Platform for expressing proteins of interest in the liver

Abstract

The present specification relates to a platform for expressing a protein of interest by artificially manipulating the liver, and more specifically, to a platform for alleviating or treating a genetic disease or improving body function by inserting a transgene (e.g., a therapeutic gene) that can have normal function and expression into a highly expressed secretory gene to replace a disease gene with abnormal function or expression, and inducing the expression of the transgene. The highly expressed secretory gene includes an HP gene or an APOC3 gene. The transgene includes a transgene that is expressed at a high level by a promoter in the liver cell genome and secreted outside the cell.

Description

Technical Field

[0001] The present invention relates to a platform for expressing a protein of interest by artificially manipulating or modifying the liver. More specifically, the present invention relates to a platform for alleviating or treating genetic disorders or improving body function by inducing the expression of a normally functioning or expressed transgene (e.g., a therapeutic gene) to replace a disease gene with abnormal function or expression using a guide nucleic acid and / or editing protein. Background Art

[0002] Gene therapy has great potential in a new era of human engineering. Gene therapy includes genome editing technologies such as gene disruption, gene modification, and insertion of transgenes (which can be regulated by specific exogenous promoters fused to them or endogenous promoters found at the genomic insertion site).

[0003] Currently, there are no suitable therapeutic agents for refractory hemophilia or lysosomal storage diseases, and replacement therapy using enzyme or protein replacement is mainly used for treatment, but this therapy is not a radical treatment. In addition, methods of expressing therapeutic genes using AAV are being conducted in clinical trials, but it can also be expected that these methods are only effective when expressed by AAV, so they cannot be radical and long-term treatments.

[0004] Therefore, there is a need for a therapeutic platform for longer-lasting and more effective treatment by inserting a true therapeutic gene into the patient's genome to enable permanent expression. Such a therapeutic platform is different from existing treatment methods that are temporary and repeated administration of therapeutic agents, and an efficient therapeutic platform can be developed using efficient target-specific programmable nucleases for gene correction to insert therapeutic genes into the patient's genome.

[0005] For this reason, the inventors aim to develop a therapeutic platform that can use target-specific programmable nucleases to continuously express genes of interest. In particular, for high efficiency, an effective platform is completed by determining the site of a gene highly expressed in an organ or tissue (e.g., liver) as an insertion site for a gene of interest. Summary of the invention

[0006] Technical issues

[0007] The present invention aims to provide a platform for expressing proteins of interest in hepatocytes using the CRISPR-Cas system.

[0008] The present invention also aims to provide a composition for expressing a protein of interest and various uses thereof, wherein the composition comprises the protein of interest or a sequence encoding the protein of interest and uses a CRISPR-Cas system.

[0009] The present invention also aims to provide cells that use the CRISPR-Cas system to express a protein of interest and methods for preparing the cells.

[0010] The present invention also aims to provide a method for expressing a protein of interest in hepatocytes using the CRISPR-Cas system.

[0011] The present invention also aims to provide a method for treating a genetic disorder, the method comprising administering to a subject to be treated a composition for expressing a protein of interest in hepatocytes.

[0012] The present invention also provides guide nucleic acids and editing proteins, which can be used to manipulate corresponding target genes to express proteins of interest in hepatocytes.

[0013] Technical Solution

[0014] In order to solve the above problems, the present invention provides a platform for expressing a protein of interest in hepatocytes. More specifically, the present invention provides a platform for improving body function and treating genetic diseases by inserting a gene encoding a protein of interest into a target gene using a CRISPR-Cas system to continuously express the protein at a high level; and also provides artificially modified cells, organs or tissues.

[0015] As used herein, "Liver biofactory platform" is a system capable of sustained expression of a protein of interest by inserting a specific transgene into liver cells, and includes all compositions, methods and uses directly or indirectly involved therein.

[0016] In one example, the artificially inserted transgene can be integrated into a secreted gene that is highly expressed in hepatocytes.

[0017] The highly expressed secretory gene is at least one selected from the group consisting of the ALB gene, FTL gene, FTH1 gene, ACTB gene, HP gene, APOC3 gene, SOD2 gene, ORM1 gene and F9 gene present in the hepatocyte genome.

[0018] Transgene expression is higher compared to before insertion, or the transgene is newly expressed in hepatocytes.

[0019] In addition, the disclosure disclosed herein relates to a composition for expressing a protein of interest in hepatocytes comprising a target-specific programmable nuclease; specifically, to a composition for manipulating genes, the composition comprising a programmable nuclease for inserting a transgene into a highly expressed secretory gene, wherein the highly expressed secretory gene is one or more genes selected from the group of highly expressed secretory genes.

[0020] In one example, the present invention provides a composition for expressing a protein of interest, the composition comprising: a guide nucleic acid corresponding to a target sequence of one or more genes selected from the group of highly expressed secreted genes expressed in the liver;

[0021] An editing protein or a nucleic acid encoding an editing protein; and

[0022] A donor comprising a nucleotide sequence encoding a protein of interest.

[0023] "Highly expressed secretory genes" refer to genes that are expressed at a predetermined level or higher in hepatocytes, and are highly expressed in hepatocytes, so that the expression product is secreted out of the hepatocytes. Highly expressed secretory genes exist at sites where they can be continuously expressed at high levels in the hepatocyte genome; and can function as safe harbor sites.

[0024] The highly expressed secretory gene may be at least one selected from the group consisting of ALB gene, FTL gene, FTH1 gene, ACTB gene, HP gene, APOC3 gene, SOD2 gene, ORM1 gene, AAVS1 gene, Rosa gene, HPRT gene and CCR5 gene.

[0025] The highly expressed secretory gene may preferably be the HP gene or the APOC3 gene.

[0026] Programmable nuclease refers to an artificially engineered nuclease. In an example, the programmable nuclease is at least one selected from the group consisting of the following nucleases: clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN).

[0027] Regarding modifications in nucleotide sequences, artificial manipulation can be performed without restriction through the use of the CRISPR-Cas system.

[0028] The “CRISPR-Cas system” refers to a system formed by the interaction between a guide nucleic acid that recognizes and binds to the sequence of a gene of interest and an editing protein that cleaves the target gene, and the CRISPR-Cas system includes the guide nucleic acid and the editing protein.

[0029] The CRISPR-Cas system can modify the target. The target can be a target nucleic acid, gene, chromosome or protein.

[0030] The editing protein is one or more proteins selected from the group consisting of: Cas9 protein derived from Streptococcus pyogenes, Cas9 protein derived from Campylobacter jejuni, Cas9 protein derived from Streptococcus thermophilus, Cas9 protein derived from Staphylococcus aureus, Cas9 protein derived from Neisseria meningitidis, and Cpf1 protein, or nucleic acids encoding the protein. In an example, the editing protein can be a Cas9 protein derived from Streptococcus pyogenes or a Cas9 protein derived from Campylobacter jejuni.

[0031] The target sequence may be a continuous nucleotide sequence of 10 bp to 25 bp located in the exon region of a highly expressed secretory gene sequence.

[0032] The target sequence may be a continuous nucleotide sequence of 10 bp to 25 bp located in the intron region of a highly expressed secretory gene sequence.

[0033] The target sequence may be at least one selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 348.

[0034] The guide nucleic acid is capable of forming a complementary bond with a portion of the nucleotide sequence of at least one gene selected from the group of highly expressed secreted genes. The guide nucleic acid may contain 0-5 nucleotides, 0-4 nucleotides, 0-3 nucleotides, or 0-2 nucleotide mismatches with the target sequence.

[0035] In one embodiment, the disclosure disclosed herein comprises a guide nucleic acid for a highly expressed secretory gene, wherein the highly expressed secretory gene is at least one selected from the group consisting of the following genes: ALB gene, FTL gene, FTH1 gene, ACTB gene, HP gene, APOC3 gene, SOD2 gene, ORM1 gene, AAVS1 gene, Rosa gene, HPRT gene and CCR5 gene.

[0036] In an example, one or more guide nucleic acids selected from the group consisting of:

[0037] A guide nucleic acid for a target sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 153 of HP gene sequences;

[0038] A guide nucleic acid directed against a target sequence selected from the group consisting of SEQ ID NO: 168 to SEQ ID NO: 348 of APOC3 gene sequences.

[0039] In one embodiment, a composition for modifying a gene provides

[0040] a guide nucleic acid corresponding to a target sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 40 and SEQ ID NO: 154-SEQ ID NO: 167; and

[0041] The composition further comprises a Cas9 protein derived from Campylobacter jejuni, or a nucleic acid encoding the Cas9 protein.

[0042] In another embodiment, the composition for modifying a gene provides

[0043] a guide nucleic acid corresponding to a target sequence selected from the group consisting of SEQ ID NO:41-SEQ ID NO:134 and SEQ ID NO:168-SEQ ID NO:332; and

[0044] The composition further comprises a Cas9 protein derived from Streptococcus pyogenes, or a nucleic acid encoding the Cas9 protein.

[0045] The guide nucleic acid may be any one of nucleotide sequences of 18-25 bp, 18-24 bp, 18-23 bp, 19-23 bp, and 20-23 bp without limitation.

[0046] The guide RNA (gRNA) can exist in the form of dual RNA (dual RNA, including crRNA and tracrRNA) or single guide RNA (sgRNA).

[0047] The disclosure disclosed herein includes a transgene, which is a gene encoding a protein of interest.

[0048] The expression of the protein of interest can function to replace an existing protein or provide a new protein. Alternatively, the protein that is lacking or missing in the body is expressed, thereby treating a disease caused by the lack or missing of the protein or enhancing the function of the corresponding protein.

[0049] For example, the protein of interest can be a protein that has different characteristics (eg, increased serum half-life) than an existing protein.

[0050] For example, the protein of interest can be a therapeutic gene for a particular disease. The therapeutic gene can be a gene that affects the expression and function of a pathogenic gene associated with a genetic disorder. The therapeutic gene can be a portion (e.g., a functional domain) of a normal gene (a mutant form of a normal gene) of a pathogenic gene associated with a genetic disorder.

[0051] The disease may be a disease that can be treated by an antibody.

[0052] The disease may be caused by mutations in a variety of genes. For example, the disease may be a disease associated with a "genetic metabolic disorder". For example, the disease may be hemophilia.

[0053] The therapeutic gene (ie the normal form of the disease-causing gene) may be selected from the group consisting of FVII, FVIII, FIX and different coagulation factors.

[0054] The therapeutic gene (i.e., the normal form of the disease-causing gene) can be selected from the group consisting of IDUA, I2S, SGSH, NAGLU, HGSNAT, GNS, GALNS, GLB1, ARSB, GUSB, HYAL, NEU, GNPTAB and MCOLN1.

[0055] The therapeutic gene (ie, the normal form of the disease-causing gene) may be selected from the group consisting of SAH1, GALC, CTSA, GLA, NAGA, β-galactosidase, hexosaminidase, GBA, SMPD1, ARSA, and SUMF.

[0056] The therapeutic gene (ie, the normal form of the disease-causing gene) may be selected from the group consisting of NPC, PPT, TPP1, CLN3, CLN6, PPT1, DNAJC5, CTSF, CLN7, CLN8 and CTSD.

[0057] The therapeutic gene (ie, the normal form of the disease-causing gene) can be GAA or LAMP2.

[0058] The therapeutic gene (ie, the normal form of the disease-causing gene) can be CTNS, CTSK, or SLC17A5.

[0059] The therapeutic gene (ie, the normal form of the disease-causing gene) may be selected from the group consisting of MAN2B, MAN2C, MANBA, AGA, FUCA1, and LAL.

[0060] The therapeutic gene (i.e., the normal form of the disease-causing gene) may be a gene encoding a protein selected from the group consisting of: methylmalonic aciduria CbIA type (MMAA) protein, methylmalonic aciduria CbIB type (MMAB) protein, methylmalonic aciduria CbIC type (MMADHC) protein, 5-methyltetrahydrofolate-homocysteine ​​methyltransferase reductase (MTRR) protein, lysosomal membrane protein domain (LMBRD 1) protein, 5-methyltetrahydrofolate-homocysteine ​​methyltransferase (MTR) protein, propionyl-CoA protein, glucose-6-phosphate transporter (G6PT) protein, glucose-6-phosphatase (G6Pase) protein, low-density lipoprotein receptor (LDLR) protein, low-density lipoprotein receptor adaptor protein 1 (LDLRAP-1 protein), N-acetylglutamate synthetase (NAGS) protein, carbamoyl phosphate synthetase 1 (CPS1) protein, ornithine carbamoyltransferase (OTC) protein, argininosuccinate synthetase (ASS) protein, argininosuccinate lyase (ASL) protein, arginase (ARG1) protein, solute carrier family 25 protein (solute carrier family 25protein), UDP glucuronosyltransferase 1 family polypeptide A1 (UGT1A1) protein, fumarylacetoacetate hydrolase (FAH), alanine-glyoxylate aminotransferase (AGXT) protein, glyoxylate reductase / hydroxypyruvate reductase (GRHPR) protein, APTase Cu(2+) transporter β (ATP7B) protein, phenylalanine hydroxylase (PAH) protein and lipoprotein lyase (LPL) protein.

[0061] The therapeutic gene may be a gene encoding an antibody. That is, the therapeutic gene may be a gene encoding a protein or polypeptide that blocks a specific factor or signal pathway related to the cause of disease through an antibody.

[0062] The therapeutic gene can be fused with a specific peptide that can penetrate into a specific tissue, such as the blood-brain barrier (BBB), or with a functional peptide that can increase the therapeutic effect of the therapeutic gene.

[0063] In addition, the present application document provides artificially modified cells, tissues or organs into which a transgene is inserted using a guide nucleic acid and / or an editing protein.

[0064] The modified cells, tissues or organs are organs or tissues in which a protein of interest is expressed by inserting a transgene into a target sequence in a genomic gene of interest using the CRISPR-Cas system. In artificially modified cells, tissues or organs, the protein of interest is continuously expressed at a certain level or higher by inserting the protein of interest into a highly expressed secretory gene position in hepatocytes.

[0065] In an exemplary embodiment of the disclosure disclosed herein,

[0066] The highly expressed secretory gene may be at least one selected from the group consisting of FTL gene, FTH1 gene, ACTB gene, HP gene, APOC3 gene, SOD2 gene, ORM1 gene and F9 gene,

[0067] Artificial insertion of transgenes into highly expressed secreted genes, and

[0068] Hepatocytes containing the protein of interest expressed by the transgene.

[0069] In one example, the highly expressed secretory gene may be the HP gene or the APOC3 gene.

[0070] The transgene may be contained within an exon or intron of a highly expressed secreted gene.

[0071] The transgene is expressed from a promoter that is natively present in the hepatocyte genome.

[0072] The hepatocyte may be a hepatic stem cell that includes, without limitation, an manipulated locus (e.g., a highly expressed secretory gene locus). Specific stem cell types that can be used with the methods and compositions of the present specification include adult stem cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs).

[0073] In another exemplary embodiment of the disclosure disclosed herein,

[0074] As a method for artificially manipulating hepatocytes to express a protein of interest by artificially inserting a transgene into the hepatocyte genome,

[0075] The method comprises:

[0076] Introducing programmable nucleases into hepatocytes to manipulate highly expressed secreted genes present in the hepatocyte genome, as well as donor sequences containing transgenes,

[0077] wherein the transgene is inserted into a highly expressed secretory gene present in the hepatocyte genome, and

[0078] The highly expressed secretory gene is one or more genes selected from FTL, FTH1, ACTB, HP, APOC3, SOD2, ORM1 and F9.

[0079] The introduction of the guide nucleic acid, editing protein and donor into the hepatocytes can be carried out by one or more means selected from liposomes, plasmids, viral vectors, nanoparticles or protein translocation domain (PTD) fusion proteins.

[0080] The guide nucleic acid, the editing protein and the donor may be encoded in the form of nucleic acid sequences, respectively, in at least one vector.

[0081] The vector may be a viral vector system. The viral vector may be at least one selected from the group consisting of adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus, herpes simplex virus and lentivirus.

[0082] Furthermore, introduction of programmable nucleases and donors into hepatocytes was performed in vitro.

[0083] The intra-subject delivery method may be systemic administration or local administration. Systemic administration may be intravenous administration.

[0084] Furthermore, the introduction of the programmable nuclease and the donor into the hepatocytes is performed ex vivo. The method of delivery in the subject can be electroporation.

[0085] In yet another exemplary embodiment of the disclosure disclosed herein,

[0086] A method for treating a specific disease is provided, the method comprising administering a composition for continuously expressing a protein of interest at a certain level or higher in hepatocytes to treat the specific disease.

[0087] The specific disease may be a disease caused by the deficiency or absence of a specific protein.

[0088] The method of treatment comprises administering to a subject to be treated a composition comprising, as an active ingredient, a composition for manipulating genes to express a transgene artificially inserted into the genome of hepatocytes.

[0089] The composition for manipulating a gene may include at least one selected from a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas) system, a zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).

[0090] For example, as a method of treating hemophilia,

[0091] A method for treating hemophilia is disclosed, the method comprising administering a composition for manipulating genes to insert a transgene F9 into an HP or APOC3 gene sequence in a hepatocyte.

[0092] The subject to be treated can be a mammal including primates (such as humans and monkeys) and rodents (such as mice and rats).

[0093] In yet another exemplary embodiment of the disclosure disclosed herein, all aspects are provided for therapeutic use for a specific disease.

[0094] [Beneficial effects of the present invention]

[0095] As disclosed herein, a platform for expressing a protein of interest in hepatocytes, where the protein of interest can be continuously expressed at high levels, and a basic therapeutic agent for improving body function and treating genetic disorders can be provided.

[0096] For example, genetic disorders caused by the lack or deletion of a specific protein can be prevented or treated by producing a therapeutic protein using the composition described herein to express the lacking or deleted protein, or by providing a new protein with different characteristics (eg, increased half-life). BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1 The figure shows the results of RNA sequencing for selecting highly expressed secreted genes.

[0098] Figure 2 A diagram of the liver biofactory platform formed by artificially manipulating high-expression secretory genes.

[0099] Figure 3 Shown are the results of T7E1 analysis used to confirm the activity of gRNA targeting haptoglobin (HP).

[0100] Figure 4 A group map of off-target sites confirmed using Digenome-seq.

[0101] Figure 5 Figure 2. Validation of off-target activity by targeted deep sequencing using NGS in a human cell line (i.e., HEK293 cells).

[0102] Figure 6 A diagram showing packaging of CRISPR-SpCas9 or CRISPR-CjCas9 and F9 gene in double AAV.

[0103] Figure 7 Shown is the primer design used to confirm HDR-mediated knock-in of the F9 gene into the hHP gene by Sanger sequencing to confirm whether knock-in of the F9 therapeutic gene has occurred in the genomic DNA of the HEK293 cell line, in which pAAV-CMV-CjCas9-U6-sgRNA and pAAV-hF9-donor were transfected.

[0104] Figure 8 A set of images showing the F9 insertion between the left arm of the homology arm and the hHP gene (left panel) and the F9 insertion between the right arm of the homology arm and the hHP gene (right panel).

[0105] Fig. 9The following sets of figures show the on-target activity ( Fig. 9 A) and the secretion level of hF9 obtained by knock-in (using supernatant samples) ( Fig. 9 B). DETAILED DESCRIPTION

[0106] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or identical to the methods and materials 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, materials, methods and embodiments are illustrative only and are not intended to be limiting.

[0107] The disclosure disclosed herein relates to a platform for expressing a protein of interest in the liver.

[0108] More specifically, the disclosure disclosed herein includes compositions for expressing a protein of interest by artificially manipulating a highly expressed secreted gene using a CRISPR-Cas system,

[0109] Manipulated cells, organs or tissues expressing a protein of interest, and

[0110] The use of the composition, cell, organ or tissue in treating, preventing or alleviating specific genetic disorders or improving body functions.

[0111] [Liver Biofactory Platform (LBP)]

[0112] One exemplary embodiment disclosed herein relates to a "platform for expressing a protein of interest".

[0113] A platform for expressing a protein of interest is a general term for a system that can continuously produce (express) a large amount of a protein of interest in a specific organ or tissue by artificially inserting a transgene into a wild-type genome, and includes aspects of compositions, uses, and methods.

[0114] In addition, the platform includes a system for secreting the produced (expressed) protein of interest out of the cell. Therefore, the produced protein of interest can not only function in the organ or tissue into which the transgene is inserted, but also in the organ or tissue into which the protein is secreted.

[0115] In an exemplary embodiment, the platform can sufficiently produce a protein of interest at a certain level or higher.

[0116] In another exemplary embodiment, the platform can produce a protein of interest that is sufficiently secreted from cells to be manipulated, even in different organs or tissues.

[0117] In the present specification, the specific organ or tissue implementing the platform for expressing a protein of interest is the liver or liver tissue.

[0118] The liver is an organ known as the human body's chemical factory, which is involved in all events that occur in the body and is responsible for the metabolism of nutrients, detoxification and immune function through natural types of enzymes. In particular, the human body can produce and store a variety of substances required for proper functioning, and then transport them throughout the body through the hepatic vein.

[0119] For example, a specific protein can be produced in large amounts by highly expressing a specific gene in the liver, and the corresponding protein can be supplied to a desired organ by secreting the produced protein throughout the body.

[0120] Hereinafter, the Liver Biofactory Platform (hereinafter referred to as "LBP") will be described.

[0121] In one aspect disclosed herein, LBP is a system for sustained production of a protein of interest in liver tissue, which system includes all materials, compositions, methods and uses that are directly or indirectly involved in secreting the produced protein out of cells.

[0122] LBP includes a system for producing a protein of interest in hepatocytes by artificially manipulating the hepatocytes (eg, the genome in the hepatocytes).

[0123] For example, LBP involves artificial modification of the hepatocyte genomic sequence.

[0124] In an exemplary embodiment, the LBP comprises cleavage of a partial sequence of a specific gene in the hepatocyte genome.

[0125] In another exemplary embodiment, LBP comprises inserting a transgene encoding a protein of interest into a cleaved hepatocyte genomic sequence.

[0126] In the disclosure disclosed herein, various types of liver-derived cells can be used for LBP.

[0127] For example, the LBP may include hepatocytes, induced pluripotent cells (iPSCs), or different types of stem cells (embryonic stem cells, hematopoietic stem cells, or mesenchymal stem cells), but the present invention is not limited thereto.

[0128] LBP can exploit specific regions in the hepatocyte genome to overexpress a protein of interest in the liver.

[0129] In one example, a transgene can be inserted into a safe harbor site in the hepatocyte genome.

[0130] A "safe harbor site" is a specific region in the genome where there are no serious side effects (such as cancer) even if a foreign gene is inserted, and where the foreign gene inserted into the specific region can be permanently and safely expressed at a high level.

[0131] In another example, a transgene can be inserted into a region that enables use of gene expression regulatory regions in the hepatocyte genome.

[0132] "Gene regulatory region" is a region that exists in the hepatocyte genome and plays a key role in the regulation of gene expression, and is a general term for regions including promoters and / or regulatory factors (enhancers, transcriptional promoters, etc.) adjacent to sequences containing genetic information. Foreign genes inserted using promoters and / or regulatory factors can be highly expressed efficiently.

[0133] LBP can produce large amounts of a protein of interest by expressing a transgene at high and sustained levels using a specific site in the genome in hepatocytes.

[0134] Highly expressed secretory gene

[0135] In the disclosure disclosed herein,

[0136] The specific site for inserting the transgene in the hepatocyte genome may be included in the partial sequence of the “highly expressed secretory gene.” In this specification, the highly expressed secretory gene is also referred to as the target gene for artificial manipulation in the hepatocyte genome.

[0137] The "highly expressed secretory gene" refers to a gene that is continuously and highly expressed in hepatocytes and secretes its expression product to the outside of the cells.

[0138] For example, a highly expressed secreted gene may be contained in any safe harbor site in the hepatocyte genome.

[0139] For example, a highly expressed secreted gene may include a promoter and regulatory factors, which may allow the gene to be continuously and highly expressed in the genome of hepatocytes.

[0140] The highly expressed secretory gene may be a wild-type gene present in the genome of hepatocytes.

[0141] The term "wild type" refers to the most common gene in nature, or the allele designated as normal. For example, the wild type may be a genotype in a normal state that does not exhibit a particular disease.

[0142] In addition, the highly expressed secretory gene may be a gene that does not function normally in the hepatocyte genome.

[0143] Here, the highly expressed secretory gene may be a gene in which one or more specific nucleotides are modified compared to the wild type. For example, the modification includes deletion, substitution and / or insertion of one or more nucleotides. The modified highly expressed secretory gene may be expressed in whole or in part, or may not be expressed at all.

[0144] In the disclosure disclosed herein,

[0145] A transgene capable of expressing the desired protein is integrated into a highly expressed secretory gene sequence.

[0146] Here, the corresponding highly expressed secretory gene may be expressed in whole or in part, that is, the transgene and the highly expressed secretory gene may be expressed together.

[0147] Alternatively, the corresponding highly expressed secreted gene can be expressed in hepatocytes and secreted into the blood.

[0148] Alternatively, the corresponding highly expressed secreted gene may not be expressed at all.

[0149] For example, the highly expressed secretory gene can be, for example, ALB gene, FTL gene, FTH1 gene, ACTB gene, HP gene, APOC3 gene, SOD2 gene, ORM1 gene, AAVS1 gene, Rosa gene or HPRT gene. In another example, the highly expressed secretory gene can be IDUA gene, IDS gene, GLA gene or GBA gene.

[0150] In an example, the highly expressed secreted gene may be a HP gene.

[0151] HP gene refers to a gene encoding haptoglobin. In one example, the HP gene may be one or more genes selected from the group consisting of the following genes, but the present invention is not limited thereto: a gene encoding human HP (e.g., NCBI accession numbers NP_001119574, NP_001305067, NP_005134) or mouse HP (NP_001316894, NP_059066), for example, an HP gene expressed by NCBI accession numbers NM_001126102, NM_005143 or NM_001318138.

[0152] In another embodiment, the highly expressed secretory gene may be the APOC3 gene.

[0153] Apolipoprotein C3 (APOC3) gene refers to a gene encoding apolipoprotein C-III, which is a component of very low density lipoprotein (VLDL). The APOC3 gene may be one or more genes selected from the group consisting of the following genes, but the present invention is not limited thereto: a gene encoding human APOC3 (e.g., NCBI Accession No. NP_000031), for example, an APOC3 gene expressed by NCBI Accession No. NM_000040.

[0154] In one example of the disclosure disclosed herein, a LBP system is provided for expressing an artificially inserted transgene in the genome of a hepatocyte.

[0155] The transgene is integrated into a highly expressed secreted gene.

[0156] The highly expressed secretory gene may be at least one selected from the group consisting of ALB gene, FTL gene, FTH1 gene, ACTB gene, HP gene, APOC3 gene, SOD2 gene, ORM1 gene and F9 gene.

[0157] The LBP system is a system in which the transgene is expressed at a higher level than before insertion, or the transgene is newly expressed in hepatocytes.

[0158] [Compositions for expressing proteins of interest]

[0159] One exemplary embodiment of the disclosure disclosed herein relates to a composition that enables a "platform for expressing a protein of interest."

[0160] The composition includes a composition of the LBP system capable of achieving expression of a transgene artificially inserted into the genome of a hepatocyte.

[0161] In an exemplary embodiment, a composition for artificially manipulating a highly expressed secretory gene is provided.

[0162] The composition for artificially manipulating a highly expressed secretory gene can modify any region in the highly expressed secretory gene.

[0163] The term "manipulated by humans (artificial modification or engineering or artificial engineering)" refers to a state in which artificial modification has been performed, rather than the original state existing in the natural state. For example, some nucleotides of a gene may be deleted or replaced, and a gene may be modified by inserting foreign nucleotides or polynucleotides.

[0164] The composition for artificially manipulating a highly expressed secretory gene comprises a programmable nuclease.

[0165] "Programmable nucleases" include all types of nucleases that recognize specific sites on chromosomes of interest to cut genomes. Specifically, programmable nucleases can be clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) systems (which are domains that recognize specific target sequences on chromosomes), transcription activator-like effector nucleases (TALENs) (wherein the cleavage domain derived from plant pathogens and the transcription activator-like (TAL) effector domain are fused), zinc finger nucleases, meganucleases, RNA-guided engineered nucleases (RGENs), Cpf1, FokI endonucleases, or combinations thereof, but the present invention is not limited thereto.

[0166] The programmable nuclease is preferably a CRISPR-Cas system, but the present invention is not limited thereto.

[0167] [CRISPR-Cas system]

[0168] Another exemplary embodiment disclosed herein relates to a composition comprising artificially manipulating a highly expressed secreted gene using a CRISPR-Cas system.

[0169] The CRISPR-Cas system can consist of a guide nucleic acid and / or an editing protein.

[0170] In an exemplary embodiment disclosed herein,

[0171] The composition for manipulating a highly expressed secretory gene may include a guide nucleic acid for manipulating a highly expressed secretory gene.

