Induction of proliferative islet precursor-like cells and differentiation of insulin-positive cells based on transient expression of Mycl

Abstract

While there have been reports of cultivating islet-like insulin-producing cells by inducing differentiation of ES / iPS cells, the technology for cultivating high-functioning, large-scale insulin-positive islet cells is not yet mature, and there are concerns about rejection and unexpected immune responses. The present invention provides islet-like cells into which the Mycl gene has been introduced, as well as methods for inducing the proliferation of islet-like cells and their differentiation into insulin-producing cells through transient expression of the Mycl gene.

Description

Technical Field

[0001] The present invention relates to an agent for promoting insulin production comprising the Mycl gene or pancreatic islet-like cells into which the Mycl gene has been introduced. Furthermore, the present invention relates to a pharmaceutical composition for preventing / treating diabetes comprising the Mycl gene or its gene product, or pancreatic islet-like cells into which the Mycl gene has been introduced. Furthermore, the present invention relates to a method for inducing proliferative pancreatic islet precursor-like cells to differentiate into insulin-producing cells by transiently expressing the Mycl gene. Background Art

[0002] Studies have shown that repeated short-term expression of cell reprogramming factors in mice leads to the proliferation of pancreatic islet cells and improved glucose tolerance (Non-Patent Document 1).

[0003] Islet transplantation is a medical treatment for diabetes that stabilizes blood sugar levels by transplanting pancreatic islet tissue. With donor shortages becoming a serious problem, particularly in Japan, there is a pressing need for the development of various methods to regenerate islet function. While recent reports have demonstrated the creation of islet-like insulin-producing cells by inducing differentiation of ES / iPS cells, the technology for functionally and efficiently cultivating large quantities of insulin-positive pancreatic islet cells remains a challenge, and concerns remain about rejection and autoimmune reactions.

[0004] [Prior art literature]

[0005] [Non-patent literature]

[0006] [Non-patent document 1] Cell, 2016, 167, 1719-1733, e12. doi: 10 / 1016 / j.cell.2016.11.052. Summary of the Invention

[0007] [Technical Problems to be Solved by the Present Invention]

[0008] The proliferation technology of pancreatic insulin-producing cells based on transient expression of the Mycl gene is expected to cultivate functional and large quantities of pancreatic insulin-producing cells from pancreatic islets from a small number of donors. Furthermore, if this technology is applied to the process of inducing pancreatic insulin cells from ES / iPS cells, it can be expected that insulin-positive cells can be efficiently prepared from stem cells. The object of the present invention is to provide a method for treating diabetes, comprising removing pancreatic islet cells from a subject and inducing pancreatic progenitor cell-like cells that can proliferate in vitro, and then returning the pancreatic islet cells to the subject. Furthermore, one object of the present invention is to provide a method for treating diabetes by transiently expressing the Mycl gene in pancreatic islet cells in vivo.

[0009] [ Technical means to solve technical problems ]

[0010] The present inventors have discovered that by introducing the Mycl gene into pancreatic islet cells and expressing it, proliferative islet precursor cell-like cells can be induced. In the presence of the Mycl gene, the proliferating cells express the Fev, Pax4, and Cck genes expressed in embryonic islet precursor cells, while also expressing somatostatin and causing them to proliferate. If Mycl gene expression is stopped, the cells differentiate into insulin-positive cells while their proliferation stops. In other words, the present inventors discovered that transient induction of Mycl gene expression can induce proliferative islet precursor cell-like cells, and that the proliferated islet precursor cell-like cells can differentiate into insulin-positive cells, thereby completing the present invention.

[0011] That is, the present invention has the following technical solutions.

[0012] [1] An insulin production promoter comprising the Mycl gene or its gene product.

[0013] [2] The insulin production promoter according to [1] above, wherein the Mycl gene comprises:

[0014] (1) a nucleic acid comprising the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3; or

[0015] (2) A nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 and encodes a polypeptide having insulin-promoting activity produced when the Mycl gene is induced for expression.

[0016] [3] The insulin production promoter according to [1] above, wherein the Mycl gene product comprises:

[0017] (1) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4; or

[0018] (2) having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, and having the effect of promoting the proliferation of pancreatic islet-like cells and / or producing a polypeptide that promotes insulin activity.

[0019] [4] An insulin production promoter, wherein the Mycl gene or its gene product is introduced into pancreatic islet cells.

[0020] [5] The insulin production promoter according to any one of [1] to [4], wherein the Mycl gene is transiently expressed.

[0021] [6] The insulin production promoter according to [4] or [5], wherein the pancreatic islet cells are derived from primary pancreatic islet cells isolated from the pancreas, cultured pancreatic islet cells, or stem cells.

[0022] [7] The insulin production promoter according to [6], wherein the stem cells are selected from iPS cells, ES cells, and adult stem cells.

[0023] [8] A pharmaceutical composition for preventing and / or treating diabetes and related diseases, comprising:

[0024] (i) Mycl gene or its gene product; a vector into which Mycl gene is integrated; and / or pancreatic islet cells into which Mycl gene or its gene product is introduced or induced to express. And

[0025] (ii) a pharmaceutically acceptable excipient, diluent or carrier.

[0026] [9] The pharmaceutical composition according to [8], wherein the diabetes and its related diseases are selected from diseases, disorders or symptoms related to type I diabetes, type II diabetes, impaired glucose tolerance, hyperglycemia, dyslipidemia, obesity or metabolic syndrome.

[0027]

[10] The pharmaceutical composition according to [8] or [9], wherein the Mycl gene comprises:

[0028] (1) a nucleic acid comprising the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3; or

[0029] (2) A nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 and encodes a polypeptide having insulin-promoting activity produced when the Mycl gene is induced for expression.

[0030]

[11] The pharmaceutical composition according to [8] or [9], wherein the Mycl gene product comprises:

[0031] (1) a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4; or

[0032] (2) A polypeptide having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 3, and having an effect of promoting pancreatic islet-like cell proliferation and / or an effect of promoting insulin production.

[0033]

[12] A kit comprising the insulin production promoter described in any one of [1] to [7] or the pharmaceutical composition described in any one of [8] to

[11] .

[0034]

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

[0035]

[14] The kit according to

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

[0036]

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

[0037]

[16] The pancreatic islet cells according to

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

[0038]

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

[16] , comprising:

[0039] (a) integrating the Mycl gene into a recombinant plasmid, a recombinant viral vector, a minicircle or an episomal vector; and

[0040] (b) a step of introducing the recombinant plasmid, recombinant viral vector, minicircle or episomal vector obtained in step (a) into pancreatic islet cells.

[0041]

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

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

[0042]

[19] A method for proliferating pancreatic islet cells, wherein the method includes the step of expressing the Mycl gene, and the pancreatic islet cells are the pancreatic islet cells described in

[15] or

[16] or the pancreatic islet cells prepared by the method described in

[17] or

[18] .

[0043]

[20] The method according to

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

[0044] Effects of the Invention

[0045] According to the present invention, diabetes or related diseases requiring insulin production can be treated by controlling the expression of exogenous or endogenous Mycl gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This indicates the cultivation of mouse ES cells in which Mycl expression can be induced.

[0047] Figure 2 Indicates pancreatic islet enlargement based on Mycl overexpression.

[0048] Figure 3 Represents the proliferation of somatostatin-positive islet-like cells based on Mycl overexpression.

[0049] Figure 4 Represents the proliferation of somatostatin-positive islet-like cells based on Mycl overexpression.

[0050] Figure 5 Represents the proliferation of somatostatin-positive islet-like cells based on Mycl overexpression.

[0051] Figure 6 This indicates that somatostatin-positive islet-like cells that proliferate based on Mycl overexpression are similar to embryonic islet progenitor cells.

[0052] Figure 7 The figure shows the induction of proliferation of islet precursor cell-like cells upon Mycl overexpression and the arrest of proliferation upon Mycl overexpression inhibition.

[0053] Figure 8 Indicates insulin-positive cell differentiation into islet precursor-like cells based on cessation of Mycl expression.

[0054] Figure 9 This indicates enhanced glucose tolerance based on transient overexpression of Mycl.

[0055] Figure 10 Shows induction of Mycl expression in isolated islets and islet enlargement in vitro.

[0056] Figure 11 The results show the induction of Mycl expression in cells dispersed from isolated pancreatic islets and the proliferation of pancreatic islet cells in vitro.

[0057] Figure 12 Indicates the cultivation of mouse ES cells capable of inducing the expression of c-Myc gene or Mycn gene.

[0058] Figure 13 The results show that expression of c-Myc or Mycn in isolated pancreatic islets in vitro leads to cell death.

[0059] Figure 14 Indicates therapeutic effect in diabetic mouse model.

[0060] Figure 15 This indicates that Mycl does not induce abnormal proliferation outside of pancreatic islets.

[0061] Figure 16Indicates pancreatic islet hyperplasia in aged mice.

[0062] Figure 17 Represents the expression of Mcyl in human pancreatic islet precursor cells.

[0063] Figure 18 Indicates confirmation of human pancreatic islet hyperplasia.

[0064] Figure 19 The results indicate that human pancreatic islet hyperplasia was confirmed by single-cell analysis. DETAILED DESCRIPTION

[0065] The present invention relates to insulin production promoters comprising the Mycl gene or its gene product, or pancreatic islet-like cells into which the Mycl gene has been introduced. The present invention also relates to pharmaceutical compositions for preventing or treating diabetes, comprising these insulin production promoters. The present invention is described in detail below.

[0066] 1. Islet-like cells and their preparation methods

[0067] (1) Islet-like cells

[0068] As used in this specification, "islet-like cells" refer to islet cells into which the Mycl gene or its gene product has been introduced. It should be noted that, in this specification, islet-like cells that are sometimes transferred to the proliferation phase by forced expression of the Mycl gene are particularly referred to as "islet precursor cell-like cells". Furthermore, by stopping the expression of this gene, it is possible to differentiate into cells with the ability to produce insulin (hereinafter also referred to as "insulin-producing cells"). That is, according to the present invention, by transient expression of the Mycl gene, the number of islet-like cells can be increased and differentiated into cells that produce insulin. Among them, when focusing on the markers expressed in each cell, "islet precursor cell-like cells" are, for example, positive for at least one, preferably two, and more preferably three or more of the genes selected from Fev, Pax4, Cck, CDK4, and Ki67. In addition, if focusing on the expressed protein, compared with the situation confirmed in normal islets, it is characterized by a reduction in the production of particularly insulin and glucagon, or a significant production of somatostatin.

