Cells having high adaptability under hypoxic conditions, and use thereof

Abstract

To provide cells having high adaptability under hypoxic conditions and a preparation method therefor.SOLUTION: The present disclosure provides cells that have high adaptability under hypoxic conditions, and that includes at least one engineered gene having an indel within a wild-type gene selected from a group consisting of HIF1AN, HIF3A, PHD2, TLR4 and PAI1. In addition, the present disclosure provides, as a method for preparing the cells having high adaptability under hypoxic conditions, a gene editing method including introducing a CRISPR / Cas9 system into the cells.SELECTED DRAWING: Figure 16

Description

[Technical field]

[0001] The present application relates to cells having high adaptability under hypoxic conditions, a method for producing the same, and a cell therapy agent containing the cells as an active ingredient. Recently, various studies have been conducted on the possibility of using stem cells, such as embryonic stem cells and adult stem cells, as cell therapy by differentiating them into various cells. Embryonic stem cells, which have pluripotent differentiation function, have attracted attention as cell therapy agents by differentiating them into various cells, but due to ethical issues in utilizing embryonic stem cells, it is difficult to actually use them as cell therapy. In order to avoid such ethical issues, active research is being conducted on the use of adult stem cells. [Background technology]

[0002] It is known that in cells placed in a hypoxic environment, the expression of HIF1 (Hypoxia-Inducible Factor 1) protein increases in order to survive. HIF1 protein is a transcriptional regulator of genes that play a role in creating an environment in which cells can survive by participating in angiogenesis, vascularization, energy metabolism, etc. in a hypoxic environment, and has two subunits, HIF1α and HIF1β. HIF1α and HIF1β are encoded by the HIF1A and HIF1B genes, respectively. [Prior art documents] [Non-patent literature]

[0003] Mahon PC, Hirota K, Semenza GL.FIH-1: a novel protein that interacts with HIF-1alpha and VHL to mediate repression of HIF-1 transcriptional activity.Genes Dev.2001;15(20):2675?2686.doi:10.1101 / gad.924501. Summary of the Invention [Problem to be solved by the invention]

[0004] The present specification provides cells that have high adaptability in a hypoxic environment.

[0005] Provided herein are compositions for genetic manipulation to produce cells that are highly adaptable to hypoxic environments.

[0006] Provided herein are genetic engineering methods for producing cells that are highly adaptable to hypoxic environments.

[0007] The present specification provides uses of cells that have high adaptability in a hypoxic environment. [Means for solving the problem]

[0008] Provided herein is an engineered cell comprising: at least one engineered gene having an indel in a wild-type gene selected from the group consisting of HIF1AN, HIF3A, PHD2, TLR4, and PAI1; In this case, the sequence of the engineered gene is different from the sequence of the wild-type gene, and the expression level of mRNA transcribed from the wild-type gene in the engineered cell is lower than that in the wild-type cell; In the engineered cells, expression levels of HIFα are characterized as being higher as compared to the wild-type cells.

[0009] In one embodiment, when the engineered cell comprises an engineered gene having an indel in the HIF1AN gene, the indel in the HIF1AN gene is located within exon 1 region of the HIF1AN gene; When the engineered cell comprises an engineered gene having an indel in the HIF3A gene, the indel in the HIF3A gene is located within one or more regions of the HIF3A gene selected from the group consisting of exon 2, exon 3, exon 4, exon 5, and exon 6; wherein the engineered cell comprises an engineered gene having an indel in a PHD2 gene, the indel in the PHD2 gene is located within exon 1 region of the PHD2 gene; When the engineered cell comprises an engineered gene having an indel in the TLR4 gene, the indel in the TLR4 gene is located within one or more regions of the TLR4 gene selected from the group consisting of exon 1, exon 2, and exon 3; Where the engineered cell comprises an engineered gene having an indel within the PAI1 gene, the engineered cell is provided with an indel within the PAI1 gene, where the indel within the PAI1 gene is located within the exon 2 region of the PAI1 gene.

[0010] In one embodiment, when the engineered cell comprises an engineered gene having an indel in the HIF1AN gene, the sequence of the engineered gene does not comprise one or more sequences selected from the group consisting of SEQ ID NOs: 1 to 9; When the engineered cell comprises an engineered gene having an indel in the HIF3A gene, the sequence of the engineered gene does not comprise one or more sequences selected from the group consisting of SEQ ID NOs: 10 to 34; When the engineered cell comprises an engineered gene having an indel in the PHD2 gene, the sequence of the engineered gene does not comprise one or more sequences selected from the group consisting of SEQ ID NOs: 35-38; When the engineered cell comprises an engineered gene having an indel in the TLR4 gene, the sequence of the engineered gene does not comprise one or more sequences selected from the group consisting of SEQ ID NOs: 39 to 59; The present invention provides an engineered cell, wherein the engineered cell comprises an engineered gene having an indel within the PAI1 gene, and the sequence of the engineered gene does not comprise one or more sequences selected from the group consisting of SEQ ID NOs: 60-71.

[0011] In one embodiment, the engineered cell is provided comprising an engineered gene having an indel within the HIF1AN gene.

[0012] In one embodiment, the engineered cell is provided wherein the indel in the HIF1AN gene is located within exon 1 of the HIF1AN gene.

[0013] In one embodiment, an engineered cell is provided in which the sequence of the engineered gene does not include one or more sequences selected from the group consisting of SEQ ID NOs: 1-9.

[0014] In one embodiment, an engineered cell is provided, wherein the expression level of FIH-1 in the engineered cell is less than 70% of the expression level of FIH-1 in the wild-type cell.

[0015] In one embodiment, engineered cells are provided, wherein the engineered cells secrete levels of VEGF and IL-8 that are greater than the levels of VEGF and IL-8 secreted by the wild-type cells.

[0016] In one embodiment, the engineered cell is provided comprising an engineered gene having an indel in the HIF3A gene.

[0017] In one embodiment, the engineered cell is provided, wherein the indel in the HIF3A gene is located in one or more regions selected from the group consisting of exon 2, exon 3, exon 4, exon 5, and exon 6.

[0018] In one embodiment, an engineered cell is provided in which the sequence of the engineered gene does not include one or more sequences selected from the group consisting of SEQ ID NOs: 10-34.

[0019] In one embodiment, the engineered cell provides an engineered cell that comprises an engineered gene with an indel in the PHD2 gene.

[0020] In one embodiment, an engineered cell is provided, wherein the indel in the PHD2 gene is located in the exon 1 region of the PHD2 gene.

[0021] In one embodiment, an engineered cell is provided in which the sequence of the engineered gene does not include one or more sequences selected from the group consisting of SEQ ID NOs: 35-38.

[0022] In one embodiment, the engineered cell provides an engineered cell that comprises an engineered gene with an indel in the TLR4 gene.

[0023] In one embodiment, the engineered cell is provided, wherein the indel in the TLR4 gene is located in one or more regions selected from the group consisting of exon 1, exon 2, and exon 3.

[0024] In one embodiment, an engineered cell is provided in which the sequence of the engineered gene does not include one or more sequences selected from the group consisting of SEQ ID NOs: 39-59.

[0025] In one embodiment, the engineered cell is provided comprising an engineered gene having an indel in the PAI1 gene.

[0026] In one embodiment, an engineered cell is provided, wherein the indel in the PAI1 gene is located in the exon 2 region of the PAI1 gene.

[0027] In one embodiment, an engineered cell is provided, wherein the sequence of the engineered gene does not include one or more sequences selected from the group consisting of SEQ ID NOs: 60-71.

[0028] In one embodiment, the engineered cells are provided as engineered cells that are mesenchymal stem cells derived from a tissue selected from the group consisting of bone marrow, adipose tissue, umbilical cord, placenta, amniotic fluid, and umbilical cord blood.

[0029] In one embodiment, the engineered cells are provided as engineered cells that are bone marrow derived mesenchymal stem cells.

[0030] Provided herein are engineered cells comprising: at least one engineered gene selected from the group consisting of an engineered HIF1AN gene, an engineered HIF3A gene, an engineered PHD2 gene, an engineered TLR4 gene, and an engineered PAI1 gene; In this case, the sequence of the engineered HIF1AN gene does not include one or more sequences selected from SEQ ID NOs: 1 to 9; The sequence of the engineered HIF3A gene does not include one or more sequences selected from SEQ ID NOs: 10 to 34; The sequence of the engineered PHD2 gene does not include one or more sequences selected from SEQ ID NOs: 35 to 38; The sequence of the engineered TLR4 gene does not include one or more sequences selected from SEQ ID NOs: 39 to 59; The sequence of the engineered PAI1 gene does not include one or more sequences selected from SEQ ID NOs: 60 to 71.

[0031] Provided herein is a gene editing composition comprising: A Cas9 protein derived from Streptococcus pyogenes or a DNA encoding the Cas9 protein, and A guide RNA or a DNA encoding the guide RNA, In this case, the guide RNA has a sequence selected from the group consisting of SEQ ID NOs: 146 to 216.

[0032] In one embodiment, the composition provides a gene editing composition comprising the Cas9 protein and the guide RNA in a ribonucleoprotein (RNP) form.

[0033] In one embodiment, the composition provides a genetic manipulation composition comprising DNA encoding the Cas9 protein and DNA encoding the guide RNA in the form of a single vector.

[0034] In one embodiment, a gene editing composition is provided, wherein the single vector is selected from the group consisting of plasmids, AAVs, etc. As used herein, a method for editing a gene in a cell includes introducing a genetic engineering composition into the cell, At this time, the composition for genetic manipulation comprises: A Cas9 protein derived from Streptococcus pyogenes, or a DNA encoding the Cas9 protein, and A guide RNA or a DNA encoding the guide RNA; In this case, the guide RNA has a sequence selected from the group consisting of SEQ ID NOs: 146 to 216.

[0035] In one embodiment, the composition provides a method for editing a gene in a cell, the composition comprising DNA encoding the Cas9 protein and DNA encoding the guide RNA in a vector form.

