Regulation of pancreatic islet cell differentiation using regulatory factors associated with n6-adenosine methylation modification of mrna

Abstract

This invention relates to the regulation of pancreatic islet cell differentiation by regulatory factors related to N6-adenosine methylation modification of mRNA. Specifically, it relates to the use of (a) the ALKBH5 gene, or its protein, or its promoter; and / or (b) the YTHDF2 gene, or its protein, or its inhibitor, for the preparation of compositions or formulations for (1) promoting the differentiation of endodermal cells into pancreatic islet precursor cells and / or islet organoids; and / or (2) preventing and / or treating diabetes. The ALKBH5 of this invention regulates the N6-adenosine methylation modification of mRNA by... 6 Modification A controls the in vitro differentiation process of human pancreatic cells. Furthermore, the method of this invention holds promise as an effective method for in vitro culture of pancreatic islet cells, and could subsequently be applied to the treatment of diabetes.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the regulation of pancreatic islet cell differentiation by regulatory factors related to N6-adenosine methylation modification of mRNA. Background Technology

[0002] Approximately 460 million people worldwide are affected by diabetes, which is caused by insufficient insulin secretion or insulin resistance, leading to abnormal blood sugar regulation. Islet transplantation holds great promise for diabetes treatment, but it faces many limitations, particularly the shortage of organ donors and post-transplant immunosuppression.

[0003] Human pluripotent stem cells (hPSCs) have enormous potential to produce insulin-secreting pancreatic β cells for the treatment of diabetes. Current differentiation methods can progressively differentiate human pluripotent stem cells into definitive endoderm cells (DE), posterior foregut cells (PF), pancreatic islet precursor cells (PP), and ultimately, human islet organoids (hILO).

[0004] However, the differentiation of human pluripotent stem cells into the pancreatic lineage depends on a complex regulatory network involving transcription factors, epigenetic regulators, and signaling pathways. Furthermore, because the directed differentiation process requires multiple intermediate steps, the large-scale production of functional pancreatic islet cells for disease modeling, drug screening, and cell therapy remains an extremely time-consuming and labor-intensive process.

[0005] Therefore, there is a need in this field to develop new methods to promote the differentiation of stem cells into pancreatic islet cells. Summary of the Invention

[0006] The purpose of this invention is to provide a method for promoting the differentiation of stem cells into pancreatic islet cells.

[0007] In a first aspect of the invention, there is provided the use of an N6-adenosine methylation inhibitor of mRNA for the preparation of a composition or formulation for (1) promoting the differentiation of endodermal cells into pancreatic islet precursor cells (PP) and / or pancreatic islet organoids; and / or (2) preventing and / or treating diabetes.

[0008] In another preferred embodiment, the N6-adenosine methylation inhibitor of the mRNA is selected from the group consisting of:

[0009] (a) the ALKBH5 gene, or its protein, or its promoters; and / or

[0010] (b) YTHDF2 inhibitor.

[0011] In another preferred embodiment, the composition or formulation is used to promote the differentiation of pancreatic islet precursor cells into pancreatic islet organoids.

[0012] In another preferred embodiment, the endoderm cells are derived from mammals, preferably humans or rodents (such as mice or rats).

[0013] In another preferred embodiment, the endoderm cells are derived from human pluripotent stem cells (hPSCs), such as human embryonic stem cells or human induced pluripotent stem cells.

[0014] In another preferred embodiment, the endoderm cells are selected from the group consisting of: fixed endoderm cells (DE), posterior foregut endoderm cells (PF), or combinations thereof; preferably posterior foregut endoderm cells (PF).

[0015] In another preferred embodiment, the endoderm cells are SOX17 and FOXA2 positive endoderm cells (DE cells); or FOXA2 and PDX1 positive endoderm cells (PF cells).

[0016] In another preferred embodiment, the endoderm cells are PDX1-positive endoderm cells.

[0017] In another preferred embodiment, the islet precursor cells are PDX1 and NKX6.1 double-positive islet precursor cells.

[0018] In another preferred embodiment, the pancreatic islet precursor cells have one or more of the following characteristics:

[0019] At least 50% of the islet precursor cells, preferably at least 60%, 70%, 80%, 90%, or 100% of the islet precursor cells, express the islet precursor cell markers PDX1 and NKX6.1.

[0020] In another preferred embodiment, the pancreatic islet precursor cells have one or more of the following characteristics:

[0021] At least 50% of the islet precursor cells, preferably at least 60%, 70%, 80%, 90%, or 100% of the islet precursor cells, express the islet precursor cell markers PDX1, NKX6.1, SOX9, and HNF6.

[0022] In another preferred embodiment, the pancreatic islet organoids include pancreatic β cells, pancreatic α cells, and / or pancreatic δ cells, etc.

[0023] In another preferred embodiment, the marker gene for pancreatic β cells is selected from the group consisting of INS, NKX6.1, PDX1, MNX1, NKX2.2, GCK, PCSK1, PCSK2, or combinations thereof.

[0024] In another preferred embodiment, the composition or formulation is also used for one or more of the following purposes:

[0025] (3) Promotes pancreatic differentiation;

[0026] (4) Promotes the expression of pancreatic islet precursor cell marker genes;

[0027] (5) Promotes the expression of marker genes in pancreatic β cells;

[0028] (6) Reduce the RNA m of pancreatic islet precursor cell marker genes 6 A content; and / or

[0029] (7) Increase the content of PDX1 and NKX6.1 double-positive pancreatic islet precursor cells in the pancreatic islet precursor cell stage.

