Method for quantifying insulin secretion capacity of human cells

Abstract

Provided is a method whereby it is possible to quickly and inexpensively quantify human insulin secreted from human cells. Specifically disclosed is a method for quantifying the insulin secretion ability of human cells, which is characterized by culturing a recombinant cell in which a gene expressing a fusion C-peptide and an insulin gene in a human cultured cell strain genome are replaced, and measuring the amount of the secreted fusion C-peptide. The fusion C-peptide is a fusion C-peptide obtained by inserting a detectable tag peptide into a C-peptide chain in a human proinsulin molecule.

Description

Technical Field

[0001] The present invention relates to a quantitative method for the insulin secretion ability of human cells and an application thereof. Background Art

[0002] Type 1 diabetes is a condition in which the pancreatic beta cells, which produce and secrete insulin, are largely ineffective. Approximately 1.1 million people suffer from it in Japan and another 1.1 million sufferers overseas, with 78,000 experiencing symptoms annually, a trend that is rapidly increasing.

[0003] The existing treatment, insulin injections, is symptomatic, but hypoglycemia can lead to lethargy and difficulty managing blood sugar. The burden of blood sugar management can negatively impact the patient's family's quality of life (QOL). Furthermore, brain-dead donors are scarce, limiting the number of patients eligible for islet transplantation and making it suitable only for adults.

[0004] Based on the above-mentioned points, regenerative transplantation of human β cells is expected, and regenerative medicine using iPS cells has attracted attention. However, issues such as increased tumor risk have prevented it from reaching the clinical stage. Meanwhile, the inventors of the present application have reported a technique for inducing the differentiation of somatic cells into β cells through direct reprogramming (Patent Documents 1-3), but the efficiency of inducing differentiation in human cells remains insufficient.

[0005] In addition, even when utilizing the β cells obtained by direct reprogramming, it is also important to develop a composition that promotes insulin secretion ability. For the screening of insulin secretion ability-promoting compositions, it is necessary to develop its screening system. As its method, a method of using human islet cells to detect proliferation markers and proliferation signals of islet cells has been reported (non-patent literature 1, 2). In addition, a screening system (non-patent literature 3) using the replication regulatory activity of rat β cells as an index has also been reported. Further, a method of replacing the C-peptide portion of the proinsulin molecule with luciferase has also been reported, and the cells of rats and mice have been used to detect the technology (patent literature 4) of insulin secretion ability.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: International Publication No. 2016 / 002937

[0009] Patent Document 2: International Publication No. 2017 / 073740

[0010] Patent Document 3: International Publication No. 2021 / 095811

[0011] Patent Document 4: U.S. Patent Application Publication No. 2020017485

[0012] Non-patent literature

[0013] Non-patent literature 1: J Biomol Screen. 2012 Apr; 17(4): 509-518

[0014] Non-patent literature 2: Am J Physiol Endocrinol Metab. 2016 Nov 1; 311(5): 859-E868

[0015] Non-patent literature 3: Endocrinology. 2018 Sep; 159(9): 3143-3157 Summary of the Invention

[0016] (1) Technical issues to be resolved

[0017] However, the methods described in Non-Patent Documents 1 and 2 are difficult to obtain from human pancreatic islet cells, and their detection only detects molecules that affect proliferation, failing to screen for components that affect all beta cell functions, including insulin secretion. Furthermore, the method described in Non-Patent Document 3 detects the replication activity of rat beta cells, failing to screen for components that affect all beta cell functions, including insulin secretion. Furthermore, the method described in Patent Document 1 incorporates a long luciferase structure fused to the C-peptide of mouse insulin, failing to accurately reflect the response to human insulin. Furthermore, the large size of the luciferase molecule may partially impair secretion, making it difficult to fully reflect insulin secretion.

[0018] Therefore, the present invention provides a method capable of rapidly and inexpensively quantifying human insulin secreted from human cells.

[0019] (2) Technical solution

[0020] In response to this, the inventors of the present application discovered that the insulin secretion capacity of human cells can be quantified rapidly and inexpensively by using a gene expressing a fusion C-peptide, which led to the completion of the present invention. The fusion C-peptide is formed by identifying a position within the C-peptide portion of the proinsulin molecule that does not hinder insulin expression / secretion and inserting a short-chain peptide that can be detected with high sensitivity into that position.

[0021] That is, the present invention provides the following inventions [1] to

[17] .

[0022] [1] A method for quantifying the insulin secretion capacity of human cells, characterized in that recombinant cells in which a gene expressing a fusion C-peptide is replaced with an insulin gene in the genome of a cultured human cell line are cultured, and the amount of secreted fusion C-peptide is measured, wherein the fusion C-peptide is a fusion C-peptide in which a detectable tag peptide is inserted into the C-peptide chain in the human proinsulin molecule.

[0023] [2] The quantitative determination method according to [1], wherein the detectable insertion position of the tag peptide is a position corresponding to the C-terminal side of 7V of the C-peptide chain.

[0024] [3] The quantitative method according to [1] or [2], wherein the detectable tag peptide is a tag peptide selected from a tag peptide detectable by chemiluminescence analysis and a tag peptide detectable by immunoassay.

[0025] [4] The quantitative method according to any one of [1] to [3], wherein the detectable tag peptide is a tag peptide selected from a tag peptide capable of binding to luciferase, a tag peptide containing DDDDK, and a tag peptide derived from hemagglutinin.

[0026] [5] The quantitative determination method according to any one of [1] to [4], wherein the detectable tag peptide is a tag peptide that can bind to luciferase.

[0027] [6] The quantitative method according to any one of [1] to [5], wherein the detectable tag peptide is a tag peptide selected from VSGWRLFKKIS (SEQ ID NO: 1), YPYDVPDYA (SEQ ID NO: 51) and YPYDVPDYA (SEQ ID NO: 51).

[0028] [7] The quantitative method according to any one of [1] to [6], wherein the human cultured cell line is: (1) a human cultured cell line; (2) a cell in which three genes, namely Ngn3, Mafa and Pdx1, are introduced into human cells; or (3) a cell in which three genes, namely Ngn3, Mafa and Pdx1, and genes of KLF9, KLF11 or KLF12, and UCN3 are simultaneously introduced into human cells.

[0029] [8] A recombinant cell, which is a recombinant cell formed by replacing the insulin gene in the genome of a human cultured cell line with a gene expressing a fusion C-peptide, wherein the fusion C-peptide is a fusion C-peptide formed by inserting a detectable tag peptide into the C-peptide chain in the human proinsulin molecule.

[0030] [9] The recombinant cell according to [8], wherein the detectable tag peptide is inserted at a position corresponding to the C-terminus of 7V of the C-peptide chain.

[0031]

[10] The recombinant cell according to [8] or [9], wherein the detectable tag peptide is a tag peptide selected from a tag peptide detectable by chemiluminescence analysis and a tag peptide detectable by immunoassay.

[0032]

[11] The recombinant cell according to any one of [8] to

[10] , wherein the detectable tag peptide is a tag peptide selected from a tag peptide capable of binding to luciferase, a tag peptide containing DDDDK, and a tag peptide derived from hemagglutinin.

[0033]

[12] The recombinant cell according to any one of [8] to

[11] , wherein the detectable tag peptide is a tag peptide that can bind to luciferase.

[0034]

[13] The recombinant cell according to any one of [8] to

[12] , wherein the detectable tag peptide is a tag peptide selected from VSGWRLFKKIS (SEQ ID NO: 1), YPYDVPDYA (SEQ ID NO: 51) and YPYDVPDYA (SEQ ID NO: 51).

[0035]

[14] The recombinant cell according to any one of [8] to

[13] , wherein the human cultured cell line is: (1) a human cultured cell line; (2) a cell obtained by introducing three genes, namely, Ngn3, Mafa, and Pdx1, into human cells; or (3) a cell obtained by simultaneously introducing three genes, namely, Ngn3, Mafa, and Pdx1, and genes of KLF9, KLF11, or KLF12, and UCN3 into human cells.

[0036]

[15] A method for screening a substance that promotes the production and secretion of human insulin, characterized in that the recombinant cell described in any one of [8] to

[14] is cultured in the presence of a test substance and the amount of secreted fusion C-peptide is measured.

[0037]

[16] A human insulin production and secretion promoter comprising a substance obtained by the screening method described in

[15] .

[0038]

[17] An insulin production and secretion promoter comprising one or two selected from valproic acid and zebularine.

[0039] (3) Beneficial effects

[0040] According to the present invention, human insulin secreted by human beta cells can be quantified rapidly and at low cost, thereby enabling rapid and low-cost screening for components that affect all beta cell functions, including insulin secretion capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Comparative results show the effect of introducing mouse insulin 1-HiBiT and insulin 2-HiBiT fusion protein expression plasmids with five different structures into the mouse pancreatic β-cell line MIN6 and culturing them for three days to increase insulin protein production in the cells. The vertical axis represents the amount of insulin-HiBiT fusion protein in the cell lysate, as measured by chemiluminescence using luciferase activity as an indicator. Error bars indicate the standard deviation of the values ​​measured for three replicates.

[0042] Figure 2 The chemiluminescence level, measured using luciferase activity as an indicator, shows the amount of insulin-HiBiT fusion protein secreted extracellularly after three days of culture in the mouse pancreatic β-cell line MIN6 following the introduction of five different mouse insulin 1-HiBiT and insulin 2-HiBiT fusion protein expression plasmids. Error bars represent the standard deviation of the values ​​measured for three replicates.

[0043] Figure 3 The results show the results of using genome editing to insert a gene encoding a human insulin-HiBiT fusion protein into the insulin genome of the human adipose-derived cell line SW872. Single cells were isolated and amplified using drug resistance as an indicator, and genomic PCR and agarose electrophoresis were used to confirm whether the genome editing occurred in the monoallelic or biallelic loci.

[0044] Figure 4 The results show the results of inducing direct reprogramming into β cells by co-expressing four transcription factors (O: Ngn3, K: Glis1, A: MafA, P: Pdx1) by inserting genes encoding three different human insulin-HiBiT fusion proteins into the genome of the human adipose-derived cell line SW872. The results also show a comparison of the amount of insulin protein produced and secreted by these cells. The vertical axis shows the amount of HiBiT protein in the cell culture medium, as measured by chemiluminescence using luciferase activity as an indicator. Error bars indicate the standard deviation of the values ​​measured for three replicates.

