Title of invention: Induction of proliferative pancreatic islet progenitor-like cells and induction of differentiation into insulin-positive cells by transient expression of Mycl
Technical field
[0001]
The present invention relates to an insulin production promoter containing the Mycl gene or pancreatic islet-like cells introduced with the Mycl gene. The present invention also relates to a pharmaceutical composition for preventing/treating diabetes, which contains the Mycl gene or its gene product, or pancreatic islet-like cells transfected with the Mycl gene. Furthermore, the present invention relates to a method for inducing proliferation-capable pancreatic islet progenitor cell-like cells by transient expression of the Mycl gene and inducing differentiation into insulin-producing cells.
Background technology
[0002]
It has been shown that repeated short-term expression of cell reprogramming factors in mouse individuals proliferates pancreatic islet cells and improves glucose tolerance (Non-Patent Document 1).
[0003]
Pancreatic islet transplantation is a medical treatment that makes it possible to keep blood sugar stable by transplanting pancreatic islet tissue for diabetes. Development of a method for regenerating pancreatic islet function is urgently required. In recent years, the establishment of pancreatic islet-like insulin-producing cells by inducing the differentiation of ES/iPS cells has been reported. Problems such as autoimmune reactions are also of concern.
prior art documents
Non-patent literature
[0004]
Non-Patent Document 1: Cell, 2016, 167, 1719-1733, e12. doi: 10/1016/j. cell. 2016. 11. 052
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0005]
The technique of expanding pancreatic islet insulin-producing cells by transient expression of the Mycl gene is expected to establish a large number of functional pancreatic islet insulin-producing cells from pancreatic islets derived from a small number of donors. Furthermore, if this technology is applied to the process of inducing pancreatic islet insulin cells from ES/iPS cells, efficient production of insulin-positive cells from stem cells can be expected. The present invention aims to provide a means for treating diabetes comprising removing islet cells from a subject, inducing proliferative islet progenitor-like cells in vitro, and then returning the islet cells to the subject. do. A further object of the present invention is to provide means for treating diabetes by transiently overexpressing the Mycl gene in pancreatic islet cells in the body.
Means to solve problems
[0006]
The present inventors discovered that proliferative pancreatic islet progenitor-like cells can be induced by introducing the Mycl gene into pancreatic islet cells and expressing the gene. In the presence of the Mycl gene, the proliferating cells express the Fev gene, the Pax4 gene, and the Cck gene, which are expressed in fetal islet progenitor cells, while simultaneously expressing somatostatin and proliferating. , differentiate into insulin-positive cells with cessation of proliferation. That is, by transiently inducing the expression of the Mycl gene, proliferative islet progenitor-like cells can be induced, and the proliferated islet progenitor-like cells can be differentiated into insulin-positive cells. I have completed my invention.
[0007]
That is, the present invention has the following aspects.
[1] An insulin production promoter containing the Mycl gene or its gene product.
[2] Mycl gene is
(1) a nucleic acid containing the base sequence represented by SEQ ID NO: 1 or 3; or
(2) a polypeptide that hybridizes under stringent conditions with a nucleic acid comprising a nucleotide sequence represented by SEQ ID NO: 1 or 3 and has an activity of promoting insulin production when the expression of the Mycl gene is induced; encoding nucleic acid
The insulin production promoter according to [1], comprising
[3]
The Mycl gene product is
(1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or 4; or
(2) has at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO: 2 or 4, and islet-like cells A polypeptide having a proliferative effect and/or an activity to promote insulin production
The insulin production promoter according to [1], comprising
[4] An insulin production promoter in which the Mycl gene or its gene product has been introduced into pancreatic islet cells.
[5] The insulin production promoter according to any one of [1] to [4], in which the Mycl gene is transiently expressed.
[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.
[7] The insulin production promoter according to [6], wherein the stem cells are selected from the group consisting of iPS cells, ES cells, and somatic stem cells.
[8] A pharmaceutical composition for preventing and/or treating diabetes and diseases related thereto,
(i) the Mycl gene or its gene product; a vector into which the Mycl gene is integrated; and/or an islet cell into which the Mycl gene or its gene product has been introduced or whose expression has been induced;
(ii) the pharmaceutical composition comprising a pharmaceutically acceptable excipient, diluent or carrier;
[9] diabetes and diseases associated therewith are selected from type I diabetes, type II diabetes, impaired glucose tolerance, hyperglycemia, dyslipidemia, obesity or metabolic syndrome-related diseases, disorders or conditions; The pharmaceutical composition of [8].
[10] Mycl gene is
(1) a nucleic acid containing the base sequence represented by SEQ ID NO: 1 or 3; or
(2) a polypeptide that hybridizes under stringent conditions with a nucleic acid comprising a nucleotide sequence represented by SEQ ID NO: 1 or 3 and has an activity of promoting insulin production when the expression of the Mycl gene is induced; encoding nucleic acid
The pharmaceutical composition according to any one of [8] or [9], comprising
[11] The Mycl gene product is
(1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or 4; or
(2) has at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO: 2 or 3, and islet-like cells Polypeptides having proliferative effects and/or insulin production-promoting effects
The pharmaceutical composition according to any one of [8] or [9], comprising
[12] A kit comprising the insulin production enhancer according to any one of [1] to [7] or the pharmaceutical composition according to any one of [8] to [11].
[13] A kit further comprising an activator for activating the Mycl gene.
[14] the active agent for activating the Mycl gene is selected from the group consisting of promoters, enhancers, promoter-activating enzymes or factors, enhancer-activating enzymes or factors, nucleic acid-protein complexes, and low-molecular-weight compounds The kit of [13], which is selected.
　
[15] Pancreatic islet cells into which the Mycl gene or its gene product has been introduced.
[16] The islet-like cells according to [15], wherein the islet cells are derived from primary islet cells isolated from the pancreas, cultured islet cells, or stem cells.
[17] A method for preparing islet-like cells according to [16],
(a) the step of integrating the Mycl gene into a recombinant plasmid, recombinant viral vector, minicircle, or episomal vector; and
(b) A step of introducing the recombinant plasmid, recombinant viral vector, minicircle, or episomal vector obtained in step (a) into pancreatic islet cells
method including.
A method for preparing islet-like cells according to [18] and [16], comprising the step of introducing RNA encoding the Mycl gene or Mycl protein into the islet cells.
[19] A method for growing the islet-like cells according to [15] or [16] or the islet-like cells prepared by the method according to [17] or [18], comprising the step of expressing the Mycl gene.
[20] The method of [19], wherein the expression of the Mycl gene is transient.
Effect of the invention
[0008]
According to the present invention, by controlling the expression of the exogenous or endogenous Mycl gene, it is possible to treat diabetes or its related diseases in which insulin production is desired.
Brief description of the drawing
[0009]
[Fig. 1] Fig. 1 shows the establishment of mouse ES cells capable of inducing Mycl expression.
[Fig. 2] Fig. 2 shows pancreatic islet expansion due to Mycl overexpression.
[Fig. 3] Fig. 3 shows proliferation of somatostatin-positive pancreatic islet cell-like cells due to Mycl overexpression.
[Fig. 4] Fig. 4 shows proliferation of somatostatin-positive pancreatic islet cell-like cells due to Mycl overexpression.
[Fig. 5] Fig. 5 shows proliferation of somatostatin-positive pancreatic islet cell-like cells due to Mycl overexpression.
[FIG. 6] Somatostatin-positive pancreatic islet cell-like cells proliferated by Mycl overexpression resemble fetal islet progenitor cells.
FIG. 7 shows proliferation induction of pancreatic islet progenitor-like cells by Mycl overexpression and proliferation arrest by Mycl overexpression cessation.
[Fig. 8] Fig. 8 shows differentiation of islet progenitor-like cells into insulin-positive cells by terminating Mycl expression.
[Fig. 9] Fig. 9 shows enhancement of glucose tolerance by transient overexpression of Mycl.
[Fig. 10] Fig. 10 shows in vitro induction of Mycl expression in isolated pancreatic islets and expansion of pancreatic islets.
[Fig. 11] Fig. 11 shows in vitro Mycl expression induction in cells dispersed from isolated pancreatic islets and proliferation of pancreatic islet cells.
FIG. 12 shows the establishment of mouse ES cells capable of inducing c-Myc gene or Mycn gene expression.
[Fig. 13] Fig. 13 shows in vitro cell death by c-Myc gene or Mycn gene expression in isolated pancreatic islets.
[Fig. 14] Fig. 14 shows therapeutic effects in a diabetic mouse model.
[Fig. 15] Fig. 15 shows that Mycl does not induce abnormal proliferation in cells other than pancreatic islets.
[Fig. 16] Fig. 16 shows pancreatic islet proliferation in aged mice.
[Fig. 17] Fig. 17 shows the expression of Mcyl in human pancreatic islet progenitor cells.
[Fig. 18] Fig. 18 shows diagrams confirming pancreatic islet hyperplasia in humans.
[Fig. 19] Fig. 19 shows the results of confirmation of pancreatic islet hyperplasia in humans by single-cell analysis.
MODE FOR CARRYING OUT THE INVENTION
[0010]
The present invention relates to an insulin production promoter comprising the Mycl gene or its gene product, or pancreatic islet-like cells into which the Mycl gene has been introduced. The present invention relates to a pharmaceutical composition for preventing/treating diabetes containing these insulin production-enhancing agents. The invention is described in detail below.
[0011]
1. Pancreatic islet-like cells and preparation method thereof
(1) Islet-like cells
As used herein, the term "pancreatic islet-like cells" refers to pancreatic islet cells into which the Mycl gene or its gene product has been introduced. In the present specification, pancreatic islet-like cells that have transitioned to the proliferation phase due to forced expression of the Mycl gene are sometimes referred to as "pancreatic islet progenitor-like cells". Furthermore, by stopping the expression of the gene, the cells can be differentiated into cells having insulin-producing ability (hereinafter also referred to as "insulin-producing cells"). That is, according to the present invention, transient expression of the Mycl gene can increase the number of pancreatic islet-like cells and differentiate them into insulin-producing cells. Here, focusing on the markers expressed in each cell, "pancreatic islet progenitor cell-like cells" include, for example, at least one, preferably two, genes selected from Fev, Pax4, Cck, CDK4, and Ki67, More preferably three or more are positive. In addition, when focusing on the expressed proteins, it is also characterized by reduced insulin and glucagon production compared to normal pancreatic islets, or marked somatostatin production.
