Mesenchymal stem cell with blood compatibility as well as preparation method and application thereof

Abstract

The present invention provides a mesenchymal stem cell having blood compatibility, and a preparation method and use thereof, the mesenchymal stem cell inducing exon skipping of an exon 5 by performing gene modification on the exon 5 of an F3 gene, thereby increasing the expression of a selective splicing tissue factor (asTF), and improving the blood compatibility of the mesenchymal stem cell. The present invention relates to a method for inhibiting thrombosis by reducing or inhibiting the expression or activity level of a full-length tissue factor (flTF), which is a blood coagulation promoter, and thereby inhibiting thrombosis.

Description

TECHNICAL FIELD

[0001] The present application provides a mesenchymal stem cell having hemocompatibility capable of intravascular administration, a preparation method thereof, and a use thereof, the mesenchymal stem cell induces exon skipping of exon 5 by gene modification of exon 5 of F3 gene, thereby increasing expression of alternative splicing factor (asTF) and reducing or inhibiting expression or activity level of full-length Tissue Factor (flTF) as a blood coagulation initiator, thereby inhibiting thrombosis reaction. BACKGROUND

[0002] The mesenchymal stem cell has self-renewal ability and multi-potency, is easy to ensure a stem cell line due to a plurality of precursor cells, and has an outstanding advantage of genetic stability as compared to omnipotent stem cells such as embryonic stem cells. Also, it is developed as a cell therapeutic agent for cartilage regeneration, myocardial infarction treatment, treatment of graft-versus-host disease, etc. due to its anti-inflammatory ability and immune-modulation ability.

[0003] The mesenchymal stem cell used as a cell therapeutic agent is administered intravenously, intravascularly, or directly to a lesion. When the mesenchymal stem cell is administered to an animal by intravascular injection, although most of it is accumulated in the lung, a part of it can move to various organs such as inflammatory sites, lymphocytes, liver, bone, brain, etc. The therapeutic mechanism of action of the mesenchymal stem cell is known to be secretion of paracrine factor, and immune-modulation is known to be the main therapeutic mechanism. Therefore, the mesenchymal stem cell administered intravascularly into a vein or an artery, etc. can cause immune-modulation in various organs in the body, so intravascular administration can be a preferred administration route.

[0004] However, it is known that unlike existing intravascularly administered bone marrow (BM)-derived mesenchymal stem cells, adipose tissue-derived, umbilical cord blood / umbilical cord-derived, perinatal tissue-derived mesenchymal stem cells overexpress tissue factor (tissue factor, CD142, TF, factor III, blood coagulation factor III, hereinafter referred to as CD142), which has a risk of inducing venous thromboembolism (VTE) in which thrombosis and embolism occur in blood vessels because it can cause coagulation of blood, and thus cannot be administered intravenously. Also, the dose of bone marrow-derived mesenchymal stem cells is limited when administered intravenously. In a hemocompatibility experiment in an in vitro / in vivo environment with human-derived mesenchymal stem cells as the subject, perinatal tissue (PT)-derived, umbilical cord blood / umbilical cord (UCB / Umbilical cord, UC)-derived mesenchymal stem cells, and adipose tissue (AT)-derived mesenchymal stem cells showed a tendency to cause strong thrombosis due to high expression of CD142.

[0005] Further, perinatal tissue-derived, umbilical cord blood / umbilical cord (UCB / Umbilical cord, UC)-derived mesenchymal stem cells, and adipose tissue (AT)-derived mesenchymal stem cells expressing CD142 not only generated thrombus due to acute blood-mediated inflammatory reaction (IBMIR) when administered intravenously, but also showed a tendency to inhibit engraftment of mesenchymal stem cells and instead increase immune response (Guido Moll et al., Intravascular Mesenchymal Stromal / Stem Cell Therapy Product Diversification: Time for New Clinical Guidelines, Trends in Molecular Medicine, VOLUME 25, ISSUE 2, P149-163, FEBRUARY 01, 2019).

[0006] An animal body has a blood coagulation system as a complex biological tool for preventing bleeding in order to minimize blood loss caused by tissue damage. The blood coagulation system is a successive amplification reaction process called a cascade reaction to an initial stimulus, and is composed of an intrinsic pathway directly induced by a stimulus and a unique extrinsic pathway. Among them, various blood coagulation factors are associated.

[0007] 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 CD142 binds to factor VII or VIIa, factor IX and factor X related to activation of prothrombin to thrombin are activated to generate thrombin in the coagulation process, and thrombin activates fibrinogen to fibrin to form a thrombus. Also, CD142 activates immune cells to induce the activity of neutrophils, platelets, and monocytes, induce the secretion of various cytokines, and activate the intrinsic immune system.

[0008] CD142 exists in two subtypes of full-length tissue factor (flTF) and alternative splicing factor (asTF). Full-length tissue factor (flTF) is a complete transmembrane protein, participates in the formation of a complex with coagulation factor VIIa, and is expressed as a whole on the cell membrane to induce blood coagulation. The N-terminal 1 to 219 amino acid residues of full-length tissue factor (flTF) are located outside the cell membrane, the transmembrane domain consists of 220-242 amino acids, is fixed on the cell membrane, and is tightly bound to phospholipids, and the C-terminal region in the cytoplasm consists of 243-263 amino acids, participates in signal transduction. Alternative splicing tissue factor (asTF) expressed due to the deletion of exon 5 by splicing is known to be secreted because it does not contain a transmembrane domain, and does not participate in blood coagulation. Alternative splicing tissue factor (asTF) contains 206 amino acids, of which 1-166 amino acids are identical to full-length tissue factor (flTF), and 167-206 amino acids are specific C-termini, do not contain a transmembrane domain, are soluble, and can be secreted into the circulatory system.

[0009] In the analysis of the characteristics of commonly used multipotent adult stem cells, the expression of differentiation phenotypes such as CD105, CD73, and CD90 was confirmed, and the absence of expression of CD45, CD34, CD14, and HLA-DR was confirmed, but the expression of tissue factor marker CD142 was rarely confirmed. However, recently, there have been many reports that the expression rate of CD142 in adipose tissue-derived mesenchymal stem cells (AT-MSCs), perinatal tissue-derived stem cells (PT-MSCs) is significantly higher than that in bone marrow-derived mesenchymal stem cells (BM-MSCs), and it is known that the expression increases postnatally depending on the culture conditions (passaging, media type, etc.). SUMMARY

[0010] PROBLEMS TO BE SOLVED BY THE INVENTION

[0011] The present application relates to a mesenchymal stem cell having hemocompatibility, a method for preparing the same, and a cell therapeutic agent using the same, and more particularly to a mesenchymal stem cell having hemocompatibility, in which the expression level of full-length tissue factor is reduced and the expression level of alternative splicing tissue factor is increased on the surface of the stem cell compared to a wild-type mesenchymal stem cell, thereby improving hemocompatibility, a method for preparing the same, and a cell therapeutic agent using the same.

[0012] MEANS FOR SOLVING THE PROBLEMS

[0013] To achieve the above object, the present application provides an artificially engineered mesenchymal stem cell,

[0014] The artificially engineered mesenchymal stem cell includes an artificially engineered F3 gene, and the artificially engineered F3 gene includes the induction of exon skipping of exon 5 by genetic modification of exon 5 of the F3 gene, and the artificially engineered mesenchymal stem cell has reduced expression levels of full-length tissue factor and increased expression levels of alternative splicing tissue factor on the surface of the stem cell compared to a wild-type mesenchymal stem cell, thereby improving hemocompatibility.

[0015] Further, the present application provides a composition for preparing a mesenchymal stem cell having hemocompatibility, which includes a guide nucleic acid or a nucleic acid encoding the same, the guide nucleic acid including a guide sequence capable of targeting a target sequence of exon 5 of an F3 gene of a mammalian cell; and

[0016] an editing protein or a nucleic acid encoding the same.

[0017] Also, the present application provides a method for preparing mesenchymal stem cells having blood compatibility, the method comprising: step (1) introducing the composition for preparing mesenchymal stem cells having blood compatibility into isolated mesenchymal stem cells; and step (2) genetically modifying exon 5 of the F3 gene located in the genome of the isolated mesenchymal stem cells, thereby editing exon 5 of the F3 gene to induce exon skipping of exon 5.

[0018] Also, the present application provides a cell therapeutic agent for vascular administration, which comprises artificially engineered mesenchymal stem cells as an effective ingredient to have the blood compatibility.

[0019] Also, the present application provides a composition for cell transplantation or biological tissue regeneration, which comprises artificially engineered mesenchymal stem cells as an effective ingredient to have the blood compatibility.

[0020] Effects of Invention

[0021] The artificially engineered mesenchymal stem cells of the present application induce exon skipping of exon 5 by artificially modifying exon 5 of the F3 gene encoding blood coagulation initiator CD142 through a gene editing technique, thereby converting the F3 gene transcriptome into a selective splicing tissue factor (asTF) transcriptome that is a splice variant of a full-length tissue factor (flTF) and expressing it. Accordingly, the expression of the full-length tissue factor (flTF) is reduced while the expression of the selective splicing tissue factor (asTF) is increased, thereby making it possible to inhibit the blood coagulation mechanism. Thus, it becomes possible to intravenously administer perinatal tissue (PT)-derived, umbilical cord blood / umbilical cord-derived mesenchymal stem cells, and adipose tissue-derived mesenchymal stem cells, which have been difficult to intravenously administer due to the risk of inducing venous thromboembolism in which thrombosis and embolism occur in veins, due to overexpression of CD142 on the surface of stem cells.

[0022] Also, it is possible to increase the administration dose of bone marrow-derived mesenchymal stem cells, which are currently used for intravenous administration routes but have a limited administration dose. At the same time, it is possible to improve the engraftment of mesenchymal stem cells and increase the efficacy of mesenchymal stem cells by reducing inflammatory reactions. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 A target position of an sgRNA of the F3 gene designed in Example 1 is shown.

[0024] Figure 2 A graph showing the results of determining the insertion / deletion efficiency of each target sequence of each guide RNA by a targeted deep sequencing method.

[0025] Figure 3 A graph showing the results of CD142 expression level of normal umbilical cord / umbilical cord-derived stem cells (UC-WT), normal umbilical cord / umbilical cord-derived stem cells (4D only) which were subjected to electroporation only without gene manipulation, umbilical cord / umbilical cord stem cells (sgSHS231 re#2) edited for SHS231 as a safe harbor gene, umbilical cord / umbilical cord-derived stem cells (sgCD142#33) edited for F3 exon 3 with one gRNA, umbilical cord / umbilical cord-derived stem cells (sgF3 ΔEx5 #2+5, #2+10, #2+14, #2+16, #3+14, #3+15, #3+16) edited for F3 exon 5 with two gRNA combinations, by the proportion of cells showing CD142 expression (CD142 positive cell population, %) and median fluorescence intensity (MFI).

[0026] Figure 4 A graph showing the results of the exon 5 deletion region of umbilical cord / umbilical cord-derived stem cells edited for F3 exon 5 with two sgRNA combinations (sgF3 ΔEx5 #2+5, sgF3 ΔEx5 #2+10) confirmed by BT-seq.

