Proliferation activation of pancreatic islet cells by activation of glucose pathway

Abstract

To provide a method that sustains proliferation of mature pancreatic islet cells by a Mycl gene and enables continuous induction of proliferation.SOLUTION: The present invention provides a method for promoting and / or sustaining, by a glucose stimulation activation pathway activator, the proliferation activity of pancreatic islet cells in which a Mycl gene or a gene product thereof has been introduced or expressed through induction. More specifically, in the method, the Mycl gene includes (1) a nucleic acid having a base sequence represented by SEQ ID NO: 1 or 3, or (2) a nucleic acid that hybridizes with, under stringent conditions, a nucleic acid having a base sequence represented by SEQ ID NO: 1 or 3, and that encodes a polypeptide having a function of sustaining and / or promoting the proliferation activity of pancreatic islet-like cells when expression of the Mycl gene is induced.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for sustaining and promoting the proliferation of pancreatic islet cells into which the Mycl gene or its gene product has been introduced or into which expression has been induced, by regulating a glucose-activated pathway. [Background technology]

[0002] The medical expenses for diabetes and its complications in the world are estimated at 90 trillion yen per year, and the pressure on medical expenses has become a major social issue. Diabetes can cause complications such as retinopathy, neuropathy, and kidney damage, and in the final stages, artificial dialysis may be required. In severe cases of end-stage diabetes where insulin secretion is almost no longer observed, blood sugar control is particularly poor, causing repeated episodes of severe hypoglycemia that may lead to coma or death. The onset of severe hypoglycemia can lead to aftereffects of brain damage, including dementia, and a worsening prognosis, so avoiding the onset of severe hypoglycemia is an important clinical issue. The mainstream treatment for diabetes is symptomatic treatment, such as administration of drugs that suppress sugar absorption and insulin preparations, and most of these do not treat the diabetes itself.

[0003] Pancreas and islet transplants are performed for patients with poor prognosis, who suffer from repeated episodes of severe hypoglycemia due to insulin secretion depletion and particularly poor glycemic control. These procedures have been shown to be effective not only in preventing the onset of severe hypoglycemia, but also in achieving insulin withdrawal and good glycemic control. However, due to a shortage of donors and a small number of facilities for isolating islets for transplantation, the number of eligible patients is extremely limited, and pancreas and islet transplants are not offered as a treatment option for early intervention for patients who simply have a history of severe hypoglycemia, high-risk groups with depleted insulin secretion, or even ordinary type I diabetes patients. In 2020, donor islet transplants were covered by insurance as "allogeneic cadaveric islet transplantation." Although treatment outcomes have improved compared to the past, there are challenges in ensuring the quality and yield of islets due to donor shortages, individual differences in donor islets, and variations in the time and method for islet isolation. These issues have led to a variety of problems, including a limited number of patients who can receive transplants, the cancellation of planned islet transplants, and the need for repeated transplants to achieve sufficient therapeutic effects such as insulin withdrawal. Although the development of cell transplantation medicine using insulin-producing cells derived from pluripotent stem cells is progressing, a major problem is that insulin-producing cells derived from pluripotent stem cells are functionally immature. In addition, due to the complexity of the differentiation induction process, it is currently difficult to induce differentiation of pluripotent stem cells into endoderm, pancreas, and pancreatic islets at low cost.

[0004] On the other hand, if a sufficient amount of donor islet cells can be proliferated, it is expected that a safe and high-quality product can be stably supplied at a relatively low cost through a relatively simple manufacturing process and quality control. In addition, since a necessary and sufficient amount of high-quality islet cells can be transplanted, there is a possibility that a sufficient therapeutic effect can be achieved by one transplant. Furthermore, by cryopreserving intermediate products and final products, planned transplant treatment is possible, and the risk of transplant discontinuation due to instability in the supply of islets can be minimized. In addition, if a sufficient amount of transplant islets can be stably supplied, it is expected that the number of patients who can be treated will increase. In addition, if it is possible to proliferate islet cells derived from pluripotent stem cells, it is expected that the cost of producing islet cells derived from pluripotent stem cells for transplantation can be reduced. From this perspective, it is expected to have a great impact on diabetes treatment strategies compared to existing treatment methods. In recent years, it has become possible to induce the proliferation of mature islet cells by introducing Mycl genes. It is expected that transplantation medicine will be realized by inducing the proliferation of islet cells by Mycl (Patent Document 1). There was a problem that the proliferation induction of mature islet cells by introducing Mycl genes is limited, and the amplification efficiency is limited. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2021 / 117840 Summary of the Invention [Problem to be solved by the invention]

[0006] Conventionally, it has been possible to impart proliferation activity to mature islet cells by expressing the Mycl gene, but the proliferation of islets is temporary, and the proliferation activity decreases after several passages, making it impossible to achieve sustained expansion. Therefore, the present invention aims to provide a method for sustaining the proliferation of mature islet cells by the Mycl gene, thereby enabling continuous induction of proliferation. [Means for solving the problem]

[0007] The present inventors have discovered that it is possible to proliferate islet cells and expand mature islet cells over the long term by regulating pathways activated by glucose, such as by activating the proliferation of mature islet cells by the Mycl gene in a glucose concentration-dependent manner, by using a glucokinase activator to activate the proliferation of mature islet cells by the Mycl gene, and by repeating short-term stimulation with high concentrations of glucose, and have thus completed the present invention.

[0008] That is, the present invention has the following aspects. [1] A method for promoting and / or sustaining the proliferation activity of pancreatic islet cells into which a Mycl gene or its gene product has been introduced or whose expression has been induced, by using an activator of a glucose-stimulated activation pathway. [2] The method according to [1], wherein the glucose-stimulated activation pathway activator is at least one selected from the group consisting of glucose, sucrose, and glucokinase activators. [3] The Mycl gene (1) a nucleic acid containing a base sequence represented by SEQ ID NO: 1 or 3; or (2) A nucleic acid that hybridizes under stringent conditions with a nucleic acid containing a base sequence represented by SEQ ID NO: 1 or 3, and encodes a polypeptide that has the function of sustaining and / or promoting the proliferation activity of pancreatic islet-like cells in a glucose concentration-dependent manner when the expression of the Mycl gene is induced. The method according to [1] or [2], comprising: [4] The Mycl gene product is (1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or 4; or (2) A polypeptide having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO: 2 or 4, and having the function of sustaining and / or promoting the proliferation activity of pancreatic islet-like cells in a glucose concentration-dependent manner. The method according to [1] or [2], comprising: [5] The method according to any one of [1] to [4], wherein the Mycl gene is transiently expressed. [6] The method according to any of [1] to [5], in which high and low concentrations of glucose are applied alternately. [7] The method according to any one of [1] to [6], wherein the islet cells are primary islet cells isolated from the pancreas, cultured islet cells, or stem cell-derived islet cells. [8] The method according to [7], wherein the stem cells are selected from the group consisting of iPS cells, ES cells, and somatic stem cells. Effect of the Invention

[0009] According to the present invention, by regulating the pathway activated by glucose, pancreatic islet cells can be proliferated and the proliferation activity of pancreatic islet-like cells can be maintained for a long period of time, thereby making it possible to treat diabetes or related diseases in which insulin production is desired. [Brief description of the drawings]

[0010] [Figure 1] 1 shows the activation of proliferation of Mycl-induced islet cells in a test by glucose. [Diagram 2] 1 shows glucose concentration-dependent activation of Mycl-induced pancreatic islet cell proliferation. [Diagram 3] FIG. 1 shows changes in the proliferation efficiency of Mycl-induced pancreatic islet cells by glucokinase activator (GKA). [Figure 4] FIG. 1 shows changes in the proliferation efficiency of Mycl-induced pancreatic islet cells by glucokinase inhibitors (GKIs). [Diagram 5] 1 shows the activation of proliferation of Mycl-induced pancreatic islet cells in vivo by glucose. [Figure 6] 1 shows a decrease in the proliferation activity of Mycl-induced pancreatic islet cells under culture conditions with a high glucose concentration. [Figure 7] 1 shows sustained proliferation activity of Mycl-induced pancreatic islet cells by high-low glucose. [Figure 8] 1 shows sustained proliferation activity of Mycl-induced pancreatic islet cells by high-low glucose. [Figure 9] 1 shows the promotion of proliferation of mature hormone-producing cells by Mycl gene expression. [Figure 10] 1 shows that Mycl gene expression and glucose cooperate to efficiently induce β-cell proliferation. [Figure 11] 1 shows recovery of diabetic mice by long-term culture of pancreatic islet cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention relates to a method for sustaining and / or promoting the proliferation activity of pancreatic islet cells (hereinafter, sometimes referred to as "pancreatic islet-like cells") into which a Mycl gene or a gene product thereof has been introduced or whose expression has been induced, in a glucose concentration-dependent manner. The present invention is described in detail below.

[0012] 1. Pancreatic islet-like cells and their preparation method (1) Pancreatic islet-like cells As used herein, the term "islet-like cells" refers to mature islet cells into which the Mycl gene or its gene product has been introduced. In this specification, islet-like cells that have been forced to express the Mycl gene and have entered the proliferation phase may be referred to as "islet precursor-like cells". Furthermore, by stopping the expression of the gene, the cells can be differentiated into islet cells including cells capable of producing insulin (hereinafter also referred to as "insulin-producing cells"). That is, according to the present invention, the number of islet-like cells can be increased by transient expression of the Mycl gene, and the cells can be differentiated into islet cells including insulin-producing cells. Here, when focusing on the markers expressed in each cell, the "islet precursor-like cells" are positive for at least one, preferably two, more preferably three or more of the genes selected from, for example, Fev, Pax4, Cck, CDK4, and Ki67. When focusing on the expressed proteins, the cells are also characterized in that the production of insulin and glucagon is particularly reduced compared to that observed in normal islets, or the production of somatostatin is prominent.

[0013] (2) Pancreatic islet cells "Pancreatic islet cells" generally refer to endocrine cells that make up about 1-2% of the total cells in the pancreas, which are a cell aggregate called a pancreatic islet, also known as an islet of Langerhans, and which control the endocrine function of the pancreas. In this specification, the term "pancreatic islet cells" is used interchangeably with "mature pancreatic islet cells". In addition, pancreatic islet cells are mainly composed of five types of cells, namely, α cells, β cells, δ cells, ε cells, and PP cells. The main cells that make up the cell aggregate called a pancreatic islet are β cells. β cells make up about 60-80% of pancreatic islet cells and secrete insulin, which allows glucose to be transferred to most cells in the body. On the other hand, α cells make up about 10-30% of pancreatic islets and secrete glucagon, which is released during starvation, which breaks down glycogen stored in the liver to maintain normal blood sugar and allows glucose to be released into the blood. δ cells make up about 5-10% of pancreatic islet cells and secrete somatostatin, which further regulates glucose concentration. Additionally, ε and PP cells secrete ghrelin and pancreatic polypeptide, respectively. Pancreatic polypeptide-producing cells (approximately 5-10% of the islet cells) release hormones that alter exocrine and gastrointestinal function. There are other islet cell types, including endothelial cells, neural cells, and progenitor cells.