[0069] (2) Pancreatic islet cells

[0070] "Islet cells," also commonly referred to as pancreatic islets, refer to the cell clusters known as islets that govern the endocrine function of the pancreas, representing approximately 1-2% of the pancreatic endocrine cells. Islet cells are primarily composed of five cell types: α cells, β cells, δ cells, ε cells, and PP cells. β cells are the primary cell type that occupies the central portion of the pancreas. β cells make up approximately 60-80% of the cell cluster and secrete insulin, which enables glucose to be transferred to most cells in the body. On the other hand, α cells, which make up approximately 10-30% of the islets, secrete glucagon, which is released during starvation and releases glucose from the liver to maintain normal blood sugar levels. δ cells, which make up approximately 5-10% of the islet cells, secrete somatostatin, which further regulates glucose levels. ε cells and PP cells secrete ghrelin and pancreatic polypeptide, respectively. Pancreatic polypeptide-producing cells (approximately 5-10% of islet cells) release hormones that alter exocrine and gastrointestinal function. There are other islet cell types, such as endothelial cells, neurons, and progenitor cells.

[0071] As used herein, islet cells include the aforementioned islet cells, islet precursor cells that are precursors of islet cells, and intermediate cells, which are cells produced during the process of islet cell generation or differentiation induction from adult stem cells / pluripotent stem cells, or islet precursor cells. Here, "intermediate cells" are preferably cells determined to differentiate into islet cells. Furthermore, in the present invention, islet cells may also be cells produced from islet-like cells or islet precursor-like cells into which the Mycl gene or its gene product has been introduced.

[0072] In type 1 diabetes, cellular infiltration, primarily of lymphocytes, is observed soon after onset. Ultimately, with the selective loss of β cells, the size of the pancreatic islets decreases, with α cells becoming the dominant form. The pancreatic islets have a reserve capacity for insulin secretion, and when 90-95% of the β cells are lost, type 1 diabetes develops. In type 2 diabetes, there are essentially no morphological changes observed in the pancreatic islets; instead, the islet's capacity for insulin secretion decreases.

[0073] In the present invention, the source or supply source of the islet cells into which the Mycl gene or its gene product has been introduced is not limited, and may be primary islet cells isolated from the pancreas of an individual or islet cells cultured by a known culture method. Cultured islet cells may include but are not limited to islet cells of strains, islet cells from stem cells (e.g., iPS cells, ES cells, adult stem cells) (see, e.g., 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 has been introduced may be islet cells obtained from islet-like cells and / or islet precursor cell-like cells, or Mycl genes or their gene products may be repeatedly introduced into islet-like cells and / or islet precursor cell-like cells into which the Mycl gene has been introduced. In particular, in the case of treating diabetes for the purpose, it is preferred to use any of the islet cells collected from a donor or the islet cells derived from stem cells. The islet cells from the donor used can be autologous or allogeneic relative to the recipient. According to the present invention, by importing the Mycl gene in such islet cells, islet precursor cell-like cells are proliferated in vitro, and by returning (transplantation) to the patient, it is possible to treat diabetes. In this case, in cases where the islet cells are allogeneic relative to the patient, immunosuppressants can be appropriately given to the patient. In addition, the transplantation of the patient can use the above-mentioned islet cells, islet precursor cell-like cells or cells with insulin production ability or any combination thereof.

[0074] In connection with the above, it has been reported that, at least in mice, when β cells are lost, α cells proliferate, and then a portion of the α cells differentiate and convert into β cells. Therefore, the islets used as the starting material for islet cells before Mycl gene introduction can be islets containing a large number of normal β cells, as well as islets that have lost a large portion of β cells. From this perspective, the present invention can also be used for gene therapy in patients with type 1 diabetes who have lost β cells, using islet-like cells, etc., that have been introduced with the Mycl gene.

[0075] (3) Pluripotent stem cells

[0076] As used in this specification, "pluripotent stem cells" are cells with the ability to self-replicate and differentiate, and refer to cells that have the ability to form all cells that constitute an organism. "Self-replication ability" refers to the ability of a single cell to produce undifferentiated cells identical to itself. "Differentiation ability" refers to the ability of a cell to differentiate. Examples of pluripotent stem cells include embryonic stem cells (ES cells), artificial pluripotent stem cells (iPS cells), Muse cells (Multi-lineage differentiating Stress Enduring cells), sperm stem cells (GS cells), and embryonic germ cells (EG cells). Pluripotent stem cells used in the present invention are preferably ES cells. The source of pluripotent stem cells can be any one of mammals, birds, fish, reptiles, and amphibians, without particular limitation. Mammals include primates (humans, monkeys, etc.), rodents (mice, rats, guinea pigs, etc.), cats, dogs, rabbits, sheep, pigs, cattle, horses, donkeys, goats, ferrets, and the like.

[0077] When used in this specification, "ES cells" refer to pluripotent stem cells that have the ability to differentiate into all tissue cells that constitute an individual in the early stages of development, and are able to be cultured in vitro. ES cells, like pluripotent stem cells in early embryos, maintain the ability to differentiate into all cells that constitute an individual, while actually being able to proliferate indefinitely. Specifically, ES cells were first described in 1981 in mouse ES cells (Proc. Natl. Acad. Sci. USA 78, 7634-7638, 1981; Nature 292, 154-156, 1981). ES cells have multi-differentiation capabilities and can generate all tissues and cell types that constitute an individual. Multipotent embryonic stem cells have been isolated from a variety of species, including rat (Iannaconns et al., Dev. Biol. 163, 288-292, 1994), hamster (Dev. Biol. 127, 224-227, 1988), rabbit (Mol. Reprod. Dev. 36, 424-433, 1999), birds, fish, pig (Reprod. Fertil. Dev. 6, 563-568, 1994), cow (Reprod. Fertil. Dev. 6, 553-562, 1994) and primates (Proc. Natl. Acad. Sci. USA 92, 7844-7848, 1995). ES cells that can be used in the present invention are not limited, and examples thereof include KH2 cells, RF8 cells, JI cells, CGR8 cells, MG1.19 cells, 129SV cells, C57 / BL6 cells, and DBA-1 cells.

[0078] Several research groups have also successfully isolated ES cells and ES cell-like stem cells from embryonic human tissue. Early examples of success are listed below (Science 282, 1145-1147, 1998; Proc. Natl. Acad. Sci. USA 95, 13726-13731, 1998; Nature Biotech., 18, 399-404, 2000). These ES cell lines are constructed by culturing the ICM isolated from blastocysts in feeder cells. Other recent studies have shown that embryos and embryonic cells can be obtained by transferring nuclei from embryonic and mature mammalian cells into enucleated oocytes.

[0079] In the present invention, any constructed ES cell line can be used. Alternatively, to prevent immune rejection when ES cells produced using the methods of the present invention are applied to an individual, it is effective to use somatic cells from the individual to create cloned embryos and construct an ES cell line from them. This method allows the construction of ES cells with the same genetic characteristics as the individual.

[0080] Alternatively, it is considered that in the preparation of somatic cell cloning, the nucleus of the somatic cell introduced into the egg becomes a state similar to that of the fertilized egg, a phenomenon called "initialization". It is reported that ES cells also have an activity similar to that of the egg (Curr.Biol., 11, 1553-1558, 2001). That is, by fusing individual somatic cells with ES cells, it is expected that somatic cells will be converted into cells such as ES cells. ES cells can be genetically manipulated in vitro, and therefore, by utilizing ES cells that have been manipulated in advance for factors involved in immune rejection such as MHC gene groups, it is expected that rejection reactions can be avoided without using methods such as somatic cell cloning embryo preparation.

[0081] As used herein, "iPS cells" refer to cells that have ES cell-like differentiation pluripotency, obtained by introducing genes for transcription factors such as Oct3 / 4, Sox2, Klf4, and c-Myc into somatic cells. Like ES cells, iPS cells can be multiplied indefinitely while maintaining their differentiation pluripotency.

[0082] The basic method for preparing iPS cells is a method of introducing four transcription factors, namely Oct3 / 4, Sox2, Klf4 and c-Myc, into cells using viruses (Takahashi K, Yamanaka S: Cell 126(4), 663-676, 2006; Takahashi, K, et al.: Cell 131(5), 861-72, 2007). In addition, examples of cells that can be used to prepare iPS cells, that is, cells that serve as a source of iPS cells, 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.

[0083] Initialization of iPS cells can be performed by methods known to those skilled in the art, for example, as summarized in Addgene's Blog / Post, "Delivery Methods for Generating iPSCs" (https: / / blog.addgene.org / delivery-methods-for-generating-ipscs). There are no limitations on the method for introducing the Mycl gene into iPS cells, and examples include 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)-type episomal vectors), or methods of directly introducing RNA (including mRNA) encoding the Mycl gene or the Mycl protein itself into cells.

[0084] As used herein, "EG cells" refers to any embryonic germline stem cell derived from primordial germ cells, and their origin is not particularly limited. Furthermore, "GS cells" as used herein refer to germline stem cells derived from testicular germ cells and are cell lines capable of in vitro culture of spermatogonial stem cells (sperm stem cells) (Cell. 119, 1001-1012, 2004). Among GS cells, mGS cells (multipotent germline stem cells) are particularly preferred, possessing properties similar to ES cells and the additional potential for differentiation and pluripotency.

[0085] (4) Mycl gene and its gene product

[0086] The Mycl (also known as "L-Myc") gene is one of the members of the Myc gene family, including the c-Myc gene and the Mycn ("N-Myc" gene). The Mycl gene is also an oncogene like the c-Myc gene, and is also known as an initialization gene. In addition, it is known that the Mycl gene is different from the c-Myc gene and has almost no transformation ability (Nakagawa, M., et al., Proc. Natl. Acad. Sci. USA, vol. 107, p. 14152-14157, 2010). The cDNA sequence information of Mycl mice and humans can be obtained by referring to the NCBI accession numbers NM_008506 and NM_001033081, respectively, and those skilled in the art can easily isolate cDNA.