[0036] Provided herein is a method for editing a gene containing target DNA in a cell, the method comprising: contacting a CRISPR / Cas9 complex with a gene comprising said target DNA; wherein the CRISPR / Cas9 complex comprises: Cas9 protein derived from Streptococcus pyogenes, and A guide RNA that targets the target DNA, wherein the guide RNA has a sequence selected from the group consisting of SEQ ID NOs: 146 to 216. Effect of the Invention

[0037] The present specification provides cells having high adaptability in a hypoxic environment and a method for producing the same. The cells having high adaptability in a hypoxic environment exhibit high adaptability and survival rate in a hypoxic environment, and therefore are suitable for use as a cell therapy agent. [Brief description of the drawings]

[0038] [Figure 1] This is experimental data confirming the presence or absence of genetic manipulation after treatment with a genetic manipulation composition targeting a part of the nucleotide sequence of the hHIF3A gene. Specifically, the occurrence rate of indels (InDel) was confirmed when the guide nucleic acid was changed. [Diagram 2] This is experimental data confirming the presence or absence of genetic manipulation after treatment with a genetic manipulation composition targeting a part of the nucleotide sequence of the hTLR4 gene. Specifically, the occurrence rate of indels when the guide nucleic acid was changed was confirmed. [Diagram 3] This is experimental data confirming the presence or absence of genetic manipulation after treatment with a genetic manipulation composition targeting a part of the nucleotide sequence of the hPAI1 gene. Specifically, the occurrence rate of indels when the guide nucleic acid was changed was confirmed. [Figure 4]This is experimental data confirming the presence or absence of genetic manipulation after treatment with a genetic manipulation composition targeting a part of the nucleotide sequence of the hHIF1AN gene. Specifically, the occurrence rate of indels (InDel) was confirmed when the guide nucleic acid was changed. [Diagram 5] This is experimental data confirming the presence or absence of genetic manipulation after treatment with a genetic manipulation composition targeting a part of the nucleotide sequence of the hPHD2 gene. Specifically, the occurrence rate of indels (InDel) was confirmed when the guide nucleic acid was changed. [Figure 6] After treating the MSCs with the genetic manipulation composition, cells suitable for this experiment were screened using Miseq. [Figure 7-9] Figures 6 and 10 show data comparing mRNA expression levels in cells selected by Miseq using qRT-PCR. [Figure 10] After treating the MSCs with the genetic manipulation composition, cells suitable for this experiment were screened using Miseq. [Figure 11-13] Figures 6 and 10 show data comparing mRNA expression levels in cells selected by Miseq using qRT-PCR. [Figure 14] After treating MSCs with the hHIF1AN gene manipulation composition, cells suitable for this experiment were screened by deep sequencing. [Figure 15] Figure 10 shows the results of a comparison of mRNA expression levels in cells selected by deep sequencing using qRT-PCR. [Figure 16] This data measured the change in MSC proliferation ability due to reactive-oxygen stress, which frequently occurs in low-oxygen conditions, and measured the number of viable cells using the CCK8 assay. [Figure 17]This data measured the change in MSC proliferation ability due to reactive oxygen stress, which frequently occurs in low oxygen conditions. The data measured the number of live cells in hHIF1AN gene knockout MSCs and control MSCs when the H2O2 concentration was 0uM and 500uM. [Figure 18] 1 is a graph comparing the indel efficiency and out-of-frame ratio by deep sequencing after manipulating the HIF1AN gene of mesenchymal stem cells according to an experimental example. Mock indicates cells that were not genetically manipulated, HIF1AN KO indicates cells in which the HIF1AN gene was manipulated, SHS231 KO indicates cells in which the SHS231 genetic sequence position was manipulated, and AAVS1 indicates cells in which the AAVS1 genetic sequence position was manipulated, and the SHS231 KO and AAVS1 were used as controls. [Figure 19] 1 is a graph comparing HIF1AN mRNA expression levels by qRT-PCR after manipulating the HIF1AN gene in mesenchymal stem cells according to an experimental example. Mock indicates cells that were not genetically manipulated, HIF1AN KO indicates cells in which the HIF1AN gene was manipulated, SHS231 KO indicates cells in which the SHS231 genetic sequence position was manipulated, and AAVS1 indicates cells in which the AAVS1 genetic sequence position was manipulated, and the SHS231 KO and AAVS1 were used as controls. [Figure 20] The results of the CCK-8 assay were shown for the viability of mesenchymal stem cells manipulated with the HIF1AN gene produced according to the experimental example against reactive nitrogen species (SNAP). Mock indicates cells that were not genetically manipulated, and HIF1AN KO indicates cells that were manipulated with the HIF1AN gene. [Figure 21-22] 1 shows the results of BrdU cell proliferation analysis to confirm the characteristics of mesenchymal stem cells manipulated with the HIF1AN gene prepared according to the experimental example. Mock indicates cells that were not genetically manipulated, HIF1AN KO indicates cells in which the HIF1AN gene was manipulated, and SHS231 KO indicates cells in which the SHS231 gene sequence position was manipulated, and the SHS231 KO was used as a control. [Figure 23-25] 1 shows the results of FACS analysis to confirm the MSC marker of mesenchymal stem cells manipulated with the HIF1AN gene prepared according to the experimental example. Mock indicates cells that were not genetically manipulated, HIF1AN KO indicates cells in which the HIF1AN gene was manipulated, and SHS231 KO indicates cells in which the SHS231 gene sequence position was manipulated, and the SHS231 KO was used as a control. [Figure 26-27] The results show the results of measuring the PDL (Population Doubling Level) and PDT (Population Doubling Time) of mesenchymal stem cells manipulated with the HIF1AN gene, which were prepared according to the respective experimental examples. Mock indicates cells that were not genetically manipulated, HIF1AN KO indicates cells in which the HIF1AN gene was manipulated, SHS231 KO indicates cells in which the SHS231 genetic sequence position was manipulated, and AAVS1 indicates cells in which the AAVS1 genetic sequence position was manipulated, and the SHS231 KO and AAVS1 were used as controls. [Figure 28] 1 shows the results of comparing the amount of cytokine secretion in the culture medium of mesenchymal stem cells manipulated with the HIF1AN gene, which were prepared according to the experimental example. Mock indicates cells that were not genetically manipulated, HIF1AN KO indicates cells in which the HIF1AN gene was manipulated, and AAVS1 indicates cells in which the AAVS1 gene sequence position was manipulated, and the AAVS1 was used as a control. [Figure 29-30] The results of Experiment 3 were performed with new MSC stock to confirm the reproducibility of the results of the experimental example. Mock is non-genetically manipulated cells, Mock H2O2 300μM+ is non-genetically manipulated cells with 300μM H2O2 added to induce oxidative stress, HIF1AN KO is HIF1AN gene-manipulated cells, HIF1AN KO H2O2 300μM+ is HIF1AN gene-manipulated cells with 300μM H2O2 added to induce oxidative stress. A: Graph comparing HIF1AN mRNA expression levels, B: Graph comparing cytokine (VEGF and IL-8) mRNA expression levels. [Diagram 31] 1 shows the results of Western blot in Experimental Example 4-1. Mock indicates cells that were not genetically manipulated, sgHIF1AN indicates cells in which the HIF1AN gene was manipulated, and sgAAVS1 indicates cells in which the AAVS1 gene sequence position was manipulated, and the sgAAVS1 was used as a control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Hereinafter, the contents of the present invention will be described in more detail with reference to the drawings, with specific implementations and examples. It should be noted that the drawings include some implementations of the present invention, but do not include all implementations. The contents of the invention disclosed in this specification may be embodied in various ways and are not limited to the specific implementations described herein. Such implementations should be considered to be provided to satisfy the legal requirements applicable to this specification. A person of ordinary skill in the art to which the invention disclosed in this specification belongs can think of many variations and other implementations of the contents of the invention disclosed in this specification. Therefore, it should be understood that the contents of the invention disclosed in this specification are not limited to the specific implementations described herein, and variations and other implementations thereon are also included in the scope of the claims. <Terminology>

[0040] medicine The term "drug" as used herein refers to an amount, level, value, number, frequency, percentage, size, magnitude, amount, weight, or length that varies by about 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% relative to a reference amount, level, value, number, frequency, percentage, size, magnitude, amount, weight, or length.

[0041] The meaning of A, T, C, G, and U The symbols A, T, C, G and U used herein are to be interpreted as understood by those skilled in the art. They can be appropriately interpreted as bases, nucleosides or nucleotides on DNA or RNA depending on the context and technology. For example, when referring to a base, they can be interpreted as adenine (A), thymine (T), cytosine (C), guanine (G) or uracil (U) themselves, respectively, when referring to a nucleoside, they can be interpreted as adenosine (A), thymine (T), cytidine (C), guanosine (G) or uridine (U), respectively, and when referring to a nucleotide in a sequence, they should be interpreted as a nucleotide containing each of the nucleosides.

[0042] Engineered The term "engineered" as used herein is used to distinguish a substance, molecule, etc., having a structure already existing in nature, and means that the substance, molecule, etc., has been artificially modified. For example, an "engineered gene" means a gene in which the structure of a gene existing in nature has been artificially modified. In addition, the term includes all meanings that a person skilled in the art can recognize, and can be appropriately interpreted according to the context.

[0043] Wild type "Wild type" means that a gene containing a naturally occurring base sequence and a protein expressed from the gene have normal functional properties. Wild type genes are those most frequently observed in a population. In this specification, when the term "wild type" is used in contrast to artificially manipulated (engineered) genes and / or cells, it can be interpreted as meaning a gene containing a naturally occurring base sequence that is "not artificially manipulated" and a cell having the same, which corresponds to the artificially manipulated gene and / or cell. In addition, the term includes all meanings that a person skilled in the art can recognize, and can be interpreted appropriately depending on the context.

[0044] Knock-out The term "knockout" or "knocked-out gene" as used herein means that the protein expressed by the wild-type gene cannot be produced through the transcription and / or translation process. For example, a cell containing knocked-out gene A may not express the mRNA and / or protein expressed by the wild-type gene A. A cell containing knocked-out gene A may have only one or more of the genes A present in the cell knocked out. In addition, the above terms include all meanings that a person skilled in the art can recognize, and can be interpreted appropriately depending on the context.

[0045] knock-down As used herein, the term "knockdown" or "knocked down gene" refers to expressing a substance in less than the wild-type gene. For example, a cell containing knocked down gene A may express less mRNA than the mRNA expressed by wild-type gene A. A cell in which gene A is knocked down may have only one gene A present in the cell knocked down, or may have two or more genes A knocked down. In addition, the term includes all meanings that a person skilled in the art would recognize, and can be interpreted appropriately according to the context.

[0046] Expression level The term "expression level" as used herein means how frequently a product expressed from a particular gene, such as mRNA and / or protein, is expressed. In general, when the expression level of a particular gene in a cell is high, it indicates that the amount of mRNA and / or a particular protein expressed from the particular gene contained in the cell, or the concentration of the expression product in the cytoplasm is high. Conversely, when the expression level of a particular gene in a cell is low, it indicates that the amount of mRNA and / or a particular protein expressed from the particular gene contained in the cell, or the concentration of the expression product in the cytoplasm is low. In this specification, when "expression level is high" or "expression level is low", the comparison target can be interpreted as the same kind of wild-type cell, unless otherwise specified. In addition, the quantitative indicator that serves as the basis for comparison should be interpreted as the most appropriate in the context. For example, the comparison target can be the absolute amount, relative amount, and / or concentration of each cell, but is not limited thereto. For example, when the expression level of gene a in a first cell is said to be higher than the expression level of gene a in a second cell, the expression product (mRNA, protein, etc.) of gene a in the first cell may be greater in absolute amount, greater in relative amount, and / or greater in concentration than the expression product of gene a in the second cell. Furthermore, the terms include all meanings that one skilled in the art would recognize and may be interpreted appropriately depending on the context.

