Cells for endovascular transplantation having blood compatibility, method for producing the same, and uses thereof

Abstract

The present invention artificially modifies the F3 gene encoding CD142, which is a blood coagulation initiation factor, by gene scissors technology, and inhibits the blood coagulation mechanism and thrombus formation reaction by reducing or suppressing the expression or activity of CD142, and proposes a blood-compatible intravascular transplantation cell, a manufacturing method thereof, and a preventive or therapeutic use for liver diseases and diabetes using the same.

Description

[Technical Field]

[0001] The present invention provides blood-compatible cells for intravascular transplantation that can be administered intravascularly by inhibiting thrombus formation reactions through a reduction or suppression of the expression or activity level of CD142, a factor that initiates blood coagulation, as well as methods for producing and using the same. [Background technology]

[0002] Animals have a complex blood coagulation system, a biological mechanism that prevents bleeding by minimizing blood loss due to tissue damage. The blood coagulation system is a continuous amplification reaction process called a cascade reaction in response to an initial stimulus, and is composed of an intrinsic pathway that is directly induced by the stimulus and a unique extrinsic pathway. Many types of blood coagulation factors are involved in this process.

[0003] Among these, CD142 is a cell membrane glycoprotein that acts in the first step of the extrinsic pathway coagulation process and plays an important role in the in vivo coagulation process. After binding to coagulation factors VII and VIIa, CD142 activates coagulation factors IX and X, which are involved in activating prothrombin to thrombin, to generate thrombin during the coagulation process. Thrombin then activates fibrinogen to fibrin, resulting in the formation of a blood clot. CD142 also activates immune cells, causing activation of neutrophils, platelets, and monocytes, inducing the secretion of various cytokines, and activating the innate immune system.

[0004] Cell transplantation is a rapidly developing treatment in regenerative medicine that uses living autologous, allogenic, or xenegenic cells in an in vitro culture environment to restore cell and tissue function through a series of steps: isolating, expanding, and selecting cells, or altering their biological properties, for therapeutic, diagnostic, and preventative purposes. Compared to whole organ transplants, cell-based therapies are relatively less invasive procedures with lower morbidity and mortality rates. Cell transplantation significantly reduces the risks associated with surgery and has the advantage that the existing organ remains viable even if the graft is not functional or the inter-organ graft is lost.

[0005] Pancreatic islet cell transplantation has been proposed as the ideal method for treating type 1 diabetes. However, its clinical application is hindered by inflammatory responses, such as acute blood-mediated inflammatory reaction (IBMIR), which limits the survival of transplanted islets. Portal vein thrombosis induced by islet transplantation is a widely known and dreaded complication. Anticoagulants such as heparin are used to prevent this. Acute blood-mediated inflammatory responses occur when transplanted islet cells are directly exposed to the blood. This activation of the blood coagulation system, including platelets and complement, leads to blood clots around the islet cells, resulting in their rapid destruction. This is due to the dual activation of the blood coagulation and complement pathways by islet cells or cells bearing CD142 (tissue factor, TF, factor III).

[0006] Hepatocyte transplantation is an alternative to liver transplantation for patients with certain liver-based metabolic diseases and acute liver failure. For transplantation, human hepatocytes are isolated from donor liver tissue using collagenase perfusion and purified using centrifugation. The transplanted cells are targeted at approximately 5% of normal liver mass and are typically administered via the hepatic portal vein or spleen. One of the major obstacles to the sustained success of this therapy is initial cell loss, with up to 70% of hepatocytes lost immediately after infusion. This is primarily due to the acute blood-mediated inflammatory response (IBMIR). Transplanted hepatocytes produce and release CD142 (tissue factor, TF, factor III), which activates the coagulation pathway, forming thrombin and fibrin clots. Thrombin further activates multiple complement proteins, leading to membrane attack complex (MAC) activation and subsequent hepatocyte death. Summary of the Invention [Problem to be solved by the invention]

[0007] The objective of the present invention is to provide blood-compatible cells for intravascular transplantation that inhibit thrombus formation reactions that may be induced by the administered cells when administered intravascularly, a method for producing the same, and a cell therapy agent using the same. [Means for solving the problem]

[0008] In order to achieve the above object, the present invention provides The present invention provides cells for intravascular transplantation that are blood compatible and can be administered intravascularly by inhibiting thrombus formation reactions through a reduction or suppression of the expression or activity level of CD142, a factor that initiates blood coagulation.

[0009] The present invention also provides artificially engineered mammalian cells for intravascular transplantation, comprising an artificially engineered F3 gene, wherein the artificially engineered F3 gene is different from the F3 gene sequence of a wild-type mammalian cell, the artificially engineered F3 gene contains one or more indels in its nucleic acid sequence, and the CD142 expression level of the artificially engineered mammalian cells is reduced compared to that of the wild-type mammalian cells.

[0010] The present invention also provides artificially engineered cells for intravascular transplantation, which are cells differentiated or derived from artificially engineered stem cells and contain an artificially engineered F3 gene, wherein the artificially engineered F3 gene differs from the F3 gene sequence of cells differentiated or derived from wild-type stem cells, and the artificially engineered F3 gene contains one or more indels within its nucleic acid sequence, and the expression level of CD142 on the surface of the cells differentiated or derived from the artificially engineered stem cells is reduced compared to cells differentiated or derived from wild-type stem cells.

[0011] The present invention also provides a blood-compatible composition for producing cells for intravascular transplantation, which comprises a guide nucleic acid or a nucleic acid encoding the same, including a guide sequence capable of targeting a target sequence in the F3 gene of a mammalian cell or stem cell; and an editor protein or a nucleic acid encoding the same.

[0012] The present invention also provides a method for producing blood-compatible cells for intravascular transplantation, comprising the steps of: (1) introducing the above-mentioned composition for producing blood-compatible cells for intravascular transplantation into isolated mammalian cells or isolated stem cells; and (2) generating an indel in the target sequence of the F3 gene located in the genome of the mammalian cells, thereby editing the F3 gene so that the expression or activity of CD142 is reduced or suppressed.

[0013] The present invention also provides a cell therapy agent for vascular administration, which contains, as an active ingredient, the above-described cells for intravascular transplantation artificially engineered to have blood compatibility.

[0014] The present invention also provides a pharmaceutical composition for preventing or treating liver diseases, which comprises, as an active ingredient, the above-described cells for intravascular transplantation artificially engineered to have blood compatibility.

[0015] The present invention also provides a pharmaceutical composition for preventing or treating diabetes, which contains, as an active ingredient, the above-described cells for intravascular transplantation artificially engineered to have blood compatibility. [Effects of the Invention]

[0016] The artificially engineered cells for intravascular transplantation of the present invention can inhibit the blood coagulation mechanism by artificially modifying the F3 gene encoding CD142, a blood coagulation initiation factor, using genetic scissors technology to reduce or suppress the expression or activity of CD142. Therefore, overexpression of CD142 on the cell surface enables the intravascular administration of cells for transplantation that previously failed to show therapeutic efficacy due to post-transplant loss caused by inflammatory reactions such as instant blood-mediated inflammatory reaction (IBMIR), and can prevent the loss of transplanted cells after transplantation, thereby improving therapeutic efficacy. [Brief explanation of the drawings]

[0017] [Figure 1] This is a graph showing the results of targeted deep sequencing of normal iPSCs and iPSCs that had been gene-edited with the target sequence of CD142#33 (sequence number 1). [Figure 2] The results of qRT-PCR were used to confirm the differentiation markers PDX-1, NKX6.1, and SOX9 in pancreatic lineage cells from the control group (normal iPSCs), which were normal iPSC-derived pancreatic lineage cells, and the experimental group (iPSC-CD142#33), which were iPSC-derived pancreatic lineage cells in which the CD142 gene had been knocked out. [Figure 3] The results of qRT-PCR were used to confirm the hepatocyte differentiation markers HNF4α, ALB (albumin), and AFP in the control group (normal iPSCs) of normal iPSC-derived hepatocytes and the experimental group (iPSC-CD142#33) of iPSC-derived hepatocytes in which the CD142 gene had been knocked out. [Figure 4] The results of ELISA analysis of the hepatocyte differentiation marker ALB (albumin) in the control group (normal iPSCs) of normal iPSC-derived hepatocytes and the experimental group (iPSC-CD142#33) of iPSC-derived hepatocytes in which the CD142 gene had been knocked out are shown. [Figure 5] The results of qRT-PCR were shown for CD142 mRNA levels in the control group (normal iPSCs), which were normal iPSC-derived pancreatic lineage cells, and the experimental group (iPSC-CD142#33), which were iPSC-derived pancreatic lineage cells in which the CD142 gene had been knocked out. [Figure 6] This graph shows the results of measuring the percentage of CD142-expressing cells (CD142 positive cell population, %) by FACS in the control group (normal iPSCs), which were normal iPSC-derived pancreatic lineage cells, and the experimental group (iPSC-CD142#33), which were iPSC-derived pancreatic lineage cells in which the CD142 gene had been knocked out. [Figure 7] The results of qRT-PCR analysis of CD142 mRNA levels in the control group (normal iPSCs) of normal iPSC-derived hepatocytes and the experimental group (iPSC-CD142#33) of iPSC-derived hepatocytes in which the CD142 gene had been knocked out are shown. [Figure 8]This graph shows the results of measuring the percentage of cells expressing CD142 (CD142 positive cell population, %) using FACS in a control group (normal iPSCs) of normal iPSC-derived hepatocytes and an experimental group (iPSC-CD142#33) of iPSC-derived hepatocytes in which the CD142 gene had been knocked out. [Figure 9] The results of ELISA analysis of CD142 protein levels in cell lysates from the control group (normal iPSCs), which are normal iPSC-derived pancreatic lineage cells, and the experimental group (iPSC-CD142#33), which are iPSC-derived pancreatic lineage cells in which the CD142 gene had been knocked out. [Figure 10] The figure shows the results of ELISA analysis of CD142 protein levels in cell lysates from the control group (normal iPSCs), which were normal iPSC-derived hepatocytes, and the experimental group (iPSC-CD142#33), which were iPSC-derived hepatocytes in which the CD142 gene had been knocked out. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention will be described in detail below.

