High-throughput gene editing technology

Abstract

One of the purposes of the present invention is to provide a method for quickly producing a gene-edited tissue stem cell in an undifferentiated state. Another one of the purposes of the present invention is to provide a therapeutic and / or prophylactic method for disease through usage of a gene-edited tissue stem cell in an undifferentiated state. According to the present invention, a gene-edited tissue stem cell is produced by a method comprising a step for editing the gene sequence of a target gene in tissue stem cells, and a step for screening the tissue stem cells having an edited gene sequence, and optionally, further comprising a step for adding a tag to the gene sequence of the target gene and / or a step for activating gene expression of the target gene in the tissue stem cells. The tissue stem cell thus produced is used in therapy / prevention of diseases.

Description

Technical Field

[0001] The present invention relates to a gene editing method, and more particularly, to a method for performing gene editing of tissue stem cells in a short period of time.

Background Art

[0002] Genome editing is a technique for modifying a target gene using a site-specific nuclease. As the nuclease, ZFN, TALEN, CRISPR / Cas9, etc. are used. Compared with conventional genetic engineering and gene therapy, the application range is very wide, and the development of therapeutic methods applying genome editing technology is also in progress (Non-Patent Document 1).

[0003] For example, CRISPR Therapeutics (Switzerland) is developing a treatment method for sickle cell anemia (CTX001) using genome editing (Non-Patent Documents 2 and 3). Sickle cell anemia is caused by a mutation in the HBB gene that gives instructions to make hemoglobin, a molecule in red blood cells that carries oxygen. In sickle cell anemia, as a result of this mutation, a deficiency of hemoglobin occurs, and the oxygen-carrying function of red blood cells decreases.

[0004] CTX001 aims to change genes using gene editing technology to increase the production of fetal hemoglobin (HbF) in the red blood cells of patients. Fetal hemoglobin is a type of hemoglobin that naturally exists in newborns and is later replaced by adult-type hemoglobin. However, sometimes fetal hemoglobin persists in adults and protects people from sickle cell anemia and β-thalassemia.

[0005] For treatment, hematopoietic stem cells, which are bone marrow-derived cells that give rise to red blood cells and white blood cells that make up the blood, are collected from a patient, and then the genes are modified so that they can produce high levels of fetal hemoglobin. More specifically, gene modification is performed to disrupt the BCL11A gene, which is a transcriptional repressor of HbF. The cells thus treated are then returned to the patient's body, and it is thought that they can produce a large amount of red blood cells containing fetal hemoglobin in the patient's body, thereby overcoming the hemoglobin deficiency caused by the disease.

[0006] However, in the treatment of diseases such as blood, immune, and cancer, many problems remain, such as the efficiency of gene editing in tissue stem cells and the difficulty of cell selection, and there are still many inadequacies in the medical application of gene editing technology.

Prior Art Documents

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0008] One object of the present invention is to provide a method for producing undifferentiated tissue stem cells edited by gene editing in a short period of time. Another object of the present invention is also to provide a method for treating and / or preventing diseases using undifferentiated tissue stem cells edited by gene editing.

Means for Solving the Problems

[0009] The present inventors have developed a technique for producing undifferentiated tissue stem cells edited by gene editing in a short period of time by applying genome editing technology. By using this technique, a method for treating and / or preventing diseases using undifferentiated tissue stem cells edited by gene editing can be provided. More specifically, the present invention includes the following aspects.

[0010] [Aspect 1] A method for producing gene-edited tissue stem cells, comprising a step of editing a gene sequence of a target gene in tissue stem cells and a step of selecting the tissue stem cells with the edited gene sequence. [Aspect 2] The method according to Aspect 1, further comprising a step of adding a tag to the gene sequence of the target gene and / or a step of activating the gene expression of the target gene in the tissue stem cells. [Aspect 3] The method according to Aspect 1 or 2, wherein the tissue stem cells are tissue stem cells isolated from a patient. [Aspect 4] The method according to any one of Aspects 1 to 3, wherein the editing of the gene sequence is performed ex vivo. [Aspect 5] The method according to any one of Aspects 1 to 4, wherein the editing of the gene sequence is performed using the CRISPR / Cas system or the TALEN system. [Aspect 6] The method according to any one of Aspects 1 to 5, wherein the target gene is a gene that is not constitutively expressed in tissue stem cells. [Aspect 7] The method according to any one of Aspects 2 to 6, wherein the activation of gene expression is performed using the CRISPRa system or the TALEN effector system. [Aspect 8] The method according to any one of Aspects 2 to 7, wherein the tissue stem cells with the edited gene sequence are selected using a tag added to the gene sequence. [Aspect 9] The method according to any one of Aspects 2 to 8, wherein the step of editing the gene sequence of the target gene in the tissue stem cells and the step of adding a tag to the gene sequence of the target gene are performed simultaneously. [Aspect 10] The method according to any one of Aspects 1 to 9, wherein the tissue stem cells with the edited gene sequence are selected within 24 hours after the cells are isolated from the patient. [Aspect 11] The method according to any one of Aspects 1 to 10, wherein the selected tissue stem cells maintain an undifferentiated state. [Aspect 12] The method according to any one of Aspects 1 to 11, further comprising the step of proliferating the selected tissue stem cells. [Aspect 13] The method according to any one of Aspects 1 to 12, further comprising the step of transplanting the selected tissue stem cells into the patient. [Aspect 14] The method according to any one of Aspects 1 to 13, wherein the tissue stem cells with the edited gene sequence are cells for use in the treatment of a disease. [Aspect 15] The method according to Aspect 14, wherein the disease is a blood disease or an immune disease. [Aspect 16] The method according to aspect 15, wherein the blood disorder or immune disorder is selected from the group consisting of ADA deficiency, X-linked severe combined immunodeficiency (SCID), other SCIDs, Wiskott-Aldrich syndrome, chronic granulomatous disease, leukocyte adhesion deficiency, familial hemophagocytic syndrome, X-linked Hyper IgM syndrome, X-linked lymphoproliferative disease, X-linked agammaglobulinemia, Hyper-IgE syndrome, sickle cell anemia, and β-thalassemia. [Aspect 17] The method according to any one of aspects 1 to 16, wherein the target gene is selected from the group consisting of ADA, IL2RG, WAS, CYBB, INTGB2, UNC13D, CD40L, SAP / SH2D1A, BTK, STAT3, and hemoglobin. [Aspect 18] A therapeutic agent for a blood disorder or immune disorder, comprising hematopoietic stem cells within 48 hours after isolation from a living body, wherein the gene sequence of the target gene has been edited. [Aspect 19] The therapeutic agent according to aspect 18, wherein the blood disorder or immune disorder is selected from the group consisting of ADA deficiency, X-linked severe combined immunodeficiency (SCID), other SCIDs, Wiskott-Aldrich syndrome, chronic granulomatous disease, leukocyte adhesion deficiency, familial hemophagocytic syndrome, X-linked Hyper IgM syndrome, X-linked lymphoproliferative disease, X-linked agammaglobulinemia, Hyper-IgE syndrome, sickle cell anemia, and β-thalassemia. [Aspect 20] The therapeutic agent according to aspect 18 or 19, wherein the target gene is selected from the group consisting of ADA, IL2RG, WAS, CYBB, INTGB2, UNC13D, CD40L, SAP / SH2D1A, BTK, STAT3, and hemoglobin. [Aspect 21] A method for producing genetically edited tissue stem cells, comprising: editing the gene sequence of the target gene in the tissue stem cells; adding a tag to the gene sequence of the target gene; activating the gene expression of the target gene in the tissue stem cells; and selecting the genetically edited tissue stem cells. comprising a method in which a step of editing a gene sequence of a target gene in tissue stem cells and a step of adding a tag to the gene sequence of the target gene are simultaneously performed using a CRISPR / Cas system or a TALEN system. [Aspect 22] The target gene is a gene that is not constitutively expressed in tissue stem cells, only in cells in which editing of the gene sequence of the target gene and addition of a tag have been performed, a target protein labeled with the tag is produced by a step of activating gene expression of the target gene, and tissue stem cells with the edited gene sequence are selected using the tag, the method according to Aspect 21. [Aspect 23] The tissue cells are tissue stem cells isolated from a patient, and the tissue stem cells with the edited gene sequence are selected within 48 hours after the cells are isolated from the patient, the method according to Aspect 21 or 22.

