Transient expression of Mycl induces proliferation-competent islet progenitor-like cells and their differentiation into insulin-positive cells

Abstract

Establishment of pancreatic islet-like insulin producing cells by inducing differentiation of ES / iPS cells has been reported. However, no technique has been developed so far for producing functional pancreatic islet insulin-positive cells in a large amount. In addition, there are concerns regarding rejection responses, accidental immune responses, etc. The present invention provides pancreatic islet-like cells having Mycl gene introduced thereinto and a method that comprises inducing proliferation of pancreatic islet-like cells by transient expression of Mycl gene and then inducing degradation thereof into insulin producing cells.

Description

[Technical field]

[0001] The present invention relates to an insulin production promoter comprising a Mycl gene or pancreatic islet-like cells introduced with the Mycl gene. The present invention also relates to a pharmaceutical composition for preventing / treating diabetes, comprising a Mycl gene or its gene product, or pancreatic islet-like cells introduced with the Mycl gene. Furthermore, the present invention relates to a method for inducing proliferation-capable islet precursor-like cells by transient expression of the Mycl gene and inducing their differentiation into insulin-producing cells. [Background technology]

[0002] It has been shown that repeated short-term expression of a cellular reprogramming factor in a mouse individual leads to proliferation of pancreatic islet cells and improvement of glucose tolerance (Non-Patent Document 1).

[0003] Pancreatic islet transplantation is a medical treatment that makes it possible to stabilize blood sugar levels by transplanting pancreatic islet tissue for diabetes, but the shortage of donors is a serious problem, especially in Japan, and there is an urgent need to develop methods for regenerating pancreatic islet function using various approaches. In recent years, the establishment of islet-like insulin-producing cells by inducing differentiation of ES / iPS cells has been reported, but the technology for generating functional and large-scale islet insulin-positive cells has not yet been established, and there are concerns about problems such as rejection and autoimmune reactions. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Cell, 2016, 167, 1719-1733, e12. doi: 10 / 1016 / j. cell. 2016. 11. 052 Summary of the Invention [Problem to be solved by the invention]

[0005] It is expected that the proliferation technique of islet insulin-producing cells by transient expression of Mycl gene will enable the establishment of functional and large amounts of islet insulin-producing cells from islets derived from a small number of donors. Furthermore, if this technique is applied to the process of inducing islet insulin cells from ES / iPS cells, it is expected that insulin-positive cells can be efficiently produced from stem cells. The present invention aims to provide a means for treating diabetes, which includes removing islet cells from a subject, inducing islet progenitor-like cells capable of proliferation in vitro, and then returning the islet cells to the subject. Furthermore, the present invention aims to provide a means for treating diabetes by transiently overexpressing the Mycl gene in islet cells in the body. [Means for solving the problem]

[0006] The present inventors have found that proliferation-capable islet precursor-like cells can be induced by introducing the Mycl gene into islet cells and expressing the gene. In the presence of the Mycl gene, the proliferating cells express somatostatin while expressing the Fev gene, Pax4 gene, and Cck gene, which are expressed in fetal islet precursor cells, and proliferate. However, when the expression of the Mycl gene is stopped, the cells differentiate into insulin-positive cells as the proliferation stops. That is, the inventors have found that proliferation-capable islet precursor-like cells can be induced by transiently inducing the expression of the Mycl gene, and the proliferated islet precursor-like cells can be differentiated into insulin-positive cells, thereby completing the present invention.

[0007] That is, the present invention has the following aspects. [1] An insulin production-promoting agent comprising a MycI gene or a gene product thereof. [2] 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 having an activity of promoting insulin production when expression of the Mycl gene is induced. The insulin production promoter according to [1], [3] The Mycl gene product (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 an effect of proliferating pancreatic islet-like cells and / or an activity of promoting insulin production. The insulin production promoter according to [1], [4] An insulin production enhancer, in which the Mycl gene or its gene product is introduced into pancreatic islet cells. [5] The insulin production enhancer according to any one of [1] to [4], wherein the Mycl gene is transiently expressed. [6] The insulin production promoter described in [4] or [5], wherein the pancreatic islet cells are primary islet cells isolated from the pancreas, cultured islet cells, or derived from stem cells. [7] The insulin production promoter described in [6], wherein the stem cells are selected from the group consisting of iPS cells, ES cells, and somatic stem cells. [8] A pharmaceutical composition for preventing and / or treating diabetes and related diseases, comprising: (i) Mycl gene or its gene product; a vector incorporating a Mycl gene; and / or a pancreatic islet cell into which a Mycl gene or its gene product has been introduced or into which expression has been induced; and (ii) The pharmaceutical composition, comprising a pharma- ceutically acceptable excipient, diluent or carrier. [9] The pharmaceutical composition according to [8], wherein the diabetes and diseases related thereto are selected from diseases, disorders or symptoms related to type I diabetes, type II diabetes, impaired glucose tolerance, hyperglycemia, dyslipidemia, obesity or metabolic syndrome.

