAM sequence, which has the nuclease activity of SpCas9, thereby allowing SpCas9 recognizing the CjCas9-specific PAM sequence Can be generated. Through the substitution or replacement of this PI domain, it is possible to design and modify a specifically recognized PAM sequence.

CRISPR  enzyme Mutant

The CRISPR enzyme is mutated such that it can enhance or inhibit various properties such as the nuclease activity of the CRISPR enzyme, the helicase activity, the ability to interact with the gRNA, and the close proximity to the target gene or nucleic acid, .

In addition, the CRISPR enzyme mutant is a non-target gene or a nucleic acid that undergoes some complementary binding with a gRNA when interacting with gRNA to form a gRNA-CRISPR enzyme complex, i.e., forming a CRISPR complex to be close to or localized to a target gene or nucleic acid And a modified or engineered CRISPR enzyme that enhances target specificity so that only the double strand or single strand of the target gene or nucleic acid can be cleaved without cleavage of double strand or single strand of non-target gene or nucleic acid that does not make complementary binding Lt; / RTI >

At this time, the effect of cleaving a double strand or a single strand of a non-target gene or nucleic acid that partially combines with the gRNA and a non-target gene or nucleic acid that does not complementarily bind to the gRNA is referred to as an off-target effect Target gene or nucleic acid having some complementary binding with the gRNA and a non-target gene or nucleic acid without complementary binding are referred to as off-target, and the number of off-targets is one Or more. In contrast, if the cleavage effect of a double strand or single strand of a target gene or nucleic acid is referred to as an on-target effect, the position or target sequence of the target gene or nucleic acid is referred to as an on-target.

The CRISPR enzyme variant is a modification of at least one amino acid of a naturally occurring CRISPR enzyme, which has at least one modification of the amino acid sequence of the CRISPR enzyme, such as nuclease activity, helicase activity, ability to interact with the gRNA, proximity to the target gene or nucleic acid, One or more of the target specificities can be altered, e.g., enhanced or inhibited, properties. At this time, the modification may be amino acid substitution, deletion, addition, or a mixture thereof.

In CRISPR enzyme mutants,

The modification may be a modification of one or more amino acids located in a region comprised of positively charged amino acids present in the naturally occurring CRISPR enzyme.

For example, the variants may include one or more amino acids of positively charged amino acids, such as lysine (K), arginine (R) and histidine (H), present in the naturally occurring CRISPR enzyme Lt; / RTI >

The modification may be a modification of one or more amino acids located in a region consisting of non-positively charged amino acids present in the naturally occurring CRISPR enzyme.

For example, the modifications may include amino acids that do not have a positive charge, such as aspartic acid D, glutamic acid E, serine S, threonine, or the like, present in the naturally occurring CRISPR enzyme Threonine, T, Asparagine, N, Glutamine, Q, Cysteine, C, Proline, P, Glysin, G, Alanine, A, Valine One or two or more of Vole, V, Isoleucine I, Leucine L, Methionine M, Phenylalanine F, Tyrosine Y and Tryptophan W, It may be a modification of an amino acid.

In another example, the modifications include amino acids that do not have charge, such as Serine, S, Threonine, T, Asparagine, N, Glutamine, Q, which are present in naturally occurring CRISPR enzymes. , Cysteine (C), proline (P), glycine (G), alanine (A), valine (V), isoleucine (I), leucine A modification of one or more amino acids of methionine (M), phenylalanine (F), tyrosine (Y) and tryptophan (T).

In addition, the modification may be a modification of one or more amino acids of an amino acid having a hydrophobic moiety present in the naturally occurring CRISPR enzyme.

For example, the modifications include glycine (Glysin, G), alanine (A), valine (V), isoleucine (I), leucine (L) , Methionine (M), phenylalanine (F), tyrosine (Y), and tryptophan (Tryptophan, W).

The modification may be a modification of one or more of the amino acids of a polar residue present in the naturally occurring CRISPR enzyme.

For example, the modifications include serine (S), threonine (T), asparagine (N), glutamine (Q) and cysteine (C) residues in the naturally occurring CRISPR enzyme. One or two or more of the following: Proline, P, Lysine, K, Arginine, R, H, Aspartic acid D and Glutamic acid E It may be a modification of an amino acid.

Alternatively, the modification may be a modification of one or more amino acids of amino acids consisting of lysine (K), arginine (R) and histidine (H) present in the naturally occurring CRISPR enzyme.

For example, the modification may be a substitution of one or more amino acids of amino acids consisting of lysine (K), arginine (R) and histidine (H) present in the naturally occurring CRISPR enzyme have.

The modification may be a modification of one or more amino acids of amino acids composed of aspartic acid (D) and glutamic acid (E) present in the naturally occurring CRISPR enzyme.

For example, the modification may be a substitution of one or more amino acids of amino acids composed of aspartic acid (D) and glutamic acid (E) present in the naturally occurring CRISPR enzyme.

These modifications include serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine (C), proline , P), glycine (G), alanine (A), valine (V), isoleucine I, leucine L, methionine M, phenylalanine, F), tyrosine (Y), and tryptophan (Tryptophan, W).

For example, the modifications include serine (S), threonine (T), asparagine (N), glutamine (Q) and cysteine (C) residues in the naturally occurring CRISPR enzyme. , Proline (P), Glysin (G), Alanine (A), Valine (V), Isoleucine (I), Leucine (L), Methionine A substitution of one or more amino acids of amino acids composed of phenylalanine (F), tyrosine (Y) and tryptophan (Tryptophan, W).

In addition, the modification may be a modification of one, two, three, four, five, six, seven or more of the amino acids present in the naturally occurring CRISPR enzyme.

In addition, in CRISPR enzyme mutants,

The modification may be a modification of one or more of the amino acids present in the RuvC domain of the CRISPR enzyme. At this time, the RuvC domain may be a RuvCI, RuvCII or RuvCIII domain.

The modification may be a modification of one or more of the amino acids present in the HNH domain of the CRISPR enzyme.

The modification may be a modification of one or more amino acids of the amino acid present in the REC domain of the CRISPR enzyme.

The modification may be a modification of one or more of the amino acids present in the PI domain of the CRISPR enzyme.

The modification may be a modification of two or more of the amino acids contained in at least two of the REC, RuvC, HNH or PI domains of the CRISPR enzyme.

In one embodiment, the variant may be a modification of two or more amino acids of the amino acids included in the REC and RuvC domains of the CRISPR enzyme.

In one embodiment, in a SpCas9 variant, the variant may be a modification of at least two of the amino acids A203, H277, G366, F539, I601, M763, D965 and F1038 amino acids contained in the REC and RuvC domains of SpCas9.

In another embodiment, the variant may be a modification of two or more amino acids of the amino acids included in the REC and HNH domains of the CRISPR enzyme.

In one embodiment, in a SpCas9 variant, the variant may be a variant of at least two of the included amino acids A203, H277, G366, F539, I601 and K890 amino acids contained in the REC and HNH domains of SpCas9.

In one example, the variant may be a modification of two or more amino acids of the amino acids included in the REC and PI domains of the CRISPR enzyme.

In one embodiment, in a SpCas9 variant, the variant may be a modification of at least two of the amino acids A203, H277, G366, F539, I601, T1102 and D1127 amino acids contained in the REC and PI domains of SpCas9.

In another example, the variant may be a modification of three or more amino acids of the amino acids included in the REC, RuvC and HNH domains of the CRISPR enzyme.

In one embodiment, in a SpCas9 variant, the variant may be a variant of at least three of the amino acids A203, H277, G366, F539, I601, M763, K890, D965 and F1038 amino acids contained in the REC, RuvC and HNH domains of SpCas9 have.

As an example, the variant may be a modification of three or more amino acids of the amino acids included in the REC, RuvC and PI domains of the CRISPR enzyme.

In one embodiment, in a SpCas9 variant, the variant is a variant of at least three of the amino acids A203, H277, G366, F539, I601, M763, D965, F1038, T1102 and D1127 amino acids contained in the REC, RuvC and PI domains of SpCas9 Lt; / RTI >

In another example, the variant may be a modification of three or more amino acids of the amino acids included in the REC, HNH and PI domains of the CRISPR enzyme.

In one embodiment, in a SpCas9 variant, the variant may be a modification of at least three of the amino acids A203, H277, G366, F539, I601, K890, T1102 and D1127 amino acids contained in the REC, HNH and PI domains of SpCas9.

For example, the variant may be a modification of three or more amino acids of the amino acids included in the RuvC, HNH and PI domains of the CRISPR enzyme.

In one embodiment, in a SpCas9 variant, the variant may be a variant of at least three of the amino acids M763, K890, D965, F1038, T1102 and D1127 amino acids contained in the RuvC, HNH and PI domains of SpCas9.

In another embodiment, the variant may be a variant of at least four of the amino acids contained in the REC, RuvC, HNH and PI domains of the CRISPR enzyme.

In one embodiment, in SpCas9 variants, the variants include at least four of the A203, H277, G366, F539, I601, M763, K890, D965, F1038, T1102 and D1127 amino acids contained in the REC, RuvC, HNH and PI domains of SpCas9 Or more.

In addition, in CRISPR enzyme mutants,

The modification may be a modification of one or more amino acids of the amino acids involved in the nuclease activity of the CRISPR enzyme.

For example, in a SpCas9 variant, the variant may be one or more variants of the amino acid group consisting of D10, E762, H840, N854, N863 and D986, or one or two of the corresponding amino acid groups of other Cas9 orthologs Or more.

Such a modification may be a modification that partially inactivates the nuclease activity of the CRISPR enzyme, and such a CRISPR enzyme variant may be a niacase.

The variant may be a variant that inactivates the nuclease activity of the RuvC domain of the CRISPR enzyme. Such a CRISPR enzyme variant may be a non-complementary strand of the target gene or nucleic acid, i.e., a strand that is not complementary to the gRNA Can not be cut.

In one specific example, in the case of SpCas9, mutation of aspartic acid at position 10 of amino acid sequence of SpCas9 to alanine, that is, mutation to D10A, inactivates the nuclease activity of the RuvC domain, so that it can be used as a niacase , Wherein the generated niacase is unable to cleave the target gene or the non-complementary strand of the nucleic acid, i.e., the strand that is not complementary to the gRNA.

In another specific example, in the case of CjCas9, mutation of the aspartic acid at position 8 of the amino acid sequence of CjCas9 to alanine, that is, mutation to D8A, inactivates the nuclease activity of the RuvC domain, , Wherein the generated niacase is unable to cleave the target gene or the non-complementary strand of the nucleic acid, i.e., the strand that is not complementary to the gRNA.

Also, the modification may be a modification that inactivates the nuclease activity of the HNH domain of the CRISPR enzyme, and such CRISPR enzyme mutants may be used to cleave a complementary strand of the target gene or nucleic acid, Can not.

In one specific example, in the case of SpCas9, mutation of histidine of amino acid sequence 840 of SpCas9 to alanine, that is, mutation to H840A, can be used as a niacase since the nuclease activity of the HNH domain is inactivated, At this time, the generated niacase can not cleave the complementary strand of the target gene or the nucleic acid, that is, the strand that makes a complementary bond to the gRNA.

In another specific example, in the case of CjCas9, mutation of histidine of amino acid sequence 559 of CjCas9 to alanine, that is, mutation to H559A, can be used as a niacase since the nuclease activity of the HNH domain is inactivated , Wherein the generated niacase can not cleave the target gene or the complementary strand of the nucleic acid, i.e., the strand that makes a complementary binding to the gRNA.

In addition, the modification may be a modification that completely inactivates the nuclease activity of the CRISPR enzyme, and such a CRISPR enzyme variant may be an inactive CRISPR enzyme.

Wherein said modification may be a modification that inactivates the nuclease activity of the RuvC and HNH domains of the CRISPR enzyme and such a CRISPR enzyme mutant is unable to cleave the double strand of the target gene or nucleic acid.

In one specific example, in the case of SpCas9, mutation of the amino acid sequence 10 aspartic acid of SpCas9 and 840 of histidine to alanine, that is, mutation to D10A and H840A, results in nuclease activity by RuvC domain and HNH domain The target gene or the double strand of the nucleic acid can not be cleaved.

In another specific example, in the case of CjCas9, mutation of the amino acid sequence of the CjCas9 amino acid sequence aspartate and the 559th histidine to alanine, that is, mutation to D8A and H559A, the nuclease by the RuvC domain and the HNH domain Since the activity is inactivated, neither the target gene nor the double strand of the nucleic acid can be cleaved.

In addition, the CRISPR enzyme variant may additionally include a functional domain in addition to the original property of the CRISPR enzyme, and such CRISPR enzyme variant may have additional characteristics in addition to the original characteristics.

The functional domain may be selected from the group consisting of methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification a domain having histone modification activity, cleavage activity or nucleic acid binding activity, or a tag or reporter gene for the separation and purification of a protein (including a peptide) But is not limited thereto.

The functional domains, peptides, polypeptides or proteins may be deaminases.

For example, an incomplete or partial CRISPR enzyme may additionally include cytidine deaminase as a functional domain. In one embodiment, a fusion protein can be generated by adding SpCas9 nicarase stanidine diamine, for example APOBEC1 (apolipoprotein B editing complex 1). The [SpCas9 niacase] - [APOBEC1] thus formed can be used for base correction or editing of base C with T or U, or for base correction or editing with base G.

The tag includes a histidine (His) tag, a V5 tag, a FLAG tag, an influenza hemagglutinin (HA) tag, a Myc tag, a VSV-G tag and a thioredoxin (Trx) tag, (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein But are not limited to, autophosphor proteins, including GFP, HcRed, DsRed, Cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and blue fluorescent protein (BFP).

Also, the functional domain may be a nuclear localization sequence or signal (NLS) or a nuclear export sequence or signal (NES).

In one example, the CRISPR enzyme may comprise one or more NLSs. Wherein the NLS is at or near the amino terminus of the CRISPR enzyme; Carboxy terminus or vicinal thereof; Or a combination thereof. The NLS can be, but is not limited to, the NLS sequence derived from: the NLS of the SV40 virus large T-antigen with the amino acid sequence PKKKRKV; NLS from nucleoplasmin (e. G., Nucleoplasmin NLS with sequence KRPAATKKAGQAKKKK); C-myc NLS with amino acid sequence PAAKRVKLD or RQRRNELKRSP; HRNPA1 M9 NLS with sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; The sequence of the IBB domain from IMPOTIN-ALPHA RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV; Sequences of myoma T protein VSRKRPRP and PPKKARED; Sequence of human p53 POPKKKPL; Sequence of mouse c-abl IV SALIKKKKKMAP; The sequences DRLRR and PKQKKRK of influenza virus NS1; The sequence of the hepatitis virus delta antigen RKLKKKIKKL; Sequence of mouse Mx1 protein REKKKFLKRR; Sequence of human poly (ADP-ribose) polymerase KRKGDEVDGVDEVAKKKSKK; And the sequence of the steroid hormone receptor (human) glucocorticoid RKCLQAGMNLEARKTKK.

In addition, a CRISPR enzyme variant may comprise a CRISPR enzyme in a split form divided into two or more fragments by dividing the CRISPR enzyme. &Quot; Split " means dividing a protein functionally or structurally or optionally into two or more.

At this time, the split type CRISPR enzyme may be a fully activated enzyme, an incomplete or partially activated enzyme, or an inactive enzyme.

For example, in the case of SpCas9, split SpCas9 can be generated by dividing the 656th tyrosine and the 657th threonine into two parts.

In addition, the split CRISPR enzyme may optionally include additional domains, peptides, polypeptides or proteins for reconstitution.

Wherein said " reconstitution " means that the split form of the CRISPR enzyme is structurally identical or similar to the wild type CRISPR enzyme.

Additional domains, peptides, polypeptides or proteins for the reconstitution include FRB and FKBP dimerization domains; Intein; ERT and VPR domains, or domains that form heterodimers in certain conditions.

For example, in the case of SpCas9, the FRC domain can be connected to one of the two parts and the FKBP domain to the other, to the split SpCas9 divided into two parts by dividing the serine between 713 and 714 glycine. Split SpCas9 thus produced can dimerize the FRB domain and the FKBP domain in the presence of rapamycin to generate a reconstituted CRISPR enzyme.

The CRISPR enzyme or CRISPR enzyme variant described in the present invention may be a polypeptide, a protein, or a nucleic acid having a sequence encoding the CRISPR enzyme or the CRISPR enzyme variant. The CRISPR enzyme or the CRISPR enzyme variant may be a codon optimized .

By " codon optimization " is meant the use of at least one codon of the native sequence to replace the nucleic acid of the host cell with a codon that is more frequently or most frequently used, It means a process of transforming the sequence. Various species have specific biases for a particular codon of a particular amino acid and codon bias (difference in codon usage between organisms) is often correlated with the efficiency of translation of the mRNA, which is dependent on the nature of the codon being translated and the availability of a particular tRNA molecule As shown in FIG. The predominance of the selected tRNA in the cell generally reflects the codons most frequently used for peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

3. Target sequence

A " target sequence " is a base sequence present in a target gene or nucleic acid, and is complementary to a guide sequence contained in the guide domain of the guide nucleic acid. The target sequence may be designed in various ways depending on the target gene or nucleic acid, that is, the base sequence that can be changed depending on the subject to be genetically modified or corrected.

The target sequence may be complementary to the guide sequence contained in the guide domain of the guide nucleic acid, and the length of the target sequence may be the same as the length of the guide sequence.

The target sequence may be from 5 to 50 nucleotide sequences.

In one embodiment, the target sequence comprises 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, Base sequence or 25 base sequences.

The target sequence may be a nucleic acid sequence complementary to a guiding sequence contained in the guiding domain of the guiding nucleic acid, and may be at least 70%, 75%, 80%, 85%, 90% or 95% complementary or complete Lt; RTI ID = 0.0 > complementary < / RTI >

As an example, the target sequence may have or contain 1 to 8 nucleotide sequences that are not complementary to the guiding sequences contained in the guiding domain of the guide nucleic acid.

In addition, the target sequence may be a nucleotide sequence located at a position close to a nucleic acid sequence recognizable by the editor protein.

In one example, the target sequence may be a sequence of 5 to 50 consecutive nucleotides located adjacent to the 5 ' end or / and the 3 ' end of the nucleic acid sequence recognizable by the editor protein.

As one specific example of the target sequence of the present invention, the target sequence for the gRNA-CRISPR enzyme complex is described below.

When a target gene or nucleic acid is targeted using the gRNA-CRISPR enzyme complex,

The target sequence is complementary to the guiding sequence contained in the guiding domain of the gRNA. The target sequence may be designed in various ways depending on the target gene or nucleic acid, that is, the base sequence that can be changed depending on the subject to be genetically modified or corrected.

In addition, the target sequence may be a nucleotide sequence located at a position close to the CRISPR enzyme, i.e., a PAM sequence recognizable by Cas9 or Cpf1.

In one example, the target sequence may be a sequence of 5 to 50 consecutive nucleotides located adjacent to the 5 'end and / or the 3' end of the PAM sequence that the CRISPR enzyme recognizes.

In one embodiment, when the CRISPR enzyme is SpCas9, the target sequence may be 5'-NGG-3 ', 5'-NAG-3' or / and 5'-NGA- C, or A, U, G, or C) sequences, or contiguous 16 to 25 nucleotide sequences located adjacent to the 3 ' end.

In another embodiment, when the CRISPR enzyme is StCas9, the target sequence is 5'-NGGNG-3 'or / and 5'-NNAGAAW-3' (W = A or T; N = A, C, or A, U, G, or C) sequences, or contiguous 16 to 25 nucleotide sequences located adjacent to the 3 ' end.

In another embodiment, when the CRISPR enzyme is NmCas9, the target sequence is 5'-NNNNGATT-3 'or / and 5'-NNNGCTT-3' (N = A, T, G or C; , G or C) contiguous 16-25 nucleotide sequences located adjacent to the 5'end and / or the 3'end of the sequence.

In one embodiment, when the CRISPR enzyme is CjCas9, the target sequence is 5'-NNNVRYAC-3 '(V = G, C or A; R = A or G; Y = C or T; Or contiguous 16 to 25 nucleotide sequences located adjacent to the 5 'end and / or the 3' end of the T, G or C; or the A, U, G, or C sequence.

In another embodiment, when the CRISPR enzyme is SmCas9, the target sequence is 5'-NGG-3 'and / or 5'-NAAR-3' (R = A or G; N = A, C, or A, U, G, or C) sequences, or contiguous 16 to 25 nucleotide sequences located adjacent to the 3 ' end.

In another embodiment, when the CRISPR enzyme is SaCas9, the target sequence is 5'-NNGRR-3 ', 5'-NNGRRT-3' or / , Contiguous 16-25 residues adjacent to the 5'end and / or the 3'end of the V, G, C or A, N = A, T, G or C; or A, Lt; / RTI >

In one embodiment, when the CRISPR enzyme is Cpf1, the target sequence may be the 5 'end of the 5'-TTN-3' (N = A, T, G or C; And contiguous 16-25 nucleotide sequences located adjacent to the 3 ' end.

4. Guide nucleic acid - Editor protein  Using the complex

The guide nucleic acid-editor protein complex can modify the target.

The subject may be a target nucleic acid, gene, chromosome or protein .

For example, the guide nucleic acid-editor protein complex can be used to control (e.g., inhibit, inhibit, decrease, increase, or promote) the expression of a protein of interest, Inhibition, inhibition, reduction, increase or promotion) or to express a new protein.

At this time, the guide nucleic acid-editor protein complex can act at DNA, RNA, gene or chromosome level.

For example, the guide nucleic acid-editor protein complex may manipulate or modify the target DNA to control (e.g., suppress, inhibit, decrease, increase, or enhance) the expression of the protein encoded by the target DNA, (E. G., Inhibit, inhibit, decrease, increase or enhance) protein activity or altered protein expression.

Alternatively, the guide nucleic acid-editor protein complex may manipulate or modify the target RNA to control (e.g., suppress, inhibit, decrease, increase, or enhance) the expression of the protein encoded by the target DNA, , Modulate (e.g., inhibit, inhibit, decrease, increase or promote) protein activity, or altered protein expression.

For example, the guide nucleic acid-editor protein complex may manipulate or modify the target gene to control (e.g., suppress, inhibit, decrease, increase, or enhance) the expression of the protein encoded by the target DNA, (E. G., Inhibit, inhibit, decrease, increase or enhance) protein activity or altered protein expression.

Alternatively, the guide nucleic acid-editor protein complex may manipulate or modify the target chromosome to control (e.g., suppress, inhibit, decrease, increase, or enhance) the expression of the protein encoded by the target DNA, , Modulate (e.g., inhibit, inhibit, decrease, increase or promote) protein activity, or altered protein expression.

The guide nucleic acid-editor protein complex can act at the transcription and translation step of the gene.

For example, the guide nucleic acid-editor protein complex may modulate (e.g., inhibit, inhibit, decrease, increase, or promote) the expression of a protein encoded by the target gene by promoting or inhibiting transcription of the target gene.

In another example, the guide nucleic acid-editor protein complex can modulate (e.g., inhibit, inhibit, reduce, increase, or promote) the expression of a protein encoded by the target gene by promoting or inhibiting translation of the target gene.

The guide nucleic acid-editor protein complex can act at the protein level.

As an example, the guide nucleic acid-editor protein complex may manipulate or modify the target protein to remove target proteins or modulate (e.g., inhibit, inhibit, reduce, increase, or enhance) protein activity.