Advantages of the Invention

[0011] According to the present invention, undifferentiated tissue stem cells edited with a gene can be produced within a short period of time. And, using such tissue stem cells, it becomes possible to treat and / or prevent diseases that have been difficult to intervene in until now.

Brief Description of the Drawings

[0012]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

[0013] Some embodiments of the present invention relate to a method for producing genetically edited tissue stem cells that can accurately select genetically edited tissue stem cells at the single-cell level in a short period of time. The method of the present invention includes a step of editing the gene sequence of a target gene in tissue stem cells and a step of selecting the genetically edited tissue stem cells, and further optionally includes a step of adding a tag to the gene sequence of the target gene and / or a step of activating the gene expression of the target gene in the tissue stem cells. Each step will be described in detail below.

[0014] [Step of Editing the Gene Sequence of a Target Gene in Tissue Stem Cells] Genome editing is a technique for introducing mutations (substitutions, insertions, deletions) into any genomic sequence using an artificial restriction enzyme that can specifically cleave a target site in the genome. When a double-strand break occurs at the target site by a restriction enzyme, in the absence of template DNA, during genome repair by the cell repair mechanism NHEJ (Non-Homologous End Joining), base pair deletions and insertions occur due to errors during end-to-end ligation. As a result, the target gene is finally knocked out due to a frameshift or the like. On the other hand, in the presence of template DNA, substitution and insertion of the template sequence into the target site occur by the repair mechanism HDR (Homology Dependent Repair) (knock-in).

[0015] In the method according to the present invention, the editing of the gene sequence can be either a knock-out that destroys the gene or a knock-in that substitutes or inserts a sequence for which introduction or modification is desired. Preferably, it is a knock-in using a desired template sequence, whereby, for example, a sequence variation that causes a disease or the like is corrected to a normal sequence or a harmless sequence.

[0016] In the method according to the present invention, main correction tools that can be used for editing the gene sequence of a target gene include, for example, ZFN systems (Zinc Finger Nucleases), TALEN systems (Transcription Activator Like Effector Nucleases), and CRISPR / Cas systems (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR Associated Protein 9), etc. These systems are highly specific genome editing tools composed of a nuclease responsible for cleaving the DNA double strand of the genome, a guide molecule that guides the nuclease to a target site on the genome, and optionally template DNA. The template DNA may be single-stranded DNA or double-stranded DNA.

[0017] The ZFN system (see, for example, Kim, Y. G., et al., 1996, Proc. Natl. Acad. Sci. USA 93:1156-1160.) is simply an artificial chimeric protein composed of two functional domains, Zinc-Finger, which is one of the DNA-binding motifs present in the DNA-binding domains of many transcription factors, and the FokI nuclease domain, and genome editing is performed using a pair of ZFNs designed to sandwich the target sequence. In the ZFN system, the Zinc-Finger, which is the DNA-binding domain, recognizes a specific gene sequence on the target gene, whereby the FokI nuclease, which is the DNA cleavage domain, causes double-strand cleavage with high specificity.

[0018] The TALEN system (see, for example, Cermak T, et al., 2011, Nucleic Acids Res 39: e82) is also an artificial chimeric protein in which a FokI nuclease domain is fused to a DNA-binding domain (TAL effector (TALE)) that recognizes an arbitrary base sequence, similar to ZFN. The DNA-binding domain of TALEN recognizes 15-20 bases, and double-strand cleavage is performed using a pair of TALENs designed for each of the sense strand and the antisense strand so as to sandwich a 14-20 base spacer that becomes the target sequence of FolI. According to the TALEN system, cleavage of a more specific target sequence than ZFN becomes possible, and theoretically any genomic sequence can be targeted. The TALEN system is also said to be superior to the CRISPR / CAS system in terms of suppression of off-target mutations.

[0019] The CRISPR / Cas system centered around CRISPR / Cas9 (see, for example, Cong, L. et al., 2013, Science, 339, 819-823.) uses complementary binding between RNA and DNA for target sequence recognition, whereas the aforementioned ZFN and TALEN use protein-DNA interactions for target sequence recognition. In type II CRISPR / Cas systems, briefly, the Cas9 nuclease responsible for double-strand cleavage is guided to the target sequence by guide RNA (a complex of crRNA and tracrRNA or a single-stranded sgRNA formed by them), and the CAS9-RNA complex cleaves the DNA sequence complementary to the guide RNA. In type V CRISPR / Cas systems, the RNA-dependent DNA nuclease Cpf1 is used. The guide RNA of the type V CRISPR / Cas system consists only of crRNA. Since the guide RNA and nuclease in the CRISPR / Cas system exist and function separately, it is excellent in convenience in that it enables the introduction of multiple mutations by introducing multiple guide RNAs. For example, components used in the CRISPR / CAS system (Cas9 gRNA, Cas9 protein, Cas9 plasmid, Cas9 carrier DNA, buffer, etc.) and components used in the CRISPR / Cas12a system (Cas12a (Cpf1) gRNA, Cas12a (Cpf1) protein, Cas12a (Cpf1) carrier DNA, buffer, etc.) can be purchased from Integrated DNA Technologies (IDT). As a tool for designing sgRNA, for example, CRISPRdirect (http: / / crispr.dbcls.jp) can be used. For the synthesis and purification of RNA, for example, mMessage mMachine (trademark) T7 UTRA Transcription Kit (Ambion, AM1345), MEGAclear (trademark) Transcription Clean-Up Kit (Ambion, AM1908), etc. can be used.

[0020] For example, when preparing guide RNA using Alt-RTM (IDT), mix 3.58 μl each of Alt-RTM crRNA (100 μM, IDT) containing the target sequence, tracrRNA (100 μM, IDT, 1072532), and Nuclease-free Duplex Buffer (IDT, 11-01-03-01), incubate at 95 °C for 5 minutes, and then cool to room temperature for use. In the preparation of sgRNA using the gRNA vector (Addgene No. 41824), the target sequence is introduced by inverse PCR to prepare gRNA template DNA. RNA is synthesized using the mMessage mMachine™ T7 UTRA Transcription Kit (Ambion, AM1345) and purified using the MEGAclear™ Transcription Clean-Up Kit (Ambion, AM1908) to a concentration of 100 μM. When using nuclease as a recombinant protein, SpCas9 (IDT, 1074182), cpf1 (IDT, 1081069) can be diluted to 10 mg / ml for use. When using nuclease as RNA, for example, referring to the methods described in the literature (Nishimasu H, et al. Science. 2018;361:1259-1262. Esvelt KM, et al. Nat Methods. 2013;10(11):1116-1121.), RNA can be synthesized using the mMessage mMachine™ T7 UTRA Transcription Kit (Ambion, AM1345) with SpCas9, SpCas-9-NG, dCas9, M-NMn-VP64 (Addgene # 80425, #41816, #48676) on the vector as template DNA, and purified by ethanol precipitation.

[0021] Although the ZFN system, TALEN system, and CRISPR / Cas system have been briefly described above, improved versions of these systems and any other genome editing tools can also be used in the present invention. From the perspective of specificity, gene sequence editing is preferably performed using the CRISPR / Cas system or the TALEN system.

[0022] When using these genome editing tools, a guide molecule-nuclease complex, or an expression plasmid of guide RNA and / or nuclease, or a virus packaged with the expression plasmid, can be introduced into target cells to express the guide molecule-nuclease complex, or guide RNA and / or nuclease in the target cells. Alternatively, the nuclease or guide molecule-nuclease complex can be introduced into the target cells as mRNA or protein, and guide RNA can be introduced as RNA. When introducing an expression plasmid or the like, an agent that induces expression can be allowed to act on the target cells. From the perspective of off-target risk due to continuous expression of the nuclease by plasmid introduction, the time lag involved in transcription and translation, and the improvement of mutagenesis efficiency, it is preferable to introduce the nuclease or guide molecule-nuclease complex as mRNA or protein, and guide RNA as RNA. The methods for introducing these RNAs, proteins, plasmids, or packaged viruses are not particularly limited, and methods known to those skilled in the art can be used. Non-limiting examples include calcium phosphate precipitation, lipofection, polymer capsules, particle guns, microinjection, electroporation, and the like. Electroporation is common in terms of the balance between simplicity and introduction efficiency.