[0027] Figure 5 A graph showing the results of full-length tissue factor (flTF) mRNA and alternatively spliced tissue factor (asTF) mRNA expression of normal umbilical cord / umbilical cord-derived stem cells (UC-WT), normal umbilical cord / umbilical cord-derived stem cells (4D only) which were subjected to electroporation only without gene manipulation, umbilical cord / umbilical cord stem cells (sgSHS231 re#2) edited for SHS231 as a safe harbor gene, umbilical cord / umbilical cord-derived stem cells (sgF3 Ex3 #33) edited for exon 3 with SEQ ID NO: 22 as a target sequence as a positive control group, by qRT-PCR analysis.

[0028] Figure 6 A graph showing the results of full-length tissue factor (flTF) expression of normal umbilical cord / umbilical cord-derived stem cells (UC-WT), umbilical cord / umbilical cord-derived stem cells (sgF3 ΔEx5 #2+5, #2+10) edited for F3 exon 5 with two gRNA combinations, by Western Blot analysis.

[0029] Figure 7 A chart showing the activity of cell lysate and conditioned media (cm) of normal umbilical cord / umbilical cord-derived stem cell (UC-WT), umbilical cord / umbilical cord-derived stem cell edited for exon 3 using SEQ ID NO: 22 as a target sequence (sgF3 Ex3 #33) as a positive control group, umbilical cord / umbilical cord-derived stem cell edited for F3 exon 5 using two gRNA combinations (sgF3 ΔEx5 #2+5, #2+10, #3+14) by tissue factor concentration.

[0030] Figure 8 A chart showing the clotting reaction time (R-time: time required for the thrombus amplitude to reach 2 mm from the start of analysis), thrombus dynamics (K-time: time required for the amplitude to reach 20 mm after the thrombus amplitude reaches 2 mm, alpha-angle: inclination of a straight line connecting R-time to 20 mm), thrombus formation strength, and thrombus maximum amplitude (MA: absolute thrombus strength) of wild-type umbilical cord blood-derived mesenchymal stem cell (WT), umbilical cord / umbilical cord-derived stem cell edited for exon 3 using SEQ ID NO: 22 as a target sequence (sgF3 #33) measured using a TEG 6s device.

[0031] Figure 9 A chart showing the PDT and PDL in each passage of normal umbilical cord / umbilical cord-derived stem cell (UC-WT), normal umbilical cord / umbilical cord-derived stem cell subjected to transfection only without gene manipulation (Mock), umbilical cord / umbilical cord-derived stem cell edited for exon 3 using SEQ ID NO: 22 as a target sequence (sgF3 Ex3 #33), and umbilical cord / umbilical cord-derived stem cell edited for F3 exon 5 using two gRNA combinations (sgF3 ΔEx5 #3+14) during long-term culture.

[0032] Figure 10A graph showing the proportion of cells expressing CD142 (CD142-positive cell population, %) and the median fluorescence intensity (MFI) for each passage of normal umbilical cord / umbilical cord-derived stem cells (UC-WT), normal umbilical cord / umbilical cord-derived stem cells subjected to transfection only without genetic manipulation (Mock), umbilical cord / umbilical cord-derived stem cells edited for exon 3 using SEQ ID NO: 22 as the target sequence (sgF3 Ex3 #33), and umbilical cord / umbilical cord-derived stem cells edited for F3 exon 5 using a combination of two gRNAs (sgF3ΔEx5 #3+14) during long-term culture.

[0033] Figure 11 A heat map of data for confirming the pattern of changes in intracellular mRNA after F3 knockout (KO) (only data with a fold change cut-off of 2.0 or more between the two groups were analyzed).

[0034] Figure 12 A heat map of data for confirming the pattern of changes in intracellular cytokines after F3 knockout (KO) (only data with a fold change cut-off of 2.0 or more between the two groups were analyzed). DETAILED DESCRIPTION

[0035] Hereinafter, the present application will be described in detail.

[0036] The term "about" used in the present specification means an amount, level, value, number, frequency, percentage, size, magnitude, quantity, weight, or length that varies by about 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 from a reference amount, level, value, number, frequency, percentage, size, magnitude, quantity, weight, or length.

[0037] The term "artificially modified" used in the present specification is a term used to distinguish from substances, molecules, etc. having structures that already exist in nature, and means applying artificial modification to the above-mentioned substances, molecules, etc. For example, "artificially modified gene" means a gene to which artificial modification is applied to the structure of a gene that exists in nature. Also, the above-mentioned term includes all meanings that can be recognized by those skilled in the art, and can be understood appropriately according to the context.

[0038] The term "wild type" as used in the present specification refers to a gene comprising a naturally occurring base sequence and a protein expressed from the gene having a normal functional property. The wild type gene has a form that is natural or has not been artificially mutated, and can be observed most frequently in a group. In the present specification, if the term "wild type" is used in contrast to an artificially modified gene and / or an artificially modified cell, it should be interpreted as the meaning of a "non-artificially modified" gene comprising a naturally occurring base sequence and a cell having the same, corresponding to the artificially modified gene and / or the artificially modified cell. Also, the above term includes all meanings that can be recognized by those skilled in the art, and can be appropriately understood according to the context.

[0039] The term "editing" as used in the present specification refers to gene editing, which means a genetic engineering that changes a nucleotide sequence of a target polynucleotide by introducing a deletion, an insertion, or a base substitution into the polynucleotide sequence, including knock-out, knock-down, and knock-in.

[0040] The term "knock-out" or "knocked-out gene" as used in the present specification refers to a mutation or an artificial modification in a wild type gene, as a result of which a protein expressed from the wild type gene cannot be produced through a transcription and / or a translation process. For example, a cell comprising a knocked-out gene A can not express mRNA and / or a protein expressed from the wild type gene A. The cell comprising a knocked-out gene A can be a cell in which only one of the gene A present in the cell is knocked out, or a cell in which two or more genes are knocked out. Also, the above term includes all meanings that can be recognized by those skilled in the art, and can be appropriately understood according to the context.

[0041] The term "knock-down" or "knocked-down gene" as used in the present specification refers to a mutation or an artificial modification in a wild type gene, as a result of which a substance is expressed in an amount less than that of the wild type gene. For example, a cell comprising a knocked-down gene A can express mRNA in an amount less than that of mRNA expressed from the wild type gene A. For another example, a cell comprising a knocked-down gene A can express a protein in an amount less than that of a protein expressed from the wild type gene A. The cell in which the gene A is knocked down can be a cell in which only one of the gene A present in the cell is knocked down, or a cell in which two or more genes are knocked down. Also, the above term includes all meanings that can be recognized by those skilled in the art, and can be appropriately understood according to the context.

[0042] In this specification, "reduced expression" means expression of mRNA and / or protein at levels lower than those measured in wild-type cells. This reduction can be approximately 5%, 10%, 15%, 20%, 30%, 50%, 60%, 70%, or 100% lower than that of cells without genetic modification or wild-type cells.

[0043] The terms "reduced activity" or "reduced activity" as used in this specification can refer to a relative reduction in the activity of a protein or enzyme when its activity is measured. Specifically, "reduced activity" or "reduced activity" refers to a protein or enzyme activity at a lower level than that of the given progenitor or wild-type cells.

[0044] In this specification, "stem cell" refers to any undifferentiated cell with the ability to differentiate into various body tissue cells; that is, a broad concept of stem cell-like characteristics. In this context, the aforementioned stem cells can be induced pluripotent stem cells, embryonic stem cells, and adult stem cells. Furthermore, these cells can be of human origin, but are not limited to this.

[0045] In this specification, "mesenchymal stem cells" refers to undifferentiated stem cells isolated from human or mammalian tissues, which can be derived from a variety of tissues. In particular, they can be umbilical cord-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, muscle-derived mesenchymal stem cells, neural-derived mesenchymal stem cells, skin-derived mesenchymal stem cells, amniotic membrane-derived mesenchymal stem cells, amniotic fluid-derived mesenchymal stem cells, perinatal tissue-derived mesenchymal stem cells, or placental-derived mesenchymal stem cells. Techniques for isolating stem cells from these tissues are well known in the art.

[0046] As used in this instruction manual, "hemocompatibility" means that contact with blood does not induce hemolysis, platelet adhesion, fibrin formation based on platelet activation and complement activation, thrombotic reactions, or embolism.

[0047] This invention modifies exon 5 of the F3 gene to artificially induce exon skipping in the F3 gene transcriptome, thereby converting the F3 gene transcriptome into an alternatively spliced ​​tissue factor (asTF) transcriptome, which is a splice variant of full-length tissue factor (flTF), and expressing it.

[0048] Therefore, the present invention provides artificially modified mesenchymal stem cells in which blood compatibility is improved by increasing the expression of alternatively spliced ​​tissue factor (asTF) and decreasing the expression of full-length tissue factor (flTF) in two subtypes of tissue factor generated by naturally occurring exon 5 exon skipping.

[0049] Mesenchymal stem cells that reduce or inhibit the expression or activity of full-length tissue factor (flTF) are mesenchymal stem cells that inhibit the thrombus coagulation mechanism by reducing or inhibiting the expression or activity of full-length tissue factor (flTF), a cell membrane glycoprotein that plays an important role in the initiation stage of extrinsic coagulation. When these mesenchymal stem cells that reduce or inhibit the expression or activity of full-length tissue factor (flTF) are administered intravenously as cell therapy agents, they can exhibit improved blood compatibility compared to wild-type mesenchymal stem cells, artificially modified mesenchymal stem cells or antibodies using existing technologies, or CD142-low-expressing mesenchymal stem cells screened by cell separation devices or magnetic beads.

[0050] Specifically, the present invention provides blood-compatible artificially modified mesenchymal stem cells, characterized in that the expression or activity of full-length tissue factor (flTF) mRNA and / or full-length tissue factor (flTF) is reduced or inhibited by artificially modifying exon 5 of the F3 gene of the mesenchymal stem cells, thereby reducing blood clotting reaction when administered intravascularly.

[0051] In one embodiment of the present invention, the aforementioned artificial modification of the F3 gene includes the induction of exon skipping of exon 5 of the F3 gene through artificial modification of the exon 5 gene.

[0052] In this invention, the "artificial modification" or "artificial alteration" of the gene's nucleic acid sequence can be achieved through modification of the nucleic acid sequence constituting the gene or through chemical modification of a single base. This can be achieved through mutation, substitution, deletion, or insertion of one or more bases into the gene, either partially or entirely, or through gene editing technologies such as the CRISPR enzyme system. As an example, the artificial modification of the gene's nucleic acid sequence can be achieved through non-homologous end joining (NHEJ) or homology-directed repair (HDR) mechanisms.

[0053] In one embodiment of the present invention, the gene modification of exon 5 of the F3 gene includes the deletion or destruction of exon 5.

[0054] In one embodiment of the present invention, the gene modification of exon 5 of the F3 gene includes the deletion of the nucleotide sequence containing the splicing acceptor immediately preceding exon 5.