[0030] In another preferred embodiment, the promotion of pancreatic differentiation refers to promoting the differentiation of pancreatic islet precursor cells into pancreatic islet organoids.

[0031] In another preferred embodiment, the pancreatic islet precursor cell marker gene is selected from the group consisting of: PDX1, NKX6.1, SOX9, HNF6, MNX1 or combinations thereof; preferably, PDX1 and NKX6.1.

[0032] In another preferred embodiment, the "reduction of RNA m of pancreatic islet precursor cell marker genes" 6 "A content" refers to the RNA m of pancreatic islet precursor cell marker genes. 6 A content C1, compared with the control group RNA m 6 Compared to C0, the ratio of A content to C0 is ≤60%, preferably ≤40%, more preferably ≤20%, and even more preferably ≤10%.

[0033] In another preferred embodiment, the marker gene for pancreatic β cells is selected from the group consisting of INS, NKX6.1, PDX1, MNX1, NKX2.2, GCK, PCSK1, PCSK2, or combinations thereof.

[0034] In another preferred embodiment, the promoter refers to a substance capable of increasing the activity and / or content of the ALKBH5 gene or its protein in vivo or in vitro; the substance may be a synthetic or natural compound, protein, nucleotide, etc.

[0035] In another preferred embodiment, the ALKBH5 promoter includes a substance that promotes ALKBH5 expression.

[0036] In another preferred embodiment, the ALKBH5 promoter includes an ALKBH5 protein promoter and / or an ALKBH5 gene promoter.

[0037] In another preferred embodiment, the promotion of ALKBH5 expression or activity refers to increasing the expression or activity of the ALKBH5 gene or protein by ≥20%, more preferably ≥50%, and even more preferably ≥70%.

[0038] In another preferred embodiment, the ALKBH5 promoter is selected from the group consisting of small molecule compounds, nucleic acids, vectors expressing ALKBH5, or combinations thereof.

[0039] In another preferred embodiment, the ALKBH5 promoter is dimethyl-α-ketoglutaric acid (dimethyl α-ketoglutaric acid) or α-ketoglutaric acid.

[0040] In another preferred embodiment, the protein comprises a full-length protein or a protein fragment.

[0041] In another preferred embodiment, the ALKBH5 gene, or its protein, is derived from mammals, more preferably from rodents (such as mice and rats), primates, and humans.

[0042] In another preferred embodiment, the ALKBH5 gene or its protein is derived from endoderm cells, morphological endoderm cells, or hindbrain endoderm cells.

[0043] In another preferred embodiment, the ALKBH5 protein further includes derivatives of the ALKBH5 protein.

[0044] In another preferred embodiment, the derivatives of the ALKBH5 protein include modified ALKBH5 protein, protein molecules whose amino acid sequences are homologous to those of the natural ALKBH5 protein and have the activity of the natural ALKBH5 protein, dimers or polymers of the ALKBH5 protein, and fusion proteins containing the amino acid sequence of the ALKBH5 protein.

[0045] In another preferred embodiment, the "protein molecule whose amino acid sequence is homologous to the natural ALKBH5 protein and has the activity of the natural ALKBH5 protein" means a protein molecule whose amino acid sequence has ≥85% homology to the ALKBH5 protein, preferably ≥90% homology, more preferably ≥95% homology, and most preferably ≥98% homology; and has the activity of the natural ALKBH5 protein.

[0046] In another preferred embodiment, the ALKBH5 protein is selected from the group consisting of:

[0047] (A) A polypeptide with the amino acid sequence shown in SEQ ID NO.1;

[0048] (B) An ALKBH5 protein derivative or its active fragment formed by substituting, deleting or adding one or more (usually 1-60, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid residues of the amino acid sequence shown in SEQ ID NO.1.

[0049] (C) An ALKBH5 protein derivative or its active fragment thereof with ≥90%, preferably ≥95%, more preferably ≥98%, and most preferably ≥99% homology to the amino acid sequence shown in SEQ ID NO.1.

[0050] In another preferred embodiment, the ALKBH5 gene encodes the ALKBH5 protein.

[0051] In another preferred embodiment, the YTHDF2 inhibitor refers to a substance capable of reducing the activity and / or content of the YTHDF2 gene or its protein in vivo or in vitro.

[0052] In another preferred embodiment, the YTHDF2 inhibitor comprises synthetic or natural compounds, proteins, nucleotides, or combinations thereof.

[0053] In another preferred embodiment, the YTHDF2 inhibitor includes a substance that inhibits the expression of YTHDF2.

[0054] In another preferred embodiment, the YTHDF2 inhibitor includes a YTHDF2 protein inhibitor and / or a YTHDF2 gene inhibitor.

[0055] In another preferred embodiment, the inhibition of YTHDF2 expression or activity refers to reducing the expression or activity of the YTHDF2 gene or protein by ≥20%, more preferably ≥50%, more preferably ≥70%, and even more preferably ≥90%.

[0056] In another preferred embodiment, the YTHDF2 inhibitor is selected from the group consisting of small molecule compounds, nucleic acids, antibodies, or combinations thereof.

[0057] In another preferred embodiment, the YTHDF2 inhibitor includes RNAi that inhibits the transcription of the YTHDF2 gene, a methylation promoter of the YTHDF2 gene, or an inhibitor that inhibits the function of the YTHDF2 protein.