[0047] Background Art - Factors affecting cell survival in hypoxic environments HIF1 protein It is known that cells placed in a hypoxic environment express a large amount of HIF1 (Hypoxia-Inducible Factor 1) protein in order to survive. HIF1 protein is a transcriptional regulator of genes that plays a role in creating an environment in which cells can survive in a hypoxic environment by participating in angiogenesis, vascularization, energy metabolism, etc., and has subunits HIF1α and HIF1β. HIF1α and HIF1β are encoded by the HIF1A and HIF1B genes, respectively. Therefore, in order for cells to survive in a hypoxic environment, the expression level of HIF1 protein or its subunits must be high or its activity must be maintained at a high level.

[0048] Factor that inhibits HIF1 protein function - FIH1 FIH-1 is a protein encoded by the HIF1AN gene and is involved in the post-translational control of HIF1α. More specifically, FIH-1 is an asparaginyl hydroxylase. FIH-1 plays a role in reducing HIF1 activity by interfering with the interaction between HIF1α and p300 / CBP, a nuclear receptor activator. FIH-1 also plays a role in inducing the degradation of HIF1α after its synthesis in cells. Furthermore, FIH-1 functions even under severe hypoxia conditions, and is therefore a protein directly related to cell survival under hypoxic conditions.

[0049] Factor that inhibits HIF1 protein function - PHD2 PHD2 is a protein that is involved in the post-translational control of HIF1α together with FIH-1. More specifically, PHD2 is a prolyl hydroxylase. PHD2 plays a role in inducing the degradation of HIF1α in cells after it is synthesized.

[0050] Factor that inhibits HIF1 protein function - HIF3A HIF3A is a protein involved in the transcription control of HIF1α and plays a role in interfering with the transcription of the HIF1α gene.

[0051] Other factors that affect cell survival in hypoxic environments TLR4 is known to be a factor involved in the innate immune response to injury. TLR4 activated under hypoxic conditions functions to induce pro-apoptotic signaling, ultimately inducing apoptosis. PAI1 is a factor that negatively regulates extracellular matrix attachment of mesenchymal stem cells (MSCs). It is known that expression of PAI1 increases under hypoxic conditions and is known to affect autograft survival of stem cells.

[0052] Background Technology - CRISPR / Cas System General The CRISPR / Cas system is a type of immune system found in prokaryotic organisms, and includes a Cas protein and a guide RNA. The detailed structure of the Cas protein or the guide RNA is described in detail in the published document WO2018 / 231018 (International Publication Number). The term "Cas protein" used herein is a general term for nucleases that can be interpreted as being used in the CRISPR / Cas system. However, for ease of explanation, the DNA cleavage process of the most commonly used CRISPR / Cas9 system will be briefly described below as an example.

[0053] Cas9 protein In the CRISPR / Cas9 complex, the protein with nuclease activity that cleaves nucleic acids is called the Cas9 protein. The Cas9 protein corresponds to Class 2, Type II in the CRISPR / Cas system classification, and examples thereof include Cas9 proteins derived from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, and Streptosporangium roseum. Among these, the Cas9 protein most commonly used for research purposes is the Cas9 protein derived from Streptococcus pyogenes.

[0054] Guide RNA In the CRISPR / Cas9 complex, an RNA that has the function of inducing the CRISPR / Cas9 complex to recognize a specific sequence contained in a target nucleic acid is called a guide RNA. The configuration of the guide RNA can be classified functionally into 1) a scaffold sequence portion and 2) a guide sequence portion. The scaffold sequence portion is a portion that interacts with the Cas9 protein and binds to the Cas9 protein to form a complex. In general, the scaffold sequence portion includes a tracrRNA and a crRNA repeat sequence portion, and the scaffold sequence is determined depending on the type of Cas9 protein to be used. The guide sequence portion is a portion that can bind complementarily to a nucleotide sequence portion of a certain length in the target nucleic acid. The guide sequence portion is a base portion that can be artificially modified and is determined by the desired target nucleotide sequence.

[0055] How the CRISPR / Cas9 complex cleaves target nucleic acid When the CRISPR / Cas9 complex contacts a target nucleic acid, the Cas9 protein recognizes a nucleotide sequence of a certain length, and a portion of the guide RNA (the guide sequence portion) binds complementarily to a portion adjacent to the PAM sequence, the target nucleic acid is cleaved by the CRISPR / Cas9 complex.

[0056] In this case, the nucleotide sequence of a certain length that the Cas9 protein recognizes is called a protospacer-adjacent motif (PAM) sequence, which is determined by the type and origin of the Cas9 protein. For example, the Cas9 protein derived from Streptococcus pyogenes can recognize the 5'-NGG-3' sequence in the target nucleic acid, where N is one of adenosine (A), thymidine (T), cytidine (C), and guanosine (G).

[0057] In order for the CRISPR / Cas9 complex to cleave the target nucleic acid, the guide sequence portion of the guide RNA must bind complementarily to a portion of the sequence adjacent to the PAM sequence, and therefore the guide sequence portion is determined by the target nucleic acid sequence, specifically the sequence portion adjacent to the PAM sequence.

[0058] When the CRISPR / Cas9 complex cleaves the target nucleic acid, it cleaves either at the PAM sequence portion of the target nucleic acid and / or at a position within the sequence portion that complementarily binds to the guide sequence.

[0059] Target strand, non-target strand The CRISPR / Cas9 complex has a cleavage activity against double-stranded DNA. In the double-stranded DNA, a strand having a protospacer that binds to the guide sequence portion is called a target strand (TS). The target strand is a complementary strand, and a strand having a protospacer that does not bind to the guide sequence portion is called a non-target strand (NTS). The guide sequence portion can bind complementarily to a protospacer sequence portion contained in the target strand (TS) of double-stranded DNA. The guide sequence and the protospacer sequence contained in the non-target strand (NTS) of double-stranded DNA are equivalent sequences. Specifically, the guide sequence is an RNA sequence, and the protospacer sequence contained in the non-target strand (NTS) only differs from the corresponding DNA sequence.

[0060] Limitations of conventional technology Recently, there has been much research into so-called "cell therapy," which uses cells themselves as a therapeutic agent for the purpose of treating, diagnosing, and / or preventing a disease. The cells used in cell therapy are cells capable of expressing a therapeutic substance or cells that have all of their original functions, and are directly administered to the site of a disease or damaged tissue or organ to treat the disease or regenerate the damaged tissue or organ. To achieve this goal, the cells administered as cell therapy must survive for a long time at the site of administration and perform normal functions. However, when cells for treatment are selected and grown outside the body to create a cell therapy and then administered into the body, there is a problem that the cells cannot adapt to the body's environment and die within a short period of time. The reasons for cell death are 1) the environment in which they are cultured outside the body is different from the environment inside the body, 2) the body is in a hypoxic environment, 3) there is a lack of nutrients inside the body, 4) the administered cells do not attach to the ECM inside the body, and 5) an immune response occurs, and among these, the death of cells due to a hypoxic environment is representative. For these reasons, conventional cell therapy agents could no longer guarantee a sufficient survival time within the body, making it difficult to achieve goals such as treating diseases and regenerating tissue.

[0061] Cells with high adaptability in low oxygen environments General This specification discloses cells having high adaptability under hypoxic environment. The type of the cells is not particularly limited as long as they can be used for cell therapy. The cells are characterized in that a wild-type gene expressing a substance that inhibits the expression of HIF1 protein is artificially manipulated. Accordingly, the expression level of the substance that inhibits the expression of HIF1 protein (negative regulator of HIF1 expression) in the cells is reduced compared to wild-type cells. As a result, the cells are characterized in that the expression level and / or activity level of HIF1 protein maintained in the cells is higher than that of wild-type cells. The cells having high adaptability under hypoxic environment disclosed in this specification are characterized in that they have high adaptability or survival ability in hypoxic environment and are suitable for use as cell therapy.

[0062] In one embodiment, the cells having high adaptability to hypoxic environments provided herein may be engineered cells. In one embodiment, the engineered cells may be engineered mesenchymal stem cells.

[0063] In one embodiment, the engineered cell may have at least one engineered gene having an indel in a wild-type gene selected from the group consisting of HIF1AN, HIF3A, PHD2, TLR4, and PAI1 that expresses an agent that inhibits expression of HIF1 protein. In one embodiment, in the engineered cell, the expression level of mRNA transcribed from the wild-type gene may be lower compared to a wild-type cell. In one embodiment, in the engineered cell, the expression level of HIFα may be higher compared to a wild-type cell.

[0064] In one embodiment, the engineered cell can comprise at least one engineered gene selected from the group consisting of an engineered HIF1AN gene, an engineered HIF3A gene, an engineered PHD2 gene, an engineered TLR4 gene, and an engineered PAI1 gene.

[0065] Cell types In one embodiment, the cells having high adaptability under hypoxic environment disclosed herein may be somatic cells, such as oocytes, epithelial cells, fibroblasts, nerve cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, macrophages, monocytes, muscle cells, B lymphocytes, T lymphocytes, dendritic cells, NK cells, regulatory T cells, germ cells, embryonic stem cells, neural stem cells, mesenchymal stem cells, adult stem cells, such as stem cells derived from tissues such as fat, uterus, bone marrow, liver, muscle, placenta, nerve, umbilical cord blood or skin (epithelium), and / or induced pluripotent stem cells (iPSCs). In one embodiment, the cells may be mesenchymal stem cells. In one embodiment, the cells may be mesenchymal stem cells derived from bone marrow, adipose tissue, umbilical cord, placenta, amniotic fluid, and / or umbilical cord blood. In one embodiment, the cells may be mesenchymal stem cells derived from bone marrow.

[0066] 1. Genotyping of Cells - Engineering the Target Gene The cells having high adaptability to a hypoxic environment are characterized in that a wild-type gene expressing a substance that inhibits the transcription, expression, and / or activity of HIF1 protein is artificially edited. In other words, the cells contain an engineered gene that expresses a substance that inhibits the transcription, expression, and / or activity of HIF1 protein.

[0067] In one embodiment, the artificially edited wild-type gene may be HIF1AN, HIF3A, PHD2, TLR4, and / or PAI1 gene. In this case, the wild-type gene may be, but is not limited to, a human gene. In one embodiment, the cell may include an engineered HIF1AN, an engineered HIF3A, an engineered PHD2, an engineered TLR4, and / or an engineered PAI1 gene. In this case, the sequence of the engineered gene is different from the sequence of the corresponding wild-type gene. Specifically, the sequence of the engineered HIF1AN is different from the sequence of the wild-type HIF1AN. Specifically, the sequence of the engineered HIF3A is different from the sequence of the wild-type HIF3A. Specifically, the sequence of the engineered PHD2 is different from the sequence of the wild-type PHD2. Specifically, the sequence of the engineered TLR4 is different from the sequence of the wild-type TLR4. Specifically, the sequence of the engineered PAI1 is different from the sequence of the wild-type PAI1.