As an example of the use of the guide nucleic acid-editor protein complex of the present invention, the manipulation or modification of the target DNA, RNA, gene or chromosome using the gRNA-CRISPR enzyme complex is described below.

Gene editing

The target gene or nucleic acid can be edited or calibrated using the above-described gRNA-CRISPR enzyme complex, i.e., CRISPR complex. At this time, the editing or correction of the target gene or the nucleic acid includes both the step of i) the cleavage or damage of the target gene or nucleic acid, and ii) the repair or repair of the damaged target gene or nucleic acid.

i) cutting or damaging the target gene or nucleic acid

i) The cleavage or damage of the target gene or nucleic acid may be a cleavage or damage of the target gene or nucleic acid using the CRISPR complex, specifically a cleavage of the target gene or the target sequence in the nucleic acid.

For example, the cleavage or deletion of a target gene or nucleic acid using the CRISPR complex may result in the cleavage or damage of the double strand of the target sequence.

In one embodiment, when wild-type SpCas9 is used, the double strand of the target sequence that makes a complementary binding to the gRNA can be cleaved.

In another embodiment, when using SpCas9 nicase (D10A) and SpCas9 nicase (H840A), the complementary single strand of the target sequence that makes a complementary binding to the gRNA is cleaved by SpCas9 nicase (D10A) The non-complementary single strand of the target sequence that makes complementary binding can be cleaved by SpCas9 nicase (H840A), and each cleavage can occur sequentially or concurrently.

In another embodiment, when two gRNAs having different target sequences from SpCas9 nicase (D10A) and SpCas9 nicase (H840A) are used, the complementary single strand of the target sequence that makes a complementary binding with the first gRNA The non-complementary single strand of the target sequence which is cleaved by SpCas9 nicase (D10A) and which makes a complementary binding with the second gRNS can be cleaved by SpCas9 nicase (H840A), and each cleavage can occur sequentially or simultaneously .

As another example, the cleavage or deletion of a target gene or nucleic acid using the CRISPR complex may be such that only a single strand of the target sequence is cut or damaged. The single strand may be a single strand complementary to a target sequence that is complementary to the gRNA, or may be an uncharged single strand of a target sequence that undergoes a complementary binding to the gRNA.

In one embodiment, when a SpCas9 nicase (D10A) is used, the complementary single strand of the target sequence that is complementary to the gRNA is cleaved by SpCas9 nicase (D10A), and the target sequence complementary to the gRNA The non-complementary single strand may not be cleaved.

In another embodiment, when using SpCas9 nicase (H840A), the non-complementary single strand of the target sequence that undergoes complementary binding to the gRNA is cleaved by SpCas9 nicase (H840A), and a target that is complementary to gRNA The complementary single strand of the sequence may not be cleaved.

As another example, cleavage or damage of a target gene or nucleic acid using the CRISPR complex may be to remove some nucleic acid fragments.

In one embodiment, when two gRNAs having different target sequences and wild-type SpCas9 are used, the double strand of the target sequence that undergoes complementary binding with the first gRNA is cleaved, and the target sequence complementary to the second gRNA Can be cleaved to remove nucleic acid fragments by the first gRNA, second gRNA and SpCas9.

In another embodiment, when two gRNAs having different target sequences and wild-type SpCas9 and SpCas9 nicase (D10A) and SpCas9 nicase (H840A) are used, the double strand of the target sequence that makes a complementary binding with the first gRNA Is cleaved by wild-type SpCas9, and the complementary single strand of the target sequence that is complementary to the second gRNA is cleaved by SpCas9 nicase (D10A) and the non-complementary single strand is cleaved by SpCas9 nicase (H840A) The nucleic acid fragments can be removed by the gRNA and the second gRNA and wild-type SpCas9, SpCas9 nicase (D10A) and SpCas9 nicase (H840A).

In another embodiment, when two gRNAs having different target sequences and SpCas9 nicase (D10A) and SpCas9 nicase (H840A) are used, the single complementary strand of the target sequence that makes a complementary binding to the first gRNA is SpCas9 The non-complementary single strand was cleaved by SpCas9 nicase (H840A) and the complementary single strand of the target sequence that was complementary to the second gRNA was cleaved by SpCas9 nicase (D10A) Bose single strand can be cleaved by SpCas9 nicase (H840A) to remove nucleic acid fragments by first gRNA and second gRNA and by SpCas9 nicase (D10A) and SpCas9 nicase (H840A).

In another embodiment, when using three gRNAs with different target sequences and wild-type SpCas9 and SpCas9 nicase (D10A) and SpCas9 nicase (H840A), the double strand of the target sequence that makes a complementary binding to the first gRNA The complementary single strand of the target sequence that is cleaved by the wild-type SpCas9 and which makes a complementary binding to the second gRNA is cleaved by SpCas9 nicase (D10A), and an uncharged single strand of the target sequence that undergoes a complementary binding with the third gRNA Can be cleaved by SpCas9 nicase (H840A) to remove the nucleic acid fragments by the first gRNA, the second gRNA and the third gRNA and the wild type SpCas9, SpCas9 nicase (D10A) and SpCas9 nicase (H840A).

As another example, when four gRNAs having different target sequences and SpCas9 nicase (D10A) and SpCas9 nicase (H840A) are used, the single complementary strand of the target sequence that makes a complementary binding with the first gRNA is SpCas9 The non-complementary single strand of the target sequence cleaved by niacase (D10A) and having a complementary binding with the second gRNA is cleaved by SpCas9 nicase (H840A), and the target sequence complementary to the third gRNA The complementary single strand is cleaved by SpCas9 nicase (D10A), and the uncharged single strand of the target sequence that is complementary to the fourth gRNA is cleaved by SpCas9 nicase (H840A) to form a first gRNA, a second gRNA, The nucleic acid fragments can be removed by the third and fourth gRNAs and SpCas9 nicase (D10A) and SpCas9 nicase (H840A).

ii ) Repair or restoration of damaged target gene or nucleic acid

The target gene or nucleic acid that is cleaved or damaged by the CRISPR complex can be repaired or restored via non-homologous terminal-binding (NHEJ) and homologous recombination repair (HDR).

ratio- Homology  End-bonding ( Non - homologous end joining , NHEJ )

NHEJ is a method of repairing or repairing double strand breakage in DNA by joining both ends of a truncated double strand or single strand together to form a DNA strand that generally has two conformal ends formed by breakage (e.g., cleavage) Repeating this frequent contact causes the broken double strand to recover if the two ends are fully bonded. NHEJ is a possible repair method in all cell cycles and occurs when there is no homologous genome to be used as a template in cells, such as the G1 phase.

In the process of repairing a damaged gene or nucleic acid using NHEJ, some insertion and / or deletion (insertion deletion) of the nucleic acid sequence is caused at the NHEJ repair site. Such insertion and / or deletion changes the reading frame, mRNA, resulting in loss of native function by undergoing nonsense mediated decay or failure to synthesize normal proteins. Or, in addition, retains the leading frame, but can result in a mutation that inserts or deletes a significant amount of sequence, thereby destroying the functionality of the protein. It is site-dependent because a mutation in an important functional domain is less likely to be tolerated than a mutation in a non-critical region of the protein.

Insertion deletion mutations generated by NHEJ are unpredictable in nature, but certain deletion deletion sequences are preferred at a given site of failure, possibly due to small regions of micro homology. Typically deletion lengths range from 1 bp to 50 bp, insertions tend to be shorter and often contain short overlapping sequences immediately surrounding the site of the breakage.

In addition, NHEJ is a mutagenic process that can be used to delete small sequence motifs if production of a specific final sequence is not required.

With such NHEJ, specific knockout of the gene targeted by the CRISPR complex can be achieved. A double strand or two single strands of a target gene or nucleic acid are cleaved using a CRISPR enzyme such as Cas9 or Cpf1 and a double strand or two single strands of the target gene or nucleic acid that has been broken are inserted and deleted by NHEJ , Which can induce a specific knockout of the target gene or nucleic acid. At this time, the position of the target gene or nucleic acid cleaved by the CRISPR enzyme may be a non-coding region or a coding region, and the position of the target gene or nucleic acid restored by the NHEJ may be a non-coding region or a cipher region.

Homogeneous recombination repair homology directed repairing , HDR )

HDR is a method that can be corrected without error by using a sequence having homology to repair or repair a damaged gene or nucleic acid as a template. In general, in order to repair or repair damaged DNA, that is, In order to restore the original information, information of the complementary base sequence which has not been modified is used, or the damaged DNA is repaired or restored by using the information of the sister chromatid powder. The most common form of HDR is homologous recombination (HR). HDR is usually a repair or repair method that occurs mainly in the S or G2 / M phase of actively dividing cells.

Instead of using complementary base sequences or sister chromatids originally possessed by cells for repairing or repairing damaged DNA using HDR, an artificially synthesized DNA template using complementary base sequence or homologous base sequence information, that is, The DNA can be repaired or repaired by providing the nucleic acid template containing the nucleotide sequence or the homologous nucleotide sequence to the cell. At this time, when nucleic acid sequences or nucleic acid fragments are added to the nucleic acid template to repair or repair the broken DNA, the nucleic acid sequence or the nucleic acid manipulation additionally contained in the broken DNA can be knocked-in. Further included nucleic acid sequences or nucleic acid manipulation may be, but are not limited to, nucleic acid sequences or fragments of nucleic acids for correcting mutated modified target genes or nucleic acids into normal genes or nucleic acids, or genes or nucleic acids desiring expression in cells .

For example, a CRISPR complex may be used to cleave a double strand or single strand of a target gene or nucleic acid and provide the cell with a nucleic acid template containing a nucleotide sequence complementary to the nucleotide sequence close to the cleavage site, The nucleotide sequence can be repaired or restored by the HDR method.

At this time, the nucleic acid template containing the complementary base sequence has a complementary base sequence of the broken DNA, that is, a truncated double strand or single strand, and further includes a nucleic acid sequence or a nucleic acid fragment to be inserted into the broken DNA . Thus, a nucleic acid sequence or a fragment of nucleic acid can be inserted at the cleaved position of the broken DNA, that is, the target gene or the nucleic acid, using the nucleic acid template containing the complementary base sequence and the nucleic acid sequence or nucleic acid fragment to be inserted. The nucleic acid sequence or nucleic acid fragment and the additional nucleic acid sequence or nucleic acid fragment to be inserted may be a nucleic acid sequence or a nucleic acid fragment for correcting a mutated target gene or nucleic acid to be a normal gene or a nucleic acid, It may be a nucleic acid. The complementary base sequence may be a base sequence that makes a complementary binding to the right and left base sequences of the broken DNA, that is, a double strand of a target gene or a truncated double strand of the nucleic acid or a single strand. Alternatively, the complementary base sequence may be a base sequence that makes a complementary binding to the 3 ' and 5 ' ends of the broken DNA, i.e., the truncated double strand or single strand of the target gene or nucleic acid. The complementary base sequence may be 15 to 3,000 base sequences, and the length or size of the complementary base sequence may be appropriately designed according to the size of the nucleic acid template or the target gene or nucleic acid. At this time, the nucleic acid template may be double-stranded or single-stranded nucleic acid, and may be linear or circular, but is not limited thereto.

In another example, a CRISPR complex is used to cleave a double strand or a single strand of a target gene or nucleic acid, and a nucleic acid template containing nucleotide sequences close to the cleavage site and a homologous base sequence is provided to the cells to cleave the target gene or nucleic acid Can be repaired or restored by the HDR method.

At this time, the nucleic acid template containing the homologous base sequence has a homologous base sequence of the broken DNA, that is, a truncated double strand or single strand, and further includes a nucleic acid sequence or a nucleic acid fragment to be inserted into the broken DNA . Thus, a nucleic acid sequence or a fragment of nucleic acid can be inserted at the cleaved position of the broken DNA, that is, the target gene or the nucleic acid, using the nucleic acid template containing the homologous base sequence and the nucleic acid sequence or the nucleic acid fragment to be inserted. The nucleic acid sequence or nucleic acid fragment and the additional nucleic acid sequence or nucleic acid fragment to be inserted may be a nucleic acid sequence or a nucleic acid fragment for correcting a mutated target gene or nucleic acid to be a normal gene or a nucleic acid, It may be a nucleic acid. The homologous base sequence may be a base sequence having homology to the right and left base sequences of the broken DNA, that is, the double strand of the target gene or the truncated double strand or single strand of the nucleic acid. Alternatively, the complementary base sequence may be a base sequence having homology with the 3 ' and 5 ' ends of the broken DNA, i.e., the truncated double strand or single strand of the target gene or nucleic acid. The homologous base sequence may be 15 to 3,000 base sequences, and the length or size of the homologous base sequence may be designed appropriately according to the size of the nucleic acid template or the target gene or nucleic acid. At this time, the nucleic acid template may be double-stranded or single-stranded nucleic acid, and may be linear or circular, but is not limited thereto.

In addition to the above NHEJ and HDR, there is a method for repairing or repairing damaged DNA.

Single strand Annealing ( Single - strand annealing , SSA )

SSA is a method of repairing double strand breaks between two repeating sequences present in a target nucleic acid, generally using a repeat number sequence of more than 30 nucleotide sequences. The single stranded protrusions containing the repeating sequence after cleavage are cut so that each single strand has a repeating sequence with respect to the double strand of the target nucleic acid at the breakage end, It is coated with RPA protein to prevent it from being annealed. RAD52 binds to each repeating subsequence on the overhang and aligns the sequence to allow annealing of the complementary repeating subsequence. After annealing, the single-stranded flap of the protrusion is cut, and the new DNA synthesis replenishes the double strand of DNA filling any gap. As a result of this repair or repair, the DNA sequence between the two repeats is deleted, and the deletion length can depend on a number of factors including the location of the two repeats used and the cleavage path or progression.

SSA uses a complementary sequence, that is, a complementary repeating sequence, similar to the HDR method, and in contrast does not require a nucleic acid template for altering or modifying the target nucleic acid sequence.

Single strand  Break repair ( Single - strand break repair , SSBA )

Single strand breaks in the genome are repaired or repaired via SSBR, a mechanism separate from the repair mechanism discussed above. When the form of DNA breakage is a single strand break, PARP1 and / or PARP2 recognize breakage and mobilize repair mechanisms. The binding and activity of PARP1 in DAN breakdown is transient and promotes the 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, and stabilizes, proteins that promote the 3'and 5'terminal processing of DNA. Termination generally involves restoring the damaged 3 'end to the hydroxylated state and / or the 5' end to the phosphate moiety, and when the ends are processed, a DNA gap fill occurs. There are two ways to fill the DNA gap: short patch repair and long patch repair, where short patch repair involves insertion of a missing base. After filling the DNA gap, the DNA ligase promotes the binding of the ends.

Mismatch  repair( Mismatch repair , MMR )

MMR works on a mismatched DNA base. MSH2 / 6 or MSH2 / 3 complexes both have ATPase activity that plays an important role in the initiation of mismatch recognition and repair, MSH2 / 6 preferentially recognizes base-base mismatches, and one or two While MSH2 / 3 identifies a mismatch of bases, it recognizes a larger mismatch preferentially.

Base Cutting Repair ( Base excision repair , BER )

BER is active throughout the cell cycle and is the repair method used to remove small non-helical-warp base damage from the genome. Damaged DNA cleaves the N-glycoside linkage that links the base to the sugar phosphatidylated backbone, cleaves the damaged base, and then cleaves the phosphodiester backbone to produce DNA single strand breaks. The damaged single-stranded end thus formed is removed, the gap generated by the removed single strand is filled with a new complementary base, and then the complementary base end and the backbone newly filled with DNA ligase are bonded to repair or repair the damaged DNA.

Nucleotide  Cutting Repair ( Nucleotide excision repair , NER )

NER is an important cleavage mechanism that eliminates large helical-shear damage from DNA and, once the damage is recognized, removes the short single stranded DNA segment containing the damaged portion, creating a single strand gap of 22-30 bases. The resulting gap is filled with a new complementary base, and then the complementary base end and backbone newly filled with DNA ligase are combined to repair or repair the damaged DNA.

Gene editing effect

Editing or calibrating a target gene or nucleic acid can largely lead to the effects of knockout, knockdown, and knockin.

Knockout

&Quot; Knockout " means inactivation of a target gene or nucleic acid, and " inactivation of a target gene or nucleic acid " means a state in which transcription and / or translation of a target gene or nucleic acid fails. Knockout can inhibit protein expression by inhibiting transcription and translation of genes that cause disease or abnormally functioning genes.

For example, when a target gene or a nucleic acid is edited or corrected using a gRNA-CRISPR enzyme complex, i.e., a CRISPR complex, a CRISPR complex can be used to cleave a target gene or a nucleic acid. Using the CRISPR complex, the damaged target gene or nucleic acid can be repaired with NHEJ or the damaged gene or nucleic acid. A damaged target gene or nucleic acid is generated by insertion deletion by NHEJ, which can induce a specific knockout of the target gene or nucleic acid.

Knockdown

&Quot; Knockdown " means reducing transcription and / or translation of a target gene or nucleic acid or reducing expression of a target protein. By controlling the expression of overexpressed genes or proteins through knockdown, the disease can be prevented from occurring or the disease can be treated.

For example, when a target gene or a nucleic acid is edited or calibrated using a gRNA-CRISPR inactivating enzyme-transcription inhibiting activity domain complex, i.e., a CRISPR inactive complex containing a transcription inhibitory activity domain, the CRISPR inactive complex is used as a target gene A knockdown in which the target gene or nucleic acid transcription is inhibited by the transcription inhibitory activity domain contained in the CRISPR inactive complex and specifically inhibits the expression of the gene or the nucleic acid can be induced.

Knockin

&Quot; Knockin " means the insertion of a specific nucleic acid or gene into a target gene or nucleic acid, wherein the " specific nucleic acid " means a gene or nucleic acid to be inserted or desired to be expressed. It can be used to correct disease by correcting the mutation gene which induces disease through meltedin or inducing normal gene expression by inserting normal gene.

In addition, melted can additionally require a donor.

For example, when a target gene or a nucleic acid is edited or corrected using a gRNA-CRISPR enzyme complex, i.e., a CRISPR complex, a CRISPR complex can be used to cleave a target gene or a nucleic acid. Using a CRISPR complex, a damaged target gene or nucleic acid can be repaired with a gene or nucleic acid that is damaged through HDR. At this time, a specific nucleic acid can be inserted into a damaged gene or nucleic acid using a donor.

The term " donor " refers to a nucleic acid sequence that aids in repairing damaged DNA or nucleic acid through HDR, wherein the template may comprise a specific nucleic acid.

The donor may be a double-stranded nucleic acid or a single-stranded nucleic acid.

The donor may be linear or circular.

The donor may comprise a nucleic acid sequence having homology to a target gene or nucleic acid.

For example, the donor may comprise a nucleic acid sequence that is homologous to the upstream and downstream base sequences, respectively, of a damaged nucleic acid at a location where the particular nucleic acid is to be inserted. The specific nucleic acid to be inserted may be located between a nucleic acid sequence homologous to the right base sequence of the damaged nucleic acid and a nucleic acid sequence homologous to the left base sequence of the damaged nucleic acid. The nucleic acid sequence having the above homology may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% You can have same sex.

The donor may optionally include ancillary nucleic acid sequences. At this time, an ancillary nucleic acid sequence may serve to increase donor stability, dissolved efficiency, or HDR efficiency.

For example, the ancillary nucleic acid sequence may be a nucleotide sequence rich in base A, T, i.e., an A-T rich domain. Or the ancillary nucleic acid sequence may be a scaffold / matrix attachment region (S / MAR).

5. Additional components

An additional construct may be included to increase the efficiency of the guide nucleic acid-editor protein complex or to improve the repair efficiency of the damaged gene or nucleic acid.

Additional constructs may optionally be used to enhance the efficiency of the guided nucleic acid-editor protein complex.

Activator ( activator )

The additional construct may be used as an activator to increase the cleavage efficiency of the target nucleic acid, gene or chromosome of the guide nucleic acid-editor protein complex.

The " activator " serves to stabilize the binding of the guide nucleic acid-editor protein complex to the target nucleic acid, gene or chromosome, or to better access the guide nucleic acid-editor protein complex to the target nucleic acid, gene or chromosome ≪ / RTI >

The activator may be a double-stranded nucleic acid or a single-stranded nucleic acid.

The activator may be linear or circular.

The activator serves to better access the "helper" and guide nucleic acid-editor protein complexes that stabilize the binding of the target nucleic acid, gene or chromosome with the guide nucleic acid-editor protein complex to the target nucleic acid, gene or chromosome It can be divided into "escortor".

The helper can increase the cleavage efficiency of the target nucleic acid, gene or chromosome of the guide nucleic acid-editor protein complex.

For example, the helper may include a nucleic acid sequence having a homology to a target nucleic acid, gene or chromosome so that when the guide nucleic acid-editor protein complex binds to a target nucleic acid, gene or chromosome, The nucleic acid sequence may further complementarily bind to the target nucleic acid, gene or chromosome so that the binding of the target nucleic acid, the gene or the chromosome with the guide nucleic acid-editor protein complex can be stabilized.

The escor can increase the cleavage efficiency of the target nucleic acid, gene or chromosome of the guide nucleic acid-editor protein complex.

For example, the escoter includes a nucleic acid sequence having homology to a target nucleic acid, a gene or a chromosome, wherein the nucleic acid sequence having homology with the escoter is part of a guide nucleic acid of the guide nucleic acid- A complementary combination can be made. As a result, an escoter having some complementary binding with the guide nucleic acid-editor protein complex can make some complementary binding with the target nucleic acid, gene or chromosome, and as a result, the escoter can convert the guide nucleic acid-editor protein complex into an accurate target nucleic acid, Or chromosomal location.

The nucleic acid sequence having the above homology may have a homology of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% Lt; / RTI >

In addition, the additional construct may be optionally used to improve the repair efficiency of the damaged gene or nucleic acid.

Assister ( Assistor )

The additional construct may be used as an assistor to improve the repair efficiency of the damaged gene or nucleic acid.

The " assistor " means a nucleic acid that participates in the repair process of a damaged gene or a nucleic acid, for example, a gene or a nucleic acid cleaved by a guide nucleic acid-editor protein complex, or plays a role in improving repair efficiency.

The acceptor may be a double-stranded nucleic acid or a single-stranded nucleic acid.

The acceptor may be linear or circular.

These assists can be divided into "NHEJ assister" which participates in the restoration process using NHEJ or improves restoration efficiency, and "HDR assister" which participates in the restoration process using HDR or improves restoration efficiency.

The NHEJ assister can participate in the repair process of the damaged gene or nucleic acid using NHEJ or improve the restoration efficiency.

For example, the NHEJ acceptor may comprise a nucleic acid sequence that is homologous to a portion of the damaged nucleic acid sequence. Herein, the nucleic acid sequence having the above homology includes a nucleic acid sequence homologous to the one-terminal (for example, 3 'terminal) nucleic acid sequence of the damaged nucleic acid sequence and the other terminal of the damaged nucleic acid sequence (for example, Terminal) nucleic acid sequence of the invention. Or a nucleic acid sequence that is homologous to the upstream and downstream base sequences of the damaged nucleic acid sequence, respectively. Such homologous nucleic acid sequences can enhance the repair efficiency of nucleic acids damaged by NHEJ by helping both parts of the damaged nucleic acid sequence to be in close proximity.

The HDR acceptor can participate in the repair process of the damaged gene or nucleic acid using HDR or improve the restoration efficiency.

