Blood-compatible intravascular transplantation cell, and preparation method and application thereof

Abstract

The present invention provides: a method for reducing or inhibiting the expression or activity of CD142 by artificially deforming an F3 gene encoding CD142, which is a starting factor for blood coagulation, by means of a gene editing technique; the present invention relates to a cell for intravascular transplantation having blood compatibility, a method for preparing the same, and a use of the same in the prevention or treatment of liver diseases and diabetes mellitus, and more particularly, to a cell for intravascular transplantation having blood compatibility.

Description

Technical Field

[0001] The present invention provides blood-compatible cells for intravascular transplantation that can be administered into a blood vessel by reducing or inhibiting the expression or activity level of CD142, an initiator of blood coagulation, to suppress thrombogenic reaction, and a method for preparing and using the cells. Background Art

[0002] In order to minimize blood loss caused by tissue damage, animals have a blood coagulation system as a complex biological tool to prevent bleeding. The blood coagulation system is a continuous amplification reaction process called a cascade reaction to an initial stimulus, which consists of an intrinsic pathway directly induced by the stimulus and a unique extrinsic pathway. It is related to a variety of blood coagulation factors.

[0003] Among them, CD142 is a cell membrane glycoprotein that acts in the initial stage of the extrinsic pathway coagulation process and plays an important role in the coagulation process in vivo. After binding to coagulation factor VII or VIIa, CD142 activates coagulation factor IX and X, which is related to the activation of prothrombin to thrombin, to generate thrombin during the coagulation process. Thrombin activates fibrinogen to fibrin to form a thrombus. In addition, CD142 stimulates the activity of neutrophils, platelets, and monocytes by activating immune cells, inducing the secretion of various cytokines and activating the intrinsic immune system.

[0004] Cell transplantation is a rapidly developing treatment method in regenerative medicine. In order to restore the function of cells and tissues, living autologous, allogenic or xenogeneic cells are cultured in an in vitro environment (in vitro) and isolated, proliferated and selected or a series of behaviors that change the biological characteristics of cells are used for therapeutic, diagnostic and preventive purposes. Compared with the transplantation of whole organs, cell-based treatment is a low morbidity / mortality and less invasive process. Cell transplantation significantly reduces the risks associated with surgery and has the advantage of preserving the original organ even if the function of the graft is insufficient or graft loss occurs.

[0005] The most ideal method for treating type 1 diabetes is islet cell transplantation, but there are still problems in clinical application because of inflammatory reactions such as acute blood mediated inflammatory reaction (IBMIR) that limit the survival of transplanted islet cells. Portal vein thrombosis induced by islet transplantation is a well-known serious complication. In order to prevent the above complications, anticoagulants such as heparin are used. Acute blood-mediated inflammatory reaction is when islet cells are transplanted through the portal vein, and the transplanted islet cells are directly exposed to the blood. Due to the activation of blood coagulation systems such as platelets and complement, blood coagulation occurs around the islet cells, causing rapid destruction of the islet cells. This is caused by the dual activation of blood coagulation and complement pathways caused by islet cells or cells that retain CD142 (tissue factor, TF, factor III, tissue factor, blood coagulation factor III).

[0006] Hepatocyte transplantation is an alternative to liver transplantation for patients with specific liver-based metabolic diseases and acute liver failure. For transplantation, human hepatocytes are isolated from donor liver tissue using collagenase perfusion technology and purified using centrifugation. The number of transplanted cells is targeted at about 5% of normal liver mass and is usually administered through the portal vein or spleen. One of the main obstacles to the continued success of this therapy is the initial cell loss, and the loss of hepatocytes can reach up to 70% immediately after perfusion. It is known that this is caused by acute blood-mediated inflammatory response (IBMIR). Transplanted hepatocytes form thrombin and fibrin coagulation by producing and releasing CD142 (tissue factor, TF, factor III, tissue factor, blood coagulation factor III) that activates the coagulation pathway. Thrombin activates the membrane attack complex (MAC, membrane attack complex) by activating multiple complement proteins and causes subsequent apoptosis of hepatocytes. Summary of the invention

[0007] Problems to be solved by the invention

[0008] An object of the present invention is to provide blood-compatible cells for intravascular transplantation in which a thrombogenic reaction induced by the administered cells can be suppressed when administered into a blood vessel, a method for preparing the cells, and a cell therapy agent using the cells.

[0009] Means for solving problems

[0010] To achieve the above object, the present invention provides blood-compatible cells for intravascular transplantation that can be administered into a blood vessel by reducing or inhibiting the expression or activity level of CD142, an initiator of blood coagulation, to suppress thrombogenic reaction.

[0011] Furthermore, the present invention provides an artificially modified cell for intravascular transplantation, which is an artificially modified mammalian cell comprising an artificially modified F3 gene, characterized in that the artificially modified F3 gene is different from the F3 gene sequence of a wild-type mammalian cell, the artificially modified F3 gene comprises one or more insertions / deletions (indels) in the nucleic acid sequence, and the expression level of CD142 on the surface of the artificially modified mammalian cell is reduced compared to that of the wild-type mammalian cell.

[0012] Furthermore, the present invention provides an artificially modified cell for intravascular transplantation, which is a cell differentiated from or derived from an artificially modified stem cell containing an artificially modified F3 gene, characterized in that the artificially modified F3 gene is different from the F3 gene sequence of a cell differentiated from or derived from a wild-type stem cell, the artificially modified F3 gene contains one or more insertions / deletions in the nucleic acid sequence, and the expression level of CD142 on the surface of the cell differentiated from or derived from the artificially modified stem cell is reduced compared to that of the cell differentiated from or derived from the wild-type stem cell.

[0013] Furthermore, the present invention provides a composition for preparing blood-compatible cells for intravascular transplantation, comprising: a guide nucleic acid or a nucleic acid encoding the same, a guide sequence comprising a target sequence of the F3 gene that can target mammalian cells or stem cells; and an editing protein or a nucleic acid encoding the same.

[0014] Furthermore, the present invention provides a method for preparing blood-compatible cells for intravascular transplantation, which comprises: step (1), introducing a composition for preparing blood-compatible cells for intravascular transplantation into isolated mammalian cells or isolated stem cells, wherein the composition comprises a guide nucleic acid capable of targeting a target sequence of the F3 gene or a nucleic acid encoding the same and an editing protein or a nucleic acid encoding the same; and step (2), editing the F3 gene by causing insertion / deletion of the target sequence of the F3 gene located in the genome of the isolated mammalian cells or isolated stem cells to reduce or inhibit the expression or activity of CD142.

[0015] Furthermore, the present invention provides a cell therapy agent for vascular administration, comprising artificially modified cells for transplantation into blood vessels as an effective ingredient so as to have the above-mentioned blood compatibility.

[0016] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating liver diseases, comprising artificially modified intravascular transplantation cells as an effective ingredient so as to have the above-mentioned blood compatibility.

[0017] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising artificially modified intravascular transplantation cells as an effective ingredient so as to have the above-mentioned blood compatibility.

[0018] Effects of the Invention

[0019] The artificially modified intravascular transplantation cells of the present invention artificially deform the F3 gene encoding CD142, which is a blood coagulation initiation factor, using gene editing technology, thereby reducing or inhibiting the expression or activity of CD142, thereby inhibiting the blood coagulation mechanism. Therefore, it is possible to intravascularly administer transplantation cells that cannot show therapeutic effects due to post-transplantation losses caused by inflammatory reactions such as acute blood-mediated inflammatory reactions caused by overexpression of CD142 on the cell surface, and the therapeutic effect can be improved by preventing the loss of transplanted cells.

[0020] Brief description of the attached figure

[0021] Figure 1 The figure is a graph showing the results of targeted deep sequencing of normal iPSCs and iPSCs with the target sequence (SEQ ID NO. 1) of gene-edited CD142#33.

[0022] Figure 2 The results of confirming the differentiation markers PDX-1, NKX6.1, and SOX9 of pancreatic lineage cells in a control group (normal iPSC) of normal iPSC-derived pancreatic lineage cells and an experimental group (iPSC-CD142#33) of iPSC-derived pancreatic lineage cells with CD142 gene knocked out by qRT-PCR are shown.

[0023] Figure 3 The diagram shows the results of confirming the differentiation markers HNF4α, ALB (Albumin), and AFP of hepatocytes in a control group of normal iPSC-derived hepatocytes (normal iPSC) and an experimental group of CD142 gene-knocked-out iPSC-derived hepatocytes (iPSC-CD142#33) by qRT-PCR.

[0024] Figure 4 The results of ELISA analysis of ALB (albumin), a differentiation marker for hepatocytes, in a control group of normal iPSC-derived hepatocytes (normal iPSC) and an experimental group of CD142 gene-knockout iPSC-derived hepatocytes (iPSC-CD142#33) are shown.

[0025] Figure 5The graph shows the results of confirming the CD142 mRNA levels in a control group (normaliPSC) of normal iPSC-derived pancreatic lineage cells and an experimental group (iPSC-CD142#33) of iPSC-derived pancreatic lineage cells in which the CD142 gene was knocked out by qRT-PCR.

[0026] Figure 6 The figures show the results of FACS measurement of the proportion of cells expressing CD142 (CD142 positive cell population, %) in a control group (normal iPSC) of normal iPSC-derived pancreatic lineage cells and an experimental group (iPSC-CD142#33) of CD142 gene-knocked-out iPSC-derived pancreatic lineage cells.