[0023] Electroporation for introducing RNA, protein, and DNA into cells can be performed, for example, using a Nepa21 pulse generator (Nepa Gene). The power transmission can be performed, for example, by applying two rectangular electric pulses (225 V, 2 ms width, 50 ms interval), followed by five pulses (30 V, 50 ms width, 50 ms interval). 2 ml of HBSS+ medium can be added to the electroporated mixture and centrifuged at 800 rpm for 3 minutes for washing. These cells may be cultured for about 24 hours using the medium used for hematopoietic stem cell collection. Furthermore, cells in which gene editing has been accurately performed can be selected by detecting at the single-cell level, such as GFP signals.

[0024] When handling tissue stem cells, it is desirable to have a working space suitable for using RNA and protein under aseptic conditions and an SPF area as an animal experiment facility. Also, as a means for efficiently extracting cells into which mutations have been introduced, equipment for detecting and selecting luminescent tags can be preferably used.

[0025] [Step of selecting tissue stem cells with edited gene sequences] The method according to the present invention includes a step of selecting, at the single-cell level, cells in which the target gene has been edited from a pool of tissue stem cells after editing of the target gene in the target cells. Examples of methods for selecting cells in which the target gene has been edited include methods based on the nucleic acid sequence of the edited target gene and methods based on the expression of the edited target gene. From the viewpoint of accurately selecting, at the single-cell level, cells that can normally express the edited target gene, it is preferable to perform the selection by detecting the expression of the target gene that has been transiently activated, for example. For detecting the expression of the activated target gene, a system that utilizes, for example, fluorescence or phosphorescence detection can be used.

[0026] [Step of activating gene expression of the target gene in tissue stem cells] The target gene is often a gene that is expressed when tissue stem cells differentiate into the corresponding tissue cells and perform various functions. That is, in many cases, the target gene is a gene that is not constitutively expressed in tissue stem cells. Therefore, in order to select cells in which the target gene has been edited based on the expression of the target gene, it is required to transiently activate the expression of the target gene. Thus, in one embodiment, the method of the present invention may further include a step of activating the gene expression of the target gene in tissue stem cells. Such activation of gene expression may be transient. By this step, only the cells in which the target gene has been edited so that it can be normally expressed can be sorted at the single-cell level. By this step, it is also possible to exclude cells in which unintended editing, such as off-target mutations, has occurred in the step of editing the gene sequence.

[0027] Examples of the activation tool for transiently activating the target gene include, for example, a method of allowing a transcriptional activator (such as VP64) to act on the promoter region existing upstream of the target gene. In one embodiment, a method that can be performed substantially simultaneously with the above-described step of editing the target gene or the step of tagging the target gene described below is preferred.

[0028] Examples of such activation tools include the CRISPRa (CRISPR activation) system and the TALEN effector system.

[0029] The CRISPRa system (see, for example, Silvana Konermann et al. Nature. 2015 Jan 29;6(7536):583-588) is, briefly, a system in which a dCas9-transcriptional activator complex, which is a fusion of a dead Cas9 (dCas9) nuclease lacking DNA cleavage activity and a transcriptional activator (such as VP16, VP64, etc.), and a guide RNA targeting the promoter site of a target gene are introduced, thereby inducing the dCas9-transcriptional activator complex at the transcription start point of the target gene and activating the expression of the target gene. As the CRISPRa system, an expression vector for the dCas9-transcriptional activator complex and the guide RNA, or a viral particle packaging the expression vector, etc. may be introduced into target cells, or the dCas9-transcriptional activator complex may be introduced into target cells as a protein and the guide RNA may be introduced into target cells as RNA. However, in the present embodiment, from the viewpoints of rapid activation and safety, it is more preferable to introduce the dCas9-transcriptional activator complex as a protein and the guide RNA as RNA into target cells respectively.

[0030] In addition, it is also preferable to use a TAL effector system in which a TAL DNA binding domain targeting the promoter region of a target gene is fused to a transcriptional activator (such as VP16, VP64, etc.). As a design tool for the TAL effector system, for example, http: / / tale-nt.cac.cornell.edu / node / add / single-tale can be used. For example, referring to the method described in the literature (Matsubara, Y, et al. Sci Rep, 4 : 5043, 2014), the TAL effector is introduced into a mammalian expression vector (pcDNA-TAL-VP-64 (Addgene, 47107)) by the Golden Gate method with reference to TAL Effector Nucleotide Targeter 2.0 (https: / / tale-nt.cac.cornell.edu / node / add / single-tale) for preparation. When the CRISPRa system is used as an activation tool, it can be designed by the same method as the above editing tool.

[0031] These activation tools can be introduced into target cells by the same method as the above editing tools.

[0032] [Step of adding a tag to the gene sequence of the target gene] The method for detecting the expression of the target gene can be appropriately selected according to the characteristics of the target gene, and examples thereof include a method using an antibody reactive with a membrane protein, a method for detecting a secreted protein by its enzyme activity, and the like.

[0033] In one embodiment, the genetically edited tissue stem cells can be sorted using the tag added to the gene sequence. For example, by adding a tag sequence that is expressed only when the target gene is edited into the normal gene of interest to the target gene, only the cells that normally express the target gene can be accurately selected at the single-cell level. Such tag sequences are not particularly limited, and examples thereof include peptide or polypeptide tags (FLAG, HA, His, Myc, V5, S, Trx, etc.), reporter genes (Luciferase, etc.), fluorescent proteins (GFP, RFP, Venus, etc.). The Venus sequence can be prepared by introducing a homologous gene of the target gene into the pVenus vector with reference to the method described in the literature (Matsubara, Y, et al. Sci Rep, 4 : 5043, 2014) and used as template DNA. Such tags can be detected using labeled antibodies, fluorescence, luminescence, or the like. In the present invention, from the viewpoints of live cell detection and mutagenesis efficiency, small molecules such as peptides with less damage to cells (FLAG, HA, etc.) and the HiBiT system (Promega) described below are preferred.

[0034] In one embodiment, from the perspective of suppressing unexpected effects on target cells, the tag may preferably be as small a molecule as possible, such as a peptide. Non-limiting examples of such tags include the HiBiT system (Promega). This system uses an 11-amino acid peptide tag (HiBiT) as the tag and a luciferase fragment (LgBiT) that binds to the tag for detection. Therefore, the influence on target cells is minimized, and target cells can be detected with high sensitivity.

[0035] The tagging tool for adding such a tag sequence to the target gene sequence is not particularly limited. For example, it may be the same as the above-described editing tool. In one embodiment, the tagging tool includes a guide RNA targeting the target gene, a Cas nuclease, and a template DNA for tag sequence insertion. Such a tagging tool can also be introduced into target cells by the same method as the above-described editing tool and activation tool. Note that the position where the tag is introduced can be any site at the N-terminus, C-terminus, or central part of the target gene. From the perspective of being able to detect the expression of the intact protein of the target gene, it is also preferable to introduce it on the C-terminal side.

[0036] [ACT Genome Editing] In some embodiments, the steps of editing the gene sequence of the target gene in the above-described tissue stem cells, activating the gene expression of the target gene in the tissue stem cells, and adding a tag to the gene sequence of the target gene are performed in combination. As used herein, "performing in combination" means performing the steps from editing to selection within a short period during which the target tissue stem cells can be maintained in vitro.

[0037] For this purpose, in one embodiment, an activation tool, a correction tool, and a tagging tool can be introduced into target cells substantially simultaneously. In the present application, the genome editing technology that combines the activation tool, the correction tool, and the tagging tool in this way is referred to as "ACT genome editing". In ACT genome editing, for example, an editing tool of the ZFN, TALEN, or CRISPR / Cas system targeting the editing site of a target gene, a tagging tool of the ZFN, TALEN, or CRISPR / Cas system targeting the translation region (C-terminal side, N-terminal, or within the translation region) of the target gene, and an activation tool of the ZFN, TALEN, or CRISPR / Cas system targeting the promoter site of the target gene are introduced into target cells substantially simultaneously. Here, the editing tool and the tagging tool may use the same system. By doing so, introduction of multiple mutations (editing and tagging of the target gene) and expression of the edited target gene can be performed in an extremely short time. As a result, tissue stem cells in which the target gene has been accurately edited can be accurately selected in a short period of time.

