Mesenchymal stem cells having acid stress resistance, a method for producing the same, and uses thereof

Abstract

The present invention provides mesenchymal stem cells that are resistant to oxidative stress by decreasing or suppressing the expression or activity level of KEAP1, a negative regulator of NrF2, and increasing the activity of NrF2, and a method for producing the same. It is provided that the mesenchymal stem cells have an increased survival rate in the body and suppressed aging, and can be used as a cell therapeutic agent with improved therapeutic effects against ischemic diseases.

Description

Technical Field

[0001] By reducing or suppressing the expression or activity level of KEAP1, a negative regulator of Nrf2, and increasing the activity of Nrf2, mesenchymal stem cells having oxidative stress resistance, a method for producing the same, and uses thereof are provided.

Background Art

[0002] Mesenchymal stem cells have self-proliferative ability and multi-potency, and since there are various types of progenitor cells, it is easy to secure stem cell lines. As multipotent stem cells, they have the advantage of being much more genetically stabilized compared to pluripotent stem cells such as embryonic stem cells. Also, due to their anti-inflammatory ability and immune regulatory ability, they have been developed as cell therapy agents for cartilage regeneration, treatment of myocardial infarction, treatment of graft-versus-host disease, etc.

[0003] However, in in-vivo pathological environments such as ischemia and inflammation, the blood supply is poor, so the oxygen concentration is low (Hypoxia), the oxidative stress due to reactive oxygen species (ROS) is high, and the in-vivo survival rate of mesenchymal stem cells is low. Also, in an oxidative stress environment, the self-renewal of stem cells is inhibited and cell aging accumulates, and aged mesenchymal stem cells show a low therapeutic effect.

[0004] Thus, in order to sustain and enhance the therapeutic effect on mesenchymal stem cells with a low in-vivo survival rate, repeated administration must be performed, but repeated administration can be a burden on patients and there is also a problem that the productivity must be increased.

[0005] Reactive oxygen species (ROS) are molecules naturally produced during cell metabolism and play important roles in normal cell functions and signaling systems. However, excessive production of ROS induces oxidative stress, causes damage to intracellular molecules, and interferes with normal cell functions and roles. Cells have a regulatory system to mitigate intracellular stress and toxicity caused by excessive reactive oxygen species (ROS), and a typical one is the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) / Kelch-like ECH-associated protein 1 (Keap1) signaling system. Keap1, which plays a major regulatory role in Nrf2, is a protein that regulates the antioxidant system in the human body. In the cytoplasm, the protein-protein interaction (PPI) of Nrf2-Keap1 is an important element in regulating the antioxidant pathway that causes many pathological conditions induced by inflammation and inflammatory mediators. Nrf2 is a transcription factor that plays a role in protecting cells from oxidative stress. In a normal state, it forms a complex with Keap1 in the cytoplasm. When Keap1 is inactivated by cytotoxic agents or oxidative stress, Nrf2 is released and enters the nucleus. Thereafter, Nrf2 is known to activate antioxidant enzymes such as HO-1, SOD, catalase, and GPx-1 / 2 to protect cells from oxidative damage.

[0006] (Patent Document 1) Republic of Korea Published Patent No. 10-2019-0069238

[0007] (Non-Patent Document 1) Xiaozhen Dai et al., Trends Mol Med. 2020 Feb;26(2):185-200, Nrf2: Redox and Metabolic Regulator of Stem Cell State and Function

[0008] (Non-Patent Document 2) D S Yoon et al., Cell Death & Disease volume 7, pagee2093 (2016), Cellular localization of NRF2 determines the self-renewal and osteogenic differentiation potential of human MSCs via the P53-SIRT1 axis

[0009] (Non-Patent Document 3) Yiling Hu et al., Front. Neurol., 18 February 2020, CRISPR / Cas9-Induced Loss of Keap1 Enhances Anti-oxidation in Rat Adipose-Der4ed Mesenchymal Stem Cells

[0010] (Non-Patent Document 4) Mohammad Mohammadzadeh et al., Cell Stress and Chaperones volume 17, pages553-565 (2012), Nrf-2 overexpression in mesenchymal stem cells reduces oxidat4e stress-induced apoptosis and cytotoxicity

[0011] (Non-Patent Document 5) Shouqin Zhang et al., J Cell Biochem. 2018 Feb;119(2):1627-1636, Nrf2 transfection enhances the efficacy of human amniotic mesenchymal stem cells to repair lung injury induced by lipopolysaccharide

Summary of the Invention

Problems to be Solved by the Invention

[0012] An object of the present invention is to provide mesenchymal stem cells having resistance to oxidative stress increased by reactive oxygen species (ROS) in an in-vivo pathological environment such as ischemia and inflammation, a method for producing the same, and a cell therapeutic agent using the same.

Means for Solving the Problems

[0013] To achieve the above object, the present invention

[0014] provides mesenchymal stem cells in which the expression or activity of KEAP1 protein that binds to and causes degradation of Nfr2 in the Nfr2-Keap1 pathway, which is a regulatory system that alleviates intracellular stress and toxicity caused by excessive reactive oxygen species (ROS), is decreased or suppressed.

[0015] Further, the present invention provides artificially engineered mesenchymal stem cells containing an artificially engineered Keap1 (Kelch-like ECH-associated protein 1) gene, wherein the artificially engineered Keap1 gene is different from the Keap1 gene sequence of wild-type mesenchymal stem cells, the artificially engineered Keap1 gene contains one or more indels in the nucleic acid sequence, and at this time, the indel is located within the protospacer-adjacent motif (PAM) sequence in the second exon region or the third exon region of the Keap1 gene, or within a continuous 5-50 nucleotide sequence adjacent to the 5′ end or the 3′ end of the PAM sequence, and the artificially engineered mesenchymal stem cells are characterized in that their oxidative stress resistance is improved.

[0016] The present invention also provides a guide nucleic acid containing a guide sequence capable of targeting a target sequence of the Keap1 (Kelch-like ECH-associated protein 1) gene of mesenchymal stem cells or a nucleic acid sequence encoding the same; and a composition for producing mesenchymal stem cells having oxidative stress resistance, which contains an editor protein or a nucleic acid sequence encoding the same.

[0017] The present invention also provides a method for producing mesenchymal stem cells having oxidative stress resistance, which includes: (1) a step of introducing the composition for producing mesenchymal stem cells having oxidative stress resistance into isolated mesenchymal stem cells; and (2) a step of editing the Keap1 gene so that an indel is generated in a target sequence of the Keap1 (Kelch-like ECH-associated protein 1) gene located in the genome of the mesenchymal stem cells, thereby reducing or suppressing the expression or activity of the Keap1 protein.

[0018] The present invention also provides a pharmaceutical composition for preventing or treating ischemic diseases, which contains, as an active ingredient, mesenchymal stem cells artificially manipulated to have the above-mentioned oxidative stress resistance.

[0019] The present invention also provides a method for treating ischemic diseases, which includes administering to a mammal a therapeutically effective amount of mesenchymal stem cells artificially manipulated to have the above-mentioned oxidative stress resistance.

[0020] The present invention also provides the use of mesenchymal stem cells artificially manipulated to have the above-mentioned oxidative stress resistance in the manufacture of a medicament for preventing or treating ischemic diseases in a mammal.

Advantages of the Invention

[0021] The artificially engineered mesenchymal stem cells of the present invention are artificially modified by gene scissors technology to the gene encoding the Keap1 protein that binds to NrF2 and causes degradation, so that the expression or activity of the Keap1 protein is reduced or suppressed, whereby the activity of NrF2 can be increased. The mesenchymal stem cells with reduced or suppressed expression or activity of the Keap1 protein show resistance to oxidative stress increased by reactive oxygen species (ROS) in in vivo pathological environments such as ischemia and inflammation, have increased survival rates in the body and suppressed aging, and can be used as a cell therapy agent with improved therapeutic effects against ischemic diseases.

Brief Description of the Drawings

[0022]

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Mode for Carrying Out the Invention

[0023] Hereinafter, the present invention will be described in detail.