[0027] Figure 7 The graph shows the results of confirming the CD142 mRNA levels in a control group (normal iPSC) of normal iPSC-derived hepatocytes and an experimental group (iPSC-CD142#33) of iPSC-derived hepatocytes in which the CD142 gene was knocked out by qRT-PCR.

[0028] Figure 8 The graph shows the results of FACS measurement of the proportion of cells expressing CD142 in a control group (normal iPSC) of normal iPSC-derived hepatocytes and an experimental group (iPSC-CD142#33) of iPSC-derived hepatocytes in which the CD142 gene was knocked out.

[0029] Fig. 9 The results of ELISA analysis of the amount of CD142 protein in cell lysates of a control group of normal iPSC-derived pancreatic lineage cells (normaliPSC) and an experimental group of CD142 gene-knocked-out iPSC-derived pancreatic lineage cells (iPSC-CD142#33) are shown.

[0030] Fig.10 The results of ELISA analysis of the amount of CD142 protein in cell lysates of a control group of normal iPSC-derived hepatocytes (normal iPSC) and an experimental group of iPSC-derived hepatocytes in which the CD142 gene was knocked out (iPSC-CD142#33) are shown.

[0031] Specific embodiments of the invention

[0032] Hereinafter, the present invention will be described in detail.

[0033] In this specification, "stem cell" refers to a general term for undifferentiated cells that have the ability to differentiate into a variety of body tissue cells, that is, stem cells with stemness. In this case, the stem cells may be induced pluripotent stem cells, embryonic stem cells (somatic cell nuclear transfer-PSC) and adult stem cells. In addition, the cells may be human-derived, but are not limited thereto.

[0034] In this specification, "differentiation" refers to the phenomenon that cells proliferate by dividing, and the structure or function of cells is specialized during the growth of the whole individual. That is, it refers to the process in which cells, tissues, etc. of an organism change into appropriate forms and functions in order to play their respective roles. For example, it includes not only the process in which pluripotent stem cells become ectoderm (cerebral cortex, midbrain, hypothalamus, etc.), mesoderm (yolk sac, etc.) and endoderm cells, but also the process in which hematopoietic stem cells become red blood cells, white blood cells, platelets, etc., that is, the precursor cells expressing specific differentiation traits can be included in differentiation.

[0035] The term "about" as used in this specification refers to an amount, level, value, number, frequency, percentage, size, size, quantity, weight or length that varies by about 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 relative to a reference amount, level, value, number, frequency, percentage, size, size, quantity, weight or length.

[0036] The term "artificial modification" used in this specification is a term used to distinguish substances, molecules, etc. with structures that already exist in nature, and refers to artificial deformation of the above substances, molecules, etc. For example, "artificially modified genes" refer to genes that have artificial deformation of the structure of genes that exist in nature. In addition, the above terms include all meanings that can be recognized by those skilled in the art and can be appropriately understood according to the context.

[0037] "Wild type" used in this specification refers to a gene comprising a naturally occurring base sequence and a protein expressed from the gene with normal functional characteristics. A wild-type gene has a natural or non-artificially mutated form and can be observed most frequently in a group. In this specification, if the term "wild type" is used in comparison with an artificially modified gene and / or an artificially modified cell, it should be interpreted as the meaning of a gene comprising a naturally occurring base sequence and a cell having the same "unartificially modified" corresponding to an artificially modified gene and / or an artificially modified cell. In addition, the above terms include all meanings that can be recognized by a person skilled in the art and can be appropriately understood according to the context.

[0038] The expression of so-called "knock-out" or "knocked gene" used in this specification refers to a mutation or artificial deformation in a wild-type gene, as a result of which the protein expressed by the wild-type gene cannot be generated by transcription and / or translation processes. For example, a cell comprising a knocked-out gene A may not be able to express mRNA and / or protein expressed by wild-type gene A. The so-called cell comprising a knocked-out A gene may be a cell in which only one of the genes A present in the cell is knocked out, or a cell in which more than two are knocked out. Furthermore, the above terms include all meanings that can be recognized by a person skilled in the art, and can be appropriately understood according to the context.

[0039] The expression of "knock-down" or "knock-down gene" used in this specification refers to a mutation or artificial deformation in a wild-type gene, which results in the expression of a substance in an amount less than that of the wild-type gene. For example, a cell comprising a knock-down gene A can express a small amount of mRNA than the mRNA expressed by the wild-type gene A. As another example, a cell comprising a knock-down gene A can express a small amount of protein than the protein expressed by the wild-type gene A. The so-called cell in which the A gene is knocked down can be a cell that only knocks down one of the genes A present in the cell, or a cell that knocks down more than two. In addition, the above terms include all meanings that can be recognized by a person skilled in the art, and can be appropriately understood according to the context.

[0040] In this specification, "reduced expression" refers to expression that is lower than the expression level of mRNA and / or protein measured in the wild type. The above reduction can be about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 50% or more, about 60% or more, about 70% or more, or about 100% or more compared to cells without genetic deformation or wild type cells.

[0041] The term "reduced activity" or "reduced activity" used in this specification may refer to a relative decrease in activity when measuring the activity of a protein or enzyme. Specifically, "reduced activity" or "reduced activity" refers to a lower level of protein or enzyme activity than a given progenitor cell or wild-type cell.

[0042] The term "hemocompatibility" as used herein means that the substance does not induce hemolysis, platelet adhesion, fibrin formation due to platelet activation and complement activation, thrombotic reaction or embolism when in contact with blood.

[0043] The present invention provides cells for intravascular transplantation that reduce or inhibit the expression or activity of CD142 (tissue factor, TF, factor III, tissue factor, blood coagulation factor III).

[0044] The above-mentioned cells for intravascular transplantation with reduced or inhibited expression or activity of CD142 are mammalian cells that inhibit the thrombus coagulation mechanism by reducing or inhibiting the expression or activity of CD142, which is a cell membrane glycoprotein that plays an important role in the blood coagulation process in the body and acts in the initial stage of the extrinsic coagulation process. When the above-mentioned mammalian cells with reduced or inhibited expression or activity of CD142 are administered as cell therapeutic agents through vascular administration, they can show improved blood compatibility than wild-type mammalian cells, mammalian cells or antibodies artificially modified by existing technologies, and CD142 low-expressing mammalian cells screened by cell sorting devices or magnetic beads.

[0045] Specifically, the present invention provides artificially modified cells for intravascular transplantation with blood compatibility, characterized in that the expression or activity of F3 mRNA and / or CD142 is reduced or inhibited by artificially modifying the F3 gene of mammalian cells, thereby reducing blood coagulation reactions when administered intravascularly.

[0046] Mammals of the present invention may be, for example, primates such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees, etc.; rodents such as mice, rats, hamsters, guinea pigs, etc.; Lagomorpha such as rabbits; Ungulates such as pigs, cattle, goats, horses, sheep, etc.; Feliformes such as dogs and cats, etc. Among them, mice, pigs, and humans can be appropriately exemplified.

[0047] According to one embodiment of the present invention, the cells for transplantation of the present invention are allogenic or autologous cells for transplantation, more specifically, allogenic cells for transplantation.

[0048] According to one example of the present invention, the mammalian cell of the present invention can be a primary hepatocyte, a primary pancreatic β cell, a primary pancreatic islet cell, a pancreatic progenitor cell, a pluripotent stem cell (PSC) derived hepatocyte, a pluripotent stem cell derived pancreatic β cell (PSC derived pancreatic beta cell), a pluripotent stem cell derived pancreatic islet cell, a pluripotent stem cell derived pancreatic lineage cell, a pluripotent stem cell derived pancreatic progenitor cell, a pluripotent stem cell derived pancreatic organoid, a pluripotent stem cell derived pancreatic liver organoid, an induced pluripotent stem cell derived hepatocyte (iPSC derived hepatocyte), an induced pluripotent stem cell derived pancreatic β cell (iPSC derived pancreatic beta cell), an induced pluripotent stem cell derived pancreatic islet cell (iPSC derived pancreatic islet cell). cell), pancreatic lineage cells derived from induced pluripotent stem cells, pancreatic progenitor cells derived from induced pluripotent stem cells, pancreatic organoids derived from induced pluripotent stem cells, pancreatic liver organoids derived from induced pluripotent stem cells, hepatocytes (induced hepatocytes, iHeps) induced from somatic cells by compounds or genetic modification, islet cells induced from somatic cells by compounds or genetic modification, or pancreatic β cells induced from somatic cells by compounds or genetic modification.

[0049] According to one example of the present invention, the transplantation cells of the present invention are cells differentiated or derived from artificially modified stem cells containing an artificially modified F3 gene, the artificially modified F3 gene is different from the F3 gene sequence of cells differentiated or derived from wild-type stem cells, the artificially modified F3 gene contains one or more insertions / deletions in the nucleic acid sequence, and the expression level of CD142 on the surface of the cells differentiated or derived from the artificially modified stem cells is reduced compared with that of the cells differentiated or derived from wild-type stem cells.

[0050] According to an embodiment of the present invention, the stem cells are induced pluripotent stem cells (iPSCs), embryonic stem cells, somatic cell nuclear transfer embryonic stem cells or adult stem cells.

[0051] According to one example of the present invention, the cells differentiated from or derived from artificially modified stem cells are pancreatic β cells, hepatocytes, pancreatic progenitor cells, islet cells, pancreatic lineage cells, pancreatic organoids or pancreatic liver organoids.

[0052] The present invention provides artificially modified cells for intravascular transplantation comprising an artificially modified nucleic acid sequence of the F3 gene.