[0012]
(2) Pancreatic islet cells
"Pancreatic islet cells" are generally called islets of Langerhans, and are cell aggregates called pancreatic islets that control the endocrine function of the pancreas, and refer to endocrine cells that account for about 1-2% of all pancreatic cells. Pancreatic islet cells are mainly composed of five types of cells: α cells, β cells, δ cells, ε cells, and PP cells. The major cells that make up cell aggregates are β cells, which occupy the central part of the pancreas. β-cells They make up about 60-80% of the coalescence and secrete insulin, which allows glucose transport to most cells in the body. Alpha cells, on the other hand, occupy about 10-30% of pancreatic islets and secrete glucagon, which is released during starvation, allowing the hepatic release of glucose to maintain normal blood glucose. Delta cells make up about 5-10% of pancreatic islet cells and secrete somatostatin, which further regulates glucose concentration. Epsilon cells and PP cells also oversecrete ghrelin and pancreatic polypeptides, respectively. Pancreatic polypeptide-producing cells (about 5-10% of pancreatic islet cells) release hormones that alter exocrine and gastrointestinal function. There are other islet cell types such as endothelial cells, neuronal cells, and progenitor cells.
[0013]
As used herein, pancreatic islet cells include the above-described islet cells and pancreatic islet progenitor cells that are progenitor cells of pancreatic islet cells. It may also be an intermediate cell that is generated up to the islet cell or islet progenitor cell that is generated in the process of inducing the differentiation of cells. Here, the “intermediate cell” is preferably a cell whose fate is determined to differentiate into a pancreatic islet cell. Furthermore, in the present invention, pancreatic islet cells may be those prepared from pancreatic islet-like cells or pancreatic islet progenitor-like cells into which the Mycl gene or its gene product has been introduced.
[0014]
In type I diabetes, cell infiltration, mainly composed of lymphocytes, is observed shortly after onset. Eventually, β-cells selectively disappear, and the pancreatic islet volume decreases, and α-cells become the main constituent. Pancreatic islets have a reserve capacity for insulin secretion, and type I diabetes develops when 90-95% of β-cells have disappeared. In type II diabetes, basically no morphological change is observed in pancreatic islets, but the insulin secretory ability of pancreatic islets is reduced.
[0015]
In the present invention, the origin or source of islet cells into which the Mycl gene or its gene product is introduced is not limited, but primary islet cells isolated from the pancreas of an individual or islet cells cultured by a known culture method. There may be. Cultured islet cells include, but are not limited to, established pancreatic islet cells, stem cell (e.g., iPS cells, ES cells, somatic stem cells)-derived pancreatic islet cells (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 is introduced may be islet-like cells obtained from pancreatic islet-like cells and/or islet progenitor-like cells, or islet-like cells into which the Mycl gene has already been introduced. The cells and/or pancreatic islet progenitor-like cells may be repeatedly introduced with the Mycl gene or its gene product. In particular, for the purpose of treating diabetes, both donor-derived pancreatic islet cells and stem cell-derived pancreatic islet cells can be preferably used. The islet cells from the donor used can be either autologous or xenogeneic to the recipient. According to the present invention, by introducing the Mycl gene into such pancreatic islet cells, it is possible to treat diabetes by proliferating pancreatic islet progenitor-like cells in vitro and returning (transplanting) them to the patient. . In this case, in cases where pancreatic islet cells are allogeneic to the patient, the patient can be administered an immunosuppressant as appropriate. For transplantation into a patient, the above-described islet-like cells, pancreatic islet progenitor-like cells, cells having insulin-producing ability, or any combination thereof can be used.
[0016]
In relation to the above, it has been reported that, at least in mice, when β-cells are shed, α-cells proliferate, and then some α-cells transdifferentiate into β-cells. Therefore, the pancreatic islet as a raw material for islet cells before the introduction of the Mycl gene may be an islet from which most of the β cells have been eliminated, in addition to a pancreatic islet containing a large number of normal β cells. From this point of view, the present invention can also be used for gene therapy of type I diabetic patients who have lost β cells by using pancreatic islet-like cells transfected with the Mycl gene.
[0017]
(3) Pluripotent stem cells
As used herein, the term "pluripotent stem cell" refers to a cell that has self-renewal ability and multipotency, and that has the ability to form all the cells that make up an organism. "Self-renewal ability" refers to the ability to generate undifferentiated cells identical to itself from one cell. "Differentiation potential" refers to the ability of a cell to differentiate. Pluripotent stem cells include, for example, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), Muse cells (Multi-lineage differentiating Stress Enduring cells), and spermatogonial stem cells. (germline stem cells: GS cells), embryonic germ cells (EG cells) and the like, but are not limited to these. Pluripotent stem cells used in the present invention are preferably ES cells. The origin of the pluripotent stem cells is not particularly limited and may be mammals, birds, fish, reptiles or amphibians. Mammals include primates (humans, monkeys, etc.), rodents (mice, rats, guinea pigs, etc.), cats, dogs, rabbits, sheep, pigs, cows, horses, donkeys, goats, ferrets, and the like.
[0018]
As used herein, the term "ES cell" refers to a pluripotent stem cell that has the ability to differentiate into all tissue cells that constitute an individual that exists in the early stage of development, and is established so that it can be cultured in vitro. means something ES cells, like pluripotent stem cells in early embryos, can be proliferated virtually unlimitedly while retaining the ability to differentiate into all cells that constitute an individual. Specifically, mouse ES cells were first described in 1981 (Proc. Natl. Acad. Sci. USA 78, 7634-7638, 1981; Nature 292, 154-156, 1981). ES cells are pluripotent and can give rise to all the tissues and cell types that make up an individual. Rats (Iannaconns et al., Dev. Biol. 163, 288-292, 1994), hamsters (Dev. Biol. 127, 224-227, 1988), rabbits (Mol. Reprod. Dev. 36, 424-433, 1993) ), birds, fish, pigs (Reprod. Fertil. Dev. 6, 563-568, 1994), bovines (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 include, but are not limited to, KH2 cells, RF8 cells, JI cells, CGR8 cells, MG1.19 cells, 129SV cells, C57/BL6 cells, DBA-1 cells, and the like. .
[0019]
Several research teams have also succeeded in isolating ES cells and ES cell-like stem cells from embryonic human tissues. Early success stories include: (Science 282, 1145-1147, 1998; Proc. Natl. Acad. Sci. USA 95, 13726-13731, 1998; Nature Biotech., 18, 399-404, 2000). These ES cell lines are established by culturing ICM isolated from blastocysts on feeder cells. Other recent studies have shown that it is possible to obtain embryos and embryonic cells by transferring nuclei from embryos and mature mammalian cells into enucleated oocytes.
[0020]
Any established ES cell line can be used in the present invention. Alternatively, in order to prevent immune rejection when ES cells produced by the method of the present invention are applied to an individual, a cloned embryo is created using the somatic cells of the individual, and an ES cell line is established therefrom. It is valid. Using this method, it is possible to establish ES cells having the same genetic elements as the individual.
[0021]
Alternatively, in the creation of somatic cell cloning, it is believed that a phenomenon called "initialization" occurs in which the nucleus of the somatic cell introduced into the egg changes to a state similar to that of the fertilized egg. It has been reported that ES cells have similar activities to those of ovum (Curr. Biol., 11, 1553-1558, 2001). In other words, it is expected that by fusing the somatic cells of an individual with ES cells, the somatic cells can be converted into cells such as ES cells. Since ES cells can be genetically manipulated in vitro, ES cells pre-manipulated with factors involved in immune rejection, such as the MHC gene group, can be used without using techniques such as somatic cell cloning. It is expected that rejection can be avoided.
[0022]
As used herein, "induced pluripotent stem (iPS) cells" are obtained by introducing transcription factor genes such as Oct3/4, Sox2, Klf4, and c-Myc into somatic cells. , means cells with pluripotency similar to ES cells. Similarly to ES cells, iPS cells can also be increased indefinitely while maintaining pluripotency.
[0023]
The basic technique for producing iPS cells is to introduce four transcription factors, 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 produce iPS cells, i.e., cells that are derived from iPS cells, include lymphocytes (T cells, B cells), fibroblasts, epithelial cells, endothelial cells, mucosal epithelial cells, Leaf stem cells, hematopoietic stem cells, adipose stem cells, dental pulp stem cells, neural stem cells can be mentioned.
[0024]
Initialization of iPS cells can be performed by a method well known to those skilled in the art, for example, Addgene's Blog/Post, "Delivery Methods for Generating iPSCs" for-generating-ipscs). Methods for introducing the Mycl gene into iPS cells include, but are not limited to, recombinant viruses (e.g., retroviruses, lentiviruses, adenoviruses, Sendai viruses, etc.), recombinant plasmids, minicircles, or episomes (e.g., oriP/ Epstein-Barr nuclear antigen-1 (EBNA1)-based episomal vector), or a method of directly introducing RNA (including mRNA) encoding the Mycl gene or Mycl protein itself into cells.
[0025]
As used herein, "EG cell" means any embryonic germ stem cell produced from primordial germ cells, and its origin is not particularly limited. As used herein, "GS cells" are germ stem cells produced from testicular germ cells, and are cell lines that allow spermatogonial stem cells (spermatogonial stem cells) to be cultured in vitro (Cell. 119, 1001-1012, 2004). Among GS cells, mGS cells (multipotent germline stem cells), which have properties similar to those of ES cells and also have pluripotency, are particularly preferred.
[0026]
(4) Mycl gene and its gene product
The Mycl (also called "L-Myc") gene is one member belonging to the family of Myc genes, which includes the c-Myc gene and Mycn ("N-Myc" gene). The Mycl gene is similar to the c-Myc gene in cancer A gene, also known as a reprogramming gene. In addition, unlike the c-Myc gene, the Mycl gene is known to have little transforming ability (Nakagawa, M., et al., Proc. Natl. Acad. Sci. USA, vol. 107, p. 14152-14157, 2010). Mouse and human cDNA sequence information for Mycl can be obtained by referring to NCBI accession numbers NM 008506 and NM 001033081, respectively, and those skilled in the art can easily isolate the cDNA.
[0027]
According to the present invention, it is preferred to use the isolated Mycl gene and its gene product. As described above, the base sequence of the Mycl gene can be identified by the NCBI accession number, but usable Mycl genes also include single-stranded or double-stranded DNA and their RNA complements. . DNA includes, for example, naturally occurring DNA, recombinant DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. DNA is preferred as the nucleic acid used in the present invention. As is well known, codons are degenerate, and some amino acids have multiple nucleotide sequences encoding one amino acid. is not particularly limited as long as it has an effect of promoting cell proliferation by expression of and cessation of its expression to promote insulin production.