[0058] In another preferred embodiment, the YTHDF2 inhibitor includes an antibody against YTHDF2, antisense RNA of YTHDF2 nucleic acid, microRNA, siRNA, shRNA, and an activity inhibitor of YTHDF2.

[0059] In another preferred embodiment, the sequence of the YTHDF2 protein is shown in SEQ ID NO.2.

[0060] In another preferred embodiment, the shRNA sequence of the YTHDF2 nucleic acid is shown in SEQ ID NO.4 or SEQ ID NO.5.

[0061] In a second aspect of the invention, a method for preparing pancreatic islet precursor cells or islet organoids in vitro is provided, comprising the steps of:

[0062] Endoderm cells are cultured in a culture system in the presence of (a) the ALKBH5 gene, or its protein or its promoter; and / or (b) the YTHDF2 gene, or its protein or its inhibitor, to differentiate the endoderm cells into islet precursor cells or islet organoids, thereby obtaining the islet precursor cells or islet organoids.

[0063] In another preferred embodiment, the islet precursor cells are PDX1 and NKX6.1 double-positive islet precursor cells.

[0064] In another preferred embodiment, the pancreatic islet precursor cells have one or more of the following characteristics:

[0065] At least 50% of the islet precursor cells, preferably at least 60%, 70%, 80%, 90%, or 100% of the islet precursor cells, express the islet precursor cell markers PDX1 and NKX6.1.

[0066] In another preferred embodiment, the pancreatic islet precursor cells have one or more of the following characteristics:

[0067] At least 50% of the pancreatic islet precursor cells, preferably at least 60%, 70%, 80%, 90%, or 100% of the pancreatic islet precursor cells, express the pancreatic islet precursor cell markers PDX1, NKX6.1, SOX9, HNF6, MNX1, or combinations thereof.

[0068] In another preferred embodiment, the islet organoids include pancreatic β cells, the marker genes of which are selected from the group consisting of INS, PDX1, NKX6.1, MNX1, NKX 2.2, GCK, PCSK1, PCSK2, or combinations thereof.

[0069] In another preferred embodiment, the ALKBH5 promoter is selected from the group consisting of small molecule compounds, promoters that promote the function of the ALKBH5 protein, or combinations thereof.

[0070] In another preferred embodiment, the ALKBH5 promoter is dimethyl-α-ketoglutaric acid.

[0071] In another preferred embodiment, the YTHDF2 inhibitor is selected from the group consisting of small molecule compounds, nucleic acids, inhibitors that inhibit the function of the YTHDF2 protein, or combinations thereof.

[0072] In another preferred embodiment, the YTHDF2 inhibitor includes an antibody against YTHDF2, antisense RNA of YTHDF2 nucleic acid, microRNA, siRNA, shRNA, and an activity inhibitor of YTHDF2.

[0073] In a third aspect of the invention, a composition is provided that (1) promotes the differentiation of endoderm cells into pancreatic islet precursor cells and / or islet organoids; and / or (2) prevents and / or treats diabetes, comprising:

[0074] (a) the ALKBH5 gene, or its protein, or its promoters; and / or (b) YTHDF2 inhibitors; and

[0075] (c) An optional pharmaceutically acceptable carrier.

[0076] In another preferred embodiment, the composition further includes additional active ingredients selected from the group consisting of:

[0077] (Y1) PDX1 gene, or its protein, or its promoter;

[0078] (Y2)NKX6.1 gene, or its protein, or its promoter;

[0079] (Y3) SOX9 gene, or its protein, or its promoter;

[0080] (Y4) MNX1 gene, or its protein or its promoter.

[0081] In another preferred embodiment, the composition comprises a pharmaceutical composition.

[0082] In another preferred embodiment, the pharmaceutical composition is liquid, solid, or semi-solid.

[0083] In another preferred embodiment, the dosage form of the pharmaceutical composition includes injectable dosage forms and oral dosage forms.

[0084] In another preferred embodiment, the oral dosage form includes tablets, capsules, films, and granules.

[0085] In another preferred embodiment, the dosage form of the pharmaceutical composition includes a sustained-release dosage form and a non-sustained-release dosage form.

[0086] In another preferred embodiment, the component (a) comprises 60-99 wt% of the total weight of the pharmaceutical composition, more preferably 75-99 wt%, and even more preferably 80-99 wt%.

[0087] In another preferred embodiment, the components (a) and (b) in the pharmaceutical composition account for 0.01-99.99 wt% of the total weight of the product composition, more preferably 0.1-90 wt%, and even more preferably 1-80 wt%.

[0088] In another preferred embodiment, the weight ratio of component (a) to component (b) is 100:1-0.01:1, more preferably 10:1-0.1:1, and even more preferably 2:1-0.5:1.

[0089] In another preferred embodiment, the composition or formulation further includes other substances that can promote the differentiation of endoderm cells into pancreatic islet precursor cells; and / or (2) substances that promote the differentiation of pancreatic islet precursor cells into pancreatic organoids.

[0090] In a fourth aspect of the invention, a kit is provided, comprising:

[0091] (a) the ALKBH5 gene, or its protein, or its promoter; and / or (b) a YTHDF2 inhibitor.

[0092] In another preferred embodiment, the kit further comprises a detection reagent for detecting biomarkers selected from the group consisting of:

[0093] (Z1)PDX1 mRNA or protein;

[0094] (Z2)NKX6.1 mRNA or protein;

[0095] (Z3)SOX9 mRNA or protein;

[0096] (Z4)MNX1 mRNA or protein.