[0053] In the present invention, the "artificial deformation" or "artificial modification" of the gene nucleic acid sequence can be achieved by deformation of the nucleic acid sequence constituting the gene or chemical deformation of a single base. This can be achieved by mutation, substitution, deletion of part or all of the gene, or insertion of more than one base into the above-mentioned gene, and can be achieved by gene editing technologies such as CRISPR enzyme system (CRISPR-enzymesystem). As an example, the artificial deformation of the above-mentioned gene nucleic acid sequence can be achieved by non-homologous end joining (NHEJ) or homology directed repair (HDR) mechanism.

[0054] For example, the artificially modified intravascular transplantation cell may be an artificially modified intravascular transplantation cell in which the F3 gene is knocked out.

[0055] In this specification, "non-homologous end joining (NHEJ)" is a method for repairing or mending double-stranded breaks in DNA by combining the two ends of a cut double-stranded or single-stranded chain. Usually, two compatible ends formed by double-stranded damage (e.g., cutting) are brought into repeated and frequent contact to completely combine the two ends, thereby restoring the damaged double strand.

[0056] In the repair process of damaged genes or nucleic acids using non-homologous end joining (NHEJ) as described above, "insertion and / or deletion" (or "InDel") of a portion of the nucleic acid sequence may occur at the NHEJ repair site, and the gene in which the insertion / deletion occurs does not have the same sequence as the wild-type gene. Such insertion and / or deletion changes the reading frame of the gene, producing a shifted transcript mRNA, resulting in the loss of the original function through nonsense mediated decay or failure in synthesizing normal proteins. Alternatively, although the reading frame is maintained, mutations that cause a considerable amount of sequence insertion or deletion may occur, thereby destroying the functionality of the protein. As another example, in the case of insertion / deletion in a transcriptional regulatory region such as a promoter region or an enhancer region of a gene, the mRNA is not transcribed or the amount of transcription is reduced, so the protein may not be expressed or the amount of expression is reduced. Alternatively, using the mutation mechanism of NHEJ, when the generation of a specific final sequence is not required, it can be used only in the deletion of a portion of the sequence motif. For example, by using two or more guide RNAs that target the intron sites of the 5' and 3' parts of a specific exon, double-stranded cleavage is induced in each intron part. Through NHEJ, if only part of the exon of a gene is deleted, the main functionality of the protein can be maintained because the other parts are expressed normally.

[0057] Where such NHEJ is utilized, gene editing techniques can be used to specifically knock out or knock down the targeted gene.

[0058] For example, a CRISPR enzyme such as Cas9 or Cpf1, one of the gene scissors, can be used to cut the double strands or two single strands of a targeted gene or a targeted nucleic acid, and the damaged double strands or two single strands of the targeted gene or the damaged targeted nucleic acid can generate insertions / deletions through NHEJ, thereby inducing specific knockout or knockdown of the targeted gene or nucleic acid.

[0059] For example, the artificially modified intravascular transplantation cell may be an artificially modified intravascular transplantation cell in which the F3 gene is knocked down.

[0060] In one embodiment of the present invention, the F3 gene of the artificially modified intravascular transplantation cell may contain one or more insertions / deletions in the nucleic acid sequence.

[0061] In one embodiment of the present invention, the artificially modified intravascular transplantation cells may not express F3 mRNA.

[0062] In one embodiment of the present invention, the expression level of mRNA transcribed from the artificially modified F3 gene of the artificially modified intravascular transplantation cell may be lower than the mRNA level transcribed from the F3 gene of the wild-type cell.

[0063] In one embodiment of the present invention, the F3 mRNA sequence of the artificially modified intravascular transplantation cells may be different from that of wild-type cells.

[0064] In one embodiment of the present invention, the expression or activity of CD142 on the surface of the artificially modified intravascular transplantation cells can be reduced compared to wild-type cells. The artificially modified intravascular transplantation cells of the present invention can reduce or lose the function of CD142 by this.

[0065] That is, in the above-mentioned artificially modified cells for intravascular transplantation, the expression or activity of CD142 can be reduced by about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 55% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 100% as compared with the expression or activity of wild-type cells.

[0066] The present invention provides a method for preparing artificially modified cells for intravascular transplantation with blood compatibility, comprising the step of reducing the expression or activity of CD142 of mammalian cells.

[0067] The increase or decrease in the expression or activity of CD142 can be achieved by artificially modifying the F3 gene, and as an example, gene editing technology can be used.

[0068] As an example, the above-mentioned gene editing technology can utilize a transcription activator-like effector (TALE) domain fused to a cleavage domain, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease, or a CRISPR enzyme system derived from regularly interspaced short palindromic repeats (CRISPR) as a microbial immune system, but is not limited thereto.

[0069] In relation to the above-mentioned TALEN, the entire contents of International Patent Publication No. WO2012 / 093833 or US Patent Publication No. 2013-0217131 are included in the present specification as references. In connection with the above-mentioned zinc finger nuclease (ZFN), Beerli et al. (2002) Nature Biotechnol. 20: 135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70: 313-340; Isalan et al, (2001) Nature Biotechnol. 19: 656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12: 632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10: 411-416, U.S. Patent Grant Nos. 7,888,121, 8,409,861, 6,479,626, 6,903,185, 7,153,949 are included in the present specification as references.

[0070] The above-mentioned "CRISPR enzyme system" is composed of guide nucleic acids and / or editing proteins.

[0071] "Guide nucleic acid" refers to a nucleic acid that recognizes a target nucleic acid, a target gene, or a target chromosome and can interact with an editing protein. In this case, the guide nucleic acid can form a complementary bond with a portion of the nucleotides of the target nucleic acid, the target gene, or the target chromosome.

[0072] The guide nucleic acid may be a DNA-targeting specific guide RNA, a DNA encoding the guide RNA, or a DNA / RNA hybrid.

[0073] The guide nucleic acid may be a guide RNA. As an example, the guide RNA may be transcribed in vitro, in particular, may be transcribed from a double-stranded oligonucleotide or a plasmid template. As another example, the guide RNA may be encoded in the form of a vector, which may be delivered to cells in an ex vivo or in vivo environment and transcribed from the vector, but is not limited thereto.

[0074] The design and structure of the above-mentioned guide RNA are well known to those skilled in the art and are described in detail in Korean Patent Nos. 10-1656236, 10-1656237, 10-1706085, 10-2052286, and 10-2182847. The full texts of the above-mentioned patents are included in this specification as reference materials of the present invention.

[0075] The guide nucleic acid may include a backbone sequence portion and a guide sequence portion. The backbone sequence portion is a portion that interacts with the Cas protein, and a complex (ribonucleoprotein (RNP)) can be formed by binding the Cas protein to the guide nucleic acid. Typically, the backbone sequence portion includes a portion of the sequence of tracrRNA and crRNA, and the backbone sequence can be determined based on which Cas protein is used.

[0076] The guide sequence portion is a nucleotide sequence portion that can complementarily bind to a portion of the sequence of either of the double strands of the targeted gene or nucleic acid. The nucleotide sequence portion that can be artificially deformed depends on the desired targeted nucleotide sequence. In this case, the guide sequence can be a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% complementarity or complete complementarity with the guide nucleic acid binding sequence of the targeted gene or nucleic acid. The guide sequence can be a sequence contained in the guide domain of the guide nucleic acid.

[0077] The guide sequence portion may be included in crRNA. As an example, the guide nucleic acid may be a dual RNA comprising two RNAs, that is, crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) as structural elements.

[0078] As another example, the guide nucleic acid may be a sgRNA (single-chain guide RNA) in which the main part of crRNA and tracrRNA are linked.

[0079] The target sequence is a nucleotide sequence of a specified length present in a targeted gene or a targeted nucleic acid, specifically, a portion of a nucleotide sequence in a targeted region that is distinguished as a regulatory region, a coding region (coding region or CDS, coding sequence) or a non-coding region (non-coding region or UTR, untranslated region) of a targeted gene, or a portion of a nucleotide sequence selected from one or more of the combinations of the above-mentioned targeted regions. The target sequence can become a target of a guide nucleic acid-editing protein complex (RNP).

[0080] In one example of the present invention, the target sequence may be a sequence contained in the first region, the second region, the third region, the fourth region or the sixth region of the exon of the wild-type F3 gene.

[0081] In one example of the present invention, the target sequence may be the sequence of sequence number 1.

[0082] The target sequence is a nucleotide sequence surrounding a protospacer-adjacent motif (PAM) sequence recognized by the editing protein, and may include all or part of the PAM, but is not limited thereto.

[0083] The target sequence can be used as a term that represents two nucleotide sequence information at the same time. For example, in the case of a targeted gene, the target sequence can represent the sequence information of the transcribed strand of the targeted gene DNA, or can represent the nucleotide sequence information of the non-transcribed strand.

[0084] The target sequence includes a guide nucleic acid binding sequence or a guide nucleic acid non-binding sequence. The guide nucleic acid binding sequence is a nucleotide sequence that has partial or complete complementarity with the guide sequence contained in the guide domain of the guide nucleic acid, and 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 can be different nucleotide sequences depending on the targeted gene or nucleic acid, that is, depending on the object of genetic manipulation or editing, and the guide nucleic acid can be designed in a variety of ways according to the targeted gene or targeting nucleic acid.