[0055] The gene modification of exon 5 of the F3 gene in this invention causes exon 5, which is the target exon, to undergo artificial skipping during splicing when the pre-mRNA of the F3 gene transcriptome matures into mRNA.

[0056] As an example, the aforementioned artificially modified mesenchymal stem cells can be artificially modified mesenchymal stem cells with exon 5 of the F3 gene knocked out.

[0057] In this specification, "non-homologous end joining (NHEJ)" is a method of repairing or restoring double-stranded DNA damage by having the two ends of a cleaved double or single strand join together. Typically, the two compatible ends formed by the double-stranded damage (e.g., cleavage) are brought into repeated and frequent contact to achieve complete joining of the two ends, thereby restoring the damaged double strand.

[0058] In the repair of damaged genes or nucleic acids using non-homologous end joining (NHEJ), a portion of the nucleic acid sequence may be "inserted" and / or "deleted" (or "InDel") at the NHEJ repair site. The gene with the insertion / deletion does not have the same sequence as the wild-type gene. Such insertions and / or deletions alter the gene's reading frame, creating frameshifted transcriptomic mRNA, which, as a result, loses its original function through nonsense-mediated decay or failure to synthesize normal protein. Alternatively, while maintaining the reading frame, mutations may cause a significant amount of sequence insertion or deletion, thus disrupting protein function. For example, in the case of insertions / deletions in transcriptional regulatory regions such as promoter or enhancer regions of a gene, mRNA may not be transcribed or the amount of transcription may be reduced, potentially leading to no protein expression or reduced protein expression. Alternatively, utilizing the mutation mechanism of NHEJ, it may be possible to use only the deletion of a portion of the sequence motif without requiring the generation of a specific final sequence. For example, by using two or more guide RNAs that target the intron regions of the 5' and 3' portions of a specific exon to induce double-strand cleavage in each intron region, and by using NHEJ, if only a portion of the exon of the gene is deleted, the main function of the protein can be maintained because the other parts are expressed normally.

[0059] In the case of such NHEJ, gene editing technology can be used to specifically knock out or knock down the targeted gene.

[0060] For example, CRISPR enzymes such as Cas9 or Cpf1, which are gene-editing scissors, can be used to cut double strands or two single strands of a target gene or target nucleic acid. The damaged double strands or two single strands of the target gene or target nucleic acid can be used to generate insertions / deletions through NHEJ, thereby inducing specific knockout or knockdown of the target gene or nucleic acid.

[0061] In one embodiment of the present invention, the F3 gene of the artificially modified mesenchymal stem cells described above may contain one or more insertions / deletions within the nucleic acid sequence of exon 5.

[0062] In one embodiment of the present invention, the above-described artificially modified mesenchymal stem cells may not exhibit expression of full-length tissue factor (flTF) mRNA.

[0063] In one embodiment of the present invention, the expression level of full-length tissue factor (flTF) mRNA transcribed from the artificially modified F3 gene of the aforementioned artificially modified mesenchymal stem cells can be lower than the expression level of full-length tissue factor (flTF) mRNA transcribed from the F3 gene of wild-type mesenchymal stem cells.

[0064] In one embodiment of the present invention, the expression level of alternative splicing tissue factor (asTF) mRNA transcribed from the artificially modified F3 gene of the aforementioned artificially modified mesenchymal stem cells can be higher than the expression level of alternative splicing tissue factor (asTF) mRNA transcribed from the F3 gene of wild-type mesenchymal stem cells.

[0065] In one embodiment of the present invention, the F3 mRNA sequence of the artificially modified mesenchymal stem cells described above may be different from that of wild-type mesenchymal stem cells.

[0066] In one embodiment of the present invention, the artificially modified mesenchymal stem cells described above exhibit reduced expression or activity of full-length tissue factor (flTF) on their cell surface compared to wild-type mesenchymal stem cells. Therefore, the artificially modified mesenchymal stem cells of the present invention can reduce or eliminate the function of full-length tissue factor (flTF).

[0067] That is, in the above-mentioned artificially modified mesenchymal stem cells, the expression or activity of full-length tissue factor (flTF) can be reduced by more than 20%, more than 30%, more than 40%, more than 50%, more than 55%, more than 60%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or more than 100% compared with wild-type mesenchymal stem cells.

[0068] In one embodiment of the present invention, the artificially modified mesenchymal stem cells described above, compared with wild-type mesenchymal stem cells, exhibit increased expression or activity of selectively spliced ​​tissue factor (asTF) on their cell surface. Therefore, the artificially modified mesenchymal stem cells of the present invention can reduce or eliminate the function of full-length tissue factor (flTF).

[0069] That is, in the above-mentioned artificially modified mesenchymal stem cells, the expression or activity of selective splicing tissue factor (asTF) can be increased by approximately 20% or more, approximately 30% or more, approximately 40% or more, approximately 50% or more, approximately 55% or more, approximately 60% or more, approximately 70% or more, approximately 75% or more, approximately 80% or more, approximately 85% or more, approximately 90% or more, approximately 95% or more, or approximately 100% compared with the expression or activity of wild-type mesenchymal stem cells.

[0070] This invention provides a method for preparing blood-compatible artificially modified mesenchymal stem cells. The method includes a step of editing exon 5 of the F3 gene of mesenchymal stem cells to induce exon skipping of exon 5.

[0071] The artificial modification of exon 5 of the F3 gene mentioned above can be used as an example of gene editing technology.

[0072] As an example, the gene editing technology described above can utilize transcription activator-like effector nucleases (TALENs), zinc-finger nucleases, or CRISPR enzyme systems derived from regularly interspaced short palindromic repeats (CRISPR) as part of a microbial immune system, but is not limited to these.

[0073] In connection with the above TALEN, the full text of International Patent Publication No. WO2012 / 093833 or U.S. Patent Publication No. 2013-0217131 is included in this specification as reference. In connection with the zinc finger nucleases (ZFNs) described above, the following references are included in this specification: Beerliet 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 Nos. 7,888,121, 8,409,861, 6,479,626, 6,903,185, and 7,153,949.

[0074] The aforementioned "CRISPR enzyme system" consists of guide nucleic acid and / or editing protein.

[0075] "Guide nucleic acid" refers to a nucleic acid that recognizes a target nucleic acid, target gene, or target chromosome and can interact with editing proteins. In this case, the guide nucleic acid can form a complementary binding with a portion of the nucleotides of the target nucleic acid, target gene, or target chromosome.

[0076] The aforementioned guide nucleic acid can be a DNA-specific guide RNA, DNA encoding the aforementioned guide RNA, or a DNA / RNA mixture.

[0077] The aforementioned guide nucleic acid can be a guide RNA. As an example, a "guide RNA" can be transcribed in vitro, particularly from an oligonucleotide double strand or plasmid template. Another example is that the aforementioned guide RNA can be encoded in the form of a vector, which can be delivered into cells and transcribed from a vector in an ex vivo or in vivo environment, but is not limited to this.

[0078] The design and structure of the aforementioned 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 text of the aforementioned patents is included in this specification as reference material for this invention.

[0079] The aforementioned guide nucleic acid may include a backbone sequence portion and a guide sequence portion. The backbone sequence portion is the part that interacts with the Cas protein, forming a complex (ribonucleoprotein (RNP)) by binding the Cas protein to the guide nucleic acid. Typically, the backbone sequence portion includes a portion of the tracrRNA and crRNA sequences, and the backbone sequence is determined depending on which Cas protein is used.

[0080] The aforementioned guide sequence is a nucleotide sequence portion capable of binding complementary to a portion of the double-stranded sequence of the target gene or nucleic acid. As a nucleotide sequence portion that can be artificially modified, it depends on the desired target nucleotide sequence. In this case, the guide sequence can be a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% complementarity to the guide nucleic acid binding sequence of the target gene or nucleic acid, or having complete complementarity. The guide sequence can be a sequence contained within a guide domain of the guide nucleic acid.

[0081] The aforementioned guide sequence portion can be contained within crRNA. As an example, the aforementioned guide nucleic acid can be a dual RNA containing two RNAs, namely, a dual RNA containing crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) as structural elements.

[0082] As another example, the aforementioned guide nucleic acid can be a sgRNA (single-chain guide RNA) in the form of crRNA linked to the main part of tracrRNA.

[0083] The target sequence described above is a nucleotide sequence of a specified length present within the target gene or target nucleic acid. Specifically, it can be a portion of a nucleotide sequence within a target region that is divided into a regulatory region, a coding region (CDS, coding sequence), or a non-coding region (UTR, untranslated region) of the target gene, or it can be a portion of a nucleotide sequence selected from one or more of the aforementioned target regions. The target sequence described above can be a target of a guide nucleic acid-editing protein complex (RNP).

[0084] In one embodiment of the present invention, the target sequence may be a sequence contained in exon 5 of the wild-type F3 gene.

[0085] In one embodiment of the present invention, the target sequence may be one or more sequences selected from SEQ ID NO: 1 to 21.

[0086] The target sequence mentioned above is the nucleotide sequence surrounding the protospacer-adjacent motif (PAM) sequence that is recognized by the edited protein. It may contain all or part of the PAM, but is not limited to this.

[0087] Target sequences can be used in terms that simultaneously represent two types of nucleotide sequence information. For example, in the case of a targeted gene, a target sequence can represent the sequence information of the transcribed strand of the target gene DNA, or it can represent the nucleotide sequence information of the non-transcribed strand.

[0088] The target sequence contains either a guide nucleic acid binding sequence or a guide nucleic acid non-binding sequence. A "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 bind complementaryally 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 target gene or nucleic acid, that is, depending on the object of gene manipulation or editing. Guide nucleic acids can be designed in a variety of ways based on the target gene or target nucleic acid.

[0089] A "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 can bind complementaryally to the guide sequence contained in the guide domain of the guide nucleic acid. Furthermore, as a nucleotide sequence complementary to the guide nucleic acid binding sequence, the guide nucleic acid non-binding sequence can bind complementaryly to the guide nucleic acid binding sequence. The guide nucleic acid binding sequence, as a part of the target sequence, can be one of two nucleotide sequences in the target sequence that have two distinct sequence orders; that is, one of two nucleotide sequences capable of complementary binding. In this case, the guide nucleic acid non-binding sequence can be any remaining nucleotide sequence in the target sequence other than the guide nucleic acid binding sequence.

[0090] The guide nucleic acid binding sequence can be the target sequence, that is, it can be a nucleotide sequence selected from 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 can be a nucleotide sequence other than the guide nucleic acid binding sequence in the target sequence, that is, it can be the remaining nucleotide sequence other than the nucleotide sequence selected from the nucleotide sequence identical to the transcribed strand and the nucleotide sequence identical to the non-transcribed strand.

[0091] The guide nucleic acid binding sequence can be the same length as the target sequence. The guide nucleic acid non-binding sequence can be the same length as either the target sequence or the guide nucleic acid binding sequence. The guide nucleic acid binding sequence can be 5 to 50 nucleotides long.

[0092] As a specific example, the guide nucleic acid binding sequence can be 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. The guide nucleic acid non-binding sequence can be 5 to 50 nucleotides.

[0093] As a specific example, the aforementioned guide nucleic acid non-binding sequence 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.