[0087] According to the present invention, it is preferred 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, and the Mycl gene that can be used also includes single-stranded or double-stranded DNA and its RNA complement. DNA includes, for example, DNA of natural origin, recombinant DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. As the nucleic acid used in the present invention, DNA is preferred. As is well known, codons have degeneracy, and there are also amino acids with multiple base sequences encoding one amino acid, but the pancreatic islet cells (i.e., pancreatic islet-like cells) into which the Mycl gene has been introduced are not particularly limited as long as they have the effect of promoting cell proliferation through the expression of the Mycl gene and promoting insulin production by stopping its expression.

[0088] In one embodiment, the Mycl gene may comprise or consist of the following nucleic acid:

[0089] (1) a nucleic acid comprising or consisting of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3; or

[0090] (2) a nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising or consisting of the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 and encodes a polypeptide having an activity of proliferating pancreatic islet-like cells into which the Mycl gene has been introduced; or

[0091] (3) A nucleic acid that hybridizes under stringent conditions with a nucleic acid comprising the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3, or consisting of the base sequence shown in SEQ ID NO: 1 or 3, and encodes a polypeptide that proliferates pancreatic islet-like cells into which the Mycl gene has been introduced, thereby having the effect of proliferating insulin-producing cells and, consequently, promoting insulin production.

[0092] As used in this specification, "stringent conditions" refers to conditions for hybridization under moderate or highly stringent conditions. Specifically, for moderately stringent conditions, for example, those skilled in the art with ordinary skills can easily determine based on the length of DNA. Basic conditions are shown in Sambrook, J et al., Molecular Cloning, A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, 7.42-7.45 (2001), about nitrocellulose filters, including 5 × SSC, 0.5% SDS, 1.0mM EDTA (pH 8.0) pre-wash solution, about 50% formamide at about 40-50 ° C, 2 × SSC-6 × SSC (or about 50% formamide at about 42 ° C in Stark's solution (Stark's solution) and other similar hybridization solutions) and about 60 ° C, 0.5 × SSC, 0.1% SDS washing conditions. For highly stringent conditions, those skilled in the art can also easily determine based on, for example, the length of DNA. Typically, such conditions are defined as including hybridization and / or washing at higher temperatures and / or lower salt concentrations than those under moderately stringent conditions, for example, with hybridization conditions as described above and a wash in 0.2X SSC, 0.1% SDS at about 68° C. Those skilled in the art will recognize that the temperature and wash solution salt concentration can be adjusted as needed based on factors such as probe length.

[0093] Homologous nucleic acids cloned using nucleic acid amplification reactions as described above or hybridization have at least 30% or more, preferably 50% or more, more preferably 70% or more, further more preferably 90% or more, further preferably 95% or more, and most preferably 98% or more identity relative to the base sequence of record in SEQ ID NO:1 or SEQ ID NO:3. It should be noted that the percent identity can be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two nucleotide sequences can be determined by comparing sequence information using the GAP computer program (GCG Wisconsin Package, Version 10.3), which is described in Devereux et al., Nucl. Acids Res., 12, 387 (1984), and available from the University of Wisconsin Genetics Computer Group (UWGCG).

[0094] In one embodiment, the gene product of the Mycl gene is a polypeptide expressed by the Mycl gene. Typically, the polypeptide can be:

[0095] (1) a polypeptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4; or

[0096] (2) A polypeptide having at least 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, and having an effect of promoting islet-like cell proliferation and / or an effect of promoting insulin production.

[0097] In one embodiment, the gene product of the Mycl gene may be a variant of the polypeptide defined above, or may be an amino acid sequence comprising one or more amino acid deletions, substitutions, insertions, and / or additions in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. The substitution may be a conservative substitution, which is a substitution in which a specific amino acid residue is replaced with a residue having similar physicochemical characteristics. Non-limiting examples of conservative substitutions include substitutions between amino acid residues containing aliphatic groups, such as substitutions of Ile, Val, Leu, or Ala; substitutions between polar residues, such as substitutions of Lys and Arg, Glu and Asp, Gln and Asn, and the like.

[0098] Variants based on amino acid deletion, substitution, insertion, and / or addition can be prepared by subjecting the Mycl gene to, for example, site-specific mutagenesis, a well-known technique (e.g., Nucleic Acid Research, Vol. 10, No. 20, p. 6487-6500, 1982). In this specification, "one or more amino acids" refers to amino acids that can be deleted, substituted, inserted, and / or added by site-specific mutagenesis. In addition, in this specification, "one or more amino acids" may refer to one or more amino acids, depending on the circumstances.

[0099] In addition to the above-mentioned site-specific mutagenesis, methods for deleting, substituting, inserting, and / or adding one or more amino acids to the amino acid sequence of a polypeptide while maintaining its activity include methods of treating the gene at the source of the mutation, and methods of selectively cleaving the gene, followed by removal, substitution, insertion, or addition of selected nucleotides, and then ligation. Although not limited to this, the gene product of the Mycl gene in the present invention can be a polypeptide having activity that promotes insulin production, comprising an amino acid sequence in which 1 to 10 amino acids are deleted, substituted, or added, preferably 9 or fewer, 7 or fewer, 5 or fewer, 3 or fewer, 2 or fewer, and more preferably 1 or fewer amino acids are deleted, substituted, or added to SEQ ID NO: 2 or SEQ ID NO: 4.

[0100] 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 with the amino acid sequence of SEQ ID NO: 2, and is a polypeptide having an effect of promoting islet-like cell proliferation and / or an effect of promoting insulin production.

[0101] The percent identity of two amino acid sequences can be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two protein sequences can be determined based on the algorithm of Needleman, SB and Wunsch, CD (J. Mol. Biol. 48:443-453, 1970) and by comparing sequence information using the GAP computer program available from the University of Wisconsin Genetics Computer Group (UWGCG). Preferred default parameters for the GAP program include: (1) the 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 weighting for end gaps.

[0102] (5) Introduction of the Mycl gene

[0103] According to the present invention, the method for importing Mycl gene into primary islet cells, cultured islet cells or stem cells (such as iPS cells, ES cells, adult stem cells) is not particularly limited, and methods well known to those skilled in the art can be used. As gene introduction means, "conversion" or "transfection" is common, which refers to the transient or stable genetic change induced in the cell after being incorporated into exogenous nucleic acid (for example, exogenous DNA or RNA for host cells). Generally, genetic change can be achieved by incorporating exogenous nucleic acid into the genome of the host cell or by maintaining exogenous nucleic acid as an additional component or independently transiently or stably. According to the present invention, as long as the Mycl gene imported can control the on / off of the expression of the gene, it can be the state integrated into the genome of the host cell, or it can also exist as an additional component, or it can also be directly present in the cytoplasm in the form of a plasmid or a vector comprising the gene.

[0104] Generally, when introducing exogenous nucleic acid (preferably DNA) into a host cell, a "vector" is used. Generally, vectors include viruses, especially attenuated viruses and / or replication-defective viruses. As viral vectors, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, Sendai viral vectors, etc. can be exemplified. In addition, the vector can include control sequences such as promoters, enhancers, ribosome binding sequences, terminators, polyadenylation sites, so that exogenous nucleic acids can be expressed. Furthermore, as needed, it can include drug resistance genes (such as kanamycin resistance genes, ampicillin resistance genes, puromycin resistance genes, etc.), thymidine kinase genes, diphtheria toxin gene selection marker sequences, fluorescent proteins, beta-glucuronidase (GUS), FLAG and other reporter gene sequences, etc. In this article, "promoter" can also include cell type specificity regulated, tissue specificity regulated, or promoter components that are inducible by external signals or reagents and are sufficient for promoter-dependent gene expression. Such components can be located in the 5' or 3' region of the natural gene. Furthermore, "functional binding" refers to the binding of a DNA sequence to a regulatory sequence so that an appropriate molecule (eg, a transcriptional activator protein) can be expressed when linked to the regulatory sequence.

[0105] In addition, there is no limitation on the introduction of the vector into the host cell, and it can be carried out by 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, cells into which the vector has been introduced can be selected based on the characteristics of the marker gene (e.g., drug resistance gene). The correct occurrence of homologous recombination in the selected cells can be confirmed by Southern blotting using a portion of the exogenous nucleic acid as a probe, etc. In this way, cells containing the gene of the target gene, specifically the Mycl gene, in a hybrid manner can be prepared.

[0106] As ES cells, the KH2 strain (Beard C, et al., Genesis, vol. 44, p. 23-28 (2006)) having an Frt sequence downstream of the Cola1 locus and expressing M2-rtTA as a reverse tetracycline-controlled transactivator under the control of the endogenous Rosa26 promoter can be used. The introduction of the Mycl gene into the ES cells can be carried out using methods well known to those skilled in the art. For example, the Mycl gene can be inserted into the pCR8-GW-TOPO vector (manufactured by Invitrogen Life Technologies) as an entry vector by TA cloning, and then an LR reaction is performed between the vector and, for example, a Colla1-TetOP-AttR1-ccdB-AttR2-ires-mCherry vector, and the TetOP-Mycl-ires-mCherry vector is used as a gene introduction vector. The "TetOP (operon)" sequence in this vector is a sequence to which a reverse tetracycline-controlled transactivator binds (tetracycline response element: TRE). Depending on the addition of a reverse tetracycline such as doxycycline (Dox) to the cells, it binds to the reverse tetracycline-controlled transactivator expressed from the host cells, inducing the expression of genes linked downstream. Furthermore, "ires" (internal ribosome entry site) is an internal ribosome recognition sequence, and "mCherry" is a gene encoding red fluorescent protein (reporter gene). By introducing this vector into KH2-ES cells along with a nucleic acid encoding flipase, the Mycl gene can be integrated into the ES cell chromosomes. In addition, in addition to the vector containing "ires-mCherry" as described above, pBSSK(-)-IRES-βgeo containing the "ires-βgeo (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 the same vector containing the IRES-Hygro (hygromycin resistance gene) cassette can also be used.