[0172] The term "guide nucleic acid" refers to a nucleotide sequence that can recognize a target nucleic acid, gene or chromosome and interact with an editing protein. Here, the guide nucleic acid can form a complementary bond with some nucleotides in the target nucleic acid, gene or chromosome.

[0173] The guide nucleic acid can form a guide nucleic acid-editing protein complex and be used to localize the guide nucleic acid-editing protein complex to a target region of a target nucleic acid, gene or chromosome.

[0174] The guide nucleic acid can be a target DNA-specific gRNA, a DNA encoding a gRNA, or a DNA / RNA mixture.

[0175] The guide nucleic acid can be gRNA.

[0176] "Guide RNA (gRNA)" can be transcribed in vitro, particularly from an oligonucleotide duplex or a plasmid template, but the present invention is not limited thereto.

[0177] The guide nucleic acid can be a continuous nucleic acid sequence.

[0178] For example, the continuous nucleic acid sequence may be (N) m , wherein N is A, T, C or G, or is A, U, C or G; and m is an integer of 1-150.

[0179] The guide nucleic acid may be two or more consecutive nucleic acid sequences.

[0180] For example, the two or more consecutive nucleic acid sequences may be (N) m Sum (N) o , wherein N represents A, T, C or G, or represents A, U, C or G; m and o are integers of 1-150, and may be the same as or different from each other.

[0181] A guide nucleic acid may comprise one or more domains.

[0182] The domain may be a functional domain, such as a leader domain, a first complementary domain, a linker domain, a second complementary domain, a proximal domain or a tail domain, but the present invention is not limited thereto.

[0183] Here, a guide nucleic acid may have more than two functional domains. Here, the two or more functional domains may be different from each other. Alternatively, the two or more functional domains contained in a guide nucleic acid may be the same. For example, a guide nucleic acid may have more than two proximal domains; as another example, a guide nucleic acid may have more than two tail domains. However, the expression "the functional domains contained in a guide nucleic acid are the same" does not mean that the sequences of the two functional domains are the same, and when these domains functionally perform the same function, they can be considered to be the same domain even if they have different sequences.

[0184] The domains are described below.

[0185] i) Guide domain

[0186] The term "guide domain" is a domain having a complementary guide sequence capable of forming a complementary binding with a target sequence on a target gene or nucleic acid, and the function is to specifically interact with the target gene or nucleic acid. For example, the guide domain can perform the function of inducing the guide nucleic acid editing protein complex to the location of a specific nucleotide sequence of the target gene or nucleic acid.

[0187] The guide domain can be a sequence of 10-35 bases.

[0188] In examples, the guide domain can be a sequence of 10-35 bases, 15-35 bases, 20-35 bases, 25-35 bases, 30-35 bases.

[0189] In another example, the guide domain can be a sequence of 10-15 bases, 15-20 bases, 20-25 bases, 25-30 bases, 30-35 bases.

[0190] The leader domain may have a leader sequence.

[0191] The guide sequence can be a nucleic acid sequence that is complementary to a target sequence on a target gene or nucleic acid, for example, having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.

[0192] The guide sequence can be a sequence of 10-25 bases.

[0193] In an example, the guide sequence can be a sequence of 10-25 bases, 15-25 bases, or 20-25 bases.

[0194] In another example, the guide sequence can be a sequence of 10-15 bases, 15-20 bases, or 20-25 bases.

[0195] Furthermore, the leader domain may comprise additional base sequences.

[0196] Additional base sequences can be used to increase or decrease the function of the guide domain.

[0197] Additional base sequences can be used to increase or decrease the function of the guide sequence.

[0198] The additional base sequence may be a sequence of 1 to 10 bases.

[0199] In one example, the additional base sequence may be a sequence of 2-10 bases, 4-10 bases, 6-10 bases, or 8-10 bases.

[0200] In another example, the additional base sequence may be a sequence of 1-3 bases, 3-6 bases, or 7-10 bases.

[0201] As specific examples, the additional base sequence may be a sequence of 1 base, 2 bases, 3 bases, 4 bases, 5 bases, 6 bases, 7 bases, 8 bases, 9 bases, or 10 bases.

[0202] In one example, the additional nucleotide sequence may be a 1-base sequence G (guanine) or a 2-base sequence GG.

[0203] The additional base sequence may be located at the 5' end of the guide sequence.

[0204] The additional base sequence may be located at the 3' end of the guide sequence.

[0205] ii) First complementary domain

[0206] The term "first complementary domain" is a nucleic acid sequence comprising a nucleic acid sequence complementary to the second complementary domain, which has sufficient complementarity to form a double strand with the second complementary domain. In one example, the first complementary domain can be a nucleic acid sequence complementary to the second complementary domain, for example, having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or higher complementarity or complete complementarity.

[0207] The first complementary domain can form a double chain with the second complementary domain through complementary binding. Here, the formed double chain can be used to form a guide nucleic acid-editing protein complex by interacting with some amino acids of the editing protein.

[0208] The first complementary domain may be a sequence of 5-35 bases.

[0209] In examples, the first complementary domain can be a sequence of 5-35 bases, 10-35 bases, 15-35 bases, 20-35 bases, 25-35 bases, or 30-35 bases.

[0210] In another example, the first complementary domain can be a sequence of 1-5 bases, 5-10 bases, 10-15 bases, 15-20 bases, 20-25 bases, 25-30 bases, or 30-35 bases.

[0211] iii) Linker domain

[0212] The term "joint domain" is a nucleic acid sequence that connects two or more domains (two or more identical or different domains). The joint domain can be connected to the two or more domains by means of a covalent bond or a non-covalent bond, or can be connected to the two or more domains by means of a covalent bond or a non-covalent bond.

[0213] The linker domain may be a sequence of 1-30 bases.

[0214] In one example, the linker domain can be a sequence of 1-5 bases, 5-10 bases, 10-15 bases, 15-20 bases, 20-25 bases, or 25-30 bases.

[0215] In another example, the linker domain can be a sequence of 1-30 bases, 5-30 bases, 10-30 bases, 15-30 bases, 20-30 bases, or 25-30 bases.

[0216] iv) Second complementary domain

[0217] The term "second complementary domain" refers to a nucleic acid sequence comprising a nucleic acid sequence complementary to the first complementary domain, which has sufficient complementarity to form a double strand with the first complementary domain.

[0218] In one example, the second complementarity domain can be a nucleic acid sequence that is complementary to the first complementarity domain, for example having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.

[0219] The second complementary domain can form a double chain with the first complementary domain through complementary binding. Here, the formed double chain can be used to form a guide nucleic acid-editing protein complex by interacting with some amino acids of the editing protein.

[0220] The second complementary domain may have a base sequence complementary to the first complementary domain and a base sequence not complementary to the first complementary domain (eg, a base sequence that does not form a double strand with the first complementary domain), and may have a longer base sequence than the first complementary domain.

[0221] The second complementary domain may have a sequence of 5-35 bases.

[0222] In examples, the second complementary domain can be a sequence of 1-35 bases, 5-35 bases, 10-35 bases, 15-35 bases, 20-35 bases, 25-35 bases, or 30-35 bases.

[0223] In another example, the second complementary domain can be a sequence of 1-5 bases, 5-10 bases, 10-15 bases, 15-20 bases, 20-25 bases, 25-30 bases, or 30-35 bases.

[0224] v) Proximal domain

[0225] The term "proximal domain" refers to a nucleic acid sequence that is located proximal to a second complementary domain.

[0226] The proximal domain may have a complementary base sequence, and may form a double strand based on the complementary base sequence.

[0227] The proximal domain may be a sequence of 1-20 bases.

[0228] In one example, the proximal domain can be a sequence of 1-20 bases, 5-20 bases, 10-20 bases, or 15-20 bases.

[0229] In another example, the proximal domain can be a sequence of 1-5 bases, 5-10 bases, 10-15 bases, or 15-20 bases.

[0230] vi) Tail domain

[0231] The term "tail domain" is a nucleic acid sequence located at one or more of the two ends of a guide nucleic acid.

[0232] The tail domain may have a complementary base sequence and may form a double strand based on the complementary base sequence.

[0233] The tail domain may be a sequence of 1-50 bases.

[0234] In one example, the tail domain can be a sequence of 5-50 bases, 10-50 bases, 15-50 bases, 20-50 bases, 25-50 bases, 30-50 bases, 35-50 bases, 40-50 bases, or 45-50 bases.

[0235] In another example, the tail domain can be a sequence of 1-5 bases, 5-10 bases, 10-15 bases, 15-20 bases, 20-25 bases, 25-30 bases, 30-35 bases, 35-40 bases, 40-45 bases, or 45-50 bases.

[0236] Meanwhile, part or all of the nucleic acid sequences contained in the domains (ie, the leader domain, the first complementary domain, the linker domain, the second complementary domain, the proximal domain and the tail domain) may optionally or additionally contain chemical modifications.

[0237] The chemical modification may be, but is not limited to, methylation, acetylation, phosphorylation, phosphorothioate linkage, locked nucleic acid (LNA), 2'-O-methyl 3' phosphorothioate (MS), or 2'-O-methyl 3' thio PACE (MSP).

[0238] A guide nucleic acid comprises one or more domains.

[0239] A guide nucleic acid may comprise a guide domain.

[0240] The guide nucleic acid may comprise a first complementarity domain.

[0241] The guide nucleic acid may comprise a linker domain.

[0242] The guide nucleic acid may comprise a second complementarity domain.

[0243] The guide nucleic acid may comprise a proximal domain.

[0244] The guide nucleic acid may comprise a tail domain.

[0245] Here, 1, 2, 3, 4, 5, 6 or more domains may be present.

[0246] A guide nucleic acid may comprise 1, 2, 3, 4, 5, 6 or more guide domains.

[0247] The guide nucleic acid may comprise 1, 2, 3, 4, 5, 6 or more first complementary domains.

[0248] A guide nucleic acid may comprise 1, 2, 3, 4, 5, 6 or more linker domains.

[0249] The guide nucleic acid may comprise 1, 2, 3, 4, 5, 6 or more second complementarity domains.

[0250] A guide nucleic acid may comprise 1, 2, 3, 4, 5, 6 or more proximal domains.

[0251] The guide nucleic acid may comprise 1, 2, 3, 4, 5, 6 or more tail domains.

[0252] Here, in the guide nucleic acid, one type of domain may be repeated.

[0253] A guide nucleic acid may comprise several domains with or without repeats.

[0254] The guide nucleic acid may comprise the same type of domain. Here, the same type of domain may have the same nucleic acid sequence or different nucleic acid sequences.

[0255] The guide nucleic acid may comprise two types of domains. Here, the two different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.

[0256] The guide nucleic acid may comprise three types of domains. Here, the three different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.

[0257] The guide nucleic acid may comprise four types of domains. Here, the four different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.

[0258] The guide nucleic acid may comprise five types of domains. Here, the five different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.

[0259] The guide nucleic acid may comprise six types of domains. Here, the six different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.

[0260] For example, the guide nucleic acid may be composed of [guide domain]-[first complementary domain]-[linker domain]-[second complementary domain]-[linker domain]-[guide domain]-[first complementary domain]-[linker domain]-[second complementary domain]. Here, the two guide domains may contain guide sequences for different or the same targets; the two first complementary domains and the two second complementary domains may have the same or different nucleotide sequences. When the guide domains contain guide sequences for different targets, the guide nucleic acid can specifically bind to two different targets; here, the specific binding can be performed simultaneously or sequentially. In addition, the linker 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 or three parts.

[0261] In an exemplary embodiment disclosed herein, the guide nucleic acid for manipulating a highly expressed secretory gene may be a gRNA for manipulating a highly expressed secretory gene.

[0262] The gRNA can be transcribed in vitro, in particular, from an oligonucleotide duplex or a plasmid template, but the present invention is not limited thereto.

[0263] The term "gRNA" used herein refers to a target DNA-specific RNA that can form a complex with a Cas protein and guide the Cas protein to the target DNA.

[0264] The gRNA may comprise multiple domains. Each domain may have a three-dimensional form or an intra-chain or inter-chain interaction of the active form of the gRNA.

[0265] In an exemplary embodiment, the single-stranded gRNA may include, from the 5' to the 3' direction, a guide domain (e.g., a domain having a guide sequence that can form complementary binding with a target gene or nucleic acid); a first complementary domain; a linker domain; a second complementary domain (the domain is a domain that can form a double-stranded nucleic acid with the first complementary domain because it has a sequence complementary to the sequence of the first complementary domain); a proximal domain; and an optional tail domain.

[0266] In another exemplary embodiment, the double gRNA may comprise a first chain and a second chain, for example, the first chain comprises a guide structure (having a guide sequence that can form complementary binding with a target gene or nucleic acid) and a first complementary domain from the 5' to 3' direction; the second chain comprises a second complementary domain (the domain is a domain that can form a double-stranded nucleic acid with the first complementary domain because it has a sequence complementary to the sequence of the first complementary domain), a proximal domain and an optional tail domain.

[0267] Here, the first strand may refer to crRNA, and the second strand may refer to tracrRNA. The crRNA may include a guide domain and a first complementary domain; the tracrRNA may include a second complementary domain, a proximal domain, and an optional tail domain.

[0268] In another exemplary embodiment, the single-stranded gRNA may include a guide domain (e.g., a domain having a guide sequence that can form complementary binding with a target gene or nucleic acid), a first complementary domain, and a second complementary domain (i.e., the second complementary domain, which is a domain capable of forming a double-stranded nucleic acid with the first complementary domain because it has a sequence complementary to the sequence of the first complementary domain) in the 5' to 3' direction.

[0269] Here, the first complementary domain may have homology with a naturally occurring first complementary domain, or may be derived from a naturally occurring first complementary domain. In addition, the first complementary domain may differ in the nucleotide sequence of the first complementary domain according to naturally occurring species, may be derived from a first complementary domain contained in a naturally occurring species, or may have partial or complete homology with a first complementary domain contained in a naturally occurring species.

[0270] In an exemplary embodiment, the first complementary domain may have partial (ie, at least 50% or more) or complete homology with the first complementary domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus or Neisseria meningitidis or a first complementary domain derived therefrom.

[0271] For example, when the first complementary domain is the first complementary domain of Streptococcus pyogenes or the first complementary domain derived therefrom, the first complementary domain may be 5'-GUUUUAGAGCUA-3' or a base sequence having partial (i.e., at least 50% or more) or complete homology with 5'-GUUUUAGAGCUA-3'. Here, the first complementary domain may further include (X) n , so that it is 5'-GUUUUAGAGCUA(X) n -3'. X may be selected from the group consisting of bases A, T, U and G; n may represent the number of bases, which is an integer from 5 to 15. Here, (X) n It can be n repeats of the same base, or a mixture of n bases A, T, U and G.

[0272] In another embodiment, when the first complementary domain is the first complementary domain of Campylobacter jejuni or the first complementary domain derived therefrom, the first complementary domain may be 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3' or 5'-GUUUUAGUCCCUU-3', or a base sequence having partial (i.e., at least 50% or more) or complete homology with 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3' or 5'-GUUUUAGUCCCUU-3'. Here, the first complementary domain may further comprise (X) n , such that it is 5'-GUUUUAGUCCCUUUUUAAAUUUCUU(X) n -3' or 5'-GUUUUAGUCCCUU(X) n -3'. X may be selected from the group consisting of bases A, T, U and G; n may represent the number of bases, which is an integer from 5 to 15. Here, (X) n It can represent n repeats of the same base, or a mixture of n bases A, T, U and G.

[0273] In another embodiment, the first complementary domain may have partial (i.e., at least 50% or more) or complete homology with the first complementary domain of the following bacteria or the first complementary domain derived therefrom: Parcubacteriabacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae (ND2006), Porphyromonas crevioricanis, Prevotelladisiens, Moraxella bovoculi (237), Smiihella sp. (SC_KO8D17), Leptospira inadai, Lachnospira (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum, or Eubacterium eligens.

[0274] For example, when the first complementary domain is the first complementary domain of Bacteria or the first complementary domain derived therefrom, the first complementary domain may be 5'-UUUGUAGAU-3' or a base sequence having partial (i.e., at least 50% or more) homology with 5'-UUUGUAGAU-3'. Here, the first complementary domain may further include (X) n , such that it is 5'-(X) n UUUGUAGAU-3'. X can be selected from the group consisting of bases A, T, U and G; n can represent the number of bases, which is an integer from 1 to 5. Here, (X) n It can represent n repeats of the same base, or a mixture of n bases A, T, U and G.

[0275] Here, the linker domain may be a nucleotide sequence for connecting the first complementary domain to the second complementary domain.

[0276] The linker domain may be covalently bonded or non-covalently bonded to each of the first complementarity domain and the second complementarity domain.

[0277] The linker domain can covalently or non-covalently link the first complementarity domain to the second complementarity domain.

[0278] The linker domain is suitable for use in a single-stranded gRNA molecule and can form a covalent bond or a non-covalent bond with the first and second strands of the dual gRNA, or be used to generate a single-stranded gRNA through a covalent or non-covalent bond between the first and second strands.

[0279] The linker domain can form a covalent bond or a non-covalent bond with the crRNA and the tracrRNA of the dual gRNA, or can be used to generate a single-stranded gRNA through a covalent or non-covalent bond between the crRNA and the tracrRNA.

[0280] Here, the second complementary domain may have homology with a naturally occurring second complementary domain, or may be derived from a naturally occurring second complementary domain. In addition, the second complementary domain may differ in the nucleotide sequence of the second complementary domain according to naturally occurring species, may be derived from a second complementary domain contained in a naturally occurring species, or may have partial or complete homology with a second complementary domain contained in a naturally occurring species.

[0281] In an exemplary embodiment, the second complementarity domain may have partial (ie, at least 50% or more) or complete homology with the second complementarity domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus, or Neisseria meningitidis, or a second complementarity domain derived therefrom.

[0282] For example, when the second complementary domain is the second complementary domain of Streptococcus pyogenes or a second complementary domain derived therefrom, the second complementary domain may be 5'- UAGC AAGU UAAAA U-3' or with 5'- UAGC AAGU UAAAA U-3' has a base sequence with partial (i.e., at least 50%) homology (the base sequence forming a double strand with the first complementary domain is underlined). Here, the second complementary domain may further include (X) n and / or (X) m , such that it is 5'-(X) n UAGC AAGU UAAAA U(X) m -3'. X can be selected from the group consisting of bases A, T, U and G; n and m can each represent the number of bases, wherein n can be an integer from 1 to 15, and m can be an integer from 1 to 6. Here, (X) n It can represent n repetitions of the same base, or a mixture of n bases A, T, U, and G. In addition, (X) m It can represent m repeats of the same base, or a mixture of m bases A, T, U, and G.

[0283] In another example, when the second complementarity domain is the second complementarity domain of Campylobacter jejuni or a second complementarity domain derived therefrom, the second complementarity domain may be 5'- AAGAAAUUUAAAAAGGGACUAAA U-3' or 5'- AAGGGACUAAAAU -3', or with 5'- AAGAAAUUUAAAAAGGGACUAAA U-3' or 5'- AAGGGACUAAAAU -3' has a partial (ie at least 50%) homology base sequence (the underlined base sequence that forms a double strand with the first complementary domain). Here, the second complementary domain may further include (X) n and / or (X) m , such that it is 5'-(X) n AAGAAAUUUAAAAAGGGACUAAA U(X) m -3' or 5'- AAGGGACUAAAA U(X) m -3'. X can be selected from the group consisting of bases A, T, U and G; n and m can each represent the number of bases, wherein n can be an integer from 1 to 15, and m can be an integer from 1 to 6. Here, (X) n It can represent n repetitions of the same base, or a mixture of n bases A, T, U, and G. In addition, (X) m It can represent m repeats of the same base, or a mixture of m bases A, T, U, and G.

[0284] In another embodiment, the second complementary domain may have partial (i.e., at least 50% or more) or complete homology with the second complementary domain of the following bacteria or the second complementary domain derived therefrom: Thriptorfactant (GWC2011_GWC2_44_17), Lachnospira (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus (BV3L6), Porphyromonas macaques, Lachnospira (ND2006), Porphyromonas crevioricanis, Prevotella saccharolyticus, Moraxella bovoculi (237), Smiihellasp. (SC_KO8D17), Leptospira oryzae, Lachnospira (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum or Eubacterium fastidious.

[0285] For example, when the second complementary domain is the second complementary domain of Bacteria or a second complementary domain derived therefrom, the second complementary domain may be 5'-AAAUU UCUAC U-3' or with 5'-AAAUU UCUAC U-3' has a base sequence with partial (i.e., at least 50%) homology (the base sequence forming a double strand with the first complementary domain is underlined). Here, the second complementary domain may further include (X) n and / or (X) m , such that it is 5'-(X) n AAAUU UCUAC U(X) m -3'. X can be selected from the group consisting of bases A, T, U and G; n and m can each represent the number of bases, wherein n can be an integer from 1 to 10, and m can be an integer from 1 to 6. Here, (X) n It can represent n repetitions of the same base, or a mixture of n bases A, T, U, and G. In addition, (X) m It can represent m repeats of the same base, or a mixture of m bases A, T, U, and G.

[0286] Here, the first complementary domain and the second complementary domain may complementarily bind.

[0287] The first complementary domain and the second complementary domain can form a double chain through complementary binding.

[0288] The resulting duplex can interact with the CRISPR enzyme.

[0289] Optionally, the first complementary domain may comprise an additional nucleotide sequence that does not form complementary binding with the second complementary domain of the second chain.

[0290] Here, the additional nucleotide sequence may be a sequence of 1 to 15 nucleotides. For example, the additional nucleotide sequence may be a sequence of 1 to 5 nucleotides, 5 to 10 nucleotides, or 10 to 15 nucleotides.

[0291] Here, the proximal domain may be located in the 3' direction of the second complementary domain.

[0292] In addition, the proximal domain may have homology with the natural proximal domain, or may be derived from the natural proximal domain. In addition, the proximal domain may differ in base sequence depending on the naturally occurring species, may be derived from the proximal domain contained in the naturally occurring species, or may have partial or complete homology with the proximal domain contained in the naturally occurring species.

[0293] In an exemplary embodiment, the proximal domain may have partial (ie, at least 50% or more) or complete homology with the proximal domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus, or Neisseria meningitidis, or a proximal domain derived therefrom.

[0294] For example, when the proximal domain is the proximal domain of Streptococcus pyogenes or a proximal domain derived therefrom, the proximal domain may be 5'-AAGGCUAGUCCG-3' or a base sequence having partial (i.e., at least 50% or more) homology with 5'-AAGGCUAGUCCG-3'. Here, the proximal domain may further include (X) n , making it 5'-AAGGCUAGUCCG(X) n -3'. X may be selected from the group consisting of bases A, T, U and G; n may represent the number of bases, which may be an integer from 1 to 15. Here, (X) n It can represent n repeats of the same base, or a mixture of n bases A, T, U and G.

[0295] In another embodiment, when the proximal domain is the proximal domain of Campylobacter jejuni or a proximal domain derived therefrom, the proximal domain may be 5'-AAAGAGUUUGC-3' or a base sequence having at least 50% or higher homology with 5'-AAAGAGUUUGC-3'. Here, the proximal domain may further comprise (X) n , making it 5'-AAAGAGUUUGC(X) n -3'. X may be selected from the group consisting of bases A, T, U and G; n may represent the number of bases, which may be an integer from 1 to 40. Here, (X) nIt can represent n repeats of the same base, or a mixture of n bases A, T, U and G.

[0296] Here, a tail domain can be optionally added to the 3' end of the first or second strand of the single-stranded gRNA or double gRNA.

[0297] In addition, the tail domain may have homology with a natural tail domain, or may be derived from a natural tail domain. In addition, the tail domain may differ in base sequence depending on the naturally occurring species, may be derived from a tail domain contained in a naturally occurring species, or may have partial or complete homology with a tail domain contained in a naturally occurring species.

[0298] In an exemplary embodiment, the tail domain may have partial (ie, at least 50% or more) or complete homology with the tail domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus, or Neisseria meningitidis, or a tail domain derived therefrom.

[0299] For example, when the tail domain is the tail domain of Streptococcus pyogenes or a tail domain derived therefrom, the tail domain may be 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' or a base sequence having partial (i.e., at least 50% or more) homology with 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3'. Here, the tail domain may further include (X) n , so that it is 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(X) n -3'. X may be selected from the group consisting of bases A, T, U and G; n may represent the number of bases, which may be an integer from 1 to 15. Here, (X) n It can represent n repeats of the same base, or a mixture of n bases (such as A, T, U, and G).

[0300] In another example, when the tail domain is the tail domain of Campylobacter jejuni or a tail domain derived therefrom, the tail domain may be 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3' or a base sequence having partial (i.e., at least 50% or more) homology to 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3'. Here, the tail domain may further comprise (X) n , so that it is 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU(X)n -3'. X may be selected from the group consisting of bases A, T, U and G; n may represent the number of bases, which may be an integer from 1 to 15. Here, (X) n It may represent n repeats of the same base, or a mixture of n bases A, T, U and G. In another embodiment, the tail domain may contain a sequence of 1-10 bases at the 3' end that participates in in vitro or in vivo transcription methods.

[0301] For example, when the T7 promoter is used for in vitro transcription of the gRNA, the tail domain can be any base sequence present at the 3' end of the DNA template. In addition, when the U6 promoter is used for in vivo transcription, the tail domain can be UUUUUU; when the H1 promoter is used for transcription, the tail domain can be UUUU; and when the pol-III promoter is used, the tail domain can contain several uracil bases or alternative bases.

[0302] The gRNA may comprise multiple domains as described above, and thus the length of the nucleotide sequence may be adjusted according to the domains contained in the gRNA; based on each domain, interactions may occur within or between chains of the three-dimensional structure or active form of the gRNA.

[0303] gRNA can refer to single-stranded gRNA (a single RNA molecule) or dual-gRNA (containing more than one RNA molecule, usually two separate RNA molecules).

[0304] Dual gRNA

[0305] Dual gRNA consists of a first strand and a second strand.

[0306] Here, the first chain can be

[0307] 5'-[leading domain]-[first complementary domain]-3'; and

[0308] The second chain can be

[0309] 5'-[Second complementary domain]-[Proximal domain]-3' or

[0310] 5'-[second complementary domain]-[proximal domain]-[tail domain]-3'.

[0311] Herein, the first strand may refer to crRNA, and the second strand may refer to tracrRNA.

[0312] Furthermore, the first strand and the second strand may optionally contain additional base sequences.

[0313] In one exemplary embodiment, the first chain may be

[0314] 5'-(N 靶标 )-(Q) m -3'; or

[0315] 5'-(X) a -(N 靶标 )-(X) b -(Q) m -(X) c -3'.

[0316] Here, N 靶标 It is a base sequence that can form complementary binding with a target sequence on a target gene or nucleic acid, and is a base sequence region that can be changed according to the target sequence on a target gene or nucleic acid.

[0317] Here, (Q) m It is a base sequence comprising a first complementary domain, which can form a complementary bond with the second complementary domain of the second chain. (Q) m It can be a sequence having partial or complete homology with the first complementary domain of a naturally occurring species; the base sequence of the first complementary domain can be changed according to the species of origin. Q can be independently selected from the group consisting of A, U, C and G; m can be the number of bases, which is an integer of 5-35.

[0318] For example, when the first complementarity domain has partial or complete homology with the first complementarity domain of Streptococcus pyogenes or a first complementarity domain derived from Streptococcus pyogenes, (Q) m It may be 5'-GUUUUAGAGCUA-3' or a base sequence having at least 50% or more homology to 5'-GUUUUAGAGCUA-3'.

[0319] In another example, when the first complementarity domain has partial or complete homology to the first complementarity domain of Campylobacter jejuni or the first complementarity domain derived from Campylobacter jejuni, (Q) m It may be 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3' or 5'-GUUUUAGUCCCUU-3, or a base sequence having at least 50% or more homology to 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3' or 5'-GUUUUAGUCCCUU-3.

[0320] In 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, (Q) mIt may be 5'-GUUUUAGAGCUGUGUUGUUUCG-3' or a base sequence having at least 50% or more homology to 5'-GUUUUAGAGCUGUGUUGUUUCG-3'.

[0321] In addition, (X) a 、(X) b 、(X) c Each is an optional additional base sequence, wherein X can be independently selected from the group consisting of A, U, C and G; a, b, c can be a base number, which is 0 or an integer of 1-20.

[0322] In an exemplary embodiment, the second strand may be 5'-(Z) h -(P) k -3'; or 5'-(X) d -(Z) h -(X) e -(P) k -(X) f -3'.

[0323] In another embodiment, the second strand may be 5'-(Z) h -(P) k -(F) i -3'; or 5'-(X) d -(Z) h -(X) e -(P) k -(X) f -(F) i -3'.

[0324] Here, (Z) h It is a base sequence containing a second complementary domain, which can form a complementary bond with the first complementary domain of the first chain. (Z) h It can be a sequence having partial or complete homology with the second complementary domain of a naturally occurring species; the base sequence of the second complementary domain can be modified according to the species of origin. Z can be independently selected from the group consisting of A, U, C and G; h can be the number of bases, which can be an integer of 5-50.