[0085] The guide nucleic acid non-binding sequence is a nucleotide sequence having partial or complete homology with the guide sequence contained in the guide domain of the guide nucleic acid, and can be complementary to the guide sequence contained in the guide domain of the guide nucleic acid. In addition, the guide nucleic acid non-binding sequence, as a nucleotide sequence complementary to the guide nucleic acid binding sequence, can be complementary to the guide nucleic acid binding sequence. The guide nucleic acid binding sequence, as a part of the nucleotide sequence in the target sequence, can be a nucleotide sequence having two different sequence orders in the target sequence, that is, a nucleotide sequence of the two nucleotide sequences that can be complementary. In this case, the guide nucleic acid non-binding sequence can be the remaining nucleotide sequence in the target sequence except the guide nucleic acid binding sequence.

[0086] The guide nucleic acid binding sequence may be a target sequence, that is, it may be a nucleotide sequence selected from the group consisting of the nucleotide sequence identical to the transcribed strand and the nucleotide sequence identical to the non-transcribed strand. In this case, the guide nucleic acid non-binding sequence may be a nucleotide sequence other than the guide nucleic acid binding sequence in the target sequence, that is, it may be the remaining nucleotide sequence other than the group consisting of the nucleotide sequence identical to the transcribed strand and the nucleotide sequence identical to the non-transcribed strand.

[0087] The length of the guide nucleic acid binding sequence can be the same as the length of the target sequence. The length of the guide nucleic acid non-binding sequence can be the same as the length of the target sequence or the guide nucleic acid binding sequence. The guide nucleic acid binding sequence can be a 5 to 50 nucleotide sequence.

[0088] As a specific example, the guide nucleic acid binding 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. The guide nucleic acid non-binding sequence can be a 5 to 50-nucleotide sequence.

[0089] As a specific example, the non-binding sequence of the guide nucleic acid can be a 16-nucleotide sequence, a 17-nucleotide sequence, an 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.

[0090] The guide nucleic acid binding sequence can partially or completely complementarily bind to the guide sequence included in the guide domain of the guide nucleic acid, and the length of the guide nucleic acid binding sequence can be the same as the length of the guide sequence.

[0091] The guide nucleic acid binding sequence may be a nucleotide sequence complementary 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% complementarity or complete complementarity.

[0092] As an example, the guide nucleic acid binding sequence may have or include a 1 to 8 nucleotide sequence that is non-complementary to the guide sequence included in the guide domain of the guide nucleic acid.

[0093] The guide nucleic acid non-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 guide nucleic acid non-binding sequence may be the same as the length of the guide sequence. As an example, the guide sequence can be designed based on a sequence having homology with the guide nucleic acid non-binding sequence.

[0094] The non-binding sequence of the guide nucleic acid may be a nucleotide sequence having homology with 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% homology or having complete homology.

[0095] As an example, the guide nucleic acid non-binding sequence may have or include 1 to 8 nucleotide sequences that have no homology with the guide sequence included in the guide domain of the guide nucleic acid. The guide nucleic acid non-binding sequence may complementarily bind to the guide nucleic acid binding sequence, and the length of the guide nucleic acid non-binding sequence may be the same as the length of the guide nucleic acid binding sequence.

[0096] The guide nucleic acid non-binding sequence may be a nucleotide sequence complementary to the guide nucleic acid binding sequence, for example, a nucleotide sequence having at least 90% or 95% complementarity or complete complementarity.

[0097] As an example, the guide nucleic acid non-binding sequence may have or include 1 to 2 nucleotide sequences that are non-complementary to the guide nucleic acid binding sequence. In addition, the guide nucleic acid binding sequence may be a nucleotide sequence adjacent to a sequence complementary to a nucleotide sequence (PAM sequence) that can be recognized by the editing protein.

[0098] As an example, the guide nucleic acid binding sequence may be a continuous sequence of 5 to 50 nucleotides located adjacent to the 5' end and / or 3' end of a sequence complementary to a nucleotide sequence (PAM sequence) that can be recognized by an editing protein.

[0099] Furthermore, the guide nucleic acid non-binding sequence may be a nucleotide sequence located adjacent to a nucleotide sequence (PAM sequence) that can be recognized by the editing protein.

[0100] As an example, the guide nucleic acid non-binding sequence may be a continuous sequence of 5 to 50 nucleotides located adjacent to the 5' end and / or 3' end of a sequence complementary to a nucleotide sequence (PAM sequence) recognizable by an editing protein.

[0101] "Editing protein" refers to a peptide, polypeptide or protein that directly binds to nucleic acid or that does not directly bind to but can interact with nucleic acid. The above-mentioned editing protein is also conceptually called "artificially modified nuclease" or RGEN (RNA-Guided Endonuclease).

[0102] In a specific example, the editing protein can be a CRISPR (Clustered regularly interspaced short palindromic repeats) enzyme. "CRISPR enzyme" is the main structural element in the CRISPR enzyme (CRISPR-enzyme) system, also known as "Cas protein (CRISPR associated protein)", which refers to a nuclease that can recognize the target sequence and cut DNA by forming a mixture or complex with the guide RNA.

[0103] CRISPR enzymes are well known in the art, with reference to Korean patents No. 10-1656236, No. 10-1656237, No. 10-1706085, No. 10-2052286, and No. 10-2182847. In this specification, in addition to native proteins, the above-mentioned CRISPR enzymes are also used with the concept of all variants that can be activated by endonucleases or nickases that work in coordination with guide RNA. In the case of activated endonucleases or nickases, targeted DNA cutting can be brought about, and genome editing can be brought about by it. And, in the case of non-activated variants, it can be used to bring about transcriptional regulation or separation of targeted DNA.

[0104] The above-mentioned CRISPR enzyme is a nucleic acid or polypeptide (or protein) having a sequence encoding the CRISPR enzyme, and type II (Type II) CRISPR enzyme or type V (Type V) CRISPR enzyme is representatively used. The above-mentioned type II CRISPR enzyme includes Cas9 (CRISPR associated protein 9) protein.

[0105] The above-mentioned Cas9 protein can be derived from various microorganisms such as Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Campylobacter jejuni, Staphylococcus aureus, Staphylococcus Auricularis, and Neisseria meningitidis.

[0106] In order to induce double-stranded DNA break, the Cas9 protein should recognize the PAM sequence, which is a nucleotide sequence of a specified length, and a portion of the guide RNA (the guide sequence portion) should complementarily bind to the complementary strand of the single-stranded DNA where the target sequence is located (the guide nucleic acid non-binding sequence).

[0107] The PAM sequence is a sequence determined by the type or origin of the Cas9 protein. For example, the Cas9 protein (SpCas9) derived from Streptococcus pyogenes can recognize the 5'-NGG-3' sequence (complementary sequence: 5'-CCN-3') in the target nucleic acid. In this case, the above-mentioned N is one of adenosine (A), thymidine (T), cytidine (C), and guanosine (G). In addition, the above-mentioned SpCas9 can recognize the 5'-NAG-3' sequence (complementary sequence: 5'-CTN-3') in the target nucleic acid with low activity.

[0108] Furthermore, the type V CRISPR enzyme is Cpf1, and the Cpf1 may be derived from Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae ), Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium, or Acidaminococcus.

[0109] CRISPR enzymes such as the above-mentioned Cas9 or Cpf1 proteins can be isolated from microorganisms existing in a natural state or produced non-naturally by a recombinant method or a synthetic method. In addition, the above-mentioned Cas protein can be a form that is easy to introduce into the cell. For example, the Cas protein can be connected to a cell-penetrating peptide or a protein transduction domain. The above-mentioned protein transduction domain can be a TAT protein derived from polyarginine or acquired immunodeficiency virus (HIV), but is not limited to this. In addition to the examples described above, multiple types of cell-penetrating peptides or protein transduction domains are well known in the art, and those skilled in the art can adopt multiple examples not limited to the above examples in this specification. In addition, the above-mentioned Cas protein can be fused with functional domains such as nuclear localization sequences or signals (NLS, nuclear localization sequence or signal). In addition, the above-mentioned Cas9 protein can be encoded in the form of a vector to be expressed in the cell.

[0110] The present invention provides a composition for preparing blood-compatible cells for intravascular transplantation, comprising: a guide nucleic acid or a nucleic acid encoding the guide sequence comprising a target sequence of an F3 gene that can target mammalian cells or stem cells; and an editing protein or a nucleic acid encoding the editing protein.

[0111] Furthermore, the composition may also selectively contain a donor or a nucleic acid encoding the donor, wherein the donor contains a specific nucleotide sequence that is desired to be inserted.

[0112] The donor refers to an exogenous nucleotide sequence that can express a specific peptide or protein and can be inserted into genomic DNA through homology directed repair (HDR).

[0113] The donor may be a double-stranded nucleic acid or a single-stranded nucleic acid. The donor may be linear or circular.

[0114] The donor may be in the form of a viral vector or a non-viral vector (eg, a plasmid).

[0115] The virus may be a DNA virus or an RNA virus. In this case, the DNA virus may be a double-stranded DNA (dsDNA) virus or a single-stranded DNA (ssDNA) virus. In this case, the RNA virus may be a single-stranded RNA (ssRNA) virus.

[0116] The above-mentioned viral vector can be one or more viral vectors selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus and herpes simplex virus (HSV).

[0117] The target sequence may be a target of the guide nucleic acid-editing protein complex, and the target sequence may include a PAM sequence recognized by the editing protein, but is not limited thereto.

[0118] The guide nucleic acid may comprise a guide domain capable of targeting a target sequence of the F3 gene.

[0119] In the present specification, the guide nucleic acid, editing protein or guide nucleic acid-editing protein complex (ribonucleoprotein, RNP) and / or donor can be delivered or introduced into a subject in various forms.