[0019] As used herein, the term "stem cell" refers broadly to undifferentiated cells that have the ability to differentiate into various types of body tissue cells, i.e., stemness. The stem cells may be induced pluripotent stem cells, embryonic stem cells, somatic cell nuclear transfer (PSC), or adult stem cells. The cells may be derived from humans, but are not limited thereto.

[0020] As used herein, "differentiation" refers to the phenomenon in which cell structure and function become specialized as cells divide and proliferate, resulting in the growth of an entire organism. In other words, it refers to the process by which cells, tissues, etc. of an organism change into morphologies and functions suited to the roles assigned to them. For example, differentiation can include the process by which pluripotent stem cells change into ectodermal (cerebral cortex, midbrain, hypothalamus, etc.), mesodermal (yolk sac, etc.), and endodermal cells, as well as the process by which hematopoietic stem cells change into red blood cells, white blood cells, platelets, etc., i.e., the process by which progenitor cells express specific differentiation traits.

[0021] As used herein, the term "about" means an amount, level, value, number, frequency, percent, dimension, size, amount, weight, or length that varies by 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 degree relative to a reference amount, level, value, number, frequency, percent, dimension, size, amount, weight, or length.

[0022] The term "artificially engineered" as used herein is used to distinguish a substance, molecule, etc. from a substance, molecule, etc. that has a structure already existing in nature, and means that the substance, molecule, etc. has been artificially modified. For example, an "artificially engineered gene" refers to a gene in which the structure of a gene existing in nature has been artificially modified. Furthermore, the term includes all meanings that a person of ordinary skill in the art would recognize, and may be interpreted appropriately depending on the context.

[0023] As used herein, "wild-type" means that a gene containing a naturally occurring nucleotide sequence and the protein expressed from that gene have normal functional properties. A wild-type gene has a form that is free of natural or artificial mutations and is the most frequently observed in a population. When the term "wild-type" is used in contrast to an artificially manipulated gene and / or an artificially manipulated cell, it can be interpreted as meaning a gene containing a naturally occurring nucleotide sequence that is "not artificially manipulated" and is the same type as the artificially manipulated gene and / or the artificially manipulated cell, and a cell containing the same. Furthermore, the term includes all meanings that would be recognized by a person of ordinary skill in the art and can be interpreted appropriately depending on the context.

[0024] As used herein, the term "knockout" or "knocked-out gene" refers to a mutation or artificial modification of a wild-type gene, such that the protein expressed by the wild-type gene cannot be produced through the transcription and / or translation processes. For example, a cell containing knocked-out gene A may be unable to express the mRNA and / or protein expressed by wild-type gene A. A cell containing knocked-out gene A may be one in which only one gene A present in the cell is knocked out, or two or more genes A are knocked out. Furthermore, the terms include all meanings that would be recognized by a person of ordinary skill in the art and may be interpreted appropriately according to the context.

[0025] As used herein, the term "knockdown" or "knocked-down gene" refers to a mutation or artificial modification of a wild-type gene, resulting in the expression of a substance in a lower amount than the wild-type gene. For example, a cell containing knocked-down gene A may express a lower amount of mRNA than the mRNA expressed by the wild-type gene A. As another example, a cell containing knocked-down gene A may express a lower amount of protein than the protein expressed by the wild-type gene A. A cell in which gene A is knocked down may be one in which only one gene A present in the cell is knocked down, or two or more genes A are knocked down. Furthermore, the terms include all meanings that a person of ordinary skill in the art would recognize and may be interpreted appropriately according to the context.

[0026] As used herein, "decreased expression" refers to expression of mRNA and / or protein at a level lower than that measured in wild-type cells. The decrease may be at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 50%, at least about 60%, at least about 70%, or at least about 100% compared to cells without the genetic mutation or wild-type cells.

[0027] As used herein, the term "decreased activity" or "decreased activity" may refer to a relative decrease in activity when measuring the activity of a protein or enzyme. Specifically, "decreased activity" or "decreased activity" refers to a lower level of protein or enzyme activity compared to a given parent cell or wild-type cell.

[0028] As used herein, "hemocompatibility" means that upon contact with blood, it does not induce hemolysis, platelet adhesion, platelet activation, and fibrin formation through complement activation, thrombus formation, or embolism.

[0029] The present invention provides cells for intravascular transplantation in which the expression or activity of CD142 (tissue factor, TF, factor III, tissue factor, blood coagulation factor 3) is reduced or suppressed.

[0030] The cells for intravascular transplantation with reduced or suppressed CD142 expression or activity are mammalian cells in which the expression or activity of CD142, a cell membrane glycoprotein that acts in the initial step of the extrinsic coagulation process and plays an important role in the in vivo blood coagulation process, has been reduced or suppressed, thereby inhibiting the thrombus coagulation mechanism.The mammalian cells with reduced or suppressed CD142 expression or activity can be used as a cell therapeutic agent and, when administered via vascular administration, can exhibit improved blood compatibility compared to wild-type mammalian cells, mammalian cells or antibodies artificially manipulated using conventional technology, and mammalian cells with low CD142 expression selected using a cell sorter or magnetic beads.

[0031] Specifically, the present invention provides artificially engineered cells for intravascular transplantation that are blood compatible and characterized by reduced blood coagulation reactions upon intravascular administration, which are obtained by artificially manipulating the F3 gene in mammalian cells to reduce or suppress the expression or activity of F3 mRNA and / or CD142.

[0032] Examples of mammals of the present invention include primates such as humans, monkeys, resuscitating monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees; rodents such as mice, rats, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, and sheep; and felines such as dogs and cats, with mice, pigs, and humans being particularly suitable.

[0033] 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.

[0034] According to an embodiment of the present invention, the mammalian cells of the present invention include primary hepatocytes, primary pancreatic beta cells, primary pancreatic islet cells, pancreatic progenitor cells, pluripotent stem cell (PSC) derived hepatocytes, pluripotent stem cell-derived pancreatic beta 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 (iPSC-derived hepatocytes), induced pluripotent stem cell-derived pancreatic beta cells, induced pluripotent stem cell-derived islet cells, and pluripotent stem cell-derived pancreatic progenitor cells. 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-hepatic organoids, hepatocytes induced from somatic cells by chemical compounds or genetic manipulation (induced hepatocytes, iHeps), pancreatic islet cells induced from somatic cells by chemical compounds or genetic manipulation, or pancreatic beta cells induced from somatic cells by chemical compounds or genetic manipulation.

[0035] According to one embodiment of the present invention, the transplant cells of the present invention are cells differentiated or derived from artificially engineered stem cells containing an artificially engineered F3 gene, wherein the artificially engineered F3 gene is different from the F3 gene sequence of cells differentiated or derived from wild-type stem cells, and the artificially engineered F3 gene contains one or more indels in its nucleic acid sequence, and the expression level of CD142 on the surface of cells differentiated or derived from the artificially engineered stem cells is reduced compared to cells differentiated or derived from wild-type stem cells.

[0036] According to one embodiment of the present invention, the stem cells are induced pluripotent stem cells (iPSCs), embryonic stem cells, somatic cell nuclear transfer embryonic stem cells (PSCs), or adult stem cells.

[0037] According to one embodiment of the present invention, the cells differentiated or derived from the artificially manipulated stem cells are pancreatic beta cells, hepatocytes, pancreatic progenitor cells, pancreatic islet cells, pancreatic lineage cells, pancreatic organoids, or pancreatic liver organoids.

[0038] The present invention provides artificially engineered cells for intravascular transplantation, which contain an artificially altered nucleic acid sequence of the F3 gene.