[0014] As used herein, the term "islet cell" includes the above-mentioned islet cell and islet precursor cell, which is a precursor cell of the islet cell, and may be an intermediate cell that is generated before reaching the islet cell or islet precursor cell generated during the process of development into the islet cell or during the process of differentiation induction from the somatic stem cell / pluripotent stem cell. Here, the "intermediate cell" is preferably a cell that is destined to differentiate into an islet cell. Furthermore, in the present invention, the islet cell may be one prepared from an islet-like cell or islet precursor cell-like cell into which the Mycl gene or its gene product has been introduced.

[0015] In type I diabetes, cellular infiltration of the pancreatic islets, mainly composed of lymphocytes, is observed shortly after the onset of the disease. Eventually, as beta cells are selectively lost, the volume of the islets decreases and alpha cells become predominant. Although the islets have a reserve capacity for insulin secretion, type I diabetes develops when the beta cells are lost and the necessary amount of insulin can no longer be secreted. On the other hand, in type II diabetes, morphological changes in the islets, such as a significant decrease in the number of beta cells, are generally not observed. In type II diabetes, the main cause of the disease is a decrease in the blood glucose regulation effect of insulin due to insulin resistance in peripheral tissues.

[0016] In the present invention, the origin or source of the islet cells into which the Mycl gene or its gene product is introduced is not limited, but may be primary islet cells isolated from an individual's pancreas, or islet cells cultured by a known culture method. The cultured islet cells may include, but are not limited to, established islet cells, and islet cells derived from stem cells (e.g., iPS cells, ES cells, somatic stem cells) (see, for example, Kimura, A., et al., Cell Chemical Biology, 2020, doi.org / 10.1016 / j.chembiol.2020.08.018). Furthermore, in the present invention, the islet cells into which the Mycl gene or its gene product is introduced may be islet cells obtained from islet-like cells and / or islet progenitor-like cells, or the Mycl gene or its gene product may be repeatedly introduced into islet-like cells and / or islet progenitor-like cells into which the Mycl gene has already been introduced. In particular, for the purpose of treating diabetes, either islet cells collected from a donor or islet cells derived from stem cells can be preferably used. The islet cells from the donor used may be either autologous or allologous to the recipient. According to the present invention, it is possible to treat diabetes by introducing the Mycl gene into such islet cells, proliferating islet precursor cell-like cells in vitro, and returning (transplanting) them to the patient. In this case, in cases where the islet cells are allologous to the patient, they can be used in combination with known methods such as administering an immunosuppressant to the patient as appropriate, matching the HLA type of the transplanted donor tissue with that of the recipient, and embedding the donor tissue in alginic acid, alginic acid derivatives, semipermeable membranes, etc. In addition, for transplantation into a patient, the above-mentioned islet-like cells, islet precursor cell-like cells, or cells having insulin production ability, or any combination thereof, can be used.

[0017] In relation to the above, it has been reported that, at least in mice, when β cells are lost, α cells proliferate, and then some of the α cells transdifferentiate into β cells. Therefore, the pancreatic islets as a source of islet cells before the introduction of the Mycl gene may be islets containing a large number of normal β cells, or may be islets from which most of the β cells have been lost. From this perspective, the present invention can also be used in gene therapy for type I diabetes patients who have lost β cells, by using islet-like cells or the like introduced with the Mycl gene.

[0018] (3) Pluripotent stem cells As used herein, the term "pluripotent stem cell" refers to a cell that has self-renewal and multi-differentiation capabilities, and has the ability to form all the cells that constitute a living body. "Self-renewal capability" refers to the ability to create undifferentiated cells identical to itself from one cell. "Differentiation capability" refers to the ability of a cell to differentiate. Examples of pluripotent stem cells include, but are not limited to, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), Muse cells (Multi-lineage differentiating Stress Enduring cells), spermatogonial stem cells (GS cells), and embryonic germ cells (EG cells). The pluripotent stem cells used in the present invention are preferably ES cells. The origin of the pluripotent stem cells may be any of mammals, birds, fish, reptiles, and amphibians, and is not particularly limited. Mammals include primates (humans, monkeys, etc.), rodents (mice, rats, guinea pigs, etc.), cats, dogs, rabbits, sheep, pigs, cows, horses, donkeys, goats, ferrets, and the like.

[0019] As used herein, "ES cells" refers to pluripotent stem cells that have the ability to differentiate into all tissue cells that constitute an individual present at an early stage of development, which have been extracted and established to be cultured in vitro. ES cells can be multiplied virtually indefinitely while retaining the ability to differentiate into all cells that constitute an individual, similar to pluripotent stem cells in early embryos. Specifically, ES cells were first described in mice in 1981 (Proc. Natl. Acad. Sci. USA 78, 7634-7638, 1981; Nature 292, 154-156, 1981). ES cells have pluripotency and can generate all tissues and cell types that constitute an individual. Pluripotent embryonic stem cells have been isolated from a wide variety of species, including rats (Iannaconns et al., Dev. Biol. 163, 288-292, 1994), hamsters (Dev. Biol. 127, 224-227, 1988), rabbits (Mol. Reprod. Dev. 36, 424-433, 1993), birds, fish, pigs (Reprod. Fertil. Dev. 6, 563-568, 1994), cows (Reprod. Fertil. Dev. 6, 553-562, 1994), and primates (Proc. Natl. Acad. Sci. USA 92, 7844-7848, 1995). Incidentally, ES cells that can be used in the present invention include, but are not limited to, KH2 cells, RF8 cells, JI cells, CGR8 cells, MG1.19 cells, 129SV cells, C57 / BL6 cells, DBA-1 cells, and the like.

[0020] Several research groups have also been successful in isolating ES cells and ES cell-like stem cells from embryonic human tissues. Early success stories include (Science 282, 1145-1147, 1998; Proc. Natl. Acad. Sci. USA 95, 13726-13731, 1998; Nature Biotech., 18, 399-404, 2000). These ES cell lines were established by culturing ICMs isolated from blastocysts on feeder cells. Other recent studies have shown that it is possible to obtain embryos and embryonic cells by transferring nuclei from embryos and mature mammalian cells into enucleated oocytes.

[0021] In the present invention, any established ES cell line can be used. Alternatively, in order to prevent immune rejection when ES cells produced by the method of the present invention are applied to an individual, it is effective to create a cloned embryo using somatic cells of the individual and establish an ES cell line from the cloned embryo. Using this method, it is possible to establish ES cells that have the same genetic elements as the individual.

[0022] Alternatively, it is believed that in the creation of somatic cell clones, a phenomenon called "initialization" occurs, in which the nucleus of the somatic cell introduced into the egg changes to a state similar to that of the nucleus of a fertilized egg. It has been reported that ES cells also have activity similar to that of eggs (Curr.Biol.,11,1553-1558,2001). In other words, it is expected that by fusing an individual's somatic cells with ES cells, it will be possible to convert somatic cells into cells similar to ES cells. Since ES cells can be genetically manipulated in vitro, it is expected that by performing this with ES cells in which factors involved in immune rejection, such as MHC genes, have been previously manipulated, it will be possible to avoid rejection reactions without using techniques such as the creation of somatic cell clone embryos.

[0023] As used herein, "induced pluripotent stem (iPS) cells" refers to cells with pluripotency similar to that of ES cells, obtained by introducing genes of transcription factors such as Oct3 / 4, Sox2, Klf4, and c-Myc into somatic cells. Like ES cells, iPS cells can also be multiplied indefinitely while retaining their pluripotency.

[0024] The basic method for producing iPS cells is to introduce four transcription factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into cells using a virus (Takahashi K, Yamanaka S: Cell 126(4), 663-676, 2006; Takahashi, K, et al: Cell 131(5), 861-72, 2007). Examples of cells that can be used to produce iPS cells, i.e., cells from which iPS cells are derived, include lymphocytes (T cells, B cells), fibroblasts, epithelial cells, endothelial cells, mucosal epithelial cells, mesenchymal stem cells, hematopoietic stem cells, adipose stem cells, dental pulp stem cells, and neural stem cells.

[0025] Reprogramming of iPS cells can be performed by methods well known to those skilled in the art, and is outlined, for example, in Addgene's Blog / Post, "Delivery Methods for Generating iPSCs" (https: / / blog.addgene.org / delivery-methods-for-generating-ipscs). Methods for introducing the Mycl gene into iPS cells include, but are not limited to, introduction methods using recombinant viruses (e.g., retroviruses, lentiviruses, adenoviruses, Sendai viruses, etc.), recombinant plasmids, minicircles, or episomes (e.g., oriP / Epstein-Barr nuclear antigen-1 (EBNA1)-based episomal vectors), or methods for directly introducing RNA (including mRNA) encoding the Mycl gene or the Mycl protein itself into cells.

[0026] As used herein, "EG cells" refer to any embryonic germline stem cell produced from a primordial germ cell, and the origin, etc., of the cell are not particularly limited. As used herein, "GS cells" refer to germline stem cells produced from testicular germ cells, and are a cell line that allows spermatogonial stem cells (spermatogonial stem cells) to be cultured outside the body (Cell. 119, 1001-1012, 2004). Of the GS cells, mGS cells (multipotent germline stem cells), which have properties similar to those of ES cells and also have pluripotency, are particularly preferred.

[0027] (4) MycI gene and its gene product The Mycl (also called "L-Myc") gene is one of the members of the Myc gene family, which includes the c-Myc gene and Mycn (the "N-Myc" gene). The Mycl gene, like the c-Myc gene, is an oncogene and is also known as a reprogramming gene. Unlike the c-Myc gene, the Mycl gene is known to have almost no transforming ability (Nakagawa, M., et al., Proc. Natl. Acad. Sci. USA, vol. 107, p.14152-14157, 2010). The mouse and human cDNA sequence information for Mycl are available under the NCBI accession numbers NM 008506 and NM The cDNA can be obtained by reference to US Pat. No. 6,233,081, and a person skilled in the art can easily isolate the cDNA.