[0068] 2. Genotyping of Cells - Characterization of Engineered Target Genes The engineered gene is characterized by having a sequence different from that of a wild-type gene. The engineered gene may be unable to express a substance that is expressed by a non-engineered wild-type gene. The engineered gene may express a substance that is expressed by a wild-type gene at a lower level than the wild-type gene.

[0069] In one embodiment, the engineered gene may be a mutated form of a wild-type gene, where the mutated form is an artificially generated mutation rather than a mutation already present in nature.

[0070] In one embodiment, the engineered gene may be a knock-out of its corresponding wild-type gene.

[0071] In one embodiment, the engineered gene may be a knock-down of its corresponding wild-type gene.

[0072] In one embodiment, the engineered gene may have one or more nucleotide insertions, deletions, substitutions, and / or inversions compared to the corresponding wild-type gene.

[0073] In one embodiment, the engineered gene may contain an indel in the corresponding wild-type gene. In this case, the indel may be generated by a non-homologous end joining (NHEJ) mechanism in a cell, and one or more nucleotides in the wild-type gene may be inserted, deleted, and / or substituted by the NHEJ mechanism. In this case, the expression "containing an indel in a gene" refers to the insertion, deletion, and / or substitution of one or more nucleotides in the wild-type gene, and / or the result thereof.

[0074] For example, an engineered A gene that contains an indel in a wild-type A gene may have one or more nucleotides inserted at any position in the wild-type A gene. In another example, an engineered A gene that contains an indel in a wild-type A gene may have one or more nucleotides deleted at any position in the wild-type A gene. In another example, an engineered A gene that contains an indel in a wild-type A gene may have one or more nucleotides replaced at any position in the wild-type A gene with a different one.

[0075] 3. Genotyping of Cells - Example of an Engineering Region within a Gene of Interest As described above, the cells having high adaptability under a hypoxic environment provided herein are characterized in that a wild-type gene expressing a substance that inhibits the transcription, expression, and / or activity of HIF1 protein is artificially edited, which means that one or more regions in the wild-type gene are engineered. In this case, the engineered region in the wild-type gene is not particularly limited as long as it can achieve the above-mentioned purpose.

[0076] In one embodiment, the cell having high adaptability to a hypoxic environment may be one in which an exon region, an intron region and / or a regulatory region in the wild-type gene is edited, and the wild-type gene may be selected from the group consisting of HIF1AN, HIF3A, PHD2, TLR4 and PAI1.

[0077] In one embodiment, the cells having high adaptability in a hypoxic environment may include an engineered gene in which one or more regions selected from exon 1, exon 2, exon 3, exon 4, exon 5, ..., and exon N of one or more wild-type genes selected from the group consisting of HIF1AN, HIF3A, PHD2, TLR4, and PAI1 genes are artificially edited. In this case, N is an arbitrary integer. In one embodiment, the cells having high adaptability in a hypoxic environment may include an engineered gene in which one or more regions selected from intron 1, intron 2, intron 3, intron 4, intron 5, ..., and intron M of one or more wild-type genes selected from the group consisting of HIF1AN, HIF3A, PHD2, TLR4, and PAI1 genes are artificially edited. In this case, M is an arbitrary integer.

[0078] In one embodiment, the cell can comprise an engineered HIF1AN gene in which an exon 1 region in a wild-type HIF1AN gene has been artificially edited. In one embodiment, the cell can comprise an engineered HIF3A gene in which one or more regions selected from the group consisting of exon 2, exon 3, exon 4, exon 5, and exon 6 in a wild-type HIF3A gene have been artificially edited. In one embodiment, the cell can comprise an engineered PHD2 gene in which an exon 1 region in a wild-type PHD2 gene has been artificially edited. In one embodiment, the cell can comprise an engineered TLR4 gene in which one or more regions selected from the group consisting of exon 1, exon 2, and exon 3 in a wild-type TLR4 gene have been artificially edited. In one embodiment, the cell can comprise an engineered PAI1 gene in which an exon 2 region in a wild-type PAI1 gene has been artificially edited.

[0079] In one embodiment, when the engineered cell comprises an engineered gene having an indel in the HIF1AN gene, the indel in the HIF1AN gene can be located in the exon 1 region of the HIF1AN gene. When the engineered cell comprises an engineered gene having an indel in the HIF3A gene, the indel in the HIF3A gene can be located in one or more regions selected from the group consisting of exon 2, exon 3, exon 4, exon 5, and exon 6 of the HIF3A gene. When the engineered cell comprises an engineered gene having an indel in the PHD2 gene, the indel in the PHD2 gene can be located in the exon 1 region of the PHD2 gene. When the engineered cell comprises an engineered gene having an indel in the TLR4 gene, the indel in the TLR4 gene can be located in one or more regions selected from the group consisting of exon 1, exon 2, and exon 3 of the TLR4 gene. Where the engineered cell comprises an engineered gene having an indel within the PAI1 gene, the indel within the PAI1 gene can be located within the exon 2 region of the PAI1 gene.

[0080] 4. Genotyping of Cells - Examples of Engineering Target Sequences In one embodiment, the cell having high adaptability in a hypoxic environment provided herein may be one in which one or more sequences selected from SEQ ID NOs: 1 to 9 in a HIF1AN gene have been artificially edited. In one embodiment, the cell comprises an engineered HIF1AN gene, and the sequence of the engineered HIF1AN gene may not include one or more sequences selected from SEQ ID NOs: 1 to 9. In other words, there may be no sequence in the sequence of the engineered HIF1AN gene that matches one or more sequences selected from SEQ ID NOs: 1 to 9.

[0081] In one embodiment, the cell having high adaptability under a hypoxic environment provided herein may be one in which one or more sequences selected from SEQ ID NOs: 10 to 34 in a HIF3A gene have been artificially edited. In one embodiment, the cell comprises an engineered HIF3A gene, and the sequence of the engineered HIF3A gene may not include one or more sequences selected from SEQ ID NOs: 10 to 34. In other words, there may be no sequence in the sequence of the engineered HIF3A gene that matches one or more sequences selected from SEQ ID NOs: 10 to 34.

[0082] In one embodiment, the cells having high adaptability under low oxygen environment provided herein may be those in which one or more sequences selected from SEQ ID NOs: 35 to 38 in the PHD2 gene have been artificially edited. In one embodiment, the cells include an engineered PHD2 gene, and the sequence of the engineered PHD2 gene may not include one or more sequences selected from SEQ ID NOs: 35 to 38. In other words, there may be no sequence in the sequence of the engineered PHD2 gene that matches one or more sequences selected from SEQ ID NOs: 35 to 38.

[0083] In one embodiment, the cells having high adaptability under a hypoxic environment provided herein may be those in which one or more sequences selected from SEQ ID NOs: 39 to 59 in a TLR4 gene have been artificially edited. In one embodiment, the cells include an engineered TLR4 gene, and the sequence of the engineered TLR4 gene may not include one or more sequences selected from SEQ ID NOs: 39 to 59. In other words, there may be no sequence in the sequence of the engineered TLR4 gene that matches one or more sequences selected from SEQ ID NOs: 39 to 59.

[0084] In one embodiment, the cell having high adaptability in a hypoxic environment provided herein may be one in which one or more sequences selected from SEQ ID NOs: 60 to 71 in the PAI1 gene have been artificially edited. In one embodiment, the cell comprises an engineered PAI1 gene, and the sequence of the engineered PAI1 gene may not include one or more sequences selected from SEQ ID NOs: 60 to 71. In other words, there may be no sequence in the sequence of the engineered PAI1 gene that matches one or more sequences selected from SEQ ID NOs: 60 to 71.

[0085] 1. Cellular Phenotype - Reduction in Expression Product of Engineered Gene The cells disclosed herein that are highly adaptable to hypoxic environments are characterized in that a wild-type gene expressing a substance that inhibits the expression of HIF1 protein is artificially manipulated to prevent or reduce the expression of the substance expressed by the wild-type gene. Thus, the cells express a reduced amount of the substance that inhibits the expression of HIF1 protein (negative regulator of HIF1 expression) compared to wild-type cells.

[0086] In one embodiment, the cell having high adaptability in a hypoxic environment is characterized by having at least one engineered gene having an indel in a gene selected from the group consisting of wild-type HIF1AN, HIF3A, PHD2, TLR4, and PAI1 genes, and the sequence of the mRNA expressed from the engineered gene may be different from the sequence of the mRNA expressed from the wild-type gene. In one embodiment, the cell having high adaptability in a hypoxic environment is characterized by having an engineered HIF1AN gene having an indel in a wild-type HIF1AN gene, and the sequence of the mRNA expressed from the engineered HIF1AN gene may be different from the sequence of the mRNA expressed from the wild-type HIF1AN gene. In one embodiment, the cell having high adaptability in a hypoxic environment is characterized by having an engineered HIF3A gene having an indel in a wild-type HIF3A gene, and the sequence of the mRNA expressed from the engineered HIF3A gene may be different from the sequence of the mRNA expressed from the wild-type HIF3A gene. In one embodiment, the cell having high adaptability in a hypoxic environment is characterized by having an engineered PHD2 gene having an indel in a wild-type PHD2 gene, and the sequence of the mRNA expressed from the engineered PHD2 gene may be different from the sequence of the mRNA expressed from the wild-type PHD2 gene. In one embodiment, the cell having high adaptability in a hypoxic environment is characterized by having an engineered TLR4 gene having an indel in a wild-type TLR4 gene, and the sequence of the mRNA expressed from the engineered TLR4 gene may be different from the sequence of the mRNA expressed from the wild-type TLR4 gene. In one embodiment, the cell having high adaptability in a hypoxic environment is characterized by having an engineered PAI1 gene having an indel in a wild-type PAI1 gene, and the sequence of the mRNA expressed from the engineered PAI1 gene may be different from the sequence of the mRNA expressed from the wild-type PAI1 gene.