[0039] In the present invention, "artificial modification" or "artificial manipulation" of a gene nucleic acid sequence can be achieved by modifying the nucleic acid sequence constituting the gene or by chemically modifying a single base. This can be achieved by mutating, substituting, or deleting part or all of the gene, or by inserting one or more bases into the gene, and can be achieved using genetic scissors technology such as the CRISPR-enzyme system. For example, the artificial modification of the gene nucleic acid sequence can be achieved by non-homologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms.

[0040] For example, the artificially engineered cells for intravascular transplantation may be those in which the F3 gene has been knocked out.

[0041] As used herein, "non-homologous end joining (NHEJ)" refers to a method of repairing or patching a double-strand break in DNA by joining together both ends of a broken double or single strand. Generally, the broken double strand is repaired when two compatible ends formed by a double-strand break (e.g., a cleavage) come into frequent contact and the two ends are completely joined.

[0042] During the repair of damaged genes or nucleic acids using NHEJ, an "insertion and / or deletion" (or "indel") of a portion of the nucleic acid sequence can occur at the NHEJ repair site, resulting in a gene with an indel that does not have the same sequence as the wild-type gene. Such insertions and / or deletions alter the reading frame of the gene, producing a frameshifted transcript mRNA, which can then undergo nonsense-mediated decay or fail to synthesize normal proteins, resulting in a loss of its original function. Alternatively, while maintaining the reading frame, they can also result in a mutation that inserts or deletes a significant amount of sequence, disrupting protein functionality. In yet another example, if an indel occurs in a transcriptional regulatory region, such as a promoter or enhancer region of a gene, mRNA transcription may be prevented or reduced, resulting in a corresponding loss of protein expression or reduced expression. Alternatively, the mutagenesis mechanism of NHEJ can be used to delete only a partial sequence motif when it is not necessary to generate a specific final sequence. For example, two or more guide RNAs targeting the intron regions in the 5' and 3' parts of a specific exon can be used to create double-strand breaks in each intron region, resulting in the deletion of only a portion of the exon of a gene by NHEJ, with the remaining exons being expressed normally and the primary functionality of the protein being maintained.

[0043] By utilizing this NHEJ, it is possible to specifically knock out or knock down a target gene using genetic scissors technology.

[0044] For example, CRISPR enzymes such as Cas9 or Cpf1, which are types of genetic scissors, are used to cut the double strand or two single strands of a target gene or target nucleic acid, and indels are generated in the damaged double strand or two single strands of the target gene or damaged target nucleic acid by NHEJ, which can induce specific knockout or knockdown of the target gene or nucleic acid.

[0045] For example, the artificially engineered cells for intravascular transplantation may be those in which the F3 gene has been knocked out.

[0046] In one embodiment of the present invention, the F3 gene of the artificially engineered intravascular transplant cells may contain one or more indels in the nucleic acid sequence.

[0047] In one embodiment of the present invention, the artificially engineered cells for intravascular transplantation may not express F3 mRNA.

[0048] In one embodiment of the present invention, the mRNA expression level transcribed from the artificially engineered F3 gene of the artificially engineered cells for intravascular transplantation may be lower than the mRNA expression level transcribed from the F3 gene of wild-type cells.

[0049] In one embodiment of the present invention, the artificially engineered cells for intravascular transplantation may have a different F3 mRNA sequence compared to wild-type cells.

[0050] In one embodiment of the present invention, the artificially engineered cells for intravascular transplantation may have reduced expression or activity of CD142 on the cell surface compared to wild-type cells, thereby reducing or eliminating the function of CD142 in the artificially engineered cells for intravascular transplantation of the present invention.

[0051] That is, the expression or activity of CD142 in the artificially engineered cells for intravascular transplantation may 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% compared to the expression or activity in wild-type cells.

[0052] The present invention provides a method for producing hemocompatible artificially engineered cells for intravascular transplantation, which comprises the step of reducing the expression or activity of CD142 in mammalian cells.

[0053] The expression or activity of CD142 can be increased or decreased by artificially modifying the F3 gene, for example, by using gene scissors technology.

[0054] For example, the genetic scissors technology can utilize, but is not limited to, TALEN (transcription act4ator-like effector nuclease), which is a fusion of a TAL effector (transcription act4ator-like effector or TALE) domain and a cleavage domain, zinc-finger nuclease, or a CRISPR-enzyme system derived from CRISPR (Clustered regularly interspaced short palindromic repeats), a microbial immune system.

[0055] The entire contents of the disclosures in International Patent Publication WO2012 / 093833 or US Patent Publication No. 2013-0217131 relating to the TALENs are incorporated herein by reference. With respect to the ZFNs, 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, and U.S. Patent Registrations 7,888,121, 8,409,861, 6,479,626, 6,903,185, and 7,153,949 may be incorporated herein by reference.

[0056] The "CRISPR-enzyme system" is composed of a guide nucleic acid and / or an editor protein.

[0057] The term "guide nucleic acid" refers to a nucleic acid that can recognize a target nucleic acid, a target gene, or a target chromosome and interact with an editor protein, where the guide nucleic acid can form a complementary bond with a portion of a nucleotide in the target nucleic acid, the target gene, or the target chromosome.

[0058] The guide nucleic acid may be a target DNA-specific guide RNA, a DNA encoding the guide RNA, or a mixed form of DNA / RNA.

[0059] The guide nucleic acid may be a guide RNA. For example, the "guide RNA" may be transcribed in vitro, particularly from a double-stranded oligonucleotide or a plasmid template. For another example, the guide RNA may be encoded in the form of a vector and transcribed from the vector after being delivered into a cell in an ex vivo or in vivo environment, but is not limited thereto.

[0060] The design and construction of the guide RNA are known to those skilled in the art and are described in detail in Korean Patents Nos. 10-1656236, 10-1656237, 10-1706085, 10-2052286, and 10-2182847, the entire texts of which are incorporated herein by reference.

[0061] The guide nucleic acid can include a scaffold sequence portion and a guide sequence portion. The scaffold sequence portion interacts with a Cas protein, allowing the Cas protein and the guide nucleic acid to bind to form a complex (ribonucleoprotein, RNP). Generally, the scaffold sequence portion includes a portion of the sequence of tracrRNA and crRNA, and the scaffold sequence is determined depending on the type of Cas protein used.

[0062] The guide sequence portion is a nucleotide sequence portion that can complementarily bind to a portion of the sequence of one strand of the double strand of a target gene or nucleic acid, and can be artificially modified and determined based on the target nucleotide sequence of interest. In this case, the guide sequence may be a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more complementarity or complete complementarity with the guide nucleic acid binding sequence of the target gene or target nucleic acid. The guide sequence may be a sequence contained in the guide domain of the guide nucleic acid.

[0063] The guide sequence portion may be included in the crRNA. For example, the guide nucleic acid may be a dual RNA containing two RNAs, i.e., a crRNA (CRISPR RNA) and a tracrRNA (trans-acting crRNA), as components.

[0064] As another example, the guide nucleic acid may be a single-chain guide RNA (sgRNA), which is a combination of the main portions of crRNA and tracrRNA.

[0065] The target sequence is a nucleotide sequence of a certain length present in a target gene or target nucleic acid, and specifically, it may be a partial nucleotide sequence within a target region divided into a regulatory region, a coding region (or CDS, coding sequence), or a non-coding region (or UTR, untranslated region) of a target gene, or one or more partial nucleotide sequences selected from a combination of the target regions. The target sequence can be a target of a guide nucleic acid-editor protein complex (RNP).

[0066] In one embodiment of the present invention, the target sequence may be a sequence contained in exon region 1, 2, 3, 4, or 6 of the wild-type F3 gene. In one embodiment of the present invention, the target sequence may be the sequence of SEQ ID NO:1.

[0067] The target sequence is a nucleotide sequence adjacent to and surrounding a protospacer-adjacent motif (PAM) sequence recognized by the editor protein, and may include, but is not limited to, the entire or a portion of the PAM.

[0068] The term "target sequence" can refer to both sets of nucleotide sequence information. For example, in the case of a target gene, the target sequence can refer to the sequence information of the transcribed strand of the target gene DNA, or the nucleotide sequence information of the non-transcribed strand.

[0069] 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 is partially or completely complementary to the guide sequence contained in the guide domain of the guide nucleic acid and can complementarily bind to the guide sequence contained in the guide domain of the guide nucleic acid. The target sequence and guide nucleic acid binding sequence are nucleotide sequences that can vary depending on the target gene or nucleic acid, i.e., the subject to be genetically manipulated or edited, and the guide nucleic acid can be designed in a variety of ways depending on the target gene or target nucleic acid.

[0070] The guide nucleic acid non-binding sequence is a nucleotide sequence that has partial or complete homology to the guide sequence contained in the guide domain of the guide nucleic acid and is unable to bind complementary to the guide sequence contained in the guide domain of the guide nucleic acid. Alternatively, the guide nucleic acid non-binding sequence is a nucleotide sequence that is complementary to the guide nucleic acid binding sequence and is able to bind complementary to the guide nucleic acid binding sequence. The guide nucleic acid binding sequence is a partial nucleotide sequence of the target sequence and may be one of two nucleotide sequences of the target sequence that have different sequence orders, i.e., two nucleotide sequences that can bind complementary to each other. In this case, the guide nucleic acid non-binding sequence may be the remaining nucleotide sequence of the target sequence excluding the guide nucleic acid binding sequence.