[0028]
In one embodiment, the Mycl gene is
(1) a nucleic acid comprising or consisting of a base sequence represented by SEQ ID NO: 1 or 3; or
(2) a polypeptide that hybridizes under stringent conditions with a nucleic acid comprising or consisting of the nucleotide sequence represented by SEQ ID NO: 1 or 3 and has the activity of proliferating pancreatic islet-like cells transfected with the Mycl gene; encoding nucleic acid; or
(3) an insulin-producing cell hybridized with a nucleic acid comprising or consisting of a nucleotide sequence represented by SEQ ID NO: 1 or 3 under stringent conditions, and by proliferating pancreatic islet-like cells introduced with the Mycl gene; may comprise/consist of a nucleic acid encoding a polypeptide that has the effect of increasing the proliferation of , and thus the effect of promoting insulin production.
[0029]
As used herein, "under stringent conditions" means hybridizing under moderately or highly stringent conditions. Specifically, moderately stringent conditions can be readily determined by one of ordinary skill in the art, eg, based on the length of the DNA. Basic conditions are described in Sambrook, J.; et al., Molecular Cloning, A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, 7.42-7.45 (2001); A pre-wash solution of 0 mM EDTA (pH 8.0), about 50% formamide at about 40-50°C, 2X SSC-6X SSC (or Stark's solution in about 50% formamide at about 42°C). 's solution), and the use of washing conditions of about 60° C., 0.5×SSC, 0.1% SDS. Highly stringent conditions can also be readily determined by one skilled in the art, eg, based on the length of the DNA. Generally, such conditions include hybridization and/or washing at higher temperatures and/or lower salt concentrations than moderately stringent conditions, e.g. Defined as 0.2×SSC, with a wash of 0.1% SDS. Those skilled in the art will recognize that the temperature and wash solution salt concentration can be adjusted as needed according to factors such as length of the probe.
[0030]
Homologous nucleic acids cloned using the above-described nucleic acid amplification reaction, hybridization, or the like have at least 30% or more, preferably 50% or more, More preferably 70% or more, even more preferably 90% or more, still more preferably 95% or more, and most preferably 98% or more. In addition, percent identity can be determined by visual inspection and mathematical calculations. Alternatively, the percent identity of two nucleic acid sequences can be determined according to Devereux et al., Nucl. Acids Res. , 12, 387 (1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG) by comparing sequence information using the GAP computer program (GCG Wisconsin Package, version 10.3). can do.
[0031]
In one embodiment, the gene product of the Mycl gene is a polypeptide expressed from the Mycl gene described above. Typically, the polypeptide is
(1) a polypeptide comprising or consisting of an amino acid sequence represented by SEQ ID NO: 2 or 4; or
(2) has at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO: 2 or 4, and islet-like cells It may be a polypeptide having a proliferative effect and/or an effect of promoting insulin production.
[0032]
In one embodiment, the gene product of the Mycl gene may be a variant of a polypeptide as defined above, wherein deletion, substitution, insertion of one or more amino acids in the amino acid sequence of SEQ ID NO: 2 or 4 and/or an amino acid sequence containing additions. Substitutions may also be conservative substitutions, which are the replacement of a particular amino acid residue with a residue having similar physico-chemical characteristics. Non-limiting examples of conservative substitutions include substitutions between aliphatic group-containing amino acid residues such as substitutions for Ile, Val, Leu or Ala for each other; Substitutions between polar residues such as substitutions and the like are included.
[0033]
Mutants due to deletion, substitution, insertion and/or addition of amino acids can be generated in the Mycl gene by site-directed mutagenesis (for example, Nucleic Acid Research, Vol. 10, No. 20, p. 6487), which is a well-known technique. -6500, 1982). As used herein, "one or more amino acids" means amino acids that can be deleted, substituted, inserted and/or added by site-directed mutagenesis. In addition, "one or more amino acids" as used herein may mean one or several amino acids depending on the case.
[0034]
In addition to the above-described site-directed mutagenesis, other methods for deleting, substituting, inserting and/or adding one or more amino acids in the amino acid sequence of a polypeptide while retaining its activity include Other methods include treatment with mutagens and selective cleavage of the gene followed by removal, substitution, insertion or addition of selected nucleotides followed by ligation. Although not limited, the gene product of the Mycl gene in the present invention is 1 to 10, preferably 9 or less, 7 or less, 5 or less, 3 or less, 2 or less in SEQ ID NO: 2 or 4. More preferably, it is a polypeptide consisting of an amino acid sequence in which one or less amino acids have been deleted, substituted or added, and having the activity of promoting insulin production.
[0035]
The variant further has 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 It is a protein comprising an amino acid sequence having a specific property, and is a polypeptide having the effect of promoting pancreatic islet-like cell proliferation and/or the effect of promoting insulin production.
[0036]
The percent identity of two amino acid sequences may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two protein sequences can be determined according to Needleman, S.; B. and Wunsch, C.; D. (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). may Preferred default parameters for the GAP program include: (1) Henikoff, S.; and Henikoff, J.; G. (Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992), a scoring matrix, blosum62; (2) a gap weight of 12; (3) a gap length weight of 4; and (4) no penalty for terminal gaps.
[0037]
(5) Introduction of Mycl gene
According to the present invention, the method for introducing the Mycl gene into primary pancreatic islet cells, cultured pancreatic islet cells, or stem cells (e.g., iPS cells, ES cells, somatic stem cells) is not particularly limited, and methods known to those skilled in the art are used. be able to. Gene transfer means are generally "transformation" or "transfection", which is a transient reaction induced in a cell after the uptake of exogenous nucleic acid (eg, DNA or RNA foreign to the host cell). means a static or stable genetic change. Generally, genetic alteration can be achieved by incorporating exogenous nucleic acid into the genome of the host cell, or by transiently or stably maintaining the exogenous nucleic acid either as an episomal component or independently. According to the present invention, the introduced Mycl gene may be integrated into the genome of the host cell, or may exist as an episomal component, as long as the on/off of expression of the gene can be controlled. Alternatively, the plasmid or vector containing the gene may exist in the cytoplasm as it is.
[0038]
A "vector" is usually used to introduce an exogenous nucleic acid (preferably DNA) into a host cell. Vectors generally include viruses, particularly attenuated and/or replication-incompetent viruses. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, Sendai viral vectors and the like. The vector may also contain regulatory sequences such as promoters, enhancers, ribosome binding sequences, terminators, polyadenylation sites, etc., to enable expression of exogenous nucleic acids. Furthermore, if necessary, drug resistance genes (e.g., kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, etc.), thymidine kinase gene, selectable marker sequences such as diphtheria toxin gene, fluorescent protein, β-glucuronidase (GUS), FLAG and the like, such as reporter gene sequences. Here, the "promoter" also includes a promoter component sufficient for promoter-dependent gene expression that is cell-type-specifically regulatable, tissue-specifically regulatable, or inducible by an external signal or agent. is intended. Such components may be located in the 5'or 3'region of the native gene. Also, "operably linked" means that the DNA sequence and regulatory sequence are linked such that expression is enabled when an appropriate molecule (e.g., a transcriptional activation protein) is bound to the regulatory sequence. means that there is
[0039]
In addition, introduction of vectors into host cells is not limited, but electroporation method (Meiner, V. et al., Proc. Natl. Acad. Sci. USA, 93: 14041-14046 (1996), etc.), the calcium phosphate method, the DEAE-dextran method, and a method using a lipid for gene transfer (lipofectamine, lipofectin, etc.). Cells into which the vector has been introduced can then be selected based on the properties of marker genes (eg, drug resistance genes). Correct occurrence of homologous recombination in the selected cells can be confirmed by Southern blotting or the like using a portion of the target exogenous nucleic acid as a probe. In this way, it is possible to prepare cells containing heterozygous genes of interest, specifically, genes obtained by knocking in a marker gene into the Mycl gene.
[0040]
As ES cells, the KH2 strain, which has a Frt sequence downstream of the Cola1 locus and expresses M2-rtTA, a reverse tetracycline-regulated transactivator under the control of the endogenous Rosa26 promoter, can be used ( Beard C, et al., Genesis, vol.44, p.23-28 (2006)). Introduction of the Mycl gene into the ES cells can be performed using a method well known to those skilled in the art. ), and, for example, the Colla1-TetOP-AttR1-ccdB-AttR2-ires-mCherry vector is subjected to an LR reaction to use the TetOP-Mycl-ires-mCherry vector as a gene transfer vector. can be done. Here, the "TetOP (operon)" sequence in the vector is the sequence to which the reverse tetracycline-regulated transactivator binds (tetracycline responsive element: TRE), and the reverse tetracycline such as doxycycline (Dox) added to the cells. A reverse tetracycline-regulated transactivator expressed from host cells binds depending on , and induces the expression of genes linked downstream thereof. Also, "ires" (internal ribosome entry site) is a ribosome internal recognition sequence, and "mCherry" is a gene (reporter gene) encoding a red fluorescent protein. By introducing the vector together with a nucleic acid encoding flipase into KH2-ES cells, the Mycl gene can be integrated into the chromosome of the ES cells. In addition to the vector containing "ires-mCherry" as described above, the "ires-βgeo (fused gene of β-galactosidase and neomycin resistance gene) cassette" (Mountford P. et al., Proc. USA, 91:4303-4307 (1994)), similar vectors containing the IRES-Hygro (hygromycin resistance gene) cassette, and the like. good.
[0041]
The present invention enables pancreatic islet-like cells to proliferate and differentiate into insulin-producing cells by controlling the transient expression of the Mycl gene. As described above, the present invention is characterized by the transient expression of the Mycl gene. When the Mycl gene is transiently expressed, the Mycl gene is introduced with the cell division of the islet-like cells. Since the number of cells is relatively decreased, the expression level of the Mycl gene can be naturally decreased or stopped over time. Thus, in one embodiment, the present invention achieves its objectives by "on" regulation of the expression of the Mycl gene. In another embodiment, it is also possible to forcibly turn off the expression of the Mycl gene (hereinafter referred to as "on/off" control).