[10] 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 having an activity of promoting insulin production when expression of the Mycl gene is induced. The pharmaceutical composition according to any one of [8] and [9],

[11] 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 3, and having the effect of proliferating pancreatic islet-like cells and / or the effect of promoting insulin production. The pharmaceutical composition according to any one of [8] and [9],

[12] A kit comprising the insulin production enhancer according to any one of [1] to [7], or the pharmaceutical composition according to any one of [8] to

[11] .

[13] A kit further comprising an activator for activating the Mycl gene.

[14] The kit described in

[13] , wherein the activator for activating the Mycl gene is selected from the group consisting of a promoter, an enhancer, an enzyme or factor that activates a promoter, an enzyme or factor that activates an enhancer, a nucleic acid protein complex, and a low molecular weight compound.

[15] A pancreatic islet cell into which the Mycl gene or its gene product has been introduced.

[16] The islet-like cells according to

[15] , wherein the islet cells are derived from primary islet cells isolated from the pancreas, cultured islet cells, or stem cells.

[17] A method for preparing pancreatic islet-like cells according to

[16] , comprising: (a) incorporating the Mycl gene into a recombinant plasmid, recombinant viral vector, minicircle, or episomal vector; and (b) introducing the recombinant plasmid, recombinant viral vector, minicircle, or episomal vector obtained in step (a) into pancreatic islet cells; The method includes:

[18] A method for preparing the islet-like cells according to

[16] , comprising the step of introducing RNA encoding the Mycl gene or Mycl protein into islet cells.

[19] A method for expanding islet-like cells according to

[15] or

[16] , or islet-like cells prepared by the method according to

[17] or

[18] , comprising a step of expressing the Mycl gene.

[20] The method according to

[19] , wherein expression of the Mycl gene is transient. Effect of the Invention

[0008] According to the present invention, diabetes or related diseases in which insulin production is desired can be treated by controlling the expression of exogenous or endogenous Mycl genes. [Brief description of the drawings]

[0009] [Figure 1] Establishment of mouse ES cells inducible for Mycl expression. [Diagram 2] 1 shows an increase in pancreatic islets due to Mycl overexpression. [Diagram 3] 1 shows proliferation of somatostatin-positive pancreatic islet-like cells due to Mycl overexpression. [Figure 4] 1 shows proliferation of somatostatin-positive pancreatic islet-like cells due to Mycl overexpression. [Diagram 5] 1 shows proliferation of somatostatin-positive pancreatic islet-like cells due to Mycl overexpression. [Figure 6] The somatostatin-positive islet-like cells proliferated by Mycl overexpression resemble fetal islet progenitor cells. [Figure 7] 1 shows the induction of proliferation of islet progenitor-like cells by Mycl overexpression and the arrest of proliferation by halting Mycl overexpression. [Figure 8] 1 shows differentiation of islet progenitor-like cells into insulin-positive cells by silencing Mycl expression. [Figure 9] 1 shows that transient overexpression of Mycl enhances glucose tolerance. [Figure 10] 1 shows induction of Mycl expression in isolated pancreatic islets in vitro and islet expansion. [Figure 11] 1 shows induction of Mycl expression in cells dispersed from isolated pancreatic islets and proliferation of pancreatic islet cells in vitro. [Figure 12] Establishment of mouse ES cells capable of inducing c-Myc gene or Mycn gene expression is shown. [Figure 13] 1 shows cell death due to c-Myc or Mycn gene expression in isolated pancreatic islets in vitro. [Figure 14] The therapeutic effect in a diabetic mouse model is shown. [Figure 15] This indicates that Mycl does not induce abnormal proliferation in any tissue other than pancreatic islets. [Figure 16] 1 shows islet proliferation in aged mice. [Figure 17] 1 shows expression of Mcyl in human islet progenitor cells. [Figure 18] This shows a diagram confirming pancreatic islet proliferation in humans. [Figure 19] This shows the results of confirming pancreatic islet proliferation in humans by single cell analysis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention relates to an insulin production enhancer comprising a Mycl gene or a gene product thereof, or a pancreatic islet-like cell into which the Mycl gene has been introduced. The present invention also relates to a pharmaceutical composition for preventing / treating diabetes, comprising the insulin production enhancer. The present invention is described in detail below.

[0011] 1. Pancreatic islet-like cells and their preparation method (1) Pancreatic islet-like cells As used herein, the term "islet-like cells" refers to islet cells into which Mycl gene or its gene product has been introduced. In this specification, islet-like cells that have been forced to express Mycl gene and have entered the proliferation phase may be referred to as "islet precursor cell-like cells". Furthermore, by stopping the expression of the gene, the cells can be differentiated into 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 cells that produce insulin. Here, when focusing on the markers expressed in each cell, the "islet precursor cell-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. In addition, 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.