[0120] In this case, the "subject" refers to: an organism into which a guide nucleic acid, an editing protein, or a guide nucleic acid-editing protein complex is introduced; an organism in which a guide nucleic acid, an editing protein, or a guide nucleic acid-editing protein complex works; or a subject or sample obtained from an organism.

[0121] The subject can be an organism that includes a targeted gene, a targeted nucleic acid, or a targeted chromosome comprising a guide nucleic acid-editing protein complex.

[0122] The organism may be an animal, an animal tissue or an animal cell. In this case, the tissue may be an eyeball, skin, liver, kidney, heart, lung, brain, muscle or blood.

[0123] The above cells may be mammalian cells.

[0124] Mammals of the present invention may be, for example, primates such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees, etc.; rodents such as mice, rats, hamsters, guinea pigs, etc.; Lagomorpha such as rabbits; Ungulates such as pigs, cattle, goats, horses, sheep, etc.; Feliformes such as dogs and cats, etc. Among them, mice, pigs, and humans can be appropriately exemplified.

[0125] According to one embodiment of the present invention, the cells for transplantation in the artificially modified blood vessels of the present invention are homologous or autologous cells, and more specifically, can be homologous cells for transplantation.

[0126] According to one example of the present invention, the mammalian cells of the present invention can be primary hepatocytes, primary pancreatic β cells, primary islet cells, pancreatic progenitor cells, pluripotent stem cell-derived hepatocytes, pluripotent stem cell-derived pancreatic β cells, pluripotent stem cell-derived islet cells, pluripotent stem cell-derived pancreatic lineage cells, pluripotent stem cell-derived pancreatic progenitor cells, pluripotent stem cell-derived pancreatic organoids, pluripotent stem cell-derived pancreatic liver organoids, induced pluripotent stem cell-derived hepatocytes, induced pluripotent stem cell-derived pancreatic β cells, induced pluripotent stem cell-derived islet cells, induced pluripotent stem cell-derived pancreatic lineage cells, induced pluripotent stem cell-derived pancreatic progenitor cells, induced pluripotent stem cell-derived pancreatic organoids, induced pluripotent stem cell-derived pancreatic liver organoids, hepatocytes induced from somatic cells by compounds or genetic modification, islet cells induced from somatic cells by compounds or genetic modification, or pancreatic β cells induced from somatic cells by compounds or genetic modification.

[0127] The above-mentioned subject or sample can be saliva, blood, liver tissue, brain tissue, hepatocytes, nerve cells, phages, macrophages, T cells, B cells, astrocytes, cancer cells or stem cells obtained from an organism containing a targeted gene, targeted nucleic acid or targeted chromosome.

[0128] The guide nucleic acid, editing protein, or guide nucleic acid-editing protein complex can be delivered or introduced into a subject in the form of DNA, RNA, or a mixture thereof.

[0129] In this case, DNA, RNA, or a mixture thereof encoding the guide nucleic acid and / or editing protein can be delivered or introduced into the subject by methods known in the art.

[0130] Alternatively, DNA, RNA, or a mixture thereof encoding the guide nucleic acid and / or editing protein can be delivered or introduced into a subject via a vector, a non-vector, or a combination thereof.

[0131] The above-mentioned vector may be a viral vector or a non-viral vector (eg, a plasmid).

[0132] The virus may be a DNA virus or an RNA virus. In this case, the DNA virus may be a double-stranded DNA virus or a single-stranded DNA virus. In this case, the RNA virus may be a single-stranded RNA virus.

[0133] The viral vector may be one or more selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus and herpes simplex virus.

[0134] The non-vector may be a guide DNA, a DNA complex or mRNA.

[0135] As an example of the present invention, the nucleic acid encoding the guide nucleic acid and / or editing protein can be delivered or introduced into a subject in the form of one or more vectors.

[0136] The above-mentioned vector may contain nucleic acids encoding guide nucleic acids and / or editing proteins. As one example, the above-mentioned vector may contain nucleic acids encoding guide nucleic acids and editing proteins at the same time. As another example, the above-mentioned vector may contain nucleic acids encoding guide nucleic acids. For example, the nucleic acids encoding the above-mentioned guide nucleic acids may all be contained in one vector, or the nucleic acids encoding the guide nucleic acids may be divided and contained in multiple vectors. As another example, the above-mentioned vector may contain nucleic acids encoding editing proteins. For example, in the case of the above-mentioned editing protein, the nucleic acids encoding the editing proteins may be contained in one vector, or the nucleic acids encoding the editing proteins may be divided and contained in multiple vectors.

[0137] The editing protein can be delivered or introduced into a subject in the form of a peptide, polypeptide or protein.

[0138] The editing protein can be delivered or introduced into a subject in the form of a peptide, polypeptide or protein by methods known in the art.

[0139] The guide nucleic acid and editing protein can be delivered or introduced into a subject in the form of a nucleic acid-protein mixture.

[0140] The guide nucleic acid and the editing protein can be delivered or introduced into the subject in the form of a guide nucleic acid-editing protein complex. For example, the guide nucleic acid can be DNA, RNA or a mixed form thereof. The editing protein can be in the form of a peptide, a polypeptide or a protein. As an example, the guide nucleic acid and the editing protein can be delivered or introduced into the subject in the form of a guide nucleic acid in the form of RNA and a guide nucleic acid-editing protein complex of an editing protein in the form of a protein, that is, a ribonucleoprotein (RNP).

[0141] Furthermore, the present invention provides a method for preparing blood-compatible cells for intravascular transplantation, which comprises: step (1), introducing the above-mentioned composition for preparing blood-compatible cells for intravascular transplantation into isolated mammalian cells or isolated stem cells; and step (2), editing the F3 gene by inserting / deleting the target sequence of the F3 gene located in the genome of the above-mentioned isolated mammalian cells or isolated stem cells to reduce or inhibit the expression or activity of CD142.

[0142] In this case, the above-mentioned "introduction" can be carried out by one or more methods selected from electroporation, lipofection, microinjection, gene gun, liposome, amphoteric liposome, plasmid, viral vector, nanoparticle, protein translocation domain (PTD) fusion protein method, immunoliposome, various ions or lipids, nucleic acid conjugates, naked DNA, artificial virus particles and DNA preparation enhanced absorption method, but is not limited to these.

[0143] As one example of the present invention, the blood-compatible cells for intravascular transplantation can be prepared by introducing the composition for preparing blood-compatible cells for intravascular transplantation into isolated mammalian cells or isolated stem cells by electroporation.

[0144] As an example of the present invention, insertion / deletion can be caused by contacting the F3 gene located in the genome of the above-mentioned mammalian cell with a CRISPR / Cas9 complex comprising a Cas9 protein derived from Streptococcus pyogenes and a guide RNA capable of targeting a target sequence of the F3 gene.

[0145] In one example of the present invention, the target sequence may be the sequence of sequence number 1.

[0146] The blood-compatible artificially modified intravascular transplantation cells of the present invention can be used as a cell therapy agent for vascular administration to treat a variety of diseases. For example, they can be used in heart disease, gastroduodenal disease, small intestine and large intestine disease, liver disease, bile duct disease, pancreatic disease, kidney disease, liver disease, lung disease, mediastinal disease, diaphragm disease, pleural disease, peritoneal disease, nervous system disease, central nervous system (CNS) disorder, peripheral arterial disease, peripheral venous disease. For example, specific diseases can be: autoimmune hepatitis, fulminant hepatitis, chronic hepatitis, viral hepatitis, alcoholic hepatitis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD), nonalcoholic fatty liver disease ... Liver diseases such as hepatitis (NAFL), liver fibrosis, liver cirrhosis, liver cancer, fatty liver, drug-induced allergic liver disease, hemochromatosis, hemochromatosis, Wilson's disease, primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), biliary atresia, liver abscess, chronic active hepatitis, chronic persistent hepatitis; myocardial infarction, heart failure, arrhythmia, palpitations, cardiomyopathy, ischemic cardiomyopathy, angina pectoris, congenital heart disease, heart valve Heart diseases such as heart disease, myocarditis, familial hypertrophic cardiomyopathy, dilated cardiomyopathy, acute coronary syndrome, arteriosclerosis, restenosis, etc.; gastroduodenal diseases such as acute gastritis, chronic gastritis, gastroduodenal ulcer, gastric cancer, duodenal cancer, etc.; small and large intestinal diseases such as ischemic enteritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, simple ulcer, intestinal Behcet's disease, small intestinal cancer, large intestinal cancer, etc.; bile duct diseases such as acute cholecystitis, acute cholangitis, chronic cholecystitis, bile duct cancer, gallbladder cancer, etc. Diseases; pancreatic diseases such as acute pancreatitis, chronic pancreatitis, and pancreatic cancer; kidney diseases such as acute nephritis, chronic nephritis, acute renal failure, and chronic renal failure; pneumonia, emphysema, pulmonary fibrosis, interstitial pneumonia, idiopathic interstitial pneumonia, desquamative interstitial pneumonia, acute interstitial pneumonia, nonspecific interstitial pneumonia, drug-induced lung disease, eosinophilic lung disease, pulmonary hypertension, tuberculosis, sequelae of tuberculosis, acute respiratory distress syndrome, cystic fibrosis, chronic closed Pulmonary diseases such as obstructive pulmonary disease, pulmonary embolism, pulmonary abscess, pneumoconiosis, aspiration pneumonia, pneumonia, pulmonary fibrosis, acute upper respiratory tract infection, chronic lower respiratory tract infection, pneumothorax, alveolar epithelial damage disease, lymphangioleiomyomas, lymphocytic interstitial pneumonia, and alveolar proteinosis; mediastinal diseases such as mediastinal tumors, mediastinal cystic diseases, and mediastinitis; diaphragmatic diseases such as diaphragmatic hernia; pleural diseases such as pleurisy, empyema, pleural tumors, carcinomatous pleurisy, and pleural mesothelioma; peritonitis, peritoneal tumors, and other peritoneal diseases;Neurological diseases including cerebral palsy syndrome including pediatric cerebral palsy, aseptic meningitis, Guillain-Barré syndrome, amyotrophic lateral sclerosis (ALS), myasthenia gravis, mononeuropathy, polyneuropathy, spinal muscular atrophy, spinal cord disorder, acute transverse myelitis, spinal cord infarction (ischemic bone marrow disease), intracranial tumors, spinal cord tumors; central nervous system disorders such as Alzheimer's disease, cognitive impairment, stroke, multiple sclerosis, Parkinson's disease, peripheral arterial diseases such as fibromuscular dysplasia, peripheral arterial disease (PAD), occlusive thromboangiitis vasculitis (Berger's disease), Kawasaki disease (KD); peripheral venous diseases such as deep vein thrombosis, chronic venous insufficiency, postphlebitic syndrome, superficial vein thrombosis; immunodeficiency diseases such as graft-versus-host disease (GVHD), secondary immunodeficiency, primary immunodeficiency diseases, B cell deficiency, T cell deficiency, combined deficiency of B and T cells, phage deficiency, complement deficiency, etc. ;