[0028] According to the present invention, it is preferable to use an isolated Mycl gene and its gene product. As described above, the base sequence of the Mycl gene can be specified by the NCBI accession number, but the usable Mycl gene also includes single-stranded or double-stranded DNA and its RNA complement. DNA includes, for example, naturally occurring DNA, recombinant DNA, chemically synthesized DNA, DNA amplified by PCR, chemically modified DNA, and combinations thereof. The nucleic acid used in the present invention is preferably DNA. As is well known, there is degeneracy in codons, and there are amino acids for which multiple base sequences exist that code for one amino acid. However, there is no particular limitation as long as the pancreatic islet cells (i.e., pancreatic islet-like cells) into which the Mycl gene has been introduced undergo cell proliferation due to the expression of the Mycl gene and have the effect of promoting insulin production due to the cessation of the expression.

[0029] In one embodiment, the Mycl gene is (1) a nucleic acid comprising or consisting of a base sequence represented by SEQ ID NO: 1 or 3; or (2) A nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising or consisting of a base sequence represented by SEQ ID NO: 1 or 3, and encodes a polypeptide that has the function of sustaining and / or promoting the proliferation activity of pancreatic islet-like cells when the expression of the Mycl gene is induced; or (3) It may contain / consist of a nucleic acid that hybridizes under stringent conditions with a nucleic acid containing or consisting of the base sequence represented by SEQ ID NO: 1 or 3, and that encodes a polypeptide that has the effect of sustainably proliferating insulin-producing cells and, in turn, the effect of sustainably promoting insulin production as a result of proliferation of pancreatic islet-like cells into which a Mycl gene has been introduced.

[0030] As used herein, "under stringent conditions" means hybridization under moderately or highly stringent conditions. Specifically, moderately stringent conditions can be easily determined by a person skilled in the art based on, for example, the length of DNA. Basic conditions are given in Sambrook, J. et al., Molecular Cloning, A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, 7.42-7.45 (2001), but include the use of a pre-wash solution of 5xSSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) for nitrocellulose filters, hybridization conditions of about 50% formamide, 2xSSC to 6xSSC (or other similar hybridization solutions such as Stark's solution in about 50% formamide at about 42°C) at about 40-50°C, and washing conditions of 0.5xSSC, 0.1% SDS at about 60°C. Highly stringent conditions can also be readily determined by those of skill in the art, for example based on the length of the DNA. Generally, such conditions include hybridization and / or washing at higher temperatures and / or lower salt concentrations than moderately stringent conditions, defined as, for example, hybridization conditions as described above, with a wash at about 68° C., 0.2×SSC, 0.1% SDS. One of skill in the art will recognize that temperature and wash solution salt concentration can be adjusted as necessary depending on factors such as the length of the probe.

[0031] The homologous nucleic acid cloned using the above-mentioned nucleic acid amplification reaction or hybridization has at least 30% or more, preferably 50% or more, more preferably 70% or more, even more preferably 90% or more, even more preferably 95% or more, and most preferably 98% or more identity to the nucleotide sequence set forth in SEQ ID NO: 1 or 3, respectively. The percent identity can be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two nucleic acid sequences can be determined by comparing sequence information using the GAP computer program (GCG Wisconsin Package, version 10.3) described in Devereux et al., Nucl. Acids Res., 12, 387 (1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG).

[0032] In one embodiment, the gene product of the Mycl gene is a polypeptide expressed from the Mycl gene described above. Typically, the polypeptide is (1) a polypeptide comprising or consisting of the amino acid sequence represented by SEQ ID NO: 2 or 4; or (2) It may be a polypeptide having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to the amino acid sequence represented by SEQ ID NO: 2 or 4, and having the function of sustaining and / or promoting the proliferation activity of pancreatic islet-like cells in a glucose concentration-dependent manner.

[0033] In one embodiment, the gene product of the Mycl gene may be a variant of the polypeptide defined above, and may be an amino acid sequence containing one or more amino acid deletions, substitutions, insertions and / or additions in the amino acid sequence of SEQ ID NO: 2 or 4. The substitution may be a conservative substitution, which is a replacement of a specific amino acid residue with a residue having similar physicochemical characteristics. Non-limiting examples of conservative substitutions include substitutions between aliphatic group-containing amino acid residues, such as substitutions between Ile, Val, Leu or Ala, substitutions between polar residues, such as substitutions between Lys and Arg, Glu and Asp, Gln and Asn, etc.

[0034] Mutants resulting from deletion, substitution, insertion and / or addition of amino acids can be prepared by subjecting the Mycl gene to, for example, the well-known technique of site-directed mutagenesis (e.g., Nucleic Acid Research, Vol. 10, No. 20, p. 6487-6500, 1982). As used herein, "one or more amino acids" refers to a number of amino acids that can be deleted, substituted, inserted and / or added by site-directed mutagenesis. In addition, as used herein, "one or more amino acids" may refer to one or several amino acids, depending on the circumstances.

[0035] In addition to the above-mentioned site-specific mutagenesis, methods for deleting, substituting, inserting, and / or adding one or more amino acids in the amino acid sequence of a polypeptide while retaining its activity include a method of treating the gene with a mutagen and a method of selectively cleaving the gene, then removing, substituting, inserting or adding selected nucleotides, and then ligating. Although not limited thereto, the gene product of the Mycl gene in the present invention may be a polypeptide having an activity of promoting insulin production, which consists of an amino acid sequence in which 1 to 10, preferably 9 or less, 7 or less, 5 or less, 3 or less, 2 or less, and more preferably 1 or less amino acids are deleted, substituted or added in SEQ ID NO: 2 or 4.

[0036] The variant is further a protein comprising an amino acid sequence having at least 80% or more, preferably 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid identity to the amino acid sequence of SEQ ID NO:2, and is a polypeptide having the effect of proliferating pancreatic islet-like cells and / or the effect of promoting insulin production.

[0037] The percent identity of two amino acid sequences may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two protein sequences may be determined by comparing sequence information using the GAP computer program based on the algorithm of Needleman, SB and Wunsch, CD (J. Mol. Biol., 48:443-453, 1970) and available from the University of Wisconsin Genetics Computer Group (UWGCG). Preferred default parameters of the GAP program include: (1) a scoring matrix, blosum62, as described in Henikoff, S. and Henikoff, JG (Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992); (2) a gap weight of 12; (3) a gap length weight of 4; and (4) no penalty for end gaps.

[0038] (5) Introduction of Mycl gene According to the present invention, the method of introducing the Mycl gene into primary pancreatic islet cells, cultured pancreatic islet cells, or stem cells (e.g., iPS cells, ES cells, somatic stem cells) is not particularly limited, and any method known to those skilled in the art can be used. Gene introduction means generally include "transformation" or "transfection", which means a transient or stable genetic change induced in a cell after incorporation of an exogenous nucleic acid (e.g., DNA or RNA foreign to a host cell). Usually, the genetic change can be achieved by incorporating an exogenous nucleic acid into the genome of the host cell, or by maintaining the exogenous nucleic acid transiently or stably as an episomal component or independently. According to the present invention, the introduced Mycl gene may be in a state of being integrated into the genome of the host cell, or may be present as an episomal component, or may be present in the cytoplasm as a plasmid or vector containing the gene, as long as the expression of the gene can be controlled on / off.

[0039] Usually, a "vector" is used to introduce an exogenous nucleic acid (preferably DNA) into a host cell. In general, the vector includes a virus, particularly an attenuated virus and / or a replication-incompetent virus. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated virus vectors, and Sendai virus vectors. In addition, the vector may include a control sequence such as a promoter, an enhancer, a ribosome binding sequence, a terminator, and a polyadenylation site so as to enable expression of the exogenous nucleic acid. Furthermore, if necessary, the vector may include a selection marker sequence such as a drug resistance gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene, etc.), a thymidine kinase gene, a diphtheria toxin gene, a reporter gene sequence such as a fluorescent protein, β-glucuronidase (GUS), or a FLAG. Here, the "promoter" is also intended to include a promoter component sufficient for promoter-dependent gene expression that is cell type-specifically controllable, tissue-specifically controllable, or inducible by an external signal or drug. Such elements may be located in the 5' or 3' regions of the native gene, and "operably linked" means that the DNA sequence is linked to a control sequence in such a way that expression is possible when appropriate molecules (e.g., transcriptional activator proteins) are linked to the control sequence.

[0040] Furthermore, the introduction of a vector into a host cell is carried out by, but not limited to, electroporation (Meiner, V. et al., Proc. Natl. Acad. Sci. USA, 93:14041-14046 (1996) etc.), calcium phosphate method, DEAE-dextran method, or a method using lipids for gene introduction (lipofectamine, lipofectin etc.). Then, the cells into which the vector has been introduced can be selected based on the characteristics of a marker gene (e.g., drug resistance gene). Correct homologous recombination in the selected cells can be confirmed by Southern blotting or the like using a part of the target exogenous nucleic acid as a probe. In this way, a cell heterozygously containing a gene in which a marker gene has been knocked into the target gene, specifically, the Mycl gene, can be produced.

[0041] As the ES cell, the KH2 strain, which has an Frt sequence downstream of the Cola1 locus and expresses the reverse tetracycline-regulated transactivator M2-rtTA under the control of the endogenous Rosa26 promoter, can be used (Beard C, et al., Genesis, vol. 44, p. 23-28 (2006)). The Mycl gene can be introduced into the ES cell using a method well known to those skilled in the art. For example, the Mycl gene is inserted into the entry vector pCR8-GW-TOPO vector (manufactured by Invitrogen Life Technologies) by TA cloning, and then the LR reaction is performed between the vector and, for example, the Colla1-TetOP-AttR1-ccdB-AttR2-ires-mCherry vector, and the TetOP-Mycl-ires-mCherry vector can be used as a gene introduction vector. Here, the "TetOP (operon)" sequence in the vector is a sequence (tetracycline response element: TRE) to which a reverse tetracycline-controlled transactivator binds, and the reverse tetracycline-controlled transactivator expressed from the host cell in response to a reverse tetracycline such as doxycycline (Dox) added to the cell binds to induce the expression of a gene linked downstream. In addition, "ires" (internal ribosome entry site) is an internal ribosome recognition sequence, and "mCherry" is a gene (reporter gene) that codes for a red fluorescent protein. By introducing the vector into KH2-ES cells together with a nucleic acid that codes for flipase, the Mycl gene can be integrated into the chromosome of the ES cells. In addition to vectors containing "ires-mCherry" as described above, vectors such as pBSSK(-)-IRES-βgeo containing an "ires-βgeo (a fusion gene of β-galactosidase and neomycin resistance gene) cassette" (Mountford P. et al., Proc. Natl. Sci. USA, 91:4303-4307 (1994)) containing a resistance gene, and a similar vector containing an IRES-Hygro (hygromycin resistance gene) cassette may also be used.