[0012] (2) Pancreatic islet cells "Pancreatic islet cells" generally refer to endocrine cells that are a cell aggregate called a pancreatic islet, also known as an islet of Langerhans, and that control the endocrine function of the pancreas, and account for approximately 1-2% of the total cells in the pancreas. 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 cell that constitutes the cell aggregate is the β cell, which occupies the center of the pancreas. β cells account for approximately 60-80% of the cell aggregate and secrete insulin, which allows glucose to be transferred to most cells in the body. On the other hand, α cells account for approximately 10-30% of the pancreatic islet and secrete glucagon, which is released during starvation and allows glucose to be released from the liver to maintain normal blood sugar. δ cells account for approximately 5-10% of the pancreatic islet cells and secrete somatostatin, which further regulates glucose concentration. In addition, ε cells 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.

[0013] 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.

[0014] In type I diabetes, cellular infiltration, mainly composed of lymphocytes, is observed soon after the onset of the disease. Eventually, as beta cells are selectively lost, the volume of the pancreatic islets decreases and alpha cells become predominant. The islets have a reserve capacity for insulin secretion, and type I diabetes develops when 90-95% of the beta cells have been lost. In type II diabetes, morphological changes are generally not observed in the islets, but the insulin secretion ability of the islets decreases.

[0015] 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 a 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, an immunosuppressant can be administered to the patient as appropriate. In addition, for transplantation into a patient, the above-mentioned islet-like cells, islet precursor cell-like cells, or cells capable of producing insulin, or any combination thereof, can be used.

[0016] 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 not only pancreatic islets containing a large number of normal β cells, but also pancreatic 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 from which β cells have been lost, by using islet-like cells or the like introduced with the Mycl gene.

[0017] (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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] (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.

[0027] 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, 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 proliferate due to the expression of the Mycl gene and have the effect of promoting insulin production due to the cessation of the expression.

[0028] 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 nucleotide sequence represented by SEQ ID NO: 1 or 3 and encodes a polypeptide having an activity of proliferating pancreatic islet-like cells into which a Mycl gene has been introduced; 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 proliferating insulin-producing cells and thus promoting insulin production as a result of proliferation of pancreatic islet-like cells into which a Mycl gene has been introduced.

[0029] 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.

[0030] 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).

[0031] 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 effect of proliferating pancreatic islet-like cells and / or the effect of promoting insulin production.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] (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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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).

[0042] 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.

[0043] As an alternative means of on / off control of Mycl gene expression, a "photoregulated viral vector" (Tahara, M., et al., PNAS, vol.116, 11587-11589, 2019) can be used, which allows precise on / off switching of viral vector gene expression and cell proliferation 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 a target gene incorporated in the vector can be controlled using blue light.

[0044] 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.

[0045] According to the present invention, the object of the present invention can be achieved by forcibly expressing the endogenous Mycl gene in cells, in addition to introducing an isolated exogenous Mycl gene. The means for expressing the 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 the expression of the Mycl gene. Examples of promoters used for replacement include the cytometry virus (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.

[0046] (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.

[0047] (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).

[0048] 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.

[0049] 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)).

[0050] 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.

[0051] 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.

[0052] 3. Method for producing pancreatic islet cells or their precursor cells having insulin-producing ability 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.

[0053] 4.Medicinal Use According to the present invention, it is possible to proliferate islet-like cells and promote insulin production by controlling the expression of the introduced Mycl gene. Thus, in one aspect, there are provided an islet cell expansion promoter, an islet function improving agent, and an insulin production promoter, each of which contains the Mycl gene or a gene product thereof as an active ingredient; a pharmaceutical composition containing the active ingredient and other pharma- ceutical acceptable ingredients (e.g., a carrier, an excipient, a disintegrant, a buffer, an emulsifier, a suspending agent, a soothing agent, a stabilizer, a preservative, a physiological saline solution, etc.); a method for preventing and / or treating diabetes patients using the islet cell expansion promoter, islet function improving agent, insulin production promoter, or pharmaceutical composition; and use of the Mycl gene for producing the islet cell expansion promoter, islet function improving agent, insulin production promoter, or pharmaceutical composition. In another aspect, there are provided a method for administering the Mycl gene or its gene product to a subject (in vivo method); and a method for transplanting into a subject pancreatic islet-like cells, pancreatic islet precursor cell-like cells, or insulin-producing cells (hereinafter sometimes simply referred to as "pancreatic islet-like cells, etc.") into which the Mycl gene or its gene product has been introduced in vitro, or any combination thereof (ex vivo method), for the purpose of preventing and / or treating diabetes.

[0054] The ex vivo method of the present invention contemplates different embodiments depending on the cells used. For example, there are (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 (the ex vivo method in the narrow sense includes both); (ii) an embodiment in which islet-like cells obtained by inducing differentiation, that is, islet-like cells obtained by introducing the Mycl gene of islet cells derived from stem cells (iPS cells, ES cells, somatic stem cells, etc.), are produced allogeneically in vitro or autologously in vitro (the ex vivo method in the broad sense); (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 embodiment in which the cells are insulin-producing cells or islet cells (not containing the Mycl gene) that are produced by differentiation from the above (i) to (iii).