[0045] Figure 5The results show a comparison of the amount of human insulin-HiBiT fusion protein produced in the human adipose-derived cell line SW872, which maintains the ability to secrete extracellular matter. Three transcription factors (O: Ngn3, A: MafA, P: Pdx1) were co-expressed and cultured in the cell line. The cell line then continued to be cultured under induction and non-induction conditions with the addition of factor K, a gene that promotes direct reprogramming into β cells. The vertical axis of the graph (a) represents the amount of human insulin protein measured by enzyme-linked immunosorbent assay, while the vertical axis of the graph (b) represents the luciferase activity of HiBiT. Error bars indicate the standard deviation of the values ​​measured for three replicates.

[0046] Figure 6 ELISA detection of proteins in the culture supernatant of human SW872 cells transfected with the plasmid alone, the human insulin expression plasmid, the human insulin-HA expression plasmid, and the human insulin-FLAG expression plasmid is shown. The results with both HA and FLAG antibodies also indicate that human insulin-HA and human insulin-FLAG are produced and secreted in the culture supernatant.

[0047] Figure 7A The results show that in a cultured cell line SW872 derived from human fat, in which the human insulin-HiBiT fusion gene that maintains the ability to secrete extracellularly is incorporated into the genome, three transcription factors (Ngn3, MafA, Pdx1) are co-expressed and cultured, and expression plasmids encoding various genes are introduced by transfection to compare the amount of human insulin-HiBiT fusion protein produced from the cells, thereby exploring the results of genes that promote direct reprogramming to β cells. The vertical axis shows the amount of HiBiT fusion protein contained in the cell culture medium as measured by chemiluminescence using luciferase activity as an indicator. The error bars represent the standard deviation of the measured values ​​of three repeated test samples. (1) Shows that the introduction of the gene encoding human KLF9 increases the amount of human insulin-HiBiT fusion protein produced.

[0048] Figure 7B (2) shows that when genes encoding human KLF9 and human UCN3 were introduced simultaneously, the amount of human insulin-HiBiT fusion protein produced was further increased.

[0049] Figure 7C(3) Three transcription factors (Ngn3, MafA, and Pdx1) were co-expressed in Huh7 cells derived from human adipose tissue and cultured. Expression plasmids encoding insulin or KLF9 and UCN3 genes were transfected and the human insulin production in the cells was compared. The vertical axis represents the insulin content in the cells. Error bars represent the standard deviation of the measured values ​​for three replicates.

[0050] Figure 7D (4) shows the results obtained when four transcription factors (Ngn3, Glis1, MafA, Pdx1) were co-expressed and cultured in Huh7 cells, and expression plasmids encoding insulin or KLF9 and UCN3 genes were transfected to compare the amount of human insulin produced from the cells.

[0051] Figure 7E (5) shows the results obtained when SW872 cells were co-expressed with four transcription factors (Ngn3, Glis1, MafA, and Pdx1) and cultured, and then transfected with expression plasmids encoding either KLF9, KLF11, or KLF12 along with the UCN3 gene, to compare the amount of human C-peptide produced from the cells. Error bars represent the standard deviation of the measured values ​​for three replicates.

[0052] Figure 8 The results show the results of a search for low-molecular-weight compounds that promote direct reprogramming of β cells by co-expressing three transcription factors (Ngn3, MafA, and Pdx1) in the human adipose-derived cell line SW872, which has the human insulin-HiBiT fusion gene incorporated into its genome. Various compounds were added to the cells and the amount of human insulin-HiBiT fusion protein produced was compared. The vertical axis shows the amount of insulin-HiBiT fusion protein contained in the cell lysate, using chemiluminescence measured using luciferase activity as an indicator. Error bars represent the standard deviation of the values ​​measured for three replicates. DETAILED DESCRIPTION

[0053] One embodiment of the present invention is a method for quantifying the insulin secretion capacity of human cells, characterized by culturing recombinant cells in which a gene expressing a fusion C-peptide is replaced with the insulin gene in the genome of a cultured human cell line, and measuring the amount of secreted fusion C-peptide. The fusion C-peptide is formed by inserting a detectable tag peptide into the C-peptide chain within the human proinsulin molecule.

[0054] In addition, another embodiment of the present invention is a recombinant cell, which is a recombinant cell obtained by replacing the insulin gene in the genome of a human cultured cell line with a gene expressing a fusion C-peptide, wherein the fusion C-peptide is a fusion C-peptide obtained by inserting a detectable tag peptide into the C-peptide chain within the human proinsulin molecule.

[0055] Another embodiment of the present invention is a method for screening a substance that promotes the production and secretion of human insulin, characterized in that the recombinant cell is cultured in the presence of a test substance, and the amount of secreted fusion C-peptide is measured.

[0056] Furthermore, another embodiment of the present invention is a human insulin production and secretion promoter comprising a substance obtained by the screening method.

[0057] Furthermore, another embodiment of the present invention is an insulin production and secretion promoter comprising one or two selected from valproic acid and zebullin.

[0058] Proinsulin, the precursor of insulin (with C-peptide bound between the A and B chains of insulin), is produced in beta cells and enzymatically broken down before secretion into separate molecules of insulin (with the A and B chains bound by a disulfide bond) and C-peptide. Therefore, the level of C-peptide in the blood is a clinical indicator of insulin secretion capacity, and known methods for its detection include ELISA, RIA, and RT-qPCR.

[0059] C-peptide in humans is a 31-amino acid peptide represented by EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ (SEQ ID NO: 2).

[0060] It is completely unclear at what position of the C-peptide chain the insertion of a detectable tag peptide will not hinder the expression / secretion of insulin. To this end, the inventors of the present application first designed a plurality of fusion peptides formed by inserting a detectable tag peptide into mouse insulin C-peptide, and selected the structure of the fusion peptide that would not hinder the production of mouse insulin. As a result, for mice, it was confirmed that the fusion peptide with the tag inserted at two locations would not hinder the expression / secretion of insulin. Among them, the insertion positions that did not hinder the expression / secretion of insulin in mice were equivalent to the positions of 7V and 13G of human insulin C-peptide (described SEQ ID NO: 2).

[0061] Next, a fusion peptide was designed in which a detectable tag peptide was inserted at two positions in the human insulin C-peptide that were identical to the two positions in the mouse insulin C-peptide that were well-characterized in mice, and the expression / secretion of human insulin was studied. The results showed that when a gene expressing a fusion C-peptide in which the tag peptide was inserted at a position corresponding to the C-terminus of 7V of the human proinsulin C-peptide chain was used, the expression / secretion of human insulin was not inhibited, and the human insulin secretion capacity could be accurately measured.

[0062] In addition, human insulin C-peptide does not need to be completely identical to the 31 amino acids, and may have 1 to 3 amino acids on the C-terminal and / or N-terminal sides, or may be missing 1 to 6 internal amino acids. For example, it can be: RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR (SEQ ID NO: 3), RREAEDLQVGGGPGAGSLQPLALEGSLQKR (SEQ ID NO: 4), EAEDLQVGGGPGAGSLQPLALEGSLQ (SEQ ID NO: 5), RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQ (SEQ ID NO: 6), EAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR (SEQ ID NO: 7).

[0063] As the tag peptide that can be detected, for example, a tag peptide selected from a tag peptide that can be detected by chemiluminescence analysis and a tag peptide that can be detected by immunoassay can be listed. Among them, as a tag peptide that can be detected by chemiluminescence analysis, a tag peptide that can bind to luciferase can be listed. In addition, as a tag peptide that can be detected by immunoassay, a peptide selected from a tag peptide containing DDDDK (FLAG tag) and a tag peptide from hemagglutinin (HA tag) can be listed.

[0064] More preferably, detectable tag peptides include tag peptides selected from VSGWRLFKKIS (SEQ ID NO: 1), YPYDVPDYA (SEQ ID NO: 51), and YPYDVPDYA (SEQ ID NO: 51).

[0065] Among them, from the perspective of rapid and low-cost measurement of the fusion C-peptide, a tag peptide capable of binding to luciferase is more preferred as the detectable tag peptide, and VSGWRLFKKIS (SEQ ID NO: 1) is even more preferred.

[0066] To implement the present invention, a gene expressing a fusion C-peptide, formed by inserting a detectable tag peptide into the C-peptide chain of a human proinsulin molecule, is first constructed. This gene is then replaced with the insulin gene in the genome of a cultured human cell line to produce recombinant cells. Conventional genome editing methods, such as ZFNs, TALENs, and CRISPR / Cas9, can be used to replace the fusion peptide gene with the insulin gene in the genome of the cultured human cell line.

[0067] Examples of the human cultured cell lines used include the liver-derived cell line Huh7 and the adipose-derived cell line SW872.

[0068] Furthermore, the present invention can also employ a method for inducing β cells having excellent insulin secretion capacity from somatic cells by direct reprogramming. In this case, the cells used are preferably: (1) cells into which the three genes Ngn3, Mafa, and Pdx1 have been introduced into human cells, or (2) cells into which the three genes Ngn3, Mafa, and Pdx1, the genes KLF9, KLF11, or KLF12, and the UCN3 gene have been simultaneously introduced into human cells.

[0069] By culturing the obtained recombinant cells and measuring the amount of secreted fusion C-peptide, the amount of human insulin secreted by human cells can be quantified.

[0070] For the culture of recombinant cells, culture media suitable for human cell lines, such as DMEM, RPMI1640, MEM, αMEM, M199, etc., can be used, and culture can be carried out under conventional conditions, such as a temperature of about 37°C and a CO2 concentration of 2-5%.

[0071] When a tag peptide capable of binding to luciferase is used as the detectable tag peptide, the amount of secreted fusion peptide, i.e., the amount of human insulin secreted in human cells, can be quantified based on the luminescence intensity of the luciferase fragments reacted in the culture medium. The luciferase fragments used and the luminescence intensity can be measured using the commercially available HiBiT system (Promaga KK).

[0072] When a tag peptide detectable by immunoassay is used as the detectable tag peptide, the amount of human insulin secreted by human cells can be quantified by assessing the amount of fusion C-peptide secreted in the culture medium by immunoassay. Examples of immunoassays include radioimmunoassay (RIA) and enzyme immunoassay (EIA), with ELISA being particularly preferred.