For example, the HDR acceptor may comprise a nucleic acid sequence that is homologous to a portion of a damaged nucleic acid sequence. Herein, the nucleic acid sequence having the above homology includes a nucleic acid sequence homologous to the one-terminal (for example, 3 'terminal) nucleic acid sequence of the damaged nucleic acid sequence and the other terminal of the damaged nucleic acid sequence (for example, Terminal) nucleic acid sequence of the invention. Or a nucleic acid sequence that is homologous to the upstream and downstream base sequences of the damaged nucleic acid sequence, respectively. The nucleic acid sequence having such homology can serve as a template for the damaged nucleic acid sequence, thereby enhancing the repair efficiency of the damaged nucleic acid by HDR.

In another example, the HDR acceptor may comprise a nucleic acid sequence that is homologous to a portion of the damaged nucleic acid sequence and a specific nucleic acid, such as a nucleic acid or gene to be inserted. At this time, the nucleic acid sequence having the homology may include a nucleic acid sequence having homology to the upstream and downstream base sequences of the damaged nucleic acid sequence, respectively. The specific nucleic acid may be located between a nucleic acid sequence homologous to the right base sequence of the damaged nucleic acid and a nucleic acid sequence homologous to the left base sequence of the damaged nucleic acid. Such homologous nucleic acid sequences and specific nucleic acids serve as donors capable of inserting a specific nucleic acid into the damaged nucleic acid, thereby increasing the efficiency of HDR for melted.

The nucleic acid sequence having the above homology may have a homology of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% Lt; / RTI >

6. Target

&Quot; Subject " means a specimen or sample obtained from an organism into which a guide nucleic acid, an editor protein or a guide nucleic acid-editor protein complex is introduced, or an organism or organism in which a guide nucleic acid, an editor protein or a guide nucleic acid-

The subject may be an organism comprising the target nucleic acid, gene, chromosome or protein of the guide nucleic acid-editor protein complex.

The organism may be a cell, tissue, plant, animal or human.

The cells may be prokaryotic or eukaryotic.

The eukaryotic cell may be a plant cell, an animal cell, a human cell, but is not limited thereto.

The tissue may be an animal such as skin, liver, kidney, heart, lung, brain, muscle, or a human body tissue.

The subject may be a specimen or a sample comprising the target nucleic acid, gene, chromosome or protein of the guide nucleic acid-editor protein complex.

The specimen or sample may be obtained from an organism including target nucleic acid, gene, chromosome or protein such as needle, blood, skin tissue, cancer cell or stem cell.

As a specific example of the object of the present invention, a target gene or a nucleic acid of a target nucleic acid-editor protein complex is described below.

For example, the target gene may be the PD-1 gene, CTLA-4 gene, TNFAIP3 gene, DGKA gene, DGKZ gene, Fas gene, EGR2 gene, PPP2R2D gene, PSGL-1 gene, KDM6A gene, and / or TET2 gene .

In one embodiment, the target sequence of each of the above genes may be at least one selected from the sequences listed in Table 1, except for the PAM sequence (T is replaced by U). This target sequence can serve as a guide for the design of the guide nucleic acid.

That is, the nucleotide sequence of the target sequence region of each gene and the targeting sequence region of the guide RNA corresponding thereto (the targeting sequence region of the guide RNA; the targeting sequence region of the guide RNA having the nucleotide sequence capable of hybridizing with the target sequence region) (The target sequence regions listed in Table 1 are shown in the state that PAM sequence 5'-NGG-3 'is contained at the 3' end)

These target sequence regions are sequences lacking a 0bp to 2bp mismatch region in the genome, except for the target sequence, and are characterized by low off-target effect and high gene correction efficiency.

The target sequence may be two or more targets simultaneously.

Two or more of the genes may be targeted simultaneously.

Two or more target sequences in a homologous gene or two or more target sequences in a heterologous gene can be simultaneously targeted.

A non-coding or coding region in the gene, such as a promoter region, an enhancer, a 3'UTR, and / or a polyadenylation signal sequence, or a transcription sequence such as an intron or exon sequence may be targeted.

The upper 50% of the genes of the genes can be targeted.

In one embodiment, DGKa or DGKz may be targeted, respectively.

In one embodiment, DGKa and DGKz can be targeted simultaneously.

In an embodiment of the present invention, a guide nucleic acid sequence corresponding to the target sequence of SEQ ID NOS: 1 to 289 is provided for an artificial manipulation of each gene.

In an embodiment of the present invention, for example, a complex forming editor protein is provided that interacts with a guide nucleic acid sequence corresponding to the target sequence of SEQ ID NOS: 1 to 289 for an artificial manipulation of each gene.

In an embodiment of the present invention, nucleic acid modification products of each gene and an expression product thereof of an artificial manipulation are provided in the target sequence regions of SEQ ID NOS: 1 to 289, respectively.

In an embodiment of the invention, for the artificial manipulation of each gene

SEQ ID NOS: 6 and 11 (A20),

SEQ ID NOS: 19, 20, 21, and 23 (DGKa)

SEQ ID NO: 25 (EGR2)

SEQ ID NO: 64 (PPP2R2D)

SEQ ID NOS: 87 and 89 (PD-1)

SEQ ID NOS: 109, 110, 111, 112, and 113 (Dgkζ)

SEQ ID NOS: 126, 128, and 129 (Tet-2)

SEQ ID NO: 182 (PSGL-1)

SEQ ID NOS: 252, 254, 257, and 264 (FAS)

SEQ ID NO: 285 (KDM6A)

A leader nucleic acid sequence corresponding to one or more of the target sequences and an interacting editor protein are provided.

SEQ ID NOS: 6 and 11 (A20), SEQ ID NOS: 19, 20, 21 and 23 (DGKa), SEQ ID NO: 25 (EGR2), SEQ ID NO: 64 (PPP2R2D), SEQ ID NOS: 87 and 89 (SEQ ID NO: 109), SEQ ID NO: 109, 110, 111, 112 and 113 (Dgkζ), SEQ ID NOs: 126, 128 and 129 (Tet-2), SEQ ID NO: 182 And 264 (FAS), and SEQ ID NO: 285 (KDM6A), and an expression product thereof.

7. Forwarding

The guide nucleic acid, the editor protein, or the guide nucleic acid-editor protein complex can be delivered or introduced into the subject in a variety of delivery methods and in various forms.

The guide nucleic acid may be introduced or introduced into a subject in the form of DNA, RNA or a mixture thereof.

The editor protein may be delivered or introduced into a subject in the form of DNA, RNA, DNA / RNA mixtures, peptides, polypeptides or proteins that encode the editor protein.

The guide nucleic acid-editor protein complex may be delivered or introduced into a subject in the form of DNA, RNA, or a mixture thereof, each encoding a guide nucleic acid and an editor protein.

The guide nucleic acid-editor protein complex may be transferred or introduced into a subject as a complex of a guide nucleic acid having a form of DNA, RNA, or a mixture thereof, and an editor protein having the form of a peptide, a polypeptide or a protein.

The modes and methods of introduction into additional constructs that can increase or inhibit the efficiency of the guide nucleic acid-editor protein complex

i) DNA , RNA  Or in the form of a mixture thereof

The form of DNA, RNA, or a mixture thereof encoding the guide nucleic acid and / or the editor protein can be delivered or introduced into the subject by methods known in the art.

Alternatively, the form of DNA, RNA, or a mixture thereof encoding the guide nucleic acid and / or the editor protein can be delivered or introduced into the subject by a vector, non-vector, or a combination thereof.

The vector may be a viral or non-viral vector (e. G., A plasmid).

The non-vector may be naked DNA, DNA complex or mRNA.

Vector based introduction

The nucleic acid sequence encoding the guide nucleic acid and / or the editor protein may be delivered or introduced into the subject by a vector.

The vector may comprise a nucleic acid sequence encoding a guide nucleic acid and / or an editor protein.

For example, the vector may comprise a guide nucleic acid and a nucleic acid sequence encoding the editor protein at the same time.

For example, the vector may comprise a nucleic acid sequence encoding a guide nucleic acid.

For example, the domain of the guide nucleic acid may be contained in one vector, or may be divided into individual vectors.

For example, the vector may comprise a nucleic acid sequence encoding an editor protein.

For example, in the case of the above-mentioned editor protein, the nucleic acid sequence encoding the editor protein may be contained in one vector, or the nucleic acid sequence encoding the editor protein may be divided into several vectors.

The vector may comprise one or more control / control components.

The regulatory / control component may include a capture motor, 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.

The promoter may be a promoter recognized by RNA polymerase II.

The promoter may be a promoter recognized by RNA polymerase III.

The promoter may be an inducible promoter.

The promoter may be a target specific promoter.

The promoter may be a virus or a non-viral promoter.

The promoter can use a suitable promoter depending on the control region (i.e., the nucleic acid sequence encoding the guide nucleic acid or the editor protein).

For example, a promoter useful for the guide nucleic acid may be a H1, EF-1a, tRNA or U6 promoter. For example, the promoter useful for the editor protein may be CMV, EF-1a, EFS, MSCV, PGK or CAG promoter.

The vector may be a viral vector or a recombinant viral vector.

The virus may be a DNA virus or an RNA virus.

At this time, the DNA virus may be a double-stranded DNA (dsDNA) virus or a single-stranded DNA (ssDNA) virus.

At this time, the RNA virus may be a single stranded RNA (ssRNA) virus.

The virus may be, but is not limited to, retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus or herpes simplex virus.

In general, the virus can infect a host (e. G., A cell), introduce a nucleic acid encoding the viral genetic information into the host, or insert a nucleic acid encoding the genetic information into the host's genome. The virus having these characteristics can be used to introduce the guide nucleic acid and / or the editor protein into the subject. The guide nucleic acid and / or the editor protein introduced using the virus can be transiently expressed in a subject (e.g., a cell). Or a guide nucleic acid and / or an editor protein introduced using a virus can be detected in a subject (for example, a cell) for a long period of time (for example, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months , 9 months, 1 year, 2 years or permanently).

The packaging capacity of the virus is at least 2kb to 50kb and may vary depending on the virus type. According to this packaging ability, it is possible to design a viral vector containing a guide nucleic acid or an editor protein singly or to design a viral vector containing both a guide nucleic acid and an editor protein. Or a viral vector containing the guide nucleic acid, the editor protein, and additional components.

In one example, the nucleic acid sequence encoding the guide nucleic acid and / or the editor protein can be delivered or introduced by the recombinant lentivirus.

In another example, a nucleic acid sequence encoding a guide nucleic acid and / or an editor protein may be delivered or introduced by a recombinant adenovirus.

In another example, the nucleic acid sequence encoding the guide nucleic acid and / or the editor protein may be delivered or introduced by recombinant AAV.

In another example, the nucleic acid sequence encoding the guide nucleic acid and / or the editor protein may be delivered or introduced by a hybrid of at least one of the hybrid viruses, e. G., The viruses described herein.

Non vector  Introduction of foundation

The nucleic acid sequence encoding the guide nucleic acid and / or the editor protein can be delivered or introduced into the subject as a non-vector.

The non-vector may comprise a nucleic acid sequence encoding a guide nucleic acid and / or an editor protein.

The non-vector may be naked DNA, DNA complex, mRNA or a mixture thereof.

The non-vector may be selected from the group consisting of electroporation, gene gun, ultrasonic perforation, magnetofection, transient cell compaction or squeezing (e.g., Lee, et al, (2012) Nano Lett . , 12, 6322-6327), lipid-mediated transfection, dendrimers, nanoparticles, calcium phosphate, silica, silicates (orthosilicates) or a combination thereof.

As an example, delivery via electroporation can be performed by mixing the cell with a nucleic acid sequence encoding a guide nucleic acid and / or an editor protein in a cartridge, chamber, or cuvette and applying electrical stimulation of defined duration and amplitude have.

In another example, non-vectors can be delivered using nanoparticles. The nanoparticles may be inorganic nanoparticles (e.g., magnetic nanoparticles, silica, etc.) or organic nanoparticles (e.g., lipids coated with polyethylene glycol (PEG)). The outer surface of the nanoparticles may be conjugated with a positively charged polymer (e. G., Polyethyleneimine, polylysine, polyserine, etc.) that allows attachment.

In certain embodiments, it can be delivered using a lipid envelope.

In certain embodiments, it can be delivered using exosomes. Exosomes are endogenous nano-vesicles that transport proteins and RNA that can deliver RNA to the brain and other target organs.

In certain embodiments, it can be delivered using a liposome. The liposomes are spherical structures composed of a single or multiple lamellar lipid bilayer surrounding the internal aqueous compartment and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes can be made from several different types of lipids; Phospholipids are most commonly used to produce liposomes as drug carriers.

And some other additives.

ii ) Peptides , Polypeptide  Or in the form of a protein

Editor proteins can be delivered or introduced into a subject by methods known in the art, such as in the form of peptides, polypeptides or proteins.

The form of the peptide, polypeptide or protein can be determined by electroporation, microinjection, transient cell compaction or squeezing (see, for example, Lee, et al, (2012) Nano Lett. , 12, 6322-6327) , Lipid-mediated transfection, nanoparticles, liposomes, peptide-mediated delivery, or a combination thereof.

The delivery of the peptide, polypeptide or protein may be delivered with a nucleic acid sequence encoding a guide nucleic acid.

In one example, delivery via electroporation can be performed by mixing the cells with a guide nucleic acid in a cartridge, chamber, or cuvette, or with a cell to introduce the editor protein without a guide nucleic acid, and applying an electrical stimulus of defined duration and amplitude .

iii ) Transfer in the form of nucleic acid-protein mixture

The guide nucleic acid and the editor protein can be transferred or introduced into the subject in the form of a guide nucleic acid-editor protein complex.

For example, the guide nucleic acid may be DNA, RNA, or a mixture thereof. The editor protein may be in the form of a peptide, polypeptide or protein.

For example, the guide nucleic acid and the editor protein can be delivered or introduced into the subject in the form of a guide nucleic acid-editor protein complex, that is, a ribonucleoprotein (RNP), in the form of an RNA in the form of a guide nucleic acid and a protein in the form of a protein.

In the present invention, the delivery of the gRNA, the CRISPR enzyme or the gRNA-CRISPR enzyme complex is described at the bottom as one embodiment of the method of delivering the guide nucleic acid and / or the editor protein to the subject.

8. Transformant

"Transformant" means a sample or sample obtained from an organism into which a guide nucleic acid, an editor protein or a guide nucleic acid-editor protein complex has been introduced, or an organism or organism in which a guide nucleic acid, an editor protein or a guide nucleic acid- do.

The transformant may be an organism into which a guide nucleic acid, an editor protein, or a guide nucleic acid-editor protein complex is introduced in the form of DNA, RNA, or a mixture thereof.

For example, the transformant may be an organism into which a vector including a nucleic acid sequence encoding a guide nucleic acid and / or an editor protein is introduced. Here, the vector may be a non-viral vector, a viral vector, or a recombinant viral vector.

As another example, the transformant may be an organism introduced in a non-vector form comprising a nucleic acid sequence encoding a guide nucleic acid and / or an editor protein. The non-vector may be a naked DNA, a DNA complex, an mRNA or a mixture thereof.

The transformant may be an organism in which the guide nucleic acid, the editor protein or the guide nucleic acid-editor protein complex is introduced in the form of a peptide, a polypeptide or a protein.

The transformant may be an organism into which a guide nucleic acid, an editor protein, or a guide nucleic acid-editor protein complex is introduced in the form of DNA, RNA, peptide, polypeptide, protein, or a mixture thereof.

For example, the transformant may be an organism into which a guide nucleic acid in an RNA form and an editor protein in a protein form are introduced into a guide nucleic acid-editor protein complex.

The transformant may be an organism comprising the target nucleic acid, gene, chromosome or protein of the guide nucleic acid-editor protein complex.

The organism may be a cell, tissue, plant, animal or human.

The cells may be prokaryotic or eukaryotic.

The eukaryotic cell may be a plant cell, an animal cell, a human cell, but is not limited thereto.

The tissue may be an animal such as skin, liver, kidney, heart, lung, brain, muscle, or a human body tissue.

The transformant may be a sample or a sample obtained from an organism or an organism into which a guide nucleic acid, an editor protein, or a guide nucleic acid-editor protein complex is introduced or expressed.

The specimen or sample may be saliva, blood, skin tissue, cancer cells or stem cells.

Also, in one embodiment, the invention is used for nucleic acid modification at target sites of PD-1, CTLA-4, TNFAIP3, DGKA, DGKZ, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A and / Lt; RTI ID = 0.0 > nucleic acid-editor < / RTI > protein complex.

In particular, it is possible to provide a gRNA molecule including a domain capable of complementary binding with a target site from the gene, for example, an isolated or non-native gRNA molecule and a DNA encoding the same. The gRNA and the DNA sequence encoding the gRNA can be designed to be complementary to the target site sequence of Table 1. [

In addition, the target site of the gRNA molecule can be altered in PD-1, CTLA-4, TNFAIP3, DGKA, DGKZ, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A and / or TET2, Strand breakage or single strand breakage; Or a third immunoregulatory element having a specific function at the target site.

In addition, when two or more gRNAs are used to locate two or more cleavage events in the target nucleic acid, such as double or single strand breaks, two or more cleavage events can be generated by the same or different Cas9 proteins.

The gRNA may be, for example,

Two or more genes from PD-1, CTLA-4, TNFAIP3, DGKA, DGKZ, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A and /

Two or more regions in the respective genes of PD-1, CTLA-4, TNFAIP3, DGKA, DGKZ, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A and /

Can independently induce double- and / or single-strand breaks of PD-1, CTLA-4, TNFAIP3, DGKA, DGKZ, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A, and / or TET2

It is also possible to induce the insertion of one exogenous nucleotide at the cleavage site of PD-1, CTLA-4, TNFAIP3, DGKA, DGKZ, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A and / or TET2.

In another embodiment of the present invention, the nucleic acid constituting the guide nucleic acid-editor protein complex is

(a) a guiding domain complementary to a target site sequence in PD-1, CTLA-4, TNFAIP3, DGKA, DGKZ, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A and / or TET2 genes as disclosed herein A sequence encoding the gRNA molecule; And

(b) a sequence encoding an editor protein.

At this time, the above (a) may exist more than two according to the target site, and (b) may use homologous or two or more kinds of editor proteins.

In an embodiment, the nucleic acid is used to reduce, reduce or suppress the expression of PD-1, CTLA-4, TNFAIP3, DGKA, DGKZ, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A, and / (E. G., Transcriptional reporter domain fusion) that is sufficiently close to the immune cell knockdown target site to target an enzyme protein or its fusion protein.

As an example of the present invention, immunological cell-expressing genes such as PD-1, CTLA-4, TNFAIP3, DGKA, DGKZ, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A , And / or manipulation of the TET2 gene may be mediated by any mechanism.

Exemplary mechanisms include, but are not limited to, non-homologous end-binding (NHEJ), microglucose-mediated end joining (MMEJ), homology-related repair (HDR), SDSA (synthetic dependent annealing) But are not limited to, penetration.

FGF, EGR2, PPP2R2D, PSGL-1, and / or PDLA-1, CTLA-4, TNFAIP3, DGKA, DGKZ, Fas, Or to manipulate the TET2 gene.

[By gene scissors Operator ]

In one embodiment of the present invention, the resultant immune system factor obtained using the above-described "guide nucleic acid-editor protein complex" includes, for example, a manipulated gene, a product expressed thereby, , Compositions, transformants, and the like.

As an embodiment,

An immunomodulatory gene or an expression protein thereof artificially engineered by a guide nucleic acid-editor protein complex; And cells comprising these.

An immunomodulatory gene or an expression protein thereof genetically engineered by a guide nucleic acid-editor protein complex; And cells comprising these.

Is a nucleic acid sequence or amino acid sequence of an immunomodulatory gene genetically engineered by a guide nucleic acid-editor protein complex.

An engineered immune regulatory gene, an expression protein thereof, a cell comprising said engineered immunomodulatory element and / or protein, said engineered immunomodulatory element, protein, and / or cell genetically engineered by a guide nucleic acid-editor protein complex .

An engineered immune regulatory gene, an expression protein thereof, a cell comprising said engineered immunomodulatory element and / or protein, said engineered immunomodulatory element, protein, and / or cell genetically engineered by a guide nucleic acid-editor protein complex Lt; RTI ID = 0.0 > and / or < / RTI >

The resulting immune factor obtained using the guide nucleic acid-editor protein complex may include two or more independently of each element, and may include two or more of each element.

E.g,

An artificially engineered immunomodulatory gene, an expression protein thereof; And a cell containing the same, at the same time,

An artificially engineered, one or more immunoregulatory genes can be provided at the same time,

An artificially engineered, one or more immunoregulatory proteins can be provided at the same time,

An artificially manipulated one or two or more kinds of immune cells can be simultaneously provided,

It is possible to simultaneously provide two or more combinations of the artificially manipulated immune elements.

An example of the resultant immune system factor obtained using the preferred "guide nucleic acid-editor protein complex" may have the following structure.

In one embodiment, where the immunomodulatory element is a gene,

The construction of an immunomodulatory gene artificially engineered by the guide nucleic acid-editor protein complex,

1bp to 40bp, 1bp to 30bp (contiguous 1bp to 50bp, 1bp to 3bp) located adjacent to the 5'and / or 3'terminus of the proto-spacer-adjacent Motif (PAM) sequence in the nucleic acid sequence constituting the immunomodulatory gene. , Preferably within the 3bp to 25bp base sequence region

Deletion or insertion of one or more nucleotides;

Substitution with one or more nucleotides that is different from the wild type gene;

Insert one or more nucleotides from outside

Lt; RTI ID = 0.0 > of < / RTI >

It may also comprise a chemical modification of one or more nucleotides in the nucleic acid sequence constituting the immunomodulatory gene.

Herein, the term "exogenous nucleotide" includes not only a nucleotide originally possessed by an immunomodulatory gene but also an exogenous nucleotide, for example, a heterologous biologically-derived or an artificially synthesized nucleotide. Oligonucleotides of a small size of 50 bp or less, as well as large-sized hundreds, thousands, or tens of thousands of bp nucleotides for the expression of proteins having specific functions. This "exogenous nucleotide" can be referred to as a donor.

The chemical modification includes methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation. For example, when a part of the functional group of the nucleotide has a hydrogen atom, a fluorine atom, O-alkyl group, -O-acyl group, and amino group, but is not limited thereto. Further, in order to increase the ability of the nucleic acid molecule to be transferred, it may be substituted with any one of -Br, -Cl, -R, -R'OR, -SH, -SR, -N3 and -CN (R = alkyl, aryl, alkylene) . Also, the phosphate backbone of at least one nucleotide may be substituted with any one of an alkylphosphonate form, a phosphoroamidate form and a boranophosphate form. In addition, the chemical modification may be characterized in that at least one kind of nucleotide contained in the nucleic acid molecule is substituted with any one of LNA (locked nucleic acid), UNA (unlocked nucleic acid), Morpholino, and PNA Wherein the chemical modification is characterized in that the nucleic acid molecule is bound to at least one selected from the group consisting of lipids, cell penetrating peptides and cell targeting ligands

In order to form the desired immune system, the nucleic acid constructing the immunoregulatory gene can be modified by the guide nucleic acid-editor protein complex.

The site containing the modification of the nucleic acid of the immunomodulatory gene, which can form the desired immune system, is referred to as a target sequence or a target site.