[0147] The term "vascular administration" refers to delivery within the patient's vascular system. As an example, it can be administered into a blood vessel that is considered a vein, and it can be administered into a blood vessel that is considered an artery. Veins include internal jugular veins, peripheral veins, coronary veins, hepatic veins, portal veins, great saphenous veins, pulmonary veins, superior vena cava, inferior vena cava, gastric veins, splenic veins, inferior mesenteric veins, superior mesenteric veins, head veins and / or femoral veins, but are not limited thereto. Arteries include coronary arteries, pulmonary arteries, splenic arteries, brachial arteries, internal carotid arteries, aortic arches, femoral arteries, peripheral arteries and / or ciliary arteries, but are not limited thereto. It can be delivered through the portal vein, umbilical vein, arterioles or capillaries, or delivered to arterioles or capillaries.

[0148] In one embodiment of the present invention, the artificially modified intravascular transplantable cells of the present invention can be used as a composition for cell transplantation or biological tissue regeneration to restore damaged cells or tissues.

[0149] In one example of the present invention, the biological tissue may be a damaged tissue selected from tissues forming ulcers or bedsores, brain tissue damaged by cell degeneration, brain tissue damaged by surgical operation, brain tissue damaged by traumatic brain disease, brain tissue damaged by inflammatory brain disease, damaged bone tissue, damaged periodontal tissue, tissue damaged by central nervous system myopathy, and tissue damaged by refractory dermatitis. The biological tissue regeneration may be the restoration of the damaged tissues, epidermal regeneration, reproduction of secretory glands or hair follicles, capillary formation of dermal tissue, wound healing, compensation for soft tissue defects, bone healing or bone regeneration, cartilage regeneration, etc., but is not limited thereto.

[0150] In one example of the present invention, the composition for cell transplantation or biological tissue regeneration can be a cell therapy composition, a gene therapy composition, a tissue engineering therapy composition, an immunotherapy composition, or a cancer prevention or treatment composition.

[0151] In this specification, "cell therapy agent" refers to a drug used for treatment, diagnosis and prevention using cells and tissues isolated, cultured and prepared by special operations from an individual (as stipulated by the U.S. Food and Drug Administration (FDA)). It refers to a drug used for the purpose of treatment, diagnosis and prevention through a series of actions such as in vitro proliferation and screening of autologous, allogeneic or xenogeneic cells or using other methods to change the biological characteristics of cells in order to restore the function of cells or tissues.

[0152] In the present specification, a "gene therapy agent" is administered to affect the expression of genetic material and refers to a drug containing cells into which genetic material has been transformed or introduced.

[0153] The composition for cell transplantation or biological tissue regeneration of the present invention can be administered by any common route that can reach the target tissue, and can be administered non-orally, for example, intraperitoneally, intravenously, intramuscularly, subcutaneously, or intradermally, but is not limited thereto.

[0154] The above-mentioned composition for cell transplantation or biological tissue regeneration can be formulated into an appropriate form together with a pharmaceutically acceptable carrier commonly used in cell therapy. "Pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not usually cause gastrointestinal disorders, dizziness, and other allergic reactions or similar reactions when applied to humans. As a pharmaceutically acceptable carrier, there are non-oral administration carriers such as water, appropriate oils, physiological saline, aqueous glucose, and ethylene glycol, and stabilizers and preservatives may also be included. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl parahydroxybenzoate or propyl parahydroxybenzoate, and trichlorobutanol. Other pharmaceutically acceptable carriers can refer to the contents recorded in the following documents: Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995. In addition, the above-mentioned composition can also be administered by any device that the cell therapy agent can move to the target cell.

[0155] For the treatment of diseases, the composition for cell transplantation or biological tissue regeneration of the present invention may include a therapeutically effective amount of cells. "Therapeutically effective amount" refers to the amount of active ingredients or pharmaceutical compositions that researchers, veterinarians, doctors or other clinical researchers believe induce biological or medical responses in tissue systems, animals or humans, including amounts that induce relief of symptoms of the disease or disorder being treated.

[0156] It should be understood by those skilled in the art that the cells included in the composition of the present invention vary depending on the desired effect. Therefore, the optimal amount of cell therapy can be easily determined by those skilled in the art, and can be adjusted according to a variety of factors including the type of disease, the severity of the disease, the content of other ingredients contained in the composition, the type of dosage form, and the patient's age, weight, general health status, gender and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrent medications. After considering all of the above factors, it is important to include the following amount, that is, the amount that achieves the maximum effect with the minimum amount without causing side effects. For example, the daily administration amount of the stem cells of the present invention is 1.0×10 5 Cells / kg body weight up to 1.0×10 30 cells / kg body weight, preferably, 1.0×10 10 Cells / kg body weight up to 1.0×10 20Cells / kg body weight once or divided into several times. However, it should be understood that the actual dosage of the active ingredient is determined by referring to the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age and gender and other related factors. Therefore, no matter from which level, the above dosage does not limit the scope of the present invention.

[0157] The present invention provides a method of treating a mammal, comprising administering a therapeutically effective amount of the artificially modified cells for intravascular transplantation. The term "mammal" used herein refers to a mammal as a subject of treatment, observation or experiment, preferably a human.

[0158] Furthermore, the present invention includes a pharmaceutical composition for preventing or treating liver diseases, comprising a therapeutically effective amount of the artificially modified intravascular transplantation cells as an active ingredient.

[0159] The composition of the present invention can be administered orally, or by non-oral routes including inhalation, intravascular, intraperitoneal, subcutaneous, rectal and topical administration. In one embodiment of the present invention, the composition can be administered via the portal vein, umbilical vein or splenic artery.

[0160] Furthermore, the present invention provides a method for preventing or treating liver diseases, comprising administering a therapeutically effective amount of the artificially modified intravascular transplantation cells to a mammal. The term "mammal" used herein refers to a mammal as a subject of treatment, observation or experiment, preferably a human.

[0161] The present invention provides the use of the artificially modified intravascular transplantation cells for preparing a medicament for preventing or treating liver diseases in mammals.

[0162] In one embodiment of the present invention, the above-mentioned liver diseases include cirrhosis, acute-on-chronic liver failure (ACLF), liver failure caused by drugs or poisoning, innate metabolic liver disease, Crigler-Najjar syndrome type I, familial hypercholesterolemia, factor VII deficiency, factor VIII deficiency (hemophilia A), phenylketonuria (PKU), glycogen storage disease type I, infantile Refsum's disease, progressive familial intrahepatic cholestasis type 2, hereditary tyrosinemia type 1, urea cycle disorder, acute liver failure, acute drug-induced liver failure, acute liver failure caused by viruses, idiopathic acute liver failure, acute liver failure caused by mushroom poisoning, acute liver failure after surgery, acute liver failure caused by acute fatty liver of pregnancy, alcoholic hepatitis or hepatic encephalopathy.

[0163] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising a pharmaceutically effective amount of the artificially modified intravascular transplantation cells as an active ingredient.

[0164] The composition of the present invention can be administered orally, or by non-oral routes including inhalation, intravascular, intraperitoneal, subcutaneous, rectal and topical administration. In one embodiment of the present invention, the composition can be administered via the hepatic portal vein.

[0165] Furthermore, the present invention provides a method for preventing or treating diabetes, comprising the step of administering a therapeutically effective amount of the artificially modified cells for intravascular transplantation to a mammal.

[0166] Furthermore, the present invention provides use of the artificially modified intravascular transplantation cells for preparing a medicament for preventing or treating diabetes in mammals.

[0167] In one embodiment of the present invention, the diabetes mellitus includes type 1 diabetes mellitus.

[0168] The present invention is described in more detail below by way of examples. It should be understood by those skilled in the art that these examples are only used to more specifically describe the present invention, and the scope of the present invention is not limited by these examples.

[0169] Example 1: Preparation of F3 knockout cells

[0170] Cell culture

[0171] iPSCs were cultured according to the manufacturer's instructions. TM (STEMCELL Technology) TeSR was used in culture dishes coated for 1 hour at room temperature (RT). TM -E8 TM (STEMCELL Technology) medium.