[0094] The guide nucleic acid binding sequence can partially or completely bind 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.

[0095] The aforementioned guide nucleic acid binding sequence can be a nucleotide sequence complementary to the guide sequence contained in the guide domain of the guide nucleic acid. For example, it can be a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% complementarity or complete complementarity.

[0096] As an example, the aforementioned guide nucleic acid binding sequence may have or contain one to eight nucleotide sequences that are not complementary to the guide sequence contained in the guide domain of the guide nucleic acid.

[0097] The guide nucleic acid non-binding sequence can 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 can be the same as the length of the guide sequence. As an example, the guide sequence can be designed based on a sequence that is homologous to the guide nucleic acid non-binding sequence.

[0098] The aforementioned guide nucleic acid non-binding sequence can be a nucleotide sequence that is homologous to the guide sequence contained in the guide domain of the guide nucleic acid. For example, it can be a nucleotide sequence with at least 70%, 75%, 80%, 85%, 90%, or 95% homology or with complete homology.

[0099] As an example, the aforementioned guide nucleic acid non-binding sequence may have or contain one to eight nucleotide sequences that are not homologous to the guide sequence contained in the guide domain of the guide nucleic acid. The guide nucleic acid non-binding sequence may bind complementary to the guide nucleic acid binding sequence, and the length of the aforementioned guide nucleic acid non-binding sequence may be the same as the length of the guide nucleic acid binding sequence.

[0100] The aforementioned guide nucleic acid non-binding sequence can be a nucleotide sequence complementary to the guide nucleic acid binding sequence, for example, it can be a nucleotide sequence with at least 90% or 95% complementarity or complete complementarity.

[0101] As an example, the aforementioned guide nucleic acid non-binding sequence may have or contain one or two nucleotide sequences that are not complementary to the guide nucleic acid binding sequence. Furthermore, the aforementioned guide nucleic acid binding sequence may be a nucleotide sequence adjacent to a sequence complementary to the nucleotide sequence (PAM sequence) that the editing protein can recognize.

[0102] As an example, the aforementioned guide nucleic acid binding sequence can be a series of 5 to 50 nucleotides located adjacent to the 5' end and / or 3' end of a sequence complementary to the nucleotide sequence (PAM sequence) that the editing protein can recognize.

[0103] Furthermore, the aforementioned guide nucleic acid non-binding sequence can be a nucleotide sequence adjacent to the nucleotide sequence (PAM sequence) that the editing protein can recognize.

[0104] As an example, the aforementioned guide nucleic acid non-binding sequence can be a continuous sequence of 5 to 50 nucleotides located adjacent to the 5' end and / or 3' end of a sequence complementary to the nucleotide sequence (PAM sequence) that the editing protein can recognize.

[0105] "Edited proteins" refer to peptides, polypeptides, or proteins that bind directly to nucleic acids or, although not directly, can interact with them. These edited proteins are also conceptually referred to as "artificially modified nucleases" or RGEN (RNA-Guided Endonuclease).

[0106] In one specific example, the editing protein mentioned above could be a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) enzyme. A "CRISPR enzyme," as a key structural element in the CRISPR enzyme system, also known as a "Cas protein (CRISPR-associated protein)," is a nuclease that can recognize target sequences and cleave DNA by forming mixtures or complexes with guide RNA.

[0107] CRISPR enzymes are well known in the art, with reference to Korean Patent Nos. 10-1656236, 10-1656237, 10-1706085, 10-2052286, and 10-2182847. In this specification, in addition to the native protein, the aforementioned CRISPR enzymes are used in the concept of including all variants that can function as endonucleases or nickases capable of activation in conjunction with guide RNA. In the case of activated endonucleases or nickases, targeted DNA cleavage can be achieved, enabling genome editing. Furthermore, in the case of inactive variants, transcriptional regulation or the separation of targeted DNA can be achieved.

[0108] The aforementioned CRISPR enzymes are nucleic acids or polypeptides (or proteins) with sequences encoding CRISPR enzymes. They are typically type II or type V CRISPR enzymes. The type II CRISPR enzymes contain the Cas9 protein (CRISPR associated protein 9).

[0109] The aforementioned Cas9 protein can originate from a variety of microorganisms, including Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Campylobacter jejuni, Staphylococcus aureus, Staphylococcus auricularis, and Neisseria meningitidis.

[0110] In order to induce double-stranded DNA break, the Cas9 protein should recognize the PAM sequence as a nucleotide sequence of a specified length, and a portion of the guide RNA (the guide sequence portion) should bind to the complementary strand of the single strand of DNA (the guide nucleotide non-binding sequence) where the target sequence is located.

[0111] The PAM sequence is determined based on the type or origin of the Cas9 protein. For example, the Cas9 protein derived from *Streptococcus pyogenes* (SpCas9) can recognize the 5'-NGG-3' sequence (complementary sequence: 5'-CCN-3') within the target nucleic acid. In this case, N is one of adenosine (A), thymidine (T), cytidine (C), or guanosine (G). Furthermore, the SpCas9 can recognize the 5'-NAG-3' sequence (complementary sequence: 5'-CTN-3') within the target nucleic acid with low activity.

[0112] Furthermore, the aforementioned type V CRISPR enzyme is Cpf1, which can be derived from Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacterium, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, and Lachnospiraceae. Cpf1 of Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium, or Acidaminococcus.

[0113] The CRISPR enzymes, such as Cas9 or Cpf1 proteins, can be isolated from microorganisms existing in a natural state or produced non-naturally through recombinant or synthetic methods. Furthermore, the Cas proteins can be in a form easily introduced into cells. For example, Cas proteins can be linked to cell-penetrating peptides or protein transduction domains. These protein transduction domains can be polyarginine or TAT proteins derived from acquired immunodeficiency virus (HIV), but are not limited to these. Besides the examples described above, various types of cell-penetrating peptides or protein transduction domains are known in the art, and those skilled in the art can employ various examples, not limited to those described above, in this specification. Furthermore, the Cas proteins can be fused with functional domains such as nuclear localization sequences or signaling sequences (NLS). Moreover, the Cas9 protein can be encoded as a vector for intracellular expression.

[0114] The present invention provides a composition for preparing blood-compatible mesenchymal stem cells, comprising: a guide nucleic acid or a nucleic acid encoding the guide nucleic acid, wherein the guide nucleic acid comprises a guide sequence capable of targeting exon 5 of the F3 gene of mesenchymal stem cells; and an editing protein or a nucleic acid encoding the editing protein.

[0115] Furthermore, the composition may selectively include a donor or a nucleic acid encoding it, wherein the donor contains a specific nucleotide sequence to be inserted.

[0116] The aforementioned donor refers to exogenous nucleotide sequences that can express specific peptides or proteins and can be inserted into genomic DNA through homology-directed repair (HDR).

[0117] The donors mentioned above can be double-stranded or single-stranded nucleic acids. The donors can be linear or circular.

[0118] The donors mentioned above can be in the form of viral vectors or non-viral vectors (e.g., plasmids).

[0119] The aforementioned viruses can be DNA viruses or RNA viruses. In this case, the DNA viruses can be double-stranded DNA (dsDNA) viruses or single-stranded DNA (ssDNA) viruses. In this case, the RNA viruses can be single-stranded RNA (ssRNA) viruses.

[0120] The aforementioned viral vectors may be selected from one or more of the following viral vectors: retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vacciniavirus, poxvirus, and herpes simplex virus (HSV).

[0121] The aforementioned non-viral vectors can be plasmids, lipid nanoparticles (LNPs), bacteriophages, naked DNA, DNA complexes, mRNA (transcriptions), or PCR amplicones.

[0122] The target sequence described above can be a target of the guide nucleic acid-editing protein complex, and the target sequence may contain a PAM (protospacer-adjacent motif) sequence recognized by the editing protein, but is not limited to this.

[0123] The aforementioned guide nucleic acid may contain a guide domain capable of targeting the target sequence of exon 5 of the F3 gene.

[0124] In this specification, guide nucleic acids, editing proteins, or guide nucleic acid-editing protein complexes (ribonucleoproteins, RNPs) and / or donors can be delivered or introduced into the target in various forms.

[0125] In this context, "subject" refers to: an organism into which guide nucleic acid, edited protein, or guide nucleic acid-edited protein complex is introduced; an organism into which guide nucleic acid, edited protein, or guide nucleic acid-edited protein complex is operated; or a test subject or sample obtained from an organism.

[0126] The aforementioned objects can be organisms containing guide nucleic acid-editing protein complexes, targeting genes, targeting nucleic acids, or targeting chromosomes.

[0127] The aforementioned organism can be an animal, animal tissue, or animal cell. In this case, the aforementioned tissue can be an eyeball, skin, liver, kidney, heart, lung, brain, muscle, or blood.

[0128] The aforementioned subjects or samples can be obtained from organisms containing target genes, target nucleic acids, or target chromosomes, such as saliva, blood, liver tissue, brain tissue, hepatocytes, nerve cells, bacteriophages, macrophages, T cells, B cells, astrocytes, cancer cells, or stem cells.

[0129] The aforementioned guide nucleic acids, editing proteins, or guide nucleic acid-editing protein complexes can be delivered or introduced into a target in the form of DNA, RNA, or a mixture thereof.

[0130] In this case, DNA, RNA, or a mixture thereof encoding guide nucleic acids and / or editing proteins can be delivered or introduced into the object using methods known in the art.

[0131] Alternatively, DNA, RNA, or a mixture thereof encoding guide nucleic acids and / or editing proteins can be delivered or introduced into the target via vectors, vector-free methods, or combinations thereof.

[0132] The aforementioned vectors can be viral vectors or non-viral vectors (e.g., plasmids).

[0133] The aforementioned viruses can be DNA viruses or RNA viruses. In this case, the DNA virus can be a double-stranded DNA virus or a single-stranded DNA virus. In this case, the RNA virus can be a single-stranded RNA virus.

[0134] The viral vectors mentioned above can be selected from one or more of retroviruses, lentiviruses, adenoviruses, adeno-associated viruses (AAV), vaccinia viruses, poxviruses, and herpes simplex viruses.

[0135] The aforementioned non-vectors can be plasmids, lipid nanoparticles (LNPs), bacteriophages, naked DNA, DNA complexes, mRNA, or PCR amplicones.

[0136] As an example of the present invention, the nucleic acid encoding the above-mentioned guide nucleic acid and / or editing protein can be delivered or introduced into the object in the form of more than one carrier.

[0137] The aforementioned vectors may contain nucleic acids encoding guide nucleic acids and / or editing proteins. As one example, the aforementioned vectors may simultaneously contain nucleic acids encoding both guide nucleic acids and editing proteins. As another example, the aforementioned vectors may contain nucleic acids encoding guide nucleic acids. For example, the nucleic acids encoding the aforementioned guide nucleic acids may all be contained in one vector, or the nucleic acids encoding guide nucleic acids may be fragmented and contained in multiple vectors. As yet another example, the aforementioned vectors may contain nucleic acids encoding editing proteins. For example, in the case of the aforementioned editing protein, the nucleic acids encoding the editing protein may be contained in one vector, or the nucleic acids encoding the editing protein may be fragmented and contained in multiple vectors.