[0038] Here, the "short period of time" may be any period during which the target tissue stem cells can be maintained in vitro, but is preferably 1 week or less, more preferably 3 days or less (72 hours or less), for example 2 days or less (48 hours or less, 36 hours or less, 24 hours or less, or 18 hours or less). Also, "substantially simultaneously" means within several hours (for example, within 2 or 3 hours). Considering the simplicity of the operation, it is preferable to introduce the activation tool, the correction tool, and the tagging tool simultaneously. However, the order of introduction or the timing may be changed to improve the efficiency and accuracy of gene editing and gene expression. For example, all components used for the activation tool, the correction tool, and the tagging tool may be introduced into the cells by a single electroporation, or the components used for the correction tool and the tagging tool may be introduced, and then the components used for the activation tool may be introduced after a lapse of time.

[0039] In conventional genome editing technologies, even when gene editing is performed using the CRISPR / Cas system or the like, it is necessary to perform single-cell cloning after gene editing and genotype each clone by PCR or the like to select cells in which the target gene has been edited, and it took about several days to several weeks to obtain the target cells. In addition, if a drug resistance gene or the like is inserted into the target gene, the target cells can be easily obtained by selecting drug-resistant strains. However, when inserting foreign DNA such as a drug resistance gene by homologous recombination between genomic DNA and a donor vector, there are also problems such as extremely low efficiency in general. However, according to the method according to the present embodiment, by performing editing, tagging, and activation substantially simultaneously, it is possible to select only cells in which the target gene has been edited with high efficiency in an extremely short time within the period during which tissue stem cells can be maintained in vitro.

[0040] In one embodiment, according to the method of the present invention, it is possible to select tissue stem cells with an edited gene sequence within 2 days, preferably within 24 hours, after the cells are isolated from a patient.

[0041] Examples of methods for selecting gene-edited tissue stem cells at the single-cell level include, but are not limited to, the limiting dilution method, the cell sorter method, etc. Gene-edited tissue stem cells may be selected using a tag added to the gene sequence. In some embodiments, the tag may constitute part or all of a system that emits fluorescence or phosphorescence. For the tag, for example, as described above, the HiBiT system of Promega Corporation (Wisconsin, USA) can be used.

[0042] [Tissue stem cells] In the method according to the present invention, the editing of the gene sequence is usually performed ex vivo in isolated tissue stem cells.

[0043] Tissue stem cells (also referred to as tissue-specific stem cells or somatic stem cells) can be classified, for example, into the skin system (epidermal stem cells, hair follicle stem cells, etc.), the digestive system (pancreatic (common) stem cells, hepatic stem cells, etc.), the bone marrow system (hematopoietic stem cells, mesenchymal stem cells (including muscle satellite cells, etc.)), the nervous system (neural stem cells, retinal stem cells, etc.) based on the site from which the cells are derived. The tissue stem cells to which the method according to the present invention can be applied are not particularly limited, but are particularly effective in the bone marrow system, especially hematopoietic stem cells, for reasons such as the pathological condition of the disease, established transplantation methods, and the tissue engraftment of transplanted cells.

[0044] Tissue stem cells may be isolated and obtained from the cells of each tissue from which they are derived, or may be obtained by inducing differentiation from more undifferentiated pluripotent stem cells such as ES cells and iPS cells. However, in the method according to the present invention, from the viewpoint of safety and the like, it is preferable to isolate them from the cells of each tissue from which they are derived. For example, hematopoietic stem cells can be isolated from bone marrow, umbilical cord blood, and peripheral blood. Such isolation methods are not particularly limited, and methods known to those skilled in the art can be used. For the method of obtaining hematopoietic stem cells, for example, the methods described in the literature (Bak RO, et al. Nat Protoc. 2018;13(2):358-376, Forraz N, et al. Stem Cells. 2004;22(1):100-108.) can be referred to. Specifically, for example, the supernatant is removed from bone marrow fluid by centrifugation (1500 rpm, 5 minutes, 4°C), 1 mL of red blood cell lysis medium (Takara Bio, 786-649) and 9 mL of RPMI-1640 medium (FUJIFILM Wako Pure Chemical Corporation, 189-02025) are added, and further centrifugation (1500 rpm, 5 minutes, 4°C) is performed to remove red blood cells. The cells washed with HBSS+ medium are sorted by lineage negative selection using an antibody. For lineage negative selection, MagniSort™ Human Hematopoietic Lineage Depletion Kit (Thermofisher Scientific, 8804-6836-74) can be used.

[0045] The extraction of mouse hematopoietic stem cells can be carried out with reference to the methods described in, for example, the literature (Ema H, et al. Nat Protoc. 2006;1(6):2979-87, Gundry MC, et al. Cell Rep. 2016;17:1453-61, Hetzel M, et al. Blood. 2018;131(5):533-545). Specifically, both femurs and tibias of the mouse are collected and recovered into a 6 cm dish containing about 6 ml of HBSS+ medium with 2% fetal bovine serum (FUJIFILM Wako Pure Chemical Corporation, 082-09365). Both bone ends are separated, a 25G needle is inserted into the cut fragment, and the cells are extruded with HBSS+ medium, and the cells are recovered through a 70 μm nylon cell strainer (FALCON, 352350). The supernatant is removed by centrifugation (1500 rpm, 5 minutes, 4°C), 1 mL of erythrocyte lysis medium (Takara Bio, 786-649), RPMI-1640 medium (FUJIFILM Wako Pure Chemical Corporation, 189-02025), and 9 ml of 10% fetal bovine serum are added, and centrifugation (1500 rpm, 5 minutes, 4°C) is further performed to remove erythrocytes. The cells washed with HBSS+ medium are subjected to lineage negative selection using antibodies. The antibodies are administered at 2 μl each per 1.0×10 7 cells as follows (Biotin anti-mouse Ter-119 / Erythroid Antibody (BioLegend, 79748), Biotin anti-mouse CD11b Antibody (BioLegend, 79749), Biotin anti-mouse Ly-6G / Ly-6C (Gr-1) Antibody (BioLegend, 79750), Biotin anti-mouse NK-1.1 Antibody (BioLegend, 108703), Biotin anti-mouse CD45R / B220 Antibody (BioLegend, 79752), Biotin anti-mouse CD127 (IL-7Rα) Antibody (BioLegend, 135005), Biotin anti-mouse CD3 Antibody (BioLegend, 79751)). The antibody reaction is carried out on ice for 60 minutes, and after washing, 1.0×10 7Suspend per cell in 500 μl of 0.5% fetal bovine serum + 1% PBS solution, and add 50 μl of Dynabeads™ M-280 Streptavidin (Invitrogen, 11205D). React further for 60 minutes on ice, and collect the negative cells not bound to the antibody using a magnetic stand. Wash with HBSS+ medium and suspend in 1×10 6 cells per 100 μl of culture solution, and culture at 37°C under 5% CO2 for 1 hour to 24 hours. The culture solution is adjusted to a final concentration of 200 mmol / L, 100 ng / ml, 50 μg / ml, 100 ng / ml, 100 ng / ml, and 1 mM of L-glutamine (FUJIFILM Wako Pure Chemical Corporation, 073-05391), mouse stem cell factor (FUJIFILM Wako Pure Chemical Corporation, 196-15581), thrombopoietin (R&D, 488-TO-005), insulin-like growth factor-2 (Cosmo Bio, 100-14), and fibroblast growth factor (FUJIFILM Wako Pure Chemical Corporation, 062-06041) using StemSpan™ SFEM medium (STEM CELL Technologies, 09600).

[0046] The extraction of human hematopoietic stem cells can be carried out, for example, with reference to the methods described in the literature (Bak RO, et al. Nat Protoc. 2018;13(2):358-376, Forraz N, et al. Stem Cells. 2004;22(1):100-108.). Specifically, for example, human bone marrow fluid is centrifuged (1500 rpm, 5 minutes, 4 °C) to remove the supernatant, 1 mL of red blood cell lysis medium (Takara Bio, 786-649), 9 mL of RPMI-1640 medium (FUJIFILM Wako Pure Chemical Corporation, 189-02025), and 10% fetal bovine serum are added, and centrifugation (1500 rpm, 5 minutes, 4 °C) is further performed to remove red blood cells. The cells washed with HBSS+ medium are subjected to lineage negative selection using an antibody. Lineage negative selection is performed using MagniSort™ Human Hematopoietic Lineage Depletion Kit (Thermofisher Scientific, 8804-6836-74). Wash with HBSS+ medium and suspend in 100 μl of culture medium per 1×10 6 cells and culture at 37 °C under 5% CO2 (from 1 hour to 24 hours). The culture medium is adjusted to a final concentration of 100 ng / ml, 0.75 μM, 35 nM, 100 ng / ml, 100 ng / ml, and 100 ng / ml of IL-6 (PeproTech, 200-06), StemRegenin1 (CellagenTech, C7710), UM171 (StemCell Technologies, 72914), Flt3L (PeproTech, 300-19), thrombopoietin (PeproTech, 300-18), and human stem cell factor (PeproTech, 300-07), respectively, using StemSpan™ SFEM II medium (StemCell Technologies, 9655).