[0107] The present invention can cause pancreatic islet-like cells to proliferate and differentiate into insulin-producing cells by controlling the transient expression of the Mycl gene. Thus, the present invention is characterized by the transient expression of the Mycl gene. However, when the Mycl gene is transiently expressed, the number of cells into which the Mycl gene is introduced decreases relatively as the islet-like cells divide, and thus the expression of the Mycl gene can be naturally reduced or stopped over time. That is, in one embodiment, the present invention achieves the purpose of the present invention by controlling the "on" expression of the Mycl gene. In addition, in another embodiment, the expression of the Mycl gene can also be forcibly set to "off" (hereinafter referred to as "on / off" control).

[0108] 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 is continuously expressed in the cell, which can cause the islet-like cells to proliferate. On the other hand, by removing the reverse tetracycline, the proliferation of the islet-like cells can be stopped, inducing differentiation into insulin-producing cells. In the present invention, the transient expression (on state) of the Mycl gene introduced into the islet-like cells is preferably 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, the islet-like cells into which the Mycl gene has been introduced preferably proliferate during the above period.

[0109] As an alternative to controlling the on / off expression of the Mycl gene, a "light-controlled viral vector" (Tahara, M., et al., PNAS, vol. 116, 11587-11589, 2019) can be used, which can precisely control the gene expression and cell proliferation of the viral vector by regulating light irradiation. This is achieved by introducing a gene encoding a light-switch protein called a magnet into the viral vector, and blue light can be used to control the expression of the target gene inserted into the vector.

[0110] Other alternative examples of controlling the on / off expression of the Mycl gene 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). However, the present invention is not limited to these methods, and the methods used in the initialization (construction) of iPS cells described above (i.e., methods for transient gene induction) can be used.

[0111] According to the present invention, in addition to importing the exogenous Mycl gene of separation, the purpose of the present invention can also be achieved by forcibly expressing the endogenous Mycl gene in the cell. It is not limited as a method for making endogenous Mycl gene expression, it is possible to replace the wild-type promoter or enhancer by using a powerful promoter, enhancer that can operably induce the expression of Mycl gene, forcibly making the method for gene expression. The example of the promoter for replacement includes the cytomegalovirus (CMV) promoter as a strong promoter, the inducible promoter that works in the presence of an inducer, etc. On the other hand, the example of the enhancer for replacement includes SV40 enhancer, herpes B virus enhancer, cytomegalovirus enhancer, alpha-fetoprotein enhancer, etc. In another embodiment, in addition to CRISPR II types (comprising CRISPR-dCas9), it is also possible to utilize the molecules that have been added to the genome recognition sequence of CRISPR-I types, TALENs, ZFNs to enable promoter and / or enhancer activation, demethylase, histone modifying enzymes, transcriptional activators, specifically VP64, p65, Rta, etc. In this specification, the aforementioned promoter, enhancer, 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.

[0112] (6) Introduction of the gene product of the Mycl gene

[0113] The Mycl gene product can be introduced into cells using conventional methods for introducing foreign genes or proteins into cells. For example, such methods are not limited and include methods using transfection reagents, methods using viruses, electroporation, particle guns, sonoporation, liposome fusion, and methods using micromanipulators or laser irradiation to create pores in the cell membrane.

[0114] (7) Preparation of chimeric mammals

[0115] ES cells can be introduced into mammals to produce chimeric mammals using methods well known to those skilled in the art. First, in the culture of ES cells into which the Mycl gene is introduced, any culture medium known to those skilled in the art can be used. For example, in the case of culturing the ES cells in feeder cells, feeder cells (such as MEF (mouse fetal fibroblasts)) can be used, and ES cell culture medium (such as 2-mercaptoethanol (2ME, GIBCO) and LIF (SIGMA) are added to knockout DMEM (GIBCO) containing 15% FBS, 50U / mL of penicillin / streptomycin, L-glutamine, and non-essential amino acids) can be used for the ES cells in the feeder cells.

[0116] Then, the above-mentioned ES cells are imported into mammals to obtain gene knockout animals (Mycl gene knock-in animals). Here, as mammals, mice are used as an example for illustration, and the breeding method of knock-in mice is well known to those skilled in the art. Specifically, the above-mentioned ES cells are injected into the blastocyst of mice (such as C57BL / 6, etc.), transplanted into the uterus of pseudo-pregnant female mice (ICR, etc.), thus chimeric mice can be obtained. Then, by mating the chimeric mice with common mice (C57BL / 6, etc.), heterozygous mutant mice that the Mycl gene is knocked into by heterozygosity can be obtained. By mating allogeneic mutant mice with each other, the homozygous mutant mice that the Mycl gene is knocked into by homozygosity can be obtained. For the preparation of the above knock-in mice, reference is made 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), Japanese Patent Application Laid-Open No. 2003-265166), etc.

[0117] Furthermore, chimeric mammals are not limited to the ES cells described above; iPS cells can also be used to create them. For example, blastocyst complementation can be used to create organs from human iPS cells in non-human mammals. For example, Nakauchi et al. generated a human pancreas derived from human iPS cells in a pancreasless cloned pig (see Nakauchu, H., et al., PNAS, Vol. 110, No. 1, 4557-4562 (2013)).

[0118] 2. Control of the proliferation and differentiation of islet-like cells

[0119] According to the present invention, the proliferation and differentiation of the above-mentioned islet-like cells prepared can be controlled in vitro and in vivo. The induction method of the Mycl gene can be selected according to the above-mentioned method for introducing the gene into the cell. For example, when the TetOP-Mycl-ires-mCherry vector as described above is used as a gene introduction vector to introduce the Mycl gene into the cell, it is dependent on reverse tetracyclines such as doxycycline (Dox) and combined with the reverse tetracycline-controlled transactivator expressed by the host cell to express the Mycl gene connected to its downstream, which can induce the proliferation of islet-like cells. The concentration of Dox added to the cell culture system can be appropriately adjusted. For example, it can be 1-100 mg / mL, etc. After the islet-like cells are proliferated by adding Dox, for example, by replacing the culture medium without Dox to stop the proliferation, it is possible to induce cell differentiation such as insulin production.

[0120] In vivo, the proliferation of islet-like cells in the pancreas can be induced by providing, for example, water containing Dox to a chimeric non-human mammal. The concentration of Dox when added to the water can be, for example, 1-100 mg / mL, preferably 2.0 mg / mL. When using chimeric non-human mice, since the pancreas grows with age until 8 weeks of age, Dox can be administered at a time when growth has ceased (e.g., after 8 weeks of age). However, there is no age restriction as long as proliferation can be induced.

[0121] 3. Preparation of pancreatic islet cells or their precursor cells with insulin-producing ability

[0122] According to the present invention, there is provided a method for manufacturing islet cells or their precursor cells with insulin production ability. Such a manufacturing method is not limited, including: using primary islet cells, cultured islet cells or islet cells derived from stem cells as raw materials, Mycl gene is introduced into the islet cells, after the cells are proliferated by the forced expression of the Mycl gene, the expression of the gene is stopped, thereby obtaining islet cells or their precursor cells with insulin production ability. As described above, the forced expression and stopping of the Mycl gene are as described above, for example, alternative means (for example, using a light-regulated viral vector) or a reverse tetracycline-controlled transactivator of doxycycline sensitivity can be utilized to control the expression of the gene on / off. In the case of use in this specification, "islet cell precursor cells" or "islet precursor cells" refer to cells (or cell groups) in the middle of differentiation to islet cells with insulin production ability after the expression of the Mycl gene stops, for example, more than one marker selected from PDX1 positive, PTF1a positive, NKX6.1 positive, Fev positive, Pax4 positive, and Cck gene group can be used as an index for identification.

[0123] 4. Drug Use

[0124] According to the present invention, by controlling the expression of the introduced Mycl gene, islet-like cells can be proliferated and insulin production can be promoted. Therefore, in one embodiment, there is provided an islet cell expansion promoter, islet function improver, or insulin production promoter containing the Mycl gene or its gene product as an active ingredient; a pharmaceutical composition containing the above-mentioned active ingredient and other pharmaceutically acceptable ingredients (e.g., a carrier, excipient, disintegrant, buffer, emulsifier, suspending agent, pain-relieving agent, stabilizer, preservative, preservative, physiological saline, etc.); a method for preventing and / or treating diabetes mellitus using the above-mentioned islet cell expansion promoter, islet function improver, insulin production promoter, or pharmaceutical composition; and the use of the Mycl gene in the manufacture of the above-mentioned islet cell expansion promoter, islet function improver, insulin production promoter, or pharmaceutical composition. On the other hand, provided are methods for administering the Mycl gene or its gene product to a subject for the purpose of preventing and / or treating diabetes (in vivo methods); and methods for transplanting islet-like cells, islet precursor cell-like cells, or insulin-producing cells (hereinafter sometimes referred to as "islet-like cells, etc."), or any combination thereof, into a subject (ex vivo methods) into which the Mycl gene or its gene product has been introduced in vitro.

[0125] The in vitro method of the present invention can be implemented in different ways depending on the cells used. Examples include: (i) methods for producing islet-like cells by gene transfer into adult pancreatic islet-derived cells in vitro or in autologous tissue (both encompassing in vitro methods in the narrow sense); (ii) methods for producing islet-like cells obtained by inducing differentiation, i.e., islet cells derived from stem cells (iPS cells, ES cells, adult stem cells, etc.) by introducing the Mycl gene in islet cells in vitro or in autologous tissue (in the broad sense of in vitro methods); (iii) methods in which the Mycl gene is functionally expressed in (i) and (ii) above, and the cells may be proliferating pancreatic progenitor cells; and (iv) methods for producing insulin-producing cells or pancreatic islet cells (not containing the Mycl gene) differentiated from (i) to (iii) above.

[0126] As one embodiment of the present invention, regardless of whether the cells are autologous or allogeneic to the patient, the transplanted islets are encapsulated and administered using known methods, specifically, methods 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 / s 41551-018-0275-1, EBioMedicine 12 (2016) 255?262., DOI: https: / / doi.org / 10.1016 / j.ebiom.2016.08.034, etc. By encapsulating the transplanted islets, the use of immunosuppressants can be eliminated or the dosage of immunosuppressants can be reduced.