[0325] For example, when the second complementarity domain has partial or complete homology with the second complementarity domain of Streptococcus pyogenes or a second complementarity domain derived therefrom, (Z) h It may be 5'-UAGCAAGUUAAAAU-3' or a base sequence having at least 50% or more homology to 5'-UAGCAAGUUAAAAU-3'.

[0326] In another example, when the second complementarity domain has partial or complete homology with the second complementarity domain of Campylobacter jejuni or a second complementarity domain derived therefrom, (Z) h It may be 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3' or 5'-AAGGGACUAAAAU-3', or a base sequence having at least 50% or more homology to 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3' or 5'-AAGGGACUAAAAU-3'.

[0327] In another example, when the second complementarity domain has partial or complete homology with the second complementarity domain of Streptococcus thermophilus or a second complementarity domain derived therefrom, (Z) h It may be 5'-CGAAACAACACAGCGAGUUAAAAU-3' or a base sequence having at least 50% or more homology to 5'-CGAAACAACACAGCGAGUUAAAAU-3'.

[0328] (P) k is a base sequence comprising a proximal domain, which may have partial or complete homology with the proximal domain of a naturally occurring species; the base sequence of the proximal domain may be modified according to the species of origin. P may be independently selected from the group consisting of A, U, C and G; k may be a base number, which is an integer of 1-20.

[0329] For example, when the proximal domain has partial or complete homology to the proximal domain of Streptococcus pyogenes or a proximal domain derived therefrom, (P) k It may be 5'-AAGGCUAGUCCG-3' or a base sequence having at least 50% or more homology to 5'-AAGGCUAGUCCG-3'.

[0330] In another embodiment, when the proximal domain has partial or complete homology to the proximal domain of Campylobacter jejuni or a proximal domain derived therefrom, (P) k It may be 5'-AAAGAGUUUGC-3' or a base sequence having at least 50% or more homology to 5'-AAAGAGUUUGC-3'.

[0331] In another example, when the proximal domain has partial or complete homology with the proximal domain of Streptococcus thermophilus or a proximal domain derived therefrom, (P) k It may be 5'-AAGGCUUAGUCCG-3' or a base sequence having at least 50% or more homology to 5'-AAGGCUUAGUCCG-3'.

[0332] (F) i It can be a base sequence comprising a tail domain, which can have partial or complete homology with the tail domain of a naturally occurring species; the base sequence of the tail domain can be modified according to the species of origin. F can be independently selected from the group consisting of A, U, C and G; i can be a base number, which is an integer of 1-50.

[0333] For example, when the tail domain has partial or complete homology to the tail domain of Streptococcus pyogenes or a tail domain derived therefrom, (F) i It may be 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' or a base sequence having at least 50% or more homology to 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3'.

[0334] In another embodiment, when the tail domain has partial or complete homology to the tail domain of Campylobacter jejuni or a tail domain derived therefrom, (F) i It may be 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3' or a base sequence having at least 50% or more homology to 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3'.

[0335] In another embodiment, when the tail domain has partial or complete homology with the tail domain of Streptococcus thermophilus or a tail domain derived therefrom, (F) i It may be 5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3' or a base sequence having at least 50% or more homology to 5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3'.

[0336] In addition, (F) i A sequence of 1-10 bases that participates in in vitro or in vivo transcription methods may be included at the 3' end.

[0337] For example, when the T7 promoter is used for in vitro transcription of the gRNA, the tail domain can be any base sequence present at the 3' end of the DNA template. In addition, when the U6 promoter is used for in vivo transcription, the tail domain can be UUUUUU; when the H1 promoter is used for transcription, the tail domain can be UUUU; and when the pol-III promoter is used, the tail domain can contain several uracil bases or alternative bases.

[0338] In addition, (X) d 、(X) e and (X) f It can be an optionally added base sequence, wherein X can be independently selected from the group consisting of A, U, C and G; d, e, f can be a base number, which is 0 or an integer of 1-20.

[0339] Single-stranded gRNA

[0340] Single-stranded gRNA can be divided into two types, first single-stranded gRNA and second single-stranded gRNA

[0341] First single-stranded gRNA

[0342] First, there is a first single-stranded gRNA, wherein the first strand or the second strand of the dual gRNA is connected by a linker domain.

[0343] Specifically, the single-stranded gRNA can be

[0344] 5'-[guide domain]-[first complementary domain]-[linker domain]-[second complementary domain]-3';

[0345] 5'-[leader domain]-[first complementary domain]-[linker domain]-[second complementary domain]-[proximal domain]-3'; or

[0346] 5'-[leader domain]-[first complementary domain]-[linker domain]-[second complementary domain]-[proximal domain]-[tail domain]-3'.

[0347] The first single-stranded gRNA may optionally contain additional nucleotide sequences

[0348] In an exemplary embodiment, the first single-stranded gRNA may be

[0349] 5'-(N 靶标 )-(Q) m -(L) j -(Z) h -3';

[0350] 5'-(N 靶标 )-(Q) m -(L) j -(Z) h -(P) k -3'; or

[0351] 5'-(N 靶标 )-(Q) m -(L) j -(Z)h -(P) k -(F) i -3'.

[0352] In another exemplary embodiment, the single-stranded gRNA may be

[0353] 5'-(X) a -(N 靶标 )-(X) b -(Q) m -(X) c -(L) j -(X) d -(Z) h -(X) e -3';

[0354] 5'-(X) a -(N 靶标 )-(X) b -(Q) m -(X) c -(L) j -(X) d -(Z) h -(X) e -(P) k -(X) f -3'; or

[0355] 5'-(X) a -(N 靶标 )-(X) b -(Q) m -(X) c -(L) j -(X) d -(Z) h -(X) e -(P) k -(X) f -(F) i -3'.

[0356] Here, N 靶标 is a nucleotide sequence complementary to a partial sequence of either strand of the double strand of the target gene or nucleic acid, and N 靶标 It is a nucleotide sequence site that can be changed according to the target sequence of the target gene or nucleic acid.

[0357] (Q) m It is a nucleotide sequence comprising a first complementary domain, which comprises a nucleotide sequence capable of forming a complementary bond with a second complementary domain. (Q) mIt can be a sequence having partial or complete homology with the first complementary domain of a naturally occurring species; the nucleotide sequence of the first complementary domain can be changed according to the species of origin. Q can be independently selected from the group consisting of A, U, C and G; m can be the number of nucleotides, which can be an integer of 5-35.

[0358] For example, when the first complementary domain is the first complementary domain of Streptococcus pyogenes or has partial or complete homology with the first complementary domain derived from Streptococcus pyogenes, (Q) m It may be 5'-GUUUUAGAGCUA-3' or a nucleotide sequence having at least 50% homology to 5'-GUUUUAGAGCUA-3'.

[0359] In another example, when the first complementarity domain is the first complementarity domain of Campylobacter jejuni or has partial or complete homology to the first complementarity domain derived from Campylobacter jejuni, (Q) m It may be 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3' or 5'-GUUUUAGUCCCUU-3', or a nucleotide sequence having at least 50% homology to 5'-GUUUUAGUCCCUUUUUAAAUUUCUU-3' or 5'-GUUUUAGUCCCUU-3'.

[0360] In another example, when the first complementary domain is the first complementary domain of Streptococcus thermophilus or has partial or complete homology with the first complementary domain derived from Streptococcus thermophilus, (Q) m It may be 5'-GUUUUAGAGCUGUGUUGUUUCG-3' or a nucleotide sequence having at least 50% homology to 5'-GUUUUAGAGCUGUGUUGUUUCG-3'.

[0361] In addition, (L) j It is a nucleotide sequence comprising a linker domain, which can generate a nucleotide sequence of a single-stranded gRNA by connecting the first complementary domain and the second complementary domain. Here, L can be independently selected from the group consisting of A, U, C and G; j can be a nucleotide number, which is an integer of 1-30.

[0362] (Z) h It is a nucleotide sequence comprising a second complementary domain, which comprises a nucleotide sequence capable of forming a complementary bond with the first complementary domain. (Z) hIt can be a sequence having partial or complete homology with the second complementary domain of a naturally occurring species; the nucleotide sequence of the second complementary domain can be changed according to the species of origin. Z can be independently selected from the group consisting of A, U, C and G; h can be the number of nucleotides, which is an integer of 5-50.

[0363] For example, when the second complementary domain is the second complementary domain of Streptococcus pyogenes or has partial or complete homology with the second complementary domain derived from Streptococcus pyogenes, (Z) h It may be 5'-UAGCAAGUUAAAAU-3' or a nucleotide sequence having at least 50% homology to 5'-UAGCAAGUUAAAAU-3'.

[0364] In another example, when the second complementarity domain is the second complementarity domain of Campylobacter jejuni or has partial or complete homology to the second complementarity domain derived from Campylobacter jejuni, (Z) h It may be 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3' or 5'-AAGGGACUAAAAU-3', or a nucleotide sequence having at least 50% homology to 5'-AAGAAAUUUAAAAAGGGACUAAAAU-3' or 5'-AAGGGACUAAAAU-3'.

[0365] In another example, when the second complementarity domain is the second complementarity domain of Streptococcus thermophilus or has partial or complete homology with the second complementarity domain derived from Streptococcus thermophilus, (Z) h It may be 5'-CGAAACAACACAGCGAGUUAAAAU-3' or a nucleotide sequence having at least 50% homology to 5'-CGAAACAACACAGCGAGUUAAAAU-3'.

[0366] (P) k It can be a nucleotide sequence comprising a proximal domain, which is a sequence having partial or complete homology with the proximal domain of a naturally occurring species; the nucleotide sequence of the proximal domain can be changed according to the species of origin. P can be independently selected from the group consisting of A, U, C and G; k can be the number of nucleotides, which is an integer of 1-20.

[0367] For example, when the proximal domain is a proximal domain of Streptococcus pyogenes or has partial or complete homology to a proximal domain derived from Streptococcus pyogenes, (P) k It may be 5'-AAGGCUAGUCCG-3' or a nucleotide sequence having at least 50% homology to 5'-AAGGCUAGUCCG-3'.

[0368] In another example, when the proximal domain is the proximal domain of Campylobacter jejuni or has partial or complete homology to the proximal domain derived from Campylobacter jejuni, (P) k It may be 5'-AAAGAGUUUGC-3' or a nucleotide sequence having at least 50% homology to 5'-AAAGAGUUUGC-3'.

[0369] In another example, when the proximal domain is the proximal domain of Streptococcus thermophilus or has partial or complete homology with the proximal domain derived from Streptococcus thermophilus, (P) k It may be 5'-AAGGCUUAGUCCG-3' or a nucleotide sequence having at least 50% homology to 5'-AAGGCUUAGUCCG-3'.

[0370] (F) i It can be a base sequence comprising a tail domain, which is a sequence having partial or complete homology with the tail domain of a naturally occurring species; the nucleotide sequence of the tail domain can be changed according to the species of origin. F can be independently selected from the group consisting of A, U, C and G; i can be a nucleotide number, which is an integer of 1-50.

[0371] For example, when the tail domain is the tail domain of Streptococcus pyogenes or has partial or complete homology to the tail domain derived from Streptococcus pyogenes, (F) i It may be 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3' or a nucleotide sequence having at least 50% homology to 5'-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3'.

[0372] In another example, when the tail domain is the tail domain of Campylobacter jejuni or has partial or complete homology to the tail domain derived from Campylobacter jejuni, (F) i It may be 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3' or a nucleotide sequence having at least 50% homology to 5'-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3'.

[0373] In another example, when the tail domain is a tail domain of Streptococcus thermophilus or has partial or complete homology to a tail domain derived from Streptococcus thermophilus, (F) iIt may be 5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3' or a nucleotide sequence having at least 50% homology to 5'-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3'.

[0374] In addition, (F) i A sequence of 1-10 nucleotides that participates in in vitro or in vivo transcription methods may be included at the 3' end.

[0375] For example, when the T7 promoter is used for in vitro transcription of the gRNA, the tail domain can be any nucleotide 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 can be UUUUUU; when the H1 promoter is used for transcription, the tail domain can be UUUU; when the pol-III promoter is used, the tail domain can be composed of several uracil nucleotides or contain nucleotides that can be substituted.

[0376] In addition, (X) a 、(X) b 、(X) c 、(X) d 、(X) e and (X) f is a nucleotide sequence that may be optionally added; X may be independently selected from the group consisting of A, U, C and G; a, b, c, d, e and f are nucleotide numbers, which may be 0 or an integer of 1-20.

[0377] Second single-stranded gRNA

[0378] The second single-stranded gRNA may be a single-stranded gRNA consisting of a guide domain, a first complementary domain, and a second complementary domain.

[0379] Here, the second single-stranded gRNA can be

[0380] 5'-[second complementary domain]-[first complementary domain]-[leader domain]-3'; or

[0381] 5'-[second complementary domain]-[linker domain]-[first complementary domain]-[guide domain]-3'.

[0382] The second single-stranded gRNA may optionally contain additional nucleotide sequence

[0383] In an exemplary embodiment, the second single-stranded gRNA may be

[0384] 5'-(Z) h -(Q) m -(N靶标 )-3'; or

[0385] 5'-(X) a -(Z) h -(X) b -(Q) m -(X) c -(N 靶标 )-3'.

[0386] In another exemplary embodiment, the single-stranded gRNA may be

[0387] 5'-(Z) h -(L) j -(Q) m -(N 靶标 )-3'; or

[0388] 5'-(X) a -(Z) h -(L) j -(Q) m -(X) c -(N 靶标 )-3'.

[0389] Here, N 靶标 is a nucleotide sequence complementary to a partial sequence of either strand of the double-stranded target gene or nucleic acid, N 靶标 It is a nucleotide sequence site that can be changed according to the target sequence of the target gene or nucleic acid.

[0390] (Q) m It is a nucleotide sequence comprising a first complementary domain, which comprises a nucleotide sequence capable of forming a complementary bond with a second complementary domain. (Q) m It can be a sequence having partial or complete homology with the first complementary domain of a naturally occurring species; the nucleotide sequence of the first complementary domain can be changed according to the species of origin. Q can be independently selected from the group consisting of A, U, C and G; m can be the number of nucleotides, which can be an integer of 5-35.

[0391] For example, when the first complementary domain is the first complementary domain of Bacillus subtilis or has partial or complete homology with the first complementary domain derived from Bacillus subtilis, (Q) m It may be 5'-UUUGUAGAU-3' or a nucleotide sequence having at least 50% homology to 5'-UUUGUAGAU-3'.

[0392] (Z) h It is a nucleotide sequence comprising a second complementary domain, which comprises a nucleotide sequence capable of forming a complementary bond with the first complementary domain. (Z) hIt can be a sequence having partial or complete homology with the second complementary domain of a naturally occurring species; the nucleotide sequence of the second complementary domain can be changed according to the species of origin. Z can be independently selected from the group consisting of A, U, C and G; h can be the number of nucleotides, which is an integer of 5-50.

[0393] For example, when the second complementary domain is a second complementary domain of Bacillus subtilis or has partial or complete homology with a second complementary domain derived from Bacillus subtilis, (Z) h It may be 5'-AAAUUUCUACU-3' or a nucleotide sequence having at least 50% homology to 5'-AAAUUUCUACU-3'.

[0394] In addition, (L) j is a nucleotide sequence having a linker domain, which is a nucleotide sequence connecting the first complementary domain and the second complementary domain. Here, L can be independently selected from the group consisting of A, U, C and G; j can be the number of nucleotides, which is an integer of 1-30.

[0395] In addition, (X) a 、(X) b and (X) c represents a nucleotide sequence that may be optionally added; X may be each independently selected from the group consisting of A, U, C and G; a, b and c may be the number of nucleotides, each of which is 0 or an integer of 1-20.

[0396] As one aspect of the disclosure herein, the guide nucleic acid is a gRNA that is capable of complementary binding to a target sequence of a highly expressed secretory gene.

[0397] The highly expressed secreted gene is the same as described above.

[0398] The gRNA is a gRNA that can bind to any sequence selected from a secretory gene that is highly expressed in secretory cells.

[0399] Highly expressed secreted genes are target genes or target sequences that are recognized by guide nucleic acids and cleaved by editing proteins.

[0400] "Target sequence" refers to a nucleotide sequence present in a target gene or nucleic acid, and in particular refers to a partial nucleotide sequence of a target region in a target gene or nucleic acid. Here, a "target region" is a site in a target gene or nucleic acid that can be modified by a guide nucleic acid-editing protein.

[0401] Hereinafter, target sequence may be used as a term for information about two nucleotide sequences. For example, in the case of a target gene, the target sequence may mean the sequence information of the transcription strand of the target gene DNA or the nucleotide sequence information of the non-transcription strand. In this application document, the guide nucleic acid using the target sequence including the two aspects is referred to as "guide nucleic acid for the target sequence".

[0402] For example, the target sequence may refer to 5'-ATCATTGGCAGACTAGTTCG-3', which is a partial nucleotide sequence in the target region of target gene A (transcribed chain), or 5'-CGAACTAGTCTGCCAATGAT-3', which is a nucleotide sequence complementary to 5'-ATCATTGGCAGACTAGTTCG-3' (non-transcribed chain).

[0403] The target sequence may be a sequence of 5-50 nucleotides.

[0404] In one embodiment, the target sequence can be a 16 nucleotide sequence, a 17 nucleotide sequence, a 18 nucleotide sequence, a 19 nucleotide sequence, a 20 nucleotide sequence, a 21 nucleotide sequence, a 22 nucleotide sequence, a 23 nucleotide sequence, a 24 nucleotide sequence, or a 25 nucleotide sequence.

[0405] The target sequence includes the binding sequence or the non-binding sequence of the guide nucleic acid.

[0406] The "binding sequence" of the guide nucleic acid is a nucleotide sequence that has partial or complete complementarity with the guide sequence in the guide domain contained in the guide nucleic acid, and it can be complementary to the guide sequence contained in the guide domain of the guide nucleic acid. The target sequence and the guide nucleic acid binding sequence are nucleotide sequences that can be changed according to the target gene or nucleic acid (i.e., the object for gene manipulation or correction), and can be designed into various types according to the target gene or nucleic acid.

[0407] The "non-binding sequence" of the guide nucleic acid is a nucleotide sequence having partial or complete homology with the guide sequence contained in the guide domain of the guide nucleic acid, which may not be complementary to the guide sequence contained in the guide domain of the guide nucleic acid. In addition, the non-guide nucleic acid binding sequence can be a nucleotide sequence that has complementarity with the guide nucleic acid binding sequence and can complementarily bind to the guide nucleic acid binding sequence.

[0408] The guide nucleic acid binding sequence may be a partial nucleotide sequence of the target sequence, which may be a nucleotide sequence having two nucleotide sequences of different sequence orders of the target sequence (i.e., two nucleotide sequences that can complementarily bind to each other). Here, the non-guide nucleic acid binding sequence may be a nucleotide sequence other than the guide nucleic acid binding sequence of the target sequence.

[0409] For example, when 5'-CGAACTAGTCTGCCAATGAT-3' (which is a nucleotide sequence complementary to a partial nucleotide sequence 5'-ATCATTGGCAGACTAGTTCG-3' in the target region of target gene A) is used as a target sequence, the guide nucleic acid binding sequence can be one of the two target sequences, i.e., 5'-ATCATTGGCAGACTAGTTC-3' or 5'-CGAACTAGTCTGCCAATGAT-3'. Here, when the guide nucleic acid binding sequence is 5'-ATCATTGGCAGACTAGTTCG-3', the non-guide nucleic acid binding sequence can be 5'-CGAACTAGTCTGCCAATGAT-3'; or when the guide nucleic acid binding sequence is 5'-CGAACTAGTCTGCCAATGAT-3', the non-guide nucleic acid binding sequence can be 5'-ATCATTGGCAGACTAGTTCG-3'.

[0410] The guide nucleic acid binding sequence may be a nucleotide sequence selected from a nucleotide sequence identical to the target sequence (i.e., the transcription strand) and a nucleotide sequence identical to the non-transcription strand. Here, the non-guide nucleic acid binding sequence may be a nucleotide sequence other than the following nucleotide sequence: a nucleotide sequence selected from a nucleotide sequence identical to the guide nucleic acid binding sequence of the target sequence (i.e., the transcription strand) and a nucleotide sequence identical to the non-transcription strand.

[0411] The length of the binding sequence can be the same as the length of the target sequence.

[0412] The length of the non-binding sequence can be the same as the length of the target sequence or the binding sequence.

[0413] The binding sequence may be a sequence of 5-50 nucleotides.

[0414] In an embodiment, the binding sequence may be a 16 nucleotide sequence, a 17 nucleotide sequence, a 18 nucleotide sequence, a 19 nucleotide sequence, a 20 nucleotide sequence, a 21 nucleotide sequence, a 22 nucleotide sequence, a 23 nucleotide sequence, a 24 nucleotide sequence, or a 25 nucleotide sequence.

[0415] The non-binding sequence may be a sequence of 5-50 nucleotides.

[0416] In embodiments, the non-binding sequence may be a 16 nucleotide sequence, a 17 nucleotide sequence, a 18 nucleotide sequence, a 19 nucleotide sequence, a 20 nucleotide sequence, a 21 nucleotide sequence, a 22 nucleotide sequence, a 23 nucleotide sequence, a 24 nucleotide sequence, or a 25 nucleotide sequence.

[0417] The guide nucleic acid binding sequence may partially or completely complementarily bind to a guide sequence contained in a guide domain of the guide nucleic acid, and the length of the guide nucleic acid binding sequence may be the same as that of the guide sequence.

[0418] The guide nucleic acid binding sequence can be a nucleotide sequence that is complementary to a guide sequence contained in the guide domain of the guide nucleic acid, for example, a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90% or 95% complementary or fully complementary.

[0419] In one example, the guide nucleic acid binding sequence may have or comprise a sequence of 1-8 nucleotides that is not complementary to a guide sequence comprised in the guide domain of the guide nucleic acid.

[0420] The non-guide nucleic acid binding sequence may have partial or complete homology with the guide sequence contained in the guide domain of the guide nucleic acid, and the length of the non-guide nucleic acid binding sequence may be the same as the length of the guide sequence.

[0421] The non-guide nucleic acid binding sequence can be a nucleotide sequence having homology to the guide sequence contained in the guide domain of the guide nucleic acid, for example, a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90% or 95% or more homology or complete homology.

[0422] In one example, the non-guide nucleic acid binding sequence can have or comprise a sequence of 1-8 nucleotides that has no homology to a guide sequence comprised in the guide domain of the guide nucleic acid.

[0423] The non-guide nucleic acid binding sequence may complementarily bind to the guide nucleic acid binding sequence, and the non-guide nucleic acid binding sequence may have the same length as the guide nucleic acid binding sequence.

[0424] The non-guide nucleic acid binding sequence can be a nucleotide sequence that is complementary to the guide nucleic acid binding sequence, for example, a nucleotide sequence that is at least 90% or 95% complementary or completely complementary.

[0425] In one example, a non-guide nucleic acid binding sequence can have or include a sequence of one or two nucleotides that is not complementary to a guide nucleic acid binding sequence.

[0426] In addition, the guide nucleic acid binding sequence may be a nucleotide sequence located adjacent to a nucleotide sequence that can be recognized by an editing protein.

[0427] In one example, the guide nucleic acid binding sequence may be a sequence of 5-50 consecutive nucleotides adjacent to the 5' end and / or 3' end of the nucleotide sequence that can be recognized by the editing protein.

[0428] In addition, the non-guide nucleic acid binding sequence can be a nucleotide sequence adjacent to a nucleotide sequence that can be recognized by an editing protein.

[0429] In one example, the non-guide nucleic acid binding sequence may be a sequence of 5-50 consecutive nucleotides adjacent to the 5' end and / or 3' end of the nucleotide sequence that can be recognized by the editing protein.

[0430] In one embodiment,

[0431] The target sequence may be a continuous sequence of 10-35 nucleotides located in the promoter region of a highly expressed secretory gene.

[0432] The target sequence can be a sequence of 10-35 nucleotides, a sequence of 15-35 nucleotides, a sequence of 20-35 nucleotides, a sequence of 25-35 nucleotides, or a sequence of 30-35 nucleotides.

[0433] The target sequence can be a sequence of 10-15 nucleotides, a sequence of 15-20 nucleotides, a sequence of 20-25 nucleotides, a sequence of 25-30 nucleotides, or a sequence of 30-35 nucleotides.

[0434] In one example, the target sequence may be a continuous sequence of 10-25 nucleotides located in the promoter region of the HP gene.

[0435] In another example, the target sequence may be a continuous sequence of 10-25 nucleotides located in the promoter region of the APOC3 gene.

[0436] In another example, the target sequence may be a continuous sequence of 10-35 nucleotides located in an intronic region of a highly expressed secreted gene.

[0437] The target sequence can be a sequence of 10-35 nucleotides, a sequence of 15-35 nucleotides, a sequence of 20-35 nucleotides, a sequence of 25-35 nucleotides, or a sequence of 30-35 nucleotides.

[0438] The target sequence can be a sequence of 10-15 nucleotides, a sequence of 15-20 nucleotides, a sequence of 20-25 nucleotides, a sequence of 25-30 nucleotides, or a sequence of 30-35 nucleotides.

[0439] In one example, the target sequence may be a contiguous sequence of 10-25 nucleotides located in an intron of the HP gene.

[0440] In another example, the target sequence may be a continuous sequence of 10-25 nucleotides located in an intron of the APOC3 gene.

[0441] In an example, the target sequence may be a contiguous sequence of 10-25 nucleotides located in an exon of a highly expressed secreted gene.

[0442] The target sequence can be a sequence of 10-35 nucleotides, a sequence of 15-35 nucleotides, a sequence of 20-35 nucleotides, a sequence of 25-35 nucleotides, or a sequence of 30-35 nucleotides.

[0443] The target sequence can be a sequence of 10-15 nucleotides, a sequence of 15-20 nucleotides, a sequence of 20-25 nucleotides, a sequence of 25-30 nucleotides, or a sequence of 30-35 nucleotides.

[0444] In an example, the target sequence may be a contiguous sequence of 10-25 nucleotides located in an exon of the HP gene.

[0445] In another example, the target sequence may be a continuous sequence of 10-25 nucleotides located in an exon of the APOC3 gene.

[0446] In an example, the target sequence may be a contiguous sequence of 10-35 nucleotides located in an enhancer of a highly expressed secreted gene.

[0447] The target sequence can be a sequence of 10-35 nucleotides, a sequence of 15-35 nucleotides, a sequence of 20-35 nucleotides, a sequence of 25-35 nucleotides, or a sequence of 30-35 nucleotides.

[0448] The target sequence can be a sequence of 10-15 nucleotides, a sequence of 15-20 nucleotides, a sequence of 20-25 nucleotides, a sequence of 25-30 nucleotides, or a sequence of 30-35 nucleotides.

[0449] In an example, the target sequence may be a contiguous sequence of 10-25 nucleotides located in the enhancer of the HP gene.

[0450] In an example, the target sequence may be a contiguous sequence of 10-25 nucleotides located in the enhancer of the APOC3 gene.

[0451] The target sequence disclosed herein may be a sequence of 10-35 consecutive nucleotides located in the coding portion, the non-coding portion, or a mixed portion thereof of a highly expressed secreted gene.

[0452] The target sequence can be a sequence of 10-35 nucleotides, a sequence of 15-35 nucleotides, a sequence of 20-35 nucleotides, a sequence of 25-35 nucleotides, or a sequence of 30-35 nucleotides.

[0453] Alternatively, the target sequence can be a sequence of 10-15 nucleotides, a sequence of 15-20 nucleotides, a sequence of 20-25 nucleotides, a sequence of 25-30 nucleotides, or a sequence of 30-35 nucleotides.

[0454] In one example, the target sequence can be a sequence of 10-25 consecutive nucleotides located in the coding portion, the non-coding portion, or a mixture thereof of the HP gene.

[0455] In another example, the target sequence can be a sequence of 10-25 consecutive nucleotides located in the coding portion, the non-coding portion, or a mixture thereof of the APOC3 gene.

[0456] The target sequence disclosed herein may be a sequence of 10-35 consecutive nucleotides located in the promoter, enhancer, 3'UTR, poly A tail, or a mixed portion thereof of a highly expressed secretory gene.

[0457] The target sequence can be a sequence of 10-35 nucleotides, a sequence of 15-35 nucleotides, a sequence of 20-35 nucleotides, a sequence of 25-35 nucleotides, or a sequence of 30-35 nucleotides.

[0458] The target sequence can be a sequence of 10-15 nucleotides, a sequence of 15-20 nucleotides, a sequence of 20-25 nucleotides, a sequence of 25-30 nucleotides, or a sequence of 30-35 nucleotides.

[0459] In one example, the target sequence may be a sequence of 10-25 consecutive nucleotides located in the promoter, enhancer, 3'UTR, poly A tail, or a mixture thereof of the HP gene.