Such a "target sequence" may be the target of the guide nucleic acid-editor protein complex, and the target sequence may include, but is not limited to, a protospacer-adjacent motif (PAM) sequence recognized by the editor protein . These target sequences can provide the operator with important criteria for the design of the guide nucleic acid.

Such modifications of the nucleic acid include "cleavage " of the nucleic acid.

"Cleavage" of 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 linkages, and can be accomplished by a variety of other methods. Both single-strand breaks and double-strand breaks are possible, and double-strand breaks can occur as a result of two distinct single-strand breaks. Cleavage of double strands can produce blunt ends or staggered ends.

When an inactivated editor protein is used, it is possible to induce a factor having a specific function to be located close to any part of the target site or immunoregulatory gene, without the above cutting process. And may include chemical modifications of one or more nucleotides in the nucleic acid sequence of an immunomodulatory gene according to this particular function.

In one embodiment, various cleavage of the nucleic acid formed by the guide nucleic acid-editor protein complex can result in various indel (insertion and deletion) by target and non-target activity.

"Indel" refers to a mutation in which some bases are inserted or deleted in the nucleotide sequence of DNA.

Indel, as described above, can be used for the homologous recombination or non-homologous end-joining (NHEJ) mechanism when the guide nucleic acid-editor protein complex cleaves the nucleic acid (DNA, RNA) Or may be introduced into the target sequence in the course of being repaired.

An artificially engineered immunomodulatory gene of the present invention is a gene that has been modified into a nucleic acid sequence of a native gene by cleavage of the nucleic acid and insertion of an inducer or a donor into the immune system, Or suppression or supplementation of the present invention.

E.g,

The expression and activity of a specific protein can be promoted by the artificially engineered immunomodulatory gene.

Specific proteins can be inactivated by the artificially engineered immunomodulatory genes.

For example, immunomodulatory genes such as PD-1, CTLA-4, TNFAIP3, DGKA (Dgkα), DGKAZ (Dgkζ), Fas, EGR2, PPP2R2D, PSGL -1 < / RTI > and / or the TET2 gene to knock down or knock out the gene.

In another example, targeted knockdown may be used to alter transcription, for example, PD-1, CTLA-4, TNFAIP3, DGKA (Dgk?), DGKAZ (Dgk?), Fas, EGR2, PPP2R2D, PSGL- Or by targeting an enzymatically inactive editor protein fused to a transcriptional repressor domain or chromatin modified protein to block, reduce or decrease the transcription of the TET2 gene.

The activity of immune cells can be regulated by the artificially engineered immunomodulatory genes. Proliferation, survival, cytotoxicity, infiltration, and cytokine-release of the immune cells can be controlled.

The above-mentioned artificially manipulated immunomodulatory genes can be used to obtain therapeutic effects such as immunological function, antitumor function, anti-inflammatory function and the like.

Depending on the constitutive characteristics of the guide nucleic acid-editor protein complex, the major PAM sequences involved in the target region of the immunomodulatory gene may be different.

Hereinafter, representative examples of editor proteins and immunomodulatory genes will be mainly described, but these are only specific examples and the present invention is not limited thereto.

For example, when the editor protein is a Cas9 protein derived from Streptococcus pyogenes, the PAM sequence is 5'-NGG-3 '(N is A, T, G, or C) The base sequence region (target region) to be cleaved is consecutive 1 bp to 25 bp, for example 17 bp to 23 bp or 21 bp to 23 bp, located adjacent to the 5 'end and / or the 3' end of the 5'-NGG- May be the base sequence region of < RTI ID = 0.0 >

Within the immunomodulatory gene

a) one or more nucleotides in the sequence of 1 bp to 25 bp, such as 17 bp to 23 bp, located adjacent to the 5 'and / or 3' end of the sequence 'NGG' (where N is A, T, C or G) Deletion of nucleotides

b) substitution with a nucleotide that is different from the wild-type gene of one or more nucleotides in a consecutive 1 bp to 25 bp, for example 17 bp to 23 bp nucleotide sequence located adjacent to the 5 'and / or 3' ends of the 'NGG'

c) insertion of one or more nucleotides into a consecutive 1 bp to 25 bp, for example 17 bp to 23 bp nucleotide sequence located adjacent to the 5 'and / or 3' ends of the 'NGG'

d) two or more combinations selected from a) to c)

Such as an artificially engineered immune regulatory gene such as artificially engineered PD-1 gene, CTLA-4 gene, TNFAIP3 gene, DGKA gene, DGKZ gene, Fas gene, EGR2 gene, PPP2R2D gene, PSGL -1 gene, the KDM6A gene, and the TET2 gene.

For example, if the editor protein is a Cas9 protein from Campylobacter jejuni, the PAM sequence is 5'-NNNNRYAC-3 'wherein N is each independently A, T, C or G and R (Target region) is adjacent to the 5 ' end and / or the 3 ' end of the 5 ' -NNNNRYAC-3 ' sequence in the target gene For example between 17 bp and 23 bp or between 21 bp and 23 bp.

Within the immunomodulatory gene

a ')' NNNNRYAC '(where N is each independently A, T, C or G, R is A or G and Y is C or T) Deletion of one or more nucleotides in the sequence of 1 bp to 25 bp, such as 17 bp to 23 bp,

substitution with a nucleotide that is different from the wild-type gene of one or more nucleotides in the sequence of 1 bp to 25 bp, for example 17 bp to 23 bp, located adjacent to the 5 ' and / or 3 ' ends of the ' NNNNRYAC &

insertion of one or more nucleotides into a consecutive base sequence region of 1 bp to 25 bp, for example 17 bp to 23 bp, located adjacent to the 5 'and / or 3' ends of the c ')' NNNNRYAC '

d ') a combination of two or more selected from the above a') to c '

Such as an artificially engineered immune regulatory gene such as artificially engineered PD-1 gene, CTLA-4 gene, TNFAIP3 gene, DGKA gene, DGKZ gene, Fas gene, EGR2 gene, PPP2R2D gene, PSGL -1 gene, the KDM6A gene, and the TET2 gene.

For example, if the editor protein is a Cas9 protein from Streptococcus thermophilus, the PAM sequence may be 5'-NNAGAAW-3 'wherein N is independently A, T, C or G and W Is either A or T, and the truncated base sequence region (target region) is a consecutive 1 bp to 25 bp region located adjacent to the 5 ' or 3 ' end of the 5 ' -NNAGAAW- 17 bp to 23 bp, or 21 bp to 23 bp.

Within the immunomodulatory gene

a '') 'NNAGAAW' (where N is independently A, T, C or G and W is A or T) contiguous 1 bp to 25 bp, for example 17 bp to 23 bp Deletion of one or more nucleotides in the nucleotide sequence

substitution of a wild type gene of one or more nucleotides in a nucleotide sequence of 1 bp to 25 bp, for example 17 bp to 23 bp, located adjacent to the 5 'and / or 3' ends of the nucleotide sequence, b '') 'NNAGAAW' ,

insertion of one or more nucleotides into a contiguous sequence of 1 bp to 25 bp, e.g. 17 bp to 23 bp, located adjacent to the 5 ' and / or 3 ' ends of the c "

d ") a combination of two or more selected from the above a '') to c '')

Such as an artificially engineered immune regulatory gene such as artificially engineered PD-1 gene, CTLA-4 gene, TNFAIP3 gene, DGKA gene, DGKZ gene, Fas gene, EGR2 gene, PPP2R2D gene, PSGL -1 gene, the KDM6A gene, and the TET2 gene.

For example, if the editor protein is a Cas9 protein from Neisseria meningitidis, the PAM sequence is 5'-NNNNGATT-3 '(where each N is independently A, T, C or G) , The base sequence region (target region) to be cleaved is consecutive 1 bp to 25 bp, for example 17 bp to 23 bp or 21 bp, located adjacent to the 5 'end and / or the 3' end of the 5'-NNNNGATT- Lt; / RTI > to 23 bp.

Within the immunomodulatory gene

a sequence of 1 bp to 25 bp, for example 17 bp to 23 bp, located adjacent to the 5 'and / or 3' ends of the sequence 'a' '' NNNNGATT '(where N is independently A, T, Deletion of one or more nucleotides in the nucleotide sequence

b '' ') of the nucleotide sequence of one or more nucleotides in the sequence of 1 bp to 25 bp, such as 17 bp to 23 bp, located adjacent to the 5' and / or 3 'ends of the sequence' NNNNGATT ' substitution,

insertion of one or more nucleotides into a contiguous 1 bp to 25 bp, such as 17 bp to 23 bp, base sequence region located adjacent to the 5 'and / or 3' ends of the c '' '' NNNNGATT '

d '' ') a combination of two or more selected from the above a' '') to c '' '

CTLA-4 gene, TNFAIP3 gene, DGKA gene, DGKZ gene, Fas gene, EGR2 gene, PPP2R2D gene, PSGL-4 gene, 1 gene, the KDM6A gene, and the TET2 gene.

For example, if the editor protein is a Cas9 protein from Streptococcus aureus, the PAM sequence is 5'-NNGRR (T) -3 ', where N is each independently A, T, C or G (T) -3 'sequence in the target gene, and the nucleotide sequence of the 5'-NNGRR (T) -3' sequence in the target gene Terminus or 3 ' end of the sequence, for example, 17 bp to 23 bp or 21 bp to 23 bp.

Within the immunomodulatory gene

(N is independently A, T, C or G, R is A or "

(Target site) of consecutive 1 bp to 25 bp, for example 17 bp to 23 bp, located adjacent to the 5'and / or 3'terminus of the nucleotide sequence of SEQ ID NO: fruition

b " ') is located adjacent to the 5' and / or 3 'ends of the 5'-NNGRR (T) -3' sequence

Substitution with a nucleotide that is different from the wild-type gene of one or more nucleotides in the base sequence region (target region) of 1 bp to 25 bp, such as 17 bp to 23 bp,

(target site) of consecutive 1 bp to 25 bp, for example 17 bp to 23 bp, located adjacent to the 5 'and / or 3' end of the 5'-NNGRR (T) Insertion of one or more nucleotides into the < RTI ID = 0.0 >

d " ")) < / RTI >

Such as an artificially engineered immune regulatory gene such as artificially engineered PD-1 gene, CTLA-4 gene, TNFAIP3 gene, DGKA gene, DGKZ gene, Fas gene, EGR2 gene, PPP2R2D gene, PSGL -1 gene, the KDM6A gene, and the TET2 gene.

For example, if the editor protein is a Cpf1 protein,

The PAM sequence is 5'-TTN-3 '(N is A, T, C or G) and the truncated base sequence region (target region) For example 15 bp to 26 bp, 17 bp to 30 bp, or 17 bp to 26 bp, contiguous to the 3 'end or 3' end of the nucleotide sequence.

The Cpf1 protein is selected from the group consisting of Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp. For example, Parcubacteria bacterium (GWC2011_GWC2_44_17), Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp (GW2011_GWA_33_10), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus methanoplasma termitum and Eubacterium eligens. . (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum, or Eubacterium eligens But is not limited thereto.

Within the immunomodulatory gene

terminus and / or 3 'end of a' '' '' '5'-TTN-3' (N is A, T, C or G)

(Target region) of 10 bp to 30 bp, for example, 15 bp to 26 bp, located adjacent to the nucleotide sequence

b " " ') 5'-TTN-3' sequences located adjacent to the 5 'and / or 3'

Substitution with a nucleotide that is different from the wild-type gene of one or more nucleotides in the base sequence region (target site) of 10 bp to 30 bp, for example, 15 bp to 26 bp,

c " '") 5'-TTN-3 '

Insertion of one or more nucleotides into a base sequence region (target region) of 10 bp to 30 bp, e.g., 15 bp to 26 bp,

d '' '' '') a '' '' ') to c' '' ''

Such as an artificially engineered immune regulatory gene such as artificially engineered PD-1 gene, CTLA-4 gene, TNFAIP3 gene, DGKA gene, DGKZ gene, Fas gene, EGR2 gene, PPP2R2D gene, PSGL -1 gene, the KDM6A gene, and the TET2 gene.

Other As a concrete example , An immune modulator For proteins ,

The artificially engineered protein includes all proteins that are involved in a new or altered immune response formed by direct or indirect action of a guide nucleic acid-editor protein complex.

For example, it may be a protein expressed by an artificially engineered immunoregulatory gene by a guide nucleic acid-editor protein complex, or other protein that is increased or decreased by the activity of such a protein, but is not limited thereto.

The artificially engineered immunomodulatory protein may have an amino acid composition and activity corresponding to that of the artificially engineered immunomodulatory gene.

(i) provide an artificially engineered protein with altered expression characteristics.

For example, consecutive 1 bp to 50 bp, 1 bp to 40 bp, 1 bp to 30 bp, and 3 bp to 30 bp sequences located adjacent to the 5 'end and / or the 3' end of the sequence of the proto-spacer- adjacent motif (PAM) , Preferably within the 3bp to 25bp base sequence region

A decrease or increase in expression amount due to deletion or insertion of one or more nucleotides;

Reduction or increase in the expression level upon substitution with one or more nucleotides that are different from the wild-type gene; ;

Decreasing or increasing the expression level due to the insertion of one or more nucleotides from the outside, or expression of the fusion protein or independent expression of the specific protein;

A decrease or increase in the expression level of a third protein affected by the expression characteristics of the above-described proteins;

≪ RTI ID = 0.0 > and / or < / RTI >

(ii) an artificially engineered protein with altered structural properties.

For example, consecutive 1 bp to 50 bp, 1 bp to 40 bp, 1 bp to 30 bp, and 3 bp to 30 bp sequences located adjacent to the 5 'end and / or the 3' end of the sequence of the proto-spacer- adjacent motif (PAM) , Preferably within the 3bp to 25bp base sequence region

Alteration of codons, alteration of amino acids, and alteration of three-dimensional structure upon deletion or insertion of one or more nucleotides;

Altering the codons with substitution with one or more nucleotides that are different from the wild-type gene, altering the amino acid, thereby altering the three-dimensional structure;

An independent structure in which one or more nucleotides derived from the outside originate, a codon change, an amino acid change and a three-dimensional structure change, or a fusion structure with a specific protein or a specific protein is separated;

 The codon change, the amino acid modification, and the three-dimensional structure modification of the third protein affected by the protein whose structural characteristics described above are changed;

≪ RTI ID = 0.0 > and / or < / RTI >

(iii) an artificially engineered protein with altered immune function characteristics.

For example, consecutive 1 bp to 50 bp, 1 bp to 40 bp, 1 bp to 30 bp, and 3 bp to 30 bp sequences located adjacent to the 5 'end and / or the 3' end of the sequence of the proto-spacer- adjacent motif (PAM) , Preferably within the 3bp to 25bp base sequence region

Activation or inactivation of a specific immune function or introduction of a new immune function by protein modification resulting from deletion or insertion of one or more nucleotides;

Activation or inactivation of a specific immune function or introduction of a new immune function by protein modification resulting from substitution with one or more nucleotides that are different from the wild type gene;

Introducing a third function into the existing immune function by activation or inactivation of a specific immune function or introduction of a new immune function, in particular by fusion expression or independent expression of a specific protein, by protein modification resulting from the insertion of one or more nucleotides from the outside Yes;

A function change of a third protein influenced by a protein whose immune function characteristic is changed as described above;

≪ RTI ID = 0.0 > and / or < / RTI >

In addition, it may comprise an artificially engineered protein by chemical modification of one or more nucleotides in the nucleic acid sequence constituting the immunomodulatory gene.

For example, one or more of the characteristics of protein expression, structure, and immune function by methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation may be altered.

For example, by chemical modification of the nucleotide, a third protein can be bonded in the nucleic acid sequence of the gene to impart a third structure and function.

In another embodiment, an artificially engineered cell is provided as the resultant immune system factor obtained using the "guide nucleic acid-editor protein complex ".

The artificially engineered cells

An immuno-regulated gene artificially engineered by a guide nucleic acid-editor protein complex; And

A protein involved in a new or altered immune response formed by direct or indirect action of a guide nucleic acid-editor protein complex

≪ / RTI > As an example, it may be an immune cell or a stem cell.

Such cells possess the functions involved in the immune function exhibited by the above-described artificially engineered immunomodulatory genes and / or proteins and the resulting intracellular mechanisms.

In another embodiment, the resultant immune system factor obtained using the "guide nucleic acid-editor protein complex" provides a composition that induces the desired immune response. May be referred to as a pharmaceutical composition or a therapeutic composition.

The composition that causes the desired immune response

An immuno-regulated gene artificially engineered by a guide nucleic acid-editor protein complex;

A protein involved in a new or altered immune response formed by direct or indirect action of a guide nucleic acid-editor protein complex; And

Cells containing said immunomodulatory genes and / or proteins

May be included as an active ingredient.

Such a composition possesses the function of participating in various mechanisms in the body derived from the immune function exhibited by an artificially engineered immunomodulatory gene, protein and / or cell described above.

Such compositions, such as cell therapy agents, may be used for the prevention and / or treatment of immune related diseases such as cancer.

[Manufacturing method]

As one embodiment of the present invention, there is provided an artificially engineered immunomodulatory element and a method for producing an immune cell comprising the same.

An explanation of artificially engineered immune modulating elements can be found in the description set forth above. Hereinafter, the above method will be described as a representative example center of manipulated immune cells.

- Cell culture

To produce manipulated immune cells, cells are first harvested from healthy donors and cultured. For example, immune cells such as T cells, NK cells, and NKT cells are collected from donors using known methods and cultured in a suitable cell culture medium.

As will be described later, some of the immunoregulatory factors expressed by the cultured immune cells are selected and manipulated artificially. For example, PD-1, CTLA-4, TNFAIP3, DGKA (Dgk?), DGKAZ (Dgk?), Fas, EGR2, PPP2R2D, PSGL-1 and / or TET2 are genetically engineered. For a detailed description of genetic manipulation, see above.

Or immune cells are transformed and then cultured to produce manipulated immune cells.

- Production method of functional immune cells

Functional immune cells can be produced by inserting or removing immunoregulatory factor proteins.

Functional immune cells can be produced by modifying genes that are immunomodulators.

Functional immune cells can produce either wild-type receptors or immunoregulatory genes by knock-down (KD) or knock-out (KO). Knockdown or knockout refers to inhibition of gene expression through cleavage of the target gene, transcriptional inhibitor of DNA, and RNA detoxification inhibitor such as complementary microRNA.

Knockdown or knockout can be achieved through microRNA.

Knockdown or knockout is preferably accomplished by the guide nucleic acid-editor protein complex of the present invention.

Knockdown or knockout can be achieved through NHEJ using gene scissors.

Knockdown or knockout can be achieved through gene scissors and HR using template nucleotides.

For example, specific target regions of PD-1, CTLA-4, TNFAIP3, DGKA (Dgkα), DGKAZ (Dgkζ), Fas, EGR2, PPP2R2D, PSGL-1 and / or TET2 genes are cut to knock down or knockout .

Functionally-functioning immune cells may include deformation of the target site,

Insertions or deletions (e. G., NHEJ-mediated insertion or deletion) of one or more nucleotides in the coding region that are very close to the coding region of the gene,

Deletion (e. G., NHEJ-mediated deletion) of the genomic sequence comprising at least a portion of the gene,

Examples of the knockdown or knockout transformation of a gene mediated by an enzymatically inactive editor protein by targeting a non-coding region of the gene, such as a promoter region, are exemplified.

Functionally-functioning immune cells can also be produced by transfection of wild-type receptors or immunoregulatory genes.

Transfection methods include the insertion of an episome containing the desired gene or a method of fusion to the genome.

Transduction can be accomplished by inserting an episome. An episome vector is a vector that acts as an exogenous genomic gene in the nucleus of a eukaryotic organism and is not fused to the genome. At this time, the episome may be a plasmid.

Transduction can be accomplished via HR using guide nucleic acid-editor protein complexes and template nucleotides.

In addition, functional immune cells can be produced by knocking out wild-type receptors or immunoregulatory genes while simultaneously transducing other wild-type receptors or immunoregulatory genes. Transfection methods include the insertion of an episome containing the desired gene or a method of fusion to the genome.

At this time, the transduced gene can be fused to the position of the knockout gene.

Transduction can be accomplished by inserting an episome.

Transduction can be accomplished via HR using guide nucleic acid-editor protein complexes and template nucleotides.

- Artificial structure addition type  How to produce immune cells

Immunoprecipitated cells can be produced by directly adding an artificial structure to the immune cells in the form of a protein.

Immunoreactive cells can be produced by transducing a gene encoding an artificial structure.

Transfection methods include the insertion of an episome containing the desired gene or a method of fusion to the genome.

Transduction can be accomplished by inserting an episome.

Transduction can be accomplished via HR using guide nucleic acid-editor protein complexes and template nucleotides

In an embodiment, there is provided a method of inactivating one or more immunomodulatory genes in an immune cell, comprising introducing (transducing) the guide nucleic acid and the editor protein into the immune cell.

In an embodiment, there is provided a method for producing transformed immune cells comprising introducing (transducing) a guide nucleic acid and an editor protein into an immune cell

- Production method of complex manipulated immune cells

Complex manipulated immune cells can be produced by a method for producing the functionally-functioning immune cells and a method for manipulating the proteins and genes described in the method for producing the artificial structure-added immune cells.

Methods of producing complex engineered immune cells include knocking out or transducing wild-type receptors or immunomodulatory elements. This step may be carried out according to the method described in the production method of functional immune cells.

The method of producing complex manipulated immune cells comprises the step of transducing an artificial structure. This step may be carried out according to the method described in the method for producing the artificial structural adnexal immune cells.

One preferred embodiment of the method of producing complex manipulated immune cells is knocking out the wild receptor of immune cells while simultaneously transducing the artificial structure.

For example, PD-1 and CTLA-4 of the immune cells are knocked out and transduced with an artificial structure.

Another example is to transfect an artificial construct by knocking out TNFAIP3 (A20), DGK-alpha, DGK-zeta, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A and / or TET2 of immune cells.

At this time, the transduced gene can be fused to the same position as the knockout gene.

A known method can be used for producing the above-described engineered immune cells. For example, a recombinant vector can be generally used.

- Immunocellular recombinant expression vector

"Expression target sequence" means a means for modifying a protein or gene of a target cell, or a nucleotide sequence encoding a gene to be newly expressed. In one embodiment of the present invention, the expression target nucleotide sequence may include a sequence encoding a guide nucleic acid and an editor protein, and an additional sequence for expression thereof.

A "recombinant vector" is a transporter that functions to transfer an expression target nucleotide sequence to a target cell, and includes, for example, a plasmid, an episome vector, a viral vector and the like.

By "recombinant expression vector" is meant an artificially constructed vector, which, as one embodiment of the recombinant vector, expresses the function of the expression target sequence linked to the recombinant vector to be expressed in the target cell.

The recombinant expression vector for immunocytochemistry is a recombinant expression vector, which is a recombinant expression vector encoding a protein or gene of an immune cell for expressing the immune cell as a manipulated immune cell, or a gene to be newly expressed.

The immune cell recombinant expression vector includes a recombinant expression vector for expressing the above-described guide nucleic acid-editor protein complex.

In an embodiment,

The engineered immune cells can be obtained by simply transforming the immune cells with a single immunocell recombinant expression vector.

The manipulated immune cells can be obtained by transforming immune cells with two or more immunocell recombinant expression vectors.

Immunocytochemical recombination expression vectors can be designed by dividing into an appropriate number of recombinant expression vectors according to the size of the base sequence to be finally expressed.