[0073] By culturing the recombinant cells in the presence of a test substance, culturing the recombinant cells, and measuring the amount of secreted fusion C-peptide, a substance that promotes the production and secretion of human insulin can be screened.

[0074] The amount of secreted C-peptide is measured according to the type of tag peptide used as described above.

[0075] As a more preferred embodiment, a method for screening substances that promote the production and secretion of human insulin is provided, wherein the recombinant cells are cultured in the presence of a test substance, and a luciferase fragment is allowed to function, thereby measuring the insulin secretion capacity of human cells.

[0076] The test substance may be a conventional compound or a gene that affects the recombinant cell. In the case of a gene, it may be a gene that has been previously introduced into the recombinant cell.

[0077] If the addition of a test compound or gene increases insulin production and secretion, the test compound or gene can be determined to be a substance that promotes insulin production and secretion in human β cells and can be used as an insulin production and secretion enhancer.

[0078] When inducing β cells with excellent insulin secretion capacity from somatic cells through direct reprogramming, it is sufficient to express the insulin secretion-promoting genes obtained through the above screening in addition to the genes described in Patent Documents 1 to 3. Examples of the genes described in Patent Documents 1 to 3 include Glis1 or Glis3 (K factor); Ngn3, Mafa, and Pdx1; or combinations of Glis1 or Glis3 with Ngn3, Mafa, and Pdx1; and also examples of simultaneously introducing Ngn3, Mafa, and Pdx1 into human cells along with KLF9 and UCN3 genes.

[0079] When expressing the above genes in somatic cells, the methods described in Patent Documents 1-3 can be used. Specifically, cultured somatic cells to be evaluated can be simultaneously infected with retroviral or lentiviral vectors containing the three genes cloned separately. These three or five genes are then co-expressed in the cells, and the cells are cultured for one to two weeks in a differentiation-inducing medium described in Patent Documents 1-3. This allows for the production of insulin-secreting β cells. Alternatively, the three or five genes can be co-expressed in cells using mRNA, episomal vectors encoding oriP-EBNA1, or single-stranded RNA viruses such as Sendai virus. Compared to the aforementioned lentiviral and retroviral methods, these methods prevent the introduced genes from being incorporated into the host genome, allowing for safer, differentiated β cells to be obtained.

[0080] The substances obtained by the screening method can be used as agents that promote the production and secretion of human insulin. Specifically, the screening method discovered that valproic acid and zabulaline can serve as components that promote the production and secretion of human insulin. That is, one embodiment of the present invention is an agent that promotes the production and secretion of insulin containing one or both of valproic acid and zabulaline.

[0081] Valproic acid is known as a treatment for epilepsy, mania, migraines, etc., but it is not known to have an insulin secretion-stimulating effect. Zebulin is a DNA methylation inhibitor, but it is not known to have an insulin secretion-stimulating effect.

[0082] Example

[0083] Next, the present invention will be described in further detail with reference to Examples, but the present invention is not limited to these Examples.

[0084] Example 1

[0085] (1) Design of mouse insulin C-peptide fusion peptide

[0086] For mouse insulin protein, the preproinsulin protein, which serves as a precursor, is first processed in the endoplasmic reticulum of pancreatic beta cells to remove the terminal signal sequence to become proinsulin, from which C-peptide is cleaved to form insulin. C-peptide and insulin molecules are produced simultaneously in equimolar amounts and secreted extracellularly, so its quantitative determination of insulin secretion capacity can be indirect. Among them, the HiBiT tag is inserted into a preferred site within the mouse proinsulin molecule, and an attempt is made to construct a C-peptide-HiBiTHiBiT fusion peptide that does not impair the C-peptide cleavage reaction, and the HiBiT tag fused with the C-peptide is secreted extracellularly, thereby enabling rapid and low-cost quantification.

[0087] First, fusion proteins were designed by inserting or replacing the HiBiT tag at five different sites in the C-peptide of mouse insulin 1 and mouse insulin 2 to investigate whether this would affect C-peptide cleavage and secretion. Simply inserting the HiBiT tag increases the molecular weight of the C-peptide fusion, potentially impairing cleavage and secretion. To address this issue, the amino acid sequences shown in SEQ ID NOs: 10, 12, 14, 16, and 18 were designed as HiBiT fusion proteins with the C-peptide region of mouse preproinsulin 1. These amino acid sequences consisted of HiBiT inserted between amino acids 69 and 70 from the N-terminus of mouse preproinsulin 1, or HiBiT substituted between amino acids 71-78, 62-68, 72-76, or 64-68 from the N-terminus of mouse preproinsulin 1. Genes encoding these amino acid sequences were cloned into expression plasmids.

[0088] These HiBiT fusion peptides with mouse preproinsulin 1 are referred to as mIns1-HiBiT1, mIns1-HiBiT2, mIns1-HiBiT3, mIns1-HiBiT4, and mIns1-HiBiT5 in the order described. The sequence of the expression plasmid encoding mIns1-HiBiT1 is shown in SEQ ID NO: 8. In addition, the base sequence of the gene encoding mIns1-HiBiT1 is shown in SEQ ID NO: 9, and its amino acid sequence is shown in SEQ ID NO: 10. The expression plasmids of mIns1-HiBiT2 to 5 are obtained by replacing the 1785th to 2141st nucleotide sequence portions of the sequence shown in SEQ ID NO: 8 with the gene sequences shown in SEQ ID NO: 11, 13, 15, and 17, respectively. These genes were commissioned to be synthesized by VectorBuilder Inc. (Tokyo, Japan) and purchased therefrom.

[0089] Similarly, the amino acid sequences shown in SEQ ID NOs: 21, 23, 25, 27, and 29 were designed as HiBiT fusion proteins with the C-peptide region of mouse preproinsulin 2. These amino acid sequences were designed to have HiBiT inserted between the 69th and 70th amino acids from the N-terminus of mouse preproinsulin, or to have HiBiT replace the peptide between amino acids 72 to 79, 62 to 68, 72 to 76, or 64 to 68, and the genes encoding these amino acid sequences were cloned into expression plasmids. These HiBiT fusion peptides with mouse preproinsulin 2 are referred to as mIns2-HiBiT1, mIns2-HiBiT2, mIns2-HiBiT3, mIns2-HiBiT4, and mIns2-HiBiT5, in the order in which they are described. The sequence of the expression plasmid encoding mIns2-HiBiT1 is shown in SEQ ID NO: 19. In addition, the base sequence of the gene encoding mIns2-HiBiT1 is shown in SEQ ID NO: 20, and its amino acid sequence is shown in SEQ ID NO: 21. The expression plasmids of mIns2-HiBiT2~5 are obtained by replacing the nucleotide sequence of positions 1786 to 2148 of the sequence shown in SEQ ID NO: 19 with the gene sequences shown in SEQ ID NOs: 22, 24, 26, and 28, respectively. These genes were synthesized by VectorBuilder Inc. (Tokyo, Japan) and purchased therefrom.

[0090] (2) Selection of a structure that does not interfere with insulin production in mice

[0091] To investigate whether the mouse insulin C-peptide fusion peptides designed in Example 1(1) affect gene expression and protein secretion in cells, expression vectors for these peptides were introduced into mouse pancreatic β cell line MIN6 cells (distributed from Osaka University) and expression dynamics were investigated. A 21-passage MIN6 cryopreserved cell was seeded in a 10 cm culture dish and cultured for 2 days in DMEM medium (Sigma D5796-500ML) containing 10% FBS and 1% antibiotics (Sigma Penicillin-Streptomycin). The cells were then freed in a 0.25 w / v% trypsin-1 mmol / l EDTA·4Na solution (containing phenol red) (WAKO 201-16945) and the cell number was counted using a hemocytometer. The cells were then seeded in a 96-well culture plate at 4,000 cells / well. The next day, after replacing the culture medium with 0.1 mL / well, 0.08 μg of the internal calibration vector pGL3-luc (Promega Corporation) was added to 0.08 μg of each of the 10 expression plasmids for mIns1-HiBiT1-5 and mIns2-HiBiT1-5, totaling 0.16 μg of DNA. The cells were then transformed using Lipofectamine 2000 (Thermo Fisher Scientific Inc.) in each well of a 96-well plate seeded with the cells according to the protocol. Culture was continued, and the culture medium was exchanged after 7 hours. Culture was continued and a glucose response test was performed 2 days later.

[0092] After suspending each well in 100 μL of 1.4 mM glucose / KRB buffer (130 mM NaCl, 5 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2) and incubating at 37°C for 1 hour, cells were suspended in 100 μL of 2.8 mM glucose / KRB buffer and incubated for 1 hour. The buffer was removed and the cells were collected (Low Glucose sample). Cells were then suspended in 0.1 mL of 16.8 mM glucose / KRB buffer and incubated for 1 hour. Following the same procedure as above, the medium was removed and the cells were collected (High Glucose sample). The remaining cells were washed with 100 μL of ice-cold PBS(-), and 50 μL of x1 Cell Lysis Buffer (Promega E194A) was added. Cells were disrupted and recovered by pipetting (Cell Lysate sample). The insulin content in each sample was compared using a HiBiT assay kit (Promega Corporation, Nano-Glo HiBiTExtracellular Detection System).

[0093] The results, such as Figure 1 As shown, no significant production of insulin-HiBiT fusion proteins was observed for mIns1-HiBiT1 to 5. However, significant production of fusion proteins of mouse insulin 2, which has a structure similar to human insulin, and HiBiT was observed in mIns2-HiBiT2 to 5. In particular, expression and secretion of the fusion proteins mIns2-HiBiT2 and mIns2-HiBiT5 were significant. In particular, glucose-responsive secretion was observed for mIns2-HiBiTHiBiT5, suggesting that it is the most optimal fusion protein structure that does not interfere with the inherent properties of C-peptide.