[0024] As used herein, the term "about" refers to an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by about 30, 25, 20, 15 , 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 with respect to a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0025] As used herein, the term "artificially engineered" is a term used to distinguish substances, molecules, etc. having a configuration already existing in nature, and means that artificial modification has been applied to the substances, molecules, etc. For example, in the case of an "artificially engineered gene", it means a gene to which artificial modification has been applied to the configuration of a gene existing in nature. Further, the term includes all meanings recognizable by a person of ordinary skill in the art and can be appropriately interpreted according to the context.

[0026] As used herein, "wild-type" means that a gene containing a naturally occurring nucleotide sequence and the protein expressed from that gene have normal functional properties. A wild-type gene has a form in which no natural or artificial mutations have occurred and is the most frequently observed in a population. If the term "wild-type" is used herein in contrast to a genetically engineered gene and / or a genetically engineered cell, it can be construed to mean a gene containing a homologous "non-engineered" naturally occurring nucleotide sequence corresponding to the genetically engineered gene and / or the genetically engineered cell, and a cell having the same. Further, the term includes all meanings recognizable by a person of ordinary skill in the art and can be appropriately interpreted depending on the context.

[0027] As used herein, the expression "knock-out" or "knocked-out gene" means that a mutation or artificial modification has occurred in a wild-type gene, as a result of which the protein expressed by the wild-type gene cannot be generated through the transcription and / or translation processes. For example, a cell containing a knocked-out gene A may not be able to express the mRNA and / or protein expressed by the wild-type gene A. A cell containing a knocked-out gene A may be one in which only one of the genes A present in the cell has been knocked out, or may be one in which two or more have been knocked out. Further, the term includes all meanings recognizable by a person of ordinary skill in the art and can be appropriately interpreted depending on the context.

[0028] As used herein, the term "knock-down" or "knock-down gene" means that a mutation or artificial modification has occurred in the wild-type gene, resulting in the expression of a substance in a smaller amount than the wild-type gene. For example, a cell containing a knock-down gene A may express a smaller amount of mRNA than the mRNA expressed by the wild-type gene A. As another example, a cell containing a knock-down gene A may express a smaller amount of protein than the protein expressed by the wild-type gene A. A cell in which the gene A is knocked down may be one in which only one of the genes A present in the cell is knocked down, or two or more of them may be knocked down. Further, the above terms include all meanings recognizable by a person of ordinary skill in the art and can be appropriately interpreted according to the context.

[0029] As used herein, "decreased expression" means showing an expression level lower than the expression level of mRNA and / or protein measured in the wild-type. The decrease may be about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 50% or more, about 60% or more, about 70% or more, or about 100% or more decreased compared to a cell without genetic modification or a wild-type cell.

[0030] As used herein, the term "decreased activity" or "decreased activity" may mean a relative decrease in the activity of a protein or enzyme when measured. Specifically, "decreased activity" or "decreased activity" means a lower level of protein or enzyme activity compared to a given parental cell or wild-type cell.

[0031] As used herein, the term "stem cell" refers to a broad concept that collectively refers to undifferentiated cells having the ability to differentiate into various types of somatic tissue cells, i.e., stemness. At this time, the stem cells may be induced pluripotent stem cells, embryonic stem cells, and adult stem cells. Further, the cells may be of human origin, but are not limited thereto.

[0032] As used herein, "mesenchymal stem cells" are undifferentiated stem cells isolated from human or mammalian tissues and can be derived from various tissues. In particular, they can be umbilical cord-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, muscle-derived mesenchymal stem cells, nerve-derived mesenchymal stem cells, skin-derived mesenchymal stem cells, amniotic membrane-derived mesenchymal stem cells, amniotic fluid-derived mesenchymal stem cells, perinatal tissue-derived mesenchymal stem cells, or placenta-derived mesenchymal stem cells. Techniques for isolating stem cells from each tissue are already known in the art.

[0033] The present invention provides mesenchymal stem cells in which the expression or activity of the KEAP1 (Kelch-like ECH-associated protein 1) protein is decreased or suppressed.

[0034] The mesenchymal stem cells in which the expression or activity of the KEAP1 (Kelch-like ECH-associated protein 1) protein is decreased or suppressed bind to Nfr2 in the Nfr2-Keap1 pathway, which is a regulatory system that alleviates intracellular stress and toxicity caused by excessive reactive oxygen species (ROS), and undergoes degradation. As a result, the expression or activity of the KEAP1 (Kelch-like ECH-associated protein 1) protein is decreased or suppressed, and the activity of NrF2 increases. This shows resistance to oxidative stress increased by reactive oxygen species (Reactive Oxygen Species: ROS) in in vivo pathological environments such as ischemia and inflammation. When the mesenchymal stem cells in which the expression or activity of the KEAP1 protein is decreased or suppressed are used as a cell therapeutic agent, the in vivo survival rate of the mesenchymal stem cells increases and aging is suppressed, and a therapeutic effect improved compared to wild-type mesenchymal stem cells or mesenchymal stem cells selected by conventional techniques can be shown.

[0035] Specifically, the present invention provides an artificially engineered mesenchymal stem cell having oxidative stress resistance, which is characterized by artificially manipulating the KEAP1 (Kelch-like ECH-associated protein 1) gene of the mesenchymal stem cell so that the expression or activity of KEAP1 mRNA and / or KEAP1 protein is decreased or suppressed, thereby having high viability in an oxidative stress environment.

[0036] The present invention provides an artificially engineered mesenchymal stem cell comprising one in which the nucleic acid sequence of the KEAP1 (Kelch-like ECH-associated protein 1) gene is artificially modified.

[0037] In the present invention, the "artificial modification" or "artificial manipulation" of the gene nucleic acid sequence can be made through the modification of the nucleic acid sequence constituting the gene or the chemical modification of a single base. This can be due to part or all of the gene being mutated, substituted, deleted, or one or more bases being inserted into the gene, and can be made using gene scissors techniques such as the CRISPR-enzyme system. As an example, the artificial modification of the gene nucleic acid sequence can be made by a non-homologous end joining (NHEJ) or homology directed repair (HDR) mechanism.

[0038] As an example, the artificially engineered mesenchymal stem cell can be one in which the KEAP1 (Kelch-like ECH-associated protein 1) gene is knocked out.

[0039] As used herein, "Non-homologous end joining (NHEJ)" is a method of repairing or mending double-strand breaks in DNA by joining both ends of the cleaved double-stranded or single-stranded DNA together. Generally, when two compatible ends formed by a double-strand break (e.g., cleavage) repeatedly come into frequent contact and the two ends are completely joined, the broken double-strand is restored.

[0040] In the process of repairing damaged genes or nucleic acids using the above NHEJ, "insertion and / or deletion" (or "InDel") of a part of the nucleic acid sequence may occur at the NHEJ repair site. The gene in which the InDel occurs does not have the same sequence as the wild-type gene. Such insertions and / or deletions alter the gene's reading frame, creating a frameshifted transcript mRNA, which as a result may experience nonsense-mediated decay or fail to synthesize normal proteins, thereby losing its original function. Alternatively or additionally, it may introduce mutations that maintain the reading frame but insert or delete a significant amount of sequence, destroying the functionality of the protein. In yet another example, when an InDel occurs in a transcriptional regulatory region such as the promoter region or enhancer region of a gene, mRNA may not be transcribed or the transcription level may decrease, and accordingly, the protein may not be expressed or the expression level may decrease. Or, when the mutagenesis mechanism of NHEJ is utilized and the generation of a specific final sequence is not required, it can be used to delete only some sequence motifs. For example, by using two or more guide RNAs targeting the intron sites at the 5' and 3' portions of a specific exon respectively to cause double-strand breaks in each intron portion, and when only a part of the exon of the gene is deleted by NHEJ, the other parts may be normally expressed and the main functionality of the protein may be maintained.

[0041] Using such NHEJ, it is possible to specifically knock out or knock down a target gene by utilizing gene scissors technology.

[0042] For example, a CRISPR enzyme such as Cas9 or Cpf1, which is a kind of gene scissors, is used to cleave the double strand or two single strands of a target gene or target nucleic acid, and indels are generated by NHEJ in the double strand or two single strands of the damaged target gene or damaged target nucleic acid, through which specific knock-out or knock-down of the target gene or nucleic acid can be induced.