[0460] In another example, the target sequence may be a sequence of 10-25 consecutive nucleotides located in the promoter, enhancer, 3'UTR, poly A tail, or a mixed portion thereof of the APOC3 gene.

[0461] The target sequence disclosed herein may be a sequence of 10-35 consecutive nucleotides located in an exon or intron or a mixed portion thereof of a highly expressed secreted gene.

[0462] The target sequence can be a sequence of 10-35 nucleotides, a sequence of 15-35 nucleotides, a sequence of 20-35 nucleotides, a sequence of 25-35 nucleotides, or a sequence of 30-35 nucleotides.

[0463] The target sequence can be a sequence of 10-15 nucleotides, a sequence of 15-20 nucleotides, a sequence of 20-25 nucleotides, a sequence of 25-30 nucleotides, or a sequence of 30-35 nucleotides.

[0464] In one example, the target sequence may be a sequence of 10-25 consecutive nucleotides located in an exon or intron of the HP gene or a mixed portion thereof.

[0465] In another example, the target sequence may be a sequence of 10-25 consecutive nucleotides located in an exon or intron or a mixed portion thereof of the APOC3 gene.

[0466] The target sequence disclosed herein may be a sequence of 10-35 consecutive nucleotides that includes or is adjacent to a mutated portion (eg, a portion that is different from the wild-type gene) of a highly expressed secreted gene.

[0467] The target sequence can be a sequence of 10-35 nucleotides, a sequence of 15-35 nucleotides, a sequence of 20-35 nucleotides, a sequence of 25-35 nucleotides, or a sequence of 30-35 nucleotides.

[0468] The target sequence can be a sequence of 10-15 nucleotides, a sequence of 15-20 nucleotides, a sequence of 20-25 nucleotides, a sequence of 25-30 nucleotides, or a sequence of 30-35 nucleotides.

[0469] In one example, the target sequence can be a sequence of 10-25 consecutive nucleotides that includes or is adjacent to a mutated portion of the HP gene (eg, a portion that differs from the wild-type gene).

[0470] In another example, the target sequence can be a sequence of 10-25 consecutive nucleotides that includes or is adjacent to a mutated portion of the APOC3 gene (eg, a portion that is different from the wild-type gene).

[0471] The target sequence disclosed herein may be a sequence of 10 to 35 consecutive nucleotides adjacent to the 5' end and / or 3' end of a protospacer adjacent motif (PAM) sequence in a nucleotide sequence of a highly expressed secreted gene.

[0472] The "protospacer adjacent motif (PAM) sequence" is a nucleotide sequence recognized by the editing protein. Here, the nucleotide sequence of the PAM sequence may vary depending on the type of editing protein and the species from which the PAM sequence is derived.

[0473] For example, the PAM sequence can be one or more of the following sequences (described in the 5' to 3' direction):

[0474] NGG (N is A, T, C or G);

[0475] NNNNRYAC (each N is independently A, T, C or G; R is A or G; Y is C or T);

[0476] NNAGAAW (N is independently A, T, C or G; W is A or T);

[0477] NNNNGATT (each N is independently A, T, C or G);

[0478] NNGRR(T) (each N is independently A, T, C or G; R is A or G; Y is C or T); and

[0479] TTN (N is A, T, C or G).

[0480] In examples, the target sequence can be a sequence of 10-35 bases, 15-35 bases, 20-35 bases, 25-35 bases, or 30-35 bases.

[0481] In examples, the target sequence can be a sequence of 10-15 bases, 15-20 bases, 20-25 bases, 25-30 bases, or 30-35 bases.

[0482] In one example, the target sequence may be a sequence of 10 to 25 consecutive nucleotides adjacent to the 5' end and / or 3' end of the PAM sequence in the nucleotide sequence of the HP gene.

[0483] In an exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' and / or 5'-NGA-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a sequence of 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NGG-3', 5'-NAG-3' and / or 5'-NGA-3' (N=A, T, G or C; or A, U, G or C) sequence in the nucleotide sequence adjacent to the HP gene.

[0484] In another exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' and / or 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a sequence of 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NGGNG-3' and / or 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C) sequence in the nucleotide sequence adjacent to the HP gene.

[0485] In another exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNNGATT-3' and / or 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a sequence of 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NNNNGATT-3' and / or 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C) sequence in the nucleotide sequence adjacent to the HP gene.

[0486] In an exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a sequence of 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C) sequence in the nucleotide sequence adjacent to the HP gene.

[0487] In another exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C), the target sequence may be a sequence of 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C) sequence in the nucleotide sequence adjacent to the HP gene.

[0488] In another exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' and / or 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C), the target sequence may be a sequence of 10-25 consecutive nucleotides at the 5' and / or 3' end of the 5'-NNGRR-3', 5'-NNGRRT-3' and / or 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C) sequence in the nucleotide sequence adjacent to the HP gene.

[0489] In an exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-TNN-3' (N = A, T, G or C; or A, U, G or C), the target sequence may be a sequence of 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-TNN-3' (N = A, T, G or C; or A, U, G or C) sequence in the nucleotide sequence adjacent to the HP gene.

[0490] In another example, the target sequence may be a sequence of 10 to 25 consecutive nucleotides adjacent to the 5' end and / or 3' end of the PAM sequence in the nucleotide sequence of the APOC3 gene.

[0491] In an exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' and / or 5'-NGA-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a sequence of 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NGG-3', 5'-NAG-3' and / or 5'-NGA-3' (N=A, T, G or C; or A, U, G or C) sequence in the nucleotide sequence adjacent to the APOC3 gene.

[0492] In another exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' and / or 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a sequence of 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NGGNG-3' and / or 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C) sequence in the nucleotide sequence adjacent to the APOC3 gene.

[0493] In another exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNNGATT-3' and / or 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C), the target sequence may be a sequence of 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NNNNGATT-3' and / or 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C) sequence in the nucleotide sequence adjacent to the APOC3 gene.

[0494] In an exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C), the target sequence may be a sequence of 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C) sequence in the nucleotide sequence of the APOC3 gene.

[0495] In another exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C), the target sequence may be a sequence of 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C) sequence in the nucleotide sequence of the APOC3 gene.

[0496] In another exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' and / or 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C), the target sequence may be a sequence of 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NNGRR-3', 5'-NNGRRT-3' and / or 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C) sequence in the nucleotide sequence of the APOC3 gene.

[0497] In an exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-TNN-3' (N = A, T, G or C; or A, U, G or C), the target sequence may be a sequence of 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-TNN-3' (N = A, T, G or C; or A, U, G or C) sequence in the nucleotide sequence of the APOC3 gene.

[0498] Hereinafter, examples of target sequences that can be used in an exemplary embodiment disclosed herein are summarized in the following table, and the target sequences shown in the following table are non-guide nucleic acid binding sequences, and from the disclosed sequences, complementary sequences (i.e., guide nucleic acid binding sequences) can be expected.

[0499] Table 1 Target sequences of HP gene and APOC3 gene

[0500]

[0501]

[0502]

[0503]

[0504]

[0505]

[0506]

[0507]

[0508]

[0509]

[0510] In another example, a composition for engineering a highly expressed secreted gene can include a guide nucleic acid and an editing protein.

[0511] For example, the composition may comprise

[0512] A guide nucleic acid directed against a target sequence of one or more genes selected from the group consisting of highly expressed secretory genes expressed in the liver; and

[0513] An editing protein or a nucleic acid encoding an editing protein.

[0514] The description concerning the highly expressed secretory gene is the same as described above.

[0515] (1) Editing proteins

[0516] The term "editing protein" refers to a peptide, polypeptide or protein that can directly bind to a nucleic acid or interact with a nucleic acid without direct binding. Editing protein also conceptually refers to "artificially engineered nuclease" or RGEN (RNA-guided endonuclease).

[0517] The editing protein may be an enzyme.

[0518] The term "enzyme" refers to a protein containing a domain capable of cleaving a nucleic acid, gene, chromosome or protein.

[0519] The enzyme may be a nuclease or a restriction enzyme.

[0520] The editing protein may include a fully active enzyme.

[0521] Here, "enzyme with full activity" refers to an enzyme with the same function as that of a wild-type enzyme, for example, a wild-type enzyme that cuts double-stranded DNA has full enzymatic activity that completely cuts double-stranded DNA. In another example, when a partial sequence of an amino acid sequence is deleted or replaced by artificially manipulating a wild-type enzyme (cutting double-stranded DNA), if the artificially manipulated enzyme variant cuts double-stranded DNA like the wild-type enzyme, the artificially manipulated enzyme variant may be an enzyme with full activity.

[0522] In addition, an enzyme with full activity includes an enzyme with improved function compared to the function of a wild-type enzyme, for example, a specific modification or manipulation of a wild-type enzyme that cleaves double-stranded DNA has improved full enzyme activity compared to the wild-type enzyme (i.e., activity to cleave double-stranded DNA).

[0523] Editing proteins may include enzymes with incomplete or partial activity.

[0524] Here, "an enzyme with incomplete or partial activity" refers to an enzyme that has a partial function of the original wild-type enzyme to cut nucleic acids, genes or chromosomes. For example, a specific modification or manipulation type of a wild-type enzyme that cuts double-stranded DNA may be a type with a first function or a type with a second function. Here, the first function may be a function of cutting the first strand of double-stranded DNA, and the second function may be a function of cutting the second strand of double-stranded DNA. Here, the enzyme with the first function or the enzyme with the second function may be an enzyme with incomplete or partial activity.

[0525] The editing protein may include an inactivated enzyme.

[0526] Here, "inactivated enzyme" refers to an enzyme in which the function of the wild-type enzyme is completely inactivated. For example, a specific modification or manipulation type of the wild-type enzyme may be a type in which both the first function and the second function are lost, that is, a type in which both the first function of cutting the first strand of double-stranded DNA and the second function of cutting the second strand of double-stranded DNA are lost. Here, an enzyme that loses both the first function and the second function may be an inactivated enzyme.

[0527] The editing protein may be a fusion protein.

[0528] Here, fusion protein refers to a protein produced by fusing an enzyme with an additional domain, peptide, polypeptide or protein.

[0529] The additional domain, peptide, polypeptide or protein may be a functional domain, peptide, polypeptide or protein having the same or different function as the enzyme.

[0530] The fusion protein may comprise an additional domain, peptide, polypeptide or protein at one or more of the N-terminus or near the enzyme, the C-terminus or near the enzyme, the middle part of the enzyme, and combinations thereof.

[0531] Here, the functional domain, peptide, polypeptide or protein may be a domain, peptide, polypeptide or protein having methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity or nucleic acid binding activity, or a tag or reporter gene for purifying and isolating proteins (including peptides), but the present invention is not limited thereto.

[0532] The functional domain, peptide, polypeptide or protein may be a deaminase.

[0533] Tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags and thioredoxin (Trx) tags; reporter genes include glutathione S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, autofluorescent proteins (including green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and blue fluorescent protein (BFP)), but the present invention is not limited thereto.

[0534] Furthermore, the functional domain, peptide, polypeptide or protein may be a nuclear localization sequence or signal (NLS) or a nuclear export sequence or signal (NES).

[0535] The NLS may be: the NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV; an NLS derived from a nucleoplasmin (e.g., a nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK); a c-myc NLS having the amino acid sequence PAAKRVKLD or RQRRNELKRSP; an hRNPA1M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; an IBB domain sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV derived from importin-α; the myoma T protein sequences VSRKRPRP and PPKKARED; the human p53 sequence POPKKKPL; and a mouse c-abl IV sequence SALIKKKKKMAP; influenza virus NS1 sequences DRLRR and PKQKKRK; hepatitis virus δ antigen sequence RKLKKKIKKL; mouse Mx1 protein sequence REKKKFLKRR; human poly (ADP-ribose) polymerase sequence KRKGDEVDGVDEVAKKKSKK; or steroid hormone receptor (human) glucocorticoid sequence RKCLQAGMNLEARKTKK, but the present invention is not limited thereto.

[0536] The additional domain, peptide, polypeptide or protein can be a non-functional domain, peptide, polypeptide or protein that does not show a specific function. Here, the non-functional domain, peptide, polypeptide or protein can be a domain, peptide, polypeptide or protein that does not affect the enzyme function.

[0537] Fusion proteins can be of the type in which a non-functional domain, peptide, polypeptide or protein is added to one or more of the amino terminus or near the amino terminus of an enzyme, the carboxyl terminus of an enzyme or near the carboxyl terminus of an enzyme, the middle portion of an enzyme, or a combination thereof.

[0538] The editing protein can be a natural enzyme or a fusion protein.

[0539] The editing protein can exist in the form of a partially modified native enzyme or a fusion protein.

[0540] The editing protein may be an artificially produced enzyme or fusion protein that does not exist in nature.

[0541] The editing protein may be present in the form of a partially modified artificial enzyme or a fusion protein that does not exist in the natural state.

[0542] Here, the modification may be substitution, deletion, addition, or a combination of the above modifications of the amino acids contained in the editing protein.

[0543] In addition, the modification may be substitution, deletion, addition, or a combination of the above modifications of some bases in the base sequence encoding the editing protein.

[0544] The guide nucleic acid and the editing protein can form a guide nucleic acid-editing protein complex.

[0545] The guide nucleic acid-editing protein complex can be formed in vitro.

[0546] The guide nucleic acid-editing protein complex can be formed in the cytoplasm of the cell.

[0547] The guide nucleic acid-editing protein complex can be formed in the nucleus of the cell.

[0548] In the guide nucleic acid-editing protein complex, the editing protein recognizes the PAM present in the target gene or nucleotide sequence.

[0549] On the guide nucleic acid-editing protein complex, the guide nucleic acid can complementarily bind to the target gene or nucleotide sequence.

[0550] When the guide nucleic acid-editing protein complex binds to the target gene or nucleotide sequence, the target gene or nucleotide sequence can be cleaved or modified by the editing protein of the guide nucleic acid-editing protein complex.

[0551] In one aspect of the disclosure disclosed herein, the editing protein may be a CRISPR enzyme.

[0552] For example, the composition may comprise

[0553] gRNA directed against a target sequence of one or more genes selected from the group consisting of highly expressed secretory genes expressed in the liver; and

[0554] CRISPR enzyme or nucleic acid encoding a CRISPR enzyme.

[0555] The description of gRNA is the same as described above.

[0556] The CRISPR enzyme can be a nuclease or a restriction enzyme that has the function of cleaving the target gene or double-stranded nucleic acid.

[0557] "CRISPR enzyme" is the main protein component of the CRISPR-Cas system, which refers to the nuclease that mixes or forms a complex with gRNA to recognize the target sequence and cut DNA.

[0558] The "CRISPR-Cas system" originates from the acquired immune system, which stores the genetic information of bacterial pathogens that invade from the outside and then cuts the genetic information; the "CRISPR-Cas system" refers to a gene correction system, which consists of a gRNA that manipulates genetic information to recognize the desired target sequence and a Cas protein that recognizes DNA, thereby removing and regulating gene function.

[0559] Cas protein can form a complex with CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), thereby expressing its activity.

[0560] Cas protein is used herein as a concept including all variants and natural proteins acting as endonucleases or nickases (activated by cooperation with gRNA). Activated endonucleases or nickases can cause cutting of target DNA, and gene correction using the cutting. In addition, inactivated variants can cause separation and transcriptional regulation of desired DNA.

[0561] Cas proteins can be fully active CRISPR enzymes.

[0562] "Fully active CRISPR enzyme" refers to an enzyme that has the same function as the original wild-type enzyme (cleaving nucleic acids, genes or chromosomes). That is, a fully active CRISPR enzyme refers to a state in which it has both the function of cleaving the first strand of double-stranded DNA and the second function of cleaving the second strand of double-stranded DNA.

[0563] When the artificially manipulated CRISPR enzyme variant is an enzyme that cuts double-stranded DNA like the wild-type enzyme, the artificially manipulated CRISPR enzyme may also be included in the enzyme with full activity.

[0564] An artificially manipulated CRISPR enzyme may be one in which a portion of the nucleotide sequence is deleted, substituted, or added.

[0565] An artificially manipulated CRISPR enzyme may be one in which one or more amino acids of the amino acid sequence are deleted, substituted or added.

[0566] The amino acid addition modification can be the N-terminus and / or C-terminus of the wild-type enzyme, or its adjacent region. In addition, the modification can be a combination thereof.

[0567] A CRISPR enzyme variant may be a fully active enzyme with improved function compared to a wild-type CRISPR enzyme.

[0568] For example, a specific modification or manipulation type of a wild-type CRISPR enzyme (i.e., a CRISPR enzyme variant) can cut the DNA double strand near a certain distance of the double-stranded DNA to be cut, or cut the DNA double strand while forming a specific binding with the double-stranded DNA. Here, the specific binding can be a binding to a DNA nucleotide sequence at an amino acid cleavage position at a specific position of the enzyme. In this case, the type of modification or manipulation can be a fully active CRISPR enzyme with reduced functional activity compared to the wild-type CRISPR enzyme.

[0569] The CRISPR enzyme may be a CRISPR enzyme with incomplete or partial activity.

[0570] "Having incomplete or partial activity" refers to a state of having the function of a wild-type CRISPR enzyme, wherein the function is one selected from a first function of cleaving the first strand of double-stranded DNA and a second function of cleaving the second strand of double-stranded DNA. The CRISPR enzyme in this state may be referred to as a CRISPR enzyme having incomplete or partial activity. In addition, a CRISPR enzyme having incomplete or partial activity may be referred to as a nickase.

[0571] "Nickase" refers to a CRISPR enzyme that has been manipulated or modified to cut only one strand of the target gene or nucleic acid double strand, and the nickase has a nuclease activity that cuts a single strand (e.g., a strand that is not complementary to the gRNA complementary to the target gene or nucleic acid or a strand complementary to it). Therefore, the nuclease activity of two nickases is required to cut the double strand.

[0572] For example, a nickase may have nuclease activity caused by a RuvC domain. That is, a nickase may not contain nuclease activity caused by a HNH domain, and thus, the HNH domain may be manipulated or modified.

[0573] In addition to the above-mentioned nuclease activity, CRISPR enzymes can also have helicase activity, which is the function of unwinding the helical structure of double-stranded nucleic acids.

[0574] Furthermore, the CRISPR enzyme may be modified such that the helicase activity of the CRISPR enzyme will be fully active, incompletely or partially active, or inactivated.

[0575] The CRISPR enzyme may be a nucleic acid or a polypeptide (or protein) having a sequence encoding a CRISPR enzyme, and is typically a type II CRISPR enzyme.

[0576] The crystal structure of the type II CRISPR enzyme was determined based on studies of more than two types of natural microbial type II CRISPR enzyme molecules (Jinek et al., Science, 343(6176):1247997, 2014) and studies of the complex of Streptococcus pyogenes Cas9 (SpCas9) and gRNA (Nishimasu et al., Cell, 156:935-949, 2014; and Anders et al., Nature, 2014, doi:10.1038 / nature13579).

[0577] The type II CRISPR enzyme can be Cas9.

[0578] "Cas9" is an enzyme that binds to gRNA to cut or modify a target sequence or position on a target gene or nucleic acid, and can be composed of a HNH domain (capable of cutting a nucleic acid chain complementary to gRNA), a RuvC domain (capable of cutting a nucleic acid chain not complementary to gRNA), a REC domain (capable of recognizing a target), and a PI domain (capable of recognizing a PAM). For the specific structural features of Cas9, reference can be made to Hiroshi Nishimasu et al. (2014) Cell 156:935-949.

[0579] Here, the RuvC domain shares structural similarity with members of the naturally occurring microbial family containing type II CRISPR enzymes, and cuts a single strand (e.g., a non-complementary strand of a target gene or nucleic acid, i.e., a strand that does not form a complementary bond with the gRNA). In the art, the RuvC domain is sometimes referred to as a RuvCI domain, a RuvCII domain, or a RuvCIII domain, generally referred to as RuvCI, RuvCII, or RuvCIII.

[0580] The HNH domain shares structural similarity with the HNH endonuclease and cuts a single strand (e.g., the complementary strand of the target nucleic acid molecule, i.e., the strand that forms a complementary bond with the gRNA). The HNH domain is located between the RuvCII and RuvCIII motifs.

[0581] Cas9 can be fully active Cas9 or inactivated Cas9.

[0582] The inactivated Cas9 may include completely inactivated Cas9 and partially inactivated Cas9 (eg, nickase).

[0583] Cas9 can be derived from a variety of microorganisms, such as Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius. salivarius), Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp.), Acetohalobiumarabaticum, Ammonifex degensii, Caldicelulosiruptor bescii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobiusthermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillusferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus or Acaryochloris marina.

[0584] Cas9 can be isolated from a naturally occurring microorganism or produced non-naturally by recombinant or synthetic methods.

[0585] Furthermore, the CRISPR enzyme may be a type V CRISPR enzyme.

[0586] Type V CRISPR enzymes contain a similar RuvC domain (corresponding to the RuvC domain of type II CRISPR enzymes), and may be composed of a Nuc domain (instead of the HNH domain of type II CRISPR enzymes), a REC domain and a WED domain (recognizing targets), and a PI domain (recognizing PAM). For the specific structural features of type V CRISPR enzymes, see Takashi Yamano et al. (2016) Cell 165: 949-962.

[0587] The V-type CRISPR enzyme can interact with the gRNA to form a gRNA-CRISPR enzyme complex, i.e., a CRISPR complex, and can allow the guide sequence to approach the target sequence containing the PAM sequence under the cooperation of the gRNA. Here, the ability of the V-type CRISPR enzyme to interact with the target gene or nucleic acid depends on the PAM sequence. The PAM sequence is a sequence present in the target gene or nucleic acid and can be recognized by the PI domain of the V-type CRISPR enzyme. The PAM sequence can be changed depending on the source of the V-type CRISPR enzyme. That is, depending on the species, there are different PAM sequences that can be specifically recognized. In one example, the PAM sequence recognized by Cpf1 can be 5'-TTN-3' (N is A, T, C or G).

[0588] However, while it is generally understood that the PAM is determined based on the source of the enzyme as described above, the PAM may vary based on the results of ongoing studies of mutants of the enzyme derived from the corresponding source.

[0589] The type V CRISPR enzyme can be Cpf1,

[0590] Cpf1 can be derived from the following microorganisms: Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospirium, C lostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroides, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium, or Acidaminococcus.

[0591] Cpf1 can be composed of a RuvC domain (similar to and corresponding to the RuvC domain of Cas9), a Nuc domain (rather than the HNH domain of Cas9), a REC domain and a WED domain (recognizing the target) and a PI domain (recognizing PAM). For the specific structural features of Cpf1, see Takashi Yamano et al. (2016) Cell 165: 949-962.

[0592] The Cpf1 may be fully active Cpf1 or inactivated Cpf1.

[0593] Inactivated Cpf1 may include completely inactivated Cpf1 and partially inactivated Cpf1 (eg, nickase).

[0594] In the Cpf1 enzyme, one, two or more of the amino acids present in the RuvC, Nuc, WED, REC and / or PI domains may be mutated.

[0595] The Cpf1 enzyme may comprise amino acids D917, E1006 or D1255 of FnCpf1; amino acids D908, E993 or D1263 of AsCpf1; amino acids D832, E925, D947 or D1180 of LbCpf1; or mutations in one or two or more amino acids in the amino acid groups corresponding to each of the different Cpf1 orthologs.

[0596] The CRISPR enzyme, Cas9 or Cpf1 protein can be isolated from naturally occurring microorganisms or produced non-naturally by recombinant or synthetic methods.

[0597] The Cas protein or the nucleic acid encoding the Cas protein can function in the nucleus.

[0598] In addition, the separated Cas protein can be easily introduced into the cell. For example, the Cas protein can be connected to a cell penetrating peptide or a protein transduction domain. The protein transduction domain can be a TAT protein derived from polyarginine or HIV, but the present invention is not limited thereto. Since various types of cell penetrating peptides or protein transduction domains other than the above types are known in the art, various examples can be applied to the present application document by those of ordinary skill in the art without restriction.

[0599] The CRISPR enzyme may be a CRISPR enzyme variant prepared by artificially manipulating or modifying a wild-type CRISPR enzyme.

[0600] In one example, a CRISPR enzyme variant can be prepared by replacing, deleting and / or adding at least one amino acid of the amino acid sequence of a wild-type CRISPR enzyme.

[0601] A CRISPR enzyme variant may be a CRISPR enzyme variant that has been artificially manipulated or modified to modify the function of a wild-type CRISPR enzyme (i.e., a first function of cleaving a first strand of double-stranded DNA and a second function of cleaving a second strand of double-stranded DNA).

[0602] Furthermore, in addition to the inherent features of the CRISPR enzyme, the CRISPR enzyme mutant may further comprise an optional functional domain, so that the CRISPR enzyme mutant may also have additional features in addition to the inherent features.

[0603] Here, the functional domain may be a domain having methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity or nucleic acid binding activity, or a tag or reporter gene for purifying and isolating proteins (including peptides), but the present invention is not limited thereto.

[0604] Tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags and thioredoxin (Trx) tags; reporter genes include glutathione S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, autofluorescent proteins (including green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and blue fluorescent protein (BFP)), but the present invention is not limited thereto.

[0605] The functional domain, peptide, polypeptide or protein may be a deaminase.

[0606] For example, an incomplete or partial CRISPR enzyme may additionally comprise a cytidine deaminase as a functional domain. In an exemplary embodiment, a cytidine deaminase (e.g., apolipoprotein B editing complex 1 (APOBEC1)) may be added to the SpCas9 nickase to generate a fusion protein. The [SpCas9 nickase]-[APOBEC1] thus formed can be used for editing or base repair from C to T or U, or from G to A.

[0607] In another example, it can be said that adenine deaminase can be further included in an incomplete or partial CRISPR enzyme as a functional domain. As an exemplary embodiment, a fusion protein can be produced by adding an adenine deaminase (e.g., TadA variant, ADAR2 variant, ADAT2 variant, etc.) to the SpCas9 nickase. Since in the [SpCas9 nickase]-[TadA variant], [SpCas9 nickase]-[ADAR2 variant] or [SpCas9 nickase]-[ADAT2 variant] prepared as described above, nucleotide A is modified to inosine, and the modified inosine is recognized as nucleotide G by the polymerase, thereby showing the effect of substantially editing or nucleotide correction of nucleotide A to G, the modified inosine can be used for editing or nucleotide correction of nucleotide A to G, or for editing or nucleotide correction of nucleotide T to C.

[0608] Furthermore, the functional domain may be a nuclear localization sequence or signal (NLS) or a nuclear export sequence or signal (NES).

[0609] In one example, the CRISPR enzyme may comprise one or more NLS. Here, the one or more NLS may be contained at or near the N-terminus of the CRISPR enzyme, at or near the C-terminus of the enzyme, or a combination thereof. The NLS may be an NLS sequence derived from the following NLSs, but the present invention is not limited thereto: the NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV; the NLS from the nucleoplasmic protein (e.g., the bipartite nucleoplasmic protein NLS having the sequence KRPAATKKAGQAKKKK); the c-myc NLS having the amino acid sequence PAAKRVKLD or RQRRNELKRSP; the hRNPA1M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV from the IBB domain of importin α; the sequences VSRKRPRP and PPKKARED of myoma T protein; the sequence POPKKKPL of human p53; the sequence of mouse c-abl The sequence of IV is SALIKKKKKMAP; the sequences of influenza virus NS1 are DRLRR and PKQKKRK; the sequence of hepatitis virus delta antigen is RKLKKKIKKL; the sequence of mouse Mx1 protein is REKKKFLKRR; the sequence of human poly (ADP-ribose) polymerase is KRKGDEVDGVDEVAKKKSKK; or the NLS sequence derived from the sequence of steroid hormone receptor (human) glucocorticoid is RKCLQAGMNLEARKTKK.

[0610] The CRISPR enzyme or CRISPR enzyme mutant described in the present invention can be a polypeptide, a protein, or a nucleic acid having a sequence encoding the polypeptide or protein, and codon optimization can be performed for the subject to be introduced with the CRISPR enzyme or CRISPR enzyme mutant.

[0611] The term "codon optimization" refers to a modification process to a nucleotide sequence that improves expression in a host cell by replacing at least one codon in the native sequence with a codon that is more common or most frequently used in the host cell while maintaining the native amino acid sequence. Various species have specific preferences for specific codons for specific amino acids, and the codon preference (differences in codon usage between different organisms) is generally related to the translation efficiency of mRNA, and it is believed that this depends on the characteristics of the codons translated and the availability of specific tRNA molecules. The dominant tRNA selected in the cell generally reflects the most frequently used codons in peptide synthesis. Therefore, genes can be customized in a given organism by optimizing gene expression based on codon optimization.

[0612] gRNA and CRISPR enzyme can form a gRNA-CIRSPR enzyme complex.

[0613] "gRNA-CRISPR enzyme complex" refers to the complex formed by the interaction between gRNA and CRISPR enzyme.

[0614] The gRNA-CRISPR enzyme complex can be formed in vitro.

[0615] The gRNA-CRISPR enzyme complex can be formed in the cytoplasm of the cell.