[0047] In particular, the tissue stem cells are preferably tissue stem cells isolated from the patient. By using patient-derived tissue stem cells, even when gene-edited tissue stem cells obtained by the method of the present invention are used for treating a patient, the problems of the presence or absence of a donor and GVHD due to transplantation can be avoided. In addition, since the pretreatment is mild, there is also an advantage that the burden on the patient is small.

[0048] The method according to the present invention may further include a step of proliferating the selected tissue stem cells as needed.

[0049] For culturing and proliferating tissue stem cells, components and conditions known in the art for maintaining the undifferentiated state and proliferation of cells can be used. For example, as a basal medium (including inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, fatty acids, etc.), SFEM medium, SFEM II, D-MEM, MEM, RPMI 1640, BME, D-MEM / F-12, Glasgow MEM, Hank's solution, mTeSR1, etc. can be mentioned. If necessary, stem cell factor, basic fibroblast growth factor (bFGF), leukocyte migration inhibitory factor (LIF), interleukin, insulin-like growth factor, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27-supplement, N2-supplement, ITS-supplement, antibiotics, etc. may be added to the medium. In addition, serum or plasma may be added to the medium. It is preferable to select each component of a grade compatible with transplantation into the patient.

[0050] In one embodiment, the gene sequence-edited tissue stem cells selected by the method according to the present invention and proliferated as needed can be transplanted into a patient. In particular, according to the present invention, since gene editing can be performed on tissue stem cells isolated from a patient and transplanted into the patient in an extremely short time, it can be applied to various tissue stem cells, especially cells such as hepatocytes and endogenous tissue stem cells (satellite cells) of skeletal muscle that are difficult to maintain in an undifferentiated state in vitro. In addition, there are also advantages such as enabling rapid treatment in response to changes in the patient's condition, such as an increase in the target gene expression level being required due to stress.

[0051] [Therapeutic agent] One aspect of the present invention also relates to a therapeutic or prophylactic agent comprising tissue stem cells in which the gene sequence of a target gene for use in the treatment of a disease has been edited.

[0052] The therapeutic and prophylactic agent according to the present invention contains tissue stem cells in which the gene sequence has been edited in an amount effective for the treatment of the disease. The tissue stem cells in which the gene sequence has been edited can particularly be those within 72 hours, 48 hours, 36 hours, 24 hours, or 18 hours after isolation from the living body. The amount of tissue stem cells for obtaining a therapeutic effect can be appropriately set by those skilled in the art in consideration of the administration route, administration form, target disease, treatment subject, treatment target site, etc. in order to obtain an appropriate therapeutic response.

[0053] Non-limiting examples of the administration regimen include administering 1×10 5 ~2×10 6 cells per kg of patient body weight per day. The administration may be a single administration or multiple administrations, or may be continuous infusion. The administration may start from a low dose and gradually increase to the target dose in consideration of the therapeutic effect. Also, of course, depending on the condition and urgency of the treatment subject, the amount may be changed to an amount outside the above range. The administration route may be local administration or systemic administration, and examples include parenteral delivery such as intravenous, intraportal, intramuscular, intraperitoneal, intratarget tissue, subcutaneous, or intradermal administration. Examples of the administration form include parenteral dosage forms such as injections, suspensions, drip infusions, and medical hydrogels.

[0054] The therapeutic agent according to the present embodiment may contain any additional components in addition to the tissue stem cells in which the gene sequence has been edited. Non-limiting examples of the additional components include pharmaceutically acceptable diluents, carriers, and other additives, such as physiological saline, isotonic solutions, buffers, soothing agents, stabilizers, preservatives, antioxidants, etc.

[0055] The therapeutic agent according to the present embodiment may be used alone or in combination with other drugs effective for the treatment of the target disease.

[0056] In this embodiment, the tissue stem cells with edited gene sequences can be hematopoietic stem cells, mesenchymal stem cells, or hepatic stem cells.

[0057] From another perspective, one aspect of the present invention relates to a method for treating or preventing a disease, including administering to a patient in need of treatment tissue stem cells with an edited gene sequence of a target gene. Further, one aspect of the present invention relates to the use of tissue stem cells with an edited gene sequence in the manufacture of a medicament for use in a method for treating or preventing a disease. The characteristics of these tissue stem cells are as described above.

[0058] [Disease groups that can be targeted] The genome editing technology using the method according to the present invention can be widely applied not only to genetic diseases but also to diseases involving the regulation of target gene expression in cells derived from tissue stem cells. The method of the present invention is effective for all modes of inheritance, including autosomal dominant inheritance, autosomal recessive inheritance, X-linked recessive inheritance, and X-linked dominant inheritance, and is particularly suitable for application to autosomal dominant genetic diseases with poor therapeutic effects by conventional gene transfer.

[0059] In conventional gene therapy for supplementing and adding target genes, there are problems such as the remaining of abnormal genes, the uncontrollability of the integration site of normal genes, and the difficulty in regulating the expression of introduced genes, and the applicable diseases are also limited. However, the method of the present invention enables the loss of function of abnormal genes or specific genes, the repair of mutations in abnormal genes, gene transfer to safe sites where carcinogenesis does not occur, and the regulation of gene expression. Furthermore, since cells with normally edited target genes can be stably maintained in the patient's body, long-term maintenance of the therapeutic effect is expected.

[0060] Among them, the method of the present invention may be particularly effective for single-gene diseases, especially those caused by deletion mutations / frameshifts and gene mutations similar thereto.

[0061] Diseases that can be targeted by the method or therapeutic agent according to the present invention include - Immunodeficiency syndromes: for example, ADA deficiency, X-linked severe combined immunodeficiency (SCID), other SCIDs, Wiskott-Aldrich syndrome, chronic granulomatous disease, leukocyte adhesion deficiency, familial hemophagocytic syndrome, X-linked Hyper IgM syndrome, X-linked lymphoproliferative disease, X-linked agammaglobulinemia, Hyper-IgE syndrome, etc. - Abnormal Hb diseases: for example, sickle cell disease, β-thalassemia, etc. - Metabolic diseases: for example, Gaucher disease, mucopolysaccharidosis, X-linked adrenoleukodystrophy, metachromatic leukodystrophy, marble bone disease, etc. - Others, Fanconi anemia, Schwachman-Diamond syndrome, Kostmann syndrome etc. (see, for example, Richard A. Morgan et al., 2017, Cell Stem Cell, 21, 574-590).

[0062] An example of the diseases that can be targeted by the method or therapeutic agent according to the present invention and their target genes is described in Table 1 below.

[0063]

Table 1

[0064] Among them, since the method according to the present invention can obtain gene-edited tissue stem cells in an extremely short period, there is a possibility of forming a non-physiological cell population in which cells with high proliferative ability are concentrated by long-term culture, a possibility of reduced colonization due to progression of differentiation, and a possibility of bacterial, viral, and mycoplasma growth by culture. By reducing these possibilities respectively, it is expected to increase the tissue colonization rate of the mutagenized cells more efficiently and safely. For these reasons, it is preferable to apply to blood diseases and immune diseases for which no treatment method has been established so far, for example, chronic granulomatous disease and Wiskott-Aldrich syndrome for which long-term engraftment is considered useful, and further diseases caused by dominant negative such as Hyper-IgE syndrome.

Examples

[0065] The present invention will be specifically described below based on examples, but the present invention is not limited to these examples at all.