[0043] As an example, the artificially engineered mesenchymal stem cells may have the KEAP1 (Kelch-like ECH-associated protein 1) gene knocked down.

[0044] In one embodiment of the present invention, the keap1 gene of the artificially engineered mesenchymal stem cells can contain one or more indels within the nucleic acid sequence.

[0045] In one embodiment of the present invention, the indel can be located within the Protospacer-Adjacent Motif (PAM) sequence in the second exon region or the third exon region of the Keap1 gene, or within a continuous 5-50 nucleotide sequence adjacent to the 5′ end or 3′ end of the PAM sequence.

[0046] In one embodiment of the present invention, the sequence of the Keap1 gene of the artificially engineered mesenchymal stem cells may not include one or more sequences selected from the group consisting of SEQ ID NOs: 1-15 and SEQ ID NOs: 49-56.

[0047] In one embodiment of the present invention, the artificially engineered mesenchymal stem cells may have no expression of Keap1 mRNA.

[0048] In one embodiment of the present invention, the mRNA transcribed from the artificially engineered Keap1 gene of the artificially engineered mesenchymal stem cells may have a lower mRNA expression level compared to the mRNA level transcribed from the Keap1 gene of wild-type mesenchymal stem cells.

[0049] In one embodiment of the present invention, the artificially engineered mesenchymal stem cells may have a different Keap1 mRNA sequence compared to wild-type stem cells.

[0050] In one embodiment of the present invention, the artificially engineered mesenchymal stem cells may have a reduced expression or activity of Keap1 protein compared to wild-type stem cells. Through this, the stem cells of the present invention may have a reduced or lost function of the Keap1 protein.

[0051] That is, in the artificially engineered mesenchymal stem cells, the expression or activity of the Keap1 protein may be reduced by about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 55% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 100% compared to the expression or activity of wild-type mesenchymal stem cells.

[0052] The present invention provides a method for producing artificially engineered mesenchymal stem cells with oxidative stress resistance, which includes the step of reducing the expression or activity of the Keap1 protein in mesenchymal stem cells.

[0053] The increase or decrease in the expression or activity of the KEAP1 protein can be achieved by artificial modification of the KEAP1 gene. For example, gene scissors technology can be utilized.

[0054] As an example, the gene scissors technology can utilize, but is not limited to, TALEN (transcription activator-like effector nuclease) in which a TAL effector (transcription activator-like effector or TALE) domain and a cleavage domain are fused, zinc-finger nuclease, or a CRISPR-enzyme system derived from the microbial immune system CRISPR (Clustered regularly interspaced short palindromic repeats).

[0055] The entire content disclosed in International Patent Publication WO2012 / 093833 or US Published Patent 2013-0217131 related to the TALEN is included in this specification as a reference. Regarding the ZFN, Beerli et al. (2002) Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416, US Patent Registrations 7,888,121, 8,409,861, 6,479,626, 6,903,185, 7,153,949 may be included in this specification as references.

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

[0057] "Guide nucleic acid" means a nucleic acid that can recognize a target nucleic acid, a target gene, or a target chromosome and interact with an editor protein. At this time, the guide nucleic acid can form a complementary bond with some nucleotides in the target nucleic acid, the target gene, or the target chromosome.

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

[0059] The guide nucleic acid can be a guide RNA. As an example, the "guide RNA" can be in vitro transcribed, particularly an oligonucleotide duplex or transcribed from a plasmid template. As another example, the guide RNA can be encrypted in the form of a vector, transmitted into cells in an ex vivo or in vivo environment, and can be transcribed from the vector, but is not limited thereto.

[0060] The design and configuration of the guide RNA are known to those skilled in the art and are described in detail in Korean Registered Patents 10-1656236, 10-1656237, 10-1706085, 10-2052286, 10-2182847, and the full texts of the registered patents are included herein as reference materials for the present invention.

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

[0062] The "guide sequence portion" is a nucleotide sequence portion that can complementarily bind to a part of the sequence of either strand of the double-strand of a target gene or nucleic acid, and is a nucleotide sequence portion that can be artificially modified, and is determined by the target nucleotide sequence of interest. At this time, the guide sequence may be a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity with the guide nucleic acid binding sequence of the target gene or target nucleic acid. The guide sequence may be a sequence contained in the guide domain of the guide nucleic acid.

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

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

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

[0066] In one embodiment of the present invention, the target sequence may be a sequence contained in exon region 2 or exon region 3 of the wild-type KEAP1 gene.

[0067] In one embodiment of the present invention, the target sequence is one or more sequences selected from SEQ ID NO: 1 to SEQ ID NO: 15.

[0068] The target sequence is a nucleotide sequence around and adjacent to a protospacer-adjacent motif (PAM) sequence recognized by an editor protein, and may include all or part of the PAM, but is not limited thereto.

[0069] The target sequence can be used as a term meaning all of the two nucleotide sequence informations. For example, in the case of a target gene, the target sequence may mean the sequence information of the transcribed strand of the target gene DNA, or may mean the nucleotide sequence information of the non-transcribed strand.

[0070] The target sequence includes a guide nucleic acid binding sequence or a guide nucleic acid non-binding sequence. The "guide nucleic acid binding sequence" is a nucleotide sequence having partial or complete complementarity with a guide sequence included in the guide domain of a guide nucleic acid, and can form a complementary bond with the guide sequence included in the guide domain of the guide nucleic acid. The target sequence and the guide nucleic acid binding sequence are nucleotide sequences that can vary depending on the target gene or nucleic acid, that is, the object to be genetically manipulated or corrected, and the guide nucleic acid can be designed in various ways depending on the target gene or target nucleic acid.

[0071] The "guide nucleic acid non-binding sequence" is a nucleotide sequence that has partial or complete homology with the guide sequence contained in the guide domain of the guide nucleic acid and cannot form a complementary bond with the guide sequence contained in the guide domain of the guide nucleic acid. Further, the guide nucleic acid non-binding sequence is a nucleotide sequence that has complementarity with the guide nucleic acid binding sequence and can form a complementary bond with the guide nucleic acid binding sequence. The guide nucleic acid binding sequence is a partial nucleotide sequence of the target sequence and can be a nucleotide sequence having two different sequence orders of the target sequence, that is, one of two nucleotide sequences that can form a complementary bond with each other. At this time, the guide nucleic acid non-binding sequence can be the remaining nucleotide sequence of the target sequence excluding the guide nucleic acid binding sequence.

[0072] The guide nucleic acid binding sequence can be a nucleotide sequence selected from among the target sequence, that is, a nucleotide sequence identical to the transcribed strand and a nucleotide sequence identical to the non-transcribed strand. At this time, the guide nucleic acid non-binding sequence can be the remaining nucleotide sequence of the target sequence excluding the guide nucleic acid binding sequence, that is, a nucleotide sequence selected from among a nucleotide sequence identical to the transcribed strand and a nucleotide sequence identical to the non-transcribed strand.

[0073] The guide nucleic acid binding sequence can be the same length as the target sequence. The guide nucleic acid non-binding sequence can be the same length as the target sequence or the guide nucleic acid binding sequence. The guide nucleic acid binding sequence can be a nucleotide sequence of 5 to 50 nucleotides.

[0074] As a specific example, the guide nucleic acid binding sequence can be a nucleotide sequence of 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides or 25 nucleotides. The guide nucleic acid non-binding sequence can be a nucleotide sequence of 5 to 50 nucleotides.

[0075] As a specific example, the guide nucleic acid non-binding sequence may be a 16-nucleotide sequence, a 17-nucleotide sequence, an 18-nucleotide sequence, a 19-nucleotide sequence, a 20-nucleotide sequence, a 21-nucleotide sequence, a 22-nucleotide sequence, a 23-nucleotide sequence, a 24-nucleotide sequence, or a 25-nucleotide sequence.

[0076] The guide nucleic acid binding sequence can form a partial or complete complementary bond with the guide sequence contained in the guide domain of the guide nucleic acid, and the length of the guide nucleic acid binding sequence may be the same as the length of the guide sequence.