[0094] (3) Design of human insulin C-peptide fusion peptide

[0095] The results of Example 1 (2) suggest that among HiBiT fusions with mouse insulin 2, which has a structure similar to human insulin, mIns2-HiBiT2 and mIns2-HiBiT5 are structures that do not hinder insulin expression and secretion. With reference to these structures, fusion proteins of human insulin and HiBiT were designed. As a negative control, hIns-HiBiT1, which is identical to mIns2-HiBiT1, was designed. As promising structures, hIns-HiBiT2 and hIns-HiBiT5, which are identical to mIns2-HiBiT2 and mIns2-HiBiT5, were designed. Their amino acid sequences are shown in SEQ ID NOs: 31, 33, and 35.

[0096] (4) Introduction into the genome of human cell lines

[0097] The DNA encoding the three human insulin-HiBiT fusion proteins designed in Example 1(3), namely, hIns-HiBiT1, hIns-HiBiT2, and hIns-HiBiT5 (shown in SEQ ID NOs: 30, 32, and 34), was commissioned and purchased from VectorBuilder Inc. (Tokyo, Japan). The gene sequences encoding these three human insulin-HiBiT fusion proteins are shown in SEQ ID NOs: 30, 32, and 34.

[0098] These genes were inserted into the insulin gene in the genome of human cell lines using genome editing technology to create a plasmid group for replacement.

[0099] First, the Pre-Donor-arm1-HiBiT-arm2 plasmid shown in SEQ ID NO: 36 was commissioned and purchased from VectorBuilder Inc. (Tokyo, Japan). Using the plasmid as a template, a PCR reaction was performed using primers consisting of the sequences shown in SEQ ID NO: 37 and SEQ ID NO: 38 to obtain a single-stranded DNA fragment. PCR was performed using PrimeSTAR TMMax DNA Polymerase (Takara Bio Inc.) was added and placed in a thermal cycler at 98°C for 2 minutes. The reaction was then repeated 25 times: 98°C for 10 seconds, 55°C for 10 seconds, and 72°C for 30 seconds, followed by 72°C for 2 minutes. Simultaneously, PCR was performed using pcDNA13.1(-) (Thermo Fisher Scientific Inc.) as a template and the primers shown in SEQ ID NO: 39 and SEQ ID NO: 40 in the same manner as above. The two single-stranded DNAs obtained were purified separately by agarose electrophoresis and mixed in a 1:1 ratio. The plasmid shown in SEQ ID NO: 41, Pre-Donor-arm1-NeoR-arm2, was generated by an infusion ligation reaction using Infusion Ligase (Takara Bio Inc.). This plasmid was then used as a template and PCR reactions were performed in the same manner as described above using the primers shown in SEQ ID NOs: 42 and 43 to obtain single-stranded DNA fragments. Simultaneously, PCR reactions were performed in the same manner as described above using the genes encoding hIns-HiBiT1, hIns-HiBiT2, and hIns-HiBiT5 shown in SEQ ID NOs: 30, 32, and 34 as templates and primers shown in SEQ ID NOs: 44 and 45 to obtain single-stranded DNA fragments containing the genes encoding hIns-HiBiT1, hIns-HiBiT2, and hIns-HiBiT5, respectively. These fragments were mixed with the single-stranded DNA fragment obtained by PCR from the plasmid Pre-Donor-arm1-NeoR-arm2, and a genome editing donor vector containing the same arm sequences as the genes encoding hIns-HiBiT1, hIns-HiBi2, and hIns-HiBi5, the neomycin resistance gene, and the insulin gene was constructed by enzyme-free ligation in the same manner as described above. The sequence of one of the donor vectors, Arm1-hIns-HiBiT1-NeoR-Arm2, which contains hIns-HiBiT1, is shown in SEQ ID NO: 46.In addition, the donor vector containing hIns-HiBiT2 and hIns-HiBiT5 is a donor vector in which the 60th to 432nd nucleotide sequence of the Arm1-hIns-HiBiT1-NeoR-Arm2 sequence of SEQ ID NO: 46 is replaced with the hIns-HiBiT2 and hIns-HiBiT5 sequences shown in SEQ ID NO: 32 and SEQ ID NO: 34.

[0100] In order to introduce the hIns-HiBiT1, hIns-HiBiT2, and hIns-HiBiT5 sequences in the donor vector into the genomes of various parts of human cells through genome editing, we commissioned VectorBuilder Inc. to synthesize and purchase a guide RNA-hCAS9 plasmid containing the same guide DNA as the insulin gene and hCAS9 as shown in SEQ ID NO: 47.

[0101] The Arm1-hIns-HiBiT1~5-NeoR-Arm2 donor vector and guide RNA-hCAS9 vector were cloned into the human adipose-derived cell line SW872 (ATCC HTB-92 TM ) and the cell line Huh7 from human liver (distributed from Osaka University) were transfected with Lipofectamine 3000 in a 1:1 ratio. The DMEM was used as a culture medium for Huh7, and Gibco DMEM / F12 (1:1) cat#11330-032 10% FBS, 1% P / S was used as a culture medium for SW872. 7 hours after transfection, the culture medium was exchanged, and 400 μM of G418 was added as a resistance marker to attempt to induce the isolation of genome-edited cells. The culture medium was exchanged once after 3 days, and after about 1 week, the cells were split and passaged using the trypsin EDTA, and 96-well plates were used to attempt to single the cells. As a result, it was believed that single clones of hIns-HiBiT1, hIns-HiBiT2, and hIns-HiBiT5 were obtained in Huh7 and SW872 cells, respectively, which had proliferated from one cell within a period of about 2 weeks.

[0102] (5) Confirmation of recombinant gene introduction

[0103] For the recombinant cell group obtained by genome editing by proliferating a single cell in the embodiment 1 (4), genomic PCR is performed to confirm whether the recombinant gene is actually introduced into the genome, or whether the genome editing is performed on a monoallelic or biallelic gene. Each single clone cell obtained by genome editing is inoculated into a 24-well culture plate and proliferated to more than 100,000 cells in each well. Genomic DNA is extracted from each cell using the Monarch Genomic DNA Purification Kit (New England Bio Labs., product number: T3010), and used as a template. PCR is performed using the primer set shown in SEQ ID NO: 48, 49, or the primer set shown in SEQ ID NO: 48, 50, and the amplified PCR band is confirmed by agarose gel electrophoresis to confirm whether the recombination is a monoallelic or biallelic gene. The PCR reaction conditions are the same as those in the embodiment. The structure of the primers and the recombinant gene is shown in Figure 3 .

[0104] As shown in the gel electrophoresis photographs, the bands obtained for the unedited allele using the primer set of SEQ ID NOs: 48 and 49 were essentially equal to the bands obtained for the recombined cells using the primer set of SEQ ID NOs: 48 and 50, indicating that both homozygous and heterozygous edited cells were constructed.

[0105] (6) Functional evaluation of human insulin-HiBiT fusion protein

[0106] The functions of the recombinant human cells expressing the human insulin-HiBiT fusion protein obtained in Example 1 (5) were evaluated for each recombinant protein, hIns-HiBiT1, hIns-HiBiT2, and hIns-HiBiT5. Using the monoallelic recombinant SW872 cells obtained, the three recombinant cells were seeded into a 24-well culture dish and allowed to proliferate. After culturing for 3 days until the cells were essentially confluent, the culture medium was replaced with a differentiation medium for differentiation into β cells. The differentiation medium was supplemented with 10 μM 2-mercaptoethanol, 10 μM ZnSO4, 10 μM cAMP, and 10 mM nicotinamide at final concentrations in the DMAM medium (Sigma D5796-500ML) containing 10% FBS and 1% antibiotics (Sigma penicillin-streptomycin). Furthermore, to induce direct reprogramming into β cells, the cells were infected with a single-stranded RNA virus, Sendai virus vector, containing the four genes described in Patent Documents 1 to 3: Glis1, Ngn3, Mafa, and Pdx1. These genes were co-expressed. Sendai virus was purchased from TOKIWA-Bio Inc.

[0107] The results, such as Figure 4 As shown, only the hIns-HiBiT5 recombinant protein promotes insulin expression from cells, and actual insulin expression and secretion can be observed. This demonstrates that the insertion position of HiBiT2, where excellent production and secretion are observed when fused to mouse insulin 2, is ineffective in humans, and that only the hIns-HiBiT5 fusion is a construct that can be used to quantify insulin expression and secretion. Furthermore, the NC in the figure represents a negative control, obtained using recombinant SW872 cells that were not infected with the Sendai virus but did not harbor the hIns-HiBiT5 fusion.

[0108] Example 2

[0109] (filter)

[0110] The human recombinant cells secreting the hIns-HiBiT5 fusion protein produced in Example 1 (5) and functionally verified in (6) are powerful tools for comprehensively exploring genes / compounds and other methods that promote the differentiation and function of β cells, which have been difficult to quantitatively determine due to high costs and time. Furthermore, the human recombinant cells produced by the present invention were verified to be effectively used in the search for new genes that promote β cell function.

[0111] Human SW873 cells producing hIns-HiBiT5, the usefulness of which was confirmed in Example 1 (6), were infected with Sendai virus vectors expressing three genes, Ngn3, Mafa, and Pdx1, and cultured for 2 days using a β-cell differentiation medium. Then, the gene described in Patent Documents 1 to 3, i.e., Glis1, which is carried on a free-type vector (Fujifilm), was added to the cells to investigate whether the differentiation of β cells, i.e., the hyperinsulin expression, was observed. As a result, 2 days after infection with Sendai virus, Glis1 carried on the free-type vector was added, and an excellent insulin production-promoting effect was observed. Among them, Figure 5 Insulin levels were measured using ELISA (Medallia Inc.) in (a), which required a 3-hour reaction time and cost over 100 yen per sample. The HiBiT fusion protein assay shown in (b) can be performed with a 10-minute reaction time and a cost of 3-50 yen per sample.

[0112] Example 3 Verification of the effectiveness of human insulin C-peptide fusion peptides based on multiple tags

[0113] The hIns-HiBiT5 structure described in Example 2 demonstrates the effective insertion position of a short peptide tag within the insulin molecule. Since the effectiveness of this peptide insertion position is not limited to the use of a HiBiT peptide, insulin production and secretion can be measured even when the HiBiT peptide in hIns-HiBiT5 is replaced with other short peptides. Specifically, constructs were constructed in which the HA tag (SEQ ID NO: 51) or FLAG tag (SEQ ID NO: 52), which are easily detectable by antibodies, were replaced, and their ability to detect insulin without impairing insulin production and secretion was investigated.