[0042]
By using reverse tetracycline as described above, the expression of the Mycl gene introduced into the host cell can be controlled on/off. That is, in the presence of reverse tetracycline, the Mycl gene continues to be expressed intracellularly and is able to proliferate islet-like cells, whereas removal of reverse tetracycline halts the proliferation of islet-like cells and increases insulin-producing cells. differentiation can be induced. In the present invention, the transient expression (on state) of the Mycl gene introduced into pancreatic islet-like cells is maintained for at least 2 days and up to 100 days, for example, 90 days, 80 days, 70 days, 60 days from the start of cell culture. A period of days is preferred. On the other hand, the islet-like cells into which the Mycl gene has been introduced preferably proliferate within the period described above.
[0043]
As an alternative to on/off control of Mycl gene expression, viral vector gene expression and cell growth can be precisely switched on/off by light irradiation, ``light-regulated viral vectors'' (Tahara, M., et al. , PNAS, vol.116, 11587-11589, 2019) can be used. This is a viral vector in which a gene encoding a photoswitch protein called a magnet has been introduced, and the expression of the target gene incorporated into the vector can be controlled using blue light.
[0044]
Another alternative example of on/off control of Mycl gene expression is episomal vector (Okita K., et al., Nat Methods 2011 May 8(5): 409-412), Sendai virus, RNA vector ( Warren L., et al., Cell Stem Cell 2010 Nov 7(5):618-630). It is not limited to these, and the method used for reprogramming (establishing) the iPS cells described above (that is, the method of temporarily inducing genes) can be used.
[0045]
According to the present invention, in addition to introducing an isolated exogenous Mycl gene, the object of the present invention can be achieved by forcibly expressing the Mycl gene endogenous to cells. Means for expressing the endogenous Mycl gene include, but are not limited to, replacing the wild-type promoter or enhancer with a strong promoter or enhancer for operably inducing the expression of the Mycl gene to force gene expression. It can be a method. Examples of promoters used for substitution include the cytometry (CMV) promoter, which is a strong promoter, and inducible promoters that function in the presence of an inducer. On the other hand, examples of enhancers used for substitution include SV40 enhancer, herpes B virus enhancer, cytomegalovirus enhancer, α-fetoprotein enhancer and the like. In another embodiment, in addition to CRISPR Type II (including CRISPR-dCas9), CRISPR-type I, TALENs, and ZFN genome recognition sequences can activate promoters and/or enhancers Demethylases, histone modifications Molecules to which enzymes, transcriptional activators, specifically VP64, p65, Rta and the like are added can be used. In the present specification, the above promoters, enhancers, enzymes and factors that activate them, or nucleic acid protein complexes or low-molecular-weight compounds may be referred to as "activators" for activating the Mycl gene. .
[0046]
(6) Introduction of the gene product of the Mycl gene
Introduction of the Mycl gene product into cells can be performed by a general method for introducing foreign genes or proteins into cells. Examples of such methods include, but are not limited to, methods using transfection reagents, methods using viruses, electroporation methods, particle gun methods, sonoporation methods, liposome fusion methods, micromanipulators, laser light irradiation, Examples include a method of introduction by forming pores in the cell membrane by .
[0047]
(7) Production of chimeric mammals
A method for introducing an ES cell into a mammal to produce a chimeric mammal can be performed using a method well known to those skilled in the art. First, any medium known to those skilled in the art can be used for culturing the ES cells into which the Mycl gene has been introduced. For example, when the ES cells are cultured on feeder cells, feeder cells (e.g., MEFs (mouse embryonic fibroblasts)) can be used, and the ES cells are cultured on the feeder cells in an ES cell culture medium (e.g., Knockout DMEM (GIBCO) containing 15% FBS, 50 U/mL penicillin/streptomycin, L-glutamine, non-essential amino acids supplemented with 2-mercaptoethanol (2ME, GIBCO) and LIF (SIGMA) is used. be able to.
[0048]
Next, the ES cells are introduced into mammals to produce knockout animals (Mycl gene knockin animals). Here, a mouse is used as an example of mammals, but methods for producing knock-in mice are well known to those skilled in the art. Specifically, chimeric mice are prepared by injecting the ES cells into blastocysts of mice (e.g., C57BL/6, etc.) and transplanting them into the uterus of pseudopregnant female mice (ICR, etc.). be able to. Thereafter, the chimeric mice and normal mice (C57BL/6, etc.) are mated to produce heterozygous mutant mice in which the Mycl gene is heterozygously knocked in. By mating heterozygous mutant mice with each other, homozygous mutant mice in which the Mycl gene is homozygous and knocked in can be obtained. For the production of the above knock-in mice, 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., al., Cell, 113: 631-642 (2003)), ECAT5 knock-in mouse (Takahashi, K., K. Mitsui, and S. Yamanaka, Nature, 423 (6939): p541-545 (2003), JP 2003 -265166) and the like.
[0049]
In addition, chimeric mammals can be produced using not only the above ES cells but also iPS cells. For example, blastocyst complementation procedures can be used to generate organs from human iPS cells in non-human mammals. For example, Nakauchi et al. generated human iPS-induced human pancreases in vivo in pancreatic cloned pigs (Nakauchu, H., et al., PNAS, Vol. 110, No. 1, 4557- 4562 (2013)).
[0050]
2. Regulation of islet-like cell proliferation and differentiation
According to the present invention, the proliferation and differentiation of the islet-like cells prepared above can be controlled in vitro and in vivo. A method for inducing the Mycl gene can be selected according to the above-described method used to introduce the gene into cells. For example, when the Mycl gene is introduced into cells using the TetOP-Mycl-ires-mCherry vector as described above as a vector for gene introduction, the reverse gene expressed from the host cells depends on a reverse tetracycline such as doxycycline (Dox). A tetracycline-regulated transactivator can bind and express the downstream linked Mycl gene to induce proliferation of islet-like cells. The concentration of Dox added to the cell culture system can be adjusted as appropriate. For example, it may be 1 to 100 mg/mL. After the islet-like cells are grown by the addition of Dox, the growth can be stopped and cell differentiation such as insulin production can be induced by, for example, replacement with Dox-free medium.
[0051]
In vivo, the proliferation of pancreatic islet-like cells can be induced in the pancreas by giving the chimeric non-human mammal, for example, Dox-containing water. added to water The concentration of Dox may be, for example, 1 to 100 mg/mL, preferably 2.0 mg/mL. When using chimeric non-mouse, the pancreas grows with increasing age until 8 weeks of age. Age in weeks is not limited as long as it can be induced.
[0052]
3. Method for producing pancreatic islet cells having insulin-producing ability or progenitor cells thereof
According to the present invention, a method for producing pancreatic islet cells or progenitor cells thereof having insulin-producing ability is provided. Such a production method is not limited, but primary pancreatic islet cells, cultured pancreatic islet cells, or pancreatic islet cells derived from stem cells are used as raw materials, the Mycl gene is introduced into the islet cells, and the cells are proliferated by forced expression of the Mycl gene. and then obtaining pancreatic islet cells or progenitor cells thereof having insulin-producing ability by terminating the expression of the gene. It should be noted that forced expression and termination of the Mycl gene can be achieved, for example, by alternative means of on/off-controlling expression of the gene (e.g., use of light-regulated viral vectors), or reverse tetracycline control of doxycycline sensitivity, as described above. Sexual transactivators are available. As used herein, “progenitor cells of pancreatic islet cells” or “pancreatic islet progenitor cells” refer to cells (or a group of cells) that are in the process of differentiating into pancreatic islet cells having insulin-producing ability after Mycl gene expression cessation. ), and can be identified using, for example, one or more markers selected from the group consisting of PDX1-positive, PTF1a-positive, NKX6.1-positive, Fev-positive, Pax4-positive, and Cck gene as indicators.
[0053]
4. Pharmaceutical use
According to the present invention, by controlling the expression of the introduced Mycl gene, pancreatic islet-like cells can be proliferated and insulin production can be promoted. Therefore, in one aspect, a pancreatic islet cell amplification promoter, pancreatic islet function improving agent, and insulin production promoter comprising the Mycl gene or its gene product as an active ingredient; , excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.); A method for preventing and/or treating a diabetic patient using a function-improving agent, an insulin production-enhancing agent, or a pharmaceutical composition; of the Mycl gene is provided. In another aspect, a method of administering the Mycl gene or its gene product to a subject for the prevention and/or treatment of diabetes (in vivo method); Provided is a method (ex vivo method) of transplanting cell-like cells or insulin-producing cells (hereinafter sometimes simply referred to as "pancreatic islet-like cells, etc.") or any combination thereof to a subject.
[0054]
It should be noted that the ex vivo method of the present invention is intended to have different aspects depending on the cells used. For example, (i) an embodiment in which pancreatic islet-like cells obtained by gene transfer into adult pancreatic islet-derived cells are produced by allogeneic in vitro or autologous in vitro (both narrowly defined ex vivo methods); Aspects of producing pancreatic islet-like cells obtained by introducing the Mycl gene of pancreatic islet-like cells derived from stem cells (iPS cells, ES cells, somatic stem cells, etc.) in vitro or autologously in vitro (broadly defined (iii) an aspect in which the Mycl gene is functionally expressed in (i) and (ii) above and can be proliferating pancreatic islet progenitor cell-like cells; (iv) from (i) to (iii) above An embodiment is an insulin-producing cell or pancreatic islet cell (not containing the Mycl gene) produced by differentiation.
[0055]
As one aspect of the present invention, cells, whether autologous or allogeneic to a patient, are treated by known methods, specifically Nature Medicine volume 22, pages 306-311 (2016). , doi: 10.1038/nm. 4030, Nature Biomedical Engineering volume 2, pages 810-821 (2018). , DOI: 10.1038/s41551-018-0275-1, EBioMedicine 12 (2016) 255?262. , DOI: https://doi. org/10.1016/j. e biom. The pancreatic islet to be transplanted may be encapsulated by the method described in 2016.08.034 or the like and then administered. By encapsulating the islets to be transplanted, it is possible to eliminate or reduce the dose of immunosuppressants.