[0071] The guide nucleic acid binding sequence may be a nucleotide sequence selected from the target sequence, i.e., the same nucleotide sequence as the transcribed strand and the same nucleotide sequence as the non-transcribed strand, and the guide nucleic acid non-binding sequence may be the remaining nucleotide sequence of the target sequence excluding the guide nucleic acid binding sequence, i.e., the same nucleotide sequence as the transcribed strand and the same nucleotide sequence as the non-transcribed strand.

[0072] The guide nucleic acid binding sequence may be identical in length to the target sequence. The guide nucleic acid non-binding sequence may be identical in length to the target sequence or the guide nucleic acid binding sequence. The guide nucleic acid binding sequence may be a sequence of 5 to 50 nucleotides.

[0073] In one embodiment, the guide nucleic acid binding sequence may be a 16-nucleotide sequence, a 17-nucleotide sequence, a 18-nucleotide sequence, a 19-nucleotide sequence, a 20-nucleotide sequence, a 21-nucleotide sequence, a 22-nucleotide sequence, a 23-nucleotide sequence, a 24-nucleotide sequence, or a 25-nucleotide sequence, and the guide nucleic acid non-binding sequence may be a 5-50-nucleotide sequence.

[0074] In one embodiment, the guide nucleic acid non-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.

[0075] The guide nucleic acid binding sequence can bind partially or completely complementary to the guide sequence contained in the guide domain of the guide nucleic acid, and the length of the guide nucleic acid binding sequence can be the same as the length of the guide sequence.

[0076] 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, at least 70%, 75%, 80%, 85%, 90%, or 95% or more complementary, or may be a completely complementary nucleotide sequence.

[0077] For example, the guide nucleic acid binding sequence may have or include a 1 to 8 nucleotide sequence that is not complementary to the guide sequence included in the guide domain of the guide nucleic acid.

[0078] The guide nucleic acid non-binding sequence may have partial or complete homology with the guide sequence included 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. For example, the guide sequence may be designed based on a sequence having homology with the guide nucleic acid non-binding sequence.

[0079] The guide nucleic acid non-binding sequence may be a nucleotide sequence having homology to the guide sequence contained in the guide domain of the guide nucleic acid, for example, a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% or more homology, or a nucleotide sequence having complete homology.

[0080] For example, the guide nucleic acid non-binding sequence may have or include a 1 to 8 nucleotide sequence that is not homologous to the guide sequence included in the guide domain of the guide nucleic acid. The guide nucleic acid non-binding sequence can 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.

[0081] The guide nucleic acid non-binding sequence may be a nucleotide sequence complementary to the guide nucleic acid binding sequence, for example, at least 90% or 95% or more complementary, or a completely complementary nucleotide sequence.

[0082] For example, the guide nucleic acid non-binding sequence may have or include one to two nucleotide sequences that are not complementary to the guide nucleic acid binding sequence, and the guide nucleic acid binding sequence may be a nucleotide sequence located adjacent to a sequence complementary to a nucleotide sequence (PAM sequence) that can be recognized by the editor protein.

[0083] For example, the guide nucleic acid binding sequence may be a contiguous sequence of 5 to 50 nucleotides located adjacent to the 5' end or / and 3' end of a sequence complementary to a nucleotide sequence (PAM sequence) that can be recognized by the editor protein.

[0084] Alternatively, 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 an editor protein.

[0085] For example, the guide nucleic acid non-binding sequence may be a contiguous 5 to 50 nucleotide sequence located adjacent to the 5' end or / and 3' end of a nucleotide sequence (PAM sequence) that can be recognized by an editor protein.

[0086] "Editor protein" refers to a peptide, polypeptide, or protein that directly binds to nucleic acids or that can interact with them without directly binding. The editor protein is also conceptually referred to as an "artificially engineered nuclease" or RGEN (RNA-Guided Endonuclease).

[0087] In one embodiment, the editor protein may be a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) enzyme. A "CRISPR enzyme" is a key protein component of the CRISPR-enzyme system, also known as a "CRISPR-associated protein," and refers to a nuclease that can recognize a target sequence and cleave DNA by mixing with or forming a complex with a guide RNA.

[0088] CRISPR enzymes are known to those skilled in the art, and reference is made to Korean Patent Registration Nos. 10-1656236, 10-1656237, 10-1706085, 10-2052286, and 10-2182847. The term "CRISPR enzyme" as used herein encompasses not only native proteins but also mutants that can act as activated endonucleases or nickases in cooperation with guide RNA. Activated endonucleases or nickases can cleave target DNA, which can be used for gene editing. Inactive mutants can be used for transcription regulation or target DNA isolation.

[0089] The CRISPR enzyme is a nucleic acid or polypeptide (or protein) having a sequence encoding the CRISPR enzyme. Typically, Type II CRISPR enzymes or Type V CRISPR enzymes are used, and an example of the Type II CRISPR enzyme is the CRISPR associated protein 9 (Cas9) protein.

[0090] The Cas9 protein may be derived from a variety of microorganisms, such as Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Campylobacter jejuni, Staphylococcus aureus, Staphylococcus auricularis, and Neisseria meningitidis.

[0091] In order for the Cas9 protein to induce a double-stranded DNA break, it must recognize a protospacer adjacent motif (PAM) sequence, which is a nucleotide sequence of a certain length, and a portion of the guide RNA (the guide sequence portion) must complementarily bind to the complementary strand (the guide nucleic acid binding sequence) of the single-stranded DNA where the target sequence is located (the guide nucleic acid non-binding sequence).

[0092] This PAM sequence is determined by the type and origin of the Cas9 protein. For example, the Streptococcus pyogenes Cas9 protein (SpCas9) can recognize the 5'-NGG-3' sequence (complementary sequence: 5'-CCN-3') in a target nucleic acid, where N is one of adenosine (A), thymidine (T), cytidine (C), and guanosine (G). SpCas9 can also recognize the 5'-NAG-3' sequence (complementary sequence: 5'-CTN-3') in a target nucleic acid with low activity.

[0093] Furthermore, the Type V CRISPR enzyme includes Cpf1, which is capable of inhibiting Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, and Lachnospiracea. e, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococ Cpf1 from C.cus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, uberibacillus, Bacillus, Brevibacilus, Methylobacterium or Acidaminococcus.

[0094] The CRISPR enzyme, such as the Cas9 or Cpf1 protein, may be isolated from a naturally occurring microorganism or produced non-naturally by recombinant or synthetic methods. The Cas protein may also be in a form that is easily introduced into cells. For example, the Cas protein may be linked to a cell-penetrating peptide or a protein transduction domain. The protein transduction domain may be, but is not limited to, polyarginine or the HIV-derived TAT protein. Since various types of cell-penetrating peptides or protein transduction domains are known in the art in addition to the examples described above, those skilled in the art may apply various examples to the present specification without being limited to the above examples. The Cas protein may also be fused to a functional domain, such as a nuclear localization sequence or signal (NLS). The Cas9 protein may also be encoded in the form of a vector and expressed in cells.

[0095] The present invention provides a blood-compatible composition for producing cells for intravascular transplantation, which comprises a guide nucleic acid or a nucleic acid encoding the same, including a guide sequence capable of targeting a target sequence in the F3 gene of a mammalian cell or stem cell; and an editor protein or a nucleic acid encoding the same.

[0096] Additionally, the composition may optionally further comprise a donor containing a specific nucleotide sequence desired to be inserted or a nucleic acid encoding the same.

[0097] The donor refers to exogenous nucleotide sequences that can express a specific peptide or protein and can be inserted into genomic DNA through homology directed repair (HDR).

[0098] The donor may be a double-stranded or single-stranded nucleic acid. The donor may be linear or circular.

[0099] The donor may be in the form of a viral vector or a non-viral vector (eg, a plasmid).

[0100] The virus may be a DNA virus or an RNA virus, where the DNA virus may be a double-stranded DNA (dsDNA) virus or a single-stranded DNA (ssDNA) virus, and the RNA virus may be a single-stranded RNA (ssRNA) virus.

[0101] The viral vector may be one or more viral vectors selected from the group consisting of retrovirus, lentivirus (Lent4irus), adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus, and herpes simplex virus (HSV).

[0102] The target sequence can be a target of the guide nucleic acid-editor protein complex, and the target sequence can include, but is not limited to, a PAM (protospacer-adjacent motif) sequence recognized by the editor protein.

[0103] The guide nucleic acid can comprise a guide domain that allows the composition to target a target sequence in the F3 gene.

[0104] Herein, the guide nucleic acid, editor protein, or guide nucleic acid-editor protein complex (ribonucleoprotein, RNP), and / or donor may be delivered or introduced into a subject in a variety of forms.