[0066] Example 1: Introduction of Gene Mutations into Mouse Hematopoietic Stem Cells Mutations were introduced into the target gene sites of mouse hematopoietic stem cells extracted from wild-type mouse femurs using the CRISPR / Cas9 system. The collection of mouse hematopoietic stem cells was performed with reference to the methods described in the literature (Ema H, et al. Nat Protoc. 2006;1(6):2979-87, Gundry MC, et al. Cell Rep. 2016;17:1453-61, Hetzel M, et al. Blood. 2018;131(5):533-545). Specifically, both femurs and tibias of the mouse were collected and recovered into a 6-cm dish containing about 6 ml of HBSS+ medium containing 2% fetal bovine serum (FUJIFILM Wako Pure Chemical Corporation, 082-09365). Both bone ends were separated, a 25G needle was inserted into the cut fragment, and the cells were extruded with HBSS+ medium, and the cells were recovered through a 70-μm nylon cell strainer (FALCON, 352350). The supernatant was removed by centrifugation (1500 rpm, 5 minutes, 4°C), 1 mL of erythrocyte lysis medium (Takara Bio, 786-649), RPMI-1640 medium (FUJIFILM Wako Pure Chemical Corporation, 189-02025), and 9 ml of 10% fetal bovine serum were added, and further centrifugation (1500 rpm, 5 minutes, 4°C) was performed to remove erythrocytes. Lineage negative selection was performed on the cells washed with HBSS+ medium using an antibody. The antibody was 1.0×10 7Per cell, 2 μl each of the following antibodies were administered (Biotin anti-mouse Ter-119 / Erythroid Antibody (BioLegend, 79748), Biotin anti-mouse CD11b Antibody (BioLegend, 79749), Biotin anti-mouse Ly-6G / Ly-6C (Gr-1) Antibody (BioLegend, 79750), Biotin anti-mouse NK-1.1 Antibody (BioLegend, 108703), Biotin anti-mouse CD45R / B220 Antibody (BioLegend, 79752), Biotin anti-mouse CD127 (IL-7Rα) Antibody (BioLegend, 135005), Biotin anti-mouse CD3 Antibody (BioLegend, 79751)). The antibodies were reacted for 60 minutes on ice. After washing, 1.0×10 7 Per cell, the cells were suspended in 500 μl of 0.5% fetal bovine serum + 1% PBS solution, and 50 μl of Dynabeads™ M-280 Streptavidin (Invitrogen, 11205D) was added. The reaction was further carried out for 60 minutes on ice, and the negative cells not bound by the antibody were collected using a magnetic stand.

[0067] The target gene was IL2RG, and the sgRNA mixture and Cas9 recombinant protein were introduced by electroporation. Interleukin-2 receptor gamma chain (Il2rg) is the causative gene of X-linked severe combined immunodeficiency (X-SCID). The sgRNA was prepared using Alt-RTM (IDT). Alt-RTM crRNA (100 μM, IDT, custom synthesis product (https: / / sg.idtdna.com / jp / site / )) containing the target sequence (aggattgatgttcaggcttc; SEQ ID NO: 1), tracrRNA (100 μM, IDT, 1072532), and Nuclease-free Duplex Buffer (IDT, 11-01-03-01) were mixed at 3.58 μl, 3.58 μl, and 7.84 μl respectively, incubated at 95°C for 5 minutes, and then cooled to room temperature. For the Cas9 recombinant protein, SpCas9 (IDT, 1074182) was diluted to 10 mg / ml for use. As for the details of cell culture and the method of introducing RNA and protein, nuclease recombinant protein (3.69 μl at 10 mg / ml) was added to the RNA mixture of sgRNA (100 μM) and incubated at room temperature for 20 minutes. To this, 7.7 μl of mouse hematopoietic stem cells and template DNA (1 mg / ml) were mixed, diluted with OptiMEM medium (ThermoFisher Scientific, 31985070), and adjusted to 100 μl. Electroporation was performed on this cell, RNA, protein, and oligonucleotide DNA mixture using a Nepa21 pulse generator (Nepa Gene). The power transmission was two rectangular electric pulses (225 V, 2 ms width, 50 ms interval), followed by five pulses (30 V, 50 ms width, 50 ms interval). 2 ml of HBSS+ medium was added to the electroporated mixture, and the mixture was centrifuged at 800 rpm for 3 minutes for washing. 6

[0068] Genomic DNA was extracted 24 hours after mutagenesis introduction (DNeasy Blood & Tissue Kit, Qiagen, 69504), and PCR amplification was performed on a genomic DNA sequence of approximately 500 bp in length flanking the target site (Quick Taq (registered trademark) HS DyeMix, Toyobo, DTM-101). The Forward primer (gagctatctgtctttaggcctggag; SEQ ID NO: 2) and Reverse primer (caacctggcctacatagtgagctc; SEQ ID NO: 3) were used. The PCR conditions were 2 minutes at 94°C, followed by 30 cycles of 30 seconds at 94°C / 30 seconds at 60°C / 30 seconds at 68°C. This PCR product was purified and denatured by treatment at 95°C for 5 minutes in NEBuffer2 (New England BioLab, B7002). Subsequently, slow annealing was performed at room temperature to form DNA mismatches at the sites where mutagenesis introduction occurred. This was subjected to DNA mismatch cleavage by treatment with T7 endonuclease I (T7EI) (New England BioLab, M0302S), which recognizes and cleaves only DNA containing mismatches, at 37°C for 15 minutes. Figure 1 shows the PCR product treated with this T7 endonuclease I electrophoresed on a 1% agarose gel, indicating the formation of DNA mismatches at the target site, i.e., successful mutagenesis introduction. Figure 2 shows the sequencing results of the cleaved PCR product (FASMAC, Big Dye terminator v3.1, 3130xl Genetic Analyzer). The primer used for sequencing had the nucleotide sequence: gagctatctgtctttaggcctggag (SEQ ID NO: 4). The PCR product after cleavage (bottom of Figure 2) is different from the PCR product before cleavage (top of Figure 2), showing that the sequence after the target site is difficult to read and indicating that cleavage occurred at the same site.

[0069] Example 2: Introduction of Tag Sequences into Target Genes Mouse hematopoietic stem cells extracted from the femurs of wild-type mice were subjected to introduction of tag sequence mutations into the target gene site using the CRISPR / Cas9 system. The target gene was IL2RG, and a mixed solution of sgRNA containing the target sequence (aggattgatgttcaggcttc; SEQ ID NO: 5), Cas9 recombinant protein, and template DNA for introducing the tag sequence (tgcatagcccttactggcctcccccatgttattctctgaagccggaagccgtgagcggctggcggctgttcaagaagattagctgaacatcaatcctttgatggaacctcaaagtcctatagtcctaagtgac; SEQ ID NO: 6) were introduced by electroporation. Genomic DNA was extracted 24 hours after the introduction of mutations (DNeasy Blood & Tissue Kit, Qiagen, 69504), a Forward primer (gtgagcggctggcggctgtt; SEQ ID NO: 7) was designed within the tag sequence, and a Reverse primer (caacctggcctacatagtgagctc; SEQ ID NO: 8) was designed approximately 300 bp downstream, and PCR amplification was performed (Quick Taq (registered trademark) HS DyeMix, Toyobo, DTM-101). The PCR conditions were 2 minutes at 94°C, followed by 30 cycles of 30 seconds at 94°C / 30 seconds at 60°C / 30 seconds at 68°C. Figure 3 shows the electrophoresis of this PCR product on a 1% agarose gel. Figure 4 shows the sequencing results of this PCR product. The primer used for sequencing had the base sequence: caacctggcctacatagtgagctc (SEQ ID NO: 9). These indicate that the tag sequence was introduced into the target site.

[0070] Example 3: ACT Genome Editing For Activation, referring to the method described in the literature (Matsubara, Y, et al. Sci Rep, 4 : 5043, 2014), the TAL effector is introduced into the mammalian expression vector (pcDNA-TAL-VP-64 (Addgene, 47107)) by the Golden Gate method with reference to TAL Effector Nucleotide Targeter 2.0 (https: / / tale-nt.cac.cornell.edu / node / add / single-tale).

[0071] For Correction and Tagging, when preparing sgRNA using Alt-RTM (IDT), mix 3.58 μl of Alt-RTM crRNA (100 μM, IDT, custom synthesized product (https: / / sg.idtdna.com / jp / site / )) containing the target sequence, 3.58 μl of tracrRNA (100 μM, IDT, 1072532), and 7.84 μl of Nuclease-free Duplex Buffer (IDT, 11-01-03-01), incubate at 95°C for 5 minutes, and then cool to room temperature.