[0056]
As one aspect of the present specification, isolated pancreatic islets can be appropriately gene-edited. Gene editing is not particularly limited, but specific examples include correction of mutated genes contained in pancreatic islet cells by genome editing, modification of molecules that cause immune reactions such as surface antigens such as HLA and GAD proteins, Beta-2 Microglobulin and Therapeutic favorable features can be added, such as induction of immune tolerance by deletion of the RFX5, RFXANK, RFXAP and CIITA genes. Although the method of gene editing is not particularly limited, for example, CRISPR-Cas9, TALEN, ZFN, CRISPR-Cas3, CRISPR-TypeI-D and variants thereof, and transposon vectors such as piggyBAC for expressing functional molecules are specific. mentioned as
[0057]
(1) Applicable disease
Diseases to which the Mycl gene or its gene product can be applied based on the above aspects are diseases in which insulin does not function sufficiently in the body (typically, insulin resistance and decreased insulin secretion). INDUSTRIAL APPLICABILITY According to the present invention, the Mycl gene or its gene product can promote insulin production, and has, for example, a blood glucose-lowering effect on diabetic patients. Indications are typically diabetes, but more specifically, severe hypoglycemia, type I diabetes (including slowly progressing type 1 diabetes or type 1.5 diabetes), type II diabetes, and glucose tolerance. Diseases, disorders or conditions associated with disorders, hyperglycemia, dyslipidemia, obesity or metabolic syndrome, or other specific mechanisms or conditions, e.g., genetic abnormalities involved in pancreatic β-cell function , genetic abnormalities involved in the transduction mechanism of insulin action, pancreatic exocrine disorders, endocrine disorders, liver disorders associated with other diseases or conditions, drugs or chemicals, infectious diseases, rare immune-mediated conditions, etc. Alternatively, gestational diabetes and the like can be mentioned. In addition, diabetic complications caused by diabetes (eg, diabetic retinopathy, diabetic neuropathy, etc.) can also be included in indication diseases. Insulin secretion deficiency caused by total pancreatic resection or partial pancreatic resection associated with pancreatitis or pancreatic cancer can also be included in the indications.
[0058]
Furthermore, although the types of diabetes that can be treated by the methods described herein are not particularly limited, the methods provide a treatment that is less likely to cause hypoglycemia, which secretes physiologic insulin dependent on blood sugar levels. can provide. For example, when used for the treatment of severe hypoglycemia, although not limited, it is known as one embodiment because it is known to those skilled in the art that donor pancreatic islets exhibit a remarkable effect on severe hypoglycemia. The method described in the literature can be used. The methods described in known literatures here are not limited, but for example, Diabetes Care 2016 Jul; 39(7): 1230-1240. , DOI: 10.2337/dc15-1988, The New England Journal of Medicine. 343 (4): 230?238. , DOI: 10.1056/NEJM200007273430401, The New England Journal of Medicine. 355 (13): 1318?1330. , DOI: 10.1056/NEJMoa061267 and the like are specifically mentioned. Alternatively, islet cells, islet-like cells, pancreatic islet progenitor cell-like cells, or insulin equivalent to, preferably more than, the amount of islet cell transplantation described in known literature, rather than the cell transplantation described in known literature A method of administering the Mycl gene or its gene product to a subject (in vivo method), which is capable of amplifying the producing cells, can be selected.
[0059]
In addition, when used for the treatment of type I diabetes, there is no particular limitation, but as one mode of treatment for type I diabetes, for example, insulin is used as needed, during fasting, after glucose load and/or after glucagon stimulation in type I diabetes patients. , Depending on the blood glucose level and / or the amount of C-peptide, the number of pancreatic islet cells, islet-like cells, islet progenitor cell-like cells, or insulin-producing cells may be adjusted, according to the method described herein , may determine dosages of agents for in vivo therapy. Type I diabetics who are insulin-depleted have an increased risk of developing hypoglycemic symptoms and are preferred for treatment with the methods described herein. Type I diabetes patients with a blood C-peptide level of 0.5 ng/mL or less, preferably 0.2 ng/mL or less, more preferably 0.1 ng/mL or less can be treated. The specific dose of the agent may be adjusted, for example, with reference to the amount of donor pancreatic islets that are usually transplanted for the indication of severe hypoglycemia. Specifically, for example, islet cells, islet-like cells, and islet progenitor cells equivalent to 500 IEQ/kg or more, preferably 1000 IEQ/kg or more, more preferably 2000 IEQ/kg or more, and even more preferably 5000 IEQ/kg or more. like cells, or insulin-producing cells. Alternatively, a method (in vivo method) of administering the Mycl gene or its gene product capable of amplifying pancreatic islet-like cells, pancreatic islet progenitor-like cells, or insulin-producing cells to a similar degree can be selected.
[0060]
In addition, when used for the treatment of type II diabetes, there is no particular limitation, but as one aspect, for example, it can be used as a therapeutic agent for insulin-dependent type II diabetes in which the amount of insulin secreted into the body is insufficient. Specifically, for example, pancreatic islet cells, pancreatic islet-like cells, pancreatic islets containing depending on the amount of insulin, blood sugar level and/or C peptide at any time, fasting, after glucose loading and/or after glucagon stimulation in type II diabetes patients The number of progenitor-like cells, or insulin-producing cells, may be adjusted and the dosage of the agent for in vivo treatment determined by the methods described herein. From the viewpoint of obtaining favorable effects in insulin-deficient pathological conditions, for example, the amount of C-peptide in the blood during fasting and/or glucagon stimulation is 0.5 ng/mL or less, preferably 0.2 ng/mL or less. , more preferably 0.1 ng/mL or less type II diabetes patients can be treated. A specific dose of the agent may be adjusted, for example, with reference to the amount of donor pancreatic islets transplanted for the indication of severe hypoglycemia. Specifically, for example, islet cells, islet-like cells, and islet progenitor cells equivalent to 500 IEQ/kg or more, preferably 1000 IEQ/kg or more, more preferably 2000 IEQ/kg or more, and even more preferably 5000 IEQ/kg or more. like cells, or insulin-producing cells. Alternatively, a method (in vivo method) of administering the Mycl gene or its gene product capable of amplifying pancreatic islet-like cells, pancreatic islet progenitor-like cells, or insulin-producing cells to a similar degree can be selected.
[0061]
In addition, when used as a therapeutic agent for slowly progressing type I diabetes, it is not limited, but similar to type I diabetes and/or type II diabetes, the amount of insulin, blood sugar level and / or C peptide in the body. Amount can be adjusted. In this case, islet cells and islet-like cells to be amplified by the method of the present specification , pancreatic islet progenitor-like cells, or insulin-producing cells are preferably derived from the patient's own cells. For example, as one aspect of the treatment of slowly progressing type I diabetes, it is preferable that autoantibodies against pancreatic islet cells in blood and HLA types are examined in advance, and before or after falling into an insulin-dependent state and/or insulin-depleted state. If the autologous islet cells can be expanded by the method of the present specification after the treatment, treatment that does not require immunosuppressants will be possible, and transition to insulin-dependent and/or insulin-depleted conditions can be prevented, or Insulin dependent states and/or insulin depleted states can be treated. In order to determine whether or not the patient has slowly progressing type I diabetes, autoantibodies against pancreatic islet cells in the blood and HLA types are not particularly limited as long as they are involved in slowly progressing type I diabetes. Known methods include determining whether islet-associated autoantibodies, such as antibodies (ICA), GAD antibodies, insulin autoantibodies (IAA), IA-2 antibodies, show overlapping or singular positivity. Furthermore, it may be confirmed by a known method such as whether or not the subject has HLA associated with slowly progressive type I diabetes such as HLA-DR4-DQA1*0301-B1*0401 as HLA.
[0062]
In addition, from the viewpoint of obtaining therapeutic effects from donor pancreatic islets, the ex vivo method is preferable for the treatment of severe hypoglycemia, but islet cells can be expanded in the body (in vivo) by the method described herein. . Since the islet cells amplified in this manner exhibit the same effect as the ex vivo method, the treatment method for expanding pancreatic islets in vivo can be preferably selected as a treatment method for severe hypoglycemia as well as the ex vivo method. . Furthermore, for the treatment of type I diabetes, type II diabetes, and indolent type I diabetes, as with the ex vivo method, the subject's sex, age, weight, condition of the affected area, condition of the cells to be used, etc., should be considered. A method of expanding islet cells in vivo (in vivo) can be selected. At this time, the method for expanding pancreatic islet cells in vivo (in vivo) can be combined with the ex vivo method.
[0063]
According to the present invention, such diseases can be prevented and/or treated. As used herein, the term "preventing" refers to preventing or delaying the onset/development of the above diseases or symptoms thereof, or reducing the risk of onset/development.
“Treatment” includes mitigation (mitigation) of symptoms characteristic of the target disease or accompanying symptoms, prevention or delay of worsening of symptoms, etc. Treatment also includes improvement of the disease. . "Prevention" means:
[0064]
(2) Insulin production promoter and pharmaceutical composition
The Mycl gene of the present invention or its gene product can be provided as a pancreatic islet cell expansion promoter, pancreatic islet function improving agent, insulin production promoter, or pharmaceutical composition for the prevention and treatment of the above indicated diseases. Here, as one aspect of the pancreatic islet cell expansion promoting agent, any one of α cells, β cells, δ cells, ε cells, and PP cells contained in pancreatic islets, preferably any two or more pancreatic islet cells It refers to an agent that proliferates. In addition, as one aspect of the islet function improving agent, it refers to an agent that, when administered, improves some or all of the functions of pancreatic islets in vivo. Blood sugar regulating action, hypoglycemic action by insulin, glucose production/release action by glucagon, gastrin, secretin, insulin and/or glucagon secretion inhibitory action by somatostatin or nutrient absorption inhibitory action in the gastrointestinal tract, appetite regulating action by ghrelin, pancreas Specific examples include the gallbladder contraction regulating action and appetite regulating action of the polypeptide. In addition, one aspect of the insulin production-enhancing agent includes, for example, an agent that promotes physiological insulin secretion in response to blood sugar levels, which is one of the functions that pancreatic islets perform in vivo. In addition, when provided as a pharmaceutical composition, in addition to the active ingredient of the Mycl gene or its gene product used in the above embodiment, other pharmaceutically acceptable ingredients (e.g., carriers, excipients, disintegration agents, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, saline, etc.). Additionally, an active agent for activating the Mycl gene may optionally be included.
[0065]
The living body-derived pancreatic islet cells into which the Mycl gene or its gene product is introduced may be pancreatic islet cells of a healthy subject, or a type I diabetic patient who has lost some or most of the β cells, or a terminal stage type II diabetic patient. It can be an islet cell derived from a patient with diabetes. In this case, the islet cells may be autologous or xenogeneic to the recipient.
[0066]
The cell preparations and pharmaceutical compositions of the present invention are obtained by, but not limited to, suspending the pancreatic islet-like cells obtained above in physiological saline or an appropriate buffer (e.g., phosphate-buffered physiological saline). . The number of cells required for treatment can be obtained by forcibly expressing the Mycl gene and appropriately proliferating the cells.