[0097] In a fifth aspect of the invention, a method is provided for (a) screening candidate compounds for treating diabetes; and / or (b) screening candidate compounds for promoting the differentiation of endoderm cells into pancreatic islet precursor cells and / or islet organoids, comprising:

[0098] (i) The test compound was added to the cell culture system as the test group, and the cell culture system without the test compound was used as the control group;

[0099] (ii) Compare the expression level and / or activity E1 of the ALKBH5 gene or its protein in the test group with the expression level and / or activity E0 in the control group;

[0100] Among them, when E1 is significantly higher than E0 in the test group, it indicates that the tested compound is (a) a candidate compound for treating diabetes; and / or (b) a candidate compound for promoting the differentiation of endoderm cells into pancreatic islet precursor cells and / or pancreatic islet organoids.

[0101] In another preferred embodiment, the cell is an endoderm cell.

[0102] In another preferred embodiment, the cells are cells cultured in vitro.

[0103] In another preferred embodiment, "significantly higher than" means that E1 is higher than E0 and there is a statistically significant difference; preferably, E1 ≥ 1.5E0, more preferably E1 ≥ 2E0, and most preferably E1 ≥ 3E0.

[0104] In another preferred embodiment, the method further includes the step of:

[0105] (iii) The test compound was added to the endoderm cell culture system as the test group, and the endoderm cell culture system without the test compound was used as the control group;

[0106] (iv) Compare the proportion of endoderm cells that transform into islet precursor cells and / or islet organoids in the test group to determine whether the compound is (a) a candidate compound for treating diabetes; and / or (b) a candidate compound for promoting the differentiation of endoderm cells into islet precursor cells and / or islet organoids.

[0107] Specifically, if the proportion (T1) of endoderm cells in the test group transforming into pancreatic islet precursor cells and / or islet organoids is significantly higher than that in the control group (T0), then the test compound is considered to be (a) a candidate compound for treating diabetes; and / or (b) a candidate compound for promoting the differentiation of endoderm cells into pancreatic islet precursor cells and / or islet organoids.

[0108] In another preferred embodiment, the proportion of endoderm cells in the test group that transforms into islet precursor cells and / or islet organoids includes detecting the proportion of endoderm cells that transform into PDX1 and NKX6.1 double-positive islet precursor cells.

[0109] In another preferred embodiment, the method includes step (v): further testing the effect of the candidate compound identified in step (iv) on the differentiation of endoderm cells into pancreatic islet precursor cells; wherein, if the candidate compound can promote the expression of pancreatic islet precursor cell marker genes and / or pancreatic β-cell marker genes; and / or reduce the m of pancreatic islet precursor cell marker genes 6 Modification A; and / or increasing the content of PDX1 and NKX6.1 double-positive pancreatic islet precursor cells; then the candidate compound is a promoter of pancreatic differentiation.

[0110] In another preferred embodiment, the pancreatic islet precursor cell marker gene is selected from the group consisting of: PDX1, NKX6.1, SOX9, HNF6, MNX1, or combinations thereof.

[0111] In another preferred embodiment, the marker gene for the pancreatic β cells is selected from the group consisting of INS, PDX1, NKX6.1, MNX1, NKX 2.2, GCK, PCSK1, PCSK2, or combinations thereof.

[0112] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0113] In a sixth aspect of the invention, a method for treating diabetes is provided, the method comprising administering a therapeutically effective amount of (a) the ALKBH5 gene, or its protein, or its promoter, and / or (b) a YTHDF2 inhibitor to a subject in need.

[0114] In another preferred embodiment, the first active ingredient (a) ALKBH5 gene, or its protein or its promoter; and / or the second active ingredient (b) YTHDF2 inhibitor are administered as a composition.

[0115] In another preferred embodiment, the composition is a pharmaceutical composition.

[0116] In another preferred embodiment, the diabetes is insulin-dependent diabetes mellitus.

[0117] In another preferred embodiment, the diabetes is type I diabetes or type II diabetes.

[0118] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0119] Figure 1 This study demonstrates the role of ALKBH5 in the formation of pancreatic islet precursor cells (PP) from WT and A5-KO (ALKBH5 knockout human pluripotent stem cell lines) human pluripotent stem cell lines (hPSCs).

[0120] Figure 2 This study demonstrates the construction of ALKBH5 knockout human pluripotent stem cell lines and the role of ALKBH5 in the formation of morphologic endoderm (DE), hindbrain endoderm (PF) cells, and pancreatic islet precursor cells (PP).

[0121] Figure 3 The phenotype of the second ALKBH5 gene knockout human pluripotent stem cell line during the formation of the defined endoderm (DE) and pancreatic islet precursor cells (PP) is shown.

[0122] Figure 4 This study demonstrates the role of ALKBH5 in the differentiation of pancreatic islet progenitor cells (PP) into human islet organoids (hILO) from WT and A5-KO (ALKBH5 gene knockout human pluripotent stem cell lines).

[0123] Figure 5This study demonstrates the effects of ALKBH5 on pancreatic β-cell formation and liver-related gene expression during the differentiation of WT and A5-KO (ALKBH5 knockout human pluripotent stem cell lines) into pancreatic islet organoids.

[0124] Figure 6 The study demonstrated the effect of ALKBH5 recovery assays on the differentiation of human pluripotent stem cells (hPSCs) into pancreatic islet precursor cells (PPs).

[0125] Figure 7 This shows m in WT and A5-KO (ALKBH5 gene knockout human pluripotent stem cell line) cells. 6 Distribution of A modifications on transcripts and the correlation between ALKBH5 and pancreatic islet progenitor cell marker gene expression.