[0127] As one method of this specification, the isolated islets can be appropriately gene-edited. There is no particular limitation on gene editing, but specifically, by correcting the mutant genes contained in the islet cells through genome editing, preferred therapeutic features can be added, such as by correcting surface antigens such as molecules that cause immune responses, such as HLA and GAD proteins, and inducing immune tolerance by deleting Beta-2 microglobulin, RFX5, RFXANK, RFXAP, and CIITA genes. There is no particular limitation on the method of gene editing, and specific examples include CRISPR-Cas9, TALEN, ZFN, CRISPR-Cas3, CRISPR-TypeI-D, and their modified forms, and transposon vectors such as piggyBAC for expressing functional molecules.

[0128] (1) Adaptation to disease

[0129] Diseases for which the Mycl gene or its gene product can be applied in the manner described above are diseases in which insulin is not fully functional in an organism (typically, insulin resistance or decreased insulin secretion). According to the present invention, the Mycl gene or its gene product can be used to promote insulin production, for example, to have a blood sugar-lowering effect in diabetic patients. Typical suitable diseases are diabetes mellitus. More specifically, diseases, disorders, or symptoms associated with severe hypoglycemia, type 1 diabetes (including slowly progressive type 1 diabetes or type 1.5 diabetes), type 2 diabetes, impaired glucose tolerance, hyperglycemia, dyslipidemia, obesity, or metabolic syndrome, as well as diseases caused by other specific mechanisms or diseases, such as genetic abnormalities related to pancreatic β-cell function, genetic abnormalities related to the insulin delivery mechanism, exocrine pancreatic diseases associated with other diseases or conditions, such as endocrine diseases, liver diseases, diseases caused by drugs or chemicals, infections, rare conditions caused by immune mechanisms, or gestational diabetes. Furthermore, diabetic complications caused by diabetes (e.g., diabetic retinopathy, diabetic neuropathy, etc.) may also be included in the indicated suitable diseases. In addition, the insulin secretion deficiency state caused by pancreatitis or pancreatic cancer due to total pancreatectomy or partial pancreatectomy can also be included in the applicable diseases.

[0130] Furthermore, the types of diabetes treatable by the methods described herein are not particularly limited. These methods can provide treatments that are less likely to cause hypoglycemia, with physiological insulin secretion dependent on blood glucose levels. For example, in the treatment of severe hypoglycemia, there are no limitations. Since those skilled in the art know that donor islets exhibit significant effects on severe hypoglycemia, methods described in known literature can be used as one approach. The methods described in the known literature mentioned here are not limited, and examples thereof include Diabetes Care 2016 Jul; 39(7): 1230-1240., DOI: 10.2337 / dc15-1988, The New England Journal of dicine. 343(4): 230-238., DOI: 10.1056 / NEJM200007273430401, and The New England Journal of Medicine. 355(13): 1318-1330., DOI: 10.1056 / NEJMoa061267. Alternatively, a method (in vivo method) of administering the Mycl gene or its gene product to a subject that can expand islet cells, islet-like cells, islet precursor cell-like cells, or insulin-producing cells to an amount equivalent to, or preferably greater than, the amount of islet cells transplanted as described in known literature can be selected instead of cell transplantation as described in known literature.

[0131] In addition, in the case of being used for the treatment of type 1 diabetes, it is not particularly limited. As an embodiment for the treatment of type 1 diabetes, for example, the amount of islet cells, islet-like cells, islet precursor cells, or insulin-producing cells contained can be adjusted according to the insulin, blood sugar level and / or C-peptide amount of the type 1 diabetes patient at any time, when fasting, after sugar load and / or after glucagon stimulation, or the amount of C-peptide. The dosage of the preparation for in vivo treatment can also be determined by the method described in this specification. From the viewpoint that the risk of type 1 diabetes patients in an insulin-depleted state presenting hypoglycemic symptoms is high, it is preferred to use the method described in this specification for treatment. For example, the amount of C-peptide in the blood when fasting and / or when glucagon is stimulated can be 0.5 ng / mL or less, preferably 0.2 ng / mL or less, more preferably 0.1 ng / mL or less of type 1 diabetes patients as objects for treatment. Specifically, for example, the dosage of the medicament can usually be adjusted with reference to the transplantation amount of the donor islets transplanted for adaptation to severe hypoglycemia. Specifically, for example, islet cells, islet-like cells, islet precursor cell-like cells, or insulin-producing cells at a concentration of 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 can be selected in which the Mycl gene or its gene product is administered to a subject (in vivo method) to amplify islet-like cells, islet precursor cell-like cells, or insulin-producing cells to the same degree.

[0132] In addition, when used for the treatment of type ii diabetes, it is not particularly limited. For example, as an 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 amount of islet cells, islet-like cells, islet precursor cells, or insulin-producing cells contained can be adjusted according to the insulin, blood sugar level, and / or C-peptide level of the type ii diabetes patient at any time, when fasting, after sugar load, and / or after glucagon stimulation. The dosage of the medicament for in vivo treatment can be determined by the method described in this specification. From the viewpoint of obtaining a preferred effect under the condition of insulin deficiency, for example, the blood C-peptide level when fasting and / or when glucagon stimulation is 0.5ng / mL or less, preferably 0.2ng / mL or less, more preferably 0.1ng / mL or less of type ii diabetes patients as objects for treatment. The dosage of the specific medicament is, for example, usually adjusted with reference to the transplantation amount of the donor islets transplanted for adaptation to severe hypoglycemia. Specifically, for example, islet cells, islet-like cells, islet precursor cell-like cells, or insulin-producing cells at a concentration of 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 can be selected in which the Mycl gene or its gene product is administered to a subject (in vivo method) to amplify islet-like cells, islet precursor cell-like cells, or insulin-producing cells to the same degree.

[0133] In addition, when used as the therapeutic agent of slow progress type 1 diabetes, it is not limited, and the amount of the medicament contained can be adjusted according to the amount of insulin, blood sugar level and / or C-peptide in the same manner as type 1 diabetes and / or type 2 diabetes.In this case, the islet cells, islet-like cells, islet precursor cell-like cells or insulin-producing cells amplified by the method of this specification sheets are preferably from the cells of the patient himself.For example, as a mode of the treatment of slow progress type 1 diabetes, it is preferred to investigate in advance the type of autoantibodies, HLA for the islet cells in the blood, if it is possible to amplify the islet cells of oneself by the method of this specification sheets before or after falling into insulin-dependent state and / or insulin-depleted state, then it is possible to carry out treatment that does not require an immunosuppressant, it is possible to prevent the transition to insulin-dependent state and / or insulin-depleted state, or it is possible to treat insulin-dependent state and / or insulin-depleted state. To determine whether or not a patient has slowly progressive type 1 diabetes, there are no particular limitations on the type of autoantibodies or HLA in the blood that are involved in slowly progressive type 1 diabetes. Examples include known methods such as confirming whether or not islet-related autoantibodies, such as islet cell antibodies (ICA), GAD antibodies, insulin autoantibodies (IAA), and IA-2 antibodies, are positive for multiple or individual islet-related autoantibodies. Furthermore, known methods can be used to confirm whether or not HLA associated with slowly progressive type 1 diabetes, such as HLA-DR4-DQA1*0301-B1*0401, is present.

[0134] In addition, from the viewpoint of confirming therapeutic effect by donor islets, for the treatment of severe hypoglycemia, it is preferably an in vitro method, but islet cells can be expanded in vivo (in vivo) by the method described in this specification sheets. By the islet cells expanded like this, it is confirmed that the same effect as the in vitro method is found. Therefore, the therapeutic method of expanding islets in vivo can also be preferably selected as the therapeutic method of severe hypoglycemia in the same manner as the in vitro method. And then, for the treatment of type i diabetes, type ii diabetes, slowly progressing type i diabetes, it is also possible to consider the state of the sex, age, body weight, affected part, the state of the cell used, etc. of the object in the same manner as the in vitro method, select the method for expanding islet cells in vivo (in vivo). At this point, it is also possible to combine the method for expanding islet cells in vivo (in vivo) with the in vitro method.

[0135] 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 / development of the above-mentioned diseases or their symptoms, or reducing the risk of the onset / development of the above-mentioned diseases or their symptoms.

[0136] "Treatment" includes alleviating the characteristic symptoms or accompanying symptoms of the target disease (relief), preventing or delaying the deterioration of symptoms, etc., and treatment also includes improvement of the disease.

[0137] (2) Insulin production promoter and pharmaceutical composition

[0138] The Mycl gene or its gene product of the present invention can be provided in the form of an agent for promoting pancreatic islet cell expansion, an agent for improving pancreatic islet function, an agent for promoting insulin production, or a pharmaceutical composition for the prevention and treatment of the above-mentioned diseases. Here, as one embodiment of the agent for promoting pancreatic islet cell expansion, it refers to an agent that causes the proliferation of any one of the α cells, β cells, δ cells, ε cells, and PP cells contained in the pancreatic islets, preferably any two or more pancreatic islet cells. In addition, as one embodiment of the agent for improving pancreatic islet function, it refers to a preparation that improves part or all of the functions of the pancreatic islets in a living body by administration. Part of the functions of the pancreatic islets can be specifically listed, for example: the blood sugar regulating effect of pancreatic islet cells, the blood sugar lowering effect of insulin, the glucose production and release effect of glucagon, the secretion inhibitory effect of gastrin, secretin, insulin and / or glucagon of somatostatin or the nutrient absorption inhibitory effect in the digestive tract, the appetite regulating effect of ghrelin, the gallbladder contraction regulating effect of pancreatic polypeptide, and the appetite regulating effect. In addition, as one form of an insulin production promoter, for example, an agent that promotes physiological insulin secretion corresponding to blood glucose levels, which is one of the functions of pancreatic islets in a living body. Furthermore, when provided as a pharmaceutical composition, in addition to the active ingredient of the Mycl gene or its gene product used in the above-mentioned manner, other pharmaceutically acceptable ingredients (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, pain-relieving agents, stabilizers, preservatives, preservatives, physiological saline, etc.) may also be included. Furthermore, depending on the specific circumstances, an active agent for activating the Mycl gene may be included.

[0139] The pancreatic islet cells of an organism into which the Mycl gene or its gene product is introduced can be derived from the pancreatic islet cells of a healthy individual, or can be derived from a patient with type 1 diabetes or end-stage type 2 diabetes who has lost some or most of their β cells. In this case, the pancreatic islet cells can be autologous or allogeneic to the recipient.