[0172] Preparation of CD142 gene-edited cells

[0173] The cultured cells were harvested using 4D-Nucleofector (Lonza) and the RNP complex was introduced by electroporation. Specifically, the RNP complex was synthesized by mixing 4 μg of Cas9 protein with 4 μg of in vitro transcribed sgRNA (prepared using T7 polymerase (New England BioLabs) according to the manufacturer's instructions) and the mixture was incubated at room temperature for 10 minutes. The above RNP complex was mixed with 1×10 treated with 20 ul of Primary P3 buffer.6 iPSCs were electroporated using the nucleofector program CA-137. As a result, iPSCs with CD142 gene knockout (CD142#33KO iPSCs) were obtained. In this case, a target sequence 5'-TCTGGGGAGTTCTCATACAG that can target CD142#33 was synthesized. AGG -3' (underline: PAM sequence) (SEQ ID NO: 1) was introduced into iPSCs and the insertion / deletion efficiency of the target sequence was confirmed by targeted deep sequencing.

[0174] Targeted deep sequencing

[0175] Blood Genomic DNA Extraction Kit (Favorgen) was used to extract genomic DNA (gDNA) according to the manufacturer's instructions in the obtained CD142#33KO iPSC. In order to amplify the target site, Phusion high-fidelity DNA polymerase PCR polymerase (NEB) was used to amplify 100ng of genomic DNA (gDNA). In order to generate a deep sequencing library, TruSeq HT dual-label primers (Dual Index Primers) (Illumina, San Diego, California (CA), the United States (USA)) were used to amplify the amplicon once more. Paired-end sequencing was performed using the Illumina Miniseq system, and the insertion / deletion frequency was calculated in "http: / / www.rgenome.net / ". The primer sequences of the target sequences used in the targeted deep sequencing are shown in Table 1.

[0176] Table 1

[0177]

[0178] Figure 1 Figure 2 is a chart showing the results of targeted deep sequencing of normal iPSCs and iPSCs with the target sequence (SEQ ID NO. 1) of gene-edited CD142#33. Figure 1 As shown in the results of targeted deep sequencing, 98% insertion / deletion was confirmed in iPSCs of the gene-edited target sequence CD142#33 (sequence number 1).

[0179] Pancreatic lineage cells differentiation

[0180] The CD142 gene knockout iPSCs (CD142#33KO iPSCs) obtained above were differentiated into pancreatic lineage cells. In this case, pancreatic lineage cells were differentiated into pancreatic lineage cells according to STEMCELL Technology TM The pancreatic progenitor kit (Catalog #05120) was used to differentiate the cells into four stages: 1) terminal endoderm; 2) primitive intestine; 3) posterior full-length endoderm; and 4) pancreatic lineage cells. Differentiated cells were confirmed by the expression of major markers of pancreatic lineage cells.

[0181] Hepatocyte differentiation

[0182] The iPSC (CD142#33KO iPSC) obtained above with the CD142 gene knocked out was differentiated into hepatocytes. In this case, hepatocyte differentiation was performed according to the Takara Bio protocol (Catalog Y30050) (https: / / www.takarabio.com / products / stem-cell-research / media-and-supplements / stem-cell-differentiation-media-and-kits / hepatocyte-differentiation-kit). Differentiated cells were confirmed by the expression of major markers of hepatocytes.

[0183] Experimental Example 1: Confirmation of pancreatic origin of iPSC (induced pluripotent stem cell) Differentiation markers for glandular lineage cells

[0184] In order to confirm whether iPSC cells differentiate well into pancreatic lineage cells, qRT-PCR was used to confirm markers that are particularly expressed in pancreatic lineage cells during differentiation. The markers used in the experiment were PDX-1 (pancreatic and duodenal homeobox 1), NKX6.1 (NK6 homeobox 1), and SOX9 (SRY-Box transcription factor 9).

[0185] mRNA was extracted from iPSCs using the RNeasy mini kit (Qiagen) according to the manufacturer's instructions. Then, 1 μg of mRNA was reverse transcribed using a cDNA reverse transcription kit (ThermoFisher Scientific). qRT-PCR was performed using QuantStudio 3 (Thermo Fisher Scientific) according to the manufacturer's instructions using PowerUp TM SYBR TM Green Master Mix was used. The gene expression level was calculated using the CT value, and GAPDH was used as the endogenous control group. Figure 2 The marker expressions of the control group (normal iPSC) of normal iPSC-derived pancreatic lineage cells and the experimental group (iPSC-CD142#33) of CD142-knockout iPSC-derived pancreatic lineage cells increased on day 14 of differentiation (D14), confirming that the cells were well differentiated into pancreatic lineage cells (N=4).

[0186] Experimental Example 2: Confirmation of differentiation markers of iPSC-derived hepatocytes

[0187] In order to confirm whether iPSC cells differentiate well into hepatocytes, markers that are particularly expressed in hepatocytes during differentiation were analyzed by qRT-PCR in the same manner as in Example 1. The markers used in the experiment were HNF4α (hepatocyte nuclear factor 4α), ALB (albumin), and AFP (alpha-fetoprotein). The results are as follows Figure 3 As shown. Figure 3 As shown, the expression of markers in the control group (normal iPSC) of normal iPSC-derived hepatocytes and the experimental group (iPSC-CD142#33) of CD142-knocked-out iPSC-derived hepatocytes increased on day 40 of differentiation (D40), confirming that they were well differentiated into hepatocytes.

[0188] Enzyme-linked immunosorbent assay (ELSIA)

[0189] In order to confirm whether the iPSC cells differentiated well into hepatocytes, ALB, a marker that is particularly highly expressed in hepatocytes during differentiation, was confirmed by ELISA.

[0190] Albumin was measured using the human albumin ELISA Kit (TaKaRa (MK132)) using cell lysate and cell culture supernatant of iPSC-derived hepatocytes. The experiment was performed according to the instructions provided by the manufacturer of the kit. The Multiskan TM FC Microplate Photometer (Thermo Scientific TM (51119000)) measured the absorbance at 405nm wavelength. The results are as follows Figure 4 shown.

[0191] like Figure 4 As shown, the expression of markers in the control group (normal iPSC) of normal iPSC-derived hepatocytes and the experimental group (iPSC-CD142#33) of CD142-knocked-out iPSC-derived hepatocytes increased on day 40 of differentiation (D40), confirming that they were well differentiated into hepatocytes.

[0192] Experimental Example 3: Confirmation of CD142 gene knockout in iPSC-derived pancreatic lineage cells

[0193] qRT-PCR

[0194] The mRNA level of CD142 gene was analyzed by qRT-PCR to confirm that CD142 was knocked out (KO) in iPSC cell-derived pancreatic lineage cells.

[0195] mRNA was extracted from iPSC-derived pancreatic lineage cells using the RNeasy micro kit (Qiagen) according to the manufacturer's instructions. Then, 1 μg of mRNA was reverse transcribed using a cDNA reverse transcription kit (Thermo Fisher Scientific). qRT-PCR was performed using QuantStudio 3 (Thermo Fisher Scientific) according to the manufacturer's instructions using PowerUp TM SYBR TM Green Master Mix was used. The gene expression level was calculated using the CT value, and GAPDH was used as the endogenous control group. Figure 5 As shown. Figure 5As shown, on day 14 of differentiation (D14), the CD142 mRNA level in the experimental group (iPSC-CD142#33) of iPSC-derived pancreatic lineage cells in which the CD142 gene was knocked out was reduced compared with the control group (normal iPSC) of normal iPSC-derived pancreatic lineage cells (N=4).

[0196] Flow cytometry analysis (FACS)

[0197] FACS analysis of CD142 expression at the cell level confirmed that CD142 was knocked out in iPSC-derived pancreatic lineage cells. After washing the cultured cells twice with phosphate buffer saline (PBS), the cells were removed with 0.05% trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA) and centrifuged at 1000 rpm for 5 minutes. The cells were suspended with 100 ul of FACS staining buffer and mixed with antibodies. After reacting at 4 degrees Celsius for 1 hour, the cells were washed twice with PBS, resuspended with 500 ul of PBS, and analyzed by flow cytometry. The results are as follows. Figure 6 shown.

[0198] Reference Figure 6 On day 40 of differentiation (D40), the CD142 positive cell population (%) was reduced in the experimental group (iPSC-CD142#33) of iPSC-derived pancreatic lineage cells in which the CD142 gene was knocked out, compared with the control group (normal iPSC) of normal iPSC-derived pancreatic lineage cells.

[0199] Experimental Example 4: Confirmation of CD142 gene knockout in iPSC-derived hepatocytes

[0200] qRT-PCR

[0201] The mRNA level of the CD142 gene was analyzed by qRT-PCR as described above to confirm that CD142 was knocked out in iPSC-derived hepatocytes. Figure 7 As shown. Figure 7 As shown, on day 40 of differentiation (D40), the CD142 mRNA level in the experimental group (iPSC-CD142#33) of iPSC-derived hepatocytes in which the CD142 gene was knocked out was reduced compared to that in the control group (normal iPSC) of normal iPSC-derived hepatocytes.

[0202] Flow cytometric analysis

[0203] The expression level of CD142 in cells was analyzed by FACS in the same manner as described above to confirm that CD142 was knocked out in iPSC-derived hepatocytes. Figure 8 shown.

[0204] Reference Figure 8 On day 40 of differentiation (D40), the experimental group of iPSC-CD142#33, iPSC-derived hepatocytes in which the CD142 gene was knocked out, showed a decreased proportion of cells expressing CD142 compared with the control group (normaliPSC), which was normal iPSC-derived hepatocytes.