[0087] In one embodiment, the cells having high adaptability in a hypoxic environment are characterized in that they have at least one engineered gene having an indel in a gene selected from the group consisting of wild-type HIF1AN, HIF3A, PHD2, TLR4, and PAI1, and the sequence of the mRNA expressed from the engineered gene is identical to the sequence of the mRNA expressed from the wild-type gene, and the expression level of the mRNA expressed from the engineered gene in the cells having high adaptability in a hypoxic environment may be even lower than that of the wild-type cells. In one embodiment, the cells having high adaptability in a hypoxic environment are characterized in that they have an engineered HIF1AN gene having an indel in the wild-type HIF1AN gene, and the sequence of the mRNA expressed from the engineered HIF1AN gene is identical to the sequence of the mRNA expressed from the wild-type HIF1AN gene, and in this case, the expression level of the mRNA expressed from the engineered HIF1AN gene in the cells having high adaptability in a hypoxic environment may be even lower than the expression level of the mRNA expressed from the wild-type HIF1AN gene in wild-type cells. In one embodiment, the cells having high adaptability in a hypoxic environment are characterized in that they have an engineered HIF3A gene having an indel in the wild-type HIF3A gene, and the sequence of the mRNA expressed from the engineered HIF3A gene is identical to the sequence of the mRNA expressed from the wild-type HIF3A gene, and in this case, the expression level of the mRNA expressed from the engineered HIF3A gene in the cells having high adaptability in a hypoxic environment may be even lower than the expression level of the mRNA expressed from the wild-type HIF1A gene in wild-type cells.In one embodiment, the cells having high adaptability in a hypoxic environment are characterized by having an engineered PHD2 gene having an indel in a wild-type PHD2 gene, and the sequence of the mRNA expressed from the engineered PHD2 gene is identical to the sequence of the mRNA expressed from the wild-type PHD2 gene, and in this case, the expression level of the mRNA expressed from the engineered PHD2 gene in the cells having high adaptability in a hypoxic environment may be even lower than the expression level of the mRNA expressed from the wild-type PHD2 gene in a wild-type cell. In one embodiment, the cells having high adaptability under hypoxic environment are characterized by having an engineered TLR4 gene having an indel in a wild-type TLR4 gene, and the sequence of mRNA expressed from the engineered TLR4 gene is identical to the sequence of mRNA expressed from the wild-type TLR4 gene, and in this case, the expression level of mRNA expressed from the engineered TLR4 gene in the cells having high adaptability under hypoxic environment may be even lower than the expression level of mRNA expressed from the wild-type TLR4 gene in wild-type cells. In one embodiment, the cells having high adaptability in a hypoxic environment are characterized in that they have an engineered PAI1 gene having an indel in a wild-type PAI1 gene, and the sequence of the mRNA expressed from the engineered PAI1 gene is identical to the sequence of the mRNA expressed from the wild-type PAI1 gene, and in this case, the expression level of the mRNA expressed from the engineered PAI1 gene in the cells having high adaptability in a hypoxic environment may be even lower than the expression level of the mRNA expressed from the wild-type PAI1 gene in a wild-type cell.

[0088] In one embodiment, the cells having high adaptability under hypoxic environment may be cells having a reduced amount of mRNA expressed from HIF1AN, HIF3A, PHD2, TLR4 and / or PAI1 genes. In one embodiment, in the cells having high adaptability under hypoxic environment, the expression level of mRNA expressed from HIF1AN, HIF3A, PHD2, TLR4 and / or PAI1 genes may be further lower than that of wild-type cells.

[0089] In one embodiment, the cells having high adaptability under hypoxic environment may be cells having reduced amounts of proteins expressed from HIF1AN, HIF3A, PHD2, TLR4 and / or PAI1 genes. In one embodiment, in the cells having high adaptability under hypoxic environment, the expression levels of proteins expressed from HIF1AN, HIF3A, PHD2, TLR4 and / or PAI1 genes may be further lower than that of wild-type cells.

[0090] In one embodiment, when the cell having high adaptability under a hypoxic environment comprises an engineered HIF1AN gene having an indel in the HIF1AN gene, the concentration of mRNA expressed from HIF1AN and / or FIH-1 contained in the cell may be lower than the concentration contained in a wild-type cell. In one embodiment, when the cell having high adaptability under a hypoxic environment comprises an engineered HIF1AN gene having an indel in the HIF1AN gene, the expression level of mRNA expressed from HIF1AN and / or FIH-1 in the cell may be lower than the expression level in a wild-type cell.

[0091] 2. Cellular phenotypes - Examples of quantitative indicators related to the reduction in expression product of engineered genes In one embodiment, the cells having high adaptability under hypoxic environment may have an mRNA expression level expressed from HIF1AN, HIF3A, PHD2, TLR4 and / or PAI1 genes that is about 0.9 times, about 0.85 times, about 0.8 times, about 0.75 times, about 0.7 times, about 0.65 times, about 0.6 times, about 0.55 times, about 0.5 times, about 0.45 times, about 0.4 times, about 0.35 times, about 0.3 times, about 0.25 times, about 0.2 times, about 0.15 times, about 0.10 times, about 0.05 times, about 0.04 times, about 0.03 times, about 0.02 times, or about 0.01 times or less compared to wild-type cells. In one embodiment, the cells having high adaptability under hypoxic environment may exhibit an mRNA expression level within the two numerical ranges selected in the preceding sentence compared to wild-type cells. For example, the cells having high adaptability to a hypoxic environment can exhibit an mRNA expression level that is about 0.6 to about 0.7 times higher than that of wild-type cells.

[0092] In one embodiment, the cells having high adaptability under hypoxic environment may have an expression level of a protein expressed from HIF1AN, HIF3A, PHD2, TLR4 and / or PAI1 gene that is about 0.9 times, about 0.85 times, about 0.8 times, about 0.75 times, about 0.7 times, about 0.65 times, about 0.6 times, about 0.55 times, about 0.5 times, about 0.45 times, about 0.4 times, about 0.35 times, about 0.3 times, about 0.25 times, about 0.2 times, about 0.15 times, about 0.10 times, about 0.05 times, about 0.04 times, about 0.03 times, about 0.02 times, or about 0.01 times or less compared to a wild-type cell. In one embodiment, the cells having high adaptability under hypoxic environment may exhibit a protein expression level within the two numerical ranges selected in the preceding sentence compared to a wild-type cell. For example, the cells having high adaptability in a hypoxic environment can exhibit a protein expression level that is about 0.6 to about 0.7 times higher than that of wild-type cells.

[0093] In one embodiment, the cells having high adaptability under hypoxic environment may have an FIH-1 expression level of about 0.9-fold, about 0.85-fold, about 0.8-fold, about 0.75-fold, about 0.7-fold, about 0.65-fold, about 0.6-fold, about 0.55-fold, about 0.5-fold, about 0.45-fold, about 0.4-fold, about 0.35-fold, about 0.3-fold, about 0.25-fold, about 0.2-fold, about 0.15-fold, about 0.10-fold, about 0.05-fold, about 0.04-fold, about 0.03-fold, about 0.02-fold, or about 0.01-fold or less compared to wild-type cells. In one embodiment, the cells having high adaptability under hypoxic environment may exhibit an FIH-1 expression level within the two numerical ranges selected in the preceding sentence compared to wild-type cells. For example, the cells having high adaptability to a hypoxic environment can exhibit an FIH-1 expression level that is about 0.6 to about 0.7 times higher than that of wild-type cells.

[0094] Cellular phenotype 3 – HIFα expression related The cells disclosed herein that have high adaptability to a hypoxic environment exhibit a high intracellular HIF1α expression level or high HIF1α activity.

[0095] In one embodiment, the cells having high adaptability to a hypoxic environment may have a higher HIF1α expression level compared to wild-type cells, where the HIF1α expression level may be, but is not limited to, the amount and / or concentration of intracellular HIF1α.

[0096] In one embodiment, the cells having high adaptability to a hypoxic environment may exhibit higher HIF1α activity compared to wild-type cells.

[0097] Cellular phenotype 4 - Quantitative indicators related to HIF1α expression In one embodiment, the cells having high adaptability in a hypoxic environment may have an expression level of HIF1α of about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2.0 times, about 2.1 times, about 2.2 times, about 2.3 times, about 2.4 times, about 2.5 times, about 2.6 times, about 2.7 times, about 2.8 times, about 2.9 times, or about 3.0 times or more, compared to wild-type cells. In one embodiment, the cells having high adaptability in a hypoxic environment may have an expression level of HIF1α within the two numerical ranges selected above, compared to wild-type cells. For example, the cells having high adaptability in a hypoxic environment may have an expression level of HIF1α of about 1.1 times to about 1.5 times, compared to wild-type cells.

[0098] In one embodiment, the cells having high adaptability in the hypoxic environment may have a HIF1α activity level of about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2.0 times, about 2.1 times, about 2.2 times, about 2.3 times, about 2.4 times, about 2.5 times, about 2.6 times, about 2.7 times, about 2.8 times, about 2.9 times, or about 3.0 times or more compared to wild-type cells. In one embodiment, the cells having high adaptability in the hypoxic environment may exhibit a HIF1α activity level within the two numerical ranges selected in the preceding sentence compared to wild-type cells. For example, the cells having high adaptability in the hypoxic environment may exhibit a HIF1α activity level of about 1.1 times to about 1.5 times compared to wild-type cells.

[0099] Cell Characteristics 1 - High survival rate in low oxygen environments The cells having high adaptability in a hypoxic environment disclosed herein are characterized by exhibiting improved adaptability or survival ability in a hypoxic environment. In this regard, a "hypoxic environment" refers to a cell that is exposed to a low concentration of oxygen (O 2 ) in a gaseous environment containing oxygen. As a result, cells with high adaptability in the hypoxic environment exhibit improved adaptability or survival when injected into a living organism (e.g., a human).

[0100] In one embodiment, the cells disclosed herein that are highly adaptable to hypoxic conditions are characterized by surviving longer than wild-type cells in hypoxic conditions.

[0101] Cell Characteristics 2 - Quantitative indicators of high survival rate in low oxygen environments In one embodiment, the hypoxic environment is about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, or about 21% O 2 In one embodiment, the hypoxic environment may refer to a gaseous environment having a percent concentration of H 2 O 2 The hypoxic environment may be an environment in which reactive oxidative stress (ROS), which frequently occurs in the hypoxic environment, is artificially induced by treatment with .

[0102] In one embodiment, when one or more of the cells having high adaptability in a hypoxic environment disclosed herein and wild-type cells are placed in the same hypoxic environment for a certain period of time, the number of cells having high adaptability in the hypoxic environment that survive is about 1.01 times, about 1.02 times, about 1.03 times, about 1.04 times, about 1.05 times, about 1.06 times, about 1.07 times, about 1.08 times, about 1.09 times, about 1.1 times, about 1.15 times the number of surviving wild-type cells. , about 1.2 times, about 1.25 times, about 1.3 times, about 1.35 times, about 1.4 times, about 1.45 times, about 1.5 times, about 1.55 times, about 1.6 times, about 1.65 times, about 1.7 times, about 1.75 times, about 1.8 times, about 1.85 times, about 1.9 times, about 1.95 times, about 2.0 times, about 2.1 times, about 2.2 times, about 2.3 times, about 2.4 times, about 2.5 times, about 2.6 times, about 2.7 times, about 2.8 times, about 2.9 times, about 3.0 times, about 4 times, about 5 times, or about 10 times or more. In one embodiment, the number of surviving cells having high adaptability in a hypoxic environment compared to the number of surviving wild type cells may be a number within two numerical ranges selected from the previous sentence. For example, the number of surviving cells having high adaptability in a hypoxic environment may be about 1.5 to about 2.5 times the number of surviving wild-type cells.