[0077] The guide nucleic acid binding sequence may be a nucleotide sequence complementary to the guide sequence contained in the guide domain of the guide nucleic acid, for example, a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, or 95% or more complementary or completely complementary.

[0078] As an example, the guide nucleic acid binding sequence can have or contain 1 to 8 nucleotide sequences that are not complementary to the guide sequence contained in the guide domain of the guide nucleic acid.

[0079] The guide nucleic acid non-binding sequence can have a partial or complete homology with the guide sequence contained in the guide domain of the guide nucleic acid, and the length of the guide nucleic acid non-binding sequence may be the same as the length of the guide sequence. As an example, the guide sequence can be designed based on a sequence having homology with the guide nucleic acid non-binding sequence.

[0080] The guide nucleic acid non-binding sequence may be a nucleotide sequence having homology with the guide sequence contained in the guide domain of the guide nucleic acid, for example, a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, or 95% or more homology or complete homology.

[0081] As an example, the guide nucleic acid non-binding sequence can have or contain 1 to 8 nucleotide sequences that are not homologous to the guide sequence contained in the guide domain of the guide nucleic acid. The guide nucleic acid non-binding sequence can complementarily bind to the guide nucleic acid binding sequence, and the guide nucleic acid non-binding sequence can be the same length as the guide nucleic acid binding sequence.

[0082] The guide nucleic acid non-binding sequence can be a nucleotide sequence complementary to the guide nucleic acid binding sequence, for example, it can be a nucleotide sequence that is at least 90% or 95% or more complementary or completely complementary.

[0083] As an example, the guide nucleic acid non-binding sequence can have or contain 1 to 2 nucleotide sequences that are not complementary to the guide nucleic acid binding sequence. Also, the guide nucleic acid binding sequence can be a nucleotide sequence at a position adjacent to a sequence complementary to the nucleotide sequence (PAM sequence) recognizable by the editor protein.

[0084] As an example, the guide nucleic acid binding sequence can be a continuous 5 to 50 nucleotide sequence located adjacent to the 5′ end and / or 3′ end of a sequence complementary to the nucleotide sequence (PAM sequence) recognizable by the editor protein.

[0085] Also, the guide nucleic acid non-binding sequence can be a nucleotide sequence at a position adjacent to the nucleotide sequence (PAM sequence) recognizable by the editor protein.

[0086] As an example, the guide nucleic acid non-binding sequence can be a continuous 5 to 50 nucleotide sequence located adjacent to the 5′ end and / or 3′ end of the nucleotide sequence (PAM sequence) recognizable by the editor protein.

[0087] "Editor protein" means a peptide, polypeptide or protein that can directly bind to nucleic acid or can interact without direct binding. Conceptually, the editor protein may also be referred to as "artificially engineered nuclease" or RGEN (RNA-Guided Endonuclease).

[0088] In one specific example, the editor protein can be a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) enzyme. "CRISPR enzyme" is the main protein component of the CRISPR-enzyme system, which may also be referred to as "Cas protein (CRISPR associated protein)", and refers to a nuclease that can mix or form a complex with guide RNA to recognize a target sequence and cleave DNA.

[0089] CRISPR enzymes are known to those skilled in the art. See Korean Registered Patents 10-1656236, 10-1656237, 10-1706085, 10-2052286, 10-2182847. As used herein, the CRISPR enzyme conceptually includes all variants that can act as an endonuclease or nickase activated in cooperation with guide RNA in addition to the native protein. When it is an activated endonuclease or nickase, it can cause target DNA cleavage, and this can be used to effect genome correction. Also, when it is an inactivated variant, it can be used to effect transcriptional regulation or separation of the target DNA.

[0090] The CRISPR enzyme is a nucleic acid or polypeptide (or protein) having a sequence encoding the CRISPR enzyme. Typically, Type II CRISPR enzymes or Type V CRISPR enzymes are often used. As the Type II CRISPR enzyme, there is Cas9 (CRISPR associated protein 9) protein.

[0091] The Cas9 protein can be derived from various microorganisms such as Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Campylobacter jejuni, Staphylococcus aureus, Staphylococcus Auricularis, and Neisseria meningitidis.

[0092] In order for the Cas9 protein to induce a double-stranded DNA break, the Cas9 protein must recognize a Protospacer Adjacent Motif (PAM) sequence, which is a nucleotide sequence of a certain length, and a part of the guide RNA (the guide sequence part) must complementarily bind to the complementary strand (the guide nucleic acid binding sequence) of the single-stranded DNA (the guide nucleic acid non-binding sequence) where the target sequence is located.

[0093] This PAM sequence is a sequence determined by the type and origin of the Cas9 protein. For example, the Cas9 protein derived from Streptococcus pyogenes (SpCas9) can recognize the 5′-NGG-3′ sequence (complementary sequence: 5′-CCN-3′) within the target nucleic acid. At this time, N is one of adenosine (A), thymidine (T), cytidine (C), and guanosine (G). In addition, the SpCas9 can recognize the 5′-NAG-3′ sequence (complementary sequence: 5′-CTN-3′) within the target nucleic acid with low activity.

[0094] In addition, the Type V CRISPR enzyme includes Cpf1, and the Cpf1 can be Cpf1 derived from Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, uberibacillus, Bacillus, Brevibacilus, Methylobacterium or Acidaminococcus.

[0095] The CRISPR enzyme such as the Cas9 or Cpf1 protein can be isolated from naturally occurring microorganisms or non-naturally produced by recombinant or synthetic methods. The Cas protein can also be in a form that is easily introduced into cells. As an example, the Cas protein can be linked to a cell-penetrating peptide or a protein transduction domain. The protein transduction domain can be, but is not limited to, polyarginine or the TAT protein derived from HIV. Since various types of cell-penetrating peptides or protein transduction domains are known in the art in addition to the examples described above, those skilled in the art can apply various examples to this specification without being limited to the above examples. Also, the Cas protein can be fused with a functional domain such as an NLS (nuclear localization sequence or signal). Also, the Cas9 protein can be encoded in the form of a vector and expressed in cells.

[0096] The present invention provides a guide nucleic acid containing a guide sequence capable of targeting a target sequence of the Keap1 (Kelch-like ECH-associated protein 1) gene of mesenchymal stem cells or a nucleic acid sequence encoding the same; and a composition for producing mesenchymal stem cells having oxidative stress resistance, comprising an editor protein or a nucleic acid sequence encoding the same.

[0097] Also, the composition can optionally further include a donor containing a specific nucleotide sequence to be inserted or a nucleic acid sequence encoding the same.

[0098] The donor means an exogenous nucleotide sequence capable of expressing a specific peptide or protein and can be inserted into genomic DNA through homology directed repair (HDR).

[0099] The donor can be double-stranded nucleic acid or single-stranded nucleic acid. The donor can be linear or circular.

[0100] The donor can be in the form of a viral vector or a non-viral vector (e.g., plasmid).

[0101] The virus can be a DNA virus or an RNA virus. At this time, the DNA virus can be a double-stranded DNA (dsDNA) virus or a single-stranded DNA (ssDNA) virus. At this time, the RNA virus can be a single-stranded RNA (ssRNA) virus.

[0102] The viral vector can be one or more viral vectors selected from the group consisting of Retrovirus, Lent4irus, Adenovirus, Adeno-associated virus (AAV), Vaccinia virus, Poxvirus, and Herpes simplex virus (HSV).

[0103] The target sequence can be a target of the guide nucleic acid-editor protein complex, and the target sequence can include, but is not limited to, a PAM (protospacer-adjacent motif) sequence recognized by the editor protein.

[0104] The guide nucleic acid composition can include a guide domain capable of targeting the target sequence of the KEAP1 gene.

[0105] As an embodiment of the present invention, the target sequence of the composition can be one or more sequences selected from SEQ ID NOs: 1 to 15.

[0106] In this specification, the guide nucleic acid, the editor protein, or the guide nucleic acid-editor protein complex (ribonucleoprotein, RNP) and / or the donor can be delivered or introduced into the subject in various forms.