[0067]
In the use of pancreatic islet cells, pancreatic islet-like cells, pancreatic islet progenitor cell-like cells, or insulin-producing cells in cell preparations and pharmaceutical compositions, dimethylsulfoxide (DMSO), serum albumin, etc. are used to protect the cells. may be included in cell preparations and pharmaceutical compositions, such as antibiotics, to prevent bacterial contamination and growth. In addition, other pharmaceutically acceptable components (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.) It may be included in cell preparations and pharmaceutical compositions. Those skilled in the art can add these factors and agents to cell preparations and pharmaceutical compositions at appropriate concentrations.
[0068]
The number of pancreatic islet cells, islet-like cells, pancreatic islet progenitor cell-like cells, or insulin-producing cells contained in the cell preparations and pharmaceutical compositions prepared above is desirable in the prevention and/or treatment of diabetes and diseases associated therewith. In order to obtain the effect of (for example, lowering of blood sugar level), it is possible to appropriately adjust the dosage in consideration of the subject's sex, age, weight, condition of the affected area, condition of the cells to be used, and the like.
[0069]
The insulin production-enhancing and pharmaceutical compositions of the present invention can be administered to various subjects, for example, mammals such as primates, humans, dogs, cats, cows, horses, pigs, and sheep are intended, preferably Human. In addition, the route of administration to a subject is not limited, but parenteral administration, such as injection or infusion, can be administered to any site in the body that can respond to glucose. Specifically, for example, it can be transplanted or administered into the pancreas, under the renal capsule, preferably subcutaneously, intraperitoneally, more preferably intravascularly, intravenously, still more preferably intraportally.
[0070]
The method of activating the administered Mycl gene in vivo is not limited, but the above-described "on" control or "on and/or off" control system of Mycl gene expression can be used. For example, the above-mentioned "photoregulated viral vector" (Tahara, M., et al., PNAS, vol.116, 11587-11589, 2019) may be used.
[0071]
Also, when targeting the Mycl gene to the pancreas, markers that are specifically expressed in pancreatic islet cells (e.g., PDX1, C-peptide, insulin, MafA, Mnx1, Pax4, Pax6, NeruroD1, Isl1, Nkx2 .2, Ngn3, HNF1a, Foxa2, Nkx6.1, Glucagon, Arx, MafB, RFX6, IRX1, IRX2, somatostatin). When selecting a method for expanding pancreatic islet cells in the body (in vivo), the route of administration to a subject is not limited; Specific examples include celiac artery and pancreatic duct.
[0072]
(3) Treatment method
According to the present invention, methods of preventing and/or treating a subject with diabetes or a disease related thereto are provided using the Mycl gene or its gene product, pancreatic islet-like cells, etc., or any combination thereof. . Furthermore, according to the present invention, in the above treatment method, an active agent for activating the Mycl gene can be administered, and administered before, simultaneously with, or after administration of the insulin production-enhancing agent or pharmaceutical composition. good too.
[0073]
(4) Kit
According to the present invention, a kit used for preventing and/or treating diabetes or a disease related thereto, containing an insulin production-enhancing agent or a pharmaceutical composition, is provided. Such kits can include instructions for administering or transplanting the insulin production-enhancing agent or pharmaceutical composition to a subject. Also, the kit may further comprise an activating agent for activating the Mycl gene.
Example
[0074]
Although the present invention will be described in more detail with reference to the following examples, the present invention is not limited by these examples.
[0075]
Method
(i) Establishment of ES cells capable of inducing expression of Myc, Mycn, and Mycl in a Dox-dependent manner
Myc, Mycn, and Mycl cDNAs were cloned from ES cell-derived cDNAs, and the cloned fragments were inserted into the pCR8-GW-TOPO vector (Invitrogen). Col1a1-TetOP-Mycl-ires-mCherry vector (hereinafter referred to as "targeting vector ) was inserted into the Col1a1 locus of KH2-ES cells using the flip-in recombination system (Beard et al., 2006). When performing Flip-in recombination, cells were suspended in high-glucose DMEM (Nacalai Tasque) medium containing 50 μg of each Myc gene-inserted targeting vector, 25 μg of pFlapase vector, and 25 mM HEPES buffer (Gibco). The suspension was electroporated into KH2-ES cells using the Gene pulser Xcell electroporation system (BIO-RAD) (Voltage: 550 V, Capacitance: 25 μF, Resistance: ∞, Cuvette: 2 times under the conditions of 4 mm or more pulse supply). Twenty-four hours after electroporation, the cells were selected with hygromycin B (Roche) at 150 μg/mL, and formed colonies were picked to establish ES cell lines capable of inducing expression of each Myc gene in a Dox-dependent manner.
[0076]
(ii) cell culture method
For the culture of feeder cells (MEF; mouse fetal fibroblasts), 10% FBS (GIBCO), 50 U/mL Penicillin-Streptomycin (P/S; Nacalai Tesque), L-glutamine (GIBCO), NEAA ( A DMEM (Nacalai Tesque) medium containing Nacalai Tesque) was used.
[0077]
For ES cell culture, knockout-DMEM (GIBCO) containing 15% FBS, 50 U/mL P/S, L-glutamine, and NEAA, 2-mercaptoethanol (2ME: GIBCO) and LIF (SIGMA) was added, gelatin-coated (SIGMA), and cultured in a dish seeded with feeder cells. When subculturing the ES cells, they were treated with 0.25% trypsin/1 mM EDTA (GIBCO) at 37°C for about 3 minutes, and about 1/10 of the volume of the cell suspension was seeded on a new dish.
[0078]
(iii) Generation of in vivo Mycl expression-inducible mice
Mouse blastocysts (ICR, E3 . 5) and transplanted into the uterus of day 2 pseudopregnant mice (Slc: ICR, Shimizu Experimental Materials) to prepare chimeric mice having cells capable of inducing Mycl expression in a Dox-dependent manner.
[0079]
(iv) administration of doxycycline
Using 8-week-old mice, a solution containing 2.0 mg/mL Dox was administered in drinking water. In cultured cells, it was added to the medium at a final concentration of 2.0 μg/mL.
[0080]
(v) Preparation of pathological specimens in mouse organs
After dissecting the mouse, each organ was placed in 4% PFA (Wako Pure Chemical Industries, Ltd.) and shaken for one day. did. The next day, a block was prepared using a spin tissue processor STR120 (Thermo SCIENTIFIC) according to the recommended protocol. Preparation of pathological specimens was entrusted to Biogate Co., Ltd.
[0081]
(vi) immunostaining
The tissue section was soaked in xylene (Wako Pure Chemical Industries) and then in 100% EtOH (Wako Pure Chemical Industries) for 30 minutes or longer each. The cells were washed with tap water for about 10 minutes, transferred into a boiled antigen retrieval solution pH 9 (Nichirei Biosciences, used at 10-fold dilution), and subjected to antigen retrieval treatment for 10 minutes. 200 μL of a primary antibody solution diluted with a blocking solution (2% BSA+1×PBS) was added to each tissue section and allowed to stand for 30 minutes to 1 hour. After washing twice with 1×PBS, two drops of the secondary antibody solution were added onto the tissue section and allowed to stand for 30 minutes. After washing twice with 1 × PBS, in the case of DAB staining, DAB solution (Nichirei Biosciences, using DAB substrate kit, 1 mL of Elix water, reagent A and B are added and mixed 1 drop each, then reagent C is added. 150 μL of the mixture (1 drop added and mixed) was added onto the tissue section, antigen-antibody reaction was performed, and then observation was performed under a microscope. In the case of fluorescent staining, one drop of the mounting material was added onto the tissue section, covered with a cover glass, and then observed under a microscope.
[0082]

・ Anti-mCherry antibody (abcam, 1/500)-anti-rabbit IgG antibody (Nichirei Biosciences)
・Anti-synaptophysin antibody (abcam, 1/500)-anti-rabbit IgG antibody (Nichirei Biosciences)
・Anti-chromogranin A antibody (DAKO, 1/500) - anti-rabbit IgG antibody (Nichirei Biosciences)
・ Anti-Ki67 antibody (abcam, 1/200) - anti-rabbit IgG antibody (Nichirei Biosciences)
・Anti-Insulin antibody (DAKO)-anti-guinea pig IgG antibody (BIOTIUM)
・Anti-somatostatin antibody (Santa Cruz, 1/300)-anti-mouse IgG antibody (BIOTIUM)
・Anti-Glucagon antibody (Santa Cruz, 1/300)-anti-mouse IgG antibody (BIOTIUM)
[0083]
(vii) RNA recovery, RNA extraction, and cDNA synthesis
For RNA recovery, after washing the cultured cells with PBS(-) (Nakalai Tasque), the cells were lysed with 350 μL of LBP buffer. For RNA extraction, NucleoSpin (registered trademark) RNA Plus (TAKARA) was used and the recommended protocol was followed. For synthesis of cDNA, Primescript single-strand cDNA synthesis kit (TAKARA) was used and the recommended protocol was followed.
[0084]
(viii) qRT-PCR
The recommended protocol was followed using the GoTaq qPCR master mix (Promega). Analysis was performed using the Stepone Plus system (Life Technologies). The following primers and PCR reaction conditions were used.
[0085]
[table 1]

[0086]
(b) PCR reaction conditions
・95°C for 2 minutes
· 95°C (15 seconds), 60°C (1 minute) [40 cycles]
・95°C (15 seconds), 60°C (1 minute), 95°C (15 seconds)
[0087]
(ix) islet isolation
Eight-week-old mice were anesthetized by intraperitoneal injection of Somnopentyl (Kyoritsu Seiyaku Co., Ltd.). After laparotomy, the opening of the duodenal common bile duct was identified, and then the upper part of the common bile duct and the intestinal tract were clamped with bulldog forceps. The common bile duct was cut at its border with the duodenum, and 2 mL of M199 medium (Gibco) containing collagenase P (Roche) (2 mg/mL) was injected. After that, the pancreas was extracted and digested in a hot water bath at 37° C. for 11 minutes and 30 seconds. It was suspended in 25 mL of M199 medium containing 10% FBS, and centrifuged twice (1000 rpm, 4°C, 2 minutes). After the supernatant was discarded and suspended in 10 mL of Histopaque (SIGMA), 10 mL of M199 medium containing 10% FBS was poured over the suspension and centrifuged (1000 rpm, 4°C, 30 minutes). The supernatant was transferred to another 50 mL tube, and 25 mL of M199 medium containing 10% FBS was added, followed by centrifugation (1000 rpm, 4°C, 2 minutes).