[0103] In one embodiment, the certain period of time may be about 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about 12 hours, about 24 hours, about 48 hours, about 72 hours, about 98 hours, about 120 hours, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or about one month or more. In one embodiment, the certain period of time may be a period within the above-mentioned two selected numerical ranges. For example, the certain period of time may be about 3 hours to about 12 hours.

[0104] Cellular Characteristics 3 – Cytokine Secretion The cells having high adaptability in a hypoxic environment disclosed herein may be cells that secrete large amounts of a particular cytokine or a particular growth factor.

[0105] In one embodiment, the cells having high adaptability in a hypoxic environment may have a higher secretion level of cytokines compared to wild-type cells. In one embodiment, the cells having high adaptability in a hypoxic environment may have a higher secretion level of VEGF and / or IL-8 compared to wild-type cells. In one embodiment, the cells having high adaptability in a hypoxic environment may contain an engineered HIF1AN gene and have a higher secretion level of VEGF and / or IL-8 compared to wild-type cells.

[0106] In one embodiment, the cells having high adaptability in a hypoxic environment may have a higher secretion level of cytokines in the hypoxic environment compared to wild-type cells. In one embodiment, the cells having high adaptability in a hypoxic environment may have a higher secretion level of VEGF and / or IL-8 in the hypoxic environment compared to wild-type cells. In one embodiment, the cells having high adaptability in a hypoxic environment may comprise an engineered HIF1AN gene and have a higher secretion level of VEGF and / or IL-8 in the hypoxic environment compared to wild-type cells.

[0107] Cell Characteristics 4 - Quantitative Indicators for Cytokine Secretion In one embodiment, the cells having high adaptability under a hypoxic environment can secrete a specific cytokine in an amount of about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times, about 1.6 times, about 1.7 times, about 1.8 times, about 1.9 times, about 2.0 times, about 2.1 times, about 2.2 times, about 2.3 times, about 2.4 times, about 2.5 times, about 2.6 times, about 2.7 times, about 2.8 times, about 2.9 times, or about 3.0 times, compared to wild-type cells. In one embodiment, the cells having high adaptability under a hypoxic environment can secrete a specific cytokine in an amount within the two numerical ranges selected above, compared to wild-type cells. For example, the amount may be about 1.1 times to about 1.5 times. In this case, the specific cytokine may be VEGF and / or IL-8, but is not limited thereto.

[0108] Cell Uses The cells disclosed in the present specification that have high adaptability to a hypoxic environment can be used as a cell therapy agent.

[0109] In one embodiment, the cells having high adaptability under a low oxygen environment can be used for the prevention, symptom relief, regeneration, or treatment of cancer, Alzheimer's disease, arthritis, for example, osteoarthritis, a degenerative joint disease caused by aging, as well as other autoimmune diseases such as rheumatoid arthritis and psoriatic arthritis, and septic arthritis caused by infection. In another embodiment, the present invention can be used to prevent, alleviate, regenerate, or treat diabetic retinopathy, damaged bone, cartilage, skin, cardiac muscle, and other organs, osteogenesis imperfecta (OI), Crohn's fistula, diabetic foot ulcer, myocardial infarction, Huntington's disease, Parkinson's disease, Lou Gehrig's disease, active rickets, Paget's disease of bone (osteitis deformans), fibrodysplasia ossificans progressiva, proarthritis, enteropathic joint inflammation, and the like.

[0110] Composition for genetic manipulation to produce cells with high adaptability in hypoxic environments Summary The genetic manipulation composition for producing cells having high adaptability in a hypoxic environment disclosed herein has a configuration capable of editing intracellular engineering target genes using a CRISPR / Cas system. The genetic manipulation composition is designed to recognize and edit a suitable intracellular target sequence so as to exhibit the genotype and / or phenotype of the cells having high adaptability in the above-mentioned hypoxic environment. In one embodiment, the genetic manipulation composition can include a Cas9 protein or a DNA encoding the same, and a guide RNA or a DNA encoding the same. In one embodiment, the Cas9 protein may be a Cas9 protein derived from Streptococcus pyogenes. In one embodiment, the sequence of the guide RNA may be a sequence selected from the group consisting of SEQ ID NOs: 146 to 216.

[0111] CRISPR / Cas system component 1 - Cas9 protein The genetic manipulation composition for producing cells having high adaptability under hypoxic conditions disclosed herein includes a Cas9 protein or a DNA encoding the same. In this case, the Cas9 protein may be a naturally occurring Cas9 protein or a Cas9 protein in which one or more domains of a naturally occurring Cas9 protein have been artificially modified. In one embodiment, the Cas9 protein may be a Cas9 protein in which the functions of some or all of the domains contained in the Cas9 protein have been inactivated. In one embodiment, the Cas9 protein may be a Cas9 protein derived from Streptococcus pyogenes.

[0112] CRISPR / Cas system component 2 - Guide RNA The genetic manipulation composition for producing cells having high adaptability in a hypoxic environment disclosed herein includes a guide RNA or a DNA encoding the guide RNA. In this case, the guide RNA can bind to the Cas9 protein to form a CRISPR / Cas complex. The configuration of the guide RNA can be roughly classified into a scaffold portion and a guide sequence portion. The scaffold portion is a portion that can interact with the Cas9 protein and bind to the Cas9 protein, and its configuration can vary depending on the type of Cas9 protein contained in the genetic manipulation composition. The guide sequence portion is a portion that can form a complementary bond with a specific sequence portion in a gene to be engineered contained in a cell, and allows the CRISPR / Cas complex to edit the specific sequence portion and / or a portion adjacent thereto. The guide sequence portion is designed to be capable of complementary binding to a predetermined target sequence.

[0113] In one embodiment, the engineered gene may be HIF1AN, HIF3A, PHD2, TLR4 and / or PAI1 genes.

[0114] In one embodiment, the scaffold portion can include a tracrRNA and a crRNA repeat portion. In one embodiment, the sequence of the tracrRNA can be the sequence of SEQ ID NO: 144. In one embodiment, the sequence of the tracrRNA can be the sequence of SEQ ID NO: 144 with about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more identical to the sequence of SEQ ID NO: 144. In one embodiment, the crRNA repeat portion can be the sequence of SEQ ID NO: 145. In one embodiment, the crRNA repeat portion can be the sequence of SEQ ID NO: 145 with about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more identical to the sequence of SEQ ID NO: 145. In one embodiment, the sequence of the scaffold portion can be the sequence of SEQ ID NO: 143. In one embodiment, the sequence of the scaffold portion can be the sequence of SEQ ID NO: 143 with about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more identical to the sequence of SEQ ID NO: 143.

[0115] In one embodiment, the guide sequence portion may be an RNA sequence that corresponds to a DNA sequence adjacent to a proto-spacer adjacent motif (PAM) sequence contained within the engineered gene. In one embodiment, the PAM sequence may be 5'-NGG'-3'.

[0116] Example of guide RNA sequence In one embodiment, the guide sequence portion included in the guide RNA may be a sequence selected from the group consisting of SEQ ID NOs: 72 to 142. In one embodiment, the sequence of the guide RNA may be a sequence selected from the group consisting of SEQ ID NOs: 146 to 216.

[0117] Form of CRISPR / Cas system components The form of each component of the CRISPR / Cas system contained in the genetic manipulation composition is not particularly limited, so long as it is a form in which a CRISPR / Cas complex can be formed and a target gene can be artificially manipulated when the genetic manipulation composition is introduced into a cell.

[0118] In one embodiment, the composition for genetic manipulation may include a ribonucleoprotein (RNP) to which the Cas9 protein and the guide RNA are bound. In one embodiment, the composition for genetic manipulation may include a vector including DNA encoding the Cas9 protein and DNA encoding the guide RNA. In one embodiment, the composition for genetic manipulation may include a single vector including DNA encoding the Cas9 protein and DNA encoding the guide RNA. In one embodiment, the single vector may be a plasmid and / or a viral vector. In one embodiment, the viral vector may be one selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus, vaccinia virus, poxvirus, and herpes simplex virus.

[0119] Intracellular genetic engineering method for producing cells with high adaptability in a hypoxic environment Summary This specification discloses an intracellular genetic manipulation method for producing cells having high adaptability in a hypoxic environment. Specifically, in order to produce cells having high adaptability in the above-mentioned hypoxic environment, the method is a method for obtaining the target cells by editing a gene expressing a substance that inhibits the expression of HIF1 protein. In one embodiment, the genetic manipulation method may include introducing a genetic manipulation composition into a cell containing a target gene. At this time, the genetic manipulation composition may be one described in the paragraph "Genetic manipulation composition for producing cells having high adaptability in a hypoxic environment". In one embodiment, the genetic manipulation method may include contacting a CRISPR / Cas9 complex with a target gene. At this time, the CRISPR / Cas9 complex may be a complex formed of a Cas9 protein derived from Streptococcus pyogenes and a guide RNA having a sequence selected from the group consisting of SEQ ID NOs: 146 to 216.

[0120] Environment for implementing genetic manipulation methods At this time, the genetic manipulation method may be performed in vitro. As described above, since the cells having high adaptability to the hypoxic environment are used as a cell therapy agent, the genetic manipulation method may be performed on cells isolated from an organism (ex vivo). In one embodiment, the genetic manipulation method may be performed in vitro on cells isolated from an organism (ex vivo). At this time, the organism may be, but is not limited to, a human or an animal.

[0121] Target Gene In the above-mentioned genetic engineering method, the gene targeted by the genetic engineering is called a target gene. The gene targeted by the above-mentioned genetic engineering method is a wild-type gene expressing a substance that inhibits the transcription, expression, and / or activity of HIF1 protein. The above-mentioned target gene may be a gene described in the paragraph "Cell Genotype 1 - Gene to be Engineered".

[0122] Compositions for genetic manipulation The composition for genetic manipulation used in the genetic manipulation method disclosed herein may be the one described in the paragraph "Composition for genetic manipulation for producing cells having high adaptability in a hypoxic environment." In one embodiment, the composition for genetic manipulation used in the genetic manipulation method may include a Cas9 protein derived from Streptococcus pyogenes or a DNA encoding the same, and one or more guide RNAs having a sequence selected from the group consisting of SEQ ID NOs: 146 to 216 or a DNA encoding the same.

[0123] Examples of means for introducing a composition for genetic manipulation In the genetic manipulation method disclosed herein, the introduction of the genetic manipulation composition into cells can be carried out in a variety of ways, and is not particularly limited as long as the method can achieve the purpose of editing the target gene by the CRISPR / Cas complex.