[0107] At this time, the "subject" means an organism into which the guide nucleic acid, the editor protein, or the guide nucleic acid-editor protein complex is introduced; an organism in which the guide nucleic acid, the editor protein, or the guide nucleic acid-editor protein complex functions; or a specimen or sample obtained from an organism.

[0108] The subject can be an organism containing the target gene, target nucleic acid, or target chromosome of the guide nucleic acid-editor protein complex.

[0109] The organism can be an animal, an animal tissue, or an animal cell. At this time, the tissue can be the eye, skin, liver, kidney, heart, lung, brain, muscle, or blood.

[0110] The cell can be a stem cell, a hepatocyte, a cardiomyocyte, an endothelial cell, or a pancreatic cell.

[0111] The specimen or sample can be obtained from an organism containing the target gene, target nucleic acid, or target chromosome, such as saliva, blood, liver tissue, brain tissue, hepatocytes, nerve cells, phagocytes, macrophages, T cells, B cells, astrocytes, cancer cells, or stem cells.

[0112] The guide nucleic acid, the editor protein, or the guide nucleic acid-editor protein complex can be delivered or introduced into the subject in the form of DNA, RNA, or a mixed form thereof.

[0113] At this time, the DNA, RNA, or a mixed form thereof encoding the guide nucleic acid and / or the editor protein can be delivered or introduced into the subject by methods known in the art.

[0114] DNA, RNA, or a mixed form thereof that encodes a guide nucleic acid and / or an editor protein can be delivered or introduced into a subject by a vector, a non-vector, or a combination thereof.

[0115] The vector can be a viral vector or a non-viral vector (e.g., plasmid).

[0116] The virus can be a DNA virus or an RNA virus. At this time, the DNA virus can be a double-stranded DNA (dsDNA) virus or a single-stranded DNA (ssDNA) virus. At this time, the RNA virus can be a single-stranded RNA (ssRNA) virus.

[0117] The viral vector can be one or more selected from the group consisting of retrovirus, lentivirus, adenovirus, adeno-associated virus (AAV), vaccinia virus, poxvirus, and herpes simplex virus.

[0118] The non-vector can be naked DNA, a DNA complex, or mRNA.

[0119] As an embodiment of the present invention, the nucleic acid sequence encoding the guide nucleic acid and / or the editor protein can be delivered or introduced into a subject in the form of one or more vectors.

[0120] The vector can contain a nucleic acid sequence encoding a guide nucleic acid and / or an editor protein. As an example, the vector can simultaneously contain a nucleic acid sequence encoding a guide nucleic acid and an editor protein. As another example, the vector can contain a nucleic acid sequence encoding a guide nucleic acid. For example, the nucleic acid sequence encoding the guide nucleic acid can be all contained in one vector or the nucleic acid sequence encoding the guide nucleic acid can be divided and contained in a plurality of vectors. As another example, the vector can contain a nucleic acid sequence encoding an editor protein. For example, in the case of the editor protein, the nucleic acid sequence encoding the editor protein can be contained in one vector or the nucleic acid sequence encoding the editor protein can be divided and contained in a plurality of vectors.

[0121] The editor protein can be transmitted or introduced into the subject in the form of a peptide, polypeptide or protein.

[0122] The editor protein can be transmitted or introduced into the subject by methods known in the art in the form of a peptide, polypeptide or protein.

[0123] The guide nucleic acid and the editor protein can be transmitted or introduced into the subject in the form of a nucleic acid-protein mixture.

[0124] The guide nucleic acid and the editor protein can be transmitted or introduced into the subject in the form of a guide nucleic acid-editor protein complex. For example, the guide nucleic acid can be DNA, RNA or a mixed form thereof. The editor protein can be in the form of a peptide, polypeptide or protein. As an example, the guide nucleic acid and the editor protein can be transmitted or introduced into the subject in the form of a guide nucleic acid-editor protein complex, that is, a ribonucleoprotein (RNP), in which the guide nucleic acid in RNA form and the editor protein in protein form.

[0125] The present invention also provides a method for producing mesenchymal stem cells having oxidative stress resistance, comprising: (1) introducing the composition for producing mesenchymal stem cells having oxidative stress resistance into isolated mesenchymal stem cells; and (2) editing the KEAP1 (Kelch-like ECH-associated protein 1) gene such that the expression or activity of Keap1 protein is reduced or suppressed by generating indels in the target sequence of the KEAP1 gene located within the genome of the mesenchymal stem cells.

[0126] At this time, the "introduction" can be carried out by one or more means selected from electroporation, lipofection, microinjection, gene gun, liposome, positive liposome, plasmid, viral vector, nanoparticles, PTD (Protein translocation domain) fusion protein method, immunoliposome, polyvalent cation or lipid:nucleic acid conjugate, naked DNA, artificial virion, and methods for improving the absorption of DNA formulations, but is not limited thereto.

[0127] As an embodiment of the present invention, mesenchymal stem cells having oxidative stress resistance can be produced by introducing the composition for producing mesenchymal stem cells having oxidative stress resistance into isolated mesenchymal stem cells by electroporation.

[0128] As an embodiment of the present invention, indels can be generated by contacting the Keap1 (Kelch-like ECH-associated protein 1) gene located within the genome of the mesenchymal stem cells with a CRISPR / Cas9 complex comprising a Cas9 protein derived from Streptococcus pyogenes and a guide RNA capable of targeting the target sequence of the Keap1 gene.

[0129] In an embodiment of the present invention, the target sequence can be one or more sequences selected from the group consisting of SEQ ID NOs: 1 to 15.

[0130] The artificially engineered mesenchymal stem cells with oxidative stress resistance of the present invention can be used as a cell therapeutic agent, included as an active ingredient of a pharmaceutical composition for the prevention or treatment of ischemic diseases such as ischemic heart diseases, peripheral artery disease, critical limb ischemia (CLI), thromboangitis obliteran, diabetic peripheral vascular disorders, osteonecrosis, mesenteric ischemia, ischemic colitis, ischemic enteritis, acute kidney injury, ischemia-reperfusion injury, ischemic hepatitis, ischemic pancreatitis, ischemic optic neuropathy, chronic obstructive pulmonary disease, Acute Respiratory Distress Syndrome (ARDS), COVID-19 infection, neonatal hypoxic-ischemic encephalopathy, and stroke.

[0131] The ischemia-reperfusion injury is generally a continuous complex result of reperfusion injury that occurs when blood circulation is restarted by reperfusion during ischemic injury where blood supply to an organ or tissue is interrupted and oxygen supply to the tissue decreases, inducing an acute inflammatory reaction.

[0132] As used herein, a "cell therapy agent" is a pharmaceutical product (as defined by the US FDA regulations) that is produced through the separation, culture, and special manipulation of cells and tissues from an individual and is used for the purposes of treatment, diagnosis, and prevention. It refers to a pharmaceutical product that is used for the purposes of treatment, diagnosis, and prevention through a series of actions such as in vitro proliferation and selection of viable autologous, allogeneic, or xenogeneic cells to restore the function of cells or tissues or other methods to change the biological characteristics of cells.

[0133] The administration route of the cell therapy agent composition of the present invention can be administered through any general route as long as it can reach the target tissue. Parenteral administration, for example, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intravascular administration can be used, but it is not limited thereto.

[0134] The composition can be formulated into a form compatible with a pharmaceutical carrier commonly used in cell therapy. "Pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not usually cause allergic reactions or similar reactions such as gastrointestinal disorders, dizziness, etc. when administered to humans. Examples of pharmaceutically acceptable carriers include carriers for parenteral administration such as water, compatible oils, saline, aqueous glucose, and glycols, and can further contain stabilizers and preservatives. Examples of compatible stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, or ascorbic acid. Examples of compatible preservatives include benzalkonium chloride, methyl- or propylparaben, and chlorobutanol. Other pharmaceutically acceptable carriers can be referred to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

[0135] Also, the composition may be administered by any device by which the cell therapy agent can move to the target cells.