[0126] Figure 8 This study identified potential targets of ALKBH5 (PDX1, NKX6.1, MNX1, and SOX9) during human pancreatic differentiation. ALKBH5 regulates the mRNA expression of key genes involved in human pancreatic differentiation and development, including PDX1, NKX6.1, MNX1, and SOX9. 6 A modification regulates the differentiation of human pancreatic cells.

[0127] Figure 9 This shows the m process during pancreatic differentiation 6 The expression of A reading protein, where m 6 The A-reading protein YTHDF2 plays an important role in pancreatic differentiation.

[0128] Figure 10 This study demonstrates the effects of YTHDF2 knockdown or the use of dimethyl α-ketoglutarate on the differentiation of human pluripotent stem cells (hPSCs) into pancreatic islet precursor cells (PPs). The overall effect is achieved via α-ketoglutarate-ALKBH5-m 6 The A-YTHDF2 pathway regulates the differentiation of human pancreatic cells.

[0129] Figure 11 The study showed that YTHDF2 knockdown or the use of dimethyl α-ketoglutarate could promote the differentiation of human pluripotent stem cells (hPSCs) into pancreatic islet progenitor cells (PPs). Detailed Implementation

[0130] Through extensive and in-depth research, the inventors have discovered for the first time that the demethylase ALKBH5 plays a crucial regulatory role in pancreatic lineage differentiation and islet organ development. Specifically, the absence of ALKBH5 leads to the reduction of mRNA expression in key transcription factors regulating pancreatic differentiation (such as PDX1, NKX6.1, MNX1, and SOX9). 6 The level of modification A increases. Subsequently, m6 The A-reading protein YTHDF2 can recognize and target the degradation of these molecules containing m 6 A-modified mRNAs can influence pancreatic differentiation. Furthermore, it has been found that ALKBH5 promoters, such as the cofactor α-ketoglutarate, can also significantly promote human pancreatic cell differentiation. Based on these findings, this invention was developed.

[0131] Specifically, this invention elucidates the relationship between pancreatic islet cell differentiation and N6-adenosine monophosphate methylation modification of mRNA by regulatory factors including the ALKBH5 gene, its protein or its promoter, and / or the YTHDF2 gene, its protein or its inhibitor.

[0132] m on mRNA 6 The dynamic changes of A modification during pancreatic differentiation indicate that the mRNA mRNA in the PP stage... 6 A levels were significantly downregulated compared to human pluripotent stem cells, definitive endoderm cells, and human pancreatic islet organoids. Further investigation was conducted into factors affecting m... 6 The expression levels of A-modified regulators during pancreatic development were investigated, revealing that the gene expression of the demethylase ALKBH5 was upregulated from the islet precursor stage, correlated with m during pancreatic differentiation. 6 The dynamic changes in A levels showed a negative correlation. Therefore, by further utilizing CRISPR precision gene editing technology, an ALKBH5 gene knockout cell line was constructed for the first time in human pluripotent stem cells. It was found that ALKBH5 knockout increases the levels of key transcription factors during pancreatic development, such as PDX1, NKX6.1, SOX9, and MNX1. 6 The level of A modification promotes the degradation of these mRNAs.

[0133] Furthermore, the addition of ALKBH5 cofactor α-ketoglutarate during differentiation can significantly improve the differentiation efficiency of human pluripotent stem cells into pancreatic islet progenitor cells. In summary, m 6 A modifications can affect mRNA metabolism by regulating mRNA m... 6 A-methylation modification can regulate pancreatic lineage cell differentiation and organ development at a new level.

[0134] Terminology Explanation

[0135] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0136] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0137] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0138] ALKBH5 and its accelerators

[0139] ALKBH5 is a member of the α-ketoglutarate-dependent dioxygenase AlkB family and is a major m 6 One of the A demethylases. In 2013, Zheng et al. first discovered that ALKBH5 is an m 6 AlKBH5 was identified as a demethylase for A and its deficiency led to abnormal sperm production and apoptosis in mouse testes. Subsequently, Zhang et al. found that ALKBH5 was associated with the hypoxic phenotype of breast cancer stem cells. Furthermore, Zhang et al. found that ALKBH5 maintained the tumorigenicity of glioblastoma stem cells by maintaining FOXM1 expression and cell proliferation. Liu et al. found that ALKBH5 could regulate cellular metabolism and inhibit viral replication. Later, Zhang et al. found that ALKBH5 promoted the proliferation of renal cancer cells by regulating AURKB expression. Recently, Shen et al. and Wang et al. respectively found that ALKBH5 plays an important role in the development of acute myeloid leukemia and could serve as an effective therapeutic target. In addition, Li et al. found that ALKBH5 modulates the response to anti-PD-1 therapy by regulating the tumor microenvironment. Besides tumorigenesis, the functions of ALKBH5 in other biological processes remain unclear. To date, the role of ALKBH5 in pancreatic differentiation has not been investigated.

[0140] In this invention, promoting the activity and / or content of the ALKBH5 gene or its protein is beneficial for: upregulating the expression of genes such as PDX1, NKX6.1, SOX9, and MNX1; and reducing the expression of mRNA on PDX1, NKX6.1, SOX9, and MNX1. 6 A Modification Level (m) 6 A-modified mRNAs with decreased relative abundance increase; which in turn promotes human pancreatic cell differentiation (promotes the differentiation of endodermal cells into islet precursor cells and then into islet cells).