[0124] In one embodiment, the introduction of the genetic manipulation composition into cells can be performed by one or more means selected from electroporation, lipopection, microinjection, gene gun, liposome, cationic liposome, EDV (EnGeneIC delivery vehicles), plasmid, viral vector, nanoparticles, PTD (protein translocation domain) fusion protein method, immunoliposome, polycation or lipid: nucleic acid conjugate, naked DNA, artificial virion, and DNA formulation enhanced absorption method. In one embodiment, when the genetic manipulation composition includes a ribonucleoprotein bound to a Cas9 protein and a guide RNA, the genetic manipulation composition can be introduced into cells by electric shock or electroporation.

[0125] Individual delivery of each component of the CRISPR / Cas system When the composition for genetic manipulation contains each component of the CRISPR / Cas system independently, the method for genetic manipulation may include a process of introducing each component of the CRISPR / Cas system into a cell individually. At this time, each component of the CRISPR / Cas system may be introduced into a cell simultaneously or sequentially in any order. In one embodiment, when the composition for genetic manipulation contains DNA encoding the Cas protein and guide RNA as separate components, the method for genetic manipulation may include a process of simultaneously introducing DNA encoding the Cas protein and guide RNA into a cell. In one embodiment, when the composition for genetic manipulation contains DNA encoding the Cas protein and guide RNA as separate components, the method for genetic manipulation may include a process of introducing DNA encoding the Cas protein and guide RNA into a cell sequentially in any order. In one embodiment, when the composition for genetic manipulation contains DNA encoding the Cas protein and DNA encoding guide RNA in two or more different vectors, the method for genetic manipulation may include a process of simultaneously introducing each vector into a cell. In one embodiment, when the composition for genetic manipulation contains DNA encoding the Cas protein and DNA encoding the guide RNA in two or more different vectors, the method for genetic manipulation can include a process of introducing each vector into a cell in any order.

[0126] Introduction of genetic engineering compositions The genetic engineering method disclosed herein for producing cells that have high adaptability in a hypoxic environment may include introducing the genetic engineering composition into the cells.

[0127] Contact between CRISPR / Cas complex and target gene The genetic engineering methods disclosed herein for producing cells with increased adaptability in hypoxic environments may include contacting a CRISPR / Cas complex with a target gene.

[0128] Results of gene manipulation method 1 - Gene editing position As a result of carrying out the genetic engineering method for producing cells having high adaptability to a hypoxic environment disclosed herein, gene editing occurs at a specific portion of a target gene in a cell, where the expression "gene editing occurs at a specific portion" means that the specific portion and / or a portion adjacent thereto is edited by the CRISPR / Cas complex.

[0129] In one embodiment, performing the genetic engineering method can result in gene editing at or adjacent to a portion of a target sequence within the target gene in a cell. In one embodiment, performing the genetic engineering method can result in gene editing at or adjacent to a portion of a target sequence contained within a HIF1AN, HIF3A, PHD2, TLR4, and / or PAI1 gene in a cell.

[0130] In one embodiment, the genetic manipulation method may result in gene editing in an exon region, an intron region, and / or a regulatory region in a HIF1AN, HIF3A, PHD2, TLR4, and / or PAI1 gene in a cell. In one embodiment, the genetic manipulation method may result in gene editing in one or more regions selected from exon 1, exon 2, exon 1, exon 2, exon 3, exon 4, exon 5, ..., exon N in a HIF1AN, HIF3A, PHD2, TLR4, and / or PAI1 gene in a cell, where N is any integer. In one embodiment, the genetic manipulation method may result in gene editing in one or more regions selected from intron 1, intron 2, intron 3, intron 4, intron 5, ..., and intron M in a HIF1AN, HIF3A, PHD2, TLR4, and / or PAI1 gene in a cell, where M is any integer. In one embodiment, as a result of carrying out the gene manipulation method, gene editing may occur at a portion of a sequence selected from the group consisting of SEQ ID NOs: 1 to 71 contained in a target gene in a cell.

[0131] Results of gene manipulation method implementation 2 - Gene editing mode As a result of carrying out the genetic engineering method for producing cells with high adaptability to a hypoxic environment disclosed herein, gene editing occurs at a specific portion of a target gene in a cell. As a result of the gene editing, the expression level of the expression product of the target gene is lower than that of a wild-type cell. In this case, the expression product may be an mRNA or a specific protein.

[0132] In one embodiment, as a result of performing the genetic manipulation method, one or more nucleotides may be inserted, deleted, substituted with other nucleotides, and / or inverted in a specific portion of the target gene and / or a portion adjacent thereto. In one embodiment, as a result of performing the genetic manipulation method, an indel may be generated in a specific portion of the target gene and / or a portion adjacent thereto. In this case, the specific portion may be a portion exemplified in the paragraph "1-Gene editing position as a result of performing the genetic manipulation method". In one embodiment, as a result of performing the genetic manipulation method, a target gene may be knocked out. In one embodiment, as a result of performing the genetic manipulation method, a target gene may be knocked down.

[0133] Pharmaceutical composition comprising cells having high adaptability in hypoxic environment The present specification discloses a pharmaceutical composition comprising cells having high adaptability in a hypoxic environment, which contains the cells having high adaptability in a hypoxic environment as an active ingredient and can be used for the diagnosis, symptom relief, regeneration, and / or treatment of a target disease.

[0134] In one embodiment, the pharmaceutical composition can be administered to the body to replace damaged cells, or to treat or regenerate damaged tissues or organs, for uses readily available to those skilled in the art.

[0135] In one embodiment, the pharmaceutical composition may further comprise a pharma- ceutically acceptable carrier, which may be, but is not limited to, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidine, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, saline, phosphate buffered saline (PBS), and / or culture medium.

[0136] In one embodiment, the pharmaceutical composition or the cells that are an active ingredient of the pharmaceutical composition may be cultured in a hypoxic environment, specifically, pre-conditioned in a hypoxic environment in vitro or in vivo.

[0137] In one embodiment, the pharmaceutical composition can be formulated as a parenteral dosage form, including, but not limited to, intranasal administration, ophthalmic administration, intravenous injection, intramuscular injection, intraperitoneal injection, transdermal administration, subcutaneous injection, intravenous injection, intramuscular injection, intracerebral tissue injection, intrahippocampal injection, intracaudate putamen (CPu) injection, intraarticular injection, intrachondral injection, or intrathoracic injection.

[0138] In one embodiment, the pharmaceutical composition can be prepared into a parenteral preparation by mixing the cells as an active ingredient with a stabilizer or buffer in water to prepare a solution or suspension. It can also be prepared in a unit dosage form such as an ampoule or vial. In one embodiment, the pharmaceutical composition may contain auxiliary agents such as preservatives, stabilizers, wetting agents or emulsifiers, salts for osmotic pressure adjustment and / or buffers, and other therapeutically useful substances, and can be prepared by a conventional mixing, granulation or coating method.

[0139] Therapeutic method using cells with high adaptability in hypoxic environments This specification discloses a therapeutic method using cells having high adaptability in a hypoxic environment or a pharmaceutical composition containing the same. When the cells having high adaptability in a hypoxic environment are administered to a living subject (e.g., a human), they can survive for a long time in the hypoxic environment at the administration site and provide a significant therapeutic effect. In this case, the therapeutic effect can be obtained by reducing the single dose or increasing the number of repeated administration cycles.

[0140] The present specification provides a method for using a pharmaceutical composition containing, as an active ingredient, cells having high adaptability under a hypoxic environment to diagnose, alleviate symptoms, regenerate, or treat a target disease by injecting the pharmaceutical composition into a target site.

[0141] In one embodiment, the pharmaceutical compositions may be used alone or in combination with procedures involving surgery, radiation therapy, hormonal therapy, chemotherapy and biological response modifiers.

[0142] In one embodiment, the appropriate dosage of the pharmaceutical composition varies depending on factors such as the formulation method, administration method, the patient's age, weight, sex, pathological condition, diet, administration time, administration route, excretion rate, and reaction sensitivity, and usually an effective dosage for the desired treatment or prevention can be easily determined and prescribed by a skilled physician.

[0143] Experimental Example The present invention will be described in more detail below with reference to experimental examples and examples. These examples are merely intended to illustrate the contents disclosed in the present specification, and it is obvious to those skilled in the art that the scope of the contents disclosed in the present specification is not to be construed as being limited by these examples.

[0144] Experimental example 1: Production of cells with high adaptability in a low-oxygen environment Experimental Example 1-1 Mesenchymal stem cell culture Human bone marrow-derived MSCs were purchased from Lonza (Walkersville, Maryland, USA). MSCs were cultured in MEM alpha 1x media (Gibco, Rockville, MD) containing 10% FBS (fetal bovine serum, Gibco) and 0.05 mg / mL gentamicin (Thermo Fisher Scientific) at 37°C and 5% CO. 2 The cells were cultured under the conditions.

[0145] Experimental Example 1-2 Guide RNA Production RNA was transcribed in vitro using the MEGA short script T7 kit (Ambion) according to the manufacturer's instructions. The template for sgRNA was prepared by annealing and extension of two complementary oligonucleotides.

[0146] The guide domains of sgRNAs for each target are shown in Tables 1 to 5 below.

[0147] [Table 1]

[0148] [Table 2]

[0149] [Table 3]

[0150] [Table 4]

[0151] [Table 5]

[0152] The sequence of nucleotides linked to each guide domain (crRNA repeats, linker (GAAA), and tracrRNA) is 5'-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-3' (SEQ ID NO: 143).

[0153] The full length sequences of the sgRNAs are shown in Tables 6 to 12 below.

[0154] [Table 6]

[0155] [Table 7]

[0156] [Table 8]

[0157] [Table 9]

[0158] [Table 10]

[0159] [Table 11]

[0160] [Table 12]

[0161] Experimental Example 1-3 Transfection MSCs were transfected with the Amaxa P1 Primary Cell 4D Nucleofector kit using Program EW-104 according to the manufacturer's instructions. 5 The cells were incubated with in vitro transcribed sgRNA (4 μg) and premixed Cas9 protein (4 μg) at room temperature for 10 minutes before transfection.

[0162] Experimental example 1-4 Targeted deep sequencing Phusion polymerase (New England BioLabs) was used to amplify DNA segments encompassing the target and potential non-target loci. The PCR amplicon products were subjected to paired-end sequencing analysis using an Illumina MiSeq.

[0163] Experimental Example 1-5 qRT-PCR Total RNA samples were obtained using the RNeasy Mini Kit (Qiagen), and 1ug of total RNA was used to synthesize cDNA using the ReverTra Ace qPCR RT Kit (Toyobo). The synthesized cDNA was subjected to quantitative RT-PCR using Power SYBR Green PCR Master Mix (Applied Biosystems) and the StepOnePlus Real Time PCR system (Appled Biosystems). The PCR temperature, time, and number of cycles were as follows: DNA denaturation at 95℃ for 10 minutes, primer annealing at 55-60℃ for 30 seconds, polymerization at 72℃ for 30 seconds, and amplification was repeated for 40 cycles. Beta-actin (ACTB) was used as a normalization control.