[0105] In this context, "subject" refers to an organism into which a guide nucleic acid, editor protein, or guide nucleic acid-editor protein complex is introduced; an organism in which a guide nucleic acid, editor protein, or guide nucleic acid-editor protein complex operates; or a specimen or sample obtained from an organism.

[0106] The subject can be an organism that contains a target gene, target nucleic acid, or target chromosome of the guide nucleic acid-editor protein complex.

[0107] The organism may be an animal, animal tissue or animal cell, where the tissue may be an eye, skin, liver, kidney, heart, lung, brain, muscle or blood.

[0108] The cell may be a mammalian cell.

[0109] Examples of mammals of the present invention include primates such as humans, monkeys, resuscitating monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees; rodents such as mice, rats, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, and sheep; and felines such as dogs and cats, with mice, pigs, and humans being particularly suitable.

[0110] According to one embodiment of the present invention, the artificially engineered cells for intravascular transplantation of the present invention are allogenic or autologous cells for transplantation, more specifically, allogenic cells for transplantation.

[0111] According to an embodiment of the present invention, the mammalian cells of the present invention include primary hepatocytes, primary pancreatic beta cells, primary pancreatic islet cells, pancreatic progenitor cells, pluripotent stem cell (PSC) derived hepatocytes, pluripotent stem cell-derived pancreatic beta 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 (iPSC-derived hepatocytes), induced pluripotent stem cell-derived pancreatic beta cells, induced pluripotent stem cell-derived islet cells, and pluripotent stem cell-derived pancreatic progenitor cells. 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-hepatic organoids, hepatocytes induced from somatic cells by chemical compounds or genetic manipulation (induced hepatocytes, iHeps), pancreatic islet cells induced from somatic cells by chemical compounds or genetic manipulation, or pancreatic beta cells induced from somatic cells by chemical compounds or genetic manipulation.

[0112] The specimen or sample may be obtained from an organism containing the target gene, target nucleic acid, or target chromosome, such as saliva, blood, liver tissue, brain tissue, hepatocytes, neurons, phagocytes, macrophages, T cells, B cells, astrocytes, cancer cells, or stem cells.

[0113] The guide nucleic acid, editor protein, or guide nucleic acid-editor protein complex can be delivered or introduced into a subject in the form of DNA, RNA, or a mixture thereof.

[0114] In this case, DNA, RNA, or a mixture thereof encoding the guide nucleic acid and / or the editor protein can be delivered or introduced into the subject by methods known in the art.

[0115] Alternatively, DNA, RNA, or a mixture thereof encoding the guide nucleic acid and / or editor protein can be delivered or introduced into a subject by a vector, a non-vector, or a combination thereof.

[0116] The vector can be a viral vector or a non-viral vector (eg, a plasmid).

[0117] The virus may be a DNA virus or an RNA virus, where the DNA virus may be a double-stranded DNA (dsDNA) virus or a single-stranded DNA (ssDNA) virus, and the RNA virus may be a single-stranded RNA (ssRNA) virus.

[0118] The viral vector can be one or more selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus, and herpes simplex virus.

[0119] The non-vector may be naked DNA, a DNA complex, or mRNA.

[0120] In one embodiment of the present invention, the guide nucleic acid and / or the nucleic acid encoding the editor protein may be delivered or introduced into a subject in the form of one or more vectors.

[0121] The vector can contain a nucleic acid encoding a guide nucleic acid and / or an editor protein. For example, the vector can contain both a guide nucleic acid and a nucleic acid encoding an editor protein. For another example, the vector can contain a nucleic acid encoding a guide nucleic acid. For example, the nucleic acids encoding the guide nucleic acid can be all contained in a single vector, or the nucleic acid encoding the guide nucleic acid can be divided and contained in multiple vectors. For another example, the vector can contain a nucleic acid encoding an editor protein. For example, in the case of the editor protein, the nucleic acid encoding the editor protein can be contained in a single vector, or the nucleic acid encoding the editor protein can be divided and contained in multiple vectors.

[0122] The editor protein can be delivered or introduced into a subject in the form of a peptide, polypeptide, or protein.

[0123] The editor protein can be delivered or introduced into a subject in the form of a peptide, polypeptide, or protein by methods known in the art.

[0124] The guide nucleic acid and editor protein can be delivered or introduced into a subject in the form of a nucleic acid-protein combination.

[0125] The guide nucleic acid and editor protein may be delivered or introduced into a subject in the form of a guide nucleic acid-editor protein complex. For example, the guide nucleic acid may be in the form of DNA, RNA, or a mixture thereof. The editor protein may be in the form of a peptide, polypeptide, or protein. For example, the guide nucleic acid and editor protein may be delivered or introduced into a subject in the form of a guide nucleic acid-editor protein complex, i.e., a ribonucleoprotein (RNP), in which the guide nucleic acid is in the form of an RNA and the editor protein is in the form of a protein.

[0126] The present invention also provides a method for producing blood-compatible cells for intravascular transplantation, comprising the steps of: (1) introducing the above-mentioned composition for producing blood-compatible cells for intravascular transplantation into isolated mammalian cells or isolated stem cells; and (2) generating an indel in the target sequence of the F3 gene located in the genome of the isolated mammalian cells or isolated stem cells, thereby editing the F3 gene so that the expression or activity of CD142 is reduced or suppressed. In this case, the "introduction" may be performed by one or more means selected from the group consisting of electroporation, lipofection, microinjection, gene gun, liposome, cationic liposome, plasmid, viral vector, nanoparticles, PTD (protein translocation domain) fusion protein method, immunoliposome, polyvalent cation or lipid:nucleic acid conjugate, naked DNA, artificial virion, and DNA formulation-enhanced absorption method, but is not limited thereto.

[0127] In one embodiment of the present invention, the composition for producing cells for intravascular transplantation having blood compatibility can be introduced into isolated mammalian cells or isolated stem cells by electroporation to produce cells for intravascular transplantation having blood compatibility.

[0128] In one embodiment of the present invention, an indel can be generated by contacting the F3 gene located in the genome of the mammalian cell with a CRISPR / Cas9 complex comprising the Streptococcus pyogenes-derived Cas9 protein and a guide RNA capable of targeting a target sequence in the F3 gene.

[0129] In one embodiment of the present invention, the target sequence may be the sequence of SEQ ID NO:1.

[0130] The blood-compatible artificially engineered mesenchymal stem cells of the present invention can be used as a cell therapy agent or pharmaceutical composition for vascular administration to treat a variety of diseases, including, for example, heart disease, gastroduodenal disease, small and large intestinal disease, liver disease, bile duct disease, pancreatic disease, kidney disease, liver disease, lung disease, mediastinal disease, diaphragmatic disease, pleural disease, peritoneal disease, nervous system disease, central nervous system (CNS) disorders, peripheral arterial disease, and peripheral venous disease. Specific diseases include, for example, autoimmune hepatitis, fulminant hepatitis, chronic hepatitis, viral hepatitis, alcoholic hepatitis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver (NAFL), liver fibrosis, liver cirrhosis, liver cancer, fatty liver, drug-induced allergic liver disease, hemochromatosis, Wilson's disease, primary biliary cirrhosis (PBC), and primary sclerosing cholangitis. Liver diseases such as primary cholangitis (PSC), biliary atresia, liver abscess, chronic active hepatitis, and chronic persistent hepatitis; heart diseases such as myocardial infarction, heart failure, arrhythmia, palpitations, cardiomyopathy, ischemic cardiomyopathy, angina pectoris, congenital heart disease, valvular heart disease, myocarditis, familial hypertrophic cardiomyopathy, dilated cardiomyopathy, acute coronary syndrome, arteriosclerosis, and restenosis; acute gastritis, chronic gastritis, and gastroduodenal ulcer Gastroduodenal diseases such as gastric cancer and duodenal cancer; small intestinal 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, and colon cancer; bile duct diseases such as acute cholecystitis, acute cholangitis, chronic cholecystitis, bile duct cancer, and gallbladder cancer; 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, pulmonary tuberculosis, sequelae of pulmonary tuberculosis, acute respiratory distress syndrome, cystic fibrosis, chronic obstructive pulmonary disease, pulmonary embolism, lung abscess, pneumoconiosis, aspiration pneumonia, pulmonary fibrosis, acute upper respiratory tract infection, chronic lower respiratory tract infection, pneumothorax, alveolar epithelial damage disease, lymphangioleiomyoma, lymphoma Pulmonary diseases such as interstitial pneumonia, pulmonary alveolar proteinosis, and pulmonary Langerhans cell granulomatosis; mediastinal diseases such as mediastinal tumors, mediastinal cystic disease, and mediastinitis; diaphragmatic diseases such as diaphragmatic hernia; pleural diseases such as pleurisy, empyema, pleural tumors, carcinomatous pleurisy, and pleural mesothelioma; peritonitis and peritoneal tumors; cerebral palsy syndromes including childhood cerebral palsy, aseptic meningitis, Guillain-Barré syndrome, and amyotrophic lateral sclerosis (amyotrophic lateral sclerosis) Neurological disorders include: ALS, myasthenia gravis, mononeuropathy, multiple neuropathy, spinal muscular atrophy, spinal cord disorders, acute transverse myelitis, spinal cord infarction (ischemic myelopathy), intracranial tumors, and spinal tumors; CNS disorders including Alzheimer's disease, cognitive impairment, stroke, multiple sclerosis, and Parkinson's disease; peripheral arterial diseases including fibromuscular dysplasia, peripheral artery disease (PAD), thromboangiitis obliterans (Buerger's disease), and Kawasaki disease (KD); peripheral venous diseases including deep vein thrombosis, chronic venous insufficiency, post-phlebitic syndrome, and superficial venous thrombosis; and immunodeficiency disorders including graft-versus-host disease (GVHD), secondary immunodeficiencies, primary immunodeficiencies, B cell deficiencies, T cell deficiencies, combined B and T cell deficiencies, phagocyte deficiencies, and complement deficiencies. ;