[0138] The aforementioned editing proteins can be delivered or introduced into the target in the form of peptides, polypeptides, or proteins.

[0139] The aforementioned editing proteins can be delivered or introduced into a target in the form of peptides, polypeptides, or proteins using methods known in the art.

[0140] The aforementioned guide nucleic acid and editing protein can be delivered or introduced into the target in a nucleic acid-protein mixed form.

[0141] The aforementioned guide nucleic acid and editing protein can be delivered or introduced into the target in the form of a guide nucleic acid-editing protein complex. For example, the guide nucleic acid can be DNA, RNA, or a mixture thereof. The editing protein can be in the form of a peptide, polypeptide, or protein. As an example, the guide nucleic acid and editing protein can be delivered or introduced into the target in the form of a guide nucleic acid-editing protein complex in RNA form and an editing protein in protein form, i.e., in the form of a ribonucleoprotein (RNP).

[0142] Furthermore, the present invention provides a method for preparing blood-compatible mesenchymal stem cells, the method comprising: step (1), introducing the above-mentioned composition for preparing blood-compatible mesenchymal stem cells into isolated mesenchymal stem cells; and step (2), modifying exon 5 of the F3 gene located in the genome of the above-mentioned isolated mesenchymal stem cells, thereby editing exon 5 of the F3 gene to induce exon skipping of exon 5.

[0143] In this case, the aforementioned “import” can be performed by one or more methods selected from electroporation, lipofection, microinjection, gene gun, liposomes, amphoteric liposomes, plasmids, viral vectors, nanoparticles, protein translocation domain (PTD) fusion protein methods, immunoliposomes, multivalent cations or lipids, nucleic acid conjugates, naked DNA, artificial viral particles, and DNA formulations to enhance absorption, but is not limited to these methods.

[0144] As an example of the present invention, blood-compatible mesenchymal stem cells can be prepared by introducing the above-mentioned composition for preparing blood-compatible mesenchymal stem cells into isolated mesenchymal stem cells via electroporation.

[0145] As an example of the present invention, insertion / deletion can be achieved by contacting the F3 gene located in the genome of the aforementioned 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 exon 5 of the F3 gene.

[0146] In one embodiment of the present invention, the target sequence may be one or more sequences selected from SEQ ID NO: 1 to SEQ ID NO: 21.

[0147] The blood-compatible, artificially modified mesenchymal stem cells of this invention can be used as intravenously administered cell therapy agents for the treatment of a variety of diseases. For example, they can be used for heart disease, gastroduodenal disease, small and large intestine disease, liver disease, bile duct disease, pancreatic disease, kidney disease, lung disease, mediastinal disease, diaphragmatic disease, pleural disease, peritoneal disease, nervous system disease, central nervous system (CNS) disorders, peripheral artery 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). Liver diseases including fatty liver (NAFL), liver fibrosis, 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, and chronic persistent hepatitis; myocardial infarction, heart failure, arrhythmia, palpitations, cardiomyopathy, ischemic cardiomyopathy, angina pectoris, and congenital heart disease. Heart diseases including valvular heart disease, myocarditis, familial hypertrophic cardiomyopathy, dilated cardiomyopathy, acute coronary syndrome, arteriosclerosis, restenosis, etc.; gastrointestinal diseases including acute gastritis, chronic gastritis, peptic ulcers, gastric cancer, duodenal cancer, etc.; small and large bowel diseases including ischemic enteritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, simple ulcers, Behcet's disease of the intestine, small bowel cancer, large bowel cancer, etc.; and acute cholecystitis, acute cholangitis, chronic cholecystitis, bile duct cancer, gallbladder cancer, etc. Biliary duct 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 diseases, eosinophilic lung disease, pulmonary hypertension, pulmonary tuberculosis, sequelae of pulmonary tuberculosis, acute respiratory distress syndrome, cystic fibrosis, and chronic... Lung diseases including obstructive lung disease, pulmonary embolism, lung abscess, pneumoconiosis, aspiration pneumonia, pneumonia, pulmonary fibrosis, acute upper respiratory tract infection, chronic lower respiratory tract infection, pneumothorax, alveolar epithelial injury, lymphangioleiomyomas, lymphointerstitial pneumonia, and pulmonary alveolar proteinosis; mediastinal diseases including mediastinal tumors, mediastinal vesicular diseases, and mediastinitis; diaphragmatic diseases including diaphragmatic hernia; pleural diseases including pleurisy, empyema, pleural tumors, carcinomatous pleurisy, and pleural mesothelioma; and peritonitis and peritoneal tumors.Neurological diseases including cerebral palsy syndrome (including infantile cerebral palsy), aseptic meningitis, Guillain-Barré syndrome, amyotrophic lateral sclerosis (ALS), myasthenia gravis, mononeuropathy, polyneuropathy, spinal muscular atrophy, spinal cord disorders, acute transverse myelitis, spinal cord infarction (ischemic myelopathy), intracranial tumors, spinal cord tumors, etc.; central nervous system disorders such as Alzheimer's disease, cognitive impairment, stroke, multiple sclerosis, Parkinson's disease, etc.; peripheral artery diseases such as fibromuscular dysplasia, peripheral artery disease (PAD), thromboangiitis obliterans (Bergler's disease), Kawasaki disease (KD), etc.; peripheral venous diseases such as deep vein thrombosis, chronic venous insufficiency, postphlebitis syndrome, superficial vein thrombosis, etc.; and immunodeficiency diseases such as graft-versus-host disease (GVHD), secondary immunodeficiency, primary immunodeficiency, B cell deficiency, T cell deficiency, combined B cell and T cell deficiency, phage deficiency, complement deficiency, etc. ;

[0148] The term "for vascular administration" as used above refers to delivery within the patient's vascular system. For example, it can be administered into vessels considered veins or vessels considered 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 veins, and / or femoral vein. Arteries include, but are not limited to, the coronary arteries, pulmonary artery, splenic artery, brachial artery, internal carotid artery, aortic arch, femoral artery, peripheral arteries, and / or ciliary arteries. Delivery can be made via the portal vein, umbilical vein, arterioles, or capillaries, or to arterioles or capillaries.

[0149] In one embodiment of the invention, the artificially modified mesenchymal stem cells of the invention can be used as a composition for cell transplantation or biological tissue regeneration, which restores damaged cells or tissues.

[0150] In one embodiment of the present invention, the aforementioned biological tissue may be a damaged tissue selected from tissues forming ulcers or bedsores, brain tissue damaged due to cell degeneration, brain tissue damaged due to surgical procedures, brain tissue damaged due to traumatic brain disease, brain tissue damaged due to inflammatory brain disease, damaged bone tissue, damaged periodontal tissue, tissue damaged due to central nervous system disease, and tissue damaged due to intractable dermatitis. Biological tissue regeneration may include the recovery of the aforementioned damaged tissues, epidermal regeneration, regeneration of secretory glands or hair follicles, capillary formation in dermal tissue, wound healing, repair of soft tissue defects, bone healing or bone regeneration, cartilage regeneration, etc., but is not limited thereto.

[0151] In one embodiment of the present invention, the above-mentioned 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.

[0152] In this specification, "cell therapy agent" refers to a drug that uses cells and tissues prepared by isolation, culture, and special manipulation from an individual for the purpose of treatment, diagnosis, and prevention (as defined by the U.S. Food and Drug Administration (FDA)). It refers to a drug used for the purpose of treatment, diagnosis, and prevention by a series of actions such as in vitro proliferation and screening of autologous, allogeneic, or xenogeneic cells or alteration of the biological characteristics of cells using other methods to restore the function of cells or tissues.

[0153] In this specification, "gene therapy agent" is a drug used to affect the expression of genetic material and refers to a drug containing modified or introduced cells with genetic material.

[0154] In one embodiment of the present invention, the above-described cell therapy agent and pharmaceutical composition can be used in mammals to treat or prevent conditions selected from myocardial infarction, heart failure, ischemic cardiomyopathy, myocarditis, ischemic enteritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, acute cholecystitis, acute cholangitis, chronic cholecystitis, acute pancreatitis, chronic pancreatitis, acute nephritis, chronic nephritis, acute renal failure, chronic renal failure, pneumonia, interstitial pneumonia, idiopathic interstitial pneumonia, and desquamation. Interstitial pneumonia, acute interstitial pneumonia, nonspecific interstitial pneumonia, drug-induced lung disease, acute respiratory distress syndrome, chronic obstructive pulmonary disease, cerebral palsy syndrome including cerebral palsy in children, amyotrophic lateral sclerosis, polyneuropathy, spinal muscular atrophy, acute transverse myelitis, stroke, multiple sclerosis, peripheral artery disease, thromboangiitis obliterans (Bergler's disease), Kawasaki disease, and graft-versus-host disease are among the following diseases, but not limited to these.

[0155] In one embodiment of the present invention, it can be used to treat or prevent the aforementioned diseases or conditions, which are selected from mammals including myocardial infarction, heart failure, ischemic cardiomyopathy, myocarditis, ischemic enteritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, acute cholecystitis, acute cholangitis, chronic cholecystitis, acute pancreatitis, chronic pancreatitis, acute nephritis, chronic nephritis, acute renal failure, chronic renal failure, pneumonia, interstitial pneumonia, idiopathic interstitial pneumonia, desquamative interstitial pneumonia, acute interstitial pneumonia, nonspecific interstitial pneumonia, drug-induced lung disease, acute respiratory distress syndrome, chronic obstructive pulmonary disease, cerebral palsy syndrome including cerebral palsy in children, amyotrophic lateral sclerosis (ALS), polyneuropathy, spinal muscular atrophy, acute transverse myelitis, stroke, multiple sclerosis, and peripheral artery disease. It is one of, but not limited to, diseases such as thromboangiitis obliterans (PAD), thromboangiitis obliterans (Bergler's disease), Kawasaki disease (KD), and graft-versus-host disease (GVHD).

[0156] The composition of the present invention for cell transplantation or biological tissue regeneration can be administered via any conventional route capable of reaching the target tissue. Non-oral administration can be used, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, but is not limited thereto.

[0157] The above-described compositions for cell transplantation or biological tissue regeneration can be formulated into suitable forms with pharmaceutically acceptable carriers commonly used in cell therapy. "Pharmaceutically acceptable" means a composition that is physiologically acceptable and generally does not cause gastrointestinal disturbances, dizziness, or other allergic reactions or similar reactions when administered to humans. Pharmaceutically acceptable carriers include, for example, non-oral carriers such as water, suitable oils, physiological saline, aqueous glucose, and ethylene glycol, and may also contain stabilizers and preservatives. Stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Suitable preservatives include benzalkonium chloride, methylparaben, propylparaben, and chlorobutanol. Further pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995. Furthermore, the above compositions can also be administered via any device capable of moving to target cells.