[0055] 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.

[0056] In one embodiment of the present specification, the isolated islets can be appropriately gene-edited. The gene editing is not particularly limited, but specifically, it can add preferable characteristics for treatment, such as correction of mutated genes contained in the 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 gene editing method is not particularly limited, but specific examples include CRISPR-Cas9, TALEN, ZFN, CRISPR-Cas3, CRISPR-TypeI-D, and modified versions thereof, and transposon vectors such as piggyBAC for expressing functional molecules.

[0057] (1) Indications The diseases to which the Mycl gene or its gene product can be applied based on the above-mentioned embodiment are diseases in which insulin does not function sufficiently in the living body (typically, insulin resistance, decreased insulin secretion). According to the present invention, the Mycl gene or its gene product can promote insulin production, and has, for example, an 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 diseases caused by 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, exocrine pancreatic diseases, endocrine diseases, liver diseases, diseases caused by drugs or chemicals, infectious diseases, rare pathologies caused by immune mechanisms, or gestational diabetes. Furthermore, diabetic complications caused by diabetes (for example, diabetic retinopathy, diabetic neuropathy, etc.) can also be included in the applicable disease. In addition, insulin secretion deficiency resulting from total or partial pancreatectomy associated with pancreatitis or pancreatic cancer can also be included in the applicable diseases.

[0058] 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 method described in the known literature here is not limited, but specifically includes, 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, etc. Alternatively, instead of the cell transplantation described in the known literature, a method (in vivo method) can be selected in which the Mycl gene or its gene product is administered to a subject, which can expand islet cells, islet-like cells, islet precursor-like cells, or insulin-producing cells to the same extent as, or preferably more than, the amount of transplanted islet cells described in the known literature.

[0059] 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 amount of islet cells, islet-like cells, islet precursor-like cells, or insulin-producing cells is equivalent to 500 IEQ / kg or more, preferably 1000 IEQ / kg or more, more preferably 2000 IEQ / kg or more, and even more preferably 5000 IEQ / kg or more. Alternatively, a method (in vivo method) of administering a Mycl gene or its gene product to a subject that can expand the amount of islet-like cells, islet precursor-like cells, or insulin-producing cells to the same extent can be selected.

[0060] 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 amount of islet cells, islet-like cells, islet precursor-like cells, or insulin-producing cells is equivalent to 500 IEQ / kg or more, preferably 1000 IEQ / kg or more, more preferably 2000 IEQ / kg or more, and even more preferably 5000 IEQ / kg or more. Alternatively, a method (in vivo method) of administering a Mycl gene or its gene product to a subject that can expand the amount of islet-like cells, islet precursor-like cells, or insulin-producing cells to the same extent can be selected.

[0061] 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.

[0062] 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.

[0063] 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 / manifestation of the above-mentioned diseases or symptoms thereof, or reducing the risk of onset / manifestation. "Treatment" includes alleviating (alleviating) symptoms characteristic of the target disease or associated symptoms, preventing or delaying the worsening of symptoms, and the improvement of the disease.

[0064] (2) Insulin production enhancer and pharmaceutical composition The Mycl gene or its gene product of the present invention can be provided as an agent for promoting proliferation of islet cells, an agent for improving islet function, an agent for promoting insulin production, or a pharmaceutical composition for the prevention and treatment of the above-mentioned applicable diseases. Here, one embodiment of the agent for promoting proliferation of islet cells refers to an agent for proliferating any one of α cells, β cells, δ cells, ε cells, and PP cells contained in a pancreatic islet, and preferably any two or more of these islet cells. In addition, one embodiment of the agent for improving islet function refers to an agent for improving a part or all of the functions of a pancreatic islet in a living body by administration. Part of the functions of a pancreatic islet specifically include, for example, 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.

[0065] 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.

[0066] The cell preparation 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.

[0067] In addition, when using pancreatic islet cells, pancreatic islet-like cells, pancreatic islet precursor-like cells, or insulin-producing cells in cell preparations and pharmaceutical compositions, dimethyl sulfoxide (DMSO), serum albumin, etc. may be contained in the cell preparations and pharmaceutical compositions to protect the cells, and antibiotics, etc. may be contained in the cell preparations and pharmaceutical compositions to prevent bacterial contamination and proliferation. 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 cell preparations and pharmaceutical compositions. Those skilled in the art can add these factors and drugs to the cell preparations and pharmaceutical compositions at appropriate concentrations.

[0068] The number of islet cells, islet-like cells, islet precursor-like cells, or insulin-producing cells contained in the cell preparations and pharmaceutical compositions 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 levels) in the prevention and / or treatment of diabetes and related diseases.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] (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 Mycl gene or its gene product, pancreatic islet-like cells, etc., or any combination thereof. Furthermore, according to the present invention, in the above-mentioned treatment method, an activator for activating Mycl gene can be administered, and may be administered before, simultaneously with, or after administration of an insulin production promoter or a pharmaceutical composition.