[0205] Example 5: Confirmation of CD142 protein levels in iPSC-derived pancreatic lineage cells and hepatocytes

[0206] ELISA

[0207] The amount of CD142 protein was analyzed by ELISA using cell lysates to confirm the reduction in the amount of protein produced by CD142 knockout in iPSC-derived pancreatic lineage cells and iPSC-derived hepatocytes.

[0208] CD142 was measured using a tissue factor activity assay kit (Human, Colorimetric, abcam (ab108906)) using cell lysates of iPSC-derived pancreatic lineage cells and hepatocytes and cell culture supernatants. The experiment was performed according to the instructions provided by the kit manufacturer. TM FC microplate reader (Thermo Scientific TM (51119000)) measured the absorbance at 405nm wavelength. The results are as follows Fig. 9 and Fig.10 shown.

[0209] like Fig. 9 As shown, on differentiation day 14 (D14), it was confirmed that the amount of CD142 protein in the experimental group (iPSC-CD142#33) of iPSC-derived pancreatic lineage cells in which the CD142 gene was knocked out was reduced in the cell lysate compared with the control group (normal iPSC) of normal iPSC-derived pancreatic lineage cells (N=4).

[0210] And, refer to Fig.10On day 40 of differentiation (D40), it was confirmed that the amount of CD142 protein in the experimental group (iPSC-CD142#33) of iPSC-derived hepatocytes in which the CD142 gene was knocked out was reduced in the cell lysate compared with the control group (normal iPSC) of normal iPSC-derived hepatocytes (N=4).

Claims

1. An artificially modified cell for intravascular transplantation, which is an artificially modified mammalian cell containing an artificially modified F3 gene, characterized in that: The artificially modified F3 gene is different from the F3 gene sequence of wild-type mammalian cells, The artificially modified F3 gene contains more than one insertion / deletion in the nucleic acid sequence, The expression level of CD142 on the surface of the artificially modified mammalian cells is reduced compared with that of wild-type mammalian cells.

2. The artificially modified cell for intravascular transplantation according to claim 1, characterized in that: In the cells for transplantation in the artificially modified blood vessels, Compared with the expression level of mRNA transcribed from the F3 gene of wild-type mammalian cells, the mRNA expression level of mRNA transcribed from the artificially modified F3 gene is lower or the sequence is different.

3. The artificially modified cell for intravascular transplantation according to claim 1, characterized in that: The insertion / deletion is located within the PAM sequence in the first, second, third, fourth or sixth region of the exon of the F3 gene, or within a continuous 5 to 50 nucleotide sequence adjacent to the 5' end or 3' end of the PAM sequence.

4. The artificially modified cell for intravascular transplantation according to claim 1, characterized in that: The mammalian cells are primary hepatocytes, primary pancreatic β cells, primary islet cells, pancreatic progenitor cells, hepatocytes derived from pluripotent stem cells, pancreatic β cells derived from pluripotent stem cells, islet cells derived from pluripotent stem cells, pancreatic lineage cells derived from pluripotent stem cells, pancreatic progenitor cells derived from pluripotent stem cells, pancreatic organoids derived from pluripotent stem cells, pancreatic liver organoids derived from pluripotent stem cells, hepatocytes derived from induced pluripotent stem cells, pancreatic β cells derived from induced pluripotent stem cells, islet cells derived from induced pluripotent stem cells, pancreatic lineage cells derived from induced pluripotent stem cells, pancreatic progenitor cells derived from induced pluripotent stem cells, pancreatic organoids derived from induced pluripotent stem cells, pancreatic liver organoids derived from induced pluripotent stem cells, hepatocytes induced from somatic cells by compounds or genetic modification, islet cells induced from somatic cells by compounds or genetic modification, or pancreatic β cells induced from somatic cells by compounds or genetic modification.

5. The artificially modified cell for intravascular transplantation according to claim 1, characterized in that: The sequence of the artificially modified F3 gene does not include the sequence of sequence number 1.

6. An artificially modified cell for intravascular transplantation, which is a cell differentiated from or derived from an artificially modified stem cell containing an artificially modified F3 gene, characterized in that: The artificially modified F3 gene is different from the F3 gene sequence of cells differentiated from or derived from wild-type stem cells, The artificially modified F3 gene contains more than one insertion / deletion in the nucleic acid sequence, The expression level of CD142 on the surface of cells differentiated from or derived from artificially modified stem cells is reduced compared to cells differentiated from or derived from wild-type stem cells.

7. The artificially modified cell for transplantation in blood vessels according to claim 6, characterized in that: The stem cells are induced pluripotent stem cells, embryonic stem cells, somatic cell nuclear transfer embryonic stem cells or adult stem cells.

8. The artificially modified cell for intravascular transplantation according to claim 6, characterized in that: The cells differentiated from or derived from artificially modified stem cells are pancreatic β cells, hepatocytes, pancreatic progenitor cells, islet cells, pancreatic lineage cells, pancreatic organoids or pancreatic liver organoids.

9. A composition for preparing blood-compatible cells for intravascular transplantation, characterized in that: Include: A guide nucleic acid or a nucleic acid encoding the same, comprising a guide sequence capable of targeting a target sequence of an F3 gene in a mammalian cell or a stem cell; and Editing proteins or nucleic acids encoding them.

10. The composition for preparing blood-compatible cells for intravascular transplantation according to claim 9, characterized in that: The composition comprises the editing protein and the guide nucleic acid in the form of ribonucleoprotein.

11. The composition for preparing blood-compatible cells for intravascular transplantation according to claim 9, characterized in that: The composition comprises a nucleic acid encoding the editing protein and a nucleic acid encoding the guide nucleic acid in the form of one or more vectors.

12. The composition for preparing blood-compatible cells for intravascular transplantation according to claim 11, characterized in that: The vector is selected from the group consisting of plasmid, retrovirus, lentivirus, adenovirus, adeno-associated virus, vaccinia virus, poxvirus and herpes simplex virus.

13. A method for preparing blood-compatible cells for intravascular transplantation, characterized in that: include: Step (1), introducing into isolated mammalian cells or isolated stem cells a composition for preparing blood-compatible cells for intravascular transplantation, wherein the composition comprises a guide nucleic acid or a nucleic acid encoding a target sequence of the F3 gene and an editing protein or a nucleic acid encoding the same; as well as Step (2) is to edit the F3 gene by inserting / deleting a target sequence of the F3 gene located in the genome of the isolated mammalian cell or isolated stem cell to reduce or inhibit the expression or activity of CD142.

14. The method for preparing blood-compatible cells for intravascular transplantation according to claim 13, characterized in that: The composition comprises a nucleic acid encoding the editing protein and a nucleic acid encoding the guide nucleic acid in the form of one or more vectors.

15. The method for preparing blood-compatible cells for intravascular transplantation according to claim 13, characterized in that: Insertion / deletion occurs in the target sequence of the F3 gene located in the genome of the isolated mammalian cell by contacting the target sequence with a CRISPR / Cas9 complex comprising a Cas9 protein derived from Streptococcus pyogenes and a guide RNA capable of targeting the target sequence of the F3 gene.

16. A cell therapy agent for vascular administration, characterized in that: The method comprises the artificially modified cells for transplantation into blood vessels according to any one of claims 1 to 8 as an effective ingredient.

17. A pharmaceutical composition for preventing or treating liver disease, characterized in that: The method comprises the artificially modified cells for transplantation into blood vessels according to any one of claims 1 to 8 as an effective ingredient.

18. The pharmaceutical composition for preventing or treating liver diseases according to claim 17, characterized in that: The liver disease is cirrhosis, acute-on-chronic liver failure, liver failure caused by drugs or poisoning, congenital metabolic liver disease, Crigler-Najjar syndrome type I, familial hypercholesterolemia, factor VII deficiency, factor VIII deficiency, phenylketonuria, glycogen storage disease type I, infantile Refsum's disease, progressive familial intrahepatic cholestasis type 2, hereditary tyrosinemia type 1, urea cycle disorder, acute liver failure, acute drug-induced liver failure, acute liver failure caused by viruses, idiopathic acute liver failure, acute liver failure caused by mushroom poisoning, acute liver failure after surgery, acute liver failure caused by acute fatty liver of pregnancy, alcoholic hepatitis or hepatic encephalopathy, and the factor VIII deficiency is hemophilia A.

19. The pharmaceutical composition for preventing or treating liver diseases according to claim 17, characterized in that: The pharmaceutical composition is administered via the portal vein, umbilical vein or splenic artery.

20. A pharmaceutical composition for preventing or treating diabetes, characterized in that: The method comprises the artificially modified cells for transplantation into blood vessels according to any one of claims 1 to 8 as an effective ingredient.

21. The pharmaceutical composition for preventing or treating diabetes according to claim 20, characterized in that: The diabetes is type 1 diabetes.

22. The pharmaceutical composition for preventing or treating diabetes according to claim 20, characterized in that: The pharmaceutical composition is administered via the hepatic portal vein.

23. A composition for cell transplantation or biological tissue regeneration, characterized in that: The method comprises the artificially modified cells for transplantation into blood vessels according to any one of claims 1 to 8 as an effective ingredient.

24. The composition for cell transplantation or biological tissue regeneration according to claim 23, characterized in that: The composition for cell transplantation or biological tissue regeneration is a cell therapy composition, a gene therapy composition, a tissue engineering therapy composition, an immunotherapy composition, or a cancer prevention or treatment composition.

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