[0158] For the treatment of disease, the compositions of the present invention for cell transplantation or biological tissue regeneration may contain a therapeutically effective amount of cells. "Therapeutically effective amount" refers to the amount of an effective ingredient or pharmaceutical composition inducing a biological or medical response in a tissue system, animal, or human, as determined by researchers, veterinarians, physicians, or other clinically recognized individuals, including amounts that induce relief of symptoms of the treated disease or disorder.

[0159] Those skilled in the art will understand that the cell content of the compositions of the present invention varies depending on the desired effect. Therefore, the optimal cell content can be easily determined by those skilled in the art, and can be adjusted based on various factors including the type and severity of the disease, the content of other components in the composition, the type of dosage form, and the patient's age, weight, general health status, sex, diet, timing of administration, route of administration, secretion rate of the composition, treatment duration, and concurrent medications. Considering all the above factors, it is important to include a quantity that achieves the maximum effect with the minimum amount without causing side effects. For example, the daily dosage of stem cells of the present invention is 1.0 × 10⁻⁶. 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 20 The dosage of the active ingredient can be administered in one dose or in several divided doses per kg of body weight. However, it should be understood that the actual dosage of the active ingredient is determined with reference to multiple relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age, and gender. Therefore, the above dosage does not limit the scope of the present invention in any way.

[0160] This invention provides a treatment method comprising the step of administering a therapeutically effective amount of the aforementioned artificially modified mesenchymal stem cells to a mammal suffering from a disease or symptom. As used herein, the term "mammal" refers to a mammal used as a subject of treatment, observation, or experimentation, preferably a human.

[0161] Furthermore, in the treatment methods of the present invention, the composition containing the cells of the present invention as an active ingredient can be administered in a conventional manner via rectal, intravenous (iv), intra-arterial, intraperitoneal, intramuscular, intrasternal, percutaneous, local, intraocular, or intradermal routes.

[0162] Furthermore, the present invention provides a cell therapy method using stem cells, the method comprising the step of administering a therapeutically effective amount of the aforementioned artificially modified mesenchymal stem cells to a mammal suffering from a disease or ailment.

[0163] In one embodiment of the present invention, the above method, by administering artificially modified mesenchymal stem cells with reduced expression levels of full-length tissue factor and increased expression levels of alternative splicing tissue factor, can enhance the efficacy of mesenchymal stem cells by suppressing venous thromboembolism (VTE) and instant blood-mediated inflammatory response (IBMIR) when used as cell therapy agents in autologous or homologous environments.

[0164] Example

[0165] The present invention will now be described in detail through embodiments. Those skilled in the art should understand that these embodiments are merely for illustrating the invention more specifically, and the scope of the invention is not limited by these embodiments.

[0166] Example 1: Preparation of CD142 gene exon 5 gene edited cells

[0167] sgRNA design

[0168] To knock out exon 5 and the splicing factor at the exon 5 start position in the F3 gene (the gene encoding CD142), sgRNAs were screened from the sequences surrounding the exon 5 region of the F3 gene using the Cas-Designer tool. From the guide sequences predicted at http: / / www.rgenome.net / cas-designer / , guide RNAs with predicted low off-target effects and mismatch values ​​of 0 (value 1) and 1,2 (value 0) were selected and designed as targets. Target sequences targeting exon 5 of F3 are shown in Table 1 below.

[0169] Table 1

[0170]

[0171]

[0172] Figure 1 This is a schematic diagram illustrating the target sites of sgRNA. (Reference) Figure 1 Using exon 5 of F3 as the benchmark, sites capable of completely deleting exon 5 (including deletion of exon 5 pre-, post-, and splicing acceptors) or disrupting exon 5 were designated as target sites, and 21 sgRNA candidate groups were selected. Guide RNAs targeting the target sequences of SEQ ID NO: 1 to 21 were synthesized for subsequent experiments. The target sequence of SEQ ID NO: 22 uses exon 3 of F3 as the target site. Guide RNAs were transcribed in vitro using T7 RNA polymerase (New England Biolabs) according to the manufacturer's instructions. The sgRNA template was prepared by annealing and extending two complementary oligonucleotides.

[0173] Cell culture

[0174] This experiment used commercially available cryopreserved umbilical cord (UC)-derived mesenchymal stem cells (UC-MSCs). After thawing according to the thawing method, they were cultured at a density of 2000-4000 cells / cm³. 2 Inoculate the cells, change the culture medium after 2-3 days, and collect the cells for subculture when confluence reaches 80-90%. This is called the first subculture. Subculture again at 2000-4000 cells / cm³. 2 Inoculation. Regarding culture medium replacement, replace with fresh culture medium every 2-3 days before subculturing.

[0175] Ribonucleoprotein (RNP) delivery

[0176] The RNP complex was introduced into collected cultured cells via electroporation using a 4D-Nucleofector (Lonza). Specifically, 4 μg of Cas9 protein and 4 μg of in vitro transcribed sgRNA (prepared using T7 polymerase (New England BioLabs) according to the manufacturer's instructions) were mixed to form the RNP complex, and the mixture was incubated at room temperature for 10 minutes. The sgRNA was used alone or in combination with the two prepared sgRNAs to knock out exon 5. The combinations are shown in Table 2 below.

[0177] Table 2

[0178]

[0179] The above RNP complex was mixed with 4×10⁴ cells that had been treated with 20 μL of primary P1 buffer. 5 Human bone marrow mesenchymal stem cells (Lonza, Cat. No. PT-2501) and umbilical cord mesenchymal stem cells (ATCC, Cat. No. PCS-500-010) were electroporated together using the EW-104 nuclear transfection program. The result was F3 KO UC-MSCs (F3 KO UC-MSCs) with exon 5 of the F3 gene edited. The treated cells were then cultured at 2000-5000 cells / cm². 2 The amount was cultured again for subsequent analysis.

[0180] Targeted deep sequencing

[0181] The insertion / deletion efficiency for each target sequence of each guide RNA was determined using targeted deep sequencing methods.

[0182] Genomic DNA (gDNA) was extracted from the obtained F3-Δexon5 KO UC-MSCs using the Blood Genomic DNA Extraction Kit (Favorgen) according to the manufacturer's instructions. To amplify the target sites, 100 ng of genomic DNA (gDNA) was amplified using Phusion High-Fidelity DNA Polymerase PCR Polymerase (NEB). To generate the deep sequencing library, the amplicons were amplified again using TruSeq HT dual index primers (Illumina, San Diego, CA, USA). Paired-end sequencing was performed using the Illumina Miseq system, and insertion / deletion frequencies were calculated at http: / / www.rgenome.net / . Primer sequences for each target sequence used in targeted deep sequencing are shown in Table 3 below.

[0183] Table 3

[0184]

[0185] Figure 2 The results of the first treatment of one sgRNA, confirmed by NGS, are shown.

[0186] In the second screening, one sgRNA was processed to identify candidate sgRNAs with high insertion / deletion ratios, and two guide sgRNAs were processed simultaneously to induce complete deletion of exon 5, including deletion of the splicing acceptor, and disruption of exon 5 before screening. However, due to the inability to perform NGS (Illumina) due to large gene deletions, the mRNA expression levels of full-length tissue factor (flTF) and alternative splicing tissue factor (asTF) were confirmed, and flow cytometry and Western blot were used as substitutes for the screening results.

[0187] Flow cytometer analysis

[0188] To confirm whether the expression of the surface factor CD142, which plays a role in the first step of the coagulation cascade, was reduced in F3 Δexon5 knockout (KO) stem cells using a combination of two sgRNAs, flow cytometry was used to confirm this in post-KO passages. Normal umbilical cord / umbilical cord-derived stem cells (UC-WT), normal umbilical cord / umbilical cord-derived stem cells that underwent electroporation without genetic manipulation (4D only), umbilical cord stem cells edited with SHS231 (a safe harbor gene) (sgSHS231 re#2), and umbilical cord / umbilical cord-derived stem cells edited with exon 3 using SEQ ID NO: 22 as the target sequence (sgF3 Ex3 #33) served as a positive control group to confirm CD142 expression.

[0189] The obtained WT, F3 KO, or F3-Δexon5 KO stem cells were washed twice with phosphate buffered saline (PBS), then the cells were removed with 0.05% trypsin-EDTA and centrifuged at 1500 rpm for 5 minutes. 1 × 10⁻⁶ cells were collected from each group. 5 Cells were collected and suspended in 100 μL of FACS staining buffer (2% FBS in PBS), and mixed with 0.5–1 μL of antibodies (CD142, CD34, CD45, CD73, CD90, CD105, CD29, CD51, CD44, CD51 / 61: Biolegend) for confirming stem cell surface markers and F3 surface markers. The mixture was incubated at 4°C for 20 minutes, then washed twice with FACS staining buffer and suspended in 300 μL of PBS for flow cytometry analysis. CD142 expression levels were determined by the percentage of cells expressing CD142 (CD142-positive cell population, %) and median fluorescence intensity (MFI). The results are presented below. Figure 3 middle.

[0190] refer to Figure 3It was confirmed that UC-WT, WT (4D only), and sgSHS231 showed high CD142 expression frequency and MFI (mean fluorescence intensity). In contrast, CD142 expression was reduced in the F3 Δexon5 KO group, which was edited for exon 5 using two sgRNA combinations. The level was comparable to that of umbilical cord / umbilical cord-derived stem cells (sgCD142#33), which served as a positive control group for editing F3 exon 3 using single gRNA.

[0191] Barcode Tagged Sequencing (BT-seq)

[0192] BT-seq was performed to identify the exon 5 deletion region in umbilical cord / umbilical cord-derived stem cells that had been edited using two sgRNA combinations (sgF3 Ex5 #2+5 and sgF3 Ex5 #2+10). To confirm the exon 5 deletion region, analysis was performed according to the manufacturer's instructions (BTSeq). TM DNA sequencing was performed at CELEMICS, Korea. The results are presented in [the document / platform / etc.]. Figure 4 middle.

[0193] Figure 4 Part A shows the results of repeating the experiment three times. Figure 4 Part B is a graph showing the average results. For example... Figure 4 Part A and Figure 4 As shown in Part B, it was confirmed that over 90% of the gene editing effects were observed in all experimental groups, and the editing trends included at least 60% large deletions, over 20% indels, and over 5% inversions.

[0194] Gene expression analysis (qPCR)

[0195] To confirm the reduction or increase in related RNA expression after knockout of F3-Δexon5 KO UC-MSCs and F3-Δexon5 KO BM-MSCs, mRNA was extracted using the RNEsay Mini Kit (Qiagen). 1 μg of mRNA was used to synthesize cDNA. The concentration and purity of the isolated RNA were determined using nano-drop. Then, RNA and RNase-free water were added to a Maximé RT premix kit containing Oligo dT to synthesize cDNA, bringing the total volume to 20 μL. Real-time PCR was performed using a QuantStudio instrument.TM 3. Real-Time PCR System.

[0196] To confirm the reduction of full-length tissue factor (flTF) gene and the increase of alternative splicing tissue factor (asTF), primers as shown in Table 4 below were used. The solution used for qPCR was prepared by adding 10 μL of the SYBR Green PCR Kit, 1 μL of each primer, 2.0 μL of template cDNA (1 μg), and 7 μL of RNase-free water to a total volume of 20 μL, and PCR was performed under the following conditions: The amplification reaction was performed as follows: pre-denaturation: 95°C, 10 min; denaturation: 95°C, 15 s; annealing & extension: 60°C, 1 min. This process was repeated 40 times.