[0073] (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

[0074] 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.

[0075] method (i) Establishment of ES cells capable of inducing the expression of Myc, Mycn, and Mycl in a Dox-dependent manner Myc, Mycn, and Mycl cDNAs were cloned from ES cell-derived cDNA, and the cloned fragments were 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 LR reaction between the pCR8-Mycl-TOPO vector containing each Myc gene and the TetOP-AttR1-ccdB-AttR2-ires-mCherry vector, and 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 into which each Myc gene was 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 ES cell lines capable of inducing the expression of each Myc gene in a Dox-dependent manner.

[0076] (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).

[0077] 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.

[0078] (iii) Generation of mice capable of inducible Mycl expression in vivo ES cells capable of inducing Mycl expression in a doxycycline (Sigma-Aldrich, hereafter sometimes referred to as "Dox")-dependent manner were injected into mouse blastocysts (ICR, E3.5) and transplanted into the uterus of pseudopregnant mice (Slc:ICR, Shimizu Experimental Materials) on day 2 of pregnancy to produce chimeric mice carrying cells capable of inducing Mycl expression in a Dox-dependent manner.

[0079] (iv) 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.

[0080] (v) 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.

[0081] (vi) 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.

[0082] <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)

[0083] (vii) 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.

[0084] (viii) 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:

[0085] [Table 1]

[0086] (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)

[0087] (ix) Pancreatic islet isolation Eight-week-old 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 Collagenase P (Roche) (2 mg / mL) was injected. The pancreas was then removed and digested in a 37°C water bath for 11 minutes and 30 seconds. The pancreas was suspended in 25 mL of M199 medium containing 10% FBS, and centrifuged twice (1000 rpm, 4°C, 2 minutes). The supernatant was discarded and suspended in 10 mL of Histopaque (SIGMA), after which 10 mL of M199 medium containing 10% FBS was poured on top, and centrifuged (1000 rpm, 4°C, 30 minutes). The supernatant was transferred to another 50 mL tube, and 25 mL of M199 medium containing 10% FBS was added, followed by centrifugation (1000 rpm, 4° C., 2 minutes).

[0088] (ix) 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.

[0089] [Table 2]

[0090] [Table 3]

[0091] (x) Pancreatic islet culture (three-dimensional culture method using gel) 20uL of Matrigel (Corning) containing isolated islets was added to a 96-well plate (Corning), and the Matrigel was gelled by incubating at 37℃ for 15 minutes. Then, 140μL of medium containing growth factors (see table below) was added, and the plate was cultured at 37℃ with 5% CO2.

[0092] [Table 4]

[0093] (xi) Pancreatic islet culture (suspension culture) 2 mL of medium was added to a 6-well plate, and a cell culture insert (Millipore) was suspended on the medium. The isolated pancreatic islets and 20 μL of medium were added to the cell culture insert, and cultured at 37° C. and 5% CO2.

[0094] (xii) 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).

[0095] (xiii) Generation of diabetic mice The weight of 8-12 week old immunodeficient mice (NOD / SCID) was measured, and a streptozocin (STZ) solution (Sigma-Aldrich) was injected intraperitoneally to the mice at 150 mg / kg. After one week, blood glucose levels were measured using an Antsense stand (Horiba), and mice with a casual blood glucose level of 250 mg / dL or higher were used as diabetic mice.

[0096] (xiv) Pancreatic islet transplantation (transplantation under the kidney capsule) Diabetic mice were anesthetized by intraperitoneal injection of Somnopentyl (Kyoritsu Seiyaku Co., Ltd.). After laparotomy, the renal capsule was incised using a syringe. A silicon tube was inserted under the renal capsule, and pancreatic islets were transplanted under the renal capsule using a Hamilton syringe. After transplantation, the silicon tube was removed, and the peritoneum and capsule were sutured.

[0097] (xv) Nephrectomy Mice with transplanted islets were anesthetized by intraperitoneal injection of Somnopentyl (Kyoritsu Seiyaku Co., Ltd.). After laparotomy, the kidney with transplanted islets was removed and the renal vein and renal artery were ligated with a ligature. After removal of the kidney, the peritoneum and capsule were sutured.

[0098] (xvi) Single-cell analysis Reanalysis was performed using the analysis data published in the GEO database (GSE101099; Byrnes LE et al., Nat Commun. 2018 Sep 25; 9(1): 3922). Seurat v2.2 and v2.3 were used for t-SNE analysis (Satija R., et al., Nat Biotechnol. 2015 33: 495-502).

[0099] Example 1: Establishment of mouse ES cells capable of inducing Mycl expression We investigated whether the ES cell line with the Mycl gene incorporated therein prepared above can express in a Dox-dependent manner. The expression of the Mycl gene can be confirmed by the expression of the mCherry gene incorporated downstream of the Mycl gene. The mCherry gene is a gene that encodes a red fluorescent protein, and the expression of the gene causes the cells to develop a red color. As shown in FIG. 1, when comparing the gene expression before and after the addition of Dox, the Mycl gene expression was significantly increased, indicating that mouse ES cells capable of inducing Mycl expression had been established.