[0088]
(ix) islet dispersal
The isolated pancreatic islet was collected in a 1.5 mL silicon tube, and 100 μL of dispersion buffer (see Table 2 below) was added. After standing at 37° C. for 15 minutes, it was dispersed by pipetting.
[0089]
[Table 2]

[0090]
[Table 3]

[0091]
(x) pancreatic islet culture (three-dimensional culture method using gel)
After adding 20 μL of Matrigel (CORNING) containing isolated pancreatic islets to a 96-well plate (CORNING), Matrigel was gelled by incubating at 37° C. for 15 minutes. Thereafter, 140 μL of a medium containing growth factors (see table below) was added and cultured at 37° C. in 5% CO 2 .
[0092]
[Table 4]

[0093]
(xi) pancreatic islet culture (floating culture)
　2 mL of medium was added to a 6-well plate, and cell culture inserts (Millipore) were suspended on the medium. Pancreatic islets isolated on a cell culture insert and 20 μL of medium were added and cultured at 37° C., 5% CO 2 .
[0094]
(xii) intraperitoneal glucose tolerance test (IPGTT)
The Mycl-expressing group and the control group were fasted for 12-16 hours. Thereafter, each mouse was weighed, and a D-glucose solution was intraperitoneally injected into the mouse so that D-glucose was 2 g/kg (mouse). After 15, 30, 60 and 120 minutes, the blood of each mouse was collected from the tail, and the blood glucose level was measured using an Antsense stand (Horiba).
[0095]
(xiii) generation of diabetic mice
　The body weight of 8- to 12-week-old immunodeficient mice (NOD/SCID) was measured, and streptozocin solution (STZ) (Sigma-Aldrich) was injected intraperitoneally into the mice at 150 mg/kg. One week later, the blood glucose level was measured using an Antsense stand (Horiba), and mice with random blood glucose levels of 250 mg/dL or more were used as diabetic mice.
[0096]
(xiv) pancreatic islet transplantation (subrenal capsule transplantation)
Diabetic mice were anesthetized by intraperitoneal injection of Somnopentyl (Kyoritsu Seiyaku Co., Ltd.). After laparotomy, the renal capsule was incised using an injection needle. A silicon tube was inserted under the renal capsule, and the islet was transplanted under the renal capsule using a Hamilton syringe. After transplantation, the silicon tube was pulled out and the peritoneum and capsule were sutured.
[0097]
(xv) nephrectomy
Mice transplanted with pancreatic islets were anesthetized by intraperitoneal injection of Somnopentyl (Kyoritsu Seiyaku Co., Ltd.). After laparotomy, the kidney to which the pancreatic islet was transplanted was removed, and the renal vein and renal artery were ligated with a ligature. After nephrectomy, the peritoneum and capsule were sutured.
[0098]
(xvi) single cell analysis
Re-analysis was performed using the analysis data published in the GEO database (GSE101099; Byrnes LE et al., Nat Commun. 2018 Sep 25;9(1):3922). Seurat v2.2 and v2.3 were used for t-SNE analysis (Satja R., et al., Nat Biotechnol. 2015 33:495-502).
[0099]
Example 1: Establishment of Mycl expression-inducible mouse ES cells
It was examined whether the ES cell line into which the Mycl gene prepared above could be expressed in a Dox-dependent manner. Expression of the Mycl gene can be confirmed by 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 causes cells to develop a red color. As shown in FIG. 1, when gene expression was compared before and after the addition of Dox, Mycl gene expression was significantly increased, indicating that Mycl expression-inducible mouse ES cells could be established.
[0100]
In addition, when the ES cell lines into which the Myc gene and the Mycn gene were incorporated were similarly examined whether gene expression was induced in a Dox-dependent manner, as shown in FIG. Each gene expression was observed.
[0101]
On the other hand, when observing the expression of Ki67 protein as an index of cell proliferation, the number of proliferating cells (Ki67-positive cells) increases as the period of Dox administration increases, and the number of Ki67-positive cells increases two weeks after stopping Dox administration. It decreased markedly and cell proliferation stopped (Fig. 7). From the above results, it was found that the Mycl gene-introduced ES cells were induced to have gene expression by Dox, resulting in cell proliferation.
[0102]
Example 2: Islet cell proliferation by Mycl overexpression (neuroendocrine tumor formation)
Dox was administered to the 8-week-old chimeric mice expressing the Mycl gene produced above for 8 weeks. Mice were sacrificed 8 weeks after Dox administration, the pancreas was removed, and pancreatic sections were evaluated histochemically. As shown by the arrows in FIG. 2, in the pancreatic tissue in which Mycl is overexpressed by Dox induction, islet cells proliferate and the islets are enlarged compared to normal pancreatic tissue.
[0103]
Example 3: Proliferation of islet progenitor-like cells by Mycl overexpression
　Four weeks after administration of Dox to the chimeric mice, the proliferation of somatostatin-positive cells was examined. As shown in Example 2, the proliferation of pancreatic islet cells can be induced by expressing the Mycl gene in pancreatic islet cells. As shown in FIGS. 3 to 6, in the presence of the Mycl gene, the proliferating cells showed gene expression similar to pancreatic islet progenitor cells, and at the same time expressed somatostatin and proliferated. Diminished.
[0104]
Example 4: Proliferation of somatostatin-positive pancreatic islet cell-like cells by Mycl overexpression
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. As shown in FIGS. 4 and 5, insulin-positive cells decreased and somatostatin-positive cells increased significantly.
[0105]
Example 5: De-differentiation into islet progenitor-like cells by Mycl overexpression
　In the same experimental system as in Example 2, gene expression analysis was performed in enlarged pancreatic islets 8 weeks after Dox administration. Pancreatic islets are thought to differentiate from islet progenitor cells with Ngn3 as a marker to pancreatic islet progenitor cells with Fev as a marker, and then differentiate into each islet cell (Fig. 6). Analysis of gene expression in pancreatic islet cells proliferated by Mycl overexpression revealed increased expression of Fev, a progenitor cell marker, and Pax4 and Cck, which are expressed at the same time (Fig. 13). This result suggested that proliferative pancreatic islet progenitor-like cells could be induced by Mycl overexpression.
[0106]
Example 6: Induction of Insulin-Positive Cells by Mycl Expression Shutdown
In the experimental system used in Example 2, Dox administration was stopped after 8 weeks, and changes in insulin-positive cells and somatostatin-positive cells were observed after 2 weeks and 4 weeks. After stopping Dox administration, as shown in FIG.After 2 weeks and 4 weeks, insulin-positive cells, which had once decreased, increased, and in contrast, somatostatin-positive cells, which had once increased, decreased (Fig. 8). This result suggested that the pancreatic islet progenitor-like cells proliferated by Mycl overexpression changed into insulin-positive cells by terminating Mycl expression.
[0107]
In addition, when observing the expression of Ki67, which is known as a target protein for cell proliferation, the number of proliferating cells (Ki-positive cells) increases as the period of Dox administration increases. The number of cells decreased significantly and cell proliferation stopped (Fig. 7).
[0108]
Example 7: Enhancement of glucose tolerance by transient overexpression of Mycl
In chimeric mice, glucose tolerance was evaluated when Dox administration was stopped. As shown on the left side of FIG. 9, glucose tolerance was enhanced in mice to which Dox administration was stopped compared to controls (“cont.”). In addition, fasting blood glucose levels were consistently maintained, suggesting that the pancreatic islets that increased in vivo were functional pancreatic islets (FIG. 9, right).
[0109]
Example 8: Proliferation induction of pancreatic islet cells in vitro by Mycl overexpression
(1) Mycl expression induction in isolated islets
The pancreas was removed from the chimeric mouse, and then the induction of Mycl expression in the isolated islets was examined. Taking the start of Dox addition as day 0, Mycl expression was observed over time, and the results are shown in FIG. Compared to the control, the Dox-added system tended to increase the number of pancreatic islets over time along with the expression of the Mycl gene.
[0110]
(2) Mycl expression induction in cells dispersed from isolated pancreatic islets
The expression of the Mycl gene was examined in the same manner as in (1) above for the cells dispersed from the isolated islets. When the Mycl gene was observed in the cells 14 days after the addition of Dox, it was found that the Mycl gene was expressed in each cell compared to the first day after the addition (Fig. 11).
[0111]
(3) Expression induction of c-Myc and Mycn in isolated pancreatic islets in vitro
It was confirmed that the c-Myc and Mycn-incorporated ES cells prepared above could be doxycycline-dependently induced to express. A chimeric mouse was produced using this ES cell. Expression of c-Myc and Mycn was induced in pancreatic islet cells isolated from the prepared chimeric mice by adding doxycycline in vitro for 1 week. As a result, it was found that the expression induction of c-Myc and Mycn promotes proliferation but induces cell death unlike Mycl (Fig. 12) (Pelengaris S, Khan M, Evan GI., Cell 2002 109(3):321-334).
[0112]
Example 9: Functional evaluation of pancreatic islet cells induced to proliferate in vitro
By transplanting pancreatic islet cells whose proliferation was induced by Mycl overexpression in vitro into diabetic mice, the functionality of the proliferated islet cells was examined. 30 isolated pancreatic islets were dispersed, and Dox was added thereto for one week to induce the proliferation of pancreatic islet cells. When these pancreatic islet cells were harvested and transplanted under the renal capsule of diabetic mice, improvement in random blood glucose was observed. Two weeks later, when the kidney transplanted with pancreatic islet cells was removed, the diabetic mice returned to hyperglycemia again. Transplantation of 300 or more pancreatic islets is generally required to lower blood glucose levels in diabetic mice. Pancreatic islet-like cells proliferated from 60 pancreatic islets successfully lowered blood glucose levels in diabetic mice.
[0113]
Example 10: Verification of therapeutic effect in diabetic mouse model by Mycl expression induction
A diabetic mouse model was created to verify the therapeutic effect of inducing Mycl expression. A diabetic mouse model was prepared by intraperitoneally administering 150 mg/kg of streptozocin (STZ) (having toxicity to pancreatic β-cells) to 8-week-old Mycl expression-inducible mice (KH2-Mycl). did. Two weeks later, Mycl expression was induced by administration of Dox for eight weeks. Expression of Mycl was silenced by stopping Dox administration for an additional two weeks. Glucose responsiveness test (IPGTT) and histological analysis were performed at this time point.
[0114]
The results are shown in FIG. In the control group (administration of STZ alone), blood glucose increased after administration of STZ, whereas in the group in which Mycl expression was induced by Dox (STZ+Mycl), blood glucose decreased after administration of Dox, and administration of Dox was stopped. Euglycemia was also observed after the treatment (upper left of FIG. 14). Furthermore, as a result of a glucose responsiveness test, a rapid decrease in blood glucose level was observed in the group in which Mycl expression was induced (FIG. 14, lower left).