[0197] Table 4

[0198]

[0199] Figure 5 The graph illustrates the expression of full-length tissue factor (flTF) mRNA and alternatively spliced ​​tissue factor (asTF) mRNA in normal umbilical cord / umbilical cord-derived stem cells (UC-WT), normal umbilical cord / umbilical cord-derived stem cells that underwent electroporation without genetic manipulation (4D only), umbilical cord stem cells that underwent SHS231 editing as a safe harbor gene (sgSHS231 re#2), and umbilical cord stem cells that underwent exon 3 editing using SEQ ID NO: 22 as a positive control (sgF3 Ex3 #33).

[0200] like Figure 5 As shown, the F3 Δexon5 KO group, which underwent F3 exon 5 editing using two sgRNA combinations, showed reduced expression of full-length tissue factor (flTF) mRNA, comparable to the positive control group, which underwent F3 knockout (KO) using SEQ ID NO: 22 as the target sequence for editing F3 exon 3 (sgCD142#33). In particular, the expression of alternatively spliced ​​tissue factor (asTF) mRNA was increased compared to UC-WT, WT (4D only), sgSHS231, and sgCD142#33.

[0201] Western Blot

[0202] The protein expression levels of alternative splicing tissue factor (asTF) and full-length tissue factor (flTF) were confirmed in cells and culture media from normal umbilical cord / umbilical cord-derived stem cells (UC-WT) and two ΔExon5 screening groups (sgF3 ΔEx5 #2+5 and #2+10). 1×10⁻⁶ cells were collected from each group. 6 Eited UC-MSCs were lysed using a lysis buffer supplemented with 100 μL of RIPA buffer and 1 μL of protein inhibitor. Lysis was performed on ice with vortexing every 5 minutes for 20 minutes. After confirming complete cell lysis, the cells were centrifuged at ~14000 xg for 15 minutes at 4°C to separate cell debris. The supernatant was transferred to a new e-tube and purified using Pierce. TM The BCA Protein Assay Kit quantifies proteins and should be stored at -80°C for later use. Load 15 µg of cell lysates into 10% Mini-PROTEAN® TGX stain-free solution. TM Stain-Free Protein Gel TM Protein gels were transferred to an NC membrane. The membrane was blocked using Bio-Rad blocking buffer, and the primary antibody was then incubated overnight at 4°C. A suitable horseradish peroxidase-conjugated secondary antibody was then ligated, and protein bands were confirmed using Westernlighting ECL.

[0203] Table 5

[0204]

[0205] Figure 6The results show the expression of full-length tissue factor (flTF) in normal umbilical cord / umbilical cord-derived stem cells (UC-WT) and umbilical cord / umbilical cord-derived stem cells with F3 exon 5 edited using two gRNA combinations (sgF3 ΔEx5 #2+5, #2+10) analyzed by Western blotting.

[0206] refer to Figure 6 We confirmed that full-length tissue factor (flTF) was endogenously expressed in UC-WT, and that the expression of full-length tissue factor (flTF) protein was reduced in all groups of sgF3 ΔEx5 #2+5 and #2+10 in three triplications.

[0207] Enzyme-linked immunosorbent assay (ELISA)

[0208] Normal umbilical cord / umbilical cord-derived stem cells (UC-WT), F3 KO, or F3-Δexon5 KO umbilical cord / umbilical cord-derived stem cells (UC-MSC) were collected. Tissue factor activity was confirmed using cells and cell culture medium. The ability of lipoprotein TF / FVIIa to activate Factor X (FX) into Factor Xa was measured according to the tissue factor activity assay instructions.

[0209] Human tissue factor (TF) activity was determined in cells and cell culture medium using the Tissue Factor Activity Assay Kit (Human, Colorimetric, Abcam) ab108906. The intermediate cleavage activity of the TF / FVIIa complex was quantified by the amount of FXa generated from a highly specific FXa substrate that releases a yellow para-nitroaniline (pNA) chromophore. The change in absorbance of pNA at 405 nm was proportional to the TF enzyme activity. Two × 10⁶ samples were used per group. 5 Cells were lysed by incubating on ice for 30 minutes with 500 μL of 50 mM Tris-buffered saline containing Triton X-100 or 0.1% Tween 20. Cell viability was confirmed by assaying according to the kit protocol.

[0210] Figure 7Charts showing the cell lysate and cell culture medium (cm) activity of normal umbilical cord / umbilical cord-derived stem cells (UC-WT), umbilical cord / umbilical cord-derived stem cells with exon 3 edited using SEQ ID NO: 22 as the target sequence (sgF3 Ex3 #33), and three ΔExon5 screening groups (sgF3 ΔEx5 #2+5, #2+10, #3+14) based on tissue factor concentration. Figure 7 As shown, in both cells and culture media, the tissue factor activity of the ΔExon5 group was reduced by 50% to 100%, similar to that of umbilical cord / umbilical cord-derived stem cells that underwent exon 3 editing using the target sequence of #33 (SEQ ID NO: 22).

[0211] Thromboelastrometry confirmation test

[0212] Collect cultured WT, F3 KO, or F3-Δexon5 KO umbilical cord / umbilical cord-derived mesenchymal stem cells, and prepare 4 × 10⁴ cells per group. 5 Cells were collected and reacted with 400 μL of fresh human whole blood prepared in a sterile test tube. The thoroughly mixed cell and whole blood sample was immediately sent to the experimental device to measure thromboelastography. Elastography was measured using a TEG 6s device (HAEMONETICS). After inserting the kit for confirming elastography into the device, the stem cell sample mixed with whole blood was thoroughly mixed and added to the sample loading chamber to confirm the reaction.

[0213] Table 6

[0214]

[0215] The coagulation reactions of normal umbilical cord / umbilical cord-derived stem cells (UC-WT), umbilical cord / umbilical cord-derived stem cells with exon 3 edited by targeting F3 exon3 #33 (sgF3Ex3 #33), and umbilical cord / umbilical cord-derived stem cells with exon 5 edited by targeting two types of ΔExon5 #3+14 (sgF3 ΔEx5 #3+14) were confirmed using the parameters in Table 6 above, measured with the TEG 6s device. The results are shown in Table 6. Figure 8 middle.

[0216] Figure 8Part A is a thromboelastographic map confirming overall clotting formation. Figure 8 Part B contains graphs showing coagulation reaction time (R time, min), thrombosis dynamics (K time, min), thrombosis dynamics (α angle), and MA (thrombosis strength).

[0217] It was confirmed that in normal umbilical cord / umbilical cord-derived stem cells (UC-WT), the clotting time of human blood was reduced. Conversely, similarly to umbilical cord / umbilical cord-derived stem cells (sgCD142#33) that were edited with exon 3 as the target sequence (sgCD142#33) as the positive control, the clotting time of human blood returned to normal in umbilical cord / umbilical cord-derived stem cells (sgF3 ΔEx5#3+14) that were edited with exon 5 as the target sequence (sgF3 ΔEx5#3+14).

[0218] Confirmation of changes in Population Doubling Level (PDL), Population Doubling Time (PDT) during long-term culture Figure 9

[0219] Following gene manipulation, long-term culture was performed to confirm whether proliferative capacity was maintained even under high passage rates. To confirm cell growth, cell numbers were measured before and after passage. The final cell yield, or the number of cells at the end of the growth period, is denoted as Ct, and the initial cell number seeded into the vessel is denoted as Ci. PDL(N) was calculated using the following mathematical formula 1.

[0220] Mathematical formula 1:

[0221]

[0222] The above culture time (hr) divided by the PDL value represents the Population Doubling Time (PDT).

[0223] Long-term culture of normal umbilical cord / umbilical cord-derived stem cells (UC-WT), normal umbilical cord / umbilical cord-derived stem cells that underwent only transfection without gene manipulation (Mock), umbilical cord / umbilical cord-derived stem cells that underwent exon 3 editing with F3 exon3 #33 as the target (sgF3 Ex3 #33), and umbilical cord / umbilical cord-derived stem cells that underwent exon 5 editing with two ΔExon5 #3+14 targets (sgF3 ΔEx5 #3+14) were compared. Figure 9 This chart illustrates the PDT and PDL of normal umbilical cord / umbilical cord-derived stem cells (UC-WT), normal umbilical cord / umbilical cord-derived stem cells that underwent only transfection without genetic manipulation (Mock), umbilical cord / umbilical cord-derived stem cells with exon 3 edited using F3 exon3 #33 as the target (sgF3 Ex3 #33), and umbilical cord / umbilical cord-derived stem cells with exon 5 edited using two ΔExon5 #3+14 targets (sgF3 ΔEx5 #3+14) at various passages during long-term culture. Confirmation of changes in tissue factor expression during long-term culture As shown, it was confirmed that sgF3ΔEx5 #3+14 maintained the same value-adding capacity as UC-WT during long-term culture until the 12th generation (P12).

[0224] Figure 10

[0225] Long-term culture was performed after gene manipulation to confirm whether CD142 expression remained low even at high passages. Normal umbilical cord / umbilical cord-derived stem cells (UC-WT), normal umbilical cord / umbilical cord-derived stem cells that underwent only transfection without gene manipulation (Mock), umbilical cord / umbilical cord-derived stem cells that underwent exon 3 editing with F3 exon3 #33 as the target (sgF3 Ex3 #33), and umbilical cord / umbilical cord-derived stem cells that underwent exon 5 editing with two ΔExon5 #3+14 targets (sgF3 ΔEx5 #3+14) were cultured long-term and compared using flow cytometry analysis as described above. Figure 10This chart illustrates the percentage of CD142-positive cells and median fluorescence intensity (MFI) at various passages for the following stem cell types during long-term culture: normal umbilical cord / umbilical cord-derived stem cells (UC-WT), normal umbilical cord / umbilical cord-derived stem cells that underwent transfection without genetic manipulation (Mock), umbilical cord / umbilical cord-derived stem cells that underwent exon 3 editing with F3 exon 3 #33 as the target (sgF3 Ex3 #33 #33), and umbilical cord / umbilical cord-derived stem cells that underwent exon 5 editing with two ΔExon 5 targets (sgF3 ΔEx5 #3+14). Reference RNA-seq (QuantSeq) It was confirmed that sgF3 ΔEx5 #3+14 maintained low expression of CD142 during long-term culture, just like sgF3 Ex3 #33, which served as a CD142 KO positive control group, until the 12th generation (P12).

[0226] Figure 11

[0227] To confirm the pattern of intracellular mRNA changes after F3 KO, RNA-seq was performed as follows.

[0228] Total RNA was isolated using the RNeasy Mini Kit (Qiagne). RNA quality was determined using the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands) via an Agilent 2100 bioanalyzer, and RNA quantity was quantified using an ND-2000 spectrophotometer (Thermo Inc., DE, USA).