[0072] For the preparation of sgRNA using the gRNA vector (Addgene No. 41824), introduce the target sequence by inverse PCR to prepare the gRNA template DNA. Synthesize RNA using the mMessage mMachine™ T7 UTRA Transcription Kit (Ambion, AM1345), purify it using the MEGAclear™ Transcription Clean-Up Kit (Ambion, AM1908), and adjust the concentration to 100 μM. When using nuclease as a recombinant protein, dilute SpCas9 (IDT, 1074182) and cpf1 (IDT, 1081069) to 10 mg / ml before use.

[0073] When using nuclease as RNA, referring to the methods described in the literature (Nishimasu H, et al. Science. 2018;361:1259-1262. Esvelt KM, et al. Nat Methods. 2013;10(11):1116-1121.), RNA is synthesized using mMessage mMachine™ T7 UTRA Transcription Kit (Ambion, AM1345) with SpCas9, SpCas-9-NG, dCas9, M-NMn-VP64 (Addgene #80425, #41816, #48676) on the vector as the template DNA, and purified by ethanol precipitation method.

[0074] Next, by simultaneously introducing the above tools of RNA and recombinant proteins for Activation, Correction, and Tagging into cells by electroporation, cells in which the editing and repair of the target gene have been performed are selected. That is, mutagenesis is performed using sgRNA targeting the target mutation site of the target gene as Correction, SpCas9, and template DNA. Furthermore, as Tagging, introduction of a labeling sequence is performed using sgRNA targeting the C-terminal side of the protein translation region, SpCas9 or SpCas9-NG, and template DNA. Only when these work well, cells in which the mutation site of the target gene has been accurately edited and can be produced as a protein due to the activation of the target gene induced by M-NMn-VP64 or a TAL effector by sgRNA targeting the promoter region of the target gene are selected based on the signal of the labeling sequence. As the labeling sequence, the aforementioned HiBit sequence, HA sequence, GFP sequence, or Venus sequence, etc. is used. The Venus sequence is prepared by referring to the method described in the literature (Matsubara, Y, et al. Sci Rep, 4 : 5043, 2014), introducing the homologous gene of the target gene into the pVenus vector, and used as the template DNA.

[0075] As details of the cell culture and the method of introducing RNA and proteins, add nuclease (3.69 μl at 10 mg / ml of recombinant protein or 2 μl at 250 ng / μl of RNA) to the RNA mixture (100 μM) of sgRNA and incubate at room temperature for 20 minutes. To this, mix 7.7 μl of tissue stem cells of 6 cells and template DNA (1 mg / ml), dilute with OptiMEM medium (ThermoFisher Scientific, 31985070) to make 100 μl. Perform electroporation on this cell, RNA, protein, and oligo DNA mixture using a Nepa21 pulse generator (Nepa Gene). The power transmission is carried out with two rectangular electric pulses (225 V, 2 ms width, 50 ms interval), followed by 5 pulses (30 V, 50 ms width, 50 ms interval). Add 2 ml of HBSS+ medium to the electroporated mixture and centrifuge at 800 rpm for 3 minutes for washing. Culture these cells for 24 hours using the medium used at the time of collecting tissue stem cells. Further, select cells in which gene editing has been accurately performed by detection at the single-cell level such as GFP signal. 6 Mix 7.7 μl of tissue stem cells of 6 cells and template DNA (1 mg / ml), dilute with OptiMEM medium (ThermoFisher Scientific, 31985070) to make 100 μl. Perform electroporation on this cell, RNA, protein, and oligo DNA mixture using a Nepa21 pulse generator (Nepa Gene). The power transmission is carried out with two rectangular electric pulses (225 V, 2 ms width, 50 ms interval), followed by 5 pulses (30 V, 50 ms width, 50 ms interval). Add 2 ml of HBSS+ medium to the electroporated mixture and centrifuge at 800 rpm for 3 minutes for washing. Culture these cells for 24 hours using the medium used at the time of collecting tissue stem cells. Further, select cells in which gene editing has been accurately performed by detection at the single-cell level such as GFP signal.

[0076] Example 4: Introduction of Target Gene Mutations (Correction) and Tag Label Insertion (Tagging) into Human Fetal Renal Cancer Cells For the human fetal renal carcinoma cell line (293FT cells (Thermofisher Scientific)), the CRISPR / Cas9 system was used to introduce the desired mutation into the target gene site and the HiBIT (Flag-added) sequence as a tag sequence. The target gene was RBP (TruB1), and a sgRNA mixture containing the target sequences (CTGCGCTGTCTAGAGTCCCT; SEQ ID NO: 10, CAAAAGTATGGCCGCTTCTG: SEQ ID NO: 11), Cas9 recombinant protein, and template DNA for introducing the tag sequence (GACCAAGAGGAAAAAGCAGACTTTGAAAATTGGGCATGGAGGGACTCTAGTGAGCGCAGCCCGAGGAGTTCTGGTTGTTGGAATTGGAAGCGGAACAAAAA; SEQ ID NO: 12, CAGCGTGCACCTCCACGATGAAACAGGTCTGGGCTACAAAAGTATGGCCGCTTCTGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGACTACAAAGACCATGACGGTGATTATAAAGATCATGACATCGATTACAAGGATGACGATGACAAGGCCGCTTCTGAGGCGGCGGTGGTGTCTTCGCCGTCTTTGAAAACAG; SEQ ID NO: 13) were introduced by electroporation.

[0077] After introducing the mutation, the luminescence of Nano-Glo was detected using the Nano-Glo® HiBiT Lytic Detection System (Promega N3030), and enrichment was performed by continuing the culture only for the cells that became positive. Proteins were extracted from these representative cells (RIPA buffer, Nacalaitesk 16488-34), and Western blotting was performed using a Flag antibody (MBL, FLA-1). Figure 5 shows the results of Western blotting with the Flag antibody. It shows that the tag sequence was frequently inserted in some cells and functioned as a peptide tag.

[0078] Cloning was performed using this cell, genomic DNA was extracted (DNeasy Blood & Tissue Kit, Qiagen, 69504), a Forward primer (GTTTTGAAAATGCCATCCCC; SEQ ID NO: 14) was designed approximately 200 bp upstream of the site of mutagenesis, and a Reverse primer (AGAAATAGCTACTTTTATGT; SEQ ID NO: 15) was designed approximately 300 bp downstream, followed by PCR amplification (Quick Taq® HS DyeMix, Toyobo, DTM-101). The PCR conditions were 2 minutes at 94°C, followed by 30 cycles of 30 seconds at 94°C / 30 seconds at 60°C / 30 seconds at 68°C. Figure 6 shows the sequencing results of one of the PCR products (FASMAC, Big Dye terminator v3.1, 3130xl Genetic Analyzer). The Forward primer (SEQ ID NO: 14) was used as the primer for sequencing. This indicates that the mutation has been introduced into the target sequence at the target site.

[0079] Example 5: Introduction of Target Gene Mutations (Correction) and Tag Label Insertion (Tagging) into Human Fetal Renal Cancer Cells For the human fetal renal carcinoma cell line (293FT cell (Thermofisher Scientific)), using the CRISPR / Cas9 system, the introduction of the desired mutation (removal of exon2) into the target gene site and the introduction of the HiBIT sequence as a tag sequence were performed, and immediately afterwards, the introduction of a stop codon was carried out. The target gene was RBP (TruB1), and a mixture of sgRNAs containing the complementary strands of the target sequences (TCCCCTTTTCCTCCCAAGTT; SEQ ID NO: 16, TTTCTCTCATAGAAGCTGGA: SEQ ID NO: 17), Cas9 recombinant protein, and template DNA for introducing the tag sequence (CTAGTAATGAGGTCATAGTCTCTTAACATGTAAAGTTTGTATAATACTTTGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCTTAATAGAAGCTGGAATGCCTTCTCCAGAATGGACCAAGAGGAAAAAGCAGAC; SEQ ID NO: 18) were introduced by electroporation.

[0080] After introducing the mutation, Nano-Glo luminescence detection was performed using the Nano-Glo® HiBiT Lytic Detection System (Promega N3030). Figure 7 shows the results of the Lytic Detection, indicating an increase in Luciferase activity in the knocked-in cells.