[0141] In this invention, the ALKBH5 promoter includes substances capable of increasing the activity and / or content of the ALKBH5 gene or its protein in vivo or in vitro.

[0142] The expression level of ALKBH5 can be increased by the following methods: secreting large amounts of ALKBH5 protein by the tissue itself, artificially overexpressing ALKBH5 protein, or artificially delivering ALKBH5 protein (e.g., using a viral vector, such as an adeno-associated virus vector) or ALKBH5 promoters.

[0143] In this invention, the ALKBH5 promoter is not particularly limited, as long as it can promote the expression of ALKBH5 or enhance the activity of ALKBH5 protein, it is within the scope of protection of this invention.

[0144] In a preferred embodiment, the ALKBH5 promoter comprises a small molecule compound, such as dimethyl-α-ketoglutaric acid or α-ketoglutaric acid.

[0145] YTHDF2 and its inhibitors

[0146] YTHDF2 as an RNA m 6 An important reading protein modified with A can specifically recognize m 6 A and resulting in m 6 A's mRNA is rapidly degraded. Through research, the inventors discovered that YTHDF2 expression is significantly higher than other reading proteins during the pancreatic islet precursor cell stage of pancreatic differentiation. Furthermore, by knocking down the YTHDF2 gene, their effect on human pancreatic cell differentiation was verified, revealing that inhibiting YTHDF2 gene expression promotes human pancreatic cell differentiation.

[0147] In this invention, inhibiting the expression and / or activity of the YTHDF2 gene or its encoded protein is beneficial for partially restoring the expression levels of PDX1, NKX6.1, MNX1 and SOX9, and is beneficial for the differentiation of pancreatic islet progenitor cells.

[0148] In this invention, the YTHDF2 inhibitor (or antagonist) that can be used in this invention includes any substance that can inhibit the expression and / or activity of the YTHDF2 gene or its encoded protein.

[0149] For example, the inhibitors of YTHDF2 include small molecule compounds, antibodies against YTHDF2, antisense RNA of YTHDF2 nucleic acid, siRNA, shRNA, miRNA, or activity inhibitors of YTHDF2.

[0150] As used in this article, “regulatory factors of N6-adenosine methylation modification” mainly refers to “inhibitors of N6-adenosine methylation of mRNA”, including the ALKBH5 gene, its protein or its promoters and / or the YTHDF2 gene, its protein or its inhibitors.

[0151] Differentiation of pancreatic islet cells

[0152] Human pluripotent stem cells can gradually differentiate into definitive endoderm (DE), pancreatic progenitor (PP), endocrine precursor (EP), and pancreatic β-like cells (PB).

[0153] As used herein, the term "islet cell" includes insulin-secreting pancreatic β cells, glucagon-secreting pancreatic α cells, and / or somatostatin-secreting pancreatic δ cells. Glucagon works in conjunction with insulin to regulate blood glucose levels. Islet cells can differentiate from pluripotent stem cells. For example, the process of human pluripotent stem cells differentiating into pancreatic β cells involves the following main stages in sequence: human pluripotent stem cells, definitive endoderm cells (DE), gastrulation cells (PG), hindbrain endoderm cells (PF), pancreatic islet precursor cells (PP), endocrine precursor cells (EP), and pancreatic β cells (PB).

[0154] As used herein, the term "island organoids" refers to models established using a 3D in vitro cell culture system that are highly similar to in vivo-derived islets and possess some of the key characteristics of islets. Islet organoids can be derived from pluripotent stem cells and / or islet precursor cells through a stepwise induction process in a 3D culture system. For example, the stepwise differentiation of human pluripotent stem cells into islet organoids involves the following main stages: human pluripotent stem cells, modeled endoderm cells, gastrulation cells, hindbrain endoderm cells, islet precursor cells, endocrine precursor cells, and islet organoids; the stepwise differentiation of islet precursor cells into islet organoids involves the following main stages: islet precursor cells, endocrine precursor cells, and islet organoids. Islet organoids can replicate the complex spatial morphology of islets and can exhibit cell-to-cell interactions and spatial morphology, as well as interactions between cells and between cells and their surrounding matrix. For example, islet organoids include insulin-secreting β cells, glucagon-secreting α cells, and / or somatostatin-secreting δ cells. Pancreatic organoids have physiological responses similar to those of the pancreas in the body, such as sensing changes in glucose concentration in the environment and secreting insulin.

[0155] As used herein, the endoderm cells of the present invention include stereotyped endoderm cells (DE) and hindbrain endoderm cells (PF); preferably hindbrain endoderm cells (PF). More preferably, the endoderm cells of the present invention express marker genes for SOX17, FOXA2, and PDX1, and are preferably PDX1-positive endoderm cells, particularly human PDX1-positive pancreatic endoderm cells.

[0156] In some embodiments of the present invention, the pancreatic islet precursor cells of the present invention that are double-positive for PDX1 and NKX6.1 account for approximately 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more than 95%.

[0157] In another preferred embodiment, the islet precursor cells have one or more of the following characteristics: at least 50% of the islet precursor cells, preferably at least 60%, 70%, 80%, 90%, or 100% of the islet precursor cells express islet precursor cell markers PDX1, NKX6.1, SOX9, HNF6, MNX1, or combinations thereof.

[0158] Pharmaceutical composition and administration method

[0159] The present invention also provides a composition that can be used to promote the differentiation of endoderm cells into pancreatic islet precursor cells and / or pancreatic islet organoids. The pharmaceutical compositions of the present invention can also treat or prevent diabetes, etc.