(Functional recombinant expression vector)

In one embodiment, there is provided a recombinant expression vector for the production of functionally-functioning immune cells.

In an embodiment, the functionally engineered recombinant expression vector comprises a recombinant base sequence for knocking out a wild receptor or immunoregulatory element gene.

The recombinant expression vector for knocking out the gene includes a recombinant expression vector for expressing the above-described guide nucleic acid-editor protein complex. At this time, the target sequence of the gRNA may have complementarity with the nucleotide sequence of the wild receptor or the immunomodulatory element. In addition, the recombinant expression vector may contain a template nucleotide for insertion at a site truncated by a guide nucleic acid-editor protein complex, if necessary.

In an embodiment, the functionally engineered recombinant expression vector comprises a recombinant base sequence for transducing a wild receptor or immunoregulatory element gene.

At this time, the functional recombinant expression vector may be an episome vector. The episome vector may comprise a promoter for expression of the gene.

In an embodiment, the functionally engineered recombinant expression vector may be one having a function to be fused to the genome of a living body. At this time, the functional recombinant expression vector may be a viral vector. At this time, the preferred viral vector may be an adeno-associated viral vector.

In an embodiment, the functionally engineered recombinant expression vector may comprise a nucleotide sequence that is homologous to the insertion site. It may be a template nucleotide for insertion in the HR process. The template nucleotide may be homologous to the sequence at the site cleaved by the guide nucleic acid-editor protein complex.

In an embodiment, the functionally-engineered recombinant expression vector may comprise the sequence for expressing the guide nucleic acid-editor protein complex as described above, either in the same vector or independently in different vectors.

In another embodiment, the functionally engineered recombinant expression vector comprises a recombinant base sequence for knocking out a wild receptor or immunoregulatory element gene and transducing another wild receptor or immunoregulatory element gene.

The recombinant base sequence for knocking out the gene includes the base sequence of the recombinant expression vector for expressing the guide nucleic acid-editor protein complex described above. At this time, the target sequence of the gRNA may have complementarity with the nucleotide sequence of the immunoregulatory factor.

The recombinant expression vector for transduction may be an episome vector. At this time, the episome vector may contain a promoter for expression of the gene.

The recombinant expression vector for transduction may be one having the function of being fused to the genome of a living body.

The recombinant expression vector for transduction may be a viral vector. At this time, the preferred viral vector is an adeno-associated viral vector.

Recombinant expression vectors for transduction may include nucleotide sequences that are homologous to the insertion site. It may be a template nucleotide for insertion in the HR process. The template nucleotide may be homologous to the sequence at the site cleaved by the guide nucleic acid-editor protein complex.

In addition, the functionally-modified recombinant expression vector may comprise a recombinant expression vector for expressing the above-described guide nucleic acid-editor protein complex.

(Recombinant expression vector of artificial structure addition type)

In one embodiment, there is provided a recombinant expression vector for producing an artificial structural addition type immune cell.

An artificial structural addition type recombinant expression vector comprises a recombinant base sequence for transducing an immunoregulatory factor gene.

In one example, the artificial structural appendectomy recombinant expression vector may be an episome vector. An episome vector is a vector that acts as an exogenous genomic gene in the nucleus of a eukaryotic organism and is not fused to the genome. At this time, the episome vector may contain a promoter for expression of the gene.

In another example, the artificial structural appendectomy recombinant expression vector may have a function to be fused to the genome of a living body.

At this time, the artificial structural addition type recombinant expression vector may be a viral vector. At this time, the preferred viral vector is an adeno-associated viral vector.

In addition, the recombinant expression vector of the artificial structure addition type may contain a nucleotide sequence homologous to the insertion site. It may be a template nucleotide for insertion in the HR process. The template nucleotide may be homologous to the sequence at the site cleaved by the guide nucleic acid-editor protein complex.

In addition, the recombinant expression vector of the artificial structure addition type may contain a recombinant expression vector for expressing the above-described guide nucleic acid-editor protein complex.

(Recombinant recombinant expression vector)

A recombinant recombinant expression vector comprises a recombinant base sequence for knocking out a wild receptor or immunoregulatory gene and transducing another artificial structural gene.

The recombinant base sequence for knocking out the gene includes the base sequence of the recombinant expression vector for expressing the guide nucleic acid-editor protein complex described above. At this time, the target sequence of the gRNA may have complementarity with the nucleotide sequence of the immunoregulatory factor.

In one example, the recombinant expression vector for transduction may be an episome vector. At this time, the episome vector may contain a promoter for expression of the gene.

In another example, the recombinant expression vector for transduction may be one having the function of being fused to the genome of a living body.

At this time, the recombinant expression vector for transduction may be a viral vector. At this time, the preferred viral vector is an adeno-associated viral vector.

In addition, the recombinant expression vector for transduction may include a nucleotide sequence homologous to the insertion site. It may be a template nucleotide for insertion in the HR process. The template nucleotide may be homologous to the sequence at the site cleaved by the guide nucleic acid-editor protein complex.

In addition, the functionally-modified recombinant expression vector may comprise a recombinant expression vector for expressing the above-described guide nucleic acid-editor protein complex.

On the other hand, a specific embodiment of the present invention provides a method for producing an immune cell comprising an artificially engineered immune modulatory element by a guide nucleic acid-editor protein complex.

In an embodiment, the cell is transformed with (a) one or more guide nucleic acids that target the PD-1, CTLA-4, TNFAIP3, DGKA (Dgk?), DGKAZ (Dgk?), Fas, EGR2, PPP2R2D, PSGL-1 and / For example, gRNA, and (b) an edible protein, e. G., A Cas9 protein, to produce a manipulated immune cell in which the sequence of the target nucleic acid of the cell has been altered.

The contacting method may be such that the guide nucleic acid and the editor protein are directly introduced into immune cells by a conventional method.

The contacting method may be to introduce each DNA molecule encoding the guide nucleic acid and the editor protein into the immune cells in a state where they are contained in one vector or a separate vector.

The contact method may be performed using a vector. The vector may be a viral vector. The viral vectors may be, for example, retroviruses, adenovirus vectors.

The methods can be delivered into immune cells using a variety of methods known in the art, such as electroporation, liposomes, viral vectors, nanoparticles, as well as the PTD (Protein Translocation Domain) fusion protein method.

The method may further include introducing a gRNA targeting a different gene into the cell, or introducing a nucleic acid encoding the gRNA into the cell.

The method may be carried out in vivo or ex vivo, for example, outside the human body.

For example, the contacting step may be performed in vitro, and the contacted cells may be returned to the body of the subject after the contacting step.

The method may use immune cells or organisms in vivo, for example, immune cells isolated from human bodies or artificially produced immune cells. As an example, it may include contacting cells from a subject suffering from cancer.

The immune cells used in the above method may be immune cells derived from mammals including rodents such as primates such as humans and monkeys, and rodents such as mice and rats. For example, NKT cells, NK cells, T cells, and the like. At this time, it may be an engineered immune cell to which an immune receptor has been added (for example, CAR (chimeric antigen receptor), or engineered TCR (T-cell receptor) addition). Said immune cell is capable of introducing an immune cell target site mutation in one or more of PD-1, CTLA-4, TNFAIP3, DGKA (Dgk?), DGKAZ (Dgk?), Fas, EGR2, PPP2R2D, PSGL-1 and / Before, after, or at the same time, an immunoreceptor (e. G., TCR or CAR).

IL-10, IL, IL-10, IL-10, IL-10, IL-10, (E.g., Minimal Essential Media, which may contain factors necessary for proliferation and viability, including TGF-beta, TGF-beta, and TNF-alpha or other additives for growth of cells known to those skilled in the art Or RPMI Media 1640 or X-vivo-10, -15, -20, (Lonza).

[Usage]

One embodiment of the invention is the use of an immunotherapeutic approach, including administration of an artificially engineered cell, such as a genetically engineered immune cell or stem cell, to a subject.

The subject to be treated may be a mammal including humans, primates such as monkeys, rodents such as mice and rats, and the like.

Pharmaceutical composition

One embodiment of the present invention is a composition for use in the treatment of diseases using an immune response. For example, it is a composition containing an artificially engineered immunoregulatory gene or immune cells containing the same. A therapeutic composition or a pharmaceutical composition or a cell treatment agent.

In an embodiment, the composition may comprise immune cells.

In an embodiment, the composition may comprise immunomodulating artificially engineered genes and / or proteins expressed thereby.

The immune cells may be immune cells that have already undergone differentiation.

The immune cells may be extracted from bone marrow or umbilical cord blood.

The immune cells may be stem cells. At this time, stem cells may be hematopoietic stem cells.

The composition may comprise manipulated immune cells.

The composition may comprise functionally-functioning immune cells.

The composition may comprise an artificial structural appendicular immune cell.

In other embodiments, the composition may further comprise additional elements.

The composition may comprise an antigen binding agent.

The composition may comprise a cytokine.

The composition may comprise a cytokine secretagogue or an inhibitor.

The composition may comprise suitable carriers for delivery of manipulated immune cells into the body.

The immune cells contained in the composition may be homologous to the patient

Treatment method

Another embodiment of the invention is a method of treating a disease in a patient, comprising the production of the composition described above and an effective amount of the composition in a patient in need thereof.

In one embodiment, adoptive immunotherapy may be used.

- Diseases to be treated

Adoptive immunotherapy may be intended to treat certain diseases.

Certain diseases may be immune diseases. At this time, the immune disease may be a disease in which the immune ability is deteriorated.

Immune diseases may be autoimmune diseases.

For example, autoimmune diseases can be classified as GVHD, Graft versus host disease, systemic lupus erythematosus, celiac disease, diabetes mellitus type 1, Graves' disease graves disease, inflammatory bowel disease, psoriasis, rheumatoid arthritis, muliple sclerosis, and the like.

Immune diseases can be hyperplastic diseases.

For example, blood malignant tumors or solid tumors. Representative hematologic malignancies include but are not limited to acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), chronic eosinophilic leukemia (CEL), myelodysplastic syndrome (MDS), non- Myeloma (MM). Examples of solid tumors are biliary cancer, bladder cancer, bone and soft tissue carcinoma, brain tumor, breast cancer, cervical cancer, colon cancer, colon cancer, colon cancer, desmoid tumor, embryonic cancer, endometrial cancer, Cancer of the head and neck, squamous cell carcinoma of the head and neck, lung cancer, malignant melanoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic duct adenocarcinoma, primary astrocytoma, primary thyroid cancer, prostate cancer, renal cancer, renal cell carcinoma, rhabdomyosarcoma , Skin cancer, soft tissue sarcoma, testicular germ cell tumor, urinary epithelial cell cancer, uterine sarcoma, or uterine cancer.

A wide range of cancers, including solid malignant tumors and hematologic malignancies, can be treated.

For example, the types of cancer that can be treated include breast, prostate, pancreas, colon and rectum adenocarcinoma; All forms of bronchogenic carcinoma of the lungs (including squamous cell carcinoma, adenocarcinoma, small cell lung cancer and non-small cell lung cancer); Myeloma; Melanoma; Liver cancer; Neuroblastoma; Papilloma; Apudoma; Isolated species; Shedding; Malignant carcinoid syndrome; Carcinoid heart disease; (Eg, walker, basal cell, basal ganglia, brown-pierce, catheter, ehrlich tumor, Krebs 2, Merkel cell, mucin, non-small cell lung, copper cell, papillary, Placental cells, and transitional cells). Additional types that can be cured

Cancer of the tissue organs; leukemia; Increased malignant tissue; Hodgkin's disease; Non-Hodgkin's lymphoma; Plasma cell, reticuloendothelioma; Melanoma; Renal cell carcinoma; Chondroblastoma; Chondromatosis; Chondrosarcoma; fibroma; Fibrosarcoma; Giant cell tumor; Tissue type; Lipoma, liposarcoma; Mesothelioma; Myxoma; Mucosal sarcoma; Osteoma; Osteosarcoma; Cholesteatoma, craniopharyngioma; Undifferentiated cell tumor; Hamartoma; Mesenchymal species; Middle ear; Muscular carcinoma; Enamel epithelium; Cementum species; Tooth; Teratoma; Thymoma; Includes trophoblastic tumors.

In addition, the following types of cancer may also be considered treatable: adenoma; Cholangiocarcinoma; Chinju; Kwon Ju - Jong; Cystic cancer; Cystadenoma; Granulosa cell tumor; Mastocytosis; Liver cancer; Han, Sun - Jong; Islet cell tumor; Leydig cell tumors; Papilloma; Sertoli cell tumor; Oocyte cell tumor; Uterine myoma; Uterine sarcoma; Myoblastoma; Myoma; Muscular carcinoma; Rhabdomyoma; Rhabdomyosarcoma; Cell tumor on the stomach; Ganglion cell tumor; Glioma; Hydroblastoma; Meningioma; Neurons; Neuroblastoma; Neuroepithelioma; Neurofibroma; Neuroma; Adrenal hyperplasia; Paranasal sinus non-chromaffin and polymorphic subspecies. Types of cancer that can be cured are also angina angioma; Vascular lymphatic proliferation with eosinophilia; Vascular sclerosis; Hemangioma; Dacryocystitis; Endothelial; Hemangioma; Perivascular cell tumor; Angiosarcoma; Lymphangioma; Lymphoma myoma; Lymphatic sarcoma; Pineal body; Carcinosarcoma; Chondrosarcoma; Leptoplasmic carcinoma; Fibrosarcoma; Angiosarcoma; Leiomyosarcoma; White blood cell species; Liposuction; Lymphatic sarcoma; Muscular carcinoma; Mucosal sarcoma; Ovarian carcinoma; Rhabdomyosarcoma; sarcoma; Neoplasm; Neurofibromatosis and cervical dysplasia.

In addition, any particular disease may be a refractory disease for which a pathogen has been known but whose treatment is unknown.

Refractory diseases can be viral infectious diseases.

A refractory disease can be a disease caused by a prion pathogen.

Any particular disease may be a bacterial disease.

Any particular disease may be an inflammatory disease.

Any particular disease may be an aging-related disorder.

- Immune enhancement treatment

For patients with significantly decreased immunity, mild infections can also have catastrophic consequences. Decreased immune function is caused by decreased function of immune cells and decreased production of immune cells. There is a permanent treatment method for activating the production of normal immune cells, while a temporary treatment method for transiently injecting immune cells may be used for the immunity-enhancing treatment for treating the decrease in the immune capacity.

The immunological capacity enhancement treatment may be to inject the therapeutic composition into the body of the patient to intensify the immune function permanently.

The immunological capacity enhancement treatment may be a method of injecting the therapeutic composition into a specific body part of the patient. At this time, a specific body part may be a part having an immune cell source tissue.

Immune enhancement therapy may be to create a new source of immune cells in the body of a patient. As an example, the therapeutic composition may include stem cells. At this time, stem cells may be hematopoietic stem cells.

The immunological capacity enhancement treatment may be to inject the therapeutic composition into the body of the patient to temporarily enhance the immune function.

The immune-enhancing treatment may be injecting the therapeutic composition into the patient's body.

At this time, the preferred therapeutic composition may comprise the differentiated immune cells.

Therapeutic compositions used in the immunological capacity-building treatment may include a certain number of immune cells.

The specific number may vary depending on the degree to which the immune ability has deteriorated.

The specific number may vary depending on the volume of the body.

The specific number can be adjusted according to the amount of cytokine secreted by the patient.

- Treatment of intractable disease

Immune cell manipulation techniques can provide a treatment for diseases for which complete treatment for HIV, prions, and cancer pathogens is not known. These diseases are difficult to treat because of the difficulty of antibody formation, the rapid progression, the inactivating immune system, and the latent nature of the pathogen in the body even though the pathogen is known. Manipulating immune cells can be a powerful means to solve these problems.

Treatment of intractable disease can be accomplished by injecting the therapeutic composition into the body. At this time, the preferred therapeutic compositions may comprise engineered immune cells. In addition, the therapeutic composition can be injected into a particular body location.

The manipulated immune cells may be those with improved recognition of the pathogen of the intended disease.

The manipulated immune cell may be one in which the intensity or activity of the immune response is enhanced.

- Gene therapy

In addition to treatment with exogenously extracted immune cells, there may be a therapeutic method that directly affects the expression of immune cells by manipulating a gene of a living body. Such a therapeutic method can be achieved by directly injecting a composition for gene correction for manipulating a gene of a living body into the body.

The composition for gene correction may comprise a guide nucleic acid-editor protein complex.

The composition for gene correction may be injected at a specific body location.

Certain body sites can be sources of immune cells. For example, it is a bone marrow.

One embodiment of the invention is directed to a method of treating an immune-related disorder by administering to a subject an effective amount of a composition comprising the components of an artificially engineered immune system as described above.

In certain embodiments, the therapeutic methods provide for the use of cell populations manipulated or modified recombinantly in vitro, e.g., via viral vectors. In a further embodiment, the modified cell population is a winter, allogeneic, or autologous cell. In any of the above-mentioned embodiments, the manipulated or modified cell population may be further formulated with a pharmaceutically acceptable carrier, diluent, or excipient as described herein.

The subject to be administered may be a mammal including humans, primates such as monkeys, rodents such as mice and rats, and the like.

Administration refers to delivering it to the subject regardless of the delivery route or mode. The administration can be carried out continuously or intermittently and parenterally.

In certain embodiments, co-administration with an adjunct therapeutic agent may involve simultaneous and / or sequential delivery of multiple agents in any order and any dosage regimen (e.g., antigen-specific recombinant host T cells and antigen expression Immunosuppressive therapies such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low-dose mycophenolic acid prodrugs, or any combination thereof).

In certain embodiments, the administration step can be repeated several times up to several weeks, several months, or up to two years.

The compositions may be administered in a manner appropriate to the disease or condition being treated or prevented, as determined by those skilled in the medical arts. Suitable dosages and the appropriate duration and frequency for administration of the composition will depend upon factors such as the condition of the patient, the size of the patient (i.e., weight, mass, body area), the type and severity of the patient's disease, the particular form of the active ingredient, Lt; / RTI >

For example, administration of the composition can be carried out in any convenient manner, such as injection, transfusion, implantation or transplantation. The route of administration may be subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenous, intralymphatic, intraperitoneal, intraperitoneal, intraperitoneally, and the like.

A single dose of the composition (a pharmaceutically effective amount for achieving a desired effect) may be in the range of 10 ^{4 to} 10 ⁹ cells per kg body weight of the subject, for example, about 10 ⁵ to 10 ⁶ cells / kg (body weight) But not limited to, the age, health and weight of the subject to be treated, the type of treatment received at the same time, the frequency of treatment, if any, and the characteristics of the desired effect .

When an artificially engineered immune modulator is controlled by the methods and compositions of the present invention, the survival, proliferation, persistency, cytotoxicity, cytokine-release ) And / or infiltration may be improved.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples.

It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments.

Example 1: Cell activation & culture and transfection

Jurkat cells (ATCC TIB-152; immortalized cell line of human T-cells) were cultured in RPMI 1640 medium supplemented with 10% (v / v) fetal bovine serum (GeneAll). Cells were then cultured in an incubator under 37% CO ₂ condition and 5.

Human Naive T-cells (STEMCELL Technology) were incubated with 10% (v / v) fetal bovine serum (GeneAll) and / or IL-2 (50 U / mL), IL- 5 ng / mL) (PEPROTECH) supplemented with X-VIVO 15 medium (Lonza). For cell activation, the concentration of cells in the medium was 1x10 6 cells / mL, respectively.

(Bead: cells; number of beads and cells) at a ratio of 3: 1, and the cells were washed with 37 and 5% CO 2 (anti- CD2 / CD3 / CD28 beads ₂ < / RTI > conditions in an incubator. After the cell activation was performed for 72 hours, the CD2 / CD3 / CD28 beads were removed using a magnet, and the cells were further cultured for 12-24 hours in the absence of beads.

In order to find a gRNA capable of knocking out a specific gene at a high efficiency, 1 x 10 ^ 6 Jurkat cells were cultured in the above-mentioned 1x10 ^ 6 cells. To the cells were added 1 g microgram of in vitro transcribed sgRNA as described in Examples 2 and 3 below, And Cas9 protein (Tulgen, Korea) were introduced by electroporation (in vitro).

A 10 uL tip of Neon Transfection System (ThermoFisher Scientific, Grand Island, NY) was used to introduce the gene under the following conditions:

Jurkats (Buffer R): 1,400 V, 20 ms, 2 pulses.

Similarly, 1 ug gRNA and 4 ug Cas9 protein (Tulgen, Korea) were introduced into 1x10 ^ 6 human primary T cells by electroporation to knock out specific genes in T cells. The gRNA used in this study is in vitro transcribed and AP (alkaline phosphatase) treated sgRNA or chemically synthesized crRNA and tracrRNA complex (Integrated DNA Technologies). For electroporation, a 10 uL tip of Neon Transfection System (ThermoFisher Scientific, Grand Island, NY) was used to introduce the gene under the following conditions:

Human primary T-cells (Buffer T): 1,550 V, 10 ms, 3 pulses;

The cells were plated in 500 ul of non-antibiotic medium and cultured in an incubator at 37 ° C and 5% CO ₂ .

Example 2: sgRNA design and synthesis

2.1: sgRNA design

The human PD-1 gene (PDCD1; NCBI Accession No. NM_005018.2), the CTLA-4 gene (NCBI Accession No. NM_001037631.2), and the human PD- DGK-DGK-alpha gene (NCBI Accession No. NM_001345.4), DGK-zeta gene (NCBI Accession No. NM_001105540.1), EGR2EGR2EGR2 gene (NCBI Accession No. NM_001270507.1) (NCBI Accession No. NM_001291310.1), PSGL-1 gene (NCBI Accession No. NM_003006.4), and TET2TET2TET2 gene (NCBI Accession No. NM_017628.4), FAS gene (NCBI Accession Nos. NM_001291415.1, NM_001291416.1, NM_001291418.1, NM_001291417.1, NM_001291421.1, or NM_021140. 3) CRISPR / Cas9 target site selection and estimation off-target assays were performed.

DNA sequences without 0-, 1-, or 2bp mismatch sites were selected as target regions of the sgRNA, except for the target sequence regions in the human genome (GRCh38 / hg38) as CRISPR / Cas9 target regions.

2.2: sgRNA synthesis

Templates for sgRNA synthesis were PCR-amplified by annealing and extending two complementary oligonucleotides.

The target site sequence used at this time, the primer sequence for amplifying the primer sequence, and the DNA target sequence (including PAM) targeted by the sgRNA obtained therefrom are summarized in Table 2 below.

In vitro transcription was performed using T7 RNA polymerase (New England Biolabs) for the template DNA (except for 'NGG' at the 3 'end of the target sequence), and RNA was synthesized according to the manufacturer's instructions, ) Was used to remove the template DNA. The RNA was purified through Expin Combo kit (GeneAll) and isopropanol precipitation.

In order to minimize the immunogenicity and degradation of sgRNA in T cell experiments, 5 'end phosphate residues were removed from alkaline phosphatase (New England Biolabs) to sgRNA synthesized by the above method, and the Expin Combo kit (GeneAll) and isopropanol The RNA was purified again by precipitation. In addition, chemically synthesized sgRNA (Trilink) or chemically synthesized dgRNA (Integrated DNA Technologies) was used in some T cell experiments.

In this example, the chemically synthesized sgRNA was modified with 2'OMe and phosphorothioate.