[0616] The gRNA-CRISPR enzyme complex can form in the nucleus of the cell.

[0617] In the gRNA-CRISPR enzyme complex, the CRISPR enzyme recognizes the PAM present in the target gene or nucleotide sequence.

[0618] In the gRNA-CRISPR enzyme complex, the gRNA can complementarily bind to the target gene or nucleotide sequence.

[0619] When the gRNA-CRISPR enzyme complex binds to a target gene or nucleotide sequence, the gene or nucleotide sequence targeted by the CRISPR enzyme of the gRNA-CRISPR enzyme complex can be cut or modified.

[0620] In another exemplary embodiment, the CRISPR-Cas system may be present in the form of a ribonucleoprotein (RNP) that forms a complex of gRNA and CRISPR enzyme.

[0621] In an exemplary embodiment of the disclosure disclosed herein, a protein of interest may be expressed by manipulating a highly expressed secretory gene.

[0622] In addition to gRNA and CRISPR enzyme, the composition for manipulating highly expressed secreted genes can further include a donor to express a protein of interest.

[0623] For example, the composition may comprise

[0624] A guide nucleic acid directed against a target sequence of one or more genes selected from the group of highly expressed secretory genes expressed in the liver;

[0625] An editing protein or a nucleic acid encoding the editing protein; and

[0626] A donor comprising a nucleotide sequence encoding a protein of interest.

[0627] "Donor" refers to an exogenous nucleotide sequence containing the sequences required for inserting a transgene into a subject. A donor is a molecule that contains not only the transgene, but also the sequences required for recombination and sequences that affect gene transcription and expression.

[0628] The donor molecule may be of nucleic acid type, ie DNA or RNA.

[0629] The donor molecule may be single-stranded or double-stranded. In some cases, the donor molecule may be a single-stranded oligonucleotide DNA template (ssODT).

[0630] The donor molecule can be in the form of a polynucleotide or a protein.

[0631] Donor molecules can be linear, branched or cyclic and of any length.

[0632] Here, when a linear donor molecule is introduced, the ends of the donor sequence may be protected by methods known to those of ordinary skill in the art. For example, one or more dideoxynucleotide residues may be added to the 3' end of the formed molecule, and a self-complementary oligonucleotide may be bound to one or both ends.

[0633] In specific exemplary embodiments, the addition of terminal amino groups and the use of, for example, phosphorothioate, phosphoramidate, O-methylribose or deoxyribose internucleotide linkages may be additionally included to protect the donor molecule from degradation, but the invention is not limited thereto.

[0634] The donor molecule can form a duplex, and also includes nucleic acids that form a triplex.

[0635] The donor sequence may have any length, for example 10 bp to 20 kb (or any integer between 10 bp and 20 kb or greater), preferably about 100 bp to 10 kb (or any integer between 100 bp and 10 kb), more preferably about 200 bp to 5 kb.

[0636] Depending on the subject to be introduced, the donor can be codon optimized. For example, when the subject is a human, it can be a donor with a human codon-optimized sequence.

[0637] In an exemplary embodiment of the disclosure disclosed herein, a transgene may be included in a donor included in a composition for manipulating a highly expressed secreted gene to express a protein of interest.

[0638] The term "transgene" refers to an exogenous nucleic acid inserted into a highly expressed secreted gene. For example, an editing protein and a guide nucleic acid can be used to insert a transgene into a genomic cleavage site in a cleaved hepatocyte.

[0639] The transgene can be DNA or RNA.

[0640] The transgene can be an exogenous nucleic acid that encodes a protein produced in wild-type hepatocytes.

[0641] The transgene may be an exogenous nucleic acid that encodes a protein produced in cells other than wild-type hepatocytes.

[0642] The transgene may be derived from a species different from that of the subject.

[0643] The transgene may be a wild-type gene. For example, when a protein is deficient or absent in a subject, the donor may contain a transgene having the same sequence as a normal gene encoding the corresponding protein (ie, a wild-type gene).

[0644] The transgene may be a mutant gene. Here, the mutation may be a deletion, substitution or addition of one or more nucleotides of the wild-type gene.

[0645] The transgene may be a fusion protein.

[0646] In one example, the protein fused to the transgene can be derived from a foreign gene.

[0647] In another example, the protein fused to the transgene can be derived from an endogenous gene.

[0648] For example, the protein fused to the transgene can be derived from a highly expressed secreted gene.

[0649] The protein fused to the transgene can be present at or near the amino (N) terminus of the foreign protein.

[0650] The protein fused to the transgene can be present at or near the carboxyl (C) terminus of the foreign protein.

[0651] The protein fused to the transgene may be present at both or near the amino (N) terminus and carboxyl (C) terminus of the foreign protein, or in a mixed form thereof.

[0652] The transgene may be a functional gene used to enhance or improve a desired specific function.

[0653] In one example, the transgene can enhance the function of an endogenous gene in hepatocytes.

[0654] In another example, the transgene can act to increase the expression of a wild-type gene. For example, when the transgene is a gene encoding a protein that is lacking in liver cells, the lacking protein can be produced in liver cells, and the function of the corresponding protein can function normally, or the corresponding disease can be treated.

[0655] In one example, a transgene can serve to correct a mutated gene.

[0656] In another example, a transgene can function to regulate the expression of a genomic sequence in a target gene.

[0657] In one example, a transgene can serve to replace an existing protein or to express a new protein.

[0658] Additionally, transgenes can be inserted into highly expressed secreted genes to partially or completely inactivate one or more target sequences.

[0659] The transgene can be a gene encoding a protein of interest.

[0660] The protein of interest can be a protein used to enhance the function of the body. For example, the protein of interest can enhance the function of metabolism.

[0661] The protein of interest may be a protein useful for preventing a disease.

[0662] The protein of interest may be a protein useful for alleviating a disease.

[0663] The protein of interest may be a protein useful in treating a disease.

[0664] An exemplary embodiment of the disclosure disclosed herein is to artificially manipulate hepatocytes to express a protein of interest for the treatment of a disease.

[0665] In the present specification, compared with existing alternative therapies (such as infusion, administration of proteins at regular intervals, and antibody-based therapies that inhibit factors associated with the corresponding diseases), the disease can be permanently or fundamentally treated by inserting a therapeutic gene into a highly expressed secretory gene to continuously express the corresponding gene at a high level to treat the disease.

[0666] The types of diseases are as follows.

[0667] The disease may be a disease that can be prevented or treated by an antibody. For example, a disease (such as HIV, Alzheimer's disease, or amyotrophic lateral sclerosis) may be treated by inserting a gene encoding an antibody into a highly expressed secretory gene and expressing the gene.

[0668] The disease may be a disease that occurs due to decreased expression of a specific gene. For example, a disease caused by lack of a secretory gene product can be treated by inserting the lacking gene into a highly expressed secretory gene and expressing the gene.

[0669] The disease can be a genetic disorder caused by the inhibition of normal function caused by mutations in a specific gene. In one example, a disease caused by multiple mutations in a single gene can be treated by inserting a wild-type gene into a highly expressed secretory gene and expressing the gene. For example, such a genetic disorder can be hemophilia.

[0670] The disease may be a disease associated with an inherited metabolic disorder.

[0671] "Inherited metabolic disorders" are diseases caused by deficiencies in enzymes or coenzymes responsible for the body's biochemical metabolic pathways. Inherited metabolic disorders refer to conditions in which deficiencies occur due to the inability to properly produce end products, and unnecessary precursors accumulate in various major organs (brain, heart, liver, kidneys, etc.), resulting in excess symptoms (such as intellectual disability).

[0672] Examples of inherited metabolic disorders may include: immunodeficiency, hypercholesterolemia, hemophilia, emphysema, cystic fibrosis, phenylketonuria, citrullinemia, methylmalonic acidemia, carnitine palmitoyltransferase deficiency, Hurler syndrome, ornithine carbamoyltransferase deficiency, and Tay-Sachs disease.

[0673] Other examples of inherited metabolic disorders include hemophilia A, hemophilia B, and hemophilia C.

[0674] For example, hemophilia A is caused by mutations in the F8 gene located on chromosome X, hemophilia B is caused by mutations in the F9 gene located near the F8 gene, and hemophilia C is caused by mutations in the F11 gene.

[0675] The disease may be a lysosomal storage disease.

[0676] Lysosomal storage diseases may include mucopolysaccharidosis, lysosomal acid lipase deficiency, glycogen storage disease, galactosemia, sickle cell anemia, cystic fibrosis, Tay-Sachs disease, phenylketonuria, albinism, medium chain acyl-CoA dehydrogenase deficiency, Farber disease, Krabbe disease, galactosialidase storage disease, gangliosidosis, alpha-galactosidase, Fabry disease, Schindler disease, Sandhoff disease, Gaucher disease, Niemann-Pick disease, sulfatidosis, metachromatic leukodystrophy, multiple sulfatase deficiency, sulfatase deficiency), Hurler syndrome, Scheie syndrome, Hurler-Scheie syndrome, Hunter syndrome, Sanfilippo syndrome, Morquio syndrome, Maroteaux-Lamy syndrome, Sly syndrome, Hyaluronidase deficiency, Mucolipidosis, Sialidosis, I-cell disease, Pseudo-Hurler Polydystrophy, Mucolipidin 1 deficiency, lipid storage disease, Santavuori-Haltia disease, Jansky-Bielschowsky disease, Batten-Spielmeyer-Vogt disease, Kufs disease, Wolman disease, alpha-mannosidosis, beta-mannosidosis, aspartylglucosaminuria, fucosidosis, cystinosis, pycnodystrophy, Salla disease, infantile free sialic acid storage disease, Pompe disease, Danon disease, or cholesterol ester storage disease.

[0677] As an exemplary embodiment of the disclosure disclosed herein, the transgene contained in the donor may be a therapeutic gene for treating a genetic disorder.

[0678] A therapeutic gene may encode a protein that can be used as a therapeutic agent for a specific genetic disorder.

[0679] The therapeutic gene may include a wild-type gene (normal gene form) or a portion (eg, a functional domain) of a disease gene (mutated form of a normal gene) associated with a specific genetic disorder.

[0680] In an exemplary embodiment, the therapeutic gene can be selected from the group consisting of IDUA, I2S, SGSH, NAGLU, HGSNAT, GNS, GALNS, GLB1, ARSB, GUSB, HYAL, NEU, GNPTAB and MCOLN1.

[0681] In another exemplary embodiment, the therapeutic gene may be selected from the group consisting of SAH1, GALC, CTSA, GLA, NAGA, β-galactosidase, hexosaminidase, GBA, SMPD1, ARSA, and SUMF.

[0682] In yet another example, the therapeutic gene (ie, the normal form of the disease-causing gene) can be selected from the group consisting of NPC, PPT, TPP1, CLN3, CLN6, PPT1, DNAJC5, CTSF, CLN7, CLN8 and CTSD.

[0683] In yet another example, the therapeutic gene (ie, the normal form of the disease-causing gene) can be GAA or LAMP2.

[0684] In yet another example, the therapeutic gene (ie, the normal form of the disease-causing gene) may be CTNS, CTSK, or SLC17A5.

[0685] In yet another example, the therapeutic gene (ie, the normal form of the disease-causing gene) can be selected from the group consisting of MAN2B, MAN2C, MANBA, AGA, FUCA1, and LAL.

[0686] In another example, the therapeutic gene (i.e., the normal form of the disease-causing gene) can be a gene encoding a protein selected from the group consisting of: methylmalonic aciduria CbIA type (MMAA) protein, methylmalonic aciduria CbIB type (MMAB) protein, methylmalonic aciduria CbIC type (MMADHC) protein, 5-methyltetrahydrofolate-homocysteine ​​methyltransferase reductase (MTRR) protein, lysosomal membrane protein domain (LMBRD 1) protein, 5-methyltetrahydrofolate-homocysteine ​​methyltransferase (MTR) protein, propionyl-CoA protein, glucose-6-phosphate transporter (G6PT) protein, glucose-6-phosphatase (G6Pase) protein, low-density lipoprotein receptor (LDLR) protein, low-density lipoprotein receptor adaptor protein 1 (LDLRAP-1 protein), N-acetylglutamate synthetase (NAGS) protein, carbamoyl phosphate synthetase 1 (CPS1) protein, ornithine carbamoyltransferase (OTC) protein, argininosuccinate synthetase (ASS) protein, argininosuccinate lyase (ASL) protein, arginase (ARG1) protein, solute carrier family 25 protein, UDP glucuronosyltransferase 1 family polypeptide A1 (UGT1A1) protein, fumarylacetoacetate hydrolase (FAH), alanine-glyoxylate aminotransferase (AGXT) protein, glyoxylate reductase / hydroxypyruvate reductase (GRHPR) protein, APTase Cu(2+) transporting β (ATP7B) protein, phenylalanine hydroxylase (PAH) protein and lipoprotein lyase (LPL) protein.

[0687] In yet another example, the therapeutic gene (ie, the normal form of the disease-causing gene) can be selected from the group consisting of FVII, FVIII, FIX, FX, FXI, FXII and other coagulation factors.

[0688] For example, in the case of hemophilia, a gene encoding a transgene can be inserted into the endogenous APOC3 locus to have high protein expression through the expression regulators of the APOC3 gene. Here, FVII, FVIII, FIX, FX, FXI and FXII are expressed at high levels, or any peptide or protein that contributes to the expression of a coagulation factor is highly expressed.

[0689] In an exemplary embodiment, the therapeutic gene may be a gene encoding an antibody.

[0690] Therapeutic genes are genes that encode proteins or polypeptides that block specific factors or signal pathways involved in the occurrence of diseases through antibodies.

[0691] For example, the therapeutic gene may be a gene encoding an antibody that binds to β-amyloid protein to treat Alzheimer's disease.

[0692] For example, for the treatment of HIV, the therapeutic gene can be a gene encoding zidovudine (AZT), didanosine (ddl), zalcitabine (ddC), lamivudine (3TC), stavudine (d4T), abacavir (TDF) or emtricitabine (FTC) among nucleoside analog reverse transcriptase inhibitors (NRTIs), a gene encoding nevirapine (NVP) or efavirenz (EFV) among non-nucleoside analog reverse transcriptase inhibitors (NNRTIs), or a gene encoding sanquinavir (SQV), ritonavir (RTV), indinavir (IDV), nelfinavir (NFV), lopinavir (LPV), atazanavir (ATV) or amprenavir (APV) among protease inhibitors.

[0693] In another exemplary embodiment, a therapeutic gene can be used to treat a genetic disorder by increasing or decreasing the expression of a wild-type gene by inserting a factor that regulates the expression of the wild-type gene.

[0694] In yet another exemplary embodiment, the therapeutic gene can be used to treat genetic disorders by deleting, adding, or replacing a portion of the sequence of a wild-type gene to produce a protein with improved or new functions.

[0695] For example, a therapeutic gene may be fused with a specific peptide that can permeate into a specific tissue, such as the blood-brain barrier (BBB), or a functional peptide that can improve the therapeutic effect of the therapeutic gene.

[0696] In an exemplary embodiment of the disclosure disclosed herein, the donor may further comprise a gene for inserting a transgene and / or synthesizing a transgene in the target genome.

[0697] Insertion and / or synthesis of the transgene in the target genome can be caused by homologous recombination (HDR) or non-homologous recombination (non-homologous end joining; NHEJ).

[0698] The term "recombination" refers to the process of exchanging genetic information between two polynucleotides, including transgene insertion by nonhomologous recombination (NHEJ) and homologous recombination (HDR).

[0699] "Non-homologous recombination (NHEJ)" disclosed herein refers to a specialized form of exchange that occurs between DNA sequences that do not share sequence homology or that do not occur in site-specific recombination sequences.

[0700] Here, the transgene can be inserted into a highly expressed secreted gene by non-homologous recombination.

[0701] For example, when the partial sequence of the transgene and the partial sequence of the target gene are identical, the identical partial sequence can be cleaved by the same site-specific nuclease, and the transgene can be inserted into the target gene deletion sequence or a site adjacent thereto based on non-homologous recombination.

[0702] "Homologous recombination (HDR)" as disclosed herein refers to a specialized form of exchange that occurs during the repair of double-strand breaks in cells, for example, by homology-directed repair mechanisms.

[0703] The donor sequence may comprise homology arms for homologous recombination of the transgene.

[0704] The homology arms may comprise sequences that are identical or complementary to the target sequence or sequences adjacent thereto.

[0705] The homology arms may comprise sequences that are homologous but not identical to the target sequence or sequences adjacent thereto, or sequences that are homologous but not completely homologous to the target sequence.

[0706] The homology arm having a sequence homologous or identical to the highly expressed secretory gene may contain at least one nucleotide that mismatches with the sequence of the highly expressed secretory gene.

[0707] In certain exemplary embodiments, the majority of donor sequences homologous to the target sequence exhibit about 80% to 99% (or any value between 80% and 90%) sequence identity relative to the genomic sequence to be replaced.

[0708] The homology arms can allow homologous recombination, allowing insertion of a sequence different from the target sequence (ie, a transgene) into the target sequence.

[0709] For example, when a sequence homologous to the donor sequence exists in the region in which the double strands are disrupted by the guide nucleic acid and the editing protein, the transgene can be inserted into the transgene sequence next to the homologous sequence by homologous recombination.

[0710] The donor molecule may contain several non-contiguous regions of homology to a highly expressed secreted gene.

[0711] The homology arm included in the donor sequence may include a nucleotide sequence having homology to a nucleotide sequence in one direction (eg, upstream) of the cleavage site of the highly expressed secretory gene.

[0712] The homology arms included in the donor sequence may include nucleotide sequences having homology to nucleotide sequences in different directions (eg, downstream) of the cleavage site of the highly expressed secretory gene.

[0713] The homology arms included in the donor sequence may include nucleotide sequences having homology to respective nucleotide sequences upstream and downstream of the cleavage site of the highly expressed secretory gene.

[0714] For example, when the transgene is inserted into a site in the target sequence that is 30 bp or more away from both sides of the cleavage site, both ends of the transgene in the donor sequence contain homology arms having sequences that are homologous to or identical to the gene sequence present at the site 30 bp away from the cleavage site.

[0715] Each homology arm has a length of at least 15 bp to about 3 kb (e.g., about 800b or bp, 850b or bp, 900b or bp, 950b or bp, 1000b or bp, 1100b or bp, 1200b or bp long).

[0716] In one example, the homology arms may be 500 bp to 1500 bp long, respectively. Preferably, the homology arms may be 800 bp to 1200 bp long, respectively.

[0717] Typically, homology arms can be provided as single-stranded or double-stranded oligonucleotides. Exemplary single-stranded or double-stranded oligonucleotides as templates can have a length of 800b or bp, 850b or bp, 900b or bp, 950b or bp, 1000b or bp, 150b or bp, 1100b or bp, 1150b or bp, 1200b or bp, or a length of at least about 800bp, 850bp, 900bp, 950bp, 1000bp, 1050bp, 1100bp, 1150bp, 1200bp, 1250bp or 1300bp.

[0718] Furthermore, in an exemplary embodiment of the disclosure disclosed herein, in addition to the transgene, the donor may further contain a gene that regulates transcription and expression.

[0719] The donor may comprise one or more genes or cDNA molecules and may encompass coding or non-coding regions.

[0720] The donor may contain regulatory sequences that control gene transcription and expression, ie, sequences encoding enhancers, insulators, internal ribosome entry sites, 2A peptides and / or polyadenylation signals, or promoters.

[0721] In addition, the donor may further comprise a reporter gene (eg, GFP) or a selectable marker.

[0722] Insertion of the transgene into a highly expressed secreted gene via the donor sequence is determined by the presence and use of homology sequences between the sequence in the donor and the target sequence in the target sequence.

[0723] [Delivery and delivery method]

[0724] In an exemplary embodiment of the disclosure disclosed herein, the guide nucleic acid, editing protein and / or donor molecule can be delivered or introduced into a subject by a vector, a non-vector, or a combination thereof comprising sequences encoding the guide nucleic acid, editing protein and / or donor molecule.

[0725] The guide nucleic acid may be delivered or introduced into a subject in the form of DNA, RNA, or a mixture thereof.

[0726] The editing protein can be delivered or introduced into the subject in the form of DNA, RNA, a DNA / RNA mixture, a peptide, a polypeptide, or a protein encoding the editing protein.

[0727] The donor may be delivered or introduced into the subject in the form of DNA, RNA, or a single-stranded oligonucleotide DNA template (ssODT).

[0728] For example, when the donor is delivered or introduced into a subject, all or part of the transgene can be inserted into the target gene by recombination.

[0729] The guide nucleic acid, editing protein, and / or donor can be delivered to a subject by a variety of methods.

[0730] The guide nucleic acid, editing protein and / or donor can be delivered through the use of a vector.

[0731] Here, the vector may be a viral vector or a non-viral vector.

[0732] The guide nucleic acid, editing protein and / or donor can be delivered through the use of a non-vector.

[0733] (1) Carrier

[0734] The vector can be a viral vector or a non-viral vector (eg, a plasmid).

[0735] The term "vector" can deliver a gene sequence into a cell. Generally, "vector constructs," "expression vectors," and "gene transfer vectors" can direct the expression of a gene of interest and refer to any nucleic acid construct capable of delivering a gene sequence into a target cell. Therefore, the term includes all cloning vehicles and vectors as well as expression vehicles and vectors.

[0736] The vector may comprise a nucleotide sequence encoding a guide nucleic acid, an editing protein and / or a donor.

[0737] Here, the guide nucleic acid, editing protein and / or donor may be DNA, RNA or a mixture thereof.

[0738] The guide nucleic acid, editing protein and / or donor can be delivered using the same or different vectors. For example, the donor can be delivered via a plasmid, while the guide nucleic acid and / or editing protein can be delivered via one or more viral vectors.

[0739] The guide nucleic acid, editing protein and / or donor can be delivered or introduced through the use of one or more vectors.

[0740] In one example, the guide nucleic acid, editing protein, and / or donor can be delivered or introduced by using the same vector.

[0741] In another example, both the guide nucleic acid and the editing protein may be contained in one vector in the form of nucleotide sequences, while the donor may be delivered or introduced by using a different vector.

[0742] In one example, both the guide nucleic acid and the donor polynucleotide can be contained in one vector, and the editing protein in the form of a nucleotide sequence can be delivered or introduced by using different vectors.

[0743] In another example, both the editing protein and the donor can be included in one vector in the form of nucleotide sequences, and the guide nucleic acid can be delivered or introduced by using a different vector.

[0744] For example, the guide nucleic acid, editing protein and / or donor can be contained in different vectors respectively.

[0745] All domains included in the guide nucleic acid may be included in one vector, or each domain may be included in a different vector.

[0746] In the case of an editing protein, the nucleotide sequence encoding the editing protein may be contained in one vector, or may be divided and then contained in several vectors.

[0747] A vector may include one or more regulatory / control components.

[0748] Here, the regulatory / control elements may include a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, an internal ribosome entry site (IRES), a splice acceptor and / or a 2A sequence.

[0749] In addition, the vector may further comprise a selection marker for selecting host cells containing the reporter gene (eg, GFP) or the vector, and the replicable vector may further comprise an origin of replication.

[0750] The vector may contain a splice acceptor (SA) sequence on the left or right side of the transgene or adjacent to the transgene.

[0751] The promoter can be endogenous to the target region or an exogenous promoter.

[0752] The promoter may be a promoter recognized by RNA polymerase II or RNA polymerase III.

[0753] The promoter may be a constitutive promoter.

[0754] The promoter may be an inducible promoter.

[0755] The promoter may be a target-specific promoter.

[0756] The promoter can be a viral promoter or a non-viral promoter.

[0757] As a promoter, an appropriate promoter can be used depending on the control region (ie, guide nucleic acid, editing protein or transgene). For example, a promoter useful for guide nucleic acid may be H1, EF-1a, tRNA or U6 promoter.

[0758] For example, a promoter useful for editing proteins may be CMV, EF-1a, EFS, MSCV, PGK or CAG promoters. For example, a promoter useful for transgenics may be APOC3 or HP promoters.

[0759] The vector may be a viral vector or a recombinant viral vector.

[0760] The virus can be a DNA virus or an RNA virus.

[0761] Here, the DNA virus may be a double-stranded DNA (dsDNA) virus or a single-stranded DNA (ssDNA) virus.

[0762] Here, the RNA virus may be a single-stranded RNA (ssRNA) virus.

[0763] The viral vector may be a retroviral vector, a lentiviral vector, an adenoviral vector, a fox virus vector, a herpes virus vector, a herpes simplex virus, a vaccinia virus, or an adeno-associated virus (AAV) vector, but the present invention is not limited thereto.

[0764] When a virus is used to introduce a guide nucleic acid, editing protein, and / or donor molecule into a target organism, the guide nucleic acid, editing protein, and / or donor molecule can be temporarily expressed in the subject. Alternatively, the guide nucleic acid, editing protein, and / or donor molecule can be expressed continuously for a long time. For example, the guide nucleic acid, editing protein, and / or donor molecule can be expressed for 1 week, 2 weeks, or 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, 1 year or 2 years, or permanently.

[0765] Depending on the type of virus, the packaging capacity of the virus can vary from at least 2 kb to 50 kb. Depending on such packaging capacity, a viral vector containing either a guide nucleic acid or an editing protein alone can be designed, or a viral vector containing both a guide nucleic acid and an editing protein can be designed. Alternatively, a viral vector containing a guide nucleic acid, an editing protein, and additional components can be designed.

[0766] For example, retroviral vectors have the packaging capacity of up to 6 kb to 10 kb of foreign sequences and consist of cis-long terminal repeats (LTRs). Such retroviral vectors are used to insert therapeutic genes into cells and provide permanent transgene expression.

[0767] In another example, AAV vectors can be administered repeatedly due to the very high transduction efficiency of AAV in a variety of cells (muscle, brain, liver, lung, retina, ear, heart, blood vessels, etc.) (regardless of whether the cells are dividing or not), the lack of pathogenicity, and because most of the viral genome can be replaced with therapeutic genes without inducing an immune response. In addition, since AAV is inserted into the chromosomes of target cells, therapeutic proteins are stably expressed for a long time. For example, it can be used to transduce nucleic acids and peptides previously produced in vitro to target nucleic acids of cells in vivo and in vitro. However, AAV is small in size, with a packaging capacity of 4.5 kb or less.

[0768] In one example, nucleotide sequences encoding guide nucleic acids, editing proteins, and / or donor molecules can be delivered or introduced into a subject via a recombinant lentivirus.

[0769] In another example, the nucleotide sequences encoding the guide nucleic acid, editing protein and / or donor molecule can be delivered or introduced via a recombinant adenovirus.

[0770] In one example, nucleotide sequences encoding guide nucleic acids, editing proteins, and / or donor molecules can be delivered or introduced via recombinant AAV.

[0771] The vector may comprise modifications such as phosphorothioate linkages of the nucleic acid, locked nucleic acid (LNA), 2'-O-methyl 3' phosphorothioate (MS) or 2'-O-methyl 3' thio PACE (MSP).

[0772] The nucleotide sequences encoding the guide nucleic acid, editing protein and / or donor molecule can be delivered or introduced with the aid of a non-viral vector.

[0773] The non-viral vector may comprise a nucleotide sequence encoding a guide nucleic acid, an editing protein and / or a donor molecule.

[0774] For example, the non-viral vector may be a plasmid and additionally contain a reporter gene and a nuclear targeting sequence. In addition, other additives may be included.

[0775] (2) Non-carrier

[0776] The guide nucleic acid, editing protein and / or donor molecule can be delivered or introduced into a subject through the use of a non-vector.

[0777] Nucleotide sequences encoding guide nucleic acids, editing proteins, and / or donor molecules can be delivered or introduced into a subject by using a non-vector.

[0778] The non-vector may be naked DNA, DNA complex, mRNA or a mixture thereof.

[0779] The non-vector can be delivered or introduced into a subject via electroporation, lipofection, microinjection, gene gun, virosomes, liposomes, immunoliposomes, lipid-mediated transfection, or a combination thereof.

[0780] In one example, the non-vector can be transferred to the subject by the positive liposome method. This method is used to form stable liposomes because DPOE is composed of positive amphiphilic molecules and neutral helper lipids. Here, since the liposome-DNA complex is positively charged, it can bind to the negatively charged cell surface and be introduced into the cell by endocytosis.

[0781] In another example, DNA can be coated with gold particles and injected into cells.

[0782] In addition, nucleic acids to be delivered to EnGeneIC delivery vehicles (EDVs) can be packaged and then delivered or introduced. Specifically, EDVs are delivered to target tissues using their bispecific antibodies, one arm of which is specific to the target tissue and the other arm is specific to EDVs. The antibody carries EDVs to the surface of target cells, whereupon EDVs can enter the cells by endocytosis.

[0783] A guide nucleic acid-editing protein complex can be formed and delivered or introduced into a subject as a mixture of nucleic acid and protein.

[0784] The guide nucleic acid and editing protein can be delivered or introduced into a subject in the form of a nucleic acid-protein mixture.