[0136] The cell therapy agent composition of the present invention can contain a therapeutically effective amount of a cell therapy agent for the treatment of diseases. "Therapeutically effective amount" means the amount of an active ingredient or pharmaceutical composition that can induce a biological or medical response in a tissue system, animal, or human considered by a researcher, veterinarian, physician, or other clinician, and this includes the amount that can induce the alleviation of symptoms of the disease or disorder to be treated.

[0137] It is obvious to those skilled in the art that the cell therapy agent contained in the composition of the present invention can be varied according to the desired effect. Therefore, the optimal content of the cell therapy agent can be easily determined by those skilled in the art, and can be adjusted by various factors such as the type of disease, the severity of the disease, the content of other components contained in the composition, the type of dosage form, and the age, weight, general health status, gender and diet of the patient, the administration time, the administration route and the secretion rate of the composition, the treatment period, and various drugs used simultaneously. It is important to include the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above elements. For example, the daily dosage of the stem cells of the present invention can be administered once or divided into several times at 1.0×10 5 ~1.0×10 30 cells / kg body weight, preferably 1.0×10 10 ~1.0×10 20 cells / kg body weight. However, it should be understood that the actual dosage of the active ingredient should be determined in light of various relevant factors such as the disease to be treated, the severity of the disease, the administration route, the weight, age, and gender of the patient, and therefore, the above dosage does not limit the scope of the present invention in any aspect.

[0138] In addition, the composition containing the cell therapy agent of the present invention as an active ingredient can be administered in a normal manner through rectal, intravenous (intravenous therapy, i.v.), intra-arterial, intraperitoneal, intramuscular, intrasternal, percutaneous, topical, intraocular, or intradermal routes.

[0139] The present invention provides a method for treating ischemic diseases, which includes administering to a mammal a therapeutically effective amount of the artificially engineered mesenchymal stem cells. As used herein, the term "mammal" refers to a mammal that is the subject of treatment, observation or experiment, preferably a human. At this time, the ischemic diseases follow those described above.

[0140] The present invention also provides the use of the artificially engineered mesenchymal stem cells in the manufacture of a medicament for preventing or treating ischemic diseases in a mammal. At this time, the ischemic diseases follow those described above. Mode for Carrying Out the Invention

[0141] Hereinafter, the present invention will be further described in detail through examples.

[0142] These examples are solely for more specifically explaining the present invention, and it will be apparent to those with ordinary knowledge in the technical field to which the present invention pertains that the scope of the present invention is not limited by these examples.

[0143] Example 1: Production of KEAP1-knocked-out mesenchymal stem cells

[0144] sgRNA Design

[0145] To knock out the KEAP1 gene, the target sequences of guide RNAs with mismatches of 1, 0, 0 and less predicted off-target effects among the guide sequences predicted by http: / / www.rgenome.net / cas-designer / were selected.

[0146] The target sequences of the KEAP1 gene are presented in Table 1 below.

[0147]

Table 1

[0148] Each guide RNA targeting the target sequences of SEQ ID NOs: 1 to 15 was synthesized and used in subsequent experiments. The indel efficiency of each target sequence for each guide RNA was measured through the following targeted deep sequencing method, and the results are shown in Figure 1.

[0149] Guide RNAs were in vitro transcribed using the MEGA short script T7 kit (Ambion) according to the manufacturer's instructions. The template for the sgRNA was produced through the annealing and extension of two complementary oligonucleotides.

[0150] RNP (Ribonucleoprotein) delivery

[0151] The RNP complex was introduced by electroporation using a 4D-Nucleofector (Lonza). Specifically, the RNP complex was formed by mixing 4 μg of Cas9 protein and 4 μg of in vitro transcribed sgRNA (produced using T7 polymerase (New England BioLabs) according to the manufacturer's protocol), and the mixture was incubated at room temperature for 10 minutes. The RNP complex was treated with 20 μl of Primary P1 buffer and 4×10 5 Electroporation was performed using the nucleofector program EW-104 together with Human Bone marrow MSC (Lonza, Cat. No. PT-2501). As a result, mesenchymal stem cells with the KEAP1 gene knocked out (KEAP1 KO MSC) were obtained.

[0152] Cells with the SHS231 gene sequence position manipulated were used as a control group for the mesenchymal stem cells artificially manipulated in this specification. Specifically, the SHS231 target sequence 5′-GATGTGCTCACTGAGTCTGA AGGA guide RNA capable of targeting -3′ (SEQ ID NO: 16) (underlined: PAM sequence) was synthesized by the experimental method described above, and the guide RNA and Cas9 protein were introduced into mesenchymal stem cells in the form of RNP to obtain MSCs in which the SHS231 gene sequence was manipulated.

[0153] Targeted deep sequencing

[0154] Genomic DNA (gDNA) was extracted from the obtained KEAP1 KO MSCs using the Blood Genomic DNA Extraction Kit (Favorgen) according to the manufacturer's protocol. 100 ng of genomic DNA (gDNA) was amplified using Phusion High-Fidelity DNA Polymerase PCR Polymerase (NEB) for amplification of the target site. The amplicons were amplified again using TruSeq HT Dual Index Primers (Illumina, San Diego, CA, USA) for the generation of deep sequencing libraries. Paired-end sequencing was performed using the Illumina Miniseq System, and the indel frequency was calculated at "http: / / www.rgenome.net / ".

[0155] As a result of targeted deep sequencing, the mutation positions of KEAP1 KO MSCs for each target sequence were shown based on the sequence of the wild-type KEAP1 gene. As an example, the targeted deep sequencing results of SEQ ID NO: 15 (sgKeap1-BR-#5) were shown as in Figure 2.

[0156] The primer sequences for each target sequence used in targeted deep sequencing are shown in Tables 2 to 4.

[0157]

Table 2

[0158]

Table 3

[0159]

Table 4

[0160] KEAP1 mRNA qRT-PCR Assay

[0161] Total RNA samples were obtained using the RNeasy Mini Kit (Qiagen), and among them, cDNA was synthesized from 1 μg of total RNA using the ReverTra Ace qPCR RT Kit (toyobo). Quantitative RT-PCR was performed on the synthesized cDNA using the 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 was carried out at 95°C for 10 minutes, primer annealing at 55 - 60°C for 30 seconds, and polymerization at 72°C for 30 seconds, and amplification was repeated for 40 cycles. Human GAPDH was used as the normalization control. The primer sequences for each gene used in qRT-PCR are shown in Tables 5 - 6.

[0162]

Table 5

[0163]

Table 6

[0164] The screened Keap1 target sgRNA showed a high frequency of indels at the target sequence (SEQ ID NO: 15) position within the Keap1 gene, and the expression of KEAP1 mRNA was also shown to be close to 0 and low. Also, when comparing WT and Control with Keap1 KO MSCs, no significant difference in the spindle-like and fibroblast-like shape was observed visually (see Figure 3).

[0165] Comparative Example 1: Production of Mesenchymal Stem Cells Edited with NRF2

[0166] Exon 2 of the NFE2L2 gene encoding the NRF2 protein is related to the KEAP1 interacting domain, and it is known that when a mutation occurs in which the NFE2L2 exon2 is deleted, the target genes of Nrf2 are activated (Leonard D Goldstein et.al., Cell Rep. 2016 Sep 6;1610:2605-2617.). To confirm whether the oxidative stress resistance of KEAP1 KO MSCs is due to KEAP1 KO or an increase in Nrf2 activity, a comparative experiment was conducted. Gene editing was performed to produce mesenchymal stem cells (Nrf2 edited MSCs) in which exon 2 of NRF2 was skipped to create Nrf2-active model cells, and a comparative experiment on the H2O2 (oxidative stress) resistance of KEAP1 KO MSCs and Nrf2 edited MSCs was carried out.

[0167] The specific experimental method is as follows.

[0168] sgRNA Design

[0169] To target the NRF2 gene, guide RNAs that can target intron 1 or intron 2 while corresponding to 1-0-0 mismatches (0, 1, 2) on the off-target profile were selected.

[0170] The target sequences of the NRF2 gene are presented in Table 7 below.

[0171]

Table 7

[0172] sgNRF2#1 - #4 target NRF2 exon2 (KEAP1 interacting exon), and sgNRF2#5 - #10 target NRF2 intron site (#6 and #9 are used simultaneously to induce large deletion of NRF2 exon2).