[0140] The cell preparation and pharmaceutical composition of the present invention are not limited and can be obtained by suspending the islet-like cells obtained above in physiological saline or an appropriate buffer (e.g., phosphate-buffered saline). It should be noted that the number of cells required for treatment can be obtained by forced expression of the Mycl gene and appropriate cell proliferation.

[0141] In addition, in the use of the cell preparation and pharmaceutical composition of islet cells, islet-like cells, islet precursor cells or insulin-producing cells, in order to protect the cells, dimethyl sulfoxide (DMSO), serum albumin etc. can be contained in the cell preparation and pharmaceutical composition, and in order to prevent the mixing and proliferation of bacteria, antibiotics etc. can be contained in the cell preparation and pharmaceutical composition. And then, other ingredients (for example, carrier, excipient, disintegrant, buffer, emulsifier, suspending agent, painless agent, stabilizer, preservative, preservative, normal saline etc.) allowed on the preparation can also be contained in the cell preparation and pharmaceutical composition. Those skilled in the art can add these factors and medicaments to the cell preparation and pharmaceutical composition at appropriate concentrations.

[0142] The number of pancreatic 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 account the subject's gender, age, weight, condition of the affected area, condition of the cells used, etc., so as to obtain the desired effect (e.g., reduction in blood sugar levels) in the prevention and / or treatment of diabetes and diseases related thereto.

[0143] The insulin production-promoting and pharmaceutical compositions of the present invention can be administered to various subjects, for example, mammals such as primates, humans, dogs, cats, cattle, horses, pigs, and sheep, preferably humans. Furthermore, the route of administration to the subject is not limited and can be administered parenterally, for example, by injection or infusion, to any location that can respond to glucose in the body. Specifically, for example, the subject can be transplanted or administered into the pancreas, under the renal capsule, preferably subcutaneously, intraperitoneally, more preferably intravascularly, intravenously, and even more preferably into the portal vein.

[0144] The method for activating the administered Mycl gene in vivo is not limited, and the aforementioned "on" control or "on and / or off" control system for Mycl gene expression can be utilized. For example, the aforementioned "light-regulated viral vector" (Tahara, M., et al., PNAS, vol. 116, 11587-11589, 2019) can also be used.

[0145] In addition, when the Mycl gene is targeted to the pancreas, markers specifically expressed in 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 used as targets for gene introduction. When selecting a method for expanding islet cells in vivo, the administration route to the subject is not limited, and specific examples include intrapancreatic, subcutaneous, and intraperitoneal administration, preferably intravascularly and intravenously, and more preferably intraceliac artery and pancreatic duct administration.

[0146] (3) Treatment methods

[0147] According to the present invention, methods are provided for preventing and / or treating a subject suffering from diabetes or a disease related thereto using the Mycl gene or its gene product, pancreatic islet-like cells, or any combination thereof. Furthermore, according to the present invention, in the above-described treatment methods, an agent for activating the Mycl gene can be administered, and can be administered before, simultaneously with, or after administration of an insulin production-promoting agent or pharmaceutical composition.

[0148] (4) Kit

[0149] According to the present invention, a kit for preventing and / or treating diabetes or a disease related thereto is provided, comprising an insulin production promoter or pharmaceutical composition. Such a kit may include instructions for administering or implanting the insulin production promoter or pharmaceutical composition to a subject. Furthermore, the kit may further include an agent for activating the Mycl gene.

[0150] Example

[0151] The present invention will be further specifically described with reference to the following examples, but the present invention is not limited to these examples.

[0152] method

[0153] (i) Construction of ES cells capable of inducing Myc, Mycn, and Mycl expression in a Dox-dependent manner

[0154] 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). Using the flip-in recombination system (Bear et al., 2006), the pCR8Mycl-TOPO vector containing each Myc gene was subjected to an LR reaction with the TetOP-AttR1-ccdB-AttR2-ires-mCherry vector to generate the Col1a1-TetOP-Mycl-ires-mCherry vector (hereinafter referred to as the "targeting vector"), which was then inserted into the Col1a1 locus of KH2-ES cells. For flip-in recombination, KH2-ES cells were electroporated using the Genepulser Xcell electroporation system (BIO-RAD) with a cell suspension in high-glucose DMEM (Nacalai Tasque) containing 50 μg of each Myc gene-incorporated targeting vector and 25 μg of the pFlapase vector, along with 25 mM HEPES buffer (Gibco). (Two pulses were applied at voltage: 550 V, capacitance: 25 μF, resistance: ∞, and cuvette: 4 mm or greater.) Twenty-four hours after electroporation, cells were selected with 150 μg / mL of hygromycin B (Roche), and the resulting cells were harvested to construct ES cell lines capable of inducible expression of each Myc gene in a Dox-dependent manner.

[0155] (ii) Cell culture method

[0156] Feeder cells (MEF; mouse embryonic fibroblasts) were cultured using DMEM (Nacalai Tesque) medium containing 10% FBS (GIBCO), 50 U / mL penicillin-streptomycin (P / S; Nacalai Tesque), L-glutamine (GIBCO), and NEAA (Nacalai Tesque).

[0157] ES cells were cultured using a medium containing 15% FBS, 50 U / mL P / S, L-glutamine, and NEAA in knockout DMEM (GIBCO) supplemented with 2-mercaptoethanol (2ME: GIBCO) and LIF (SIGMA). The cells were gelatin-coated (SIGMA) and cultured in a culture dish seeded with feeder cells. For passaging, ES cells were treated with 0.25% trypsin / 1 mM EDTA (GIBCO) at 37°C for approximately 3 minutes, and approximately 1 / 10 of the cell suspension was seeded onto a new culture dish.

[0158] (iii) Preparation of mice capable of inducing Mycl expression in vivo

[0159] ES cells capable of inducing Mycl expression in a doxycycline-dependent manner (Sigma-Aldrich, sometimes referred to as "Dox") were injected into mouse blastocysts (ICR, E3.5) and transplanted into the uterus of pseudopregnant day 2 mice (Slc: ICR, Shimizu Laboratory Materials), thereby culturing chimeric mice with cells capable of inducing Mycl expression in a Dox-dependent manner.

[0160] (iv) Administration of doxycycline

[0161] A solution containing 2.0 mg / mL of Dox was administered to the drinking water of 8-week-old mice and added to the culture medium of cultured cells to a final concentration of 2.0 μg / mL.

[0162] (v) Preparation of pathological specimens from various mouse organs

[0163] After dissecting the mice, each organ was shaken in 4% PFA (Wako Pure Chemical Industries, Ltd.) for one day. The next day, the organs were transferred to 70% EtOH (Wako Pure Chemical Industries, Ltd. diluted with 100% EtOH) and shaken for another day. The next day, blocks were prepared using a rotary tissue processor STR120 (Thermo Scientific) according to the recommended protocol. Preparation of pathological specimens was outsourced to Biogate.

[0164] (vi) Immunostaining

[0165] The tissue sections were immersed in xylene (Wako Pure Chemical Industries, Ltd.), and then immersed in 100% EtOH (Wako Pure Chemical Industries, Ltd.) for more than 30 minutes each. Rinse with tap water for about 10 minutes, move to boiling antigen activation solution pH 9 (Nichirei Biosciences, used with a 10-fold dilution), and perform antigen activation treatment for 10 minutes. 200 μL of the primary antibody solution diluted at each rate with blocking solution (2% BSA + 1×PBS) was added to the tissue sections and allowed to stand for 30 minutes to 1 hour. After washing twice with 1×PBS, 2 drops of the secondary antibody solution were added to the tissue sections and allowed to stand for 30 minutes. After washing twice with 1×PBS, in the case of DAB staining, 150 μL of DAB solution (Nichirei Biosciences, using a DAB substrate kit, a solution prepared by adding 1 drop each of reagents A and B to 1 mL of Elix water and mixing, followed by adding 1 drop of reagent C and mixing) was added to the tissue section for antigen-antibody reaction and then observed under a microscope. In the case of fluorescent staining, 1 drop of sealing material was added to the tissue section, covered with a coverslip, and observed under a microscope.

[0166] <Primary Antibody Used (Dilution Ratio) - Secondary Antibody>

[0167] Anti-mCherry antibody (abcam, 1 / 500) - anti-rabbit IgG antibody (Nichirei Biosciences)

[0168] Anti-Synaptophysin antibody (abcam, 1 / 500) - Anti-rabbit IgG antibody (Nichirei Biosciences)

[0169] Anti-chromatin A antibody (DAKO, 1 / 500) - Anti-rabbit IgG antibody (Nichirei Biosciences)

[0170] Anti-Ki67 antibody (abcam, 1 / 200) - anti-rabbit IgG antibody (Nichirei Biosciences)

[0171] Anti-insulin antibody (DAKO) - Anti-guinea pig IgG antibody (BIOTIUM)

[0172] Anti-somatostatin antibody (Santacruz, 1 / 300) - Anti-mouse IgG antibody (Biotinum)

[0173] Anti-glucagon antibody (Santacruz, 1 / 300) - anti-mouse IgG antibody (BIOTIUM)

[0174] (vii) RNA recovery, RNA extraction and cDNA synthesis

[0175] For RNA recovery, cultured cells were washed with PBS(-) (Nakalai Tasque) and then lysed with 350 μL of LBP buffer. RNA extraction was performed using NucreoSpin (registered trademark) RNA Plus (TAKARA) according to the recommended protocol. cDNA synthesis was performed using the Primescript Single-Stranded cDNA Synthesis Kit (TAKARA) according to the recommended protocol.

[0176] (viii) qRT-PCR

[0177] GoTaq qPCR Master Mix (Promega) was used following the recommended protocol. Analysis was performed using the Stepone Plus system (Life Technologies). The following primers and PCR reaction conditions were used.