[0100] We also investigated whether gene expression was induced in a Dox-dependent manner in ES cell lines incorporating the Myc gene and the Mycn gene, and as shown in Figure 12, gene expression was observed in both cases, similar to the induction of Mycl expression.

[0101] On the other hand, when the expression of Ki67 protein was observed as an indicator of cell proliferation, the number of proliferating cells (Ki67-positive cells) increased with the duration of Dox administration, and two weeks after Dox administration was stopped, the number of Ki67-positive cells decreased significantly and cell proliferation ceased (Figure 7).The above results demonstrated that ES cells into which the Mycl gene was introduced had gene expression induced by Dox, resulting in cell proliferation.

[0102] Example 2: Proliferation of pancreatic islet cells by overexpression of Mycl (formation of neuroendocrine tumors) Dox was administered for 8 weeks to the 8-week-old chimeric mice expressing the Mycl gene prepared above. Eight weeks after Dox administration, the mice were sacrificed, the pancreas was removed, and the pancreatic sections were histochemically evaluated. As shown by the arrows in Figure 2, in the pancreatic tissue in which Mycl was overexpressed by induction with Dox, islet cells proliferated and the islets were enlarged compared to normal pancreatic tissue.

[0103] Example 3: Expansion of islet progenitor-like cells by overexpression of Mycl Four weeks after the administration of Dox to the chimeric mice, the proliferation of somatostatin-positive cells was examined. As shown in Example 2, the proliferation of pancreatic islet cells can be induced by expressing the Mycl gene in pancreatic islet cells. As shown in Figures 3 to 6, in the presence of the Mycl gene, the proliferating cells showed gene expression similar to that of pancreatic islet precursor cells, and at the same time, they expressed somatostatin and proliferated, but in contrast, the number of insulin-positive cells decreased.

[0104] Example 4: Overexpression of Mycl induces proliferation of somatostatin-positive islet-like cells The proliferation of somatostatin-positive cells and insulin-positive cells was examined 8 weeks after Dox administration in the same experimental system as in Example 2. As shown in Figures 4 and 5, the number of insulin-positive cells decreased, and the number of somatostatin-positive cells increased significantly.

[0105] Example 5: Dedifferentiation into pancreatic islet progenitor-like cells by Mycl overexpression In the same experimental system as in Example 2, gene expression analysis was performed in hypertrophied islets 8 weeks after Dox administration. It is believed that islets differentiate from islet progenitor cells markered by Ngn3 to islet progenitor cells markered by Fev, and then to each type of islet cell (Figure 6). When gene expression was analyzed in islet cells proliferated by overexpression of Mycl, increased expression of the progenitor cell marker Fev, as well as Pax4 and Cck, which are expressed at the same time, was observed (Figure 13). This result suggested that overexpression of Mycl could induce islet progenitor-like cells capable of proliferation.

[0106] Example 6: Induction of insulin-positive cells by silencing Mycl expression In the experimental system used in Example 2, Dox administration was stopped after 8 weeks, and the increase or decrease in insulin-positive cells and somatostatin-positive cells was observed 2 and 4 weeks after the cessation of Dox administration. As shown in Figure 5, 2 and 4 weeks after the cessation of Dox administration, the insulin-positive cells, which had decreased once, increased, and in contrast, the somatostatin-positive cells, which had increased once, decreased (Figure 8). This result suggests that the islet progenitor-like cells that proliferated due to Mycl overexpression were changed to insulin-positive cells by cessation of Mycl expression.

[0107] In addition, when the expression of Ki67, which is known to be a target protein for cell proliferation, was observed, the number of proliferating cells (Ki-positive cells) increased with increasing duration of Dox administration, and two weeks after cessation of Dox administration, the number of Ki-positive cells significantly decreased and cell proliferation ceased (Figure 7).

[0108] Example 7: Enhancement of glucose tolerance by transient overexpression of Mycl Glucose tolerance was evaluated in chimeric mice when Dox administration was stopped. As shown in Figure 9 (left), glucose tolerance was improved in mice in which Dox administration was stopped, compared to the control ("cont."). Furthermore, fasting blood glucose levels were consistently maintained, suggesting that the expanded islets in vivo were functional islets (Figure 9 (right)).

[0109] Example 8: Overexpression of Mycl induces proliferation of pancreatic islet cells in vitro (1) Induction of Mycl expression in isolated pancreatic islets The pancreas was removed from the chimeric mice, and the induction of Mycl expression was examined in the isolated islets. The start of Dox addition was set as day 0, and Mycl expression was observed over time. The results are shown in Figure 10. Compared to the control, in the Dox-added system, there was a tendency for the number of islets to increase over time, along with the expression of the Mycl gene.