[0173] sgRNAs targeting SEQ ID NOs: 43 - 48 of the target sequences were synthesized respectively, and the indel efficiency for the NRF2 gene was measured by the same targeted deep sequencing method as in Example 1, and the results are shown in Figure 4.

[0174] Two in - vitro transcribed sgRNAs capable of targeting the target sequences of SEQ ID NO: 44 and SEQ ID NO: 47 of the target sequences respectively were introduced into one mesenchymal stem cell simultaneously with the editor protein by the same electroporation method as in Example 1, thereby producing MSCs in which exon2 of the Nrf2 gene was skipped.

[0175] Example 2: Production of mesenchymal stem cells with KEAP1 knocked out

[0176] sgRNA design

[0177] The target sequences of the KEAP1 gene were selected by the same method as in Example 1 and are shown in Table 8 below.

[0178]

Table 8

[0179] RNP (Ribonucleoprotein) Delivery, Targeted Deep Sequencing, and KEAP1 mRNA qRT-PCR Assay

[0180] RNP (Ribonucleoprotein) delivery, targeted deep sequencing, and KEAP1 mRNA qRT-PCR assay were performed in the same manner as in Example 1 above.

[0181] The primer sequences for each target sequence used in targeted deep sequencing are shown in Table 9.

[0182]

Table 9

[0183] The indel efficiency of each target sequence for each guide RNA targeting the target sequences of SEQ ID NO: 15 and SEQ ID NOs: 49 - 56 was measured through the following targeted deep sequencing method, and the results are shown in Figure 5 (in Figure 5, #BR5 is synthetic sgRNA (synthego) and the rest are in vitro transcribed RNA (IVT-RNA)).

[0184] Figure 6 is a graph showing the comparison of the expression levels of mRNA transcribed from the KEAP1 gene in mesenchymal stem cells in which the KEAP1 gene was knocked out using sgRNAs targeting each target sequence of the KEAP1 gene, by using qRT-PCR.

[0185] The indel efficiency of each target sequence for each guide RNA targeting the target sequences of SEQ ID NO: 15 and SEQ ID NO: 50 in mesenchymal stem cells P6 with KEAP1 knocked out (Figure 8(a)), and the expression levels of mRNA transcribed from the KEAP1 gene measured by using qRT-PCR in mesenchymal stem cells P6 and P7 with KEAP1 knocked out (Figure 8(b)) are shown in Figure 8.

[0186] Experimental Example 1: Analysis of Cell Viability and Growth Rate

[0187] CCK8 Cell Viability assay under oxidat4e stress

[0188] To confirm the viability of cells under oxidative stress (Oxidat4e stress), the viable cells in each group were measured using Cell Counting Kit-8 (CCK-8). Specifically, cells cultured in a 96-well plate were treated with H2O2 at different concentrations and further cultured for 24 h. Then, 10 μL of CCK-8 solution was added to each well and reacted for 2 h. Subsequently, the absorbance was measured at 450 nm using an ELISA reader.

[0189] PDL (Population Doubling Level), PDT (Population Doubling Time)

[0190] To examine the growth degree of cells, the cell numbers before and after subculture were measured. The value obtained by dividing the Harvest cell count number (C t ) by the Seeding cell count number (C i ) was regarded as the Growth rate, and PDL (n) was calculated using the formula as shown in the following Mathematical Formula 1.

[0191]

Number

[0192] The value obtained by dividing the culture time (hr) by PDL was expressed as the Population Doubling time (PDT).

[0193] The growth rate was observed to be slightly higher in KEAP1 KO MSCs than in WT, but there was no significant difference in viability in the general environment (see Fig. 9(a)). On the other hand, as a result of gradually increasing the concentration of hydrogen peroxide and adding it (creating an oxidative stress environment), KEAP1 KO MSCs showed high viability even when a considerable amount of hydrogen peroxide was administered (450 μM) (see Fig. 9(b)).

[0194] NRF2 exon2 skip edited MSCs (simultaneous targeting of sgNRF2-#6+#9) showed a similar survival effect up to 400 uM of H2O2 like KEAP1 KO MSCs (targeting sgKeap1-BR#5), but KEAP1 KO MSCs showed a higher survival effect at 450 uM of H2O2. This indicates that KEAP1 knockout has other survival effects on H2O2 resistance in addition to NRF2 activation (see Fig. 10).

[0195] As a result of comparing the H2O2 resistance by the KEAP1 target sequence, mesenchymal stem cells with the target sequence of SEQ ID NO: 15 (sgKeap1-BR#5) knocked out showed a relatively higher survival effect at a high concentration of H2O2 (see Figs. 11 and 12).

[0196] The survival rate (Fig. 13(a)), growth rate (Fig. 13(b)), PDL (Population Doubling Level) (Fig. 13(c)), and Population Doubling time (PDT) (Fig. 13(d)) of stem cells with the target sequences of SEQ ID NO: 15 (sgKeap1-BR#5) and SEQ ID NO: 50 (sgKEAP1-exon3-#23) knocked out are shown in Fig. 13.

[0197] Experimental Example 2: Cell cycle analysis

[0198] BrdU (Bromodeoxyuridine) Assay

[0199] The cultured cells were treated with 10 μM BrdU for 1 hour before harvesting. After treatment with BrdU, the cells were fixed with 3% formaldehyde diluted in PBS at 4°C for 1 hour, then 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 unwind the DNA double strands, and then washed with PBS. After treatment with a blocking solution (30% FBS, 1% BSA, 0.01% Tween 20 in PBS) at room temperature for 30 minutes, the cells were harvested. Anti-BrdU mouse IgG diluted 100-fold in PBS was reacted at 4°C for 30 minutes. After washing with 0.2% tween 20 diluted in PBS and centrifuging, the collected cells were finally diluted with PBS and analyzed by flow cytometry.

[0200] FACS analysis

[0201] The KEAP1 KO MSCs treated with BrdU as described above were washed twice with phosphate buffer saline (PBS), and the cells were detached from the culture plate using 0.05% trypsin-EDTA and centrifuged at 1,000 rpm for 5 minutes. The cells were suspended in 100 μl of FACS staining buffer, mixed with the antibody, reacted at 4°C for 1 hour, washed twice with PBS, suspended in 500 μl of PBS, and analyzed by FACS.

[0202] As a result of observing the cell cycle by BrdU assay, it was found that the KEAP1 KO MSCs frequently showed the S phase, indicating that cell proliferation occurred actively (see Figure 14).

[0203] Experimental Example 3: Telomere length analysis (Telomere length qPCR)

[0204] Genomic DNA (gDNA) samples of KEAP1 KO MSC were obtained using the Blood Genomic DNA Extraction Kit (FAVORGEN). Among them, 5 ng of gDNA was used to measure the Telomere Length value with the Absolute Human Telomere Length Quantification qPCR Assay Kit (Sciencell). Quantitative RT-PCR was performed on the Reference human genomic DNA in the kit and the experimental gDNA sample using the StepOnePlus Real Time PCR system (Appled biosystems) to compare their lengths. The PCR temperature, time, and number of cycles were as follows. DNA denaturation was carried out at 95°C for 10 minutes, primer annealing at 52°C for 20 seconds, and polymerization at 72°C for 45 seconds, and amplification was repeated for 32 cycles.

[0205] As a result of telomere length analysis, it was found that, as a result of the anti-aging effect of KEAP1 knockout, cell proliferation was active and at the same time, the length of telomeres became longer (see Figure 15).

[0206] Experimental Example 4: Proteomics Data Verification Using Cytokine Analysis

[0207] Cytokine analysis was performed according to the manufacturer's manual using the Human XL Cytokine Array Kit (Cat. no. ARY022, R & D systems, USA). First, the array separation membrane was placed in a 4-well multi-dish and blocked with array buffer 6 on a rocking platform shaker for one hour. After blocking, the separation membrane was incubated overnight with the KEAP1 KO MSC culture solution sample on a rocking platform shaker while maintaining a temperature of 2 - 8°C. Thereafter, the separation membrane was washed three times with 1X wash buffer for 10 minutes each. 1.5 ml of the diluted Detection Antibody Cocktail was added to each well and incubated on a shaker for one hour. Thereafter, it was washed three times with 1X wash buffer. After washing, 2 ml of Streptavidin-HRP was added to each well and reacted for 30 minutes. Finally, 1 ml of the Chemi Reagent Mix solution was evenly sprayed onto each separation membrane, and autoradiography was performed. The obtained X-ray film was scanned, and the pixel density on the film was analyzed using image analysis software (Image J).