[0131] The term "vascular administration" refers to delivery into a patient's vascular system. Examples include administration into blood vessels considered to be veins and administration into blood vessels considered to be arteries. Veins include, but are not limited to, the internal jugular vein, peripheral veins, coronary veins, hepatic veins, portal vein, great saphenous vein, pulmonary veins, superior vena cava, inferior vena cava, gastric vein, splenic vein, inferior mesenteric vein, superior mesenteric vein, cephalic vein, and / or femoral vein. Arteries include, but are not limited to, the coronary artery, pulmonary artery, splenic artery, brachial artery, internal carotid artery, aortic arch, femoral artery, peripheral artery, and / or ciliary artery. It may be delivered to bovine arteries or capillaries via the hepatic portal vein, umbilical vein, small arteries or capillaries.

[0132] In one embodiment of the present invention, the artificially engineered cells for intravascular transplantation of the present invention can be used as a cell transplant or tissue regeneration composition for restoring damaged cells or tissues.

[0133] In one embodiment of the present invention, the biological tissue may be a damaged tissue selected from tissue with ulcer or pressure sore formation, brain tissue damaged by cell degeneration, brain tissue lost due to surgical manipulation, 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 disease, and tissue damaged by intractable dermatitis. The biological tissue regeneration may be restoration of the damaged tissue, epidermal regeneration, reproduction of secretory glands or hair follicles, microvascularization of dermal tissue, wound healing, correction of soft tissue defects, bone healing or bone regeneration, cartilage regeneration, etc., but is not limited thereto.

[0134] In one embodiment of the present invention, the composition for cell transplantation or biological tissue regeneration may be a cell therapy composition, a gene therapy composition, a tissue engineering therapy composition, an immunotherapy composition, or a cancer prevention or treatment composition.

[0135] As used herein, "cell therapy" refers to a pharmaceutical product (as defined by the U.S. FDA) that is used for therapeutic, diagnostic, and preventive purposes using cells and tissues isolated from an individual, cultured, and specially processed, and that is used for therapeutic, diagnostic, and preventive purposes through a series of actions such as expanding and selecting living autologous, allogeneic, or xenogeneic cells in vitro to restore the function of cells or tissues, or by changing the biological properties of cells in other ways.

[0136] As used herein, the term "gene therapy agent" refers to a pharmaceutical product that is administered to affect the expression of genetic material and contains cells into which genetic material has been transformed or introduced.

[0137] The composition for cell transplantation or biological tissue regeneration of the present invention can be administered via any common route as long as it can reach the target tissue, including, but not limited to, parenteral administration, such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, and intravascular administration.

[0138] The cell transplant or tissue regeneration composition may be formulated in a suitable form with pharmaceutical carriers commonly used in cell therapy. "Pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not typically cause allergic or similar reactions, such as gastrointestinal upset or dizziness, when administered to humans. Pharmaceutically acceptable carriers include parenteral carriers such as water, suitable oils, saline, aqueous glucose, and glycol, and may additionally contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methylparaben or propylparaben, and chlorobutanol. Other pharmaceutically acceptable carriers may be found in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995). The composition may also be administered by any device capable of delivering the cell therapy agent to target cells.

[0139] The cell transplantation or tissue regeneration composition of the present invention can contain a therapeutically effective amount of cells for treating a disease. The term "therapeutically effective amount" refers to an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal, or human that is contemplated by a researcher, veterinarian, physician, or other clinician, including an amount that induces symptomatic relief of the disease or disorder being treated.

[0140] It is obvious to those skilled in the art that the amount of cells contained in the composition of the present invention can be varied depending on the desired effect. Therefore, the optimal cell content can be easily determined by those skilled in the art and can be adjusted depending on various 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, the patient's age, weight, general health condition, sex, and diet, the administration time, the administration route, the secretion rate of the composition, the treatment period, and any other drugs used concomitantly. Taking all of the above factors into consideration, it is important to include an amount that can achieve the maximum effect at the minimum dose without side effects. For example, the daily dose of the stem cells of the present invention is 1.0 x 10 5 ~1.0×10 30 cells / kg body weight, preferably 1.0 x 10 10 ~1.0×10 20 The cells / kg body weight can be administered in one or several divided doses. However, it should be understood that the actual dose of an effective amount should be determined in light of various relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's body weight, age, and sex, and therefore, the dose does not limit the scope of the present invention in any way.

[0141] The present invention provides a method of treatment comprising administering to a mammal a therapeutically effective amount of the artificially engineered cells for intravascular transplantation. As used herein, the term mammal refers to a mammal, preferably a human, that has been the object of treatment, observation, or experiment.

[0142] The present invention also includes a pharmaceutical composition for preventing or treating liver diseases, which contains a therapeutically effective amount of the artificially engineered cells for intravascular transplantation as an active ingredient.

[0143] The compositions of the present invention can be administered orally or parenterally, including by inhalation, intravenous, intraperitoneal, subcutaneous, rectal, and topical administration. In one embodiment of the present invention, the compositions can be administered via the hepatic portal vein, umbilical vein, or splenic artery.

[0144] The present invention also provides a method for preventing or treating liver disease, comprising administering to a mammal a therapeutically effective amount of the artificially engineered cells for intravascular transplantation. As used herein, the term "mammal" refers to a mammal that has been the subject of treatment, observation, or experiment, preferably a human.

[0145] The present invention also provides the use of the artificially engineered intravascular transplant cells for use in the manufacture of a medicament for the prevention or treatment of liver disease in a mammal.

[0146] In one embodiment of the present invention, the liver disease includes cirrhosis; acute-on-chronic liver failure (ACLF); drug- or poisoning-induced liver failure; congenital metabolic liver disease; Crigler-Nager syndrome type 1; familial hypercholesterolemia; factor VII deficiency; factor VIII deficiency (hemophilia A); phenylketonuria (PKU); glycogen storage disease type 1; infantile Refsum's disease; progressive familial intrahepatic cholestasis type 2; hereditary tyrosinemia type 1; urea cycle disorders; acute liver failure; acute drug-induced liver failure; virus-induced acute liver failure; idiopathic acute liver failure; mushroom poisoning-induced acute liver failure; post-operative acute liver failure; acute liver failure due to acute fatty liver of pregnancy; alcoholic hepatitis; or hepatic encephalopathy.

[0147] The present invention also includes a pharmaceutical composition for preventing or treating diabetes, which contains a therapeutically effective amount of the artificially engineered cells for intravascular transplantation as an active ingredient.

[0148] The compositions of the present invention can be administered orally or parenterally, including by inhalation, intravenous, intraperitoneal, subcutaneous, rectal, and topical administration. In one embodiment of the present invention, the compositions can be administered via the hepatic portal vein.

[0149] The present invention also provides a method for preventing or treating diabetes, which comprises administering to a mammal a therapeutically effective amount of the artificially engineered cells for intravascular transplantation.

[0150] The present invention also provides the use of the artificially engineered cells for intravascular transplantation for the manufacture of a medicament for the prevention or treatment of diabetes in a mammal.

[0151] In one embodiment of the present invention, the diabetes includes type 1 diabetes.

[0152] Hereinafter, the present invention will be described in more detail through examples. These examples are only for more specifically explaining the present invention, and it will be self-evident to those with ordinary knowledge in the technical field to which the present invention belongs that the scope of the present invention is not limited by these examples.

[0153] [Example 1: Production of F3 knockout cells] <Cell culture> iPSCs were cultured according to the manufacturer's manual. Vitronectin XF TM (STEMCELL Technology) was used to coat the culture dish at RT (room temperature) for 1 hour, and TeSR TM -E8 TM (STEMCELL Technology) medium was used for culturing.