[0160] The pharmaceutical compositions of the present invention comprise: (a) the ALKBH5 gene, or its protein, or an promoter thereof; and / or (b) the YTHDF2 gene, or its protein, or an inhibitor thereof; and (c) a pharmaceutically acceptable carrier.

[0161] This invention also provides a pharmaceutical composition comprising a safe and effective amount of the promoter of this invention (ALKBH5 gene, or its protein, or its promoter, such as a small molecule compound, for example a cofactor that promotes the demethylase activity of the ALKBH5 protein, etc.); and / or the inhibitor of this invention (such as an antibody, compound, CRISPR reagent, antisense sequence (such as siRNA), or inhibitor); and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of this invention can be formulated as injections, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets, and capsules are preferably manufactured under aseptic conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram to 10 milligrams per kilogram of body weight per day.

[0162] "Pharmaceutically acceptable carriers" refer to carriers used for the administration of therapeutic agents, including various excipients and diluents.

[0163] This term refers to pharmaceutical carriers that are not essential active ingredients themselves and do not cause excessive toxicity upon administration. Suitable carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in compositions may contain liquids such as water, saline, or buffer solutions. Additionally, these carriers may contain auxiliary substances such as fillers, lubricants, flow aids, wetting agents or emulsifiers, pH buffers, etc. The carriers may also contain cell transfection reagents.

[0164] The main advantages of this invention include

[0165] (1) This invention is the first to construct a human pluripotent stem cell line with ALKBH5 gene knockout, and discovers that ALKBH5 plays a crucial role in pancreatic lineage differentiation. Mechanistically, it was found that ALKBH5 knockout affects the m... 6 A modifies the level, then through m 6 The A recognition protein YTHDF2 mediates the degradation of these mRNAs, thereby affecting the differentiation of pancreatic lineage cells.

[0166] (2) This invention is the first to discover that dm-αKG significantly improves the differentiation efficiency of human pluripotent stem cells into pancreatic islet precursor cells. Specifically, through αKG-ALKBH5-m 6 Pathway A regulates pancreatic lineage cell differentiation and organ development at the level of RNA methylation modification.

[0167] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0168] Terminology Explanation:

[0169] hPSC: Human pluripotent stem cells

[0170] DE: Determined endoderm cells

[0171] PF: Post-foregut cells

[0172] PP: pancreatic islet precursor cells

[0173] hILO: Human pancreatic islet organoids

[0174] A5-KO: ALKBH5 knockout human pluripotent stem cell line 1

[0175] A5-KO2: ALKBH5 knockout human pluripotent stem cell line 2

[0176] WT: Wild-type human pluripotent stem cell line

[0177] RT-qPCR: Reverse transcription-quantitative polymerase chain reaction

[0178] FACS: Fluorescence-activated cell sorting assay

[0179] KEGG: Kyoto Encyclopedia of Genetics and Genomes

[0180] GO: Gene Ontology

[0181] A5 WT Wild type ALKBH5

[0182] A5 Mut ALKBH5 with mutation at the H204 site

[0183] shDF2-1: shRNA1 that knocks down the YTHDF2 gene

[0184] shDF2-2: shRNA2 that knocks down the YTHDF2 gene

[0185] shNC: shRNA control

[0186] αKG: α-Ketoglutarate

[0187] dm-αKG: Dimethyl-αKG (α-ketoglutarate dimethyl ester)

[0188] Actin D: Actin D

[0189] Experimental methods

[0190] 3D Differentiation Method for Obtaining Pancreatic Islet Progenitor Cells from Human Pluripotent Stem Cells

[0191] The specific steps are as follows:

[0192] 1) Culture human pluripotent stem cells in a 10 cm culture dish for four days using human pluripotent stem cell culture medium, changing the medium daily;

[0193] 2) After rinsing the cells with phosphate buffer, digest the cells into a single-cell state using Accutase digestive enzyme;

[0194] 3) Seed cells into low-adsorption six-well plates, with 5.5 × 10⁶ cells per well. 6 Add 5.5 ml of DO medium to each cell;

[0195] 4) Place the cell culture plate on a rotating shaker at 100 rpm;

[0196] 5) Change the culture medium daily to D1 to D11 and culture for 11 consecutive days to obtain human pancreatic islet precursor cells.

[0197] 6) The D0 medium contains 88% DMEM, 10% KSR, 1% NEAA, a mixture of 1% penicillin and streptomycin, 0.055 mM β-mercaptoethanol, 10 ng / mL bFGF, 10 ng / mL activin A, and 10 ng / mL heregulin B; the D1 medium contains 99% RPMI, 1% P / S, 1:5000 ITS-X, 0.2% FBS, 100 ng / mL activin A, and 3 μM CHIR99021; the D2 medium contains 99% RPMI, 1% P / S, 1:2000 ITS-X, 0.2% FBS, and 100 ng / mL activin A; the D3 medium contains 99% RPMI, a mixture of 1% penicillin and streptomycin, 1:1000 ITS-X, 0.2% FBS, 25 ng / mL KGF, and 2.5 μM A83-01; The D4-5 medium contains 99% RPMI, a mixture of 1% penicillin and streptomycin, 1:1000 ITS-X, 0.4% FBS, and 25 ng / mL KGF; The D6-7 medium contains 97% DMEM, a mixture of 1% penicillin and streptomycin, 1X B27, and 3 nM TTNPB; The D8 medium contains 97% DMEM, a mixture of 1% penicillin and streptomycin, 1X B27, 3 nM TTNPB, and 50 ng / mL EGF; The D9-11 medium contains 97% DMEM, a mixture of 1% penicillin and streptomycin, 1X B27, 50 ng / mL KGF, and 50 ng / mL EGF.