[0785] Alternatively, the guide nucleic acid and editing protein can be delivered or introduced into a subject in the form of a guide nucleic acid-editing protein complex.

[0786] The guide nucleic acid may be DNA, RNA or a mixture thereof. In addition, the editor protein may be in the form of a polynucleotide or a protein.

[0787] In an exemplary embodiment, the RNA-based guide nucleic acid and the protein-based editing protein can be delivered or introduced into a subject in the form of a guide nucleic acid-editing protein complex (ie, a ribonucleoprotein (RNP)).

[0788] (3) Delivery method

[0789] In vivo delivery methods

[0790] The guide nucleic acid and editing protein can be administered directly to a subject in the form of a vector or non-vector (containing nucleic acids encoding the guide nucleic acid and editing protein) or in the form of a guide nucleic acid-editing protein complex (ribonucleoprotein (RNP)).

[0791] Furthermore, the donor molecule can be administered directly to a subject in the form of a vector or non-vector comprising a nucleotide sequence encoding each or both of the guide nucleic acid and / or editing protein.

[0792] The injection can be carried out by delivery or introduction into a subject by systemic administration or local administration. However, the present invention is not limited thereto.

[0793] For example, systemic administration may be by intravenous, intraperitoneal, intramuscular or subcutaneous injection. Intravenous systemic administration is preferred.

[0794] When the guide nucleic acid, editing protein, and / or donor molecule are contained in two or more different vectors, the vectors can be administered via different routes (intramuscular injection, tail vein injection, other intravenous injection, and / or intraperitoneal injection).

[0795] Furthermore, when the guide nucleic acid, editing protein, and / or donor molecule are contained in two or more different vectors, the vectors can be delivered simultaneously or in a random order.

[0796] Ex vivo or in vitro delivery methods

[0797] The guide nucleic acid, editing protein, and / or donor molecule can be delivered into cells explanted from an individual subject or into universal hematopoietic stem cells, which can then be re-transplanted into the subject.

[0798] Regarding the delivery method, delivery into a subject can be achieved by: (i) contacting the guide nucleic acid, editing protein, and / or donor molecule with cells, and (ii) directly administering the contacted cells into the subject.

[0799] Specifically, in the intracellular contact step, the guide nucleic acid and the editing protein can be introduced into the cell in the form of a vector or non-vector comprising a nucleotide sequence encoding the guide nucleic acid and / or the editing protein, or in the form of a guide nucleic acid-editing protein complex (ribonucleoprotein (RNP)). Alternatively, the donor molecule can be introduced into the cell in the form of a vector or non-vector comprising a nucleotide sequence encoding each or both of the guide nucleic acid and / or the editing protein.

[0800] For example, when the guide nucleic acid and the editing protein are introduced into the cell in the form of a vector, the contacting step (i) can be performed by one or more methods selected from electroporation, liposomes, nanoparticles and protein translocation domain (PTD)-fusion protein methods.

[0801] In another example, when the guide nucleic acid and the editing protein are introduced into the cell in the form of a vector, the contacting step (i) can be performed with the aid of one or more selected from the group consisting of a non-viral vector (which is a plasmid) and a viral vector.

[0802] Step (ii) is for directly administering the contacted cells obtained in step (i) to a subject, and the cells may be delivered or introduced into a subject by systemic administration or local administration. However, the present invention is not limited thereto.

[0803] For example, systemic administration may be intravenous, intraperitoneal, intramuscular or subcutaneous injection. Preferably, intravenous systemic administration is used.

[0804] The disclosures disclosed herein include subjects modified by introducing or delivering a guide nucleic acid, editing protein, and / or donor into a subject.

[0805] A modified subject can be obtained by inserting a transgene into the genome of a hepatocyte.

[0806] One specific example of a modified subject is an artificially manipulated hepatocyte.

[0807] The modified hepatocytes may be hepatocytes comprising an artificially manipulated high-expression secretory gene.

[0808] The artificially manipulated highly expressed secretory gene may be a gene in which a transgene is inserted into the highly expressed secretory gene.

[0809] The artificially manipulated gene may have a transgene introduced into the coding region of a highly expressed secretory gene.

[0810] In one example, the transgene can be in an exon region of a highly expressed secreted gene.

[0811] For example, among highly expressed secretory genes, the coding sequence of the APOC3 gene may have exon 1, exon 2, exon 3, and exon 4.

[0812] The transgene may be located in exon 1 of the APOC3 gene.

[0813] The transgene may be located in exon 2 of the APOC3 gene.

[0814] The transgene may be located in exon 3 of the APOC3 gene.

[0815] The transgene may be located in exon 4 of the APOC3 gene.

[0816] In another example, in a highly expressed secretory gene, the coding sequence of the HP gene may have exon 1, exon 2, exon 3, exon 4, and exon 5.

[0817] Here, the transgene may be located in exon 1 of the HP gene.

[0818] Here, the transgene may be located in exon 2 of the HP gene.

[0819] Here, the transgene may be located in exon 3 of the HP gene.

[0820] Here, the transgene may be located in exon 4 of the HP gene.

[0821] Here, the transgene may be located in exon 5 of the HP gene.

[0822] Furthermore, the artificially manipulated gene may be in a form in which the transgene is inserted into a non-coding sequence of a highly expressed secretory gene.

[0823] The transgene can be introduced into a region located in a promoter, enhancer, intron, 3'UTR, poly A tail, or a mixture thereof.

[0824] In one example, a transgene can be inserted into an intronic region of a highly expressed secreted gene.

[0825] For example, in a highly expressed secreted gene, three introns, such as intron 1, intron 2, and intron 3, may be located in the coding sequence of the APOC3 gene.

[0826] Here, the transgene may be located in intron 1 of the APOC3 gene.

[0827] Here, the transgene may be located in intron 2 of the APOC3 gene.

[0828] Here, the transgene may be located in intron 3 of the APOC3 gene.

[0829] In another example, in a highly expressed secreted gene, intron 1, intron 2, intron 3 and intron 4 may be located in the coding sequence of the HP gene.

[0830] Here, the transgene may be located in intron 1 of the HP gene.

[0831] Here, the transgene may be located in intron 2 of the HP gene.

[0832] Here, the transgene may be located in intron 3 of the HP gene.

[0833] Here, the transgene may be located in intron 4 of the HP gene.

[0834] The transgene can be inserted into an exon, an intron, or both.

[0835] In one example, the transgene can be located in both the exonic and intronic regions of the APOC3 gene.

[0836] The transgene may be located in both the exon 1 region and the intron 1 region of the APOC3 gene.

[0837] The transgene may be located in both the intron 1 region and the exon 2 region of the APOC3 gene.

[0838] The transgene may be located in both the exon 2 region and the intron 2 region of the APOC3 gene.

[0839] The transgene may be located in both the intron 2 region and the exon 3 region of the APOC3 gene.

[0840] The transgene may be located in both the exon 3 region and the intron 3 region of the APOC3 gene.

[0841] The transgene may be located in both the intron 3 region and the exon 4 region of the APOC3 gene.

[0842] In another example, the transgene can be located in both the exonic and intronic regions of the HP gene.

[0843] The transgene can be located in both the exon 1 region and the intron 1 region of the HP gene.

[0844] The transgene can be located in both the intron 1 region and the exon 2 region of the HP gene.

[0845] The transgene can be located in both the exon 2 region and the intron 2 region of the HP gene.

[0846] The transgene can be located in both the intron 2 region and the exon 3 region of the HP gene.

[0847] The transgene may be located in both the exon 3 region and the intron 3 region of the HP gene.

[0848] The transgene can be located in both the intron 3 region and the exon 4 region of the HP gene.

[0849] The transgene may be located in both the exon 4 region and the intron 4 region of the HP gene.

[0850] The transgene can be located in both the intron 4 region and the exon 5 region of the HP gene.

[0851] Furthermore, the transgene can be inserted into a region comprising a mutated portion of a highly expressed secretory gene (eg, a portion that differs from the wild-type gene).

[0852] Furthermore, the transgene may be inserted into a sequence of 10 to 35 consecutive nucleotides at the 5' end and / or 3' end of the PAM sequence in the nucleotide sequence adjacent to the highly expressed secretory gene.

[0853] Here, the PAM sequence may be, for example, one or more of the following sequences (described in the 5' to 3' direction):

[0854] NGG (N is A, T, C or G);

[0855] NNNNRYAC (each N is independently A, T, C or G; R is A or G; Y is C or T);

[0856] NNAGAAW (N is independently A, T, C or G; W is A or T);

[0857] NNNNGATT (each N is independently A, T, C or G);

[0858] NNGRR(T) (each N is independently A, T, C or G; R is A or G; Y is C or T); and

[0859] TTN (N is A, T, C or G).

[0860] Here, the transgene may be in a sequence of 10-35 nucleotides, 15-35 nucleotides, 20-35 nucleotides, 25-35 nucleotides, or 30-35 nucleotides.

[0861] Alternatively, the transgene can be in a sequence of 10-15 nucleotides, 15-20 nucleotides, 20-25 nucleotides, 25-30 nucleotides, or 30-35 nucleotides.

[0862] In an exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' and / or 5'-NGA-3' (N = A, T, G or C; or A, U, G or C), the transgene may be located in the 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NGG-3', 5'-NAG-3' and / or 5'-NGA-3' (N = A, T, G or C; or A, U, G or C) sequence in the nucleotide sequence adjacent to the HP gene.

[0863] In another exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' and / or 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C), the transgene may be located in 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NGGNG-3' and / or 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C) sequence in the nucleotide sequence adjacent to the HP gene.

[0864] In another exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNNGATT-3' and / or 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C), the transgene may be located in 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NNNNGATT-3' and / or 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C) sequence in the nucleotide sequence adjacent to the HP gene.

[0865] In an exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C), the transgene can be located in 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C) sequence in the nucleotide sequence adjacent to the HP gene.

[0866] In another exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C), the transgene may be located in 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NAAR-3' (R = A or G; N = A, T, G or C, or A, U, G or C) sequence in the nucleotide sequence adjacent to the HP gene.

[0867] In another exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' and / or 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C), the transgene may be located in the 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NNGRR-3', 5'-NNGRRT-3' and / or 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C) sequence in the nucleotide sequence adjacent to the HP gene.

[0868] In an exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N = A, T, G or C; or A, U, G or C), the transgene may be located in 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-TTN-3' (N = A, T, G or C; or A, U, G or C) sequence in the nucleotide sequence adjacent to the HP gene.

[0869] In another example, the transgene may be in a sequence of 10-25 consecutive nucleotides adjacent to the 5' end and / or 3' end of the PAM sequence in the nucleotide sequence of the APOC3 gene.

[0870] In an exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NGG-3', 5'-NAG-3' and / or 5'-NGA-3' (N=A, T, G or C; or A, U, G or C), the transgene may be located in 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NGG-3', 5'-NAG-3' and / or 5'-NGA-3' (N=A, T, G or C; or A, U, G or C) sequence in the nucleotide sequence adjacent to the APOC3 gene.

[0871] In another exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NGGNG-3' and / or 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C), the transgene may be located in 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NGGNG-3' and / or 5'-NNAGAAW-3' (W=A or T; N=A, T, G or C, or A, U, G or C) sequence in the nucleotide sequence adjacent to the APOC3 gene.

[0872] In another exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNNGATT-3' and / or 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C), the transgene may be located in 10-25 consecutive nucleotides adjacent to the 5' end and / or 3' end of the 5'-NNNNGATT-3' and / or 5'-NNNGCTT-3' (N=A, T, G or C; or A, U, G or C) sequence in the nucleotide sequence of the APOC3 gene.

[0873] In an exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C), the transgene may be located in 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NNNVRYAC-3' (V=G, C or A; R=A or G; Y=C or T; N=A, T, G or C, or A, U, G or C) sequence in the nucleotide sequence adjacent to the APOC3 gene.

[0874] In another exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NAAR-3' (R=A or G; N=A, T, G or C, or A, U, G or C), the transgene may be located in 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NAAR-3' (R=A or G; N=A, T, G or C, or A, U, G or C) sequence in the nucleotide sequence adjacent to the APOC3 gene.

[0875] In another exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-NNGRR-3', 5'-NNGRRT-3' and / or 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C), the transgene may be located in 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-NNGRR-3', 5'-NNGRRT-3' and / or 5'-NNGRRV-3' (R=A or G; V=G, C or A; N=A, T, G or C, or A, U, G or C) sequence in the nucleotide sequence adjacent to the APOC3 gene.

[0876] In an exemplary embodiment, when the PAM sequence recognized by the editing protein is 5'-TTN-3' (N = A, T, G or C; or A, U, G or C), the transgene may be located in 10-25 consecutive nucleotides at the 5' end and / or 3' end of the 5'-TTN-3' (N = A, T, G or C; or A, U, G or C) sequence in the nucleotide sequence adjacent to the APOC3 gene.

[0877] The modified hepatocytes may be hepatocytes containing a protein expressed by an artificially manipulated highly expressed secretory gene (hereinafter referred to as "artificially expressed protein").

[0878] In artificially expressed proteins, highly expressed secretory genes and / or transgenes are expressed.

[0879] In one example, a hepatocyte can comprise a protein produced by expression of a transgene.

[0880] In another example, hepatocytes may contain both a protein produced by expression of a transgene and a protein produced by expression of an artificially manipulated high-expression secretory gene.

[0881] In addition, the expression levels of highly expressed secretory genes can be regulated by artificial manipulation.

[0882] In one example, the expression level of the highly expressed secretory gene may be higher than the expression level before the highly expressed secretory gene is artificially manipulated.

[0883] In another example, the expression level of the highly expressed secretory gene may be lower than the expression level before the highly expressed secretory gene is artificially manipulated.

[0884] Furthermore, the protein of interest expressed may be the wild-type protein of the gene present in hepatocytes.

[0885] In one example, a protein expressed in hepatocytes at a predetermined level or lower can show an increased expression level by expressing a protein of interest.

[0886] In another example, when a gene present in a hepatocyte is mutated and has abnormal function, a wild-type gene may be expressed so that it can have normal function. In this case, a mutant protein and a wild-type protein may exist simultaneously in the hepatocyte. Alternatively, a mutant protein in the hepatocyte may not be expressed, but a wild-type protein may be expressed.

[0887] Alternatively, the protein of interest expressed may be a novel protein not expressed in hepatocytes.

[0888] In one example, the protein of interest expressed can be a therapeutic protein. Here, a therapeutic protein is a protein that is not naturally expressed in hepatocytes and can be used to treat, alleviate or prevent disease (including genetic disorders).

[0889] The protein of interest expressed may be a mutant protein (produced by partially modifying a wild-type protein).

[0890] In one example, the protein of interest expressed may be a protein expressed by partially modifying the nucleotides of a part of the wild-type gene.

[0891] For example, some functions of a mutant protein may be enhanced compared to the wild-type protein.

[0892] The expressed protein of interest may be a fusion protein.

[0893] In one example, the protein of interest may be a fusion protein expressed by combining an endogenous gene with an exogenous gene in a subject.

[0894] In another example, the protein of interest may be a fusion protein expressed by combining foreign genes.

[0895] For example, the protein of interest may be a fusion protein expressed by combining different endogenous genes.

[0896] Thus, the disclosure disclosed herein includes hepatocytes modified by artificially manipulated high-expression secretory genes.

[0897] Additionally, the disclosure disclosed herein includes liver tissue modified by the modified hepatocytes.

[0898] Additionally, the disclosure disclosed herein includes a liver modified by modified liver tissue.

[0899] Here, the liver may be partially or completely modified.

[0900] [Methods for expressing proteins of interest]

[0901] One exemplary embodiment of the disclosure disclosed herein relates to a method of expressing a protein of interest in the liver.

[0902] The methods are performed in vivo, ex vivo, or in vitro.

[0903] In some embodiments, the method comprises (i) introducing a programmable nuclease and a transgene into an organ, tissue or cell.

[0904] In an exemplary embodiment, the method may include step (i): introducing into a subject

[0905] (a) a guide nucleic acid that forms a complementary bond with a nucleotide sequence of one or more genes selected from the group consisting of highly expressed secretory genes, or has a sequence identical to a nucleotide sequence of one or more genes selected from the group consisting of highly expressed secretory genes;

[0906] (b) an editing protein or a nucleic acid encoding the editing protein; and

[0907] (c) A donor comprising a transgene.

[0908] The one or more genes may be selected from highly expressed secretory genes, such as FTL, FTH1, ACTB, HP, APOC3, SOD2, ORM1 and F9. Preferably, the gene may be an APOC3 gene or a HP gene.

[0909] The guide nucleic acid (a) may include: a guide nucleic acid that may have the same sequence as the target sequence SEQ ID No: 1 to SEQ ID No: 348, or a guide nucleic acid that may form a complementary bond with the target sequence SEQ ID No: 1 to SEQ ID No: 348.

[0910] The editing protein (b) includes one or more editing proteins selected from the group consisting of: Cas9 protein derived from Streptococcus pyogenes, Cas9 protein derived from Campylobacter jejuni, Cas9 protein derived from Streptococcus thermophilus, Cas9 protein derived from Staphylococcus aureus, Cas9 protein derived from Neisseria meningitidis and Cpf1 protein.

[0911] The transgene (c) may be a gene encoding a protein of interest. For example, the transgene may be a therapeutic gene.

[0912] The description of the therapeutic gene is as described above.

[0913] The description of the composition used for gene manipulation is as described above.

[0914] The step of introducing can be performed in vivo.

[0915] Here, (a), (b) and (c) may be delivered via a carrier or a non-carrier.

[0916] The description of the carrier, non-carrier and delivery method is the same as described above.

[0917] The method of expressing a protein of interest in the liver may further comprise confirming transgene expression in the modified organ, tissue or cell obtained by step (i).

[0918] Here, in step (ii),

[0919] The organ or tissue modified in step (i) may be an organ or tissue in which a transgene is inserted into the genome of the organ or tissue in its natural state.

[0920] The organ or tissue modified in step (i) may comprise a transgene.

[0921] Here, the transgene contained in the modified organ or tissue may be expressed in the modified organ or tissue.

[0922] The expression of the transgene in the modified organ or tissue can be confirmed by the expression of transgene mRNA or protein.

[0923] PCR can be used as a method to confirm transgene mRNA expression.

[0924] Western blot, ELISA or IP can be used as a method to confirm transgenic protein expression.

[0925] In another example, the method can be performed ex vivo or in vitro. Here, methods for preparing artificially manipulated hepatocytes can be used in order to express a protein of interest.

[0926] In an exemplary embodiment, the method may be a method for preparing manipulated animal cells expressing a protein of interest, the method comprising contacting:

[0927] (a) animal cells; and

[0928] (b) A composition for manipulating genes to artificially insert a transgene into one or more genes selected from highly expressed secretory genes (such as ALB, FTL, FTH1, ACTB, HP, APOC3, SOD2, ORM1 and F9).

[0929] Here, the animal cell (a) may be a somatic cell or a stem cell of human origin.

[0930] Specifically, the somatic cell of human origin may be a hepatocyte.

[0931] The composition (b) for gene manipulation is as follows.

[0932] The contacting step may include introducing the composition for gene manipulation (b) into the animal cell (a).

[0933] The animal cell used in this method may be an animal cell derived from a mammal (including primates such as humans and monkeys, and rodents such as mice and rats).

[0934] [use]

[0935] Examples of the disclosure disclosed herein include therapeutic uses.

[0936] For example, a therapeutic use may include administering a composition for inserting a transgene into the genome of a hepatocyte of a subject, or a hepatocyte having the transgene inserted therein.

[0937] The subject to be treated can be a mammal, for example, a primate (eg, a human or a monkey) and a rodent (eg, a mouse or a rat).

[0938] (1) Pharmaceutical composition

[0939] An exemplary embodiment of the present specification includes a composition for treating a disease by highly expressing a therapeutic gene and a method for preparing the composition.

[0940] For example, the composition may be a composition comprising a therapeutic gene and a nuclease artificially manipulated to artificially insert the transgene into the genome of a hepatocyte. The composition may be referred to as a therapeutic composition or a pharmaceutical composition.

[0941] In an exemplary embodiment, the composition may comprise a programmable nuclease.

[0942] The programmable nuclease may be one of a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas) system, a zinc finger nuclease (ZFN), and a transcription activator-like effector nuclease (TALEN).

[0943] In an exemplary embodiment, the composition may include a therapeutic gene.

[0944] The therapeutic gene may be a gene encoding a protein or enzyme that is deficient in the body.

[0945] For example, the therapeutic gene may be F9. Here, the composition may be a composition for treating hemophilia.

[0946] In addition to the programmable nuclease and the therapeutic gene, the composition may further include a pharmaceutically acceptable excipient and / or other compounds known in the art. For example, the composition may include water, salt, dextran, glycerol, ethanol, and combinations thereof.

[0947] In addition, wetting agents, emulsifiers, pH buffers, stabilizers or other agents that enhance the efficacy of the pharmaceutical composition may be included as minor additives.

[0948] 2) Treatment methods

[0949] One exemplary embodiment described in the present specification is a method for treating a specific disease, the method comprising administering an effective amount of the above composition to a patient in need of the composition.

[0950] For example, methods of treating diseases that are treatable by antibodies are included.

[0951] For example, methods of treating diseases caused by genetic mutations are included.

[0952] For example, methods of treating inherited metabolic disorders are included.

[0953] For example, methods of treating diseases caused by the absence or deficiency of a particular protein are included.

[0954] For example, the disease may be one that can be treated by permanently expressing a therapeutic gene by injecting a therapeutic composition into a patient.

[0955] The treatment methods may be carried out by delivering the therapeutic composition to the patient via systemic administration.

[0956] The treatment method can be implemented by injecting the therapeutic composition into a specific body part of the patient. Here, the specific body part can be an organ containing cells that can highly express the therapeutic gene to secrete cells.

[0957] For example, the cell can be a hepatocyte.

[0958] For example, the cell may be a stem cell.

[0959] The treatment method may be a treatment method that affects gene expression in animal cells by directly manipulating genes using programmable nucleases. Such treatment methods may be performed by directly injecting a composition for manipulating genes into the body, the composition comprising a programmable nuclease for manipulating genes in a living body and a therapeutic gene.

[0960] Programmable nucleases can be guide nucleic acids and / or editing proteins.

[0961] The therapeutic gene may be F9.

[0962] The composition used for gene manipulation is as described above.

[0963] The subject to which the composition is administered may be any mammal, including primates (eg, humans, monkeys, etc.) and rodents (eg, mice, rats, etc.).

[0964] Administration of the composition may be by any convenient method, such as injection, infusion, implantation or transplantation. The composition may be administered subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intravenously, intralymphatically or intraperitoneally.

[0965] The compositions are preferably administered systemically by intravenous administration.

[0966] In another example, administration can be administration to any one or more of the following organs of the subject being treated: kidney; digestive system, including stomach, pancreas, duodenum, ileum and / or colon; heart; lung; brain, particularly neurons and / or CNS in general; eye, including retinal tissue; ear, including inner ear; skin; muscle; bone; and / or liver.

[0967] The organ is preferably the liver.

[0968] In an exemplary embodiment,

[0969] During delivery to humans using AAV, the dosage of the composition (pharmaceutically effective amount to achieve the desired effect) may be about 0.01-10 mL, each mL containing about 1×10 8 Up to 1×10 18 In an exemplary embodiment of the present specification, the AAV content is generally within the following concentration range: about 1×10 5 Up to 1×10 50 AAV genome, about 1×10 8 Up to 1×10 20 AAV genome, about 1×10 10 Up to 1×10 16 genome AAV, or about 1 × 10 11 Up to 1×10 16 Genome AAV. Such AAV concentrations can be delivered in a carrier solution of about 0.001-100 mL, about 0.01-50 mL, or about 0.05-10 mL. However, the dosage of the composition can be appropriately specified taking into account the age, health status, and weight of the subject being administered; the type of treatment being received, the frequency of treatment, and the characteristics of the desired effect, but the present invention is not limited thereto.

[0970] In one exemplary embodiment, the method of treatment may be a method of treating hemophilia.

[0971] The treatment method can be performed in vivo.

[0972] The method comprises administering to the subject a composition comprising as an active ingredient a composition for manipulating genes to express a transgene artificially inserted into the genome of hepatocytes,

[0973] The composition for manipulating genes comprises any of the following programmable nucleases: clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system, zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN),

[0974] The artificially inserted transgene is located in one or more gene sequences of the HP gene and the APOC3 gene, and

[0975] The transgenic plant is F9.

[0976] Hemophilia may be permanently treated through gene editing therapy using programmable nucleases.

[0977] By inserting a transgene into the genome of hepatocytes and highly expressing the gene using the methods and compositions described in this specification, the expression of the deficient protein can be increased, the final genetic product can be produced through the normal operation of the biochemical metabolic pathway, and the body function can be improved.

[0978] Example

[0979] Hereinafter, the present specification will be further described in detail with reference to examples. These examples are provided only to illustrate the present application document, and should not be interpreted as limiting the protection scope of the present application document to these examples.

[0980] Example 1: RNA sequencing

[0981] Human primary cells were cultured in 24-well plates containing William's medium for 2 days, and RNA was then isolated using the RNeasy MiniKit (Qiagen).

[0982] Samples having an RNA Integrity Number (RIN) of 7 or more were selected using Agilent 2100 BioAnalyzer and subjected to RNA enrichment using 1 μg of RNA and magnetic beads with oligo dT, followed by construction of a cDNA library by short fragmentation and reverse transcription.

[0983] Subsequently, sequencing adapters were ligated using the TrueSeq RNA sample prep kit (Illumina, CA), and the cDNA library size (350-450 bp) and quality were checked using electrophoresis and the Agilent High Sensitivity DNA Kit (Agilent Technology, CA), followed by sequencing using Illumina HiSeq2500 (Illumina, CA).

[0984] Then, the reads filtering out low-quality reads corresponding to 10% or more skipped bases (marked as “N”) and quality scores less than 20 (accounting for 40% or more) were mapped to the human reference genome using the Tophat program, and the FPKM value of each transcript was calculated using the Cufflinks v2.1.1 program.

[0985] From the FPKM results, the transcriptional activities of haptoglobin and APOC3 encoding secretory proteins and F9, IDUA, IDS, GLA, and GBA corresponding to therapeutic genes for each disease were confirmed.

[0986] like Figure 1 As shown, the haptoglobin gene and the APOC3 gene exhibited transcriptional activities approximately 170 times or more higher than those of other therapeutic genes.

[0987] Example 2: Design of sgRNA

[0988] The CRISPR RGEN tool (Institute for Basic Science, South Korea) was used to identify the CRISPR / Cas9 target sites of the human HP1 gene and APOC3 gene. The target site for each gene may vary depending on the type of CRISPR enzyme, and the target sequences of the HP gene and APOC3 gene for CjCas9 are summarized in Tables 2 and 3, and the target sequences of the HP gene and APOC3 gene for SpCas9 are summarized in Tables 4 and 5.

[0989] Table 2

[0990]

[0991] Table 3

[0992]

[0993] Table 4

[0994]

[0995] Table 5

[0996]

[0997] Example 3: gRNA activity verification and off-target analysis

[0998] 3.1 T7E1 analysis

[0999] 250 ng of sgRNA-expressing vector and 750 ng of Cas9-expressing vector (in which the corresponding gRNA sequence was cloned) were transfected into HEK293 cell line using Lipofectamine 2000 or by electroporation. In addition, 1 μg of in vitro transcribed sgRNA and 4 μg of Cas9 were mixed in the form of RNP complex and transfected into Jurkat cell line by electroporation.

[1000] After about 2 to 3 days, genomic DNA is extracted and subjected to PCR to amplify the on-target site, followed by T7E1 analysis to confirm activity, or additional PCR is performed, which connects the sequencing primers for next-generation sequencing and the adapter specific for the TruSeq HT DualIndex primer. The activity of the gRNA is checked with the T7E1-cleaved band.

[1001] like Figure 3 As shown, the gRNA activity of target sequences #1 to #11 of the HP gene (shown in Table 2) was studied using Cjcas9, and T7E1 activity was shown in target sequences #8, #9, #10, and #11.

[1002] 3.2 Confirm the insertion and deletion (indel) rate (%) of the target sequence

[1003] The reads obtained by paired sequencing were then analyzed to evaluate the activity of gRNA by detecting insertions or deletions (indels) at on-target sites on the genome, and the results are shown in Tables 2, 3, 4, and 5.

[1004] As shown in Tables 2 and 4, it was confirmed that all indels occurred in the corresponding target sequence of the HP gene, and the indel rate (%) was as high as 86.4%, confirming a high gene editing efficiency.

[1005] In addition, in Tables 3 and 5, it was confirmed that all indels occurred in the corresponding target sequence of the APOC3 gene, and the indel rate (%) was as high as 96.2%, confirming a high gene editing efficiency.

[1006] In the gRNAs targeting individual genes, DNA target sites with high activity according to the type of Cas9 are marked with bold letters.