[0110] (2) Induction of Mycl expression in cells dispersed from isolated pancreatic islets The expression of the Mycl gene was examined in cells dispersed from isolated islets in the same manner as in (1) above. When the Mycl gene was observed in cells 14 days after Dox addition, it was found that the Mycl gene was expressed in each cell compared to the cells on the 1st day after addition (Figure 11).

[0111] (3) Induction of c-Myc and Mycn expression in isolated pancreatic islets in vitro It was confirmed that the ES cells prepared above incorporating c-Myc and Mycn could be induced to express in a doxycycline-dependent manner. Chimeric mice were produced using these ES cells. In pancreatic islet cells isolated from the chimeric mice produced, the expression of c-Myc and Mycn was induced in vitro by adding doxycycline for one week. As a result, it was found that induction of the expression of c-Myc and Mycn promoted proliferation, but induced cell death, unlike Mycl (Figure 12) (see Pelengaris S, Khan M, Evan GI., Cell 2002; 109(3):321-334).

[0112] Example 9: Functional evaluation of pancreatic islet cells induced to proliferate in vitro The functionality of the expanded islet cells was examined by transplanting them into diabetic mice after in vitro induction of proliferation by overexpressing Mycl. Thirty isolated islets were dispersed and proliferation was induced by adding Dox for one week. The islet cells were collected and transplanted under the renal capsule of diabetic mice, and blood glucose improved over time. Furthermore, when the kidney into which the islet cells were transplanted was removed two weeks later, the diabetic mice reverted to hyperglycemia. Generally, more than 300 islet transplants are required to lower blood glucose levels in diabetic mice. We succeeded in lowering blood glucose levels in diabetic mice using islet-like cells expanded from 60 islets.

[0113] Example 10: Verification of the therapeutic effect of Mycl expression induction in a diabetic mouse model A diabetic mouse model was created to examine the therapeutic effect of inducing Mycl expression. A diabetic mouse model was created by intraperitoneally administering 150 mg / kg of streptozocin (STZ), which is toxic to pancreatic β cells, to 8-week-old mice (KH2-Mycl) capable of inducing Mycl expression. Two weeks later, Mycl expression was induced by Dox administration for eight weeks. Mycl expression was stopped by ceasing Dox administration for another two weeks. At this point, an intraperitoneal glucose tolerance test (IPGTT) and histological analysis were performed.

[0114] The results are shown in Figure 14. In the control group (STZ alone), blood glucose increased after STZ administration, whereas in the group in which Mycl expression was induced with Dox (STZ+Mycl), blood glucose decreased after Dox administration and remained normal even after Dox administration was stopped (Figure 14, top left). Furthermore, a glucose response test showed a rapid decrease in blood glucose levels in the group in which Mycl expression was induced (Figure 14, bottom left).

[0115] Furthermore, histological analysis of the expression of insulin (Ins), glucagon (Gog), and somatostatin (Sst) showed that the percentage of insulin-positive cells was reduced in the control group (STZ alone was administered). On the other hand, in the group in which Mycl expression was induced (STZ+Mycl), an increase in pancreatic islet cells and the percentage of insulin-producing cells was observed. Based on these results, it is expected that induction of Mycl expression will have a therapeutic effect on diabetes.

[0116] Example 11: Mycl does not induce abnormal proliferation of other organs than pancreatic islets In order to induce the expression of the MYC family genes ("Myc", "Mycn", and "Mycl") in vivo in a Dox-dependent manner, we first established ES cells capable of inducing the expression of these genes (Figure 15, top). We produced chimeric mice by injecting these ES cells into blastocysts. When these chimeric mice reached 4 weeks of age, we added Dox for 4 weeks to induce the expression of the MYC family genes.

[0117] Induction of Myc expression induced tumor formation in the liver, and induction of Mycn expression induced tumor formation in the liver and intestine, whereas induction of Mycl expression did not result in abnormal proliferation, at least in the liver and small intestine (Fig. 15, bottom).This suggests that Mycl proliferates specifically in pancreatic islets.

[0118] Example 12: Pancreatic islet proliferation in aged mice Pancreatic islet cells were isolated from aged mice (115 weeks old) and infected with lentivirus capable of inducing the expression of Mycl and pZsGreenDR vector (Takara, cat#632428) (control group) in a Dox-dependent manner. The zsGreenDR used is a short-lived green fluorescent protein in which a proteolytic signal is fused to the C-terminus of zsGreen.

[0119] The day after infection, the islet cells were transferred to three-dimensional culture using Matrigel, and at the same time, Dox was administered to induce the expression of Mycl and zsGreenDR. The islet cells in which zsGreenDR expression was induced showed no change in morphology even after 7 days (Figure 16, right). On the other hand, induction of Mycl expression led to an increase in islet cells (Figure 16, left). These results demonstrated that islet proliferation by induction of Mycl expression is possible even in very old mice.