The chemically modified example used in this embodiment as DGKα sgRNA # 11 is 5'-2 'OMe (C ( ps) U (ps) C (ps)) UCA AGC UGA GUG GGU CC U UU UAG AGC UAG AAA UAG CAA GUU AAA AUA AGG CUA GUC CGU UAU CAA CUU GAA AAA GUG GCA CCG AGU CGG UGC 2'OMe (U (ps) U (ps) U (2'OMe = 2'-methly RNA and ps = phosphorothioate).

A20 sgRNA # 1 used in this embodiment in the other examples, G CUUGUGGCGCUGAAAACGAA GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU (bold is Im sequence that hybridizes to the target sequence region; sgRNA is that the bold sequence target sequence (note that for the other target gene and the other target sequence, T Or the 3 nucleotides at the 3 ' end of the sequence and the 3 nucleotides at the 5 ' end have been subjected to a modification introduced with a 2'-OMe modification and a phosphorothioate backbone).

[Table 2]

2.3 Deep sequencing

On-target and off-target sites were PCR-amplified to 200-300 bp size using Hipi Plus DNA polymerase (Elpis-bio). The PCR product obtained by the above method was designated Mi-seq. (Illumina) equipment and analyzed by Cas Analyzer of CRISPR RGEN tool (www.rgenome.net). Insertions / deletions within 5 bp of the CRISPR / Cas9 cleavage site were considered to be mutations derived from RGEN.

As shown in Table 4 and Table 6, when the CRISPR-Cas9 was delivered as a result of deep sequencing, it was confirmed that the indel mutation occurred at high efficiency in various immune cells.

Example 3 Preparation of sgRNAs

3.1. Screening of sgRNAs in Jurkat cells

The activity of sgRNAs targeting the exons of A20, DGKα, EGR2, PPP2R2D, EGR2, PPP2r2dPPP2R2D, PD-1, CTLA-4, DGKζ, PSGL-1, KDM6A, FAS and TET2TET2TET2 obtained by the method described in Example 2 Jurkat cells.

Each of the sgRNAs obtained in Example 2 above was tested by comparing the indel ratio in Jurkat cells transfected with Cas9 by the method of Example 1 to that of Jurkat cells without transduction. Table 3 shows the number of mismatch sites having the similar target sequences in the CRISPR / Cas9 target sequence and the human genome. The indel ratio of each sgRNA is shown in Table 4. Among the gRNAs that target each gene, the DNA target region of those with good activity is displayed in bold.

[Table 3]

[Table 4] Activity of each sgRNA in the Jurkat cell relative to the target sequence

3.2. Human primary T cells ( human primary  T- cells ) In sgRNAs  Selection

Based on the results of sgRNA activity in the Jurkat cells obtained in Example 3.1 above, sgRNAs with relatively high activity in Jurkat cells (see bold in Table 3 and Table 4) were selected to show activity in human primary T-cells .

Single or dual gRNA and Cas9 were transferred to human primary T cells. The CRISPR / Cas9 target sequences tested are shown in Table 5, and the indel ratios by sgRNA are summarized in Table 6, respectively.

[Table 5] Target sequences and mismatches in human primary T-cells

[Table 6] Activity of each gRNA on the target sequence in human primary T immune cells

Based on the results of sgRNA activity in Jurkat cells obtained in Example 3.1 above, PSGL-1 # 17 sgRNA having a relatively high activity in Jurkat cells was selected to test its activity in human primary T-cells

In addition, activated human primary T cells were transfected with 4 ug of Sp [beta] through electroporation (Neon, Thermo Scientific). Cas9 protein and 1 ug of in vitro transcribed AP-treated sgRNA. Five days later, gDNA was isolated from each T cell and extracted, and the indel efficiency was analyzed by targeted deepd sequencing (FIG. 18A). In addition, PSGL-1 expression on T cell surface was analyzed by flow cytometry (Attune Flow cytometry, Thermo Scientific) to confirm PSGL-1 knockout (Fig. 18B, C).

FIGS. 17a to 17c show the results of analysis for hPSGL-1 sgRNA screening in Jurkat cells. The results are shown in FIG. 17. The results are shown in FIG. 17a and FIG. 17b. (17b, 17c) showing the degree of expression of PSGL-1.

FIG. 18 shows the results of hPSGL-1 knockout (KO) experiments in human primary T cells showing (A) the indel efficiency and (B) the degree of T cell not expressing PSGL-1 after knockout And (C) the degree of expression of PSGL-1 expressed on the T cell surface after knockout. As a result, it was confirmed that PSGL-1 was effectively knocked out through Cas9 protein and gRNA complex delivery, and PSGL-1, which is a surface protein, could not be observed by flow cytometry.

Example 4: Activation of Jurkat cells and promotion of cytokine secretion test

In the Jurkat cells into which the Cas9 protein and the sgRNA are introduced, the genomic DNA sequence corresponding to the target region of the introduced sgRNA is cleaved, and the nucleotide sequence of the cleaved DNA sequence is deleted due to deletion, insertion and substitution of NHEJ Is knocked out.

Jurkat cells transfected with Cas9 protein and sgRNA by electroporation as described in Example 1 were cultured for 7 days after electroporation and activated using CD3 dynabeads (Miltenyi Biotec) or CD3 / 28 dynabeads (Miltenyi Biotec) .

After 24 hours, the expression of IL-2 receptor CD25 and the level of IFN-gamma released were analyzed by flow cytometry and ELISA, respectively.

First, the expression level of CD25, an IL-2 receptor, was measured by flow cytometry. Jurkat cells transfected with Cas9 protein and sgRNA were cultured for 7 days after each introduction and re-stimulated using CD3 or CD3 / 28 dynabeads (Miltenyi Biotec) at a ratio of 3: 1 (bead: cells; Expression of CD25 was measured.

Phenotypic analysis was performed at 1 day after cell activation. The bead-re-stimulated (activated) cells were washed with PBS (phosphate-buffered saline) supplemented with 1% (v / v) fetal bovine serum (FBS) At < RTI ID = 0.0 > 4 C < / RTI >

The obtained cells were washed and resuspended in PBS, followed by flow cytometry on BD ACCURI C6 (BD Biosciences) and the level of CD25 expression was measured by median fluorescence intensity (MFI) obtained from this.

For comparison, flow cytometry was performed in the same manner on wild-type cells in which Cas9 protein and sgRNA were not introduced, and cells not treated with CD3 or CD3 / 28 dynabeads.

The obtained CD25 expression level (CD25 MFI) is shown in FIG. 1 to FIG.

Figure 1 shows the expression of CD25 MFI in DGK-alpha knockout cells using sgRNA (# 11; denoted DGK-alpha # 11) for DGK-alpha,

Figure 2 shows the expression of CD25 MFI in cells knocked out of the A20 gene using sgRNA (# 11; denoted as A20 # 11) against A20,

Figure 3 shows CD25 MFI in cells where the EGR2 gene was knocked out using sgRNA (# 1; denoted EGR2 # 1) for EGR2, and

Figure 4 shows CD25 MFI in cells knocked out of the PPP2R2D gene using sgRNA (# 10; designated PPP2R2D # 10) for PPP2R2D, respectively.

As shown in FIGS. 1 to 4, in the case of cells not treated with CD3 or CD3 / 28 dynabeads, the presence or absence of knockout of the genes did not affect CD25 expression level, whereas CD3 or CD3 / 28 dynabeads , It was confirmed that the expression level of CD25 was markedly increased when the genes were knocked out as compared with the wild type.

In addition, secretion levels of IFN-gamma, a kind of cytokine, were tested by ELISA.

After re-stimulated Jurkat cells were activated for 36 h using CD3 or CD3 / 28 dynabeads as described previously, the culture medium was collected and diluted to 1/100 or 1 (w / v) using a diluent buffer (provided by ELISA kit, Biolegend) / 200 ratio, color developed using an ELISA kit (BioLegend), and quantified using a spectrophotometer (MULTISCAN GO, Thermo Scientific).

For comparison, ELISA was performed in the same manner on wild type cells into which Cas9 protein and sgRNA were not introduced.

The results obtained are shown in Fig.

Figure 5 shows the IFN-gamma level in the cell culture medium in which the DGK-alpha gene was knocked out, sgRNA (# 11; A20 (Denoted as EGR2 # 1) for EGR2, IFN-gamma level in the cell culture medium in which the A20 gene was knocked out, and the expression level of the EGR2 gene in the cell culture medium knocked out IFN-gamma level (IFN-gamma level units: pg / ml).

As shown in FIG. 5, when the genes were knocked out, the secretion amount of IFN-gamma was significantly increased as compared with the wild type.

Example 5: Activation of human primary T-cells and cytokine secretion enhancement test

With reference to the method described in Example 4 above, human primary T-cells transfected with Cas9 protein and sgRNA were activated with CD3 beads (bead: cell ratio of 1: 1, 2: 1, and 3: 1, respectively After 2 days, secretion levels of IFN-gamma and IL-2 were measured by ELISA (IFN-gamma or IL-2 ELISA kit; Biolegend).

The obtained results are shown in Fig. 6 and Fig.

Figure 6 shows the IFN-gamma level in the cell culture medium in which the DGK-alpha gene was knocked out, the sgRNA (# 8) for DGK-alpha using the sgRNA (# 11; denoted as DGK-alpha # IFN-gamma level in the cell culture medium in which the DGK-alpha gene was knocked out, sgRNA (# 5; DGK-zeta #) for DGK-zeta, 5), the IFN-gamma level in the cell culture medium in which the DGK-zeta gene was knocked out, and the cell culture in which the A20 gene was knocked out using sgRNA (indicated by # 11; A20 # 11) The IFN-gamma level in the medium is shown (IFN-gamma level units: pg / ml).

FIG. 7 is a graph showing the effect of DGK-alpha gene knockout on IL-2 levels in DGK-alpha knockout cell culture medium using DGKalpha # 11 and DGK-alpha # 8 + 2 level in the cell culture medium in which the DGK-zeta gene was knocked out using DGK-zeta # 5 and the level of IL-2 in the cell culture medium in which the A20 gene was knocked out using A20 # 11 (IL-2 level units: pg / ml).

In Figures 6 and 7, "AAVSl" was used as a negative control for cells where the AAVSl site was cleaved into the CRISPR system.

As shown in FIGS. 6 and 7, when the genes were knocked out, the secretion amount of cytokines such as IFN-gamma and IL-2 was significantly increased as compared with the wild type.

These results, which showed an increase in CD25 expression and cytokine secretion in Jurkat and human primary T cells, indicate that the TCR mediated activation signal was increased when the genes were knocked out. Can be strengthened.

Example 6: CAR-T cell activation and cytokine secretion enhancement test

Human peripheral blood T cells (pan-T cells) were purchased from STEMCELL TECHNOLOGIES. For cell culture, X-VIVO 15 medium supplemented with 50 U / mL of hIL-2 and 5 ng / mL of hIL-7 was used. Anti-CD3 / 28 Dynabeads (ThermoFisher Scientific) was used to activate the cells, with a ratio of beads to cells of 3: 1.

After 24 hours of activation, T cells were mixed with 139-CAR lentivirus for 48 hours on retronectin coated plates. 139-CAR is a CAR capable of specifically recognizing EGFRvIII and inducing an immune response. Then, 10 g of tracr / crRNA (Integrated DNA Technologies) chemically synthesized with 40 g of recombinant S. pyogenes Cas9 protein (Tulgen, Korea) was introduced into the cells by electroporation of 4D-Nucleofecter (Lonza).

For in vitro experiments, U87vIII cancer cells pre-stained with Cell Trace (ThermoFisher Scientific) were co-cultured with 139 CAR-T in appropriate proportions, with 10 ng / mL TGF-β1 or 0.5 μg / mL PGE2 Was added or not added. After co-culturing with cancer cell lines, cells were stained with 7-aminoactinomycin D (7-AAD) for cytotoxicity experiments. Dyed samples were collected on an Attune NxT Acoustic Focusing Cytometer and analyzed with FlowJo.

The cytotoxicity was calculated by the formula [(% lysis sample -% lysis minimum) / (% lysis max [100%] -% lysis minimum]? 100%. 139-CAR-T cells stained with CellTrace were co-cultured with the target cancer cell line U87vIII for 139 cell-proliferation experiments of CAR-T cells, and 139 CAR- The dilution of Cell Trace in T cells was measured by flow cytometry.

According to the experimental design (FIG. 8A), the Indel effect of DGKα and DGKζ on 139 CAR-T cells delivered with a single Cas9 / gRNA ribonucleoprotein (RNP) complex targeting DGKα or DGKζ was 75.9% and 93.5% (Fig. 8A, B).

Two gRNAs targeting DGKα and DGKζ, respectively, were introduced into cells by electroporation to produce dual-negative 139 CAR-T cells of DGKα and DGKζ. As a result, the Inder effects of DGKα and DGKζ were 49.2%, 92.4 % (Fig. 8A, B).

No significant effects of off-target on the respective gRNA of DGKa and DGK were confirmed using the targeted deep-sequencing (Fig. 8b).

In addition, it was observed that DGKa, DGKζ, and DGKαζ KO 139 CAR-T cells significantly increased cytotoxicity, cytokine production and proliferative capacity compared to wild type 139 CAR-T cells (FIGS. 9A, ).

Interestingly, DGKαζ KO 139 CAR-T cells showed significantly increased cytokine release compared to DGKα or DGKζ single KO 139 CAR-T cells, which is thought to be a synergistic effect of DGKα and DGKζ. It is considered that the effector function increase of such DGKs KO 139 CAR-T cells is attributed to the increase of the CD3-terminal signal, namely ERK1 / 2 and high expression of CAR after antigen exposure (Fig. 10A, B).

In addition, despite the strongly activated signal in DGKs KO 139 CAR-T cells, no increase in basal cytokines was observed in the absence of target cancer cells, suggesting high safety of DGKs KO (FIG. 11A). In addition, the expression of PD-11 and TIM-33, which are exhaustion markers, were not increased in DGKs KO 139 CAR-T cells compared with 139 CAR-T cells, (Fig. 11B).

The anti-cancer effect of 139 CAR-T cells was markedly impaired by treatment with signaling 1 immunosuppressive inhibitors such as TGF-β1 and PGE2, whereas in the case of DGKαζ KO 139 CAR-T cells, cytotoxicity And cytokine release was maintained (Fig. 12A, B).

These results indicate that T cell function can be activated by removing DGK using CRISPR / Cas9.

In other words, it was confirmed that the removal of DGK enhanced the CD3 terminal signal, thereby enhancing the anticancer function and the proliferation of CAR-T cells.

In addition, knockout (KO) CAR-T cells of DGKαζ (two isoforms) did not show a significant increase in exhaustion markers and were less responsive to immunosuppressive solubility factors such as TGF-β and prostaglandin E2 (PGE2).

Thus, it was confirmed that DGK KO by CRISPR / Cas9 can enhance the increased effector function of T cells.

Example 7: NK (Natural Killer) cell activation and cytokine secretion enhancement test

7.1 NK 92 cell line and human primary NK (human primary NK) cell culture

NK92 cell lines were purchased from ATCC (CRL-2407), Primary NK cells were purchased from STEMCELL TECHNOLOGY and cultured according to the protocol provided.

NK92 cells were cultured in RPMI 1640 supplemented with 10% FCS supplemented with 100 μg / ml streptomycin, 100 U / ml penicillin, 2 mM UltraGlutamine I, 200-300 U / ml IL-2 and 10 U / ml IL- and cultured in RPMI 1640 (WellGene) medium containing fetal calf serum.

7.2 Introduction by electroporation

In order to knock out DGKα and DGKζ in NK92 cell line, we used the Neon electroporator (Thermo Fisher Scientific) to perform the ceiling drilling at 1200V, 10ms, and 3 pulse. For primary NK cells, 1200 V, 20 ms, and 3 pulses were used.

4 μg of recombinant S. pyogenes Cas9 protein (Toolgen, Korea) and 1 μg of chemically synthesized tracr / crRNA (Integrated DNA Technologies) were incubated for 20 minutes to obtain a Cas9 RNP complex.

2 × 10 ^ 5 resuspended in R buffer NK92 cells were electroporated by adding (contacting) to a pre-incubated Cas9 RNP complex. After that, the cells were cultured in the medium at 4 x 10 < cells / mL.

The crRNA targeting sequences used in the experiments were as follows:

DGKα: CTCTCAAGCTGAGTGGGTCC

DGKζ: ACGAGCACTCACCAGCATCC.

7.3 In vitro killing assays

To analyze the cytotoxicity of NK92 cells and primary NK cells, cells were co-cultured with CellTrace Far Red (Invitrogen) stained Raji cells or 1x10 5 K562 on U-bottom 96 plates. Cells were harvested 18 hours after co-culture and stained with 7-AAD and analyzed by flow cytometry. All cytotoxicity experiments were performed 3 times.

The results are shown in Fig. (FIG. 13A and B). (B) Killing of NK-92 through measurement of 7-AAD-positive Raji cells (FIG. killing activity, the cytotoxicity was increased by DGKα knockout.

In particular, these results confirm that the immune function can be effectively manipulated against NK cells, which are known to be difficult to genetically manipulate.

Example 8: NKT (Natural Killer) cell activation and cytokine secretion enhancement test

8.1 NKT cell culture

Human PBMC were purchased from STEMCELL TECHNOLOGY (Canada). These cells were plated at a concentration of 1 × 10 6 cells / ml in 10% FBS supplemented RPMI medium supplemented with 1000 U / ml interferon-γ (Pepro Tech). 50 ng / ml anti-human OKT-3 (Biolegend) was added to the culture medium for 5 days and 400 U / ml IL-2 (Pepro Tech) for 20 days.

8.2 Introduction by electroporation

In order to knock out DGKα, DGKζ and PD1 from NKT cell line, we used a neon electroporator (Thermo Fisher Scientific) to perform the ceiling drilling at 1550 V, 10 ms, and 3 pulse conditions.

4 μg of recombinant S. pyogenes Cas9 protein (Tulgen, Korea) and 1 μg of chemically synthesized tracr / crRNA (Integrated DNA Technologies) were incubated for 20 minutes to obtain a Cas9 RNP complex.

2 × 10 ^ 5 resuspended in R buffer NKT cells were electroporated by adding (contacting) to a pre-incubated Cas9 RNP complex. After that, the cells were cultured in the medium at 4 x 10 < cells / mL.

The crRNA targeting sequences used in the experiments were as follows:

DGKα: CTCTCAAGCTGAGTGGGTCC

DGKζ: ACGAGCACTCACCAGCATCC.

PD-11: GTCTGGGCGGTGCTACAACTGGG

7.3 In vitro killing assays

To analyze the cytotoxicity of NKT cells, 2x10 ^ 44 U87vIII cells stained with CellTrace Far Red (Invitrogen) and NKT cells were co-cultured in U-bottom 96-well plates. Cells were harvested 18 hours after co-culture and stained with 7-AAD and analyzed by flow cytometry. All cytotoxicity experiments were performed 3 times.

As a result, it was confirmed that knockout of DGK? And DGK? Efficiently occurred in CRISPR / Cas9 system in human NKT cells as shown in Fig.

Indel efficiency was confirmed by deep sequencing (Fig. 14A), and CRISPR / Cas9 treated NKT cells were analyzed by trypan blue staining to confirm cell growth (Fig. 14B) and viability maintained (Viability = Viable cell number / Total cell number). The knockout of DGKα and DGKζ occurred well, and Western blotting was performed to confirm the expression at the protein level (FIG. 14D)

Further, as shown in Fig. 15, it was confirmed that the knockout of DGK? And DGK? Improved the effector function of the NAT cell.

U87vIII, H460 and K562 cells were treated with a cell trace (Thermo fisher) and cultured for 18 hours at a ratio of E: T (effector cell: target cell ratio) of 20: 1 in a 96-well plate. Analysis of cancer cell death by flow cytometry showed that knockout of DGKα and DGKζ increased NKT killing activity of the corresponding NKT cells. The knockout of each of DGKα and DGKζ also had an effect of increasing the killing activity, but it was confirmed that the killing activity was further improved when the two genes were knocked out simultaneously (FIG. 15A).

On the other hand, IFN-secretion was confirmed by ELISA (IFN-kit, Biolegend), and knockout of DGKα and DGKζ increased the IFN-releasing ability of the corresponding cells. The knockout of DGKα and DGKζ also had the effect of enhancing IFN-secretion, but it was confirmed that IFN-secretion was further enhanced when both genes were knocked out simultaneously (FIG. 15B).

On the other hand, as shown in FIG. 16, it was confirmed that the knockout of CRISPR / Cas9 mediated PD-1 in human NKT cells enhanced the effector function of NKT cells. PD-1 knockout was induced by using CRISPR-Cas9 in PD NKT cells, and knockout efficiency of PD-1 was analyzed by targeted deep sequencing. U87vIII cells and NKT cells were co-cultured to confirm the function of NKT cells as anti-cancer effectors through PD-1 knockout. U87vIII cells were treated with Cell Trace (Thermo fisher) for 96 hours at a ratio of E: T = 50: 1 for 18 hours, and 7-AAD positive cancer cells were analyzed by flowcytometry for killing activity.

As a result, it was confirmed that the high indel efficiency in the PD-1 gene by CRISPR / Cas9 was confirmed (Fig. 16A), thereby improving cytotoxicity (Fig. 16B)

Overall, the above results show that knockout of CRISPR / Cas9-mediated immunomodulatory genes, such as DGK, can have a significant immune-enhancing effect in various types of immune cells.

These biological effects of DGK knockout show that immune cells including T cells, NK cells and NKT can be developed as immunotherapeutic agents in the form of clinically applicable cells.

An effective immune cell therapeutic can be obtained by an artificially functioning immune system comprising an artificially engineered immunomodulatory component and cells comprising it.

For example, when an artificially engineered immune modulator is modulated by the methods and compositions of the present invention, the survival, proliferation, persistency, cytotoxicity, cytokine secretion cytokine-release and / or infiltration may be improved.