[1007] 3.3 Off-target analysis

[1008] For off-target analysis of the selected gRNAs, first, a list of off-targets with 3 base mismatches was selected by an in silico approach using the Cas-Offinder of CRISPR RGEN Tools, and the mutations at specific sites in the genome corresponding to each off-target were verified in the human cell line HEK293 by targeted deep sequencing.

[1009] Second, human whole genome DNA treated with gRNA and Cas9 protein overnight at 37°C was subjected to whole genome sequencing, and then the potential list was determined by Digenome-seq. After that, the mutation of a specific site in the genome of each off-target candidate was verified by targeted deep sequencing from the human cell line HEK293.

[1010] like Figure 4 and Figure 5 As shown, as a result of deep sequencing, ten off-targets of HP1-Cj and six off-targets of APOC3-Cj were detected, there were no significant off-targets, and it was confirmed that indel mutations occurred with high efficiency.

[1011] Example 4: Construction of vectors and donors

[1012] For SpCas9 application, vectors (pAAV-EFS-SpCas9) and vectors (pAAV-hF9-donor-U6-sgRNA) were synthesized and constructed separately; pAAV-EFS-SpCas9 contained EFS (which is a promoter for mammalian expression), human codon-optimized SpCas9 (with NLS and HA tags at the C-terminus or N-terminus), and BGHA between the inverted tandem repeats (ITRs) of AAV2; pAAV-hF9-donor-U6-sgRNA contained a U6 promoter, sgRNA sequence, and a human codon-optimized F9 donor between the inverted tandem repeats (ITRs) of AAV2.

[1013] For CjCas9 application, vectors (pAAV-CMV-CjCas9-U6-sgRNA) and vectors (pAAV-hF9-donor) were synthesized and constructed separately; pAAV-CMV-CjCas9-U6-sgRNA contained CMV (which is a mammalian expression promoter), human codon-optimized CjCas (with NLS and HA tags at the C-terminus or N-terminus), and BGHA, U6 promoter and sgRNA sequences between the ITRs of AAV2; pAAV-hF9-donor contained a human codon-optimized F9 donor between the ITRs of AAV2. That is, for CjCas9, considering the packaging capacity of AAV, U6 and sgRNA can be delivered together with the sequence expressing Cas9.

[1014] The donor introduced into each vector consists of a splice acceptor (SA)-human codon-optimized F9-bGHA between homology arms, each of which is 800-1200 bp long and is present on both sides of the double-strand break generated by Cas9 in the human HP1 or APOC3 genome ( Figure 6 ).

[1015] Example 5: Confirmation of HDR in human cell lines

[1016] 500 ng of pAAV-CMV-CjCas9-U6-sgRNA targeting the human HP1 genome and 500 ng of pAAV-hF9-donor were transfected into HEK293 cell line using Lipofectamine 2000.

[1017] After 3 days, genomic DNA was extracted, and PCR bands were obtained using corresponding primers from the external regions of the homology arms of the HP1 target site and the region knocked in by HDR and cloned into the TA vector.

[1018] Subsequently, plasmids were extracted from the resulting colonies and subjected to Sanger sequencing to confirm the flanking knock-in junction sequences.

[1019] Figure 8Results showing confirmation of knock-in at the junction of the left and right arms and confirmation of knock-in occurring exactly in the region with double-strand breaks at both arms without F9 sequence modification. The gRNA sequences are Cj9: 5'-TCCAGGAAAGAGAAACCTCCC-3'; and Cj10: 5'-CATTCAGGAAAGTACATTGGC-3'.

[1020] Example 6: AAV construction

[1021] To produce AAV, the pseudotyped AAV capsid vector, constructed pAAV-EFS-SpCas9, pAAV-hF9-donor-U6-sgRNA, pAAV-CMV-CjCas9-U6-sgRNA or pAAV-hF9-donor, and pHelper vector were simultaneously transfected into HEK293 cells at a molar ratio of 1:1:1.

[1022] After 72 hours, viral particles obtained by cell fusion were isolated and purified by step-gradient ultracentrifugation using iodixanol (Sigma-Aldrich), and quantification of AAV was performed by titration using qPCR.

[1023] Example 7: Confirmation of high expression of KI (knock-in) genes and indels from hepatocytes

[1024] 6×10^5 hepatocytes were maintained in 24-well plates and then infected with 5×10^11ug of AAV6-EFS-SpCas9 and 5×10^11ug of AAV6-hF9-donor-APOC3-Sp.

[1025] 5′-CCTAAGCCTGAAGAATGAGG-3′ was used as the sequence of APOC3-Sp target. On the first day (2D), 5D, 8D, and finally 10D after infection, the culture medium was replaced and culture supernatant samples were prepared; and genomic DNA was extracted from hepatocytes in the last 10D.

[1026] Using a primer set (Forward: 5'-ACGGAAAATATCAAGAAGTA-3', Reverse: 5'-CAGCAAGCCCTGTCCTGCTGG-3'), an amplicon targeting APOC3-Sp was obtained, and an additional PCR was performed to connect the primer-specific adapter and TruSeq HT Dual Index primers.

[1027] Reads obtained by paired sequencing are then analyzed to assess CRISPR activity by the detection of indels at the targeted genomic locations.

[1028] The high expression of knocked-in human F9 was detected by ELISA against F9 protein secreted from the hepatocyte culture supernatant using Factor IX Human ELISA Kit (Abcam).

[1029] like Fig. 9 As shown in A, the double AAV-treated group (hAPOC3-Sp) showed an average of 9.6% indels compared to the non-AAV-treated control group and the donor-only group (D-only-hAPOC3-Sp).

[1030] In addition, if Fig. 9 As shown in B, the secretion level of hF9 in the control group was 42.5 ng / mL on average, in the donor-only group it was 74.1 ng / mL, and in the double AAV-treated group it was 378.2 ng / mL.

[1031] For statistical analysis, Student's t-test was used. For significance, * represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.

[1032] Sequence Listing Free Text

[1033] SEQ ID NO: 1 to SEQ ID NO: 153 represent a target sequence list of HP gene.

[1034] SEQ ID NO: 154 to SEQ ID NO: 348 represent a target sequence list of the APOC3 gene. <110> Turkin Co., Ltd. <120> Liver Biofactory Platform <130> OPP17-036-NP-PCT <150> US 62 / 520,103 <151> 2017-06-15 <150> US 62 / 662,907 <151> 2018-04-26 <160> 348 <170> KoPatentIn 3.0 <210> 1 <211> twenty two <212> DNA <213> Homo sapiens <400> 1 aaagaggaaa atatctgcta at 22 <210> 2 <211> 22 <212> DNA <213> Homo sapiens <400> 2 aaggcactta gatcttataa aa 22 <210> 3 <211> 22 <212> DNA <213> Homo sapiens <400> 3 ttctattaaa atagtttcta gg 22 <210> 4 <211> 22 <212> DNA <213> Homo sapiens <400> 4 ctcactaaca aatgccaacc at 22 <210> 5 <211> 22 <212> DNA <213> Homo sapiens <400> 5 ttagtgagat ggtgaactgg ca 22 <210> 6 <211> 22 <212> DNA <213> Homo sapiens <400> 6 aggtgaatta ttataaaata ct 22 <210> 7 <211> 22 <212> DNA <213> Homo sapiens <400> 7 ggaaaatatc aagaagtaga gg 22 <210> 8 <211> 22 <212> DNA <213> Homo sapiens <400> 8 ctccaggaaa gagaaacctc cc 22 <210> 9 <211> twenty two <212> DNA <213> Homo sapiens <400> 9 gcattcagga aagtacattg gc 22 <210> 10 <211> twenty two <212> DNA <213> Homo sapiens <400> 10 gaaattgccc ccacacctgc cc 22 <210> 11 <211> twenty two <212> DNA <213> Homo sapiens <400> 11 aagaaattgc ccccacacct gc 22 <210> 12 <211> twenty two <212> DNA <213> Homo sapiens <400> 12 tcaaaaatgt aacctgaagg aa 22 <210> 13 <211> twenty two <212> DNA <213> Homo sapiens <400> 13 tagcagatat tttcctcttt aa 22 <210> 14 <211> twenty two <212> DNA <213> Homo sapiens <400> 14 atgtgttact attagtcttc ct 22 <210> 15 <211> 22 <212> DNA <213> Homo sapiens <400> 15 atgtacaata aggaagacta at 22 <210> 16 <211> 22 <212> DNA <213> Homo sapiens <400> 16 acacaattaa ttgactagta cc 22 <210> 17 <211> 22 <212> DNA <213> Homo sapiens <400> 17 acaattaatt gactagtacc tg 22 <210> 18 <211> 22 <212> DNA <213> Homo sapiens <400> 18 aattaattga ctagtacctg gg 22 <210> 19 <211> 22 <212> DNA <213> Homo sapiens <400> 19 atcccaggta tgtgtgta tc 22 <210> 20 <211> 22 <212> DNA <213> Homo sapiens <400> 20 ttgactagta cctgggatac ac 22 <210> 21 <211> 22 <212> DNA <213> Homo sapiens (mankind) <400> 21 acctgggata cacactata cc <210> 22 <211> 22 <212> DNA <213> Homo sapiens (mankind) <400> 22 fathercctgg gatacatcta at <210> 23 <211> 22 <212> DNA <213> Homo sapiens (mankind) <400> 23 atttcctaaa ggtgaattat ta <210> 24 <211> 22 <212> DNA <213> Homo sapiens (mankind) <400> 24 aaggttcctt aatatata tt <210> 25 <211> 22 <212> DNA <213> Homo sapiens (mankind) <400> 25 tggagggctc ctgtattatt gc <210> 26 <211> 22 <212> DNA <213> Homo sapiens (mankind) <400> 26 ctcagtttct ggctgcattc ag <210> 27 <211> 22 <212> DNA <213> Homo sapiens <400> 27 catacacact ttagcagctt ct 22 <210> 28 <211> 22 <212> DNA <213> Homo sapiens <400> 28 ccaagaaatt gcccccacac ct 22 <210> 29 <211> 22 <212> DNA <213> Homo sapiens <400> 29 agtgctagga ccaagaaatt gc 22 <210> 30 <211> 22 <212> DNA <213> Homo sapiens <400> 30 ggtgtggggg caatttcttg gt 22 <210> 31 <211> 22 <212> DNA <213> Homo sapiens <400> 31 atccacacac acatgcatgt ac 22 <210> 32 <211> 22 <212> DNA <213> Homo sapiens <400> 32 gcatccacac acacatgcat gt 22 <210> 33 <211> 22 <212> DNA <213> Homo sapiens <400> 33 atgcatccac acacacatgc at 22 <210> 34 <211> twenty two <212> DNA <213> Homo sapiens <400> 34 gcatgcatcc acacacacat gc 22 <210> 35 <211> twenty two <212> DNA <213> Homo sapiens <400> 35 atgcatgcat ccacacacac at 22 <210> 36 <211> twenty two <212> DNA <213> Homo sapiens <400> 36 tggaaagcta gtctccctgc tt 22 <210> 37 <211> twenty one <212> DNA <213> Homo sapiens <400> 37 agacccgaga gggtcagagt g 21 <210> 38 <211> twenty one <212> DNA <213> Homo sapiens <400> 38 atcccactct gaccctctcg g 21 <210> 39 <211> twenty one <212> DNA <213> Homo sapiens <400> 39 tctcgggtct gcactctctc t 21 <210> 40 <211> twenty one <212> DNA <213> Homo sapiens <400> 40 agggcactgg ctgaatccac t 21 <210> 41 <211> 20 <212> DNA <213> Homo sapiens <400> 41 ggttacattt ttgactttat 20 <210> 42 <211> 20 <212> DNA <213> Homo sapiens <400> 42 ctgggataca cactaatacc 20 <210> 43 <211> 20 <212> DNA <213> Homo sapiens <400> 43 gcaagtagtg cccgaatggt 20 <210> 44 <211> 20 <212> DNA <213> Homo sapiens <400> 44 ttgttagtga gatggtgaac 20 <210> 45 <211> 20 <212> DNA <213> Homo sapiens <400> 45 gaactggcag acggcacctg 20 <210> 46 <211> 20 <212> DNA <213> Homo sapiens <400> 46 aactggcaga cggcacctgt 20 <210> 47 <211> 20 <212> DNA <213> Homo sapiens <400> 47 ctcagacacc gcaaagatag 20 <210> 48 <211> 20 <212> DNA <213> Homo sapiens <400> 48 cactatcttt gcggtgtctg 20 <210> 49 <211> 20 <212> DNA <213> Homo sapiens <400> 49 actatctttg cggtgtctga 20 <210> 50 <211> 20 <212> DNA <213> Homo sapiens <400> 50 atctttgcgg tgtctgaggg 20 <210> 51 <211> 20 <212> DNA <213> Homo sapiens <400> 51 agaaaggcac ataggtggag 20 <210> 52 <211> 20 <212> DNA <213> Homo sapiens <400> 52 gcagaaatag aacaaagaaa 20 <210> 53 <211> 20 <212> DNA <213> Homo sapiens <400> 53 agaacaaaga aacgggcaaa 20 <210> 54 <211> 20 <212> DNA <213> Homo sapiens <400> 54 gaacaaagaa acgggcaaat 20 <210> 55 <211> 20 <212> DNA <213> Homo sapiens <400> 55 caggagtgtc tttttccttc 20 <210> 56 <211> 20 <212> DNA <213> Homo sapiens <400> 56 aagtcaaaaa tgtaacctga 20 <210> 57 <211> 20 <212> DNA <213> Homo sapiens <400> 57 gttacatttt tgactttata 20 <210> 58 <211> 20 <212> DNA <213> Homo sapiens <400> 58 gatgccagga agcctaccac 20 <210> 59 <211> 20 <212> DNA <213> Homo sapiens <400> 59 ggatgccagg aagcctacca 20 <210> 60 <211> 20 <212> DNA <213> Homo sapiens <400> 60 ataatatac tcaggatgcc 20 <210> 61 <211> 20 <212> DNA <213> Homo sapiens <400> 61 atctgctaat aaatatactc 20 <210> 62 <211> 20 <212> DNA <213> Homo sapiens <400> 62 cttattgtac atttttaaag 20 <210> 63 <211> 20 <212> DNA <213> Homo sapiens <400> 63 ctctttaaaa atgtacaata 20 <210> 64 <211> 20 <212> DNA <213> Homo sapiens <400> 64 acaattaatt gactagtacc 20 <210> 65 <211> 20 <212> DNA <213> Homo sapiens <400> 65 caattaattg actagtacct 20 <210> 66 <211> 20 <212> DNA <213> Homo sapiens <400> 66 caggtattag tgtgtatccc 20 <210> 67 <211> 20 <212> DNA <213> Homo sapiens <400> 67 tgggatacac actaatacct 20 <210> 68 <211> 20 <212> DNA <213> Homo sapiens <400> 68 gccttaatta gatgtatccc 20 <210> 69 <211> 20 <212> DNA <213> Homo sapiens <400> 69 acctgggata catctaatta 20 <210> 70 <211> 20 <212> DNA <213> Homo sapiens <400> 70 agtttctagg ccagacacgg 20 <210> 71 <211> 20 <212> DNA <213> Homo sapiens <400> 71 aatagtttct aggccagaca 20 <210> 72 <211> 20 <212> DNA <213> Homo sapiens <400> 72 agaagcaagt agtgcccgaa 20 <210> 73 <211> 20 <212> DNA <213> Homo sapiens <400> 73 ctaacaaatg ccaaccattc 20 <210> 74 <211> 20 <212> DNA <213> Homo sapiens <400> 74 actaacaaat gccaaccatt 20 <210> 75 <211> 20 <212> DNA <213> Homo sapiens <400> 75 gttggcattt gttagtgaga 20 <210> 76 <211> 20 <212> DNA <213> Homo sapiens <400> 76 tgagatggtg aactggcaga 20 <210> 77 <211> 20 <212> DNA <213> Homo sapiens <400> 77 ggccatgggc attgacccac 20 <210> 78 <211> 20 <212> DNA <213> Homo sapiens <400> 78 cacctgtggg tcaatgccca 20 <210> 79 <211> 20 <212> DNA <213> Homo sapiens <400> 79 aaaagcagga cggtggccat 20 <210> 80 <211> 20 <212> DNA <213> Homo sapiens <400> 80 caaaagcagg acggtggcca 20 <210> 81 <211> 20 <212> DNA <213> Homo sapiens <400> 81 ggtgtccaaa agcaggacgg 20 <210> 82 <211> 20 <212> DNA <213> Homo sapiens <400> 82 actggtgtcc aaaagcagga 20 <210> 83 <211> 20 <212> DNA <213> Homo sapiens <400> 83 catggccacc gtcctgcttt 20 <210> 84 <211> 20 <212> DNA <213> Homo sapiens <400> 84 gagaactggt gtccaaaagc 20 <210> 85 <211> 20 <212> DNA <213> Homo sapiens <400> 85 ttggacacca gttctcttcc 20 <210> 86 <211> 20 <212> DNA <213> Homo sapiens <400> 86 tgaaacccca aaatgccaga 20 <210> 87 <211> 20 <212> DNA <213> Homo sapiens <400> 87 aataattcac ctttaggaaa 20 <210> 88 <211> 20 <212> DNA <213> Homo sapiens <400> 88 tttcagatac catttcctaa 20 <210> 89 <211> 20 <212> DNA <213> Homo sapiens <400> 89 ttttatataata attcaccttt 20 <210> 90 <211> 20 <212> DNA <213> Homo sapiens (Wise Man) <400> 90 atatataatt ttaaacacgt 20 <210> 91 <211> 20 <212> DNA <213> Homo sapiens (Wise Man) <400> 91 aatatataat tttaaacacg 20 <210> 92 <211> 20 <212> DNA <213> Homo sapiens (Wise Man) <400> 92 tgtttaaaat tatatattta 20 <210> 93 <211> 20 <212> DNA <213> Homo sapiens (Wise Man) <400> 93 ttgatatttt ccgtaataaa 20 <210> 94 <211> 20 <212> DNA <213> Homo sapiens (Wise Man) <400> 94 atttaaggaa ccttttatta 20 <210> 95 <211> 20 <212> DNA <213> Homo sapiens (Wise Man) <400> 95 cggaaaatat caagaagtag 20 <210> 96 <211> 20 <212> DNA <213> Homo sapiens (Wise Man) <400> 96 aactcagaga tgggaacttt 20 <210> 97 <211> 20 <212> DNA <213> Homo sapiens <400> 97 taactcagag atgggaactt 20 <210> 98 <211> 20 <212> DNA <213> Homo sapiens <400> 98 aatgtagata actcagagat 20 <210> 99 <211> 20 <212> DNA <213> Homo sapiens <400> 99 aaatgtagat aactcagaga 20 <210> 100 <211> 20 <212> DNA <213> Homo sapiens <400> 100 ttttattacc actatctttg 20 <210> 101 <211> 20 <212> DNA <213> Homo sapiens <400> 101 caataataca ggagccctcc 20 <210> 102 <211> 20 <212> DNA <213> Homo sapiens <400> 102 aagtacattg gcaataatac 20 <210> 103 <211> 20 <212> DNA <213> Homo sapiens <400> 103 ctgcattcag gaaagtacat 20 <210> 104 <211> 20 <212> DNA <213> Homo sapiens <400> 104 ctcagtttct ggctgcattc 20 <210> 105 <211> 20 <212> DNA <213> Homo sapiens <400> 105 ggaggggtgg gctcagtttc 20 <210> 106 <211> 20 <212> DNA <213> Homo sapiens <400> 106 aggcacatag gtggaggggt 20 <210> 107 <211> 20 <212> DNA <213> Homo sapiens <400> 107 aaggcacata ggtggagggg 20 <210> 108 <211> 20 <212> DNA <213> Homo sapiens <400> 108 cataaaggca cataggtgga 20 <210> 109 <211> 20 <212> DNA <213> Homo sapiens <400> 109 atagaaaggc acataggtgg 20 <210> 110 <211> 20 <212> DNA <213> Homo sapiens <400> 110 gggatagaaa ggcacatagg 20 <210> 111 <211> 20 <212> DNA <213> Homo sapiens <400> 111 agagggatag aaaggcacat 20 <210> 112 <211> 20 <212> DNA <213> Homo sapiens <400> 112 gcttctgcag aattcccagc 20 <210> 113 <211> 20 <212> DNA <213> Homo sapiens <400> 113 ccagcaagcc ctgtcctgct 20 <210> 114 <211> 20 <212> DNA <213> Homo sapiens <400> 114 tccagcaagc cctgtcctgc 20 <210> 115 <211> 20 <212> DNA <213> Homo sapiens <400> 115 tgcagaattc ccagcaggac 20 <210> 116 <211> 20 <212> DNA <213> Homo sapiens <400> 116 gcagaattcc cagcaggaca 20 <210> 117 <211> 20 <212> DNA <213> Homo sapiens <400> 117 cccagcagga cagggcttgc 20 <210> 118 <211> 20 <212> DNA <213> Homo sapiens <400> 118 gacagggctt gctggaagct 20 <210> 119 <211> 20 <212> DNA <213> Homo sapiens <400> 119 agaagctgct aaagtgtgta 20 <210> 120 <211> 20 <212> DNA <213> Homo sapiens <400> 120 gaagctgcta aagtgtgtat 20 <210> 121 <211> 20 <212> DNA <213> Homo sapiens <400> 121 ctgctaaagt gtgtatgggc 20 <210> 122 <211> 20 <212> DNA <213> Homo sapiens <400> 122 aaagtgtgta tgggcaggtg 20 <210> 123 <211> 20 <212> DNA <213> Homo sapiens <400> 123 aagtgtgtat gggcaggtgt 20 <210> 124 <211> 20 <212> DNA <213> Homo sapiens <400> 124 agtgtgtatg ggcaggtgtg 20 <210> 125 <211> 20 <212> DNA <213> Homo sapiens <400> 125 gtgtgtatgg gcaggtgtgg 20 <210> 126 <211> 20 <212> DNA <213> Homo sapiens <400> 126 caggtgtggg ggcaatttct 20 <210> 127 <211> 20 <212> DNA <213> Homo sapiens <400> 127 agtcgatata tggaagtgct 20 <210> 128 <211> 20 <212> DNA <213> Homo sapiens <400> 128 cagaaaagaa agtcgatata 20 <210> 129 <211> 20 <212> DNA <213> Homo sapiens <400> 129 catatatcga ctttcttttc 20 <210> 130 <211> 20 <212> DNA <213> Homo sapiens <400> 130 tttcttttct ggctgctaag 20 <210> 131 <211> 20 <212> DNA <213> Homo sapiens <400> 131 ttcttttctg gctgctaagt 20 <210> 132 <211> 20 <212> DNA <213> Homo sapiens <400> 132 ttttctggct gctagtggg 20 <210> 133 <211> 20 <212> DNA <213> Homo sapiens <400> 133 actgcagaga gaagacaagg 20 <210> 134 <211> 20 <212> DNA <213> Homo sapiens <400> 134 ggcactgcag agagaagaca 20 <210> 135 <211> 20 <212> DNA <213> Homo sapiens <400> 135 tgaaggaaaa agacactcct 20 <210> 136 <211> 20 <212> DNA <213> Homo sapiens <400> 136 aggttacatt tttgacttta 20 <210> 137 <211> 20 <212> DNA <213> Homo sapiens <400> 137 gtggtaggct tcctggcatc 20 <210> 138 <211> 20 <212> DNA <213> Homo sapiens <400> 138 tatctgctaa taaatatact 20 <210> 139 <211> 20 <212> DNA <213> Homo sapiens <400> 139 gaagactaat agtaacacat 20 <210> 140 <211> 20 <212> DNA <213> Homo sapiens <400> 140 Against the odds: 20 <210> 141 <211> 20 <212> DNA <213> Homo sapiens (people) <400> 141 ctgggataca cactaatacc 20 <210> 142 <211> 20 <212> DNA <213> Homo sapiens (people) <400> 142 Tatagag Caagtagtgc 20 <210> 143 <211> 20 <212> DNA <213> Homo sapiens (people) <400> 143 tgaactggca gacggcacct 20 <210> 144 <211> 20 <212> DNA <213> Homo sapiens (people) <400> 144 tcagatacca ttcctaaag 20 <210> 145 <211> 20 <212> DNA <213> Homo sapiens (people) <400> 145 Dad's dad's 20 <210> 146 <211> 20 <212> DNA <213> Homo sapiens (people) <400> 146 cggaaat chapter 20 <210> 147 <211> 20 <212> DNA <213> Homo sapiens <400> 147 taagcccaaa gttcccatct 20 <210> 148 <211> 20 <212> DNA <213> Homo sapiens <400> 148 attattgcca atgtactttc 20 <210> 149 <211> 20 <212> DNA <213> Homo sapiens <400> 149 atagaaaggc acataggtgg 20 <210> 150 <211> 20 <212> DNA <213> Homo sapiens <400> 150 ccagcaagcc ctgtcctgct 20 <210> 151 <211> 20 <212> DNA <213> Homo sapiens <400> 151 ttttctggct gctagtggg 20 <210> 152 <211> 20 <212> DNA <213> Homo sapiens <400> 152 gtgtgtacat gcatgtgtgtgt 20 <210> 153 <211> 20 <212> DNA <213> Homo sapiens <400> 153 actgcagaga gaagacaagg 20 <210> 154 <211> 22 <212> DNA <213> Homo sapiens <400> 154 ccagcccagc cagcaagcct gg 22 <210> 155 <211> 22 <212> DNA <213> Homo sapiens <400> 155 cttcaggtta tgatgagggg tg 22 <210> 156 <211> 22 <212> DNA <213> Homo sapiens <400> 156 gggaggggtg tcacttgccc aa 22 <210> 157 <211> 22 <212> DNA <213> Homo sapiens <400> 157 acccctgtg tagctttggg ca 22 <210> 158 <211> 22 <212> DNA <213> Homo sapiens <400> 158 aagcctgaag aatgagggggg ga 22 <210> 159 <211> 22 <212> DNA <213> Homo sapiens <400> 159 tggagagggc cagaaatcac cc 22 <210> 160 <211> 22 <212> DNA <213> Homo sapiens <400> 160 gagaggcca gaaatcaccc aa 22 <210> 161 <211> 22 <212> DNA <213> Homo sapiens <400> 161 gaaaacccac cagactgaac at 22 <210> 162 <211> 22 <212> DNA <213> Homo sapiens <400> 162 aaggagtagg ggccggctcc ct 22 <210> 163 <211> 22 <212> DNA <213> Homo sapiens <400> 163 tggggaccctg gggtgcccct ca 22 <210> 164 <211> 22 <212> DNA <213> Homo sapiens <400> 164 tcctgcaagg aagtgtcctg tg 22 <210> 165 <211> 22 <212> DNA <213> Homo sapiens <400> 165 ggaacagagg tgccatgcag cc 22 <210> 166 <211> 22 <212> DNA <213> Homo sapiens <400> 166 caacaaggag tacccggggc tg 22 <210> 167 <211> 22 <212> DNA <213> Homo sapiens <400> 167 gagcgccagg agggcaacaa ca 22 <210> 168 <211> 20 <212> DNA <213> Homo sapiens <400> 168 tctgctcagt tcatccctag 20 <210> 169 <211> 20 <212> DNA <213> Homo sapiens <400> 169 ctgctccagg taatgccctc 20 <210> 170 <211> 20 <212> DNA <213> Homo sapiens <400> 170 agaagcactt gctagagcta 20 <210> 171 <211> 20 <212> DNA <213> Homo sapiens <400> 171 ggggcacccg tccagctccg 20 <210> 172 <211> 20 <212> DNA <213> Homo sapiens <400> 172 cttcaggtta tgatgagggg 20 <210> 173 <211> 20 <212> DNA <213> Homo sapiens <400> 173 gttcttcagg tttgatgag 20 <210> 174 <211> 20 <212> DNA <213> Homo sapiens <400> 174 cccgggccctc catgttcttc 20 <210> 175 <211> 20 <212> DNA <213> Homo sapiens <400> 175 aggttcccccc ctcattcttc 20 <210> 176 <211> 20 <212> DNA <213> Homo sapiens <400> 176 cctaagcctg aagaatgagg 20 <210> 177 <211> 20 <212> DNA <213> Homo sapiens <400> 177 agccctaagc ctgaagaatg 20 <210> 178 <211> 20 <212> DNA <213> Homo sapiens <400> 178 gggtaggact gggctgtcta 20 <210> 179 <211> 20 <212> DNA <213> Homo sapiens <400> 179 agcccagtcc taccccagac 20 <210> 180 <211> 20 <212> DNA <213> Homo sapiens <400> 180 ggtgatttct ggccctctcc 20 <210> 181 <211> 20 <212> DNA <213> Homo sapiens <400> 181 cggagatcag tccagaccgc 20 <210> 182 <211> 20 <212> DNA <213> Homo sapiens <400> 182 gcgagggatc gaggcccaaa 20 <210> 183 <211> 20 <212> DNA <213> Homo sapiens <400> 183 tccctttcc cctccccaga 20 <210> 184 <211> 20 <212> DNA <213> Homo sapiens <400> 184 ccaggtaatg ccctctgggg 20 <210> 185 <211> 20 <212> DNA <213> Homo sapiens <400> 185 cctctctttc ccctccccag 20 <210> 186 <211> 20 <212> DNA <213> Homo sapiens <400> 186 caggtaatgc cctctgggga 20 <210> 187 <211> 20 <212> DNA <213> Homo sapiens <400> 187 aggtaatgcc ctctggggag 20 <210> 188 <211> 20 <212> DNA <213> Homo sapiens <400> 188 gccctctggg gaggggaaag 20 <210> 189 <211> 20 <212> DNA <213> Homo sapiens <400> 189 ctctggggag gggaaagagg 20 <210> 190 <211> 20 <212> DNA <213> Homo sapiens <400> 190 tctggggagg 20 years <210> 191 <211> 20 <212> DNA <213> Homo sapiens (mankind) <400> 191 gggagggga agaggagggg <210> 192 <211> 20 <212> DNA <213> Homo sapiens (mankind) <400> 192 aggggaaga ggaggggagg <210> 193 <211> 20 <212> DNA <213> Homo sapiens (mankind) <400> 193 aggaggggag gagggag <210> 194 <211> 20 <212> DNA <213> Homo sapiens (mankind) <400> 194 ggaggggagg rich <210> 195 <211> 20 <212> DNA <213> Homo sapiens (mankind) <400> 195 gaggggagga ggatgaagag <210> 196 <211> 20 <212> DNA <213> Homo sapiens (mankind) <400> 196 ggaggatgaa gaggggcaag <210> 197 <211> 20 <212> DNA <213> Homo sapiens <400> 197 cttgctggct gggctgggca 20 <210> 198 <211> 20 <212> DNA <213> Homo sapiens <400> 198 gcttgctggc tgggctgggc 20 <210> 199 <211> 20 <212> DNA <213> Homo sapiens <400> 199 ccaggcttgc tggctgggct 20 <210> 200 <211> 20 <212> DNA <213> Homo sapiens <400> 200 tccaggcttg ctggctgggc 20 <210> 201 <211> 20 <212> DNA <213> Homo sapiens <400> 201 cttctccagg cttgctggct 20 <210> 202 <211> 20 <212> DNA <213> Homo sapiens <400> 202 gcttctccag gcttgctggc 20 <210> 203 <211> 20 <212> DNA <213> Homo sapiens <400> 203 aagtgcttct ccaggcttgc 20 <210> 204 <211> 20 <212> DNA <213> Homo sapiens <400> 204 ccagcccag ccagcaagcc 20 <210> 205 <211> 20 <212> DNA <213> Homo sapiens <400> 205 gctctagcaa gtgcttctcc 20 <210> 206 <211> 20 <212> DNA <213> Homo sapiens <400> 206 cctccccaga gggcattacc 20 <210> 207 <211> 20 <212> DNA <213> Homo sapiens <400> 207 tgctagagct aaggaagcct 20 <210> 208 <211> 20 <212> DNA <213> Homo sapiens <400> 208 agctaaggaa gcctcggagc 20 <210> 209 <211> 20 <212> DNA <213> Homo sapiens <400> 209 tgctccaggt aatgccctct 20 <210> 210 <211> 20 <212> DNA <213> Homo sapiens <400> 210 aaggaagcct cggagctgga 20 <210> 211 <211> 20 <212> DNA <213> Homo sapiens <400> 211 aggaagcctc ggagctggac 20 <210> 212 <211> 20 <212> DNA <213> Homo sapiens <400> 212 aggttatgat gaggggtggg 20 <210> 213 <211> 20 <212> DNA <213> Homo sapiens <400> 213 caggttatga tgaggggtgg 20 <210> 214 <211> 20 <212> DNA <213> Homo sapiens <400> 214 tcaggttatg atgaggggtg 20 <210> 215 <211> 20 <212> DNA <213> Homo sapiens <400> 215 ttcaggttat gatgaggggt 20 <210> 216 <211> 20 <212> DNA <213> Homo sapiens <400> 216 gctccaggta atgccctctg 20 <210> 217 <211> 20 <212> DNA <213> Homo sapiens <400> 217 tgttcttcag gttatgatga 20 <210> 218 <211> 20 <212> DNA <213> Homo sapiens <400> 218 atgttcttca ggttatgatg 20 <210> 219 <211> 20 <212> DNA <213> Homo sapiens <400> 219 tcatcataac ctgaagaaca 20 <210> 220 <211> 20 <212> DNA <213> Homo sapiens <400> 220 tcataacctg aagaacatgg 20 <210> 221 <211> 20 <212> DNA <213> Homo sapiens <400> 221 acctgaagaa catggaggcc 20 <210> 222 <211> 20 <212> DNA <213> Homo sapiens <400> 222 ctgaagaac atggaggccc 20 <210> 223 <211> 20 <212> DNA <213> Homo sapiens <400> 223 gaagaacatg gaggccccggg 20 <210> 224 <211> 20 <212> DNA <213> Homo sapiens <400> 224 aagaacatgg aggccccggga 20 <210> 225 <211> 20 <212> DNA <213> Homo sapiens <400> 225 agaacatgga ggcccggggag 20 <210> 226 <211> 20 <212> DNA <213> Homo sapiens <400> 226 gggcaagtga cacccctccc 20 <210> 227 <211> 20 <212> DNA <213> Homo sapiens <400> 227 tgggcaagtg acacccctcc 20 <210> 228 <211> 20 <212> DNA <213> Homo sapiens <400> 228 cccaccccct gtgtagcttt 20 <210> 229 <211> 20 <212> DNA <213> Homo sapiens <400> 229 ccccaccccc tgtgtagctt 20 <210> 230 <211> 20 <212> DNA <213> Homo sapiens <400> 230 tcacttgccc aaagctacac 20 <210> 231 <211> 20 <212> DNA <213> Homo sapiens <400> 231 cacttgccca aagctacaca 20 <210> 232 <211> 20 <212> DNA <213> Homo sapiens <400> 232 acttgcccaa agctacacag 20 <210> 233 <211> 20 <212> DNA <213> Homo sapiens <400> 233 cttgcccaaa gctacacagg 20 <210> 234 <211> 20 <212> DNA <213> Homo sapiens <400> 234 gcccaaagct acacaggggg 20 <210> 235 <211> 20 <212> DNA <213> Homo sapiens <400> 235 cccaaagcta cacagggggt 20 <210> 236 <211> 20 <212> DNA <213> Homo sapiens <400> 236 ccaaagctac acagggggtg 20 <210> 237 <211> 20 <212> DNA <213> Homo sapiens <400> 237 agctacacag ggggtggggc 20 <210> 238 <211> 20 <212> DNA <213> Homo sapiens <400> 238 acagggggtg gggctggaag 20 <210> 239 <211> 20 <212> DNA <213> Homo sapiens <400> 239 ctggaagtgg ctccaagtgc 20 <210> 240 <211> 20 <212> DNA <213> Homo sapiens <400> 240 atgagggggg aacctgcact 20 <210> 241 <211> 20 <212> DNA <213> Homo sapiens <400> 241 ctaagcctga agaatgaggg 20 <210> 242 <211> 20 <212> DNA <213> Homo sapiens <400> 242 ccctaagcct gaagaatgag 20 <210> 243 <211> 20 <212> DNA <213> Homo sapiens <400> 243 gccctaagcc tgaagaatga 20 <210> 244 <211> 20 <212> DNA <213> Homo sapiens <400> 244 ccccctcatt cttcaggctt 20 <210> 245 <211> 20 <212> DNA <213> Homo sapiens <400> 245 cccctcattc ttcaggctta 20 <210> 246 <211> 20 <212> DNA <213> Homo sapiens <400> 246 tcattcttca ggcttagggc 20 <210> 247 <211> 20 <212> DNA <213> Homo sapiens <400> 247 ttcttcaggc ttagggctgg 20 <210> 248 <211> 20 <212> DNA <213> Homo sapiens <400> 248 tccctgtctg gggtaggact 20 <210> 249 <211> 20 <212> DNA <213> Homo sapiens <400> 249 ttccctgtct ggggtaggac 20 <210> 250 <211> 20 <212> DNA <213> Homo sapiens <400> 250 tcagtttccc tgtctggggt 20 <210> 251 <211> 20 <212> DNA <213> Homo sapiens <400> 251 gcccagtcct accccagaca 20 <210> 252 <211> 20 <212> DNA <213> Homo sapiens <400> 252 aggcctcagt ttccctgtct 20 <210> 253 <211> 20 <212> DNA <213> Homo sapiens <400> 253 caggcctcag tttccctgtc 20 <210> 254 <211> 20 <212> DNA <213> Homo sapiens <400> 254 taccccagac agggaaactg 20 <210> 255 <211> 20 <212> DNA <213> Homo sapiens <400> 255 gtgtgtcttt gggtgatttc 20 <210> 256 <211> 20 <212> DNA <213> Homo sapiens <400> 256 ccaacatgct gtgtgtcttt 20 <210> 257 <211> 20 <212> DNA <213> Homo sapiens <400> 257 gccaacatgc tgtgtgtctt 20 <210> 258 <211> 20 <212> DNA <213> Homo sapiens <400> 258 cccaaagaca cacagcatgt 20 <210> 259 <211> 20 <212> DNA <213> Homo sapiens <400> 259 aagacacaca gcatgttggc 20 <210> 260 <211> 20 <212> DNA <213> Homo sapiens <400> 260 acacagcatg ttggctggac 20 <210> 261 <211> 20 <212> DNA <213> Homo sapiens <400> 261 agcatgttgg ctggactgga 20 <210> 262 <211> 20 <212> DNA <213> Homo sapiens <400> 262 acatcaaggc acctgcggtc 20 <210> 263 <211> 20 <212> DNA <213> Homo sapiens <400> 263 actgaacatc aaggcacctg 20 <210> 264 <211> 20 <212> DNA <213> Homo sapiens <400> 264 acccaccaga ctgaacatca 20 <210> 265 <211> 20 <212> DNA <213> Homo sapiens <400> 265 aggtgccttg atgttcagtc 20 <210> 266 <211> 20 <212> DNA <213> Homo sapiens <400> 266 tgccttgatg ttcagtctgg 20 <210> 267 <211> 20 <212> DNA <213> Homo sapiens <400> 267 gccttgatgt tcagtctggt 20 <210> 268 <211> 20 <212> DNA <213> Homo sapiens <400> 268 ccaaagggag gtgggtggga 20 <210> 269 <211> 20 <212> DNA <213> Homo sapiens <400> 269 aggcccaaag ggaggtgggt 20 <210> 270 <211> 20 <212> DNA <213> Homo sapiens <400> 270 gaggcccaaa gggaggtggg 20 <210> 271 <211> 20 <212> DNA <213> Homo sapiens <400> 271 atcgaggcc aaagggaggt 20 <210> 272 <211> 20 <212> DNA <213> Homo sapiens <400> 272 gatcgaggcc caaagggagg 20 <210> 273 <211> 20 <212> DNA <213> Homo sapiens <400> 273 ccatcccacc cacctccctt 20 <210> 274 <211> 20 <212> DNA <213> Homo sapiens <400> 274 catcccacccc acctcccttt 20 <210> 275 <211> 20 <212> DNA <213> Homo sapiens <400> 275 agggatcgag gcccaaaggg 20 <210> 276 <211> 20 <212> DNA <213> Homo sapiens <400> 276 ggcgagggat cgaggcccaa 20 <210> 277 <211> 20 <212> DNA <213> Homo sapiens <400> 277 tggtgagggg cgagggatcg 20 <210> 278 <211> 20 <212> DNA <213> Homo sapiens <400> 278 gggggactgg tgaggggcga 20 <210> 279 <211> 20 <212> DNA <213> Homo sapiens <400> 279 agggggactg gtgaggggcg 20 <210> 280 <211> 20 <212> DNA <213> Homo sapiens <400> 280 tcagaagggg gactggtgag 20 <210> 281 <211> 20 <212> DNA <213> Homo sapiens <400> 281 ctcagaaggg ggactggtga 20 <210> 282 <211> 20 <212> DNA <213> Homo sapiens <400> 282 tctcagaagg gggactggtg 20 <210> 283 <211> 20 <212> DNA <213> Homo sapiens <400> 283 cgggctctca gaagggggac 20 <210> 284 <211> 20 <212> DNA <213> Homo sapiens <400> 284 taatacgggc tctcagaagg 20 <210> 285 <211> 20 <212> DNA <213> Homo sapiens <400> 285 ctaatacggg ctctcagaag 20 <210> 286 <211> 20 <212> DNA <213> Homo sapiens <400> 286 gcttaatacgg gctctcagaa 20 <210> 287 <211> 20 <212> DNA <213> Homo sapiens <400> 287 tgctaatacg ggctctcaga 20 <210> 288 <211> 20 <212> DNA <213> Homo sapiens <400> 288 ggccggctcc ctgctaatac 20 <210> 289 <211> 20 <212> DNA <213> Homo sapiens <400> 289 gggccggctc cctgctaata 20 <210> 290 <211> 20 <212> DNA <213> Homo sapiens <400> 290 ttctgagagc ccgtattagc 20 <210> 291 <211> 20 <212> DNA <213> Homo sapiens (mankind) <400> 291 tctgagagcc cgtattagca <210> 292 <211> 20 <212> DNA <213> Homo sapiens (mankind) <400> 292 agcccgtatt agcagggagc <210> 293 <211> 20 <212> DNA <213> Homo sapiens (mankind) <400> 293 ctgccagaag gagtagggc <210> 294 <211> 20 <212> DNA <213> Homo sapiens (mankind) <400> 294 gggtctgcca gaggagtag <210> 295 <211> 20 <212> DNA <213> Homo sapiens (mankind) <400> 295 tgggtctgcc average <210> 296 <211> 20 <212> DNA <213> Homo sapiens (mankind) <400> 296 ctgggtctgc cagaaggagt <210> 297 <211> 20 <212> DNA <213> Homo sapiens <400> 297 gagccggccc ctactccttc 20 <210> 298 <211> 20 <212> DNA <213> Homo sapiens <400> 298 ccttagctgg gtctgccaga 20 <210> 299 <211> 20 <212> DNA <213> Homo sapiens <400> 299 ccttctggca gacccagcta 20 <210> 300 <211> 20 <212> DNA <213> Homo sapiens <400> 300 cctaaggtag aaccttagct 20 <210> 301 <211> 20 <212> DNA <213> Homo sapiens <400> 301 ccctaaggta gaaccttagc 20 <210> 302 <211> 20 <212> DNA <213> Homo sapiens <400> 302 cccagctaag gttctacctt 20 <210> 303 <211> 20 <212> DNA <213> Homo sapiens <400> 303 ccagctaagg ttctacctta 20 <210> 304 <211> 20 <212> DNA <213> Homo sapiens <400> 304 cagctaaggt tctaccttag 20 <210> 305 <211> 20 <212> DNA <213> Homo sapiens <400> 305 gggaggtggc gtggcccta 20 <210> 306 <211> 20 <212> DNA <213> Homo sapiens <400> 306 ccctccctgg ggaggtggcg 20 <210> 307 <211> 20 <212> DNA <213> Homo sapiens <400> 307 tggaccccctc cctggggagg 20 <210> 308 <211> 20 <212> DNA <213> Homo sapiens <400> 308 aggggccacg ccacctcccc 20 <210> 309 <211> 20 <212> DNA <213> Homo sapiens <400> 309 ggggccacgc cacctcccca 20 <210> 310 <211> 20 <212> DNA <213> Homo sapiens <400> 310 ctctggaccc ctccctgggg 20 <210> 311 <211> 20 <212> DNA <213> Homo sapiens <400> 311 gccacgccac ctccccaggg 20 <210> 312 <211> 20 <212> DNA <213> Homo sapiens <400> 312 ccacgccacc tccccaggga 20 <210> 313 <211> 20 <212> DNA <213> Homo sapiens <400> 313 tgcctctgga cccctccctg 20 <210> 314 <211> 20 <212> DNA <213> Homo sapiens <400> 314 cacgccacct ccccagggag 20 <210> 315 <211> 20 <212> DNA <213> Homo sapiens <400> 315 atgcctctgg acccctccct 20 <210> 316 <211> 20 <212> DNA <213> Homo sapiens <400> 316 catgccctctg gacccctccc 20 <210> 317 <211> 20 <212> DNA <213> Homo sapiens <400> 317 ctccccaggg aggggtccag 20 <210> 318 <211> 20 <212> DNA <213> Homo sapiens <400> 318 cagggagggg tccagaggca 20 <210> 319 <211> 20 <212> DNA <213> Homo sapiens <400> 319 accccaggtc cccatgcctc 20 <210> 320 <211> 20 <212> DNA <213> Homo sapiens <400> 320 agggaggggt ccagaggcat 20 <210> 321 <211> 20 <212> DNA <213> Homo sapiens <400> 321 gggaggggtc cagaggcatg 20 <210> 322 <211> 20 <212> DNA <213> Homo sapiens <400> 322 ggtccagagg catggggacc 20 <210> 323 <211> 20 <212> DNA <213> Homo sapiens <400> 323 gtccagaggc atggggacct 20 <210> 324 <211> 20 <212> DNA <213> Homo sapiens <400> 324 tccagaggca tggggacctg 20 <210> 325 <211> 20 <212> DNA <213> Homo sapiens <400> 325 tgtcctgtga ggggcacccc 20 <210> 326 <211> 20 <212> DNA <213> Homo sapiens <400> 326 ggacctgggg tgcccctcac 20 <210> 327 <211> 20 <212> DNA <213> Homo sapiens <400> 327 gcaaggaagt gtcctgtgag 20 <210> 328 <211> 20 <212> DNA <213> Homo sapiens <400> 328 tgcaaggaag tgtcctgtga <210> 329 <211> 20 <212> DNA <213> Homo sapiens (mankind) <400> 329 ctgcaagga gtgtcctgtg <210> 330 <211> 20 <212> DNA <213> Homo sapiens (mankind) <400> 330 20. ctcacaggac acttccttgc <210> 331 <211> 20 <212> DNA <213> Homo sapiens (mankind) <400> 331 atggcacctc tgttcctgca <210> 332 <211> 20 <212> DNA <213> Homo sapiens (mankind) <400> 332 acacttcctt gcaggacag <210> 333 <211> 20 <212> DNA <213> Homo sapiens (mankind) <400> 333 gaggggaag aggaggggag <210> 334 <211> 20 <212> DNA <213> Homo sapiens (mankind) <400> 334 taggedcc tcggagctgg <210> 335 <211> 20 <212> DNA <213> Homo sapiens <400> 335 atgttcttca ggttatgatg 20 <210> 336 <211> 20 <212> DNA <213> Homo sapiens <400> 336 gaagaacatg gaggccccggg 20 <210> 337 <211> 20 <212> DNA <213> Homo sapiens <400> 337 cacttgccca aagctacaca 20 <210> 338 <211> 20 <212> DNA <213> Homo sapiens <400> 338 ttcctccagc cctaagcctg 20 <210> 339 <211> 20 <212> DNA <213> Homo sapiens <400> 339 caggcctcag tttccctgtc 20 <210> 340 <211> 20 <212> DNA <213> Homo sapiens <400> 340 agccaacatg ctgtgtgtct 20 <210> 341 <211> 20 <212> DNA <213> Homo sapiens <400> 341 gtgccttgat gttcagtctg 20 <210> 342 <211> 20 <212> DNA <213> Homo sapiens <400> 342 gaggcccaaa gggaggtggg 20 <210> 343 <211> 20 <212> DNA <213> Homo sapiens <400> 343 ggacgaggc ccaaagggag 20 <210> 344 <211> 20 <212> DNA <213> Homo sapiens <400> 344 agggggactg gtgaggggcg 20 <210> 345 <211> 20 <212> DNA <213> Homo sapiens <400> 345 ccttagctgg gtctgccaga 20 <210> 346 <211> 20 <212> DNA <213> Homo sapiens <400> 346 cccctaaggt agaaccttag 20 <210> 347 <211> 20 <212> DNA <213> Homo sapiens <400> 347 gccacgccac ctccccaggg 20 <210> 348 <211> 20 <212> DNA <213> Homo sapiens <400> 348 ggtccagagg catggggacc 20