[0042] The present invention is capable of controlling the transient expression of the Mycl gene, thereby allowing the proliferation of islet-like cells and their differentiation into insulin-producing cells. Thus, the present invention is characterized by the transient expression of the Mycl gene. When the Mycl gene is transiently expressed, the number of cells into which the Mycl gene has been introduced relatively decreases with cell division of the islet-like cells, and the expression level of the Mycl gene can be naturally decreased or stopped over time. That is, in one embodiment, the object of the present invention is achieved by controlling the expression of the Mycl gene "on." In addition, in another embodiment, it is also possible to forcibly turn off the expression of the Mycl gene (hereinafter referred to as "on / off" control).

[0043] As described above, by using reverse tetracycline, the expression of the Mycl gene introduced into the host cell can be controlled to be on / off. That is, in the presence of reverse tetracycline, the Mycl gene continues to be expressed in the cell, and the islet-like cells can be proliferated, while by removing reverse tetracycline, the proliferation of the islet-like cells can be stopped and differentiation into insulin-producing cells can be induced. In the present invention, the transient expression (on state) of the Mycl gene introduced into the islet-like cells is preferably for a period of at least 2 days to a maximum of 100 days, for example, 90 days, 80 days, 70 days, or 60 days, from the start of cell culture. On the other hand, it is preferable that the islet-like cells into which the Mycl gene has been introduced proliferate for the above-mentioned period.

[0044] As an alternative means of controlling the expression of the Mycl gene, we can use a "photoregulated viral vector" (Tahara, M., et al., PNAS, vol. 116, 11587-11589, 2019), which can precisely switch on / off the gene expression and cell proliferation of the viral vector by light irradiation. This is a viral vector in which a gene encoding a photoswitch protein called magnet has been introduced, and the expression of the target gene incorporated in the vector can be controlled using blue light.

[0045] Other examples of alternative methods for controlling the expression of the Mycl gene on / off include episomal vectors (Okita K., et al., Nat Methods 2011 May 8(5):409-412), Sendai virus, and RNA vectors (Warren L., et al., Cell Stem Cell 2010 Nov 7(5):618-630). Without being limited thereto, the above-mentioned method used for reprogramming (establishment) of iPS cells (i.e., a method for transiently inducing a gene) can be used.

[0046] According to the present invention, the object of the present invention can be achieved by forcibly expressing an endogenous Mycl gene in a cell, in addition to introducing an isolated exogenous Mycl gene. The means for expressing an endogenous Mycl gene is not limited, but may be a method of forcibly expressing the gene by replacing a wild-type promoter or enhancer with a strong promoter or enhancer for operably inducing expression of the Mycl gene. Examples of promoters used for replacement include the cytomegalovirus (CMV) promoter, which is a strong promoter, and an inducible promoter that functions in the presence of an inducer. On the other hand, examples of enhancers used for replacement include the SV40 enhancer, herpes B virus enhancer, cytomegalovirus enhancer, and α-fetoprotein enhancer. In another embodiment, in addition to CRISPR Type II (including CRISPR-dCas9), a molecule in which a demethylase, a histone modification enzyme, a transcription activator, specifically VP64, p65, Rta, etc., capable of activating a promoter and / or enhancer is added to the genome recognition sequence of CRISPR-type I, TALEN, or ZFN can be used. In this specification, the above-mentioned promoters, enhancers, enzymes and factors that activate them, or nucleic acid protein complexes or low molecular weight compounds may be referred to as "activators" for activating the Mycl gene.

[0047] (6) Introduction of the gene product of the Mycl gene The Mycl gene product can be introduced into cells by a general method for introducing foreign genes or proteins into cells, including, but not limited to, a method using a transfection reagent, a method using a virus, electroporation, a particle gun method, sonoporation, a liposome fusion method, and an introduction method in which pores are formed in the cell membrane by a micromanipulator or laser light irradiation.

[0048] (7) Creation of chimeric mammals A method for introducing ES cells into a mammal to produce a chimeric mammal can be carried out using a method well known to those skilled in the art. First, any medium known to those skilled in the art can be used for culturing the ES cells into which the Mycl gene has been introduced. For example, when the ES cells are cultured on feeder cells, feeder cells (e.g., MEF (mouse embryonic fibroblasts)) can be used, and the ES cells on the feeder cells can be cultured in a medium for ES cells (e.g., a medium obtained by adding 2-mercaptoethanol (2ME, GIBCO) and LIF (SIGMA) to knockout DMEM (GIBCO) containing 15% FBS, 50 U / mL penicillin / streptomycin, L-glutamine, and non-essential amino acids).

[0049] Next, the ES cells are introduced into a mammal to produce a knockout animal (Mycl gene knock-in animal). Here, a mouse is used as an example of a mammal, but the method of producing a knock-in mouse is well known to those skilled in the art. Specifically, the ES cells are injected into a blastocyst of a mouse (e.g., C57BL / 6, etc.) and transplanted into the uterus of a pseudopregnant female mouse (ICR, etc.), to produce a chimeric mouse. Thereafter, the chimeric mouse is crossed with a normal mouse (C57BL / 6, etc.) to produce a heterozygous mutant mouse in which the Mycl gene is knocked in heterozygously. By crossing heterozygous mutant mice with each other, a homozygous mutant mouse in which the Mycl gene is knocked in homozygously can be obtained. For the generation of the above knock-in mice, please refer to ECAT3 knock-in mice (Tokuzawa, Y., et al., Molecular and Cellular Biology, 23(8):2699-2708(2003)), ECAT4 knock-in mice (Mitsui, K., et al., Cell, 113:631-642(2003)), ECAT5 knock-in mice (Takahashi, K., K. Mitsui, and S. Yamanaka, Nature, 423(6939):p541-545(2003), JP 2003-265166 A), etc.

[0050] Chimeric mammals can be produced not only from ES cells but also from iPS cells. For example, organs can be produced from human iPS cells in non-human mammals using the blastocyst complementation method. For example, Nakauchi et al. have generated a human pancreas induced from human iPS cells in the body of an apancreatic cloned pig (see Nakauchu, H., et al., PNAS, Vol. 110, No. 1, 4557-4562 (2013)).

[0051] 2. Regulation of proliferation and differentiation of pancreatic islet-like cells According to the present invention, the proliferation and differentiation of the islet-like cells prepared as described above can be controlled in vitro and in vivo. The method of inducing the Mycl gene can be selected according to the above-mentioned method used for introducing the gene into cells. For example, when the Mycl gene is introduced into cells using the above-mentioned TetOP-Mycl-ires-mCherry vector as a gene introduction vector, a reverse tetracycline-controlled transactivator expressed from a host cell depending on reverse tetracycline such as doxycycline (Dox) binds to the Mycl gene linked downstream, thereby inducing proliferation of the islet-like cells. The concentration of Dox added to the cell culture system can be appropriately adjusted. For example, it may be 1 to 100 mg / mL. After the proliferation of the islet-like cells by adding Dox, the proliferation can be stopped by, for example, replacing the medium with one not containing Dox, and cell differentiation such as insulin production can be induced.

[0052] In vivo, the proliferation of islet-like cells in the pancreas can be induced by, for example, giving water containing Dox to a chimeric non-human mammal. The concentration of Dox when added to water may be, for example, 1 to 100 mg / mL, and is preferably 2.0 mg / mL. When using a chimeric non-mouse, the pancreas grows with age up to 8 weeks after birth, so Dox can be administered when growth stops, for example, after 8 weeks of age, but the age is not limited as long as proliferation can be induced.

[0053] 3. Method for producing pancreatic islet cells or their precursor cells capable of producing insulin According to the present invention, a method for producing islet cells or precursor cells thereof having insulin-producing ability is provided. Such a production method includes, but is not limited to, using primary islet cells, cultured islet cells, or islet cells derived from stem cells as a raw material, introducing Mycl gene into the islet cells, proliferating the cells by forced expression of Mycl gene, and then stopping the expression of the gene to obtain islet cells or precursor cells thereof having insulin-producing ability. As described above, the forced expression and stopping of the Mycl gene can be achieved, for example, by using an alternative means for controlling the expression of the gene on / off (for example, the use of a photoregulated viral vector) or a doxycycline-sensitive reverse tetracycline-regulated transactivator. As used herein, "precursor cells of pancreatic islet cells" or "pancreatic islet progenitor cells" refer to cells (or groups of cells) that are in the process of differentiating into pancreatic islet cells capable of producing insulin after the cessation of expression of the Mycl gene, and can be identified, for example, using one or more markers selected from the group consisting of PDX1 positivity, PTF1a positivity, NKX6.1 positivity, Fev positivity, Pax4 positivity, and Cck gene as indicators.

[0054] 4. Method for Proliferating Islet-like Cells In the past, the efficiency of expanding mature islet cells by Mycl gene introduction was limited in some cases. In particular, when the proliferation of islet-like cells was performed for a long period of time, the efficiency of expansion was sometimes found to be low. According to the present invention, further proliferation of mature islet cells by Mycl gene can be induced by providing a higher concentration of glucose stimulation. Therefore, a method for culturing islet cells into which Mycl gene or its gene product has been introduced or its expression has been induced using a medium containing a high concentration of glucose, that is, a method for sustaining and / or promoting the proliferation activity of islet cells into which Mycl gene or its gene product has been introduced or its expression has been induced in a glucose concentration-dependent manner is provided. Glucose concentration-dependent means that further proliferation of the islet cells can be induced by a higher concentration of glucose stimulation within a certain concentration range (e.g., 10 g / L or less). In one embodiment, the islet-like cells are cultured in a medium containing a high concentration of glucose (sometimes simply referred to as a "high glucose medium") in accordance with the transient expression (on state) of the Mycl gene. A high glucose medium refers to a medium having a glucose concentration of 3 g / L or more, preferably 3.5 to 10 g / L, more preferably 4 to 7 g / L, and even more preferably 4 to 6 g / L.