[0114] (1) Preparation of hIns-HA and hIns-FLAG expression plasmids

[0115] Using the primers shown in SEQ ID NO: 53 and 54, a PCR reaction was performed using human pancreatic islet DNA (healthy donor) cDNA (Product No. HIcDNA133dT and HIcDNA-171dT, Primer Corporation) as a template to obtain a single-stranded DNA fragment. PCR was performed using PrimeSTAR TMMax DNA Polymerase (Takara Bio Inc.) was incubated at 98°C for 2 minutes in a thermal cycler, followed by 25 cycles of 98°C for 10 seconds, 55°C for 10 seconds, and 72°C for 30 seconds, followed by 2 minutes at 72°C. Simultaneously, the expression plasmid pEBMulti-Hyg (FUJIFILM Wako Pure Chemical Corporation, product number 050-08121, 11,029 base pairs) was cleaved by adding the restriction endonuclease XhoI (New England Biolabs) and incubating at 37°C for 1 hour. The two single-stranded DNAs were purified separately by agarose electrophoresis and mixed at a 1:1 ratio. An INFUSION ligation reaction was performed using INFUSION ligase (Takara BioInc.) to create an expression plasmid, pEBMulti-hIns, containing the human insulin cDNA (complementary DNA: DNA consisting of the sequence from the start codon to the stop codon of a gene translated into protein) shown in SEQ ID NO: 55 cloned into the Xho I site within the multiclone of pEBMulti-Hyg. This plasmid expresses the human insulin precursor shown in SEQ ID NO: 56.

[0116] Next, the pEBMulti-hIns prepared above was subjected to PCR reaction as a template using the primers shown in SEQ ID NOs: 57 and 58 to obtain a single-stranded DNA fragment. The PCR conditions were the same as described above. In the same manner, the pEBMulti-hIns was subjected to PCR reaction as a template using the primers shown in SEQ ID NOs: 61 and 62 to obtain a single-stranded DNA fragment. The two single-stranded DNAs obtained were purified by agarose electrophoresis, and INFUSION ligase (Takara BioInc.) was used to produce an expression plasmid, pEBMulti-hIns-HA, and pEBMulti-hIns-FLAG, cloned into the XhoI site of the multiclone of pEBMulti-Hyg with hIns-HA shown in SEQ ID NO: 59 and hIns-FLAG shown in SEQ ID NO: 63.

[0117] (2) Functional evaluation of human insulin-HA and FLAG fusion proteins

[0118] SW872 cells seeded in a 24-well culture dish were infected with a substance (TOKIWA-Bio inc.) in which the four genes Glis1, Ngn3, Mafa and Pdx1 were carried on a single-stranded RNA virus, i.e., a Sendai virus vector, in the same differentiation medium as in the above-mentioned embodiment, so that the four genes were co-expressed. After 4 hours, the culture medium was replaced with the aforementioned differentiation medium for β cells. After 24 hours of culture, the expression plasmid obtained in (1), pEBMulti-hIns-HA, pEBMulti-hIns-FLAG, and pEBMulti-Hyg and pEBMulti-hIns as negative controls were transfected using Lipofectamine 3000. After 7 hours, the culture medium was exchanged for 2 days of continuous culture, and 100 μL of the culture supernatant was recovered. Whether these culture supernatants contained the fusion proteins of HA or FLAG expressed and secreted by the cells was detected by ELISA using antibodies. First, as primary antibodies, anti-HA antibody (HA-Tag Rabbit mAb (Cell Signaling)) and anti-FLAG antibody (DYKDDDDK Tag Rabbit mAb (Cell Signaling)) were used, each diluted to 100 ng / well with carbonate / bicarbonate buffer (pH 9.8) and immobilized on a MaxiSorp 96-well plate (manufactured by Nunc Co., Ltd.). After reacting overnight at 4°C, the plates were washed three times with TBS (Tris-Buffered Saline). A TBS solution containing 3% bovine serum albumin (BSA) was added to each well at 250 μL / well and allowed to stand at room temperature for 2 hours. The plates were washed three times with TBS, and each of the recovered culture supernatants was added at 50 μL / well and allowed to stand at room temperature for 1 hour.

[0119] After washing three times with TBS-T, a solution of Anti-rabbit IgG and HRP-linked Antibody (cell signaling) diluted to 10,000 times with 1% BSA / TBS-T was added at 50 μL / well and allowed to stand at room temperature for 1 hour. After that, the cells were washed four times with TBS-T, and TMB Peroxidase Substrate (TMB Peroxidase EIA Complex Matrix Kit Bio-Rad) was added. The reaction was stopped with 1 mol / L phosphoric acid solution, and the absorbance at 450 nm was measured using an absorbance reader. The results were as follows: Figure 6As shown, the culture supernatants of cells expressing hIns-HA and hIns-FLAG showed more pronounced signals than those from cells transfected with only the vector as a negative control or from cells expressing hIns. This indicates that hIns-HA and hIns-FLAG, like hIns1-HiBiT5, do not interfere with the production and secretion of the insulin fusion protein. This demonstrates that the peptide insertion position of hIns1-HiBiT5 remains effective even when the peptide type differs. HiBiTHiBiT.

[0120] Example 4 Identification of Insulin Production-Promoting Genes Based on Screening

[0121] The above-mentioned Example 2 shows that the human recombinant cells produced by the present invention can actually be effectively used for the search for new genes that promote β-cell function. In particular, using the same method as in the above-mentioned Example 3 (1), multiple human genomes that may promote direct reprogramming into β-cells were amplified by PCR from a human cDNA library and cloned into the multiple cloning site of the expression plasmid pEBMulti-Hyg used in the above-mentioned Example 3. Then, human SW872 cells producing hIns-HiBiT5, the usefulness of which was confirmed in the above-mentioned Example 1 (6), were infected with Sendai virus vectors (TOKIWA-Bio Inc.) expressing three genes, namely Ngn3, Mafa and Pdx1, and cultured in β-cell differentiation medium for 24 hours. The various human genes cloned into pEBMulti-Hyg were transmitted to the virus-infected cells using Lipofectamine 3000. After culturing in differentiation medium for 5 days, the insulin content in each sample was compared using the HiBiT assay kit (Promega Corporation, Nano-Glo HiBiTExtracellular Detection System).

[0122] As a result, as shown in (1) of FIG7 , when the human KLF9 gene (SEQ ID NO: 71) was co-expressed in recombinant SW872 cells expressing the three genes Ngn3, Mafa, and Pdx1, enhanced production of the insulin-HiBiT fusion protein was observed. When the same experiment was performed on recombinant SW872 cells expressing the three genes Ngn3, Mafa, and Pdx1 used herein, which had been cultured and expanded for a long period of time, it was found that when the human UCN3 gene (SEQ ID NO: 67) was co-expressed in addition to the human KLF9 gene, production of the insulin-HiBiT fusion protein was further significantly promoted. Among them, the liver-derived cell line Huh7 was infected with a Sendai virus vector (TOKIWA-Bio Inc.) expressing three genes, Ngn3, Mafa, and Pdx1, and cultured in a β-cell differentiation medium for 24 hours. Then, the expression plasmids of the human KLF9 gene and the human UCN3 gene were transfected using Lipofectamine 3000, and the insulin production was measured 5 days later. The expression plasmids of the human KLF9 gene and the human UCN3 gene were prepared by PCR and INFUSION ligation using the primers shown in SEQ ID NOs: 65, 66 and SEQ ID NOs: 69, 70 in the same manner as described in Example 3 (1). As a negative control, pEBMulti-Hyg was used, and as a positive control, pEBMulti-hIns was used. 12,000 cells transfected with each plasmid were treated with acidic ethanol (a mixture of hydrochloric acid and ethanol), and the supernatant of the treated cell extract was diluted 5-fold with a buffer solution and the insulin content was measured. Insulin was quantified using HISCL HI-1000 (Sysmex Corporation). As shown in (3) of FIG7 , for human cells in which the human KLF9 gene and the human UCN3 gene were co-expressed, an increase in insulin production of more than 20 times was observed compared to when only the three genes Ngn3, Mafa, and Pdx1 were expressed. Furthermore, in an experiment under the same conditions, when Huh7 cells were infected with a Sendai virus vector (TOKIWA-Bio inc.) expressing four genes formed by adding Glis1 to Ngn3, Mafa, and Pdx1, as shown in (4) of FIG7 , the co-expression of the human KLF9 gene and the human UCN3 gene induced a more significant promotion of insulin production. Based on the above results, it can be seen that for the previous direct reprogramming method to become human β cells, the co-expression of the additional KLF9 gene and the human UCN3 gene can play a role in strongly promoting the production and secretion of human insulin.

[0123] Next, the base sequence information of the open reading frame of the human KLF11 gene and the KLF12 gene, which belong to the same family gene as the human KLF9 gene, was obtained by referring to the reference sequence database accession number NM_001198851.2 and NM_007249.5 of the National Center for Biotechnology Information, respectively. The cDNAs of these genes were cloned using the same method as the method for cloning the human KLF9 gene in the multiple cloning site of the expression plasmid pEBMulti-Hyg in the above example. Specifically, primers that bind to the 30 bases from the start codon of each of the three genes at the 3' end of the base sequence CCTCACTAAAGGGGTACC and primers that bind to the complementary chain sequence 30 bases upstream of the stop codon at the 3' end of the base sequence AAGCTTATCGATACCGTC were used to prepare each expression plasmid by PCR and INFUSION ligation reaction in the same manner as described in Example 3 (1). In the same manner as described above, the human adipose-derived cell line SW872 was infected with a Sendai virus vector (TOKIWA-Bio Inc.) expressing four genes, Ngn3, Mafa, Pdx1, and Glis1. After culturing in a β-cell differentiation medium for 24 hours, the cells were co-transfected with expression plasmids for the human KLF9 gene, the human KLF11 gene, or the human KLF11 gene and the human UCN3 gene using Lipofectamine 3000. Five days later, the amount of human C-peptide produced in the culture medium was measured, which is proportional to the amount of insulin produced. As a negative control, pEBMulti-Hyg was used. C-peptide was quantified using HISCL HI-1000 (Sysmex Corporation). As shown in (5) of Figure 7, the co-expression of the human KLF11 gene or the human KLF12 gene with the human UCN3 gene resulted in an increase in C-peptide production, similar to that of human KLF9, compared to the negative control. Based on this result, it can be seen that the KLF11 gene, which belongs to the same family as the KLF9 gene, or the human KLF12 gene can also promote the acquisition of human insulin production and secretion ability based on the previous direct reprogramming method to become human β cells through co-expression with the human UCN3 gene, just like the KLF9 gene.