[0120] Example 13: Expression of Mcyl in human islet progenitor cells In a paper submitted in 2020 (JR Alvarez-Dominguez, et al., Circadian Entrainment Triggers Maturation of Human In Vitro Islets., Cell Stem Cell, 26(1), 108-122, 2020), we observed the molecular profile when human iPS cells were gradually induced to differentiate into human pancreatic beta cells. Therefore, using this dataset, we observed gene expression of the MYC family (mentioned above) and H3K27ac (a chromatin mark that generally indicates transcriptional activation) (Figure 17).

[0121] The expression of Myc family genes was high in human pluripotent stem cells (hPSCs) and tended to decrease with differentiation, but Mycl expression was transiently increased in endocrine precursor cells (ENs), suggesting that Mycl may contribute to the proliferation of islet cells in human islet cells as well as in mice.

[0122] Example 14: Confirmation of pancreatic islet proliferation in humans Human islet cells were infected with lentivirus capable of inducing the expression of Mycl and zsGreenDR (control group) in a Dox-dependent manner. The next day, the islet cells were transferred to three-dimensional culture using Matrigel and simultaneously administered Dox to induce the expression of Mycl and zsGreenDR, respectively.

[0123] Although no change in morphology was observed in islet cells in which zsGreenDR expression was induced even after 7 days, an increase in islet cells was observed when MYCL expression was induced (Figure 18, bottom left). Furthermore, immunostaining revealed that Ki67 (a cell proliferation marker) was detected in cells in which mCherry expression was confirmed (equivalent to MYCL expression) in the MYCL overexpression group (Figure 18, bottom right).

[0124] Example 15: Confirmation of pancreatic islet proliferation in humans by single cell analysis Human pancreatic islet cells were infected with lentiviruses capable of inducing the expression of Mycl, MYCL, and zsGreenDR (control group) in a Dox-dependent manner. The next day, the cells were transferred to suspension culture using cell culture inserts, and the expression of Mycl and zsGreenDR was induced by administering Dox at the same time. After 7 days, dead cells were removed, and single-cell RNA-seq was performed according to standard methods.

[0125] As a result of single-cell RNA-seq, α cells, β cells, δ cells, pancreatic duct cells ("PP"), mesenchymal cells, vascular endothelial cells, and stellate cells were detected (Figure 19, left). Of these, we focused only on β cells and analyzed the expression of CDK4, a marker of cell proliferation. As a result, induction of zsGreenDR expression did not affect CDK4, whereas induction of Mycl and MYCL expression induced CDK4 expression. This demonstrated that induction of Mycl expression can also lead to cell proliferation in human islet cells. [Industrial Applicability]

[0126] If the technology of the present invention is applied to islets isolated from a donor, it is possible to increase the number of islet insulin-positive cells in a test tube, and the shortage of islet donors in the present invention may be resolved. By using this technology in a differentiation induction system for islet insulin-positive cells from ES / iPS cells, it is possible to efficiently induce differentiation of islet insulin-positive cells from pluripotent stem cells. Furthermore, by applying this technology to a technology for producing islets from pluripotent stem cells by blastocyst complementation, it is possible to increase the number of insulin-positive cells repopulating in the produced organ. Although the present invention requires transient expression of Mycl in donor-derived islet cells or pluripotent stem cell-derived islet cells, it is considered possible to establish a large number of islet cells without genetic modification by introducing episomal vectors, RNA, etc. In addition, it is expected that the islet cells grown using this technology can be grown and maintained as an islet stock cell line. It is possible that insulin-positive cells can be supplied stably and in large quantities over the long term by using islet stock cell lines. It is expected that inexpensive and high-quality islets can be provided to many diabetes patients in the future.

[0127] 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 pharmaceutical composition for preventing and / or treating diabetes and related diseases, comprising: (i) Mycl gene or its gene product; a vector incorporating the Mycl gene; and / or a pancreatic islet cell into which the Mycl gene or its gene product has been introduced or into which expression has been induced; and (ii) The pharmaceutical composition comprising a pharma- ceutically acceptable excipient, diluent or carrier.

2. 2. The pharmaceutical composition of claim 1, wherein the diabetes and related diseases are selected from type I diabetes, type II diabetes, impaired glucose tolerance, hyperglycemia, dyslipidemia, obesity or diseases, disorders or conditions related to metabolic syndrome.

3. The Mycl gene is (1) a nucleic acid comprising 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 complementary to the base sequence represented by SEQ ID NO: 1 or 3, and encodes a polypeptide having an activity of promoting insulin production when expression of the Mycl gene is induced.

3. The pharmaceutical composition according to claim 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 3, and having the effect of proliferating pancreatic islet-like cells and / or the effect of promoting insulin production.

3. The pharmaceutical composition according to claim 1 or 2, comprising:

5. A kit comprising the pharmaceutical composition according to any one of claims 1 to 4.

6. The kit of claim 5, further comprising an activator for activating the Mycl gene.

7. The kit of claim 6, wherein the activator for activating the Mycl gene is selected from the group consisting of a promoter, an enhancer, an enzyme or factor that activates a promoter, an enzyme or factor that activates an enhancer, a nucleic acid protein complex, and a small molecule compound.

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