[0178] [Table 1]

[0179] (a) Primers

[0180]

[0181] (b) PCR reaction conditions

[0182] 95°C, 2 minutes

[0183] 95°C (15 seconds), 60°C (1 minute) [40 cycles]

[0184] 95℃ (15 seconds), 60℃ (1 minute), 95℃ (15 seconds)

[0185] (ix) Islet Isolation

[0186] 8-week-old mice were anesthetized by intraperitoneal injection of sodium pentobarbital (Kyoritsu Pharmaceutical Co., Ltd.). After opening the abdomen, the common bile duct opening in the duodenum was identified, and the upper part of the common bile duct and the intestine were stopped with Brudeck forceps. The common bile duct was cut at the border between the common bile duct and the duodenum, and 2 mL of M199 culture medium (Gibco) containing Colalgenase P (Roche) (2 mg / mL) was injected. Thereafter, the pancreas was removed and digested in a 37°C hot water bath for 11 minutes and 30 seconds. It was suspended with 25 mL of M199 culture medium containing 10% FBS and centrifuged twice (1000 rpm, 4°C, 2 minutes). The supernatant was discarded, and after being suspended with 10 mL of Histopaque (SIGMA), 10 mL of M199 culture medium containing 10% FBS was injected thereon 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 further added, followed by centrifugation (1000 rpm, 4°C, 2 minutes).

[0187] (ix) Islet dispersion

[0188] The isolated islets were collected in a 1.5 mL silicon tube, 100 μL of a dispersion buffer solution (see Table 2 below) was added, and the tube was allowed to stand at 37° C. for 15 minutes, and then dispersed by pipetting.

[0189] [Table 2] Dispersion buffer

[0190] Solution 1 10ml Solution 2 10ml 0.1M EGTA 4ml Hepes 0.1909g BSA 0.04g <![CDATA[H2O]]> 20ml

[0191] (pH adjusted to 7.4)

[0192] [Table 3]

[0193]

[0194] (x) Pancreatic islet culture (three-dimensional culture method using gel)

[0195] After adding 20 μL of Matrigel (Corning) containing isolated pancreatic islets to a 96-well plate (Corning), the plates were incubated at 37°C for 15 minutes to gel the Matrigel. Then, 140 μL of a growth factor-containing culture medium (see the table below) was added, and the plates were cultured at 37°C under 5% CO2.

[0196] [Table 4]

[0197]

[0198]

[0199] (xi) Pancreatic islet culture (suspension culture)

[0200] 2 mL of culture medium was added to a 6-well plate, and cell culture inserts (Millipore) were suspended in the culture medium. Isolated islets and 20 μL of culture medium were added to the cell culture inserts, and cultured at 37°C under 5% CO 2 .

[0201] (xii) Intraperitoneal glucose tolerance test (IPGTT)

[0202] The Mycl-expressing and control groups were fasted for 12-16 hours. The mice were then weighed and injected intraperitoneally with a D-glucose solution at 2 g / kg (mouse). Blood was drawn from the tail of each mouse 15, 30, 60, and 120 minutes later, and blood glucose levels were measured using an Antons stage (Horiba).

[0203] (xiii) Breeding of diabetic mice

[0204] 8-12 week old immunodeficient (NOD / SCID) mice were weighed and injected intraperitoneally with 150 mg / kg of streptozotocin (STZ) (Sigma-Aldrich). One week later, blood glucose levels were measured using an Antons stage (Horiba). Mice with a blood glucose level consistently above 250 mg / dL were designated as diabetic mice.

[0205] (xiv) Islet transplantation (subrenal transplantation)

[0206] Diabetic mice were anesthetized by intraperitoneal injection of sodium pentobarbital (Kyoritsu Pharmaceutical Co., Ltd.). After laparotomy, the renal capsule was incised using a needle. A silicone tube was inserted beneath the renal capsule, and the islets were transplanted using a Hamilton syringe. After transplantation, the silicone tube was removed, and the peritoneum and tunica were sutured.

[0207] (xv) Kidney removal

[0208] Mice transplanted with islets were anesthetized by intraperitoneal injection of sodium pentobarbital (Kyoritsu Pharmaceutical Co., Ltd.). After laparotomy, the kidneys with the transplanted islets were removed, and the renal vein and artery were ligated with ligatures. After kidney removal, the peritoneum and epithelium were sutured.

[0209] (xvi) Single-cell analysis

[0210] Reanalysis was performed using publicly available analysis data from the GEO database (GSE101099; Byrnes LE et al., Nat Commun. 2018 Sep 25; 9(1): 3922). Seurat v2.2 and v2.3 (Satija R. et al., Nat Biotechnol. 2015 33: 495-502) were used for t-SNE analysis.

[0211] Example 1: Construction of mouse ES cells capable of inducible Mycl expression

[0212] The ES cell line with the Mycl gene inserted above was studied to see whether it could express in a Dox-dependent manner. The expression of the Mycl gene can be confirmed by the expression of the mCherry gene integrated downstream of the gene. The mCherry gene is a gene that encodes a red fluorescent protein, and the expression of this gene makes the cells appear red. Figure 1 As shown, when the gene expression before Dox addition was compared with the gene expression after Dox addition, Mycl gene expression was significantly increased, indicating that mouse ES cells capable of inducible Mycl expression can be cultivated.

[0213] In addition, the ES cell lines in which Myc and Mycn genes were integrated were also investigated to see whether gene expression could be induced in a Dox-dependent manner. Figure 12 As shown, the expression of each gene was observed similarly to the induction of Mycl expression.

[0214] On the other hand, when the expression of Ki67 protein, an indicator of cell proliferation, was observed, the number of proliferating cells (Ki67-positive cells) increased with the extension of the Dox administration period, and the number of Ki67-positive cells decreased significantly 2 weeks after the cessation of Dox administration, and cell proliferation stopped ( Figure 7 ). The above results indicate that ES cells into which the Mycl gene has been introduced achieve cell proliferation after gene expression is induced by Dox.

[0215] Example 2: Proliferation of pancreatic islet cells based on Mycl overexpression (formation of neuroendocrine tumors)

[0216] The 8-week-old chimeric mice expressing the Mycl gene prepared above were administered Dox for 8 weeks. After 8 weeks of Dox administration, the mice were sacrificed, the pancreas was removed, and the pancreatic sections were subjected to histochemical evaluation. Figure 2 As indicated by the middle arrow, it was found that in the pancreatic tissue overexpressing Mycl by Dox induction, islet cells proliferated and islets became hypertrophic compared to normal pancreatic tissue.

[0217] Example 3: Proliferation of pancreatic islet precursor-like cells based on Mycl overexpression

[0218] After 4 weeks of Dox administration to 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. Figures 3 to 6 As shown, proliferating cells showed gene expression similar to that of pancreatic islet precursor cells in the presence of the Mycl gene, and simultaneously expressed somatostatin, leading to their proliferation. In contrast, insulin-positive cells decreased.

[0219] Example 4: Proliferation of somatostatin-positive islet-like cells based on Mycl overexpression

[0220] In the same experimental system as in Example 2, the proliferation of somatostatin-positive cells and insulin-positive cells was examined 8 weeks after Dox administration. Figure 4 and Figure 5 As shown, insulin-positive cells decreased, while somatostatin-positive cells increased significantly.

[0221] Example 5: Dedifferentiation into pancreatic islet precursor cell-like cells based on Mycl overexpression

[0222] In the same experimental system as in Example 2, gene expression in the hypertrophic pancreatic islets after 8 weeks of Dox administration was analyzed. It is believed that pancreatic islets are derived from pancreatic progenitor cells marked by Ngn3, which differentiate into various pancreatic islet cells through pancreatic progenitor cells marked by Fev ( Figure 6 Analysis of gene expression in pancreatic islet cells proliferated by overexpression of Mycl revealed an increase in the expression of Fev, a marker of progenitor cells, and Pax4 and Cck, which are expressed at the same time ( Figure 13 ). These results indicate that Mycl overexpression can induce proliferative pancreatic islet progenitor-like cells.

[0223] Example 6: Induction of insulin-positive cells based on cessation of Mycl expression

[0224] In the experimental system used in Example 2, Dox administration was stopped after 8 weeks, and the increase or decrease of insulin-positive cells and somatostatin-positive cells was observed after 2 weeks and 4 weeks. Figure 5 As shown, 2 weeks and 4 weeks after the cessation of Dox administration, the decreased insulin-positive cells increased, whereas the increased somatostatin-positive cells decreased ( Figure 8 ) This result indicates that pancreatic islet progenitor-like cells that proliferate by Mycl overexpression are transformed into insulin-positive cells by stopping Mycl expression.

[0225] Furthermore, when the expression of Ki67, a target protein known to have cell proliferation ability, was observed, the number of proliferating cells (Ki-positive cells) increased as the duration of Dox administration prolonged, and the number of Ki-positive cells decreased significantly 2 weeks after Dox administration was stopped, and cell proliferation stopped ( Figure 7 ).

[0226] Example 7: Enhancement of Glucose Tolerance Based on Transient Overexpression of Mycl

[0227] When Dox administration was terminated, the glucose tolerance of the chimeric mice was evaluated. Figure 9 As shown on the left, it can be seen that the glucose tolerance of mice that stopped Dox administration was improved compared to the control ("cont."). In addition, the fasting blood sugar level was maintained, suggesting that the enlarged islets in the body are functional islets ( Figure 9 right).

[0228] Example 8: Induction of pancreatic islet cell proliferation in vitro based on Mycl overexpression

[0229] (1) Induction of Mycl expression in isolated pancreatic islets

[0230] The pancreas was removed from the chimeric mice, and the induction of Mycl expression was studied in the isolated pancreatic islets. The start day of Dox addition was set as day 0, and Mycl expression was observed over time. The results are shown in FIG. Figure 10 Compared with the control, in samples treated with Dox, pancreatic islets tended to increase in size over time as Mycl gene expression increased.

[0231] (2) Induction of Mycl expression in isolated pancreatic islets into dispersed cells

[0232] The expression of Mycl gene in the cells dispersed from the isolated pancreatic islets was examined in the same manner as in (1). When Mycl gene was observed in cells on the 14th day after Dox addition, it was found that Mycl gene was expressed in each cell compared to the 1st day after Dox addition ( Figure 11 ).

[0233] (3) Induction of c-Myc and Mycn expression in isolated pancreatic islets in vitro

[0234] It was confirmed that the ES cells integrated with c-Myc and Mycn cultured above can be induced to express in a doxycycline-dependent manner. Chimeric mice were cultured using these ES cells. In pancreatic islet cells isolated from the cultured chimeric mice, the expression of c-Myc and Mycn was induced by adding doxycycline in vitro for 1 week. The results showed that although the expression induction of c-Myc and Mycn promoted proliferation, it induced cell death ( Figure 12 ) (See Pelengaris S, Khan M, Evan GI., Cell 2002; 109(3): 321-334).