[0154] <Production of CD142 gene-edited cells> The RNP complex was introduced by electroporation by harvesting the cultured cells using 4D-Nucleofector (Lonza). Specifically, the RNP complex was formed by mixing 4 μg of Cas9 protein and 4 μg of in vitro transcribed sgRNA (T7 polymerase (New England BioLabs)) prepared according to the manufacturer's protocol, and incubating the mixture at room temperature for 10 minutes. The RNP complex was treated with 20 μl of Primary P3 buffer and 1×10 6Electroporation was performed using the nucleofector program CA-137 with iPSCs. As a result, iPSCs with a knockout of the CD142 gene (CD142#33 KO iPSCs) were obtained. A guide RNA targeting the CD142#33 target sequence 5'-TCTGGGGAGTTCTCATACAGAGG-3' (underlined: PAM sequence) (SEQ ID NO: 1) was synthesized and introduced into the iPSCs. Targeted deep sequencing was used to confirm the indel efficiency of the target sequence.

[0155] Targeted deep sequencing Genomic DNA (gDNA) was extracted from the harvested CD142#33 KO iPSCs using the Blood Genomic DNA Extraction Kit (Favorgen) according to the manufacturer's protocol. To amplify the target region, 100 ng of gDNA was amplified using Phusion High Fidelity DNA Polymerase PCR Polymerase (NEB). To generate a deep sequencing library, the amplicon was amplified again using TruSeq HT Dual Index Primers (Illumina, San Diego, CA, USA). Paired-end sequencing was performed using the Illumina Miniseq System, and indel frequencies were calculated using http: / / www.rgenome.net / . The primer sequences for each target sequence used in targeted deep sequencing are listed in Table 1.

[0156] [Table 1]

[0157] Figure 1 is a graph showing the results of targeted deep sequencing of normal iPSCs and iPSCs gene-edited with the target sequence of CD142#33 (SEQ ID NO: 1). As shown in the targeted deep sequencing results in Figure 1, 98% indels were confirmed in iPSCs gene-edited with the target sequence of CD142#33 (SEQ ID NO: 1).

[0158] <Pancreatic lineage cell differentiation> The CD142 gene knockout iPSCs (CD142#33 KO iPSCs) obtained above were differentiated into pancreatic lineage cells. At this time, pancreatic lineage cell differentiation was performed by STEMCELL Technology. TM This was performed according to protocol (catalog #05120) (https: / / www.stemcell.com / products / stemdiff-pancreaticprogenitor-kit.html#section-protocols-and-documentation ), which differentiates through four stages: 1) terminal endoderm, 2) primitive intestine, 3) posterior full-length endoderm, and 4) pancreatic lineage cells. Differentiated cells were identified by the expression of key markers of pancreatic lineage cells.

[0159] <Hepatocyte differentiation> The CD142 gene knockout iPSCs (CD142#33 KO iPSCs) obtained above were differentiated into hepatocytes 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 key hepatocyte markers.

[0160] [Experimental Example 1: Confirmation of differentiation markers of pancreatic lineage cells derived from iPSC (induced pluripotent stem cell) cells] To confirm whether iPSCs were successfully differentiated into pancreatic lineage cells, markers that are particularly highly expressed in pancreatic lineage cells during differentiation were identified by qRT-PCR. 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).

[0161] mRNA was extracted from iPSCs using the RNeasy mini kit (Qiagen) according to the manufacturer's protocol. 1 μg of mRNA was then reverse transcribed using a cDNA reverse transcription kit (Thermo Fisher Scientific). qRT-PCR was performed using the QuantStudio 3 (Thermo Fisher Scientific) with PowerUp according to the manufacturer's protocol. TM SYBR TMThe experiment was performed using Green Master Mix. Gene expression levels were calculated using C values, and GAPDH was used as an endogenous control. The results are shown in Figure 2. Both the control group (normal iPSCs), which were normal iPSC-derived pancreatic lineage cells, and the experimental group (iPSC-CD142#33), which were iPSC-derived pancreatic lineage cells with the CD142 gene knocked out, showed increased expression of all markers by day 14 (D14), confirming successful differentiation into pancreatic lineage cells (N=4).

[0162] [Experimental Example 2: Confirmation of differentiation markers of iPSC (induced pluripotent stem cell)-derived hepatocytes] To confirm whether iPSC cells had successfully differentiated into hepatocytes, markers that are particularly highly expressed in hepatocytes during differentiation were examined by qRT-PCR using the same method as in Example 1. The markers used in the experiment were hepatocyte nuclear factor 4 alpha (HNF4α), albumin (ALB), and alpha-fetoprotein (AFP). The results are shown in Figure 3. As shown in Figure 3, both the control group (normal iPSCs), which were hepatocytes derived from normal iPSCs, and the experimental group (iPSC-CD142#33), which were hepatocytes derived from iPSCs with the CD142 gene knocked out, showed increased expression of all markers by day 40 (D40), confirming successful differentiation into hepatocytes.

[0163] <ELSIA(Enzyme-linked immunosorbent assay)> To confirm whether iPSC cells were successfully differentiated into hepatocytes, ALB (albumin), a marker that is particularly highly expressed in hepatocytes during differentiation, was examined by ELISA.

[0164] Albumin was measured using cell lysates and cell culture supernatants from iPSC-derived hepatocytes using the Human Albumin EIA Kit (TaKaRa (MK132)). The experiment was carried out according to the protocol provided by the kit manufacturer. Absorbance was measured using a Multiscan TM FC Microplate Photometer (Thermo Scientific TM The measurements were performed at a wavelength of 405 nm using a (51119000)) instrument, and the results are shown in Figure 4.

[0165] As can be seen in Figure 4, both the control group (normal iPSCs) of normal iPSC-derived hepatocytes and the experimental group (iPSC-CD142#33) of iPSC-derived hepatocytes with the CD142 gene knocked out showed increased expression of all markers on day 40 (D40), confirming successful differentiation into hepatocytes.

[0166] [Experimental Example 3: Confirmation of CD142 gene knockout in pancreatic lineage cells derived from iPSC (induced pluripotent stem cell) cells] <qrt-pcr> The knockout of CD142 in iPSC-derived pancreatic lineage cells was confirmed by analyzing the mRNA level of the CD142 gene using qRT-PCR.

[0167] mRNA was extracted from iPSC-derived pancreatic lineage cells using the RNeasy mini kit (Qiagen) according to the manufacturer's protocol. 1 μg of mRNA was then reverse transcribed using a cDNA reverse transcription kit (Thermo Fisher Scientific). qRT-PCR was performed using the QuantStudio 3 (Thermo Fisher Scientific) with PowerUp according to the manufacturer's protocol. TM SYBR TM The experiment was performed using Green Master Mix. Gene expression levels were calculated using C values, and GAPDH was used as an endogenous control. The results are shown in Figure 5. As shown in Figure 5, CD142 mRNA levels were reduced in the experimental group (iPSC-CD142#33), which were iPSC-derived pancreatic lineage cells in which the CD142 gene had been knocked out, compared to the control group (normal iPSCs), which were normal iPSC-derived pancreatic lineage cells on day 14 (D14) of differentiation (N=4).

[0168] <FACS(Flow cytometry analysis、フローサイトメトリー)> CD142 knockout (KO) in iPSC-derived pancreatic lineage cells was analyzed by FACS for cell level CD142 expression. Cultured cells were washed twice with phosphate buffered saline (PBS) and then detached using 0.05% trypsin-EDTA and centrifuged at 1,000 rpm for 5 minutes. Cells were suspended in 100 μl of FACS staining buffer, mixed with antibody, and incubated at 4°C for 1 hour. After washing twice with PBS, the cells were resuspended in 500 μl of PBS and analyzed by FACS. The results are shown in Figure 6.

[0169] Referring to Figure 6, the percentage of cells expressing CD142 (CD142 positive cell population, %) was reduced in the experimental group (iPSC-CD142#33), which were iPSC-derived pancreatic lineage cells in which the CD142 gene had been knocked out, compared to the control group (normal iPSCs), which were normal iPSC-derived pancreatic lineage cells on differentiation day 40 (D40).

[0170] [Experimental Example 4: Confirmation of CD142 gene knockout in iPSC (induced pluripotent stem cell) derived hepatocytes] <qrt-pcr> The knockout of CD142 in iPSC-derived hepatocytes was confirmed by analyzing the mRNA levels of the CD142 gene using qRT-PCR, as described above. The results are shown in Figure 7. As shown in Figure 7, CD142 mRNA levels were reduced in the experimental group (iPSC-CD142#33), which were iPSC-derived hepatocytes with the CD142 gene knocked out, compared to the control group (normal iPSCs), which were normal iPSC-derived hepatocytes at day 40 (D40) of differentiation.

[0171] <FACS(Flow cytometry analysis、フローサイトメトリー)> The CD142 knockout (KO) in iPSC-derived hepatocytes was analyzed for cell level CD142 expression using FACS in the same manner as above, and the results are shown in Figure 8.

[0172] Referring to Figure 8, the percentage of cells expressing CD142 (CD142 positive cell population, %) was reduced in the experimental group (iPSC-CD142#33), which were iPSC-derived hepatocytes with the CD142 gene knocked out, compared to the control group (normal iPSCs), which were normal iPSC-derived hepatocytes on day 40 (D40) of differentiation.