[0124] Example 5 Screening of low molecular weight compounds

[0125] In the same manner as in Example 4, human SW872 cells producing hIns-HiBiT5 were infected with Sendai virus vectors expressing three genes, Ngn3, Mafa and Pdx1, and cultured in β-cell differentiation medium for 24 hours. Various low-molecular compounds were added to the cells at a final concentration of 0.3 to 30 nM, and after culturing in differentiation medium for 3 days, the cells were further cultured in differentiation medium to which the same compounds were added at the same concentration for 2 days. The insulin content in each sample was compared using the HiBiT analysis kit to investigate whether there were low-molecular compounds that promoted differentiation into β cells. The compounds were screened using a variety of commercially available compounds and a library of 9,600 compounds provided by the Institute for Pharmaceutical Research, University of Tokyo. As a result, about 0.2% of the compounds promoted the production of insulin-HiBiT fusion protein. For two of them, the insulin production-promoting effect was actually investigated using Huh7 cells, and the results were as follows. Figure 8 As shown, valproic acid (Sigma-Aldrich Product No. P4543, CAS No. 1069-66-5) promoted insulin production from Huh7 cells at 1-30 nM, and zebulin (Tokyo Chemical Industry Co., Ltd. Product No. Z0022, CAS No. 3690-10-6) promoted insulin production from Huh7 cells at 0.3-10 nM. The quantification method of insulin was carried out in the same manner as in Example 4. Separately, a 100 mM solution of valproic acid was prepared in ethanol, precipitated, and added to the cells at the specified concentration. A 100 mM solution of zebulin was prepared in dimethyl sulfoxide, precipitated, and added to the specified cells.

[0126] The above results indicate that the addition of valproic acid or zebulin, or both, to conventional direct reprogramming methods into human β cells can be used as a promoter for promoting the production and secretion of human insulin from cells.

[0127] SEQ ID NO: 1

[0128] HiBiT amino acid sequence

[0129] VSGWRLFKKIS

[0130] SEQ ID NO: 2

[0131] C-peptide-like sequences

[0132] EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ

[0133] SEQ ID NO: 3

[0134] C-peptide-like sequences

[0135] RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR

[0136] SEQ ID NO: 4

[0137] C-peptide-like sequences

[0138] RREAEDLQVGGGPGAGSLQPLALEGSLQKR

[0139] SEQ ID NO: 5

[0140] C-peptide-like sequences

[0141] EAEDLQVGGGPGAGSLQPLALEGSLQ

[0142] SEQ ID NO: 6

[0143] C-peptide-like sequences

[0144] RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQ

[0145] SEQ ID NO: 7

[0146] C-peptide-like sequences

[0147] EAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR

[0148] SEQ ID NO: 8

[0149] mIns1-HiBiT1 expression plasmid

[0150]

[0151] SEQ ID NO: 9

[0152] mIns1-HiBiT1 encoding gene

[0153] ATGGCCCTGTTGGTGCACTTCCTACCCCTGCTGGCCCTGCTTGCCCTCTGGGAGCCCAAACCCACCCAGGCTTTTGTCAAACAGCATCTTTGTGGTCCCCACCTGGTAGAGGCTCTCTACCTGGTGTGTGGGGAGCGTGGCTTCTTCTACACACCCAAGTCCCGCCGTGAAGTGGAGGACCCACAAGTGGAACAACTGGAGCTGGGAGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGAAGCCCCGGGGACCTTCAGACCTTGGCGTTGGAGGTGGCCCGGCAGAAGCGTGGCATTGTGGATCAGTGCTGCACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAAC

[0154] SEQ ID NO: 10

[0155] mIns1-HiBiT1 amino acid sequence

[0156] MALLVHFLPLLALLALWEPKPTQAFVKQHLCGPHLVEALYLVCGERGFFYTPKSRREVEDPQVEQLELGVSGWRLFKKISGSPGDLQTLALEVARQKRGIVDQCCTSICSLYQLENYCN

[0157] SEQ ID NO: 11

[0158] mIns1-HiBiT2 encoding gene

[0159] ATGGCCCTGTTGGTGCACTTCCTACCCCTGCTGGCCCTGCTTGCCCTCTGGGAGCCCAAACCCACCCAGGCTTTTGTCAAACAGCATCTTTGTGGTCCCCACCTGGTAGAGGCTCTCTACCTGGTGTGTGGGGAGCGTGGCTTCTTCTACACACCCAAGTCCCGCCGTGAAGTGGAGGACCCACAAGTGGAACAACTGGAGCTGGGAGGAGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGCGTTGGAGGTGGCCCGGCAGAAGCGTGGCATTGTGGATCAGTGCTGCACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAAC

[0160] SEQ ID NO: 12

[0161] Amino acid sequence of mIns1-HiBiT2

[0162] MALLVHFLPLLALLALWEPKPTQAFVKQHLCGPHLVEALYLVCGERGFFYTPKSRREVEDPQVEQLELGGVSGWRLFKKISALEVARQKRGIVDQCCTSICSLYQLENYCN

[0163] SEQ ID NO: 13

[0164] Coding gene of mIns1-HiBiT3

[0165] ATGGCCCTGTTGGTGCACTTCCTACCCCTGCTGGCCCTGCTTGCCCTCTGGGAGCCCAAACCCACCCAGGCTTTTGTCAAACAGCATCTTTGTGGTCCCCACCTGGTAGAGGCTCTCTACCTGGTGTGTGGGGAGCGTGGCTTCTTCTACACACCCAAGTCCCGCCGTGAAGTGGAGGACCCAGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGAGGAAGCCCCGGGGACCTTCAGACCTTGGCGTTGGAGGTGGCCCGGCAGAAGCGTGGCATTGTGGATCAGTGCTGCACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAAC

[0166] SEQ ID NO: 14

[0167] Amino acid sequence of mIns1-HiBiT3

[0168] MALLVHFLPLLALLALWEPKPTQAFVKQHLCGPHLVEALYLVCGERGFFYTPKSRREVEDPVSGWRLFKKISGGSPGDLQTLALEVARQKRGIVDQCCTSICSLYQLENYCN

[0169] SEQ ID NO: 15

[0170] Encoding gene of mIns1-HiBiT4

[0171] ATGGCCCTGTTGGTGCACTTCCTACCCCTGCTGGCCCTGCTTGCCCTCTGGGAGCCCAAACCCACCCAGGCTTTTGTCAAACAGCATCTTTGTGGTCCCCACCTGGTAGAGGCTCTCTACCTGGTGTGTGGGGAGCGTGGCTTCTTCTACACACCCAAGTCCCGCCGTGAAGTGGAGGACCCACAAGTGGAACAACTGGAGCTGGGAGGAAGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCACCTTGGCGTTGGAGGTGGCCCGGCAGAAGCGTGGCATTGTGGATCAGTGCTGCACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAAC

[0172] SEQ ID NO: 16

[0173] Amino acid sequence of mIns1-HiBiT4

[0174] MALLVHFLPLLALLALWEPKPTQAFVKQHLCGPHLVEALYLVCGERGFFYTPKSRREVEDPQVEQLELGGSVSGWRLFKKISTLALEVARQKRGIVDQCCTSICSLYQLENYCN

[0175] SEQ ID NO: 17

[0176] Encoding gene of mIns1-HiBiT5

[0177] ATGGCCCTGTTGGTGCACTTCCTACCCCTGCTGGCCCTGCTTGCCCTCTGGGAGCCCAAACCCACCCAGGCTTTTGTCAAACAGCATCTTTGTGGTCCCCACCTGGTAGAGGCTCTCTACCTGGTGTGTGGGGAGCGTGGCTTCTTCTACACACCCAAGTCCCGCCGTGAAGTGGAGGACCCACAAGTGGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGAGGAAGCCCCGGGGACCTTCAGACCTTGGCGTTGGAGGTGGCCCGGCAGAAGCGTGGCATTGTGGATCAGTGCTGCACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAAC

[0178] SEQ ID NO: 18

[0179] Amino acid sequence of mIns1-HiBiT5

[0180] MALLVHFLPLLALLALWEPKPTQAFVKQHLCGPHLVEALYLVCGERGFFYTPKSRREVEDPQVVSGWRLFKKISGGSPGDLQTLALEVARQKRGIVDQCCTSICSLYQLENYCN

[0181] SEQ ID NO: 19

[0182] Expression plasmid of mIns2-HiBiT1

[0183]

[0184] SEQ ID NO: 20

[0185] mIns2-HiBiT1 encoding gene

[0186] ATGGCCCTGTGGATGCGCTTCCTGCCCCTGCTGGCCCTGCTCTTCCTCTGGGAGTCCCACCCCACCCAGGCTTTTGTCAAGCAGCACCTTTGTGGTTCCCACCTGGTGGAGGCTCTCTACCTGGTGTGTGGGGAGCGTGGCTTCTTCTACACACCCATGTCCCGCCGTGAAGTGGAGGACCCACAAGTGGCACAACTGGAGCTGGGTGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGAGGCCCGGGAGCAGGTGACCTTCAGACCTTGGCACTGGAGGTGGCCCAGCAGAAGCGTGGCATTGTAGATCAGTGCTGCACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAAC

[0187] SEQ ID NO: 21

[0188] mIns2-HiBiT1 amino acid sequence

[0189] MALWMRFLPLLALLFLWESHPTQAFVKQHLCGSHLVEALYLVCGERGFFYTPMSRREVEDPQVAQLELGVSGWRLFKKISGGPGAGDLQTLALEVAQQKRGIVDQCCTSICSLYQLENYCN