Claims

1. An engineered human hepatocyte, comprising: an engineered genome containing an artificially inserted transgene, in, The transgene is inserted into a region of the wild-type APOC3 gene consisting of a continuous 70 bp sequence, wherein the continuous 70 bp sequence comprises SEQ ID NO: 160 or SEQ ID NO: 176, Thus, the transgene is located in intron 1 of APOC3, a highly expressed secretory gene in hepatocytes. wherein the transgene is expressed by the endogenous promoter of the APOC3 gene, wherein the transgene is expressed at a higher level than before insertion, or the transgene is newly expressed in the engineered hepatocytes.

2. The engineered human hepatocyte according to claim 1, wherein The transgene is inserted into the genome of human hepatocytes by cleavage mediated by the CRISPR / Cas9 system of the Cas9 protein and a guide nucleic acid targeting the sequence of SEQ ID NO: 160 or SEQ ID NO:

176.

3. The engineered human hepatocyte according to claim 2, wherein The protein produced by expressing the transgene is present in the engineered human hepatocytes or is secreted outside of the engineered human hepatocytes.

4. The engineered human hepatocyte according to claim 2, wherein The transgene was inserted into a region consisting of a continuous 70 bp sequence comprising SEQ ID NO:160 in the wild-type APOC3 gene.

5. The engineered human hepatocyte according to claim 2, wherein The transgene was inserted into a region consisting of a continuous 70 bp sequence comprising SEQ ID NO:176 in the wild-type APOC3 gene.

6. The engineered human hepatocyte according to claim 2, wherein: The transgene is a gene encoding a protein of interest.

7. The engineered human hepatocyte of claim 6, wherein: The protein of interest is an antibody.

8. The engineered human hepatocyte according to claim 6, wherein The protein of interest is any one selected from the following: IDUA, I2S, SGSH, NAGLU, HGSNAT, GNS, GALNS, GLB1, ARSB, GUSB, HYAL, NEU, GNPTAB, MCOLN1, SAH1, GALC, CTSA, GLA, NAGA, β-galactosidase, hexosaminidase, GBA, SMPD1, ARSA, SUMF, NPC, PPT, TPP1, CLN3, CLN6, PPT1, DNAJC5, CT SF, CLN7, CLN8, CTSD, GAA, LAMP2, CTNS, CTSK, SLC17A5, MAN2B, MAN2C, MANBA, AGA, FUCA1, LAL, methylmalonic aciduria CbIA type (MMAA) protein, methylmalonic aciduria CbIB type (MMAB) protein, methylmalonic aciduria CbIC type (MMADHC) protein, 5-methyltetrahydrofolate-homocysteine ​​methyltransferase reductase (MTRR), lysosomal membrane protein domain (LMBRD 1), 5-methyltetrahydrofolate-homocysteine ​​methyltransferase (MTR), propionyl-CoA protein, glucose-6-phosphate transporter (G6PT) protein, glucose-6-phosphatase (G6Pase), low-density lipoprotein receptor (LDLR), low-density lipoprotein receptor adaptor protein 1 (LDLRAP-1 protein), N-acetylglutamate synthetase (NAGS), carbamoyl phosphate synthetase 1 (CPS1), ornithine carbamoyltransferase (OTC), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), arginase (ARG1), solute carrier family 25 protein, UDP glucuronosyltransferase 1 family polypeptide A1 (UGT1A1), fumarylacetoacetate hydrolase (FAH), alanine-glyoxylate aminotransferase (AGXT), glyoxylate reductase / hydroxypyruvate reductase (GRHPR), APTase Cu(2+) transporting beta (ATP7B) protein, phenylalanine hydroxylase (PAH), lipoprotein lyase (LPL), factor VII, factor VIII, factor IX, factor X, factor XI, and factor XII.

9. The engineered human hepatocyte of claim 2, wherein: The transgene is a gene encoding a therapeutic protein for treating the disease of interest.

10. The engineered human hepatocyte of claim 9, wherein: The disease of interest is any one selected from immunodeficiency and lysosomal storage disease.

11. The engineered human hepatocyte of claim 9, wherein: The disease of interest is any one selected from HIV, Alzheimer's disease, amyotrophic lateral sclerosis, hypercholesterolemia, hemophilia, emphysema, cystic fibrosis, phenylketonuria, citrullinemia, methylmalonic acidemia, carnitine palmitoyltransferase deficiency, Hurler syndrome, ornithine carbamoyltransferase deficiency and Tay-Sachs disease.

12. A composition for inserting a transgene into intron 1 of the APOC3 gene on the genome of a human hepatocyte, the composition comprising: a guide nucleic acid or a nucleic acid encoding said guide nucleic acid, an editing protein or a nucleic acid encoding the editing protein, and a donor or a nucleic acid encoding said donor, in, The guide nucleic acid comprises a guide domain corresponding to a target sequence located in intron 1 of the APOC3 gene present in the hepatocyte genome, wherein the target sequence is a sequence of SEQ ID NO: 160 or SEQ ID NO: 176, wherein the guide domain comprises 0-5 nucleotides that are mismatched with the target sequence, Wherein, the donor comprises the transgene.

13. The composition of claim 12, wherein The target sequence is the sequence of SEQ ID NO: 160, wherein the editing protein is the Cas9 protein derived from Campylobacter jejuni.

14. The composition of claim 12, wherein The target sequence is the sequence of SEQ ID NO: 176, wherein the editing protein is the Cas9 protein derived from Streptococcus pyogenes.

15. The composition of claim 12, wherein The guide nucleic acid, the editing protein and the donor are respectively encoded in at least one vector in the form of nucleotide sequences.

16. The composition of claim 15, wherein The vector is a viral vector, and the viral vector is at least one selected from the group consisting of adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus, herpes simplex virus and lentivirus.

17. The composition of claim 12, wherein The transgene is a gene encoding a protein of interest.

18. The composition of claim 17, wherein The protein of interest is an antibody.

19. The composition of claim 17, wherein The protein of interest is any one selected from the following: IDUA, I2S, SGSH, NAGLU, HGSNAT, GNS, GALNS, GLB1, ARSB, GUSB, HYAL, NEU, GNPTAB, MCOLN1, SAH1, GALC, CTSA, GLA, NAGA, β-galactosidase, hexosaminidase, GBA, SMPD1, ARSA, SUMF, NPC, PPT, TPP1, CLN3, CLN6, PPT1, DNAJC5, CT SF, CLN7, CLN8, CTSD, GAA, LAMP2, CTNS, CTSK, SLC17A5, MAN2B, MAN2C, MANBA, AGA, FUCA1, LAL, methylmalonic aciduria CbIA type (MMAA) protein, methylmalonic aciduria CbIB type (MMAB) protein, methylmalonic aciduria CbIC type (MMADHC) protein, 5-methyltetrahydrofolate-homocysteine ​​methyltransferase reductase (MTRR), lysosomal membrane protein domain (LMBRD 1), 5-methyltetrahydrofolate-homocysteine ​​methyltransferase (MTR), propionyl-CoA protein, glucose-6-phosphate transporter (G6PT) protein, glucose-6-phosphatase (G6Pase), low-density lipoprotein receptor (LDLR), low-density lipoprotein receptor adaptor protein 1 (LDLRAP-1 protein), N-acetylglutamate synthetase (NAGS), carbamoyl phosphate synthetase 1 (CPS1), ornithine carbamoyltransferase (OTC), argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), arginase (ARG1), solute carrier family 25 protein, UDP glucuronosyltransferase 1 family polypeptide A1 (UGT1A1), fumarylacetoacetate hydrolase (FAH), alanine-glyoxylate aminotransferase (AGXT), glyoxylate reductase / hydroxypyruvate reductase (GRHPR), APTase Cu(2+) transporting beta (ATP7B) protein, phenylalanine hydroxylase (PAH), lipoprotein lyase (LPL), factor VII, factor VIII, factor IX, factor X, factor XI, and factor XII.

20. The composition of claim 12, wherein The transgene is a gene encoding a therapeutic protein for treating the disease of interest.

21. The engineered composition of claim 20, wherein: The disease of interest is any one selected from immunodeficiency and lysosomal storage disease.

22. The composition of claim 20, wherein The disease of interest is any one selected from HIV, Alzheimer's disease, amyotrophic lateral sclerosis, hypercholesterolemia, hemophilia, emphysema, cystic fibrosis, phenylketonuria, citrullinemia, methylmalonic acidemia, carnitine palmitoyltransferase deficiency, Hurler syndrome, ornithine carbamoyltransferase deficiency and Tay-Sachs disease.

23. A method for modifying the genome of a human hepatocyte by inserting a transgene into intron 1 of the APOC3 gene on the genome of the human hepatocyte in vitro or ex vivo, the method comprising: The guide nucleic acid or nucleic acid encoding the guide nucleic acid, the editing protein or nucleic acid encoding the editing protein, and the donor or nucleic acid encoding the donor of the composition of any one of claims 12 to 22 are introduced into human hepatocytes.

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