[0055] In one embodiment using a high glucose medium, the high glucose medium and the low glucose medium may be alternately applied (used) by culturing the cells in the high glucose medium for a certain period of time, then culturing them in the low glucose medium for a certain period of time, and then culturing them again in the high glucose medium for a certain period of time. By culturing the cells by alternately applying (using) the high glucose medium and the low glucose medium, the proliferation activity of the islet-like cells can be maintained for a long period of time. The term "low glucose medium" is used interchangeably with "low concentration glucose medium" and specifically refers to a medium having a glucose concentration of less than 3 g / L, preferably 0.1 to 2.8 g / L, more preferably 0.5 to 2.5 g / L, and even more preferably 1 to 2 g / L. The timing of replacing the high glucose medium with the low glucose medium can be appropriately adjusted, and can be, for example, adjusted to the timing of passage of the islet-like cells (for example, but not limited to, once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, or once every 7 days). By changing the glucose concentration in this way, the proliferation activity of the islet-like cells can be maintained for a long period of time, for example, 1 month or more, 3 months or more, 6 months or more, 8 months or more, or 1 year or more. From the viewpoint of preferably maintaining the function when the islet-like cells are matured into islet cells, for example, glucose-dependent insulin secretion ability, the culture period by changing the glucose concentration is less than 1 year, preferably less than 8 months, more preferably less than 6 months, even more preferably less than 3 months, and even more preferably less than 1 month.

[0056] By regulating a pathway activated by glucose, not limited to glucose, it is possible to proliferate pancreatic islet cells into which the Mycl gene or its gene product has been introduced or whose expression has been induced. In this specification, regulating a pathway activated by glucose includes a method of activating a glucose-stimulated activation pathway and a method of alternately repeating a period in which the glucose-stimulated activation pathway is activated and a period in which the glucose-stimulated activation pathway is not activated. The glucose-stimulated activation pathway refers to a signal transduction pathway stimulated by glucose, and in one embodiment, for example, refers to the GK pathway. In the present invention, the substance that activates the glucose-stimulated activation pathway, which is added in order to maintain and / or promote the proliferation activity of islet-like cells in a concentration-dependent manner due to stimulation of the Mycl pathway, is not particularly limited as long as it activates the glucokinase pathway, and examples thereof include, in addition to the above-mentioned glucose, sucrose, glucokinase activators (known Cpd A (Merck Millipore, CAS 603108-44-7; Sigma-Aldrich, 346021), glucokinase activator II (Sigma-Aldrich, 500487), glucokinase activator III (Merck Millipore, CAS 300353-13-3; Sigma-Aldrich, 509665), and the like.

[0057] The high glucose medium and the low glucose medium can be prepared by appropriately mixing commercially available basal medium for cell culture. The basal medium for cell culture mainly contains carbon sources, nitrogen sources, and also amino acids (e.g., essential amino acids), vitamins (e.g., water-soluble vitamins, fat-soluble vitamins), and other micronutrients, inorganic salts, proteins (e.g., albumin, transferrin, insulin), reducing agents (e.g., glutathione), trace elements (e.g., ammonium metavanadate, manganese chloride, sodium selenite), and other substances (e.g., ethanolamine, hypoxanthine sodium, fatty acids, putrescine, pyruvic acid, thymidine, phenol red), and the components and concentrations of the components can be adjusted as appropriate. The basal medium that can be used for preparing the medium may be appropriately selected from commercially available basal media containing the above-mentioned concentration of glucose based on the ingredient table published by the manufacturer, etc. In addition, it is possible to separately prepare specific components (e.g., glucose-stimulated activation pathway activators) and add them at preferred concentrations, or to purchase an individually customized medium from a medium manufacturer. Examples of basal media that can be used include commercially available DMEM-F12, DMEM, RPMI1640, and the like.

[0058] The basal medium may further contain a growth factor. Examples of growth factors that are preferably added include, but are not limited to, EGF, FGF-10, Gastrin, Noggin, R-spondin, B27 supplement, etc. For example, it is generally known that FGF-10 has the effect of proliferating cells (including islet precursor cells), but whether or not it is added, it can maintain the proliferation activity of islet-like cells by regulating the pathway activated by glucose. For this reason, in the present specification, in the method of culturing the proliferation method of islet cells using a glucose-stimulated activation pathway activator, FGF10 may or may not be added, but it is preferable to add FGF10 from the viewpoint of more stable proliferation.

[0059] It is also possible to amplify pancreatic islet cells in which Mycl gene or its gene product has been introduced or induced to be expressed in vivo. Specifically, it is sufficient to regulate the pathway activated by glucose in vivo. In one embodiment, instead of the "high glucose medium" and the "low glucose medium", a buffer solution adjusted to have the above-mentioned glucose concentration as the final concentration may be administered in vivo. In this case, the term "high glucose medium" may be distinguished as "high glucose buffer solution" and the term "low glucose medium" as "low glucose buffer solution". The timing of replacement of the "high glucose buffer solution" and the "low glucose buffer solution" to be applied is not limited, and may be, for example, once a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days. The buffer solution may also be in the form of a drink containing glucose, sucrose, or the like, which is expected to cause a sufficient increase in blood glucose when ingested by a living body.

[0060] 5.Medicinal Use According to the present invention, by controlling the expression of the introduced Mycl gene and regulating the pathway activated by glucose, it is possible to increase the proliferation activity of islet-like cells, maintain the proliferation activity of islet-like cells for a long period of time, and promote insulin production along with the proliferation of islet-like cells. Thus, in one aspect, there are provided an insulin production promoter comprising, as active ingredients, islet-like cells whose proliferation activity has been maintained or promoted for a long period of time by Mycl gene or its gene product and a glucose-stimulated activation pathway activator such as glucose; a pharmaceutical composition comprising the active ingredient and other pharma- ceutical acceptable ingredients (e.g., carrier, excipient, disintegrant, buffer, emulsifier, suspending agent, soothing agent, stabilizer, preservative, antiseptic, physiological saline, etc.); a method for preventing and / or treating diabetes patients using the insulin production promoter or pharmaceutical composition; and use of the Mycl gene and the glucose-stimulated activation pathway activator for producing the insulin production promoter or pharmaceutical composition.

[0061] In another aspect, there is provided a method (ex vivo method) for transplanting islet-like cells, the proliferation activity of which is enhanced by controlling the expression of an introduced Mycl gene and regulating a pathway activated by glucose, and the proliferation activity of the islet-like cells is maintained and promoted for a long period of time, into a subject, for the purpose of preventing and / or treating diabetes. Note that the ex vivo method of the present invention contemplates different aspects depending on the cells used. For example, examples include: (i) an embodiment in which islet-like cells obtained by introducing a gene into cells derived from adult pancreatic islets are produced allogeneically in vitro or autologously in vitro (a narrow definition of the ex vivo method including both); (ii) an embodiment in which islet-like cells obtained by inducing differentiation, i.e., islet-like cells obtained by introducing the Mycl gene into islet cells derived from stem cells (iPS cells, ES cells, somatic stem cells, etc.), are produced allogeneically in vitro or autologously in vitro (a broad definition of the ex vivo method); (iii) an embodiment in which the Mycl gene is functionally expressed in the above (i) and (ii) to produce proliferating islet precursor cell-like cells; and (iv) an insulin-producing cell or islet cell (not containing the Mycl gene) that is produced by differentiation from the above (i) to (iii).

[0062] In one embodiment of the present invention, regardless of whether the cells are autologous or allogeneic to the patient, the islets to be transplanted may be encapsulated and administered by a known method, specifically, the method described in Nature Medicine volume 22, pages 306-311 (2016)., doi: 10.1038 / nm.4030, Nature Biomedical Engineering volume 2, pages 810-821 (2018)., DOI: 10.1038 / s41551-018-0275-1, EBioMedicine 12 (2016) 255-262., DOI: https: / / doi.org / 10.1016 / j.ebiom.2016.08.034, etc. By encapsulating the islets to be transplanted, it is possible to avoid the use of immunosuppressants or to reduce the dosage of the immunosuppressants.

[0063] In one embodiment of the present specification, isolated pancreatic islets can be appropriately gene-edited. The gene to be edited is not particularly limited, but specifically, it is possible to add a preferable feature for treatment, such as correction of a mutated gene contained in pancreatic islet cells by genome editing, modification of molecules that cause immune reactions, such as surface antigens such as HLA and GAD proteins, and induction of immune tolerance by deletion of Beta-2 Microglobulin, RFX5, RFXANK, RFXAP, and CIITA genes. The method of gene editing is not particularly limited, but specific examples include CRISPR-Cas9, CRISPR-Cas12, TALEN, ZFN, CRISPR-Cas3, CRISPR-TypeI-D, MAD7, and modified versions thereof, and transposon vectors such as piggyBAC for expressing functional molecules.

[0064] (1) Indications The diseases to which the present invention can be applied based on the above-mentioned embodiment are diseases in which insulin does not function sufficiently in the living body. Typical examples of such diseases include pathologies in which reduced secretion is observed, type I diabetes, insulin-resistant states in which insulin is relatively insufficient, type II diabetes, and type 1.5 diabetes, which is an intermediate pathology between type I diabetes and type II diabetes. According to the present invention, by controlling the expression of the introduced Mycl gene and regulating the pathway activated by glucose, it is possible to promote insulin production by the islet-like cells whose proliferation activity is maintained and promoted for a long period of time, and, for example, have the effect of lowering blood glucose in diabetic patients. The applicable disease is typically diabetes, but more specifically, severe hypoglycemia, type I diabetes (including slowly progressing type 1 diabetes or type 1.5 diabetes), type II diabetes, impaired glucose tolerance, hyperglycemia, dyslipidemia, obesity, or diseases, disorders or symptoms related to metabolic syndrome, and other specific mechanisms or diseases, such as genetic abnormalities involved in pancreatic β-cell function, genetic abnormalities involved in the transmission mechanism of insulin action, pancreatic exocrine diseases, endocrine diseases, liver diseases, diseases caused by drugs or chemicals, infectious diseases, rare pathologies caused by immune mechanisms, or gestational diabetes, etc., as associated with other diseases or conditions. In addition, diabetic complications caused by diabetes (e.g., diabetic retinopathy, diabetic neuropathy, etc.) can also be included in the applicable disease. In addition, insulin secretion deficiency caused as a result of total pancreatectomy or partial pancreatectomy associated with pancreatitis or pancreatic cancer can also be included in the applicable disease.

[0065] Furthermore, the type of diabetes that can be treated by the method described herein is not particularly limited, but the method can provide a treatment that secretes physiological insulin depending on blood glucose level and is less likely to cause hypoglycemia. For example, when used to treat severe hypoglycemia, but is not limited to this, a method described in a known literature can be used as one embodiment, since it is known to those skilled in the art that donor islets show a significant effect on severe hypoglycemia. The methods described in known publications herein are not limited to, but specifically include, for example, Diabetes Care 2016 Jul; 39(7):1230-1240., DOI:10.2337 / dc15-1988, The New England Journal of Medicine.343(4):230-238., DOI:10.1056 / NEJM200007273430401, The New England Journal of Medicine.355(13):1318-1330., DOI:10.1056 / NEJMoa061267, and the like.