[0235] Example 9: Functional evaluation of pancreatic islet cells induced to proliferate in vitro

[0236] The functionality of the proliferated islet cells was studied by transplanting them into diabetic mice through in vitro induced proliferation of Mycl overexpression. 30 isolated islets were dispersed and Dox was added for 1 week to induce the proliferation of islet cells. The islet cells were recovered and transplanted under the renal capsule of diabetic mice, and it was found that blood sugar improved at any time. In addition, the kidneys transplanted with islet cells were removed after 2 weeks, and the diabetic mice recovered hyperglycemia as a result. Generally speaking, in order to lower the blood sugar level of diabetic mice, more than 300 islets need to be transplanted. The blood sugar level of diabetic mice was successfully lowered using islet-like cells proliferated from 60 islets.

[0237] Example 10: Verification of the therapeutic effect of Mycl expression induction on diabetic mouse model

[0238] A diabetic mouse model was established to verify the therapeutic effect of inducing Mycl expression. Eight-week-old mice (KH2-Mycl) with inducible Mycl expression were intraperitoneally administered 150 mg / kg of streptozotocin (STZ), which is toxic to pancreatic β cells. Two weeks later, Mycl expression was induced by Dox administration for 8 weeks. Mycl expression was stopped by stopping Dox administration for an additional 2 weeks. At this point, a glucose responsiveness test (IPGTT) and histological analysis were performed.

[0239] 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 by Dox (STZ+Mycl), blood glucose decreased after Dox administration and became normal even after Dox administration was stopped ( Figure 14 ). Furthermore, a sugar responsiveness test was performed, and a rapid decrease in blood sugar levels was observed in the group in which Mycl expression was induced ( Figure 14 lower left).

[0240] Furthermore, histological analysis of insulin ("Ins"), glucagon ("Gog"), and somatostatin ("Sst") expression revealed a decrease in the proportion of insulin-positive cells in the control group (STZ alone). On the other hand, in the group in which Mycl expression was induced (STZ+Mycl), enlargement of pancreatic islet cells and an increase in the proportion of insulin-producing cells were observed. These results suggest that inducing Mycl expression can be expected to have therapeutic effects on diabetic diseases.

[0241] Example 11: Mycl does not induce abnormal proliferation outside of pancreatic islets

[0242] To induce the expression of MYC family genes ("Myc", "Mycn" and "Mycl") in a Dox-dependent manner in vivo, ES cells capable of inducible expression of these genes were first cultured ( Figure 15 The ES cells were injected into blastocysts to create chimeric mice. Dox was added to the chimeric mice for four weeks starting at four weeks of age to induce expression of MYC family genes.

[0243] Myc expression induces tumor formation in the liver, Mycn expression induces tumor formation in the liver and intestine, while Mycl expression induces no abnormal proliferation at least in the liver and small intestine ( Figure 15 This indicates that Mycl has a specific proliferation effect in pancreatic islets.

[0244] Example 12: Islet proliferation in aged mice

[0245] Islet cells were isolated from aged mice (115 weeks old) and transfected with a streptovirus capable of inducibly expressing Mycl and the pZsGreenDR vector (Takara, cat# 632428) (control group) in a Dox-dependent manner. The zsGreenDR vector used is a short-lived green fluorescent protein with a proteolysis signal fused to the C-terminus of zsGreen.

[0246] On the second day after infection, the islet cells were transferred to a three-dimensional culture using Matrigel and Dox was administered to induce the expression of Mycl and zsGreenDR. Even after 7 days, no morphological changes were found in the islet cells expressing induced zsGreenDR ( Figure 16 On the other hand, the enlargement of pancreatic islet cells was observed by inducing Mycl expression ( Figure 16 Left). These results indicate that pancreatic islet proliferation can be induced by Mcyl expression even in very old mice.

[0247] Example 13: Expression of Mcyl in human pancreatic islet precursor cells

[0248] In a paper published in 2020 (JR Alvarez-Dominguez, et al., Circadian Entrainment Triggers Maturation of Human In Vitro Islets., Cell Stem Cell, 26(1), 108-122, 2020), the molecular profile of human pancreatic β cells was observed when human iPS cells were differentiated in stages. Therefore, this dataset was used to observe the expression of genes of the MYC family (as described above) and H3K27ac (a chromatin mark that generally indicates transcriptional activation) ( Figure 17 ).

[0249] While Myc family gene expression increases in human pluripotent stem cells (hPSCs) and tends to decrease with differentiation, Mycl expression has been observed to transiently increase in endocrine precursor cells (i.e., pancreatic islet precursor cells). This suggests that, similar to mice, Mycl may contribute to islet cell proliferation in human islets.

[0250] Example 14: Confirmation of human pancreatic islet proliferation

[0251] Human pancreatic islet cells were infected with a 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 a three-dimensional culture using Matrigel and Dox was administered to induce the expression of Mycl and zsGreenDR.

[0252] The morphology of islet cells induced by expression of zsGreenDR did not change even after 7 days, but when MYCL was expressed, the islet cells were enlarged ( Figure 18 Lower left). Furthermore, the results of immunostaining showed that Ki67 (a cell proliferation marker) was detected in cells in which mCherry expression (synonymous with MYCL expression) was confirmed in the MYCL overexpression group ( Figure 18 lower right).

[0253] Example 15: Confirmation of human islet proliferation by single cell analysis

[0254] Human pancreatic islet cells were infected with lentiviruses that induce the expression of Mycl, MYCL, and zsGreenDR (control) in a Dox-dependent manner. The next day, the cells were transferred to suspension culture using cell culture inserts and simultaneously administered with Dox to induce the expression of Mycl and zsGreenDR, respectively. Seven days later, dead cells were removed, and single-cell RNA-seq was performed according to conventional methods.

[0255] The results of single-cell RNA-seq detected α cells, β cells, δ cells, pancreatic duct cells ("PP"), mesenchymal cells, vascular endothelial cells, and astrocytes ( Figure 19 Left). Specifically, analysis of CDK4 expression, a cell proliferation marker, focused solely on β cells revealed no effect of zsGreenDR expression induction on CDK4. However, induction of Mycl and MYCL expression induced CDK4 expression. This suggests that induction of Mycl expression can also promote cell proliferation in human pancreatic islet cells.

[0256] [Industrial Applicability]

[0257] If the technology of the present invention is applied to islets isolated from donors, the number of insulin-positive islet cells can be proliferated in vitro, and the problem of insufficient islet donors in the present invention may be solved. By using this technology in the differentiation induction system of islet insulin-positive cells from ES / iPS cells, it is possible to effectively induce islet insulin-positive cells from pluripotent stem cells. Furthermore, by applying the technology of cultivating islets from pluripotent stem cells using the blastocyst complement method, the number of insulin-positive cells in the cultivated organs can be increased. The present invention requires transient expression of Mycl in islet cells from donors and islet cells from pluripotent stem cells, but it is believed that by introducing additional vectors, RNA, etc., it is also possible to cultivate islet cells in large quantities without genetic modification. In addition, it is expected that islet cells proliferated using this technology can be maintained and proliferated in the form of islet reserve cell lines. Islet reserve cell lines have the potential to provide long-term, stable and large quantities of insulin-positive cells. It is expected that in the future, many diabetic patients will be able to provide cheap and high-quality islets.

[0258] All publications and patent documents cited in this specification are incorporated herein by reference in their entirety. It should be noted that, while specific embodiments of the present invention are described in this specification for illustrative purposes, various modifications may be made without departing from the spirit and scope of the present invention, as will be readily understood by those skilled in the art.

Claims

1. Use of the Mycl gene or its gene product in the preparation of an insulin production promoter, a pharmaceutical composition for preventing and / or treating diabetes and related diseases, or an agent for pancreatic islet cell proliferation; The related diseases are impaired glucose tolerance, hyperglycemia, diabetic complications, impaired insulin secretion or metabolic syndrome; The Mycl gene is: (1) a nucleic acid having a base sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 3; or (2) A nucleic acid that hybridizes under stringent conditions with a nucleic acid represented by a base sequence such as SEQ ID NO: 1 or a complementary sequence to SEQ ID NO: 3 and encodes a polypeptide having insulin-promoting activity produced when the Mycl gene is induced to express.

2. The use according to claim 1, wherein The Mycl gene product is a polypeptide with an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO:

4.

3. The use according to claim 1, wherein: The Mycl gene or its gene product is introduced into pancreatic islet cells.

4. The use according to claim 1, wherein The Mycl gene is transiently expressed.

5. The use according to claim 3 or 4, wherein: The pancreatic islet cells are derived from primary pancreatic islet cells isolated from the pancreas, cultured pancreatic islet cells or stem cells.

6. The use according to claim 5, wherein: The stem cells are selected from iPS cells, ES cells and adult stem cells.

7. The use according to claim 1, wherein: The pharmaceutical composition comprises: (i) Mycl gene or its gene product; a vector incorporating the Mycl gene; and / or pancreatic islet cells into which the Mycl gene or its gene product has been introduced or induced to express; and (ii) a pharmaceutically acceptable excipient, diluent or carrier.

8. The use according to claim 7, wherein: The diabetes and related diseases thereof are type I diabetes, type II diabetes, impaired glucose tolerance, hyperglycemia or metabolic syndrome.

9. The use according to claim 8, wherein: Type 1 diabetes includes slowly progressive type 1 diabetes and type 1.5 diabetes.

10. A method for proliferating pancreatic islet cells, comprising: The method comprises the step of expressing the Mycl gene; the method is not used to treat humans or animals; the Mycl gene is: (1) a nucleic acid having a base sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 3; or (2) A nucleic acid that hybridizes under stringent conditions with a nucleic acid represented by a base sequence such as SEQ ID NO: 1 or a complementary sequence to SEQ ID NO: 3 and encodes a polypeptide having insulin-promoting activity produced when the Mycl gene is induced to express.

11. The method according to claim 10, characterized in that The expression of the Mycl gene is transient.

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