<110> ToolGen Incorporation <120> Engineered-immune regulatory factor and Immunity Activation          thereby <130> OPP17-041 <150> KR 10-2016-0103308 <151> 2016-08-12 <150> US 62 / 502,822 <151> 2017-05-08 <160> 289 <170> KoPatentin 3.0 <210> 1 <211> 23 <212> DNA <213> Homo Sapiens <400> 1 cttgtggcgc tgaaaacgaa cgg 23 <210> 2 <211> 23 <212> DNA <213> Homo Sapiens <400> 2 atgccacttc tcagtacatg tgg 23 <210> 3 <211> 23 <212> DNA <213> Homo Sapiens <400> 3 gccacttctc agtacatgtg ggg 23 <210> 4 <211> 23 <212> DNA <213> Homo Sapiens <400> 4 gccccacatg tactgagaag tgg 23 <210> 5 <211> 23 <212> DNA <213> Homo Sapiens <400> 5 tcagtacatg tggggcgttc agg 23 <210> 6 <211> 23 <212> DNA <213> Homo Sapiens <400> 6 gggcgttcag gacacagact tgg 23 <210> 7 <211> 23 <212> DNA <213> Homo Sapiens <400> 7 cacagacttg gtactgagga agg 23 <210> 8 <211> 23 <212> DNA <213> Homo Sapiens <400> 8 ggcgctgttc agcacgctca agg 23 <210> 9 <211> 23 <212> DNA <213> Homo Sapiens <400> 9 cacgcaactt taaattccgc tgg 23 <210> 10 <211> 23 <212> DNA <213> Homo Sapiens <400> 10 cggggctttg ctatgatact cgg 23 <210> 11 <211> 23 <212> DNA <213> Homo Sapiens <400> 11 ggcttccaca gacacaccca tgg 23 <210> 12 <211> 23 <212> DNA <213> Homo Sapiens <400> 12 tgaagtccac ttcgggccat ggg 23 <210> 13 <211> 23 <212> DNA <213> Homo sapiens <400> 13 ctgtacgaca cggacagaaa tgg 23 <210> 14 <211> 23 <212> DNA <213> Homo Sapiens <400> 14 tgtacgacac ggacagaaat ggg 23 <210> 15 <211> 23 <212> DNA <213> Homo Sapiens <400> 15 cacggacaga aatgggatcc tgg 23 <210> 16 <211> 23 <212> DNA <213> Homo Sapiens <400> 16 gatgcgagtg gctgaatacc tgg 23 <210> 17 <211> 23 <212> DNA <213> Homo Sapiens <400> 17 gagtggctga atacctggat tgg 23 <210> 18 <211> 23 <212> DNA <213> Homo Sapiens <400> 18 agtggctgaa tacctggatt ggg 23 <210> 19 <211> 23 <212> DNA <213> Homo Sapiens <400> 19 attgggatgt gtctgagctg agg 23 <210> 20 <211> 23 <212> DNA <213> Homo Sapiens <400> 20 atgaaagaga ttgactatga tgg 23 <210> 21 <211> 23 <212> DNA <213> Homo Sapiens <400> 21 ctctgtctct caagctgagt ggg 23 <210> 22 <211> 23 <212> DNA <213> Homo Sapiens <400> 22 tctctcaagc tgagtgggtc cgg 23 <210> 23 <211> 23 <212> DNA <213> Homo Sapiens <400> 23 ctctcaagct gagtgggtcc ggg 23 <210> 24 <211> 23 <212> DNA <213> Homo Sapiens <400> 24 caagctgagt gggtccgggc tgg 23 <210> 25 <211> 23 <212> DNA <213> Homo Sapiens <400> 25 ttgacatgac tggagagaag agg 23 <210> 26 <211> 23 <212> DNA <213> Homo Sapiens <400> 26 gactggagag aagaggtcgt tgg 23 <210> 27 <211> 23 <212> DNA <213> Homo Sapiens <400> 27 gagacgggag caaagctgct ggg 23 <210> 28 <211> 23 <212> DNA <213> Homo Sapiens <400> 28 agagacggga gcaaagctgc tgg 23 <210> 29 <211> 23 <212> DNA <213> Homo Sapiens <400> 29 tggtttctag gtgcagagac ggg 23 <210> 30 <211> 23 <212> DNA <213> Homo Sapiens <400> 30 taagtgaagg tctggtttct agg 23 <210> 31 <211> 23 <212> DNA <213> Homo Sapiens <400> 31 tgcccatgta agtgaaggtc tgg 23 <210> 32 <211> 23 <212> DNA <213> Homo Sapiens <400> 32 gaacttgccc atgtaagtga agg 23 <210> 33 <211> 23 <212> DNA <213> Homo Sapiens <400> 33 tccattgacc ctcagtaccc tgg 23 <210> 34 <211> 23 <212> DNA <213> Homo Sapiens <400> 34 tatgccttct gggtagcagc tgg 23 <210> 35 <211> 23 <212> DNA <213> Homo Sapiens <400> 35 tgagtgcagg catcttgcaa ggg 23 <210> 36 <211> 23 <212> DNA <213> Homo Sapiens <400> 36 gagtgcaggc atcttgcaag ggg 23 <210> 37 <211> 23 <212> DNA <213> Homo Sapiens <400> 37 gatgaggctg tggttgaagc tgg 23 <210> 38 <211> 23 <212> DNA <213> Homo Sapiens <400> 38 ccactggcca caggacccct ggg 23 <210> 39 <211> 23 <212> DNA <213> Homo Sapiens <400> 39 gggacatggt gcacacaccc agg 23 <210> 40 <211> 23 <212> DNA <213> Homo Sapiens <400> 40 gagtacaggt ggtccaggtc agg 23 <210> 41 <211> 23 <212> DNA <213> Homo Sapiens <400> 41 gcggagagta caggtggtcc agg 23 <210> 42 <211> 23 <212> DNA <213> Homo Sapiens <400> 42 gcggtggcgg agagtacagg tgg 23 <210> 43 <211> 23 <212> DNA <213> Homo Sapiens <400> 43 tctcctgcac agccagaata agg 23 <210> 44 <211> 23 <212> DNA <213> Homo Sapiens <400> 44 acgcagaagg gtcctggtag agg 23 <210> 45 <211> 23 <212> DNA <213> Homo Sapiens <400> 45 aggtggtggg taggccagag agg 23 <210> 46 <211> 23 <212> DNA <213> Homo Sapiens <400> 46 cccaagccag ccacggaccc agg 23 <210> 47 <211> 23 <212> DNA <213> Homo Sapiens <400> 47 acctgggtcc gtggctggct tgg 23 <210> 48 <211> 23 <212> DNA <213> Homo Sapiens <400> 48 aagagacctg ggtccgtggc tgg 23 <210> 49 <211> 23 <212> DNA <213> Homo Sapiens <400> 49 ggatcattgg gaagagacct ggg 23 <210> 50 <211> 23 <212> DNA <213> Homo Sapiens <400> 50 gggatcattg ggaagagacc tgg 23 <210> 51 <211> 23 <212> DNA <213> Homo Sapiens <400> 51 caggatagtc tgggatcatt ggg 23 <210> 52 <211> 23 <212> DNA <213> Homo Sapiens <400> 52 ggaaagaatc caggatagtc tgg 23 <210> 53 <211> 23 <212> DNA <213> Homo Sapiens <400> 53 cagtgccaga gagacctaca tgg 23 <210> 54 <211> 23 <212> DNA <213> Homo Sapiens <400> 54 ctgtaccatg taggtctctc tgg 23 <210> 55 <211> 23 <212> DNA <213> Homo Sapiens <400> 55 agagacctac atggtacagc tgg 23 <210> 56 <211> 23 <212> DNA <213> Homo Sapiens <400> 56 ctgggccagc tgtaccatgt agg 23 <210> 57 <211> 23 <212> DNA <213> Homo Sapiens <400> 57 agggaaaggg cttacggtct ggg 23 <210> 58 <211> 23 <212> DNA <213> Homo Sapiens <400> 58 cagggaaagg gcttacggtc tgg 23 <210> 59 <211> 23 <212> DNA <213> Homo Sapiens <400> 59 tctggagatc ttcttgcaac agg 23 <210> 60 <211> 23 <212> DNA <213> Homo Sapiens <400> 60 ctccggttca tgactttgaa agg 23 <210> 61 <211> 23 <212> DNA <213> Homo Sapiens <400> 61 gtcttccatc ttcgtctttc agg 23 <210> 62 <211> 23 <212> DNA <213> Homo Sapiens <400> 62 gaagacttcg agacccattt agg 23 <210> 63 <211> 23 <212> DNA <213> Homo Sapiens <400> 63 tcgagaccca tttaggatca cgg 23 <210> 64 <211> 23 <212> DNA <213> Homo Sapiens <400> 64 gtagcgccgt gatcctaaat ggg 23 <210> 65 <211> 23 <212> DNA <213> Homo Sapiens <400> 65 cgtagcgccg tgatcctaaa tgg 23 <210> 66 <211> 23 <212> DNA <213> Homo Sapiens <400> 66 catttaggat cacggcgcta cgg 23 <210> 67 <211> 23 <212> DNA <213> Homo Sapiens <400> 67 ggtcccaata ttgaagccca tgg 23 <210> 68 <211> 23 <212> DNA <213> Homo Sapiens <400> 68 gatccatggg cttcaatatt ggg 23 <210> 69 <211> 23 <212> DNA <213> Homo Sapiens <400> 69 agatccatgg gcttcaatat tgg 23 <210> 70 <211> 23 <212> DNA <213> Homo Sapiens <400> 70 gcttctacca taagatccat ggg 23 <210> 71 <211> 23 <212> DNA <213> Homo Sapiens <400> 71 cgcttctacc ataagatcca tgg 23 <210> 72 <211> 23 <212> DNA <213> Homo Sapiens <400> 72 gcatttgcaa aaattcgccg tgg 23 <210> 73 <211> 23 <212> DNA <213> Homo Sapiens <400> 73 atgacctgag aattaattta tgg 23 <210> 74 <211> 23 <212> DNA <213> Homo Sapiens <400> 74 ccatgcactc ccagacatcg tgg 23 <210> 75 <211> 23 <212> DNA <213> Homo Sapiens <400> 75 gcactggtgc gggtggaact cgg 23 <210> 76 <211> 23 <212> DNA <213> Homo Sapiens <400> 76 acacgttgca ctggtgcggg tgg 23 <210> 77 <211> 23 <212> DNA <213> Homo Sapiens <400> 77 cgaacacgtt gcactggtgc ggg 23 <210> 78 <211> 23 <212> DNA <213> Homo Sapiens <400> 78 acgaacacgt tgcactggtg cgg 23 <210> 79 <211> 23 <212> DNA <213> Homo Sapiens <400> 79 tgtagacgaa cacgttgcac tgg 23 <210> 80 <211> 23 <212> DNA <213> Homo Sapiens <400> 80 gcgcatgtca cacaggcgga tgg 23 <210> 81 <211> 23 <212> DNA <213> Homo Sapiens <400> 81 aggagcgcat gtcacacagg cgg 23 <210> 82 <211> 23 <212> DNA <213> Homo Sapiens <400> 82 ccgaggagcg catgtcacac agg 23 <210> 83 <211> 23 <212> DNA <213> Homo Sapiens <400> 83 cctgtgtgac atgcgctcct cgg 23 <210> 84 <211> 23 <212> DNA <213> Homo Sapiens <400> 84 cgactggcca gggcgcctgt ggg 23 <210> 85 <211> 23 <212> DNA <213> Homo Sapiens <400> 85 accgcccaga cgactggcca ggg 23 <210> 86 <211> 23 <212> DNA <213> Homo Sapiens <400> 86 caccgcccag acgactggcc agg 23 <210> 87 <211> 23 <212> DNA <213> Homo Sapiens <400> 87 gtctgggcgg tgctacaact ggg 23 <210> 88 <211> 23 <212> DNA <213> Homo Sapiens <400> 88 ctacaactgg gctggcggcc agg 23 <210> 89 <211> 23 <212> DNA <213> Homo Sapiens <400> 89 cacctaccta agaaccatcc tgg 23 <210> 90 <211> 23 <212> DNA <213> Homo Sapiens <400> 90 cggtcaccac gagcagggct ggg 23 <210> 91 <211> 23 <212> DNA <213> Homo Sapiens <400> 91 gccctgctcg tggtgaccga agg 23 <210> 92 <211> 23 <212> DNA <213> Homo Sapiens <400> 92 cggagagctt cgtgctaaac tgg 23 <210> 93 <211> 23 <212> DNA <213> Homo Sapiens <400> 93 cagcttgtcc gtctggttgc tgg 23 <210> 94 <211> 23 <212> DNA <213> Homo Sapiens <400> 94 aggcggccag cttgtccgtc tgg 23 <210> 95 <211> 23 <212> DNA <213> Homo Sapiens <400> 95 ccgggctggc tgcggtcctc ggg 23 <210> 96 <211> 23 <212> DNA <213> Homo Sapiens <400> 96 cgttgggcag ttgtgtgaca cgg 23 <210> 97 <211> 23 <212> DNA <213> Homo Sapiens <400> 97 cataaagcca tggcttgcct tgg 23 <210> 98 <211> 23 <212> DNA <213> Homo Sapiens <400> 98 ccttggattt cagcggcaca agg 23 <210> 99 <211> 23 <212> DNA <213> Homo Sapiens <400> 99 ccttgtgccg ctgaaatcca agg 23 <210> 100 <211> 23 <212> DNA <213> Homo Sapiens <400> 100 cactcacctt tgcagaagac agg 23 <210> 101 <211> 23 <212> DNA <213> Homo Sapiens <400> 101 ttccatgcta gcaatgcacg tgg 23 <210> 102 <211> 23 <212> DNA <213> Homo Sapiens <400> 102 ggccacgtgc attgctagca tgg 23 <210> 103 <211> 23 <212> DNA <213> Homo Sapiens <400> 103 ggcccagcct gctgtggtac tgg 23 <210> 104 <211> 23 <212> DNA <213> Homo Sapiens <400> 104 aggtccgggt gacagtgctt cgg 23 <210> 105 <211> 23 <212> DNA <213> Homo Sapiens <400> 105 ccgggtgaca gtgcttcggc agg 23 <210> 106 <211> 23 <212> DNA <213> Homo Sapiens <400> 106 ctgtgcggca acctacatga tgg 23 <210> 107 <211> 23 <212> DNA <213> Homo Sapiens <400> 107 caactcattc cccatcatgt agg 23 <210> 108 <211> 23 <212> DNA <213> Homo Sapiens <400> 108 ctagatgatt ccatctgcac ggg 23 <210> 109 <211> 23 <212> DNA <213> Homo Sapiens <400> 109 ggctaggagt cagcgacata tgg 23 <210> 110 <211> 23 <212> DNA <213> Homo Sapiens <400> 110 gctaggagtc agcgacatat ggg 23 <210> 111 <211> 23 <212> DNA <213> Homo Sapiens <400> 111 ctaggagtca gcgacatatg ggg 23 <210> 112 <211> 23 <212> DNA <213> Homo Sapiens <400> 112 gtactgtgta gccaggatgc tgg 23 <210> 113 <211> 23 <212> DNA <213> Homo Sapiens <400> 113 acgagcactc accagcatcc tgg 23 <210> 114 <211> 23 <212> DNA <213> Homo Sapiens <400> 114 aggctccagg aatgtccgcg agg 23 <210> 115 <211> 23 <212> DNA <213> Homo Sapiens <400> 115 acttacctcg cggacattcc tgg 23 <210> 116 <211> 23 <212> DNA <213> Homo Sapiens <400> 116 caccctgggc acttacctcg cgg 23 <210> 117 <211> 23 <212> DNA <213> Homo Sapiens <400> 117 gtgccgtaca aaggttggct ggg 23 <210> 118 <211> 23 <212> DNA <213> Homo Sapiens <400> 118 ggtgccgtac aaaggttggc tgg 23 <210> 119 <211> 23 <212> DNA <213> Homo Sapiens <400> 119 ctctcctcag taccacagca agg 23 <210> 120 <211> 23 <212> DNA <213> Homo Sapiens <400> 120 cctggggcct ccgggcgcgg agg 23 <210> 121 <211> 23 <212> DNA <213> Homo Sapiens <400> 121 agtactcacc tggggcctcc ggg 23 <210> 122 <211> 23 <212> DNA <213> Homo Sapiens <400> 122 agggtctcca gcggccctcc tgg 23 <210> 123 <211> 23 <212> DNA <213> Homo Sapiens <400> 123 gcaagtactt acgcctcctt ggg 23 <210> 124 <211> 23 <212> DNA <213> Homo Sapiens <400> 124 ttgcggtaca tctccagcct ggg 23 <210> 125 <211> 23 <212> DNA <213> Homo Sapiens <400> 125 tttgcggtac atctccagcc tgg 23 <210> 126 <211> 23 <212> DNA <213> Homo Sapiens <400> 126 gcaaaacctg tccactctta tgg 23 <210> 127 <211> 23 <212> DNA <213> Homo Sapiens <400> 127 ttggtgccat aagagtggac agg 23 <210> 128 <211> 23 <212> DNA <213> Homo Sapiens <400> 128 ggtgcaagtt tcttatatgt tgg 23 <210> 129 <211> 23 <212> DNA <213> Homo Sapiens <400> 129 acctgatgca tataataatc agg 23 <210> 130 <211> 23 <212> DNA <213> Homo Sapiens <400> 130 acctgattat tatatgcatc agg 23 <210> 131 <211> 23 <212> DNA <213> Homo Sapiens <400> 131 cagagcacca gagtgccgtc tgg 23 <210> 132 <211> 23 <212> DNA <213> Homo Sapiens <400> 132 agagcaccag agtgccgtct ggg 23 <210> 133 <211> 23 <212> DNA <213> Homo Sapiens <400> 133 agagtgccgt ctgggtctga agg 23 <210> 134 <211> 23 <212> DNA <213> Homo Sapiens <400> 134 aggaaggccg tccattctca ggg 23 <210> 135 <211> 23 <212> DNA <213> Homo Sapiens <400> 135 ggatagaacc aaccatgttg agg 23 <210> 136 <211> 23 <212> DNA <213> Homo Sapiens <400> 136 tctgttgccc tcaacatggt tgg 23 <210> 137 <211> 23 <212> DNA <213> Homo Sapiens <400> 137 ttagtctgtt gccctcaaca tgg 23 <210> 138 <211> 23 <212> DNA <213> Homo Sapiens <400> 138 gtctggcaaa tgggaggtga tgg 23 <210> 139 <211> 23 <212> DNA <213> Homo Sapiens <400> 139 cagaggttct gtctggcaaa tgg 23 <210> 140 <211> 23 <212> DNA <213> Homo Sapiens <400> 140 ttgtagccag aggttctgtc tgg 23 <210> 141 <211> 23 <212> DNA <213> Homo Sapiens <400> 141 acttctggat gagctctctc agg 23 <210> 142 <211> 23 <212> DNA <213> Homo Sapiens <400> 142 agagctcatc cagaagtaaa tgg 23 <210> 143 <211> 23 <212> DNA <213> Homo Sapiens <400> 143 ttggtgtctc catttacttc tgg 23 <210> 144 <211> 23 <212> DNA <213> Homo Sapiens <400> 144 ttctggcttc ccttcataca ggg 23 <210> 145 <211> 23 <212> DNA <213> Homo Sapiens <400> 145 caggactcac acgactattc tgg 23 <210> 146 <211> 23 <212> DNA <213> Homo Sapiens <400> 146 ctactttctt gtgtaaagtc agg 23 <210> 147 <211> 23 <212> DNA <213> Homo Sapiens <400> 147 gactttacac aagaaagtag agg 23 <210> 148 <211> 23 <212> DNA <213> Homo Sapiens <400> 148 gtctttctcc attagccttt tgg 23 <210> 149 <211> 23 <212> DNA <213> Homo Sapiens <400> 149 aatggagaaa gacgtaactt cgg 23 <210> 150 <211> 23 <212> DNA <213> Homo Sapiens <400> 150 atggagaaag acgtaacttc ggg 23 <210> 151 <211> 23 <212> DNA <213> Homo Sapiens <400> 151 tggagaaaga cgtaacttcg ggg 23 <210> 152 <211> 23 <212> DNA <213> Homo Sapiens <400> 152 tttggttgac tgctttcacc tgg 23 <210> 153 <211> 23 <212> DNA <213> Homo Sapiens <400> 153 tcactcaaat cggagacatt tgg 23 <210> 154 <211> 23 <212> DNA <213> Homo Sapiens <400> 154 atctgaagct ctggattttc agg 23 <210> 155 <211> 23 <212> DNA <213> Homo Sapiens <400> 155 gcttcagatt ctgaatgagc agg 23 <210> 156 <211> 23 <212> DNA <213> Homo Sapiens <400> 156 cagattctga atgagcagga ggg 23 <210> 157 <211> 23 <212> DNA <213> Homo Sapiens <400> 157 aaggcagtgc taatgcctaa tgg 23 <210> 158 <211> 23 <212> DNA <213> Homo Sapiens <400> 158 gcagaaactg tagcaccatt agg 23 <210> 159 <211> 23 <212> DNA <213> Homo Sapiens <400> 159 accgcaatgg aaacacaatc tgg 23 <210> 160 <211> 23 <212> DNA <213> Homo Sapiens <400> 160 tgtggttttc tgcaccgcaa tgg 23 <210> 161 <211> 23 <212> DNA <213> Homo Sapiens <400> 161 cataaatgcc attaacagtc agg 23 <210> 162 <211> 23 <212> DNA <213> Homo Sapiens <400> 162 attagtagcc tgactgttaa tgg 23 <210> 163 <211> 23 <212> DNA <213> Homo Sapiens <400> 163 cgatgggtga gtgatctcac agg 23 <210> 164 <211> 23 <212> DNA <213> Homo Sapiens <400> 164 actcacccat cgcatacctc agg 23 <210> 165 <211> 23 <212> DNA <213> Homo Sapiens <400> 165 ctcacccatc gcatacctca ggg 23 <210> 166 <211> 23 <212> DNA <213> Homo Sapiens <400> 166 agcaacagga ggagttgcag agg 23 <210> 167 <211> 23 <212> DNA <213> Homo Sapiens <400> 167 ccagtaggat cagcaacagg agg 23 <210> 168 <211> 23 <212> DNA <213> Homo Sapiens <400> 168 ctcctgttgc tgatcctact ggg 23 <210> 169 <211> 23 <212> DNA <213> Homo Sapiens <400> 169 ggcccagtag gatcagcaac agg 23 <210> 170 <211> 23 <212> DNA <213> Homo Sapiens <400> 170 ttgctgatcc tactgggccc tgg 23 <210> 171 <211> 23 <212> DNA <213> Homo Sapiens <400> 171 tggcaacagc ttgcagctgt ggg 23 <210> 172 <211> 23 <212> DNA <213> Homo Sapiens <400> 172 cttgggtccc ctgcttgccc ggg 23 <210> 173 <211> 23 <212> DNA <213> Homo Sapiens <400> 173 gtcccctgct tgcccgggac cgg 23 <210> 174 <211> 23 <212> DNA <213> Homo Sapiens <400> 174 ctccggtccc gggcaagcag ggg 23 <210> 175 <211> 23 <212> DNA <213> Homo Sapiens <400> 175 tctccggtcc cgggcaagca ggg 23 <210> 176 <211> 23 <212> DNA <213> Homo Sapiens <400> 176 gtctccggtc ccgggcaagc agg 23 <210> 177 <211> 23 <212> DNA <213> Homo Sapiens <400> 177 gcttgcccgg gaccggagac agg 23 <210> 178 <211> 23 <212> DNA <213> Homo Sapiens <400> 178 ggtggcctgt ctccggtccc ggg 23 <210> 179 <211> 23 <212> DNA <213> Homo Sapiens <400> 179 cggtggcctg tctccggtcc cgg 23 <210> 180 <211> 23 <212> DNA <213> Homo Sapiens <400> 180 catattcggt ggcctgtctc cgg 23 <210> 181 <211> 23 <212> DNA <213> Homo Sapiens <400> 181 atctaggtac tcatattcgg tgg 23 <210> 182 <211> 23 <212> DNA <213> Homo Sapiens <400> 182 ataatctagg tactcatatt cgg 23 <210> 183 <211> 23 <212> DNA <213> Homo Sapiens <400> 183 ttatgatttc ctgccagaaa cgg 23 <210> 184 <211> 23 <212> DNA <213> Homo Sapiens <400> 184 atttctggag gctccgtttc tgg 23 <210> 185 <211> 23 <212> DNA <213> Homo Sapiens <400> 185 actgacacca ctcctctgac tgg 23 <210> 186 <211> 23 <212> DNA <213> Homo Sapiens <400> 186 ctgacaccac tcctctgact ggg 23 <210> 187 <211> 23 <212> DNA <213> Homo Sapiens <400> 187 accactcctc tgactgggcc tgg 23 <210> 188 <211> 23 <212> DNA <213> Homo Sapiens <400> 188 aacccctgag tctaccactg tgg 23 <210> 189 <211> 23 <212> DNA <213> Homo Sapiens <400> 189 ctccacagtg gtagactcag ggg 23 <210> 190 <211> 23 <212> DNA <213> Homo Sapiens <400> 190 gctccacagt ggtagactca ggg 23 <210> 191 <211> 23 <212> DNA <213> Homo Sapiens <400> 191 ggctccacag tggtagactc agg 23 <210> 192 <211> 23 <212> DNA <213> Homo Sapiens <400> 192 cctgctgcaa ggcgttctac tgg 23 <210> 193 <211> 23 <212> DNA <213> Homo Sapiens <400> 193 ccagtagaac gccttgcagc agg 23 <210> 194 <211> 23 <212> DNA <213> Homo Sapiens <400> 194 cgttctactg gcctggatgc agg 23 <210> 195 <211> 23 <212> DNA <213> Homo Sapiens <400> 195 tctactggcc tggatgcagg agg 23 <210> 196 <211> 23 <212> DNA <213> Homo Sapiens <400> 196 ccacggagct ggccaacatg ggg 23 <210> 197 <211> 23 <212> DNA <213> Homo Sapiens <400> 197 cgtggacagg ttccccatgt tgg 23 <210> 198 <211> 23 <212> DNA <213> Homo Sapiens <400> 198 gtccacggat tcagcagcta tgg 23 <210> 199 <211> 23 <212> DNA <213> Homo Sapiens <400> 199 gaccactcaa ccagtgccca cgg 23 <210> 200 <211> 23 <212> DNA <213> Homo Sapiens <400> 200 ggagtggtct gtgcctccgt ggg 23 <210> 201 <211> 23 <212> DNA <213> Homo Sapiens <400> 201 ggcacagaca actcgactga cgg 23 <210> 202 <211> 23 <212> DNA <213> Homo Sapiens <400> 202 gacaactcga ctgacggcca cgg 23 <210> 203 <211> 23 <212> DNA <213> Homo Sapiens <400> 203 aactcgactg acggccacgg agg 23 <210> 204 <211> 23 <212> DNA <213> Homo Sapiens <400> 204 cacagaaccc agtgccacag agg 23 <210> 205 <211> 23 <212> DNA <213> Homo Sapiens <400> 205 ggtagtaggt tccatggaca ggg 23 <210> 206 <211> 23 <212> DNA <213> Homo Sapiens <400> 206 tggtagtagg ttccatggac agg 23 <210> 207 <211> 23 <212> DNA <213> Homo Sapiens <400> 207 tcttttggta gtaggttcca tgg 23 <210> 208 <211> 23 <212> DNA <213> Homo Sapiens <400> 208 atggaaccta ctaccaaaag agg 23 <210> 209 <211> 23 <212> DNA <213> Homo Sapiens <400> 209 aacagacctc ttttggtagt agg 23 <210> 210 <211> 23 <212> DNA <213> Homo Sapiens <400> 210 gggtatgaac agacctcttt tgg 23 <210> 211 <211> 23 <212> DNA <213> Homo Sapiens <400> 211 tgtgtcctct gttactcaca agg 23 <210> 212 <211> 23 <212> DNA <213> Homo Sapiens <400> 212 gtgtcctctg ttactcacaa ggg 23 <210> 213 <211> 23 <212> DNA <213> Homo Sapiens <400> 213 gtagttgacg gacaaattgc tgg 23 <210> 214 <211> 23 <212> DNA <213> Homo Sapiens <400> 214 tttgtccgtc aactacccag tgg 23 <210> 215 <211> 23 <212> DNA <213> Homo Sapiens <400> 215 ttgtccgtca actacccagt ggg 23 <210> 216 <211> 23 <212> DNA <213> Homo Sapiens <400> 216 tgtccgtcaa ctacccagtg ggg 23 <210> 217 <211> 23 <212> DNA <213> Homo Sapiens <400> 217 gtccgtcaac tacccagtgg ggg 23 <210> 218 <211> 23 <212> DNA <213> Homo Sapiens <400> 218 ctctgtgaag cagtgcctgc tgg 23 <210> 219 <211> 23 <212> DNA <213> Homo Sapiens <400> 219 cctgctggcc atcctaatct tgg 23 <210> 220 <211> 23 <212> DNA <213> Homo Sapiens <400> 220 ccaagattag gatggccagc agg 23 <210> 221 <211> 23 <212> DNA <213> Homo Sapiens <400> 221 ggccatccta atcttggcgc tgg 23 <210> 222 <211> 23 <212> DNA <213> Homo Sapiens <400> 222 caccagcgcc aagattagga tgg 23 <210> 223 <211> 23 <212> DNA <213> Homo Sapiens <400> 223 agtgcacacg aagaagatag tgg 23 <210> 224 <211> 23 <212> DNA <213> Homo Sapiens <400> 224 tatcttcttc gtgtgcactg tgg 23 <210> 225 <211> 23 <212> DNA <213> Homo Sapiens <400> 225 cttcgtgtgc actgtggtgc tgg 23 <210> 226 <211> 23 <212> DNA <213> Homo Sapiens <400> 226 ggcggtccgc ctctcccgca agg 23 <210> 227 <211> 23 <212> DNA <213> Homo Sapiens <400> 227 gcggtccgcc tctcccgcaa ggg 23 <210> 228 <211> 23 <212> DNA <213> Homo Sapiens <400> 228 aattacgcac ggggtacatg tgg 23 <210> 229 <211> 23 <212> DNA <213> Homo Sapiens <400> 229 tgggggagta attacgcacg ggg 23 <210> 230 <211> 23 <212> DNA <213> Homo Sapiens <400> 230 gtgggggagt aattacgcac ggg 23 <210> 231 <211> 23 <212> DNA <213> Homo Sapiens <400> 231 ggtgggggag taattacgca cgg 23 <210> 232 <211> 23 <212> DNA <213> Homo Sapiens <400> 232 taattactcc cccaccgaga tgg 23 <210> 233 <211> 23 <212> DNA <213> Homo Sapiens <400> 233 agatgcagac catctcggtg ggg 23 <210> 234 <211> 23 <212> DNA <213> Homo Sapiens <400> 234 gagatgcaga ccatctcggt ggg 23 <210> 235 <211> 23 <212> DNA <213> Homo Sapiens <400> 235 tgagatgcag accatctcgg tgg 23 <210> 236 <211> 23 <212> DNA <213> Homo Sapiens <400> 236 ggatgagatg cagaccatct cgg 23 <210> 237 <211> 23 <212> DNA <213> Homo Sapiens <400> 237 atctcatccc tgttgcctga tgg 23 <210> 238 <211> 23 <212> DNA <213> Homo Sapiens <400> 238 tcatccctgt tgcctgatgg ggg 23 <210> 239 <211> 23 <212> DNA <213> Homo Sapiens <400> 239 ctcaccccca tcaggcaaca ggg 23 <210> 240 <211> 23 <212> DNA <213> Homo Sapiens <400> 240 gagggcccct cacccccatc agg 23 <210> 241 <211> 23 <212> DNA <213> Homo Sapiens <400> 241 gggccctctg ccacagccaa tgg 23 <210> 242 <211> 23 <212> DNA <213> Homo Sapiens <400> 242 ccctctgcca cagccaatgg ggg 23 <210> 243 <211> 23 <212> DNA <213> Homo Sapiens <400> 243 cccccattgg ctgtggcaga ggg 23 <210> 244 <211> 23 <212> DNA <213> Homo Sapiens <400> 244 gcccccattg gctgtggcag agg 23 <210> 245 <211> 23 <212> DNA <213> Homo Sapiens <400> 245 ggacaggccc ccattggctg tgg 23 <210> 246 <211> 23 <212> DNA <213> Homo Sapiens <400> 246 ccgggctctt ggccttggac agg 23 <210> 247 <211> 23 <212> DNA <213> Homo Sapiens <400> 247 ctgtccaagg ccaagagccc ggg 23 <210> 248 <211> 23 <212> DNA <213> Homo Sapiens <400> 248 tggcgtcagg cccgggctct tgg 23 <210> 249 <211> 23 <212> DNA <213> Homo Sapiens <400> 249 cgggcctgac gccagagccc agg 23 <210> 250 <211> 23 <212> DNA <213> Homo Sapiens <400> 250 caacaaccat gctgggcatc tgg 23 <210> 251 <211> 23 <212> DNA <213> Homo Sapiens <400> 251 gagggtccag atgcccagca tgg 23 <210> 252 <211> 23 <212> DNA <213> Homo Sapiens <400> 252 catctggacc ctcctacctc tgg 23 <210> 253 <211> 23 <212> DNA <213> Homo Sapiens <400> 253 agggctcacc agaggtagga ggg 23 <210> 254 <211> 23 <212> DNA <213> Homo Sapiens <400> 254 ggagttgatg tcagtcactt ggg 23 <210> 255 <211> 23 <212> DNA <213> Homo Sapiens <400> 255 tggagttgat gtcagtcact tgg 23 <210> 256 <211> 23 <212> DNA <213> Homo Sapiens <400> 256 agtgactgac atcaactcca agg 23 <210> 257 <211> 23 <212> DNA <213> Homo Sapiens <400> 257 gtgactgaca tcaactccaa ggg 23 <210> 258 <211> 23 <212> DNA <213> Homo Sapiens <400> 258 actccaaggg attggaattg agg 23 <210> 259 <211> 23 <212> DNA <213> Homo Sapiens <400> 259 cttcctcaat tccaatccct tgg 23 <210> 260 <211> 23 <212> DNA <213> Homo Sapiens <400> 260 tacagttgag actcagaact tgg 23 <210> 261 <211> 23 <212> DNA <213> Homo Sapiens <400> 261 ttggaaggcc tgcatcatga tgg 23 <210> 262 <211> 23 <212> DNA <213> Homo Sapiens <400> 262 agaattggcc atcatgatgc agg 23 <210> 263 <211> 23 <212> DNA <213> Homo Sapiens <400> 263 gacagggctt atggcagaat tgg 23 <210> 264 <211> 23 <212> DNA <213> Homo Sapiens <400> 264 tgtaacatac ctggaggaca ggg 23 <210> 265 <211> 23 <212> DNA <213> Homo Sapiens <400> 265 gtgtaacata cctggaggac agg 23 <210> 266 <211> 23 <212> DNA <213> Homo Sapiens <400> 266 cgtacctgtg caactcctgt tgg 23 <210> 267 <211> 23 <212> DNA <213> Homo Sapiens <400> 267 gatctactgg aattcctaat ggg 23 <210> 268 <211> 23 <212> DNA <213> Homo Sapiens <400> 268 gagtcagctg ttggcccatt agg 23 <210> 269 <211> 23 <212> DNA <213> Homo Sapiens <400> 269 ctgcctacaa actcagtctc tgg 23 <210> 270 <211> 23 <212> DNA <213> Homo Sapiens <400> 270 gggcaggcag gacggactcc agg 23 <210> 271 <211> 23 <212> DNA <213> Homo Sapiens <400> 271 ggagtccgtc ctgcctgccc tgg 23 <210> 272 <211> 23 <212> DNA <213> Homo Sapiens <400> 272 gagtccgtcc tgcctgccct ggg 23 <210> 273 <211> 23 <212> DNA <213> Homo Sapiens <400> 273 gaaaagggtc cattggccaa agg 23 <210> 274 <211> 23 <212> DNA <213> Homo Sapiens <400> 274 gcctgcagaa aagggtccat tgg 23 <210> 275 <211> 23 <212> DNA <213> Homo Sapiens <400> 275 ttgatgtgct acagggaaca tgg 23 <210> 276 <211> 23 <212> DNA <213> Homo Sapiens <400> 276 agcgttcttg atgtgctaca ggg 23 <210> 277 <211> 23 <212> DNA <213> Homo Sapiens <400> 277 cagcgttctt gatgtgctac agg 23 <210> 278 <211> 23 <212> DNA <213> Homo Sapiens <400> 278 ctgtagcaca tcaagaacgc tgg 23 <210> 279 <211> 23 <212> DNA <213> Homo Sapiens <400> 279 tgtagcacat caagaacgct ggg 23 <210> 280 <211> 23 <212> DNA <213> Homo Sapiens <400> 280 ataggcaata atcatataac agg 23 <210> 281 <211> 23 <212> DNA <213> Homo Sapiens <400> 281 agtgcgtttc gctgcaggta agg 23 <210> 282 <211> 23 <212> DNA <213> Homo Sapiens <400> 282 gagtgagtgc gtttcgctgc agg 23 <210> 283 <211> 23 <212> DNA <213> Homo Sapiens <400> 283 gtcaggtttg tgcggttatg agg 23 <210> 284 <211> 23 <212> DNA <213> Homo Sapiens <400> 284 cgctgctggt caggtttgtg cgg 23 <210> 285 <211> 23 <212> DNA <213> Homo Sapiens <400> 285 aaacctgacc agcagcgcag agg 23 <210> 286 <211> 23 <212> DNA <213> Homo Sapiens <400> 286 ccagcagcgc agaggagccg tgg 23 <210> 287 <211> 23 <212> DNA <213> Homo Sapiens <400> 287 ccacggctcc tctgcgctgc tgg 23 <210> 288 <211> 23 <212> DNA <213> Homo Sapiens <400> 288 ccaactatct aactccactc agg 23 <210> 289 <211> 23 <212> DNA <213> Homo Sapiens <400> 289 cctgagtgga gttagatagt tgg 23