[0190] SEQ ID NO: 22

[0191] mIns2-HiBiT2 encoding gene

[0192] ATGGCCCTGTGGATGCGCTTCCTGCCCCTGCTGGCCCTGCTCTTCCTCTGGGAGTCCCACCCCACCCAGGCTTTTGTCAAGCAGCACCTTTGTGGTTCCCACCTGGTGGAGGCTCTCTACCTGGTGTGTGGGGAGCGTGGCTTCTTCTACACACCCATGTCCCGCCGTGAAGTGGAGGACCCACAAGTGGCACAACTGGAGCTGGGTGGAGGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCTTGGCACTGGAGGTGGCCCAGCAGAAGCGTGGCATTGTAGATCAGTGCTGCACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAAC

[0193] SEQ ID NO: 23

[0194] Amino acid sequence of mIns2-HiBiT2

[0195] MALWMRFLPLLALLFLWESHPTQAFVKQHLCGSHLVEALYLVCGERGFFYTPMSRREVEDPQVAQLELGGGVSGWRLFKKISLALEVAQQKRGIVDQCCTSICSLYQLENYCN

[0196] SEQ ID NO: 24

[0197] Coding gene of mIns2-HiBiT3

[0198] ATGGCCCTGTGGATGCGCTTCCTGCCCCTGCTGGCCCTGCTCTTCCTCTGGGAGTCCCACCCCACCCAGGCTTTTGTCAAGCAGCACCTTTGTGGTTCCCACCTGGTGGAGGCTCTCTACCTGGTGTGTGGGGAGCGTGGCTTCTTCTACACACCCATGTCCCGCCGTGAAGTGGAGGACCCAGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGTGGAGGCCCGGGAGCAGGTGACCTTCAGACCTTGGCACTGGAGGTGGCCCAGCAGAAGCGTGGCATTGTAGATCAGTGCTGCACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAAC

[0199] SEQ ID NO: 25

[0200] Amino acid sequence of mIns2-HiBiT3

[0201] MALWMRFLPLLALLFLWESHPTQAFVKQHLCGSHLVEALYLVCGERGFFYTPMSRREVEDPVSGWRLFKKISGGGPGAGDLQTLALEVAQQKRGIVDQCCTSICSLYQLENYCN

[0202] SEQ ID NO: 26

[0203] Coding gene of mIns2-HiBiT4

[0204] ATGGCCCTGTGGATGCGCTTCCTGCCCCTGCTGGCCCTGCTCTTCCTCTGGGAGTCCCACCCCACCCAGGCTTTTGTCAAGCAGCACCTTTGTGGTTCCCACCTGGTGGAGGCTCTCTACCTGGTGTGTGGGGAGCGTGGCTTCTTCTACACACCCATGTCCCGCCGTGAAGTGGAGGACCCACAAGTGGCACAACTGGAGCTGGGTGGAGGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCCTTCAGACCTTGGCACTGGAGGTGGCCCAGCAGAAGCGTGGCATTGTAGATCAGTGCTGCACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAAC

[0205] SEQ ID NO: 27

[0206] Amino acid sequence of mIns2-HiBiT4

[0207] MALWMRFLPLLALLFLWESHPTQAFVKQHLCGSHLVEALYLVCGERGFFYTPMSRREVEDPQVAQLELGGGVSGWRLFKKISLQTLALEVAQQKRGIVDQCCTSICSLYQLENYCN

[0208] SEQ ID NO: 28

[0209] Coding gene of mIns2-HiBiT5

[0210] ATGGCCCTGTGGATGCGCTTCCTGCCCCTGCTGGCCCTGCTCTTCCTCTGGGAGTCCCACCCCACCCAGGCTTTTGTCAAGCAGCACCTTTGTGGTTCCCACCTGGTGGAGGCTCTCTACCTGGTGTGTGGGGAGCGTGGCTTCTTCTACACACCCATGTCCCGCCGTGAAGTGGAGGACCCACAAGTGGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGTGGAGGCCCGGGAGCAGGTGACCTTCAGACCTTGGCACTGGAGGTGGCCCAGCAGAAGCGTGGCATTGTAGATCAGTGCTGCACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAAC

[0211] SEQ ID NO: 29

[0212] Amino acid sequence of mIns2 - HiBiT5

[0213] MALWMRFLPLLALLFLWESHPTQAFVKQHLCGSHLVEALYLVCGERGFFYTPMSRREVEDPQVVSGWRLFKKISGGGPGAGDLQTLALEVAQQKRGIVDQCCTSICSLYQLENYCN

[0214] SEQ ID NO: 30

[0215] Coding gene of hIns - HiBiTT1

[0216] ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTGCTGGCCCTCTGGGGACCTGACCCAGCCGCAGCCTTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTACCTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCCAAGACCCGCCGGGAGGCAGAGGACCTGCAGGTGGGGCAGGTGGAGCTGGGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTTGGCCCTGGAGGGGTCCCTGCAGAAGCGTGGCATTGTGGAACAATGCTGTACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAACTAAGAATTCA

[0217] SEQ ID NO: 31

[0218] Amino acid sequence of hIns-HiBiT1

[0219] MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGVSGWRLFKKISGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN

[0220] SEQ ID NO: 32

[0221] Coding gene of hIns-HiBiT2

[0222] ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTGCTGGCCCTCTGGGGACCTGACCCAGCCGCAGCCTTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTACCTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCCAAGACCCGCCGGGAGGCAGAGGACCTGCAGGTGGGGCAGGTGGAGCTGGGCGGGGGCGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCTTGGCCCTGGAGGGGTCCCTGCAGAAGCGTGGCATTGTGGAACAATGCTGTACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAACTAAGAATTCA

[0223] SEQ ID NO: 33

[0224] hIns-HiBiT2 amino acid sequence

[0225] MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGVSGWRLFKKISLALEGSLQKRGIVEQCCTSICSLYQLENYCN

[0226] SEQ ID NO: 34

[0227] hIns-HiBiT5 coding gene

[0228] ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTGCTGGCCCTCTGGGGACCTGACCCAGCCGCAGCCTTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTACCTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCCAAGACCCGCCGGGAGGCAGAGGACCTGCAGGTGGTGAGCGGCTGGCGGCTGTTCAAGAAGATTAGCGGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTTGGCCCTGGAGGGGTCCCTGCAGAAGCGTGGCATTGTGGAACAATGCTGTACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAACTAAGAATTCA

[0229] SEQ ID NO: 35

[0230] Amino acid sequence of hIns-HiBiT5

[0231] MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVVSGWRLFKKISGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN

[0232] SEQ ID NO: 36

[0233] Pre-Donor-arm1-HiBiT-arm2 plasmid

[0234]

[0235] SEQ ID NO: 37

[0236] Select neoRdonorF

[0237] CCGATCCCATGGTTTAGTTCCTCACCTTG

[0238] SEQ ID NO: 38

[0239] Select neoRdonorR

[0240] CTTCTGAGCGGGACTCTGGGGTTC

[0241] SEQ ID NO: 39

[0242] Origin inFneoRr

[0243] AAACCATGGGATCGGCCATTGAACAAG

[0244] SEQ ID NO: 40

[0245] Origin inFneoRf

[0246] AGTCCCGCTCAGAAGAACTCGTCAAGAAGG

[0247] SEQ ID NO: 41

[0248] Pre-Donor-Arm1-NeoR-Arm2

[0249]

[0250] SEQ ID NO: 42

[0251] DonorR

[0252] CCATGGATCCGGCAGAAGGAC

[0253] SEQ ID NO: 43

[0254] Thanksgiving DonorF

[0255] CTAAGAATTCAAGGCCTCTCG

[0256] SEQ ID NO: 44

[0257] Building hInsHiBiTF

[0258] CTGCCGGATCCATGGCCCTGTG

[0259] SEQ ID NO: 45

[0260] Building hInsHiBiTR

[0261] CGAGAGGCCTTGAATTCTTAG

[0262] SEQ ID NO: 46

[0263] Arm1-hIns-HiBiT1-NeoR-Arm2

[0264]

[0265] SEQ ID NO: 47

[0266] Guide RNA-hCAS9 Plasmid

[0267]

[0268] SEQ ID NO: 48

[0269] Primer Ins1

[0270] GTCTCCCAGATCACTGTCCTTC

[0271] SEQ ID NO: 49

[0272] Primer Ins2r

[0273] TCTTCCCCATCTCCTGACTATG

[0274] SEQ ID NO: 50

[0275] Primer HiBiTR

[0276] AATCTTCTTGAACAGCCGCCAG

[0277] SEQ ID NO: 51

[0278] HA tag

[0279] YPYDVPDYA

[0280] SEQ ID NO: 52

[0281] FLAG tag

[0282] DYKDDDDK

[0283] SEQ ID NO: 53

[0284] Primer pEB-Ins F

[0285] CCTCACTAAAGGGGTACCATGGCCCTGTGGATGCGCCTCCTGCC

[0286] SEQ ID NO: 54

[0287] Primer pEB-Ins R

[0288] AAGCTTATCGATACCGTCTCACTAGTTGCAGTAGTTCTCCAGCTGGTAG

[0289] SEQ ID NO: 55

[0290] hIns encoding genes

[0291] ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTGCTGGCCCTCTGGGGACCTGACCCAGCCGCAGCCTTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTACCTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCCAAGACCCGCCGGGAGGCAGAGGACCTGCAGGTGGGGCAGGTGGAGCTGGGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTTGGCCCTGGAGGGGTCCCTGCAGAAGCGTGGCATTGTGGAACAATGCTGTACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAACTAG

[0292] SEQ ID NO: 56

[0293] hIns amino acid sequence

[0294] MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN

[0295] SEQ ID NO: 57

[0296] Primer Ins-HA R

[0297] CTGGAACATCGTATGGGTACACCTGCAGGTCCTCTGCCTCCC

[0298] SEQ ID NO: 58

[0299] Primer HA-Ins F

[0300] CCATACGATGTTCCAGATTACGCTGGCGGGGGCCCTGGTGCAGGCAG

[0301] SEQ ID NO: 59

[0302] hIns-HiBiT5-HA coding gene

[0303] ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTGCTGGCCCTCTGGGGACCTGACCCAGCCGCAGCCTTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTACCTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCCAAGACCCGCCGGGAGGCAGAGGACCTGCAGGTGTACCCATACGATGTTCCAGATTACGCTGGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTTGGCCCTGGAGGGGTCCCTGCAGAAGCGTGGCATTGTGGAACAATGCTGTACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAACTAA

[0304] SEQ ID NO: 60

[0305] hIns-HiBiT5-HA amino acid sequence

[0306] MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVYPYDVPDYAGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN

[0307] SEQ ID NO: 61

[0308] Primer Ins-FlagR

[0309] CGTCGTCATCCTTGTAATCCACCTGCAGGTCCTCTGCCTCCC

[0310] SEQ ID NO: 62

[0311] Primer Flag-InsF

[0312] ACAAGGATGACGACGATAAGGGCGGGGGCCCTGGTGCAGGCAG

[0313] SEQ ID NO: 63

[0314] hIns-HiBiT5-FLAG coding gene

[0315] ATGGCCCTGTGGATGCGCCTCCTGCCCCTGCTGGCGCTGCTGGCCCTCTGGGGACCTGACCCAGCCGCAGCCTTTGTGAACCAACACCTGTGCGGCTCACACCTGGTGGAAGCTCTCTACCTAGTGTGCGGGGAACGAGGCTTCTTCTACACACCCAAGACCCGCCGGGAGGCAGAGGACCTGCAGGTGGATTACAAGGATGACGACGATAAGGGCGGGGGCCCTGGTGCAGGCAGCCTGCAGCCCTTGGCCCTGGAGGGGTCCCTGCAGAAGCGTGGCATTGTGGAACAATGCTGTACCAGCATCTGCTCCCTCTACCAGCTGGAGAACTACTGCAACTAAGAATTCA

[0316] SEQ ID NO: 64

[0317] hIns-HiBiT5-FLAG amino acid sequence

[0318] MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREAEDLQVDYKDDDDKGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYCN

[0319] SEQ ID NO: 65

[0320] hUcn3

[0321] Primer hUcn3 F

[0322] CCTCACTAAAGGGGTACCATGCTGATGCCGGTCCACTTCCTGC

[0323] SEQ ID NO: 66

[0324] Primer hUcn3 R

[0325] AAGCTTATCGATACCGTCCTACTTCTTCCTCCCAATTTGCGCCATCAG

[0326] SEQ ID NO: 67

[0327] hUcn3 coding gene

[0328] ATGCTGATGCCGGTCCACTTCCTGCTGCTCCTGCTGCTGCTCCTGGGGGGCCCCAGGACAGGCCTCCCCCACAAGTTCTACAAAGCCAAGCCCATCTTCAGCTGCCTCAACACCGCCCTGTCTGAGGCTGAGAAGGGCCAGTGGGAGGATGCATCCCTGCTGAGCAAGAGGAGCTTCCACTACCTGCGCAGCAGAGACGCCTCTTCGGGAGAGGAGGAGGAGGGCAAAGAGAAAAAGACTTTCCCCATCTCTGGGGCCAGGGGTGGAGCCAGAGGCACCCGGTACAGATACGTGTCCCAAGCACAGCCCAGGGGAAAGCCACGCCAGGACACGGCCAAGAGTCCCCACCGCACCAAGTTCACCCTGTCCCTCGACGTCCCCACCAACATCATGAACCTCCTCTTCAACATCGCCAAGGCCAAGAACCTGCGTGCCCAGGCGGCCGCCAATGCCCACCTGATGGCGCAAATTGGGAGGAAGAAGTAG

[0329] SEQ ID NO: 68

[0330] Amino acid sequence of hUcn3

[0331] MLMPVHFLLLLLLLLGGPRTGLPHKFYKAKPIFSCLNTALSEAEKGQWEDASLLSKRSFHYLRSRDASSGEEEEGKEKKTFPISGARGGARGTRYRYVSQAQPRGKPRQDTAKSPHRTKFTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQIGRKK

[0332] SEQ ID NO: 69[[ID=1"]]

[0333] hKLF9

[0334] Primer hKLF9 F

[0335] CCTCACTAAAGGGGTACCATGTCCGCGGCCGCCTACATGGACTTCG

[0336] SEQ ID NO: 70

[0337] Primer hKLF9 R

[0338] AAGCTTATCGATACCGTCTCACAAAGCGTTGGCCAGCGCCTTTTTCGATC

[0339] SEQ ID NO: 71

[0340] hKLF9 coding gene

[0341] ATGTCCGCGGCCGCCTACATGGACTTCGTGGCTGCCCAGTGTCTGGTTTCCATTTCGAACCGCGCTGCGGTGCCGGAGCATGGGGTCGCTCCGGACGCCGAGCGGCTGCGACTACCTGAGCGCGAGGTGACCAAGGAGCACGGTGACCCGGGGGACACCTGGAAGGATTACTGCACACTGGTCACCATCGCCAAGAGCTTGTTGGACCTGAACAAGTACCGACCCATCCAGACCCCCTCCGTGTGCAGCGACAGTCTGGAAAGTCCAGATGAGGATATGGGATCCGACAGCGACGTGACCACCGAATCTGGGTCGAGTCCTTCCCACAGCCCGGAGGAGAGACAGGATCCTGGCAGCGCGCCCAGCCCGCTCTCCCTCCTCCATCCTGGAGTGGCTGCGAAGGGGAAACACGCCTCCGAAAAGAGGCACAAGTGCCCCTACAGTGGCTGTGGGAAAGTCTATGGAAAATCCTCCCATCTCAAAGCCCATTACAGAGTGCATACAGGTGAACGGCCCTTTCCCTGCACGTGGCCAGACTGCCTTAAAAAGTTCTCCCGCTCAGACGAGCTGACCCGCCACTACCGGACCCACACTGGGGAAAAGCAGTTCCGCTGTCCGCTGTGTGAGAAGCGCTTCATGAGGAGTGACCACCTCACAAAGCACGCCCGGCGGCACACCGAGTTCCACCCCAGCATGATCAAGCGATCGAAAAAGGCGCTGGCCAACGCTTTGTGA

[0342] SEQ ID NO: 72

[0343] hKLF9 amino acid sequence

[0344] MSAAAYMDFVAAQCLVSISNRAAVPEHGVAPDAERLRLPEREVTKEHGDPGDTWKDYCTLVTIAKSLLDLNKYRPIQTPSVCSDSLESPDEDMGSDSDVTTESGSSPSHSPEERQDPGSAPSPLSLLHPGVAAKGKHASEKRHKCPYSGCGKVYGKSSHLKAHYRVHTGERPFPCTWPDCLKKFSRSDELTRHYRTHTGEKQFRCPLCEKRFMRSDHLTKHARRHTEFHPSMIKRSKKALANAL

Claims

1. A method for quantifying the insulin secretion capacity of human cells, characterized in that: Recombinant cells in which a gene expressing a fusion C-peptide is substituted for the insulin gene in the genome of a cultured human cell line are cultured, and the amount of secreted fusion C-peptide is measured. The fusion C-peptide is formed by inserting a detectable tag peptide into the C-peptide chain within the human proinsulin molecule.

2. The quantitative method according to claim 1, wherein The detectable insertion position of the tag peptide is the position corresponding to the C-terminal side of 7V of the C-peptide chain.

3. The quantitative method according to claim 1 or 2, wherein The detectable tag peptide is a tag peptide selected from the group consisting of a tag peptide detectable by chemiluminescence analysis and a tag peptide detectable by immunoassay.

4. The quantitative method according to any one of claims 1 to 3, wherein The detectable tag peptide is a tag peptide selected from a tag peptide capable of binding to luciferase, a tag peptide containing DDDDK, and a tag peptide derived from hemagglutinin.

5. The quantitative method according to any one of claims 1 to 4, wherein The detectable tag peptide is a tag peptide that can bind to luciferase.

6. The quantitative method according to any one of [1] to [5], wherein The detectable tag peptide is a tag peptide selected from VSGWRLFKKIS (SEQ ID NO: 1), YPYDVPDYA (SEQ ID NO: 51), and YPYDVPDYA (SEQ ID NO: 51).

7. The quantitative method according to any one of claims 1 to 6, wherein The human cultured cell line is: (1) a human cultured cell line; (2) a cell formed by introducing three genes, Ngn3, Mafa and Pdx1, into human cells; or (3) a cell formed by simultaneously introducing three genes, Ngn3, Mafa and Pdx1, and genes for KLF9, KLF11 or KLF12, and UCN3 into human cells.

8. A recombinant cell, wherein the gene expressing a fusion C-peptide is replaced with the insulin gene in the genome of a cultured human cell line, wherein the fusion C-peptide is a fusion C-peptide formed by inserting a detectable tag peptide into the C-peptide chain within the human proinsulin molecule.

9. The recombinant cell according to claim 8, wherein The detectable insertion position of the tag peptide is the position corresponding to the C-terminal side of 7V of the C-peptide chain.

10. The recombinant cell according to claim 8 or 9, wherein The detectable tag peptide is a tag peptide selected from the group consisting of a tag peptide detectable by chemiluminescence analysis and a tag peptide detectable by immunoassay.

11. The recombinant cell according to any one of claims 8 to 10, wherein The detectable tag peptide is a tag peptide selected from a tag peptide capable of binding to luciferase, a tag peptide containing DDDDK, and a tag peptide derived from hemagglutinin.

12. The recombinant cell according to any one of claims 8 to 11, wherein The detectable tag peptide is a tag peptide that can bind to luciferase.

13. The recombinant cell according to any one of claims 8 to 12, wherein The detectable tag peptide is a tag peptide selected from VSGWRLFKKIS (SEQ ID NO: 1), YPYDVPDYA (SEQ ID NO: 51), and YPYDVPDYA (SEQ ID NO: 51).

14. The recombinant cell according to any one of claims 8 to 13, wherein The human cultured cell line is: (1) a human cultured cell line; (2) a cell formed by introducing three genes, Ngn3, Mafa and Pdx1, into human cells; or (3) a cell formed by simultaneously introducing three genes, Ngn3, Mafa and Pdx1, and genes for KLF9, KLF11 or KLF12, and UCN3 into human cells.

15. A method for screening a substance that promotes the production and secretion of human insulin, characterized in that: The recombinant cell according to any one of claims 8 to 14 is cultured in the presence of a test substance, and the amount of secreted fusion C-peptide is measured.

16. A human insulin production and secretion promoter comprising a substance obtained by the screening method according to claim 15.

17. An insulin production and secretion promoter comprising one or two selected from valproic acid and zebullin.

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