[0081] Example 6: Introduction of Target Gene Mutations (Correction), Tag Label Insertion (Tagging), and Activation into Human Fetal Renal Cancer Cells For the human fetal renal carcinoma cell line (293FT cells (Thermofisher Scientific)), the CRISPR / Cas9 system was used to introduce the target mutation into the target gene site and the HiBIT sequence as a tag sequence, followed by activation. The target gene was STAT3, and first, a cell line of mutant STAT3 was established. At this time, a mixture of sgRNAs containing the complementary strand of the target sequence (GTTGTGGTGATCTCCAACAT; SEQ ID NO: 19), Cas9 recombinant protein, and template DNA for introducing the mutation (CCCAGCTCAGTCCCCACTCCCTCCGCAGACCCACTCCTTGCCAGTTGTGTAGATCTCCAACATCTGTCAGATGCCAAATGCCTGGGCGTCCATCCTGTGGTA; SEQ ID NO: 20) were introduced by electroporation.

[0082] After introducing the mutation, the mutated cells were cloned by the limiting dilution method. Genomic DNA of the cloned mutated cells was extracted, and the introduction of the mutation was confirmed by sequencing. Figure 8 shows the sequencing results at this time. In the PCR at this time, a Forward primer (GCAGCAGGTGTGGTTTATGG; SEQ ID NO: 21) and a Reverse primer (ACCATCCCTCATCTAAACAA; SEQ ID NO: 22) were used, and the PCR conditions were 94°C for 2 minutes, followed by 30 cycles of 94°C for 30 seconds / 60°C for 30 seconds / 68°C for 30 seconds. Sequencing was performed using the Forward primer.

[0083] For the mutant cells obtained here, sgRNA for reverting the mutation (similar to SEQ ID NO: 19) and sgRNA for adding a tag sequence (with the target sequence TGCGCTACCTCCCCCATGTG; including SEQ ID NO: 23), Cas9 recombinant protein, template DNA for reverting the mutation (CCCAGCTCAGTCCCCACTCCCTCCGCAGACCCACTCCTTGCCAGTTGTGGTGATCTCCAACATCTGTCAGATGCCAAATGCCTGGGCGTCCATCCTGTGG; SEQ ID NO: 24) and template DNA for introducing the tag sequence (CCCTCACCTTTGACATGGAGTTGACCTCGGAGTGCGCTACCTCCCCCATGGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCTGAGGAGCTGAGAACGGAAGCTGCAGAAAGATACGACTGAGGCGCCTACC; SEQ ID NO: 25) were introduced by electroporation. After introducing the mutation, the STAT3 gene was activated. sgRNAs targeting the sites 1000 bp, 500 bp, and 200 bp upstream of the transcription start factor of the STAT3 gene (UUGUUGAGUAGGAGAAUCUC; SEQ ID NO: 26, UUUAAAAAAUGAGUGUGGCA; SEQ ID NO: 27, UCAAGGCCACCCUGGGCAAC; SEQ ID NO: 28 respectively) and mRNA obtained from the dCas9-VP64 vector (addgene, #47107) were introduced using the Fugene HD Transfection reagent (Promega, E2311). 24 hours later, luminescence detection of Nano-Glo was performed using the Nano-Glo® HiBiT Lytic Detection System (Promega N3030). The mRNA was synthesized using the mMESSAGE mMACHINE T7 Ultra (Invitrogen, AM1345) with the dCas9-VP64 vector as the template and purified using the MEGA Clear Transcription clean kit (Invitrogen, AM1908). Figure 9 shows the results of the Lytic Detection.Activation increased the Luciferase activity of HiBIT, indicating that mutagenesis was performed and the change was emphasized by gene activation.

[0084] Furthermore, PCR was performed on these cells using the Forward primer (GCAGCAGGTGTGGTTTATGG; SEQ ID NO: 21) and the Reverse primer (ACCATCCCTCATCTAAACAA; SEQ ID NO: 22), and the product was sequenced using the Forward primer to examine the efficiency of reverting the mutation. Figure 10 shows the sequencing results at this time. There is a waveform overlap in the target sequence, indicating that cells with the reverted mutation are included.

[0085] Example 7: Introduction of Tag Sequences for Gene Mutation Repair in Mouse Hematopoietic Stem Cells For SCID mouse hematopoietic stem cells extracted from the femurs of C.B-17 / Icr-SCID mice (CLEA Japan) with Prkdc gene mutations, the CRISPR / Cas9 system was used to repair the target gene mutation and introduce a tag sequence into the target gene. The target gene was Prkdc, and the sgRNA for repairing the mutation (including gcuuagcguauuuuauguug; SEQ ID NO: 29), the sgRNA for adding the tag sequence (including acaccacagacuuuacaucc; SEQ ID NO: 30), the Cas9 recombinant protein, the template DNA for repairing the mutation (gatcatggattcaagaaataaatgtaacggaaaagaattggtatccacaacataaaatacgctatgctaagagaaagttagcaggggccaacccagctgt; SEQ ID NO: 31), and the template DNA for introducing the tag sequence (cagaccccaatatccttggcaggacttgggaaggatgggagccctggatgCGGGACCACATGGTGCTGCACGAGTACGTGAACGCCGCCGGCATCACATAAtaaagtctgtggtgtcaccaatcataaagcattctgtctccgagaggacc; SEQ ID NO: 32) were introduced by electroporation. GFP-11 was used as the tag sequence.

[0086] Genomic DNA was extracted 48 hours after mutagenesis introduction (DNeasy Blood & Tissue Kit, Qiagen, 69504), and PCR amplification was performed using the Forward primer (CAAACTTTGAATTCACAGTCATGAGTGAC; SEQ ID NO: 33) and the Reverse primer (GAGGTCCTCTCGGAGACAGAATG; SEQ ID NO: 34) (Quick Taq (registered trademark) HS DyeMix, Toyobo, DTM-101). The PCR conditions were 2 minutes at 94°C, followed by 30 cycles of 30 seconds at 94°C / 30 seconds at 60°C / 30 seconds at 68°C. Figure 11 shows the electrophoresis of this PCR product on a 1% agarose gel. Figure 12 shows the sequencing results of this PCR product. The primer used for sequencing was the Forward primer (SEQ ID NO: 33). These show an overlap of the target tag sequence at the target site, indicating that the tag sequence was introduced with an efficiency of about 50%.

[0087] Furthermore, 48 hours after mutagenesis introduction, GFP1-10 was introduced into the cells using adeno-associated virus, and the cells were observed 24 hours later. The preparation and introduction of adeno-associated virus were carried out by introducing GFP1-10 (SEQ ID NO: 35) into pAAV-CMV (Takara, 6673) using the AAVpro (registered trademark) Helper Free System (Takara, 6673). Figure 13 shows the fluorescence of these cells. It shows that there are cells in which GFP-11 was introduced into the cells and the fluorescence of GFP was obtained by binding to AAV-derived GFP1-10.

[0088] This specification shows preferred embodiments of the present invention, but it is obvious to those skilled in the art that such embodiments are provided for illustrative purposes only, and those skilled in the art will be able to make various modifications, changes, and substitutions without departing from the present invention. It should be understood that various alternative embodiments of the invention described in this specification can be used in practicing the present invention. Also, the content described in all publications, including patents and patent application documents referred to in this specification, should be construed as being incorporated herein by reference in the same manner as the content specifically set forth herein.

Industrial Applicability

[0089] The present invention enables early radical treatment of blood and immune diseases for which there has been no sufficient treatment method until now.

Claims

1. A method for producing gene-edited tissue stem cells, comprising: a step of editing the gene sequence of a target gene in tissue stem cells isolated from a patient ex vivo; a step of adding a tag sequence to the gene sequence of the target gene; a step of activating the gene expression of the target gene in the tissue stem cells; and a step of selecting the tissue stem cells with the edited gene sequence wherein the step of editing the gene sequence of the target gene in the tissue stem cells and the step of adding the tag sequence to the gene sequence of the target gene are simultaneously performed using the CRISPR / Cas system or the TALEN system; the target gene is a gene that is not expressed in the tissue stem cells; only in the cells in which the gene sequence of the target gene has been edited and the tag sequence has been added, a target protein labeled with the tag at the N-terminus or C-terminus is produced by the step of activating the gene expression of the target gene, and the tissue stem cells with the edited gene sequence are selected using the tag; the tissue stem cells with the edited gene sequence are selected within 48 hours after isolating the tissue stem cells from the patient Method.

2. The method according to claim 1, wherein the target gene is selected from the group consisting of ADA, IL2RG, WAS, CYBB, INTGB2, UNC13D, CD40L, SAP / SH2D1A, BTK, STAT3, and hemoglobin.

Images