[0164] Experimental Example 2: Measuring the viability of cells with high adaptability in a low-oxygen environment Experimental Example 2-1 Viability measurement under oxidative stress environment We measured the change in the proliferation ability of MSCs due to reactive oxidative stress (ROS), which frequently occurs under hypoxic conditions. 2 O 2 24 hours before treatment with hydrogen peroxide, 1x10 4 After 24 hours, 500 μM H 2 O 2 The mixture was incubated with H 2 O 2 After 24 hours of treatment, cell viability was measured using a CCK-8 cell proliferation kit (Dojindo, USA).

[0165] The results of the experiment are shown in FIG.

[0166] Experimental Example 2-2 Measurement of cell viability in response to SNAP (Reactive Nitrogen Species) To confirm the viability of cells depending on the concentration of S-Nitroso-N-acetyl-DL-penicillamine (SNAP, N3398) (Sigma), Cell Counting Kit-8 (CCK-8) was used to measure the viability of cells in each group. Cells cultured in a 96-well plate were treated with SNAP at concentrations of 0-500 μM and cultured for 24 h, after which 10 μL of CCK-8 solution was added per well and incubated for 2 h. The absorbance was measured at 450 nm using an ELISA reader.

[0167] The results of the experiment are shown in FIG.

[0168] Experimental example 3: Confirmation of the characteristics of cells with high adaptability in a low-oxygen environment Experimental Example 3-1 Experimental cells The subject cells for the experiment were human bone marrow-derived mesenchymal stem cells cultured in Experimental Example 1-1, treated with the composition for hHIF1AN gene manipulation produced in Experimental Example 1-2, and then screened for cells suitable for this experiment by deep sequencing. As controls for this experiment, cells with a manipulated SHS231 gene sequence position (SHS231 KO) and / or cells with a manipulated AAVS1 gene sequence position (AAVS1) were used.

[0169] Experimental Example 3-2 BrdU cell proliferation analysis The cells selected in Experimental Example 3-1 were treated with 10uM BrdU for 1 hour before harvesting. After BrdU treatment, the cells were fixed with 3% formaldehyde diluted in PBS at 4℃ for 1 hour, harvested, and treated with 1% Triton X-100 at room temperature for 5 minutes. The cells were centrifuged again, washed with PBS, treated with 4N HCL at room temperature for 10 minutes to break the DNA double strand, and then washed with PBS. The cells were harvested after treating with blocking solution (30% FBS, 1% BSA, 0.01% Tween 20 in PBS) at room temperature for 30 minutes. Anti-BrdU mouse IgG diluted 100 times in PBS was reacted for 30 minutes at 4℃. The cells were washed with 0.2% Tween 20 diluted in PBS, centrifuged, harvested, and finally diluted with PBS and analyzed with a flow cytometer.

[0170] The results of the BrdU cell proliferation assay are shown in FIG.

[0171] Experimental Example 3-3 FACS analysis MSCs selected in Experimental Example 3-1 were washed twice with phosphate buffered saline (PBS), detached using 0.05% trypsin-EDTA, and centrifuged at 1,000 rpm for 5 minutes. The cells were suspended in 100 ul of FACS staining buffer, mixed with antibodies, and incubated at 4°C for 1 hour. The cells were then washed twice with PBS, suspended in 500 ul of PBS, and analyzed by FACS.

[0172] The results of the FACS analysis are shown in FIGS.

[0173] Experimental Example 3-4: Verification of proteomics data through cytokine array Cytokine array was performed using Human XL Cytokine Array Kit (Cat. no. ARY022, R & D systems, USA). The method followed the manufacturer's instructions. The array membrane was first immersed in a 4-well multi-dish with array buffer 6 and blocked for one hour on a rocking platform shaker. After blocking, the array membrane was incubated overnight in a rocking platform shaker at 2-8°C with the desired sample volume. The array membrane was then washed three times for 10 minutes with 1X washing buffer. Then, 1.5 mL of diluted detection antibody cocktail was added per well and incubated on a shaker for one hour. The array membrane was then washed three times again with 1X washing buffer. After washing, the array membrane was incubated with 2 mL of 1X Streptavidin-HRP for 30 minutes. Finally, 1 mL of Chemi Reagent mix was evenly applied to each membrane, and autoradiography was performed. The pixel density of the X-ray films was scanned and analyzed using image analysis software (Image J).

[0174] The results of the analysis are shown in FIG.

[0175] Experimental Example 3-5 Confirmation of PDL (Population Doubling Level) and PDT (Population Doubling Time) To confirm cell growth, the number of cells was measured before and after subculture. The growth rate was calculated by dividing the number of harvested cells (Ct) by the number of seeded cells (Ci), and the PDL (N) was calculated using the following formula. JPEG2024160283000014.jpg18153The above culture time (hr) divided by the PDL was shown as Population Doubling time (PDT).

[0176] The PDL and PDT confirmation results are shown in FIGS.

[0177] Experimental Example 4 Results Reproduction Experiment Experimental Example 4-1 Results Reproduction experiment In order to confirm the reproducibility of the results of Experimental Example 3, the experiments of Experimental Examples 1 to 3 were repeated using a new MSC stock. The results of the repeated experiments are shown in Figures 29 to 30.

[0178] Experimental Example 4-2 Western blot analysis To induce a hypoxic environment, stem cells (MSCs) were cultured in 1% O 2The cells were cultured in a hypoxic chamber to maintain the concentration of 0.1% NaCl. To perform Western blot, the MSCs were harvested and then treated with lysis buffer containing PBS, 1% Triton X-100, and a mixture of Phosphatase Inhibitor Cocktail II, III, and complete protease inhibitors. The lysates were centrifuged at 100,000g for 20 minutes at 4°C. The supernatant (nonionic dtergent-insoluble) from the centrifugation was collected. For total lysates, the cells were harvested, washed twice with PBS, and lysed in Pierce RIPA buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 1% NP40, 1% SDS, and 0.5% sodium deoxycholate, and a protease inhibitor mixture; Thermo Scientific) on ice for 30 minutes. Protein concentration was measured using a BCA protein assay kit (Thermo Scientific). Equal amounts of protein (10-20 μg) were resolved on 8%-16% SDS-PAGE and transferred to nitrocellulose (NC) membranes. After washing with TBST (Tris-buffer solution-Tween 20; 10 mM Tris-HCl [pH 7.6], 150 mM NaCl, and 0.05% Tween-20), the membranes were blocked with 5% skim milk for 1 h and incubated with the appropriate primary antibodies at the dilutions recommended by the supplier. The membranes were washed with TBST, and the primary antibodies were detected with HRP-conjugated secondary antibodies. Immunoblot signals were visualized by Chemiluminescence (Pierce, USA).

[0179] The Western blot results are shown in FIG. [Industrial Applicability]

[0180] The present specification provides cells having high adaptability to hypoxic conditions and a method for producing the same. The cells having high adaptability to hypoxic conditions can be used as a cell therapeutic agent. The method for producing cells having high adaptability to hypoxic conditions can produce industrially applicable cells having high adaptability to hypoxic conditions.

Claims

1. 1. An engineered human cell comprising: An engineered gene with an indel in the HIF1AN (Hypoxia-inducible factor 1-alpha inhibitor) gene, In this case, the sequence of the engineered gene is different from the wild-type sequence of the HIF1AN gene, The mRNA transcribed from the engineered gene in the engineered human cell comprises: The mRNA is characterized by having a different sequence compared to the mRNA transcribed from the wild-type HIF1AN gene in wild-type human cells; The engineered human cell is characterized in that it has a higher survival rate in a hypoxic environment as compared to a wild-type human cell.

2. The engineered human cell of claim 1 , wherein the indel in the HIF1AN gene is located within exon 1 of the HIF1AN gene.

3. The engineered human cell of any one of claims 1 to 2, wherein the sequence of the engineered gene does not include one or more sequences selected from the group consisting of SEQ ID NOs: 1 to 9.

4. The engineered human cell of any one of claims 1 to 3, wherein the expression level of FIH-1 in the engineered human cell is less than 70% of the expression level of FIH-1 in the wild-type human cell.

5. The engineered human cell of any one of claims 1 to 4, wherein the engineered human cell secretes VEGF and IL-8 at levels higher than the levels of VEGF and IL-8 secreted by the wild-type human cell.

6. 6. The engineered human cell of any one of claims 1 to 5, characterized in that the engineered human cell is a mesenchymal stem cell derived from a tissue selected from the group consisting of bone marrow, adipose tissue, umbilical cord, placenta, amniotic fluid, and umbilical cord blood.

7. The engineered human cell of claim 6, wherein the engineered human cell is a bone marrow derived mesenchymal stem cell.

8. 1. A genetic engineering composition comprising: A Cas9 protein derived from Streptococcus pyogenes or a DNA encoding the Cas9 protein, and A guide RNA or a DNA encoding the guide RNA; wherein the guide RNA comprises a sequence selected from the group consisting of SEQ ID NOs: 146 and 152-154.

9. 9. The genetic engineering composition of claim 8, wherein the composition comprises the Cas9 protein and the guide RNA in a ribonucleoprotein (RNP) form.

10. 9. The genetic engineering composition of claim 8, wherein the composition comprises DNA encoding the Cas9 protein and DNA encoding the guide RNA in a single vector form.

11. The genetic engineering composition of claim 10, wherein the single vector is selected from the group consisting of plasmids, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, vaccinia viruses, poxviruses and herpes simplex viruses.

12. An in vitro or ex vivo genetic engineering method comprising the steps of: introducing a gene editing composition into a human cell, At this time, the gene editing composition comprises: A Cas9 protein derived from Streptococcus pyogenes or a DNA encoding the Cas9 protein, and A guide RNA or a DNA encoding the guide RNA; wherein the sequence of the guide RNA comprises a sequence selected from the group consisting of SEQ ID NOs: 146 and 152 to 154, in vitro or ex vivo genetic engineering method.

13. 13. The method of claim 12, wherein the composition comprises DNA encoding the Cas9 protein and DNA encoding the guide RNA in a vector form.

14. An in vitro or ex vivo genetic engineering method comprising the steps of: contacting a CRISPR / Cas9 complex with a gene comprising target DNA; wherein the CRISPR / Cas9 complex comprises: Cas9 protein derived from Streptococcus pyogenes, and a guide RNA targeting the target DNA; wherein the guide RNA comprises a sequence selected from the group consisting of SEQ ID NOs: 146 and 152-154, in vitro or ex vivo genetic engineering method.

Images