[0229] To determine the RNA of the control and sample RNA, library construction was performed using the QuantSeq 3' mRNA-Seq Library Prep Kit (Lexogen, Inc., Austria) according to the manufacturer's instructions. 500 ng of total RNA was prepared and reverse transcribed by hybridizing the RNA with oligo-dT primers containing Illumina platform-compatible sequences at the 5' end. After digesting the RNA template, second-strand synthesis was initiated using random primers containing Illumina-compatible adapter sequences at the 5' end. Magnetic beads were used to purify the double-stranded library. All adapter sequences were appended for cluster generation to amplify the library. Purification was performed on the completed double-stranded library polymerase chain reaction (DPCR) structural elements. High-throughput sequencing (single-terminal 75-bit sequence) was then performed (NextSeq 500, Illumina Inc.). QuantSeq 3' mRNA-Seq interpretation was performed using Bowtie2 (Langmead and Salzberg, 2012). The Bowtie2 index is generated from genomic composite sequences or transcripts. Sequencing files are used to composite transcripts, infer proportions, and detect differential gene expression. Coverage is used in Badtools (Quinlan AR, 2010) to identify genes that are unique and differentially expressed based on counts in most sorts. RC (Read Count) data are processed using Bioconductor (Gentleman et al., 2004) and EdgeR within R (R development Core Team, 2020) using a TMM+CPM normalization method. Gene classification is based on searches using the DAVID database (http: / / david.abcc.ncifcrf.gov / ) and Medline databases (http: / / www.ncbi.nlm.nih.gov / ) engines.

[0230] Heat map analysis was used to analyze the similarity between samples and between genes based on expression similarity. Analysis was performed when the fold change was greater than 2, the p-value was less than 0.05, and the standardized data (log 2) value was greater than 4.

[0231] Antibody microarrayPart A shows the genes expressed in both umbilical cord / umbilical cord-derived stem cells (sgF3 Ex3 #33) that were edited with F3 exon3 #33 as the target (sgF3 ΔEx5 #3+14) and umbilical cord / umbilical cord-derived stem cells (sgF3 ΔEx5 #3+14) that were edited with a combination of two gRNAs to F3 exon5 (sgF3 ΔEx5 #3+14). Part B shows the results of the screening list of genes expressed only in sgF3 ΔEx5 #3+14.

[0232] Figure 12

[0233] To confirm the changes in cytokines in WT UC-MSCs and F3 KO cells, a cytokine array was performed.

[0234] Antibody array analysis of gene-edited mesenchymal stem cells was performed using Full Moon Biosystems. Cell lysates were prepared using a Protein Extraction Kit (Full Moon BioSystems). After separating excess supernatant from the lysates and biotinylating, the lysates were cultured for 2 hours at room temperature using Phospho Explorer Antibody Arrays (Full Moon BioSystems). The array slides were washed with washing buffer (Full Moon BioSystems) and then rinsed with deionized water. The slides were then co-cultured with Cy3-Streptavidin for 45 minutes at room temperature, followed by washing and drying. The arrays were scanned using a GenePix Array Scanner (Molecular Devices). Image quantification was performed using GenePix Pro (Molecular Devices). Signal intensity data for each point on the array were extracted from the array images. Two replicas were printed for each antibody to determine the average signal intensity of the replicas. The data were then normalized using the median (signal intensity) of all antibodies on the slide. Finally, the fold change between the control and treated samples was determined using the normalized data (signal intensity of the treated sample divided by the signal intensity of the control sample).

[0235] Heat map analysis was used to analyze the similarity of secretomes between samples based on expression similarity. Analysis was performed when the fold change was greater than 1.5, the p-value was less than 0.05, and the normalized data (log 2) value was greater than 4.

[0236] ​ Part A shows the secretome factors secreted in umbilical cord / umbilical cord-derived stem cells (sgF3 Ex3 #33) that have edited exon 3 with F3 exon3 #33 as the target, and in umbilical cord / umbilical cord-derived stem cells (sgF3 ΔEx5 #3+14) that have edited exon 5 with two gRNAs in combination. Part B shows the list of results that only selected factors analyzed in sgF3 ΔEx5 #3+14.

Claims

1. An artificially modified mesenchymal stem cell comprising an artificially modified F3 gene, characterized in that, The artificially modified F3 gene includes the induction of exon skipping in exon 5 of the F3 gene through gene modification of exon 5. Compared with wild-type mesenchymal stem cells, the artificially modified mesenchymal stem cells show reduced expression levels of full-length tissue factors and increased expression levels of selective splicing tissue factors on the stem cell surface, thereby improving blood compatibility.

2. The artificially modified mesenchymal stem cells according to claim 1, characterized in that, The genetic modification of exon 5 of the F3 gene includes the deletion or disruption of exon 5.

3. The artificially modified mesenchymal stem cells according to claim 1, characterized in that, The gene modification of exon 5 of the F3 gene includes the deletion of the nucleotide sequence containing the splice acceptor immediately preceding exon 5.

4. The artificially modified mesenchymal stem cells according to claim 1, characterized in that, The gene modifications in exon 5 of the F3 gene include insertions / deletions. The insertion / deletion is located in exon 5 of the F3 gene within a motif adjacent to the original spacer sequence or within a consecutive sequence of 5 to 50 nucleotides adjacent to the 5' or 3' end of the motif adjacent to the original spacer sequence.

5. The artificially modified mesenchymal stem cells according to claim 1, characterized in that, In the artificially modified mesenchymal stem cells, Compared with the mRNA expression level transcribed from the F3 gene of wild-type mesenchymal stem cells, the full-length tissue factor mRNA expression level transcribed from the artificially modified F3 gene is lower, or has a different sequence.

6. The artificially modified mesenchymal stem cells according to claim 1, characterized in that, In the artificially modified mesenchymal stem cells, The expression level of alternatively spliced ​​tissue factor mRNA transcribed from the artificially modified F3 gene is higher or has a different sequence than the expression level of mRNA transcribed from the F3 gene of wild-type mesenchymal stem cells.

7. The artificially modified mesenchymal stem cells according to claim 1, characterized in that, The sequence of the artificially modified F3 gene does not include more than one sequence selected from SEQ ID NO: 1 to 21.

8. The artificially modified mesenchymal stem cells according to claim 1, characterized in that, Artificially modified mesenchymal stem cells are derived from adipose tissue, bone marrow, umbilical cord, placenta, amniotic fluid, amnion, tissue, umbilical cord blood, or perinatal tissue.

9. A composition for preparing blood-compatible mesenchymal stem cells, characterized in that, It includes: A guide nucleic acid or a nucleic acid encoding thereon, said guide nucleic acid comprising a guide sequence capable of targeting a target sequence of exon 5 of the F3 gene in mammalian cells; and Edit proteins or the nucleic acids that encode them.

10. The composition for preparing blood-compatible mesenchymal stem cells according to claim 9, characterized in that, The target sequence is one or more sequences selected from SEQ ID NO: 1 to 21.

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

12. The composition for preparing blood-compatible mesenchymal stem cells according to claim 9, characterized in that, The composition comprises, in one or more carrier forms, nucleic acids encoding the editing protein and the guide nucleic acid.

13. The composition for preparing blood-compatible mesenchymal stem cells according to claim 12, characterized in that, The vector is selected from plasmids, lipid nanoparticles, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, vaccinia viruses, poxviruses, and herpes simplex viruses.

14. A method for preparing blood-compatible mesenchymal stem cells, characterized in that, It includes: Step (1) introduces a composition for preparing blood-compatible mesenchymal stem cells into isolated mesenchymal stem cells, the composition comprising a guide nucleic acid or nucleic acid encoding a target sequence capable of targeting exon 5 of the F3 gene and an editing protein or nucleic acid encoding a target sequence. as well as Step (2) involves gene modification of exon 5 of the F3 gene located in the genome of the isolated mesenchymal stem cells, thereby editing exon 5 of the F3 gene to induce exon skipping of exon 5.

15. The method for preparing blood-compatible mesenchymal stem cells according to claim 14, characterized in that, The genetic modification of exon 5 of the F3 gene includes the deletion or disruption of exon 5.

16. The method for preparing blood-compatible mesenchymal stem cells according to claim 14, characterized in that, The gene modification of exon 5 of the F3 gene includes the deletion of the nucleotide sequence containing the splice acceptor immediately preceding exon 5.

17. The method for preparing blood-compatible mesenchymal stem cells according to claim 14, characterized in that, The gene modifications in exon 5 of the F3 gene include insertions / deletions. The insertion / deletion is located in exon 5 of the F3 gene within a motif adjacent to the original spacer sequence or within a consecutive sequence of 5 to 50 nucleotides adjacent to the 5' or 3' end of the motif adjacent to the original spacer sequence.

18. The method for preparing blood-compatible mesenchymal stem cells according to claim 14, characterized in that, The target sequence is one or more sequences selected from SEQ ID NO: 1 to 21.

19. The method for preparing blood-compatible mesenchymal stem cells according to claim 14, characterized in that, The composition comprises, in one or more carrier forms, nucleic acids encoding the editing protein and nucleic acids encoding the guide sequence.

20. The method for preparing blood-compatible mesenchymal stem cells according to claim 14, characterized in that, Insertion / deletion occurs in the target sequence by contacting the exon 5 target sequence of the F3 gene located in the genome of the isolated mesenchymal stem 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 exon 5 of the F3 gene.

21. A cell therapy agent for intravenous administration, characterized in that, It contains artificially modified mesenchymal stem cells as an active ingredient, as described in any one of claims 1 to 8.

22. The cell therapy agent for intravenous administration according to claim 15, characterized in that, The cellular therapeutic agent for intravenous administration is used to treat a disease selected from myocardial infarction, heart failure, ischemic cardiomyopathy, myocarditis, ischemic enteritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, acute cholecystitis, acute cholangitis, chronic cholecystitis, acute pancreatitis, chronic pancreatitis, acute nephritis, chronic nephritis, acute renal failure, chronic renal failure, pneumonia, interstitial pneumonia, idiopathic interstitial pneumonia, desquamative interstitial pneumonia, acute interstitial pneumonia, nonspecific interstitial pneumonia, drug-induced lung disease, acute respiratory distress syndrome, chronic obliterative lung disease, cerebral palsy syndrome including cerebral palsy in children, amyotrophic lateral sclerosis, polyneuropathy, spinal muscular atrophy, acute transverse myelitis, stroke, multiple sclerosis, peripheral artery disease, thromboangiitis obliterans, Kawasaki disease, and graft-versus-host disease, wherein the thromboangiitis obliterans is Burger's disease.

23. A composition for cell transplantation or biological tissue regeneration, characterized in that, It contains artificially modified mesenchymal stem cells as an active ingredient, as described in any one of claims 1 to 8.

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.

25. A cell therapy method using stem cells, characterized in that, It includes the step of inhibiting thrombus formation by administering a therapeutically effective amount of artificially modified mesenchymal stem cells according to any one of claims 1 to 8 into the blood vessels of a mammal suffering from a disease or condition.

26. Use of any one of the artificially modified mesenchymal stem cells according to claims 1 to 8 in the preparation of a medicament for inhibiting thrombosis when administered intravascularly to mammals suffering from disease or ailment.

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