[0066] In addition, when used for the treatment of type I diabetes, although there is no particular limitation, as one embodiment of the treatment for type I diabetes, for example, the number of islet cells, islet-like cells, islet precursor-like cells, or insulin-producing cells contained may be adjusted according to the insulin, blood glucose level, and / or C-peptide level of a type I diabetes patient at any time, fasting, after glucose loading, and / or after glucagon stimulation, and the dosage of the agent for in vivo treatment may be determined by the method described herein. Type I diabetes patients in an insulin-depleted state are at high risk of exhibiting hypoglycemic symptoms, and from the viewpoint that treatment using the method described herein is preferable, for example, type I diabetes patients with a blood C-peptide level of 0.5 ng / mL or less, preferably 0.2 ng / mL or less, more preferably 0.1 ng / mL or less in fasting and / or glucagon stimulation can be treated. The specific dosage of the agent may be adjusted, for example, with reference to the transplantation amount of donor islets that are usually transplanted for the indication of severe hypoglycemia. Specifically, for example, the cells are pancreatic islet cells, pancreatic islet-like cells, pancreatic islet precursor-like cells, or insulin-producing cells equivalent to 500 IEQ / kg or more, preferably equivalent to 1000 IEQ / kg or more, more preferably equivalent to 2000 IEQ / kg or more, and even more preferably equivalent to 5000 IEQ / kg or more.

[0067] In addition, when used for the treatment of type II diabetes, there is no particular limitation, but as one embodiment, it can be used as a therapeutic agent for type II diabetes in an insulin-dependent state in which the amount of insulin secreted into the body is insufficient. Specifically, for example, the number of islet cells, islet-like cells, islet precursor-like cells, or insulin-producing cells contained in the type II diabetes patient may be adjusted according to the amount of insulin, blood glucose level, and / or C-peptide at any time, fasting, after glucose loading, and / or after glucagon stimulation, and the dosage of the agent for in vivo treatment may be determined by the method described herein. From the viewpoint of obtaining a favorable effect in a pathological condition in which insulin is insufficient, for example, type II diabetes patients with a blood C-peptide level of 0.5 ng / mL or less, preferably 0.2 ng / mL or less, more preferably 0.1 ng / mL or less in fasting and / or glucagon stimulation can be treated. The specific dosage of the agent may be adjusted, for example, with reference to the transplantation amount of donor islets that are usually transplanted for the indication of severe hypoglycemia. Specifically, for example, the cells are pancreatic islet cells, pancreatic islet-like cells, pancreatic islet precursor-like cells, or insulin-producing cells equivalent to 500 IEQ / kg or more, preferably equivalent to 1000 IEQ / kg or more, more preferably equivalent to 2000 IEQ / kg or more, and even more preferably equivalent to 5000 IEQ / kg or more.

[0068] In addition, when used as a therapeutic agent for slowly progressing type I diabetes, the amount of the agent contained can be adjusted according to the amount of insulin, blood glucose level and / or C peptide in the body in the same manner as type I diabetes and / or type II diabetes, but is not limited thereto. In this case, the islet cells, islet-like cells, islet precursor-like cells, or insulin-producing cells expanded by the method of the present specification are preferably derived from the patient's own cells. For example, as one embodiment of the treatment of slowly progressing type I diabetes, preferably, the type of autoantibodies or HLA against islet cells in the blood is examined in advance, and if the patient's own islet cells can be expanded by the method of the present specification before or after falling into an insulin-dependent state and / or an insulin-depleted state, treatment without the need for immunosuppressants becomes possible, and the transition to an insulin-dependent state and / or an insulin-depleted state can be prevented, or the insulin-dependent state and / or an insulin-depleted state can be treated. To determine whether or not a patient has slowly progressing type I diabetes, any known method can be used, as long as the type of autoantibody against islet cells in the blood or HLA is involved in slowly progressing type I diabetes, including, for example, whether or not islet-related autoantibodies such as islet cell antibody (ICA), GAD antibody, insulin autoantibody (IAA), and IA-2 antibody are positive in combination or alone. Furthermore, it may be confirmed by a known method, such as whether or not the patient has HLA associated with slowly progressing type I diabetes, such as HLA-DR4-DQA1*0301-B1*0401.

[0069] From the viewpoint that the therapeutic effect is observed by the donor islets, the ex vivo method is preferable for the treatment of severe hypoglycemia, but the method described herein can expand islet cells in vivo. Since the expanded islet cells in this way have the same effect as the ex vivo method, the treatment method of expanding islets in vivo can be preferably selected as a treatment method for severe hypoglycemia, as well as the ex vivo method. Furthermore, for the treatment of type I diabetes, type II diabetes, and slowly progressing type I diabetes, as with the ex vivo method, a method of expanding islet cells in vivo can be selected taking into consideration the subject's sex, age, weight, condition of the affected area, condition of the cells used, etc. At this time, the method of expanding islet cells in vivo can be combined with the ex vivo method.

[0070] According to the present invention, such diseases can be prevented and / or treated. As used herein, the term "prevent" refers to preventing or delaying the onset / onset of the above-mentioned disease or its symptoms, or reducing the risk of onset / onset. "Treatment" includes alleviating (alleviating) symptoms characteristic of the target disease or associated symptoms, preventing or delaying the worsening of symptoms, etc. "Prevention" refers to preventing or delaying the onset / onset of a disease (disorder) or its symptoms, or reducing the risk of onset / onset. On the other hand, "improvement" refers to alleviating (alleviating), improving, remission, or curing (including partial curing) a disease (disorder) or its symptoms.

[0071] (2) Insulin production enhancer and pharmaceutical composition The insulin production promoter or pharmaceutical composition containing as active ingredients the islet-like cells whose proliferation activity has been maintained or promoted for a long period of time by the Mycl gene or its gene product and the glucose-stimulated activation pathway activator of the present invention can be provided for the prevention and treatment of the above-mentioned applicable diseases. Here, one embodiment of the islet cell proliferation promoter refers to an agent that proliferates any one of α cells, β cells, δ cells, ε cells, and PP cells contained in a pancreatic islet, preferably any two or more of the islet cells. In addition, one embodiment of the islet function improver refers to an agent that improves a part or all of the functions of a pancreatic islet in a living body by administration, and examples of the part of the functions of a pancreatic islet include blood glucose regulation by islet cells, blood glucose lowering effect by insulin, glucose production / release effect by glucagon, secretion suppression effect of gastrin, secretin, insulin and / or glucagon by somatostatin or nutrient absorption suppression effect in the digestive tract, appetite regulation effect by ghrelin, and gallbladder contraction regulation and appetite regulation effect by pancreatic polypeptide. One embodiment of the insulin production promoter is, for example, an agent that promotes physiological insulin secretion in response to blood glucose levels, which is one of the functions of pancreatic islets in vivo. When provided as a pharmaceutical composition, it may contain other pharma- ceutically acceptable ingredients (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, physiological saline, etc.) in addition to the active ingredient Mycl gene or its gene product used in the above embodiment. Furthermore, it may contain an activator for activating the Mycl gene, if necessary.

[0072] The pancreatic islet cells derived from a living body into which the Mycl gene or its gene product is introduced may be those of a healthy individual, or may be those of a type I diabetes patient or a patient with terminal type II urinary catheter disease in which some or most of the β cells have been lost. In this case, the pancreatic islet cells may be autologous or allogeneic to the recipient.

[0073] The insulin production enhancer and pharmaceutical composition of the present invention can be obtained by suspending the above-obtained islet-like cells in physiological saline or an appropriate buffer solution (e.g., phosphate-buffered saline), although this is not limited thereto. The number of cells required for treatment can be obtained by forcibly expressing the Mycl gene and appropriately proliferating the cells.

[0074] Furthermore, when used in insulin production promoters and pharmaceutical compositions, dimethyl sulfoxide (DMSO), serum albumin, etc. may be contained in the insulin production promoters and pharmaceutical compositions to protect the cells, and antibiotics, etc. may be contained in the insulin production promoters and pharmaceutical compositions to prevent bacterial contamination and growth. Furthermore, other components that are acceptable for formulation (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, physiological saline, etc.) may be contained in the insulin production promoters and pharmaceutical compositions. Those skilled in the art can add these factors and drugs to the insulin production promoters and pharmaceutical compositions at appropriate concentrations.

[0075] The number of pancreatic islet cells, pancreatic islet-like cells, pancreatic islet precursor-like cells, or insulin-producing cells contained in the insulin production promoter and pharmaceutical composition prepared above can be appropriately adjusted taking into consideration the subject's sex, age, weight, condition of the affected area, condition of the cells used, etc., so as to obtain the desired effect (e.g., lowering blood glucose level) in the prevention and / or treatment of diabetes and related diseases.

[0076] The insulin production promoting and pharmaceutical composition of the present invention can be administered to various subjects, for example, mammals such as primates, humans, dogs, cats, cows, horses, pigs, sheep, etc., and preferably humans. The route of administration to the subject is not limited, but can be parenteral administration, for example, administration by injection or infusion to any location in the body that can respond to glucose. Specifically, for example, it can be implanted or administered into the pancreas, under the kidney capsule, preferably subcutaneously, intraperitoneally, more preferably intravascularly, intravenously, and even more preferably into the portal vein.

[0077] The method for activating the administered Mycl gene in vivo is not limited, but the above-mentioned system for "on" control or "on and / or off" control of Mycl gene expression can be used. For example, the above-mentioned "light-controlled viral vector" (Tahara, M., et al., PNAS, vol. 116, 11587-11589, 2019) may be used.

[0078] In addition, when the purpose is to target the Mycl gene to the pancreas, a marker specifically expressed in pancreatic islet cells (e.g., PDX1, C-peptide, insulin, MafA, Mnx1, Pax4, Pax6, NeruroD1, Isl1, Nkx2.2, Ngn3, HNF1a, Foxa2, Nkx6.1, glucagon, Arx, MafB, RFX6, IRX1, IRX2, somatostatin) can be targeted for gene transfer. When selecting a method for expanding pancreatic islet cells in vivo, specific examples of the administration route to a subject include, but are not limited to, intrapancreatic, subcutaneous, intraperitoneal, preferably intravascular, intravenous, and even more preferably intraperitoneal artery and intrapancreatic duct.

[0079] (3) Treatment method According to the present invention, there is provided a method for preventing and / or treating a subject having diabetes or a disease related thereto, using islet-like cells whose proliferation activity has been maintained or promoted for a long period of time by Mycl gene or its gene product and a glucose-stimulated activation pathway activator. Furthermore, according to the present invention, in the above-mentioned treatment method, an activator for activating the Mycl gene can be administered, and may be administered before, simultaneously with, or after administration of an insulin production promoter or a pharmaceutical composition. Furthermore, in order to expand in vivo islet cells into which Mycl gene or its gene product has been introduced or whose expression has been induced, the concentration of glucose or the like in the body may be adjusted by food, drinking water, intravenous drip, or the like.