Claims (34)

At least one artificially engineered immunomodulatory gene selected from the group consisting of PD-1, CTLA-4, A20, Dgk ?, Dgk ?, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A and Tet2 in which the nucleic acid sequence has been modified.
The method according to claim 1,
Wherein said strain in said nucleic acid sequence is artificially caused by a guide nucleic acid-editor protein complex.
The method according to claim 1,
Wherein the immunomodulatory gene is A20, Dgk ?, Dgk ?, EGR2, PSGL-1 or KDM6A.
The method according to claim 1,
The gene is an immunoregulatory gene engineered by a guide nucleic acid-editor protein complex,
In a sequence sequence of 1 bp to 50 bp adjacent to the 5 'end and / or the 3' end of the PAM (proto-spacer-adjacent motif) sequence in the nucleic acid sequence constituting the immunomodulatory gene
Deletion or insertion of one or more nucleotides;
Substitution with one or more nucleotides that is different from the wild type gene;
Insert one or more nucleotides from outside
A variant of one or more of the nucleic acids, or
The chemical modification of one or more nucleotides in the nucleic acid sequence constituting said immunomodulatory gene
Lt; RTI ID = 0.0 &gt; immunoregulatory &lt; / RTI &gt; gene.
The method according to claim 1,
Wherein the modification of the nucleic acid occurs in the promoter region of the gene.
The method according to claim 1,
Wherein the modification of the nucleic acid occurs in the exon region of the gene.
The method according to claim 1,
Wherein the modification of the nucleic acid occurs in the intron region of the gene.
The method according to claim 1,
Wherein the modification of the nucleic acid occurs in the enhancer region of the gene.
5. The method of claim 4,
Wherein said PAM sequence is at least one of the following sequences: an artificially engineered immunomodulatory gene (described in 5 'to 3' direction).
NGG (wherein N is A, T, C or G);
NNNNRYAC wherein each N is independently A, T, C or G, R is A or G, and Y is C or T;
NNAGAAW wherein N is each independently A, T, C or G and W is A or T;
NNNNGATT, where each N is independently A, T, C or G;
NNGRR (T) wherein each N is independently A, T, C or G, R is A or G, and Y is C or T; And
TTN (where N is A, T, C or G).
1 to 289 of the nucleic acid sequence of one or more genes selected from among PD-1, CTLA-4, A20, Dgk ?, Dgk ?, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A and Tet2. Forming guide nucleic acid
11. The method of claim 10,
At least one guide nucleic acid selected from the group consisting of:
A guide nucleic acid capable of forming a complementary bond to each of the target sequences of SEQ ID NOS: 6 and 11 in the A20 gene nucleic acid sequence;
A guide nucleic acid capable of forming a complementary bond to each of the target sequences of SEQ ID NOS: 19, 20, 21, and 23 in the DGKa gene nucleic acid sequence;
A guide nucleic acid capable of forming a complementary bond to the target sequence of SEQ ID NO: 25 in the EGR2 gene nucleic acid sequence;
A guide nucleic acid capable of forming a complementary bond to the target sequence of SEQ ID NO: 64, respectively, of the PPP2R2D gene nucleic acid sequence;
A guide nucleic acid capable of forming a complementary bond to the target sequence of SEQ ID NO: 87 and 89, respectively, in the PD-1 gene nucleic acid sequence;
A guide nucleic acid capable of forming a complementary bond to a target sequence of SEQ ID NO: 109, 110, 111, 112 and 113, respectively, in the Dgkζ gene nucleic acid sequence;
A guide nucleic acid capable of forming a complementary bond in each of the target sequences of SEQ ID NOS: 126, 128 and 129 in the Tet-2 gene nucleic acid sequence;
A guide nucleic acid capable of forming a complementary bond to the target sequence of SEQ ID NO: 182 in the PSGL-1 gene nucleic acid sequence;
A guide nucleic acid capable of forming a complementary bond to each of the target sequences of SEQ ID NOS: 252, 254, 257 and 264 in the FAS gene nucleic acid sequence; And
A guide nucleic acid capable of forming a complementary bond, respectively, in the target sequence of SEQ ID NO: 285 of the KDM6A gene nucleic acid sequence.
11. The method of claim 10,
Wherein the guide nucleic acid is a nucleotide of 18 to 23 bp.
One or more artificially engineered immunomodulatory genes selected from the group consisting of PD-1, CTLA-4, A20, Dgk ?, Dgk ?, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A and Tet2, Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;
14. The method of claim 13,
Wherein said immune cells are at least one cell selected from the group consisting of T cells, CAR-T cells, NK cells and NKT cells.
14. The method of claim 13,
Wherein said immune cell is an artificially engineered immune cell such that the activity of said immunoregulatory gene is suppressed or inactivated
14. The method of claim 13,
Wherein said immune cells further comprise a chimeric antigen receptor (CAR). &Lt; RTI ID = 0.0 &gt;
15. The method of claim 14,
Wherein said T cells further comprise a chimeric antigen receptor (CAR) or engineered TCR (T-cell receptor).
14. The method of claim 13,
Wherein said immune cells further comprise a guide nucleic acid-editor protein complex or nucleic acid sequence encoding them.
19. The method of claim 18, wherein the editor protein is
Cas9 protein derived from Streptococcus pyogenes, Cas9 protein derived from Campylobacter jejuni, Cas9 protein derived from Streptococcus thermophilus, Streptococcus aureus (Streptococcus aureus) ), A Cas9 protein derived from Neisseria meningitidis, and a Cpf1 protein. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt;
19. The method of claim 18,
1 to 289 of the nucleic acid sequence of one or more genes selected from the group consisting of PD-1, CTLA-4, A20, Dgk ?, Dgk ?, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A and Tet2. Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt; respectively.
14. The method of claim 13,
Wherein said immune cells comprise at least one of an artificially engineered Dgk &lt; alpha &gt; and Dgk [omega] genes which have undergone a modification in the nucleic acid sequence.
14. The method of claim 13,
Wherein said immune cells contain an artificially engineered Dgk &lt; alpha &gt; and Dgk &lt; z &gt; genes which have undergone a modification in the nucleic acid sequence.
1 to 289 of the nucleic acid sequence of one or more genes selected from the group consisting of PD-1, CTLA-4, A20, Dgk ?, Dgk ?, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A and Tet2 A guide nucleic acid capable of forming a linkage; And
Editor protein or nucleic acid encoding it
&Lt; / RTI &gt;
24. The method of claim 23, wherein the editor protein is
Cas9 protein derived from Streptococcus pyogenes, Cas9 protein derived from Campylobacter jejuni, Cas9 protein derived from Streptococcus thermophilus, Streptococcus aureus (Streptococcus aureus) ), A Cas9 protein derived from Neisseria meningitidis, and a Cpf1 protein.
24. The method according to claim 23,
Within a sequence of 1 bp to 50 bp nucleotide sequence located adjacent to the 5 'end and / or the 3' end of the PAM (proto-spacer-adjacent Motif) sequence in the nucleic acid sequence constituting the immunomodulatory gene
Deletion or insertion of one or more nucleotides;
Substitution with one or more nucleotides that is different from the wild type gene;
Insert one or more nucleotides from outside
A variant of one or more of the nucleic acids, or
The chemical modification of one or more nucleotides in the nucleic acid sequence constituting said immunomodulatory gene
Wherein the composition comprises a gene-knocking composition
25. The method of claim 24,
Wherein said PAM sequence is at least one of the following sequences (described in 5 'to 3' direction):
NGG (wherein N is A, T, C or G);
NNNNRYAC wherein each N is independently A, T, C or G, R is A or G, and Y is C or T;
NNAGAAW wherein N is each independently A, T, C or G and W is A or T;
NNNNGATT, where each N is independently A, T, C or G;
NNGRR (T) wherein each N is independently A, T, C or G, R is A or G, and Y is C or T; And
TTN (where N is A, T, C or G).
Immune cells isolated from the human body
1 to 289 of the nucleotide sequence of one or more genes selected from the group consisting of (a) PD-1, CTLA-4, A20, Dgk ?, Dgk ?, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A and Tet2 A guide nucleic acid capable of forming a complementary bond to each of the amino acids; And
(b) Cas9 protein derived from Streptococcus pyogenes, Cas9 protein derived from Campylobacter jejuni, Cas9 protein derived from Streptococcus thermophilus, Streptococcus aureus A Cas9 protein derived from Streptococcus aureus, a Cas9 protein derived from Neisseria meningitidis, and an edible protein selected from the group consisting of Cpf1 protein
And contacting the immune cells with an antigen.
28. The method of claim 27,
The guide nucleic acid and the editor protein
Are each present in one or more vectors in the form of nucleic acid sequences, or
A method for artificially manipulating an immune cell characterized by the formation of a complex by binding of a guide nucleic acid and an editor protein.
28. The method of claim 27,
Wherein the step of contacting is carried out in vitro.
28. The method of claim 27,
Wherein the step of contacting is carried out by one or more methods selected from electroporation, liposomes, plasmids, viral vectors, nanoparticles and protein translocation domain (PTD) fusion protein methods.
31. The method of claim 30,
Wherein the viral vector is at least one selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus and herpes simplex virus.
The present invention provides information on the sequence of the immune cell target position in a subject by sequencing one or more genes among PD-1, CTLA-4, A20, Dgk ?, Dgk ?, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A and Tet2 Way.
A method for building a library using information provided through the method of claim 32.
1 to 289 of the nucleotide sequence of one or more genes selected from the group consisting of (a) PD-1, CTLA-4, A20, Dgk ?, Dgk ?, Fas, EGR2, PPP2R2D, PSGL-1, KDM6A and Tet2 A guide nucleic acid capable of forming a complementary bond to each of the amino acids;
(b) Cas9 protein derived from Streptococcus pyogenes, Cas9 protein derived from Campylobacter jejuni, Cas9 protein derived from Streptococcus thermophilus, Streptococcus aureus A Cas9 protein derived from Streptococcus aureus, a Cas9 protein derived from Neisseria meningitidis, and a Cpf1 protein, or a nucleic acid encoding the same
&Lt; / RTI &gt;

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