[0173] Example 5: Confirmation of CD142 protein levels in iPSC (induced pluripotent stem cell)-derived pancreatic lineage cells and hepatocytes <ELSIA(Enzyme-linked immunosorbent assay)> The amount of CD142 protein produced by knockout (KO) of CD142 in iPSC-derived pancreatic lineage cells and iPSC-derived hepatocytes was analyzed by ELISA using cell lysates.

[0174] CD142 was measured using cell lysates and cell culture supernatants of iPSC-derived pancreatic lineage cells and hepatocytes using a Tissue Factor Activity Assay Kit (Human, Colorimetric, Abcam (ab108906)). The experiment was carried out according to the protocol provided by the kit manufacturer. Absorbance was measured using a Multiscan TM FC Microplate Photometer (Thermo Scientific TM The measurements were performed at a wavelength of 405 nm using a (51119000)) instrument, and the results are shown in Figures 9 and 10.

[0175] As shown in Figure 9, it was confirmed that the amount of CD142 protein in the cell lysate was reduced in the experimental group (iPSC-CD142#33), which were iPSC-derived pancreatic lineage cells in which the CD142 gene had been knocked out, compared to the control group (normal iPSCs), which were normal iPSC-derived pancreatic lineage cells on differentiation day 14 (D14) (N=4).

[0176] Referring to Figure 10, it was confirmed that the amount of CD142 protein in the cell lysate of the experimental group (iPSC-CD142#33), which were iPSC-derived hepatocytes in which the CD142 gene had been knocked out, was reduced compared to the control group (normal iPSC), which were normal iPSC-derived hepatocytes on differentiation day 40 (D40) (N=4).

Claims

1. An artificially engineered mammalian cell containing an artificially engineered F3 gene, wherein the artificially engineered F3 gene is different from the F3 gene sequence of wild-type mammalian cells, the artificially engineered F3 gene contains one or more indels (insertions or deletions) within the nucleic acid sequence, and the expression level of CD142 on the surface of the artificially engineered mammalian cell is reduced compared to that of wild-type mammalian cells, characterized by the artificially engineered cell for intravascular transplantation.

2. Within the artificially engineered cell for intravascular transplantation, the mRNA transcribed from the artificially engineered F3 gene has a lower mRNA expression level or a different sequence compared to the mRNA expression level of the mRNA transcribed from the F3 gene of wild-type mammalian cells, characterized by the artificially engineered cell for intravascular transplantation according to Claim 1.

3. The indel is located within the protospacer-adjacent motif (PAM) sequence in the first, second, third, fourth, or sixth exon region of the F3 gene, or within a continuous 5-50 nucleotide sequence adjacent to the 5' or 3' end of the PAM sequence, characterized by the artificially engineered cell for intravascular transplantation according to Claim 1.

4. The mammalian cells are primary hepatocytes, primary pancreatic β cells, primary pancreatic islet cells, pancreatic progenitor cells, pluripotent stem cell (PSC)-derived hepatocytes, PSC-derived pancreatic β cells, PSC-derived pancreatic islet cells, PSC-derived pancreatic lineage cells, PSC-derived pancreatic progenitor cells, PSC-derived pancreatic organoids, PSC-derived pancreatic-liver organoids, induced pluripotent stem cell (iPSC)-derived hepatocytes, iPSC-derived pancreatic β cells, iPSC-derived pancreatic islet cells, iPSC-derived pancreatic lineage cells, iPSC-derived pancreatic progenitor cells, iPSC-derived pancreatic organoids, iPSC-derived pancreatic-liver organoids, hepatocytes induced from somatic cells (induced hepatocytes, iHeps) by a compound or gene manipulation, pancreatic islet cells induced from somatic cells by a compound or gene manipulation, or pancreatic β cells induced from somatic cells by a compound or gene manipulation, the artificially manipulated cells for intravascular transplantation according to claim 1.

5. The artificially manipulated intravascular transplantation cells according to claim 1, wherein the sequence of the artificially manipulated F3 gene does not include the sequence of SEQ ID NO:

1.

6. Cells differentiated or derived from artificially manipulated stem cells containing an artificially manipulated F3 gene, wherein the artificially manipulated F3 gene is different from the F3 gene sequence of cells differentiated or derived from wild-type stem cells, and the artificially manipulated F3 gene contains one or more indels within the nucleic acid sequence. Cells for intravascular transplantation that have been artificially manipulated, characterized in that the expression level of CD142 on the cell surface differentiated or derived from the artificially manipulated stem cells is decreased compared to cells differentiated or derived from wild-type stem cells. **Claim 7** The cells for intravascular transplantation that have been artificially manipulated according to claim 6, wherein the stem cells are induced pluripotent stem cells (iPSCs), embryonic stem cells, somatic cell nuclear transfer embryonic stem cells (Somatic cell nuclear transfer-PSC) or adult stem cells. **Claim 8** The cells for intravascular transplantation that have been artificially manipulated according to claim 6, wherein the cells differentiated or derived from the artificially manipulated stem cells are pancreatic β cells, hepatocytes, pancreatic progenitor cells, pancreatic islet cells, pancreatic lineage cells, pancreatic organoids or pancreatic-liver organoids. **Claim 9** A guide nucleic acid containing a guide sequence capable of targeting a target sequence of the F3 gene of mammalian cells or stem cells, or a nucleic acid encoding the same; and a composition for producing cells for intravascular transplantation having blood compatibility, comprising an editor protein or a nucleic acid encoding the same. **Claim 10** The composition The composition for producing cells for intravascular transplantation having blood compatibility according to claim 9, which contains the editor protein and the guide nucleic acid in the form of ribonucleoprotein (RNP). **Claim 11** The composition The composition for producing cells for intravascular transplantation having blood compatibility according to claim 9, which contains a nucleic acid encoding the editor protein and a nucleic acid encoding the guide nucleic acid in the form of one or more vectors. **Claim 12** The composition for producing cells for intravascular transplantation having blood compatibility according to claim 11, wherein the vector is selected from the group consisting of plasmids, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, vaccinia viruses, poxviruses and herpes simplex viruses. **Claim 13** (1) Introducing a composition for producing cells for intravascular transplantation having blood compatibility, which contains a guide nucleic acid capable of targeting a target sequence of the F3 gene or a nucleic acid encoding the same, and an editor protein or a nucleic acid encoding the same, into isolated mammalian cells or isolated stem cells; and (2) A method for producing cells for intravascular transplantation with blood compatibility, comprising the step of editing the F3 gene such that the expression or activity of CD142 is reduced or suppressed by generating an indel in the target sequence of the F3 gene located within the genome of the isolated mammalian cells or the isolated stem cells.

14. The method for producing cells for intravascular transplantation with blood compatibility according to claim 13, wherein the composition contains the nucleic acid encoding the editor protein and the nucleic acid encoding the guide sequence in the form of one or more vectors.

15. The method for producing cells for intravascular transplantation with blood compatibility according to claim 13, characterized in that an indel occurs in the target sequence by contacting the target sequence of the F3 gene located within the genome of the isolated mammalian cells with a CRISPR / Cas9 complex containing 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, comprising the artificially engineered cells for intravascular transplantation according to any one of claims 1 to 8 as an active ingredient.

17. A pharmaceutical composition for preventing or treating liver diseases, comprising the artificially engineered cells for intravascular transplantation according to any one of claims 1 to 8 as an active ingredient.

18. The liver disease is cirrhosis; acute-on-chronic liver failure (ACLF); drug- or toxin-induced liver failure; congenital metabolic liver disease; Crigler-Najjar syndrome type 1; familial hypercholesterolemia; factor VII deficiency; factor VIII deficiency (hemophilia A); phenylketonuria (PKU); glycogen storage disease type I; infantile Refsum disease; progressive familial intrahepatic cholestasis type 2; hereditary tyrosinemia type 1; urea cycle disorder; acute liver failure; acute drug-induced liver failure; virus-induced acute liver failure; idiopathic acute liver failure; acute liver failure induced by poisonous mushroom poisoning; postoperative acute liver failure; acute liver failure due to acute fatty liver of pregnancy; alcoholic hepatitis; or hepatic encephalopathy. The pharmaceutical composition for treating or preventing liver diseases according to claim 17.

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

20. A pharmaceutical composition for preventing or treating diabetes, comprising, as an active ingredient, the artificially manipulated intravascular transplantation cells according to any one of Claims 1 to 8.

21. The diabetes is type 1 diabetes, and the pharmaceutical composition for preventing or treating diabetes according to Claim 20.

22. The pharmaceutical composition is administered through the portal vein, and the pharmaceutical composition for preventing or treating diabetes according to Claim 20.

23. A composition for cell transplantation or biological tissue regeneration, comprising, as an active ingredient, the artificially manipulated intravascular transplantation cells according to any one of Claims 1 to 8.

24. The composition for cell transplantation or biological tissue regeneration is a cell therapy agent composition, a gene therapy agent composition, a tissue engineering therapy agent composition, an immunotherapy agent composition, or a cancer prevention or treatment agent composition, and the composition for cell transplantation or biological tissue regeneration according to Claim 23.

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