[0080] (4) Kit According to the present invention, there is provided a kit for preventing and / or treating diabetes or a disease related thereto, comprising an insulin production promoter or a pharmaceutical composition. Such a kit may include an instruction manual for administering or transplanting the insulin production promoter or pharmaceutical composition to a subject. The kit may further include an activator for activating the Mycl gene. EXAMPLES

[0081] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples in any way.

[0082] method (i) Establishment of ES cells capable of inducing Mycl expression in a doxycycline (Dox)-dependent manner As an ES cell for introducing the Mycl gene, we used the KH2 strain, which has an Frt sequence downstream of the Cola1 locus and expresses the reverse tetracycline-regulated transactivator M2-rtTA under the control of the endogenous Rosa26 promoter (Beard C, et al., Genesis, vol. 44, p. 23-28 (2006)). The Mycl cDNA was cloned from the cDNA derived from the ES cell, and the cloned fragment was inserted into the pCR8-GW-TOPO vector (Invitrogen). The Col1a1-TetOP-Mycl-ires-mCherry vector (hereafter referred to as the "targeting vector") was generated by performing LR reaction between the pCR8-Mycl-TOPO vector containing the Mycl gene and the TetOP-AttR1-ccdB-AttR2-ires-mCherry vector, and was inserted into the Col1a1 locus of KH2-ES cells using the flip-in recombination system (Beard et al., 2006). When performing flip-in recombination, 50μg of the targeting vector with the Mycl gene inserted, 25μg of the pFlapase vector, and a cell suspension suspended in high glucose DMEM (Nacalai Tasque) medium containing 25mM HEPES buffer (Gibco) were electroporated into KH2-ES cells using a Gene pulser Xcell electroporation system (BIO-RAD) (voltage: 550 V, capacitance: 25μF, resistance: ∞, cuvette: 4mm or more, pulsed twice). 24 hours after electroporation, the cells were selected with 150μg / mL of hygromycin B (Roche), and the formed colonies were picked up to establish an ES cell line capable of inducing the expression of the Mycl gene in a Dox-dependent manner.

[0083] (ii) Cell culture method Feeder cells (MEF; mouse embryonic fibroblasts) were cultured in DMEM (Nacalai Tesque) medium containing 10% FBS (GIBCO), 50 U / mL Penicillin-Streptomycin (P / S; Nacalai Tesque), L-glutamine (GIBCO), and NEAA (Nacalai Tesque).

[0084] ES cells were cultured in a medium containing 15% FBS, 50 U / mL P / S, L-glutamine, and NEAA, 2-mercaptoethanol (2ME: GIBCO), and LIF (SIGMA), on a gelatin-coated (SIGMA) dish seeded with feeder cells. For subculturing ES cells, the cells were treated with 0.25% trypsin / 1 mM EDTA (GIBCO) at 37°C for about 3 minutes, and about 1 / 10 the volume of the cell suspension was seeded on a new dish.

[0085] (iii) Generation of mice capable of inducible Mycl expression in vivo (KH2-Mycl) We generated chimeric mice carrying cells capable of inducing Dox-dependent Mycl expression by injecting ES cells capable of inducing Dox-dependent Mycl expression into mouse blastocysts (ICR, E3.5) and transplanting them into the uterus of pseudopregnant mice (Slc:ICR, Shimizu Experimental Materials) on day 2. These mice were capable of inducing the expression of the Mycl gene and the red fluorescent protein mCherry systemically by administration of Dox.

[0086] (iv) Mice capable of tracking pancreatic islet cells (Ins1-ires-CreERT2; Gcg-CreERT2, Sst-ires-CreERT2) We generated mice in which tamoxifen-inducible Cre recombinase (CreERT2) was introduced downstream of the endogenous promoter of the gene Gcg, which is expressed specifically in alpha cells, and downstream of the endogenous genes for the gene Ins1, which is expressed specifically in beta cells, and the gene Sst, which is expressed specifically in delta cells.

[0087] (v) Administration of doxycycline Eight-week-old mice were administered a solution containing 2.0 mg / mL of Dox in their drinking water. For cultured cells, Dox was added to the medium at a final concentration of 2.0 μg / mL.

[0088] (vi) Preparation of pathological specimens from mouse organs After dissection of the mice, each organ was shaken in 4% PFA (Wako Pure Chemicals) for one day, and the next day transferred to 70% EtOH (diluted from 100% EtOH by Wako Pure Chemicals) and shaken for another day. The next day, blocks were prepared using a spin tissue processor STR120 (Thermo Scientific) according to the recommended protocol. Preparation of pathological specimens was outsourced to Biogate Corporation.

[0089] (vii) Immunostaining The tissue sections were immersed in xylene (Wako Pure Chemicals) and then in 100% EtOH (Wako Pure Chemicals) for 30 minutes or more each. They were washed with tap water for about 10 minutes, transferred into boiled antigen retrieval solution pH 9 (Nichirei Biosciences, used at 10-fold dilution), and antigen retrieval treatment was performed for 10 minutes. 200 μL of primary antibody solution diluted with blocking solution (2% BSA + 1×PBS) at various dilutions was added onto the tissue sections and left to stand for 30 minutes to 1 hour. After washing twice with 1×PBS, 2 drops of secondary antibody solution were added onto the tissue sections and left to stand for 30 minutes. After washing twice with 1xPBS, in the case of DAB staining, 150μL of DAB solution (Nichirei Biosciences, DAB substrate kit used, 1mL of Elix water was mixed with 1 drop each of Reagent A and B, then 1 drop of Reagent C was added and mixed) was added to the tissue section, and the antigen-antibody reaction was performed, followed by observation under a microscope. In the case of fluorescent staining, 1 drop of mounting material was added to the tissue section, a cover glass was placed, and then observation under a microscope was performed.

[0090] <Primary antibody used (dilution ratio) - secondary antibody> Anti-mCherry antibody (Abcam, 1 / 500) - Anti-rabbit IgG antibody (Nichirei Biosciences) - Anti-Synaptophysin antibody (Abcam, 1 / 500) - Anti-rabbit IgG antibody (Nichirei Biosciences) Anti-Chromogranin A antibody (DAKO, 1 / 500) - Anti-Rabbit IgG antibody (Nichirei Biosciences) Anti-Ki67 antibody (Abcam, 1 / 200) - Anti-rabbit IgG antibody (Nichirei Biosciences) - Anti-Insulin antibody (DAKO) - Anti-Guinea Pig IgG antibody (BIOTIUM) ·Anti-Somatostatin antibody (Santa cruz, 1 / 300) - anti-mouse IgG antibody (BIOTIUM) ·Anti-Glucagon antibody (Santa cruz, 1 / 300) - anti-mouse IgG antibody (BIOTIUM)

[0091] (viii) RNA recovery, RNA extraction, and cDNA synthesis For RNA collection, the cultured cells were washed with PBS(-) (Nakalai Tasque) and then lysed with 350 μL of LBP buffer. RNA was extracted using NucreoSpin® RNA Plus (TAKARA) according to the recommended protocol. cDNA was synthesized using Primescript single-stranded cDNA synthesis kit (TAKARA) according to the recommended protocol.

[0092] (ix) qRT-PCR The GoTaq qPCR Master Mix (Promega) was used according to the recommended protocol. Analysis was performed using the Stepone Plus system (Life Technologies). The primers and PCR reaction conditions used were as follows:

[0093] [Table 1]

[0094] (b) PCR reaction conditions 95°C for 2 minutes 95℃ (15 seconds), 60℃ (1 minute) [40 cycles] ·95℃(15 seconds), 60℃(1 minute), 95℃(15 seconds)

[0095] (x) Pancreatic islet isolation Eight-week-old KH2-Mycl mice were anesthetized by intraperitoneal injection of Somnopentyl (Kyoritsu Seiyaku Co., Ltd.). After laparotomy, the opening of the duodenum-common bile duct was identified, and the upper part of the common bile duct and the intestine were clamped with bulldog clamps. The common bile duct was incised at the border with the duodenum, and 2 mL of M199 medium (Gibco) containing Colgenase P (Roche) (2 mg / mL) was injected. The pancreas was then removed, transferred to a 50 mL tube, and digested in a 37°C water bath for 14 minutes and 30 seconds. The pancreas was suspended in 25 mL of cold M199 medium containing 10% FBS, and centrifuged twice (1000 rpm, 4°C, 2 minutes). The supernatant was discarded, and the cells were suspended in 10 mL of Histopaque (SIGMA), after which 10 mL of cold M199 medium containing 10% FBS was poured on top and centrifuged (1000 rpm, 4°C, 30 min).The supernatant was transferred to another 50 mL tube, and 25 mL of M199 medium containing 10% FBS was added, followed by density gradient centrifugation using Lymphoprep (1000 rpm, 4°C, 2 min) to separate the islets.

[0096] (xi) Dispersion of pancreatic islets The isolated islets were collected in a 1.5 mL silicon tube, and 100 μL of dispersion buffer (see Table 2 below) was added. After standing at 37° C. for 15 minutes, the islets were dispersed by pipetting.

[0097] [Table 2]

[0098] [Table 3]

[0099] (x) Mouse primary islet cell culture The isolated islets were transferred to a 1.5 mL tube, and islet cells were isolated using TrypLE. After washing with RPMI medium, the cell count was measured. The islet cells were seeded onto a 96-well plate, and 30 μL of Matrigel was added onto them. The cells and Matrigel were mixed with the tip of a pipette, and the 96-well plate was placed in a CO2 incubator at 37°C for 30 minutes to crosslink the gel. Then, 120 μL of medium was added, and the plate was cultured at 37°C and 5% CO2. A KEYENCE BZ-710 was used to observe and image the islet cells.

[0100] (xi) Passage of islet cells TrypLE (120 μL) was added to the matrigel in which the islet cells were cultured, and the gel was broken down by pipetting. The islet cells were transferred to a 1.5 mL tube, 300 μL of TrypLE was added, and the tube was left to stand at 37°C for 10 minutes in a CO2 incubator. The tube was then centrifuged at 200 g for 2 minutes, washed with RPMI medium, and the cell count was measured. The islet cells were seeded on a 96-well plate, and three-dimensional culture was then performed using matrigel. The islet cells were passaged once a week, and the culture medium used was RPMI1640 (nacalai tasque; 09892-15) to which 45 w / v% D(+)-glucose solution (Wako; 079-05511) was added so that the glucose concentration was at an appropriate final concentration (e.g., 4.5 g / L, 4.5 g / L, etc.).