[0208] KEAP1 KO MSCs showed increased expression of many cytokines such as Osteopontin (OPN), Angiopoietin, IL-8, VEGF, and uPAR, and among them, the expression of Osteopontin and Angiopoietin increased significantly (see Figure 16). The transient increase in Osteopontin (OPN) promotes angiogenesis and shows advantages in wound treatment. Also, since Angiopoietin-1, whose expression increased significantly, shows strong vascular protective effects such as preventing plasma leakage / vascular inflammation / endothelial cell necrosis, it is expected to have a positive impact on various cardiovascular diseases.

[0209] Experimental Example 5: Confirmation of in vivo viability

[0210] Since MSCs mainly accumulate in the lungs during intravenous (IV) administration, the results of collecting, staining, and analyzing lung tissues after IV administration of KEAP1 knockout MSCs to normal mice are shown in Fig. 11. Specifically, the control group and KEAP1 KO MSCs were stained with Vybrant DiD cell-labeling solution (ThermoFisher) to track the degree of accumulation in each part of the body during IV route administration in vivo. The stained MSCs in each experimental group were intravenously injected into 6-8-week-old C57Bl / 6 mice (Orient Bio) through the tail vein, with only 2×10 5 cells per mouse. On the first and second days after injection, the lungs of the mice were excised, and ex vivo imaging for DiD was performed using the FOBI Fluorescence In vivo imaging system (CELLGENTEK). The residual degree of MSCs was measured through DiD fluorescence intensity measurement.

[0211] Referring to Fig. 17, KEAP1 knockout mesenchymal stem cells showed excellent viability in the in vivo environment compared to the comparison group. KEAP1 knockout mesenchymal stem cells, which exhibit such excellent effects, are considered to be usable for the treatment of various ischemic and inflammatory diseases in in vivo environments such as the heart, blood vessels, lungs, and brain.

Claims

1. An artificially engineered mesenchymal stem cell containing an artificially engineered Keap1 (Kelch-like ECH-associated protein 1) gene, wherein the artificially engineered Keap1 gene is different from the Keap1 gene sequence of wild-type mesenchymal stem cells, the artificially engineered Keap1 gene contains one or more indels (insertions or deletions) within the nucleic acid sequence, at this time, the indel is located within the protospacer-adjacent motif (PAM) sequence in the third exon region of the Keap1 gene, or within a continuous 5-50 nucleotide sequence adjacent to the 5' or 3' end of the PAM sequence, the sequence of the artificially engineered Keap1 gene, characterized by not containing SEQ ID NO: 15 or SEQ ID NO: 50, the artificially engineered mesenchymal stem cell is characterized by improved oxidative stress resistance, an artificially engineered mesenchymal stem cell.

2. Within the artificially engineered mesenchymal stem cell, the mRNA transcribed from the artificially engineered Keap1 gene has a lower mRNA expression level or a different sequence compared to the mRNA expression level transcribed from the Keap1 gene of wild-type mesenchymal stem cells, the artificially engineered mesenchymal stem cell according to claim 1.

3. The artificially engineered mesenchymal stem cell according to claim 1, characterized by having high viability in an oxidative stress environment.

4. The artificially engineered mesenchymal stem cell according to claim 1, characterized by being derived from adipose, bone marrow, umbilical cord, placenta, amniotic fluid, amnion, tissue, umbilical cord blood or perinatal tissue.

5. A guide nucleic acid containing a guide sequence capable of targeting the target sequence of SEQ ID NO: 15 or SEQ ID NO: 50 of the Keap1 (Kelch-like ECH-associated protein 1) gene of mesenchymal stem cells, or a nucleic acid sequence encoding the same; and a composition for producing mesenchymal stem cells having oxidative stress resistance, containing an editor protein or a nucleic acid sequence encoding the same.

6. The composition according to claim 5, wherein the composition contains the editor protein and the guide nucleic acid in the form of a ribonucleoprotein (RNP).

7. The composition The composition for producing mesenchymal stem cells having oxidative stress resistance according to claim 5, which contains, in the form of one or more vectors, a nucleic acid sequence encoding the editor protein and a nucleic acid sequence encoding the guide nucleic acid.

8. The composition for producing mesenchymal stem cells having oxidative stress resistance according to claim 7, wherein the vector is selected from the group consisting of a plasmid, a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a vaccinia virus, a poxvirus, and a herpes simplex virus.

9. (1) A guide nucleic acid capable of targeting the target sequence of SEQ ID NO: 15 or SEQ ID NO: 50 of the Keap1 (Kelch-like ECH-associated protein 1) gene or a nucleic acid sequence encoding the same; and introducing the composition for producing mesenchymal stem cells having oxidative stress resistance containing an editor protein or a nucleic acid sequence encoding the same into isolated mesenchymal stem cells; and (2) Editing the Keap1 gene so that the expression or activity of the Keap1 (Kelch-like ECH-associated protein 1) protein is reduced or suppressed by generating an indel in the target sequence of the Keap1 (Kelch-like ECH-associated protein 1) gene located in the genome of the mesenchymal stem cells. A method for producing mesenchymal stem cells having oxidative stress resistance, comprising the steps of:

10. The method for producing mesenchymal stem cells having oxidative stress resistance according to claim 9, wherein the indel is generated so as to be located within a protospacer-adjacent motif (PAM) sequence in the third exon region of the Keap1 gene, or within a continuous 5-50 nucleotide sequence adjacent to the 5' end or 3' end of the PAM sequence.

11. The composition according to claim 9, wherein the composition contains, in the form of one or more vectors, a nucleic acid sequence encoding the editor protein and a nucleic acid sequence encoding the guide nucleic acid.

12. The method for producing mesenchymal stem cells having oxidative stress resistance according to claim 9, wherein indels are generated by contacting a target sequence of the Keap1 (Kelch-like ECH associated protein 1) gene located within the genome of the mesenchymal stem cells with a CRISPR / Cas9 complex comprising a Cas9 protein derived from Streptococcus pyogenes and a guide RNA capable of targeting the target sequence of the Keap1 gene.

13. A pharmaceutical composition for preventing or treating ischemic diseases, comprising, as an active ingredient, the artificially engineered mesenchymal stem cells according to any one of claims 1 to 4.

14. The ischemic diseases are ischemic heart diseases, peripheral artery diseases, critical limb ischemia (CLI), thromboangiitis obliterans, diabetic peripheral vascular diseases, osteonecrosis, mesenteric ischemia, ischemic colitis, ischemic enteritis, acute kidney injury, ischemia-reperfusion injury, ischemic hepatitis, ischemic pancreatitis, ischemic optic neuropathy, chronic obstructive pulmonary disease, acute respiratory distress syndrome (ARDS), COVID-19 infection, neonatal hypoxic-ischemic encephalopathy, or stroke. The pharmaceutical composition for preventing or treating ischemic diseases according to claim 13.

15. A method for treating ischemic diseases, comprising administering to a non-human mammal a therapeutically effective amount of the artificially engineered mesenchymal stem cells according to any one of claims 1 to 4.

16. The ischemic disease is ischemic heart diseases, peripheral artery disease, critical limb ischemia (CLI), thromboangiitis obliterans, diabetic peripheral vascular disease, osteonecrosis, mesenteric ischemia, ischemic colitis, ischemic enteritis, acute kidney injury, ischemia-reperfusion injury, ischemic hepatitis, ischemic pancreatitis, ischemic optic neuropathy, chronic obstructive pulmonary disease, acute respiratory distress syndrome (ARDS), COVID-19 infection, neonatal hypoxic-ischemic encephalopathy, or stroke, the method for treating an ischemic disease according to claim 15.

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