[0115]
Furthermore, histological analysis of the expression of insulin (“Ins”), glucagon (“Gog”), and somatostatin (“Sst”) showed that the proportion of insulin-positive cells decreased in the control group (only STZ was administered). Was. On the other hand, in the group in which the expression of Mycl was induced (STZ+Mycl), an increase in pancreatic islet cells and an increase in the ratio of insulin-producing cells were observed. Based on the above results, the induction of Mycl expression is expected to have a therapeutic effect on diabetes.
[0116]
Example 11: Mycl does not induce hyperplasia other than pancreatic islets
In order to induce the expression of MYC family genes (“Myc”, “Mycn”, and “Mycl”) in vivo in a Dox-dependent manner, ES cells capable of inducing the expression of these genes were first established (Fig. 15, upper row). ). A chimeric mouse was produced by injecting this ES cell into a blastocyst. The expression of the MYC family gene was induced by adding Dox for 4 weeks after the chimeric mice reached 4 weeks of age.
[0117]
Myc expression induction resulted in tumor formation in the liver, and Mycn expression induction resulted in tumor formation in the liver and intestinal tract, while Mycl expression induction did not result in abnormal proliferation at least in the liver and small intestine (Fig. 15, lower panel). This suggested that Mycl proliferates specifically in pancreatic islets.
[0118]
Example 12: Pancreatic Islet Hyperplasia in Aged Mice
Pancreatic islet cells were isolated from aged mice (115 weeks old) and infected with a lentivirus capable of inducing expression of Mycl and pZsGreenDR vectors (Takara, cat#632428) (control group) in a Dox-dependent manner. The zsGreenDR used is a short-lived green fluorescent protein with a proteolytic signal fused to the C-terminus of zsGreen.
[0119]
On the day after infection, the pancreatic islet cells were transferred to three-dimensional culture using Matrigel, and Dox was simultaneously administered to induce the expression of Mycl and zsGreenDR. Pancreatic islet cells in which expression of zsGreenDR was induced showed no change in morphology even after 7 days (Fig. 16, right). On the other hand, by inducing the expression of Mycl, an increase in pancreatic islet cells was observed (FIG. 16, left). From this result, it was found that pancreatic islet proliferation by induction of Mcyl expression is possible even in very old mice.
[0120]
Example 13: Expression of Mcyl in Human Pancreatic Islet Progenitor Cells
In a paper submitted in 2020 (JR Alvarez-Dominguez, et al., Circadian Entrainment Triggers Maturation of Human In Vitro Islets., Cell Stem Cell, 26(1), 108-122, 2020), from human iPS cells to stage We observed the molecular profile when human pancreatic β-cells were specifically induced to differentiate. We therefore used this dataset to observe gene expression of the MYC family (described above) and H3K27ac, a chromatin mark that generally indicates transcriptional activation (Figure 17).
[0121]
Expression of Myc family genes was high in pluripotent stem cells (“hPSCs”) and tended to decrease with differentiation, whereas Mycl transiently increased in “EN” (endocrine progenitor cells, i.e. pancreatic islet progenitor cells). Increased expression was observed. This suggests the possibility that Mycl contributes to the proliferation of human islet cells as well as mouse islet cells.
[0122]
Example 14: Confirmation of pancreatic islet hyperplasia in humans
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. On the next day, the pancreatic islet cells were transferred to three-dimensional culture using Matrigel, and Dox was simultaneously administered to induce the expression of Mycl and zsGreenDR, respectively.
[0123]
The pancreatic islet cells induced to express zsGreenDR showed no change in morphology even after 7 days, but an increase in islet cells was observed when the expression of MYCL was induced (Fig. 18, lower left). Furthermore, as a result of immunostaining, in the MYCL overexpressed group, Ki67 (cell proliferation marker) was detected in cells in which mCherry expression was observed (synonymous with MYCL expression) (Fig. 18 lower right).
[0124]
Example 15: Confirmation of pancreatic islet proliferation in humans by single cell analysis
Human pancreatic islet cells were infected with lentiviruses capable of inducing the expression of Mycl, MYCL, and zsGreenDR (control group) in a Dox-dependent manner. The following day, the cells were shifted to suspension culture using cell culture inserts, and Dox was simultaneously administered to induce the expression of Mycl and zsGreenDR, respectively. Seven days later, after removing dead cells, single-cell RNA-seq was performed according to a standard method.
[0125]
As a result of single-cell RNA-seq, α cells, β cells, δ cells, pancreatic duct cells ("PP"), mesenchymal cells, vascular endothelial cells, and astrocytes were detected (Fig. 19 left). Among them, we focused only on β cells and analyzed the expression of CDK4, which is a marker of cell proliferation. Expression was induced. From this, it was found that human pancreatic islet cells can also proliferate by inducing Mycl expression.
Industrial applicability
[0126]
By applying the technology of the present invention to islets isolated from donors, it is possible to increase the number of islet insulin-positive cells in vitro, and it is possible that the shortage of islet donors in the present invention can be resolved. By using this technology in the differentiation-inducing system of pancreatic islet insulin-positive cells from ES/iPS cells, it becomes possible to efficiently induce the differentiation of pancreatic islet insulin-positive cells from pluripotent stem cells. Furthermore, it is possible to increase the number of insulin-positive repopulation in the produced organ by applying it to pancreatic islet production technology from pluripotent stem cells by the blastocyst complementation method. In the present invention, it is necessary to transiently express Mycl in donor-derived pancreatic islet cells and pluripotent stem cell-derived pancreatic islet cells. It may also be possible to establish pancreatic islet cells. It is also expected that pancreatic islet cells proliferated using this technique can be proliferated and maintained as pancreatic islet stock cell lines. Pancreatic islet stock cell lines may provide a long-term, stable, and large-scale supply of insulin-positive cells. In the future, it is expected that we can provide inexpensive and high-quality pancreatic islets to many diabetic patients.
[0127]
All publications and patent documents cited herein are hereby incorporated by reference in their entirety. Although specific embodiments of the invention have been described herein for purposes of illustration, it will be appreciated by those skilled in the art that various modifications may be made without departing from the spirit and scope of the invention. will be easily understood.
The scope of the claims
[Claim 1]
An insulin production promoter containing the Mycl gene or its gene product.
[Claim 2]
The Mycl gene
(1) a nucleic acid containing the base sequence represented by SEQ ID NO: 1 or 3; or
(2) When the nucleic acid containing the nucleotide sequence represented by SEQ ID NO: 1 or 3 is hybridized under stringent conditions, and the expression of the Mycl gene is induced a nucleic acid encoding a polypeptide having activity to promote insulin production
The insulin production-enhancing agent according to claim 1, comprising
[Claim 3]
The Mycl gene product is
(1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or 4; or
(2) has at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO: 2 or 4, and islet-like cells A polypeptide having a proliferative effect and/or an activity to promote insulin production
The insulin production-enhancing agent according to claim 1, comprising
[Claim 4]
An insulin production promoter in which the Mycl gene or its gene product has been introduced into pancreatic islet cells.
[Claim 5]
The insulin production-enhancing agent according to any one of claims 1 to 4, wherein the Mycl gene is transiently expressed.
[Claim 6]
The insulin production promoter according to claim 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.
[Claim 7]
The insulin production promoter according to claim 6, wherein the stem cells are selected from the group consisting of iPS cells, ES cells, and somatic stem cells.
[Claim 8]
A pharmaceutical composition for preventing and/or treating diabetes and diseases related thereto,
(i) the Mycl gene or its gene product; a vector into which the Mycl gene is integrated; and/or an islet cell into which the Mycl gene or its gene product has been introduced or whose expression has been induced;
(ii) the pharmaceutical composition comprising a pharmaceutically acceptable excipient, diluent or carrier;
[Claim 9]
8. Diabetes and diseases associated therewith are selected from diseases, disorders or conditions associated with type I diabetes, type II diabetes, impaired glucose tolerance, hyperglycemia, dyslipidemia, obesity or metabolic syndrome. The pharmaceutical composition according to .
[Claim 10]
The Mycl gene
(1) a nucleic acid containing the base sequence represented by SEQ ID NO: 1 or 3; or
(2) a polypeptide that hybridizes under stringent conditions with a nucleic acid comprising a nucleotide sequence represented by SEQ ID NO: 1 or 3 and has an activity of promoting insulin production when the expression of the Mycl gene is induced; encoding nucleic acid
10. The pharmaceutical composition according to any one of claims 8 or 9, comprising
[Claim 11]
The Mycl gene product is
(1) a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2 or 4; or
(2) has at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the amino acid sequence represented by SEQ ID NO: 2 or 3, and islet-like cells Polypeptides having proliferative effects and/or insulin production-promoting effects
10. The pharmaceutical composition according to any one of claims 8 or 9, comprising
[Claim 12]
A kit comprising the insulin production-enhancing agent according to any one of claims 1 to 7 or the pharmaceutical composition according to any one of claims 8 to 11.
[Claim 13]
A kit that further contains an activator for activating the Mycl gene.
[Claim 14]
The active agent for activating the Mycl gene is selected from the group consisting of promoters, enhancers, promoter-activating enzymes or factors, enhancer-activating enzymes or factors, nucleic acid-protein complexes, and low-molecular-weight compounds. 14. The kit of claim 13.
[Claim 15]
A pancreatic islet cell into which the Mycl gene or its gene product has been introduced.
[Claim 16]
The islet-like cells according to claim 15, wherein the islet cells are derived from primary islet cells isolated from the pancreas, cultured islet cells, or stem cells.
[Claim 17]
A method for preparing islet-like cells according to claim 16,
(a) the step of integrating the Mycl gene into a recombinant plasmid, recombinant viral vector, minicircle, or episomal vector; and
(b) A step of introducing the recombinant plasmid, recombinant viral vector, minicircle, or episomal vector obtained in step (a) into pancreatic islet cells
method including.
[Claim 18]
A method for preparing the islet-like cells according to claim 16, comprising the step of introducing RNA encoding the Mycl gene or Mycl protein into the islet cells.
[Claim 19]
A method for growing the islet-like cells according to claim 15 or 16, or the islet-like cells prepared by the method according to claim 17 or 18, comprising a step of expressing the Mycl gene.
[Claim 20]
The method according to claim 19, wherein the expression of the Mycl gene is transient.