[0101] (xii) Cell fate tracing experiments 0.2 mL of tamoxifen (20 mg / mL) was administered intraperitoneally to 8-12 week-old lineage-traceable mice (KH2-Mycl; Ins1 / Gcg / Sst-CreERT2; R26-mTmG) five times a week. One week later, pancreatic islets were isolated from the mice. After isolating islet cells using TrypLE, 1.0 × 10 5Cells were seeded in AggreWell at 100 cells / well. 4-OHT (100nmol / mL) was added to the culture medium and cultured for 2 days. The islet cells were harvested and cultured in 3D with Matrigel. After one week, the cells were imaged with KEYENCE BZ-710 and the percentage of GFP-positive cells was evaluated by FACS.

[0102] (xiii) Islet cell transplantation experiments Diabetes was induced in immunodeficient mice (NOD / ShiJic-scid Jcl) by intraperitoneal administration of streptozotocin (100 mg / kg) three times a week. 5 × 10 islet cells (passaged 18 times) were cultured under the kidney capsule of these mice. 5 The cells were transplanted using a Hamilton syringe. Blood glucose levels were measured weekly and were measured randomly.

[0103] (ix) Intraperitoneal glucose tolerance test (IPGTT) The Mycl expression group and the control group were fasted for 12 to 16 hours. After that, the weight of each mouse was measured, and D-glucose solution was injected intraperitoneally to the mouse so that D-glucose was 2 g / kg (mouse). After 15, 30, 60, and 120 minutes, blood was collected from the tail of each mouse, and blood glucose levels were measured using an Antsense stand (Horiba).

[0104] Example 1: Glucose stimulates proliferation of Mycl-induced islet cells in vitro The expression of the Mycl gene can be confirmed by the expression of the mCherry gene inserted downstream of the gene. The mCherry gene is a gene that encodes a red fluorescent protein, and expression of the gene causes cells to develop a red color. The glucose concentration in the medium was changed to examine the difference in the proliferation activity of Mycl-induced islet cells. It was found that the proliferation efficiency of Mycl-expressing islet cells was increased under culture conditions of a high glucose medium (D-glucose; 4.5 g / L) compared to culture conditions of a low glucose medium (D-glucose; 1.0 g / L) (see Figure 1).

[0105] We investigated whether the proliferation of the above-mentioned Mycl-induced islet cells was dependent on glucose concentration. The glucose concentration in the medium was set to 1.0, 2.0, and 4.5 g / L, and Mycl-induced islet cells were cultured in the same manner as above, and the culture efficiency was evaluated. A comparison of mCherry expression and relative cell numbers revealed that the proliferation efficiency of Mycl-expressing islet cells increased in a glucose concentration-dependent manner (see Figure 2).

[0106] We used a drug that activates glucokinase, an enzyme involved in glucose metabolism (glucokinase activator; GKA), to examine the effect of the drug on the proliferation efficiency of Mycl-expressing islet cells. As is clear from Figure 3, the addition of GKA (50 μM) increased the proliferation efficiency of Mycl-expressing islet cells compared to the absence of GKA (DMSO).

[0107] Next, we used a drug (glucokinase inhibitor; GKI) that inhibits the activity of glucokinase, an enzyme involved in glucose metabolism, to examine the effect of the drug on the proliferation efficiency of Mycl-expressing islet cells. As is clear from Figure 4, the addition of GKI (10 mM) reduced the proliferation efficiency of Mycl-expressing islet cells compared to the absence of GKI (DMSO).

[0108] Example 2: Glucose stimulates proliferation of Mycl-induced pancreatic islet cells in vivo Using mice capable of tracking islet cells (Ins1-ires-CreERT2; Gcg-CreERT2, Sst-ires-CreERT2), we investigated the activation of Mycl-induced islet cell proliferation in vivo by glucose. In Example 1 (in vitro), glucose was added to the medium, but in this example, sucrose (glucose + fructose) was used instead of glucose. In addition, the proliferation activity of islet cells was evaluated by immunostaining. The results are shown in Figure 5, and in the Dox induction + sucrose administration system, significant proliferation of islet cells was observed compared to other experimental systems. This shows that the administration of sucrose (glucose + fructose) promotes islet proliferation by the Mycl gene in vivo as well.

[0109] Example 3: Effect of continuous culture in the presence of glucose on the proliferation activity of Mycl-induced islet cells The effect of the continuous presence of glucose in the medium on the proliferation activity of Mycl-induced islet cells was examined. A high glucose concentration was used for the culture. The cells were passaged a total of four times, with one culture cycle lasting 7 days. It was observed that the activation of cell proliferation by Mycl induction decreased with repeated passages (see Figure 6). This demonstrated that the proliferation ability of Mycl-expressing islet cells decreased with continued culture, even under culture conditions with a high glucose concentration, suggesting the involvement of so-called "glucotoxicity."

[0110] Example 4: Possibility of sustained Mycl-induced islet cell proliferation by adjusting glucose concentration As shown in Example 3, continuous culture in a medium with a high glucose concentration suggested the involvement of so-called "glucotoxicity," so we investigated whether cells could be cultured continuously for a long period of time by adjusting the glucose concentration. Activation of cell proliferation was examined by alternating between high and low glucose concentrations every 24 hours. As shown in Figure 7, under such culture conditions, the proliferation activity of Mycl-induced islet cells was found to persist for at least 20 passages (about 22 weeks). This demonstrated that the high proliferation ability of Mycl-induced islet cells can be maintained by repeating high glucose and low glucose (hereinafter referred to as "high-low glucose"), enabling sustained proliferation induction.

[0111] The change in cell proliferation over time was examined by counting the number of Mycl-induced islet cells in a high-low glucose culture system. As shown in Figure 8, the high proliferation ability of Mycl-induced islet cells was maintained by repeating high glucose and low glucose, and it was found that sustained proliferation induction was possible.

[0112] Example 5: Tracking test of pancreatic islet cells The expression of the gene Ins1, which is specifically expressed in β cells derived from pancreatic islet cells, the gene Gcg, which is specifically expressed in α cells, and the gene Sst, which is specifically expressed in cells, was tracked. Ins1-positive cells (β cells), Gcg-positive cells (α cells), and Sst-positive cells (δ cells) were labeled by standard methods to examine whether each cell proliferated. More specifically, the expression intensity of each gene was compared by expression of the reporter gene mGFP (see Figure 9). The data in Figure 9 show that the Mycl gene promotes the proliferation of mature hormone-producing cells, β cells, α cells, and δ cells.

[0113] Furthermore, when the proliferation efficiency of mature hormone-producing cells was examined by FACS analysis, it was found that Ins1-positive cells (β cells) proliferated more efficiently by culturing under the Mycl gene and high glucose concentration (Figure 10).

[0114] Example 6: Recovery of Diabetic Mice We investigated the change in blood glucose levels in diabetic mice with Mycl-induced islet cells grown for a long period under high-low glucose conditions (alternating high and low glucose conditions). As shown in Figure 11, transplantation of Mycl-induced islet cells into diabetic mice normalized blood glucose levels. This improved glucose tolerance in an intraperitoneal glucose tolerance test (IPGTT), confirming that the islet cells grown for a long period have high functionality.

[0115] <Summary> According to the results of the above examples, first, it was found that the proliferation activity of mature islet cells induced by Mycl gene induction depends on glucose concentration (see Figures 1 and 2). In fact, it was shown that proliferation of islet cells is promoted by glucokinase activator (GKA), which activates glucokinase, a molecule of the intracellular metabolic pathway of glucose, and is suppressed by glucokinase inhibitor (GKI), which is an inhibitor of glucokinase (see Figures 3 and 4). It was also shown that the proliferation of islet cells is strongly promoted when Mycl gene is expressed in vivo while adding glucose (see Figure 5). On the other hand, it was found that the cell proliferation activity is reduced due to glucose toxicity when cultured continuously even under high glucose culture conditions (see Figure 6). It was shown that high proliferation activity can be maintained continuously by repeating high glucose conditions and low glucose conditions to avoid glucose toxicity, and proliferation was induced for at least six months or more (see Figures 7 and 8). The proliferation of mature β cells was promoted by high glucose (see Figures 9 and 10). The continuously expanded islet cells produced insulin in response to glucose in vivo and reduced blood glucose levels in diabetic animal models, demonstrating their high functionality (see Figure 11). [Industrial Applicability]

[0116] The present invention enables the induction of sustained proliferation of islet cells, and is expected to lead to a complete cure of diabetes by islet cell transplantation. In addition, by activating the Mycl gene together with glucose in vivo, it is possible to efficiently amplify islet cells in vivo, and it is expected that diabetes can be completely cured by gene therapy.

[0117] All publications and patents cited herein are incorporated by reference in their entirety. Although specific embodiments of the invention have been described herein for purposes of illustration, those skilled in the art will readily understand that various modifications may be made without departing from the spirit and scope of the invention.

Claims

1. A method for promoting and / or sustaining the proliferative activity of pancreatic islet cells into which the Mycl gene or its gene product has been introduced or whose expression has been induced, using a glucose-stimulated activation pathway activator.

2. The method according to claim 1, wherein the glucose-stimulated activation pathway activator is selected from the group consisting of glucose, sucrose, and glucokinase activators, to the extent that at least one is selected from the group.

3. The Mycl gene, (1) Nucleic acids comprising the base sequence represented by SEQ ID NO: 1 or 3; or (2) A nucleic acid encoding a polypeptide that hybridizes under stringent conditions with a nucleic acid containing a nucleotide sequence complementary to the nucleotide sequence represented by Sequence ID No. 1 or 3, and which has the function of sustaining and / or promoting the proliferation activity of pancreatic islet-like cells when the Mycl gene is expressed. The method according to claim 1 or 2, including the method described in claim 1 or 2.

4. Mycl gene product, (1) Polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or 4; or (2) Polypeptides having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO: 2 or 4, and having the function of sustaining and / or promoting the proliferation activity of pancreatic islet-like cells. The method according to claim 1 or 2, including the method described in claim 1 or 2.

5. The method according to claim 1 or 2, wherein the Mycl gene is transiently expressed.

6. The method according to claim 1 or 2, wherein a high-glucose medium and a low-glucose medium are applied alternately.

7. The method according to claim 1 or 2, wherein the islet cells are primary islet cells isolated from the pancreas, cultured islet cells, or stem cell-derived cells.

8. The method according to claim 7, wherein the stem cells are selected from the group consisting of iPS cells, ES cells, and somatic stem cells.

Images