[0198] 2D differentiation method for obtaining pancreatic islet precursor cells from human pluripotent stem cells

[0199] The specific steps are as follows:

[0200] 1) Seed human pluripotent stem cells into 12-well plates, with 5 × 10⁶ cells per well. 5 One cell;

[0201] 2) After culturing for 48 hours, rinse the cells with phosphate buffer and replace with D1 differentiation medium to begin differentiation;

[0202] 3) Change the culture medium daily to new medium from D1 to D14, and culture continuously for 14 days to obtain human pancreatic islet precursor cells.

[0203] 4) The D1 medium contains 99% RPMI, a mixture of 1% penicillin and streptomycin, 100 ng / mL activin A, and 3 μM CHIR99021; the D2 medium contains 99% RPMI, a mixture of 1% penicillin and streptomycin, 0.2% FBS, and 100 ng / mL activin A; the D3 medium contains 99% RPMI, a mixture of 1% penicillin and streptomycin, 2% FBS, and 100 ng / mL activin A; the D4-6 mediums contain 99% RPMI, a mixture of 1% penicillin and streptomycin, 0.5X B27, 0.5X N2, 0.05% BSA, and 50 ng / mL KGF; the D7-8 medium contains 97% DMEM, a mixture of 1% penicillin and streptomycin, 1X B27, 0.05% BSA, 0.25 mM vitamin C, 50 ng / mL KGF, and 0.1 μM LDN-193189, 0.1 μM GDC-0449, 2 μM retinoic acid; the D9-14 medium contained 97% DMEM, 1% penicillin and streptomycin mixture, 1X B27, 0.05% BSA, 0.25 mM vitamin C, 0.1 μM LDN-193189, and 50 ng / mL EGF.

[0204] Experimental study on the effect of αKG on human pancreatic differentiation

[0205] The specific experimental steps are as follows:

[0206] 1) Seed human pluripotent stem cells into 12-well plates, with 5 × 10⁶ cells per well. 5 One cell;

[0207] 2) After culturing for 48 hours, rinse the cells with phosphate buffer and replace with D1 differentiation medium to begin differentiation;

[0208] 3) Change the culture medium daily to D1 to D14, culture continuously for 14 days, and add 4mM dm-αKG to the D4-D14 medium to obtain human pancreatic islet precursor cells.

[0209] 4) The D1 medium contains 99% RPMI, a mixture of 1% penicillin and streptomycin, 100 ng / mL activin A, and 3 μM CHIR99021; the D2 medium contains 99% RPMI, a mixture of 1% penicillin and streptomycin, 0.2% FBS, and 100 ng / mL activin A; the D3 medium contains 99% RPMI, a mixture of 1% penicillin and streptomycin, 2% FBS, and 100 ng / mL activin A; the D4-6 mediums contain 99% RPMI, a mixture of 1% penicillin and streptomycin, 0.5X B27, 0.5X N2, 0.05% BSA, 50 ng / mL KGF, and 4 mM dm-αKG; the D7-8 medium contains 97% DMEM, a mixture of 1% penicillin and streptomycin, 1X B27, 0.05% BSA, 0.25 mM vitamin C, 50 ng / mL KGF, and 0.1 μM LDN-193189, 0.1 μM MDC-0449, 2 μM retinoic acid, 4 mM dm-αKG; the D9-14 medium contained 97% DMEM, 1% penicillin and streptomycin mixture, 1X B27, 0.05% BSA, 0.25 mM vitamin C, 0.1 μM LDN-193189, 50 ng / mL EGF, 4 mM dm-αKG.

[0210] m 6 A-seq

[0211] For samples from the hPSC, DE, PP, and hILO stages, TRIzol was used. TM Total RNA was extracted using reagents (Invitrogen), and mRNA was further enriched using the GenElute mRNA Miniprep Kit (Sigma). 6 The A-seq experiment was performed according to a previously published protocol (Dominissini, D., Nat. Protoc. 8, 176-189 (2013)). In short, the mRNA was broken down into approximately 100-nt fragments and processed with 5 μg m... 6 Immunoprecipitation (IP) was performed using antibody A (SYSY, Cat#202003). The input and IP products were used to construct a library (Illumina). For samples from WT and A5-KO PPs, due to limited starting materials, 2-10 μg of total RNA was extracted, fragmented, and directly immunoprecipitated. First, rRNA in the input fragment was removed using the rRNA Depletion Kit (NEB), and then... The Stranded Total RNA-Seq Kit v2 (Takara) was used to prepare libraries from the input and IP fragments. All prepared libraries were then sequenced on an Illumina Hiseq X10 system, with paired-end reads of 150 bp.

[0212] m 6 A-RIP-qPCR

[0213] Through m 6 Total RNA was extracted, fragmented, and immunoprecipitated using antibody A. Both input and IP fragments were reverse transcribed (Takara), and RT-qPCR experiments were performed using an iTaq Universal SYBR Green Supermix (Bio-Rad) on a Bio-Rad CFX96Connect Real-Time system. GAPDH was selected as the m-value for computation. 6 A negative control with enrichment levels, as shown below: input and m 6 The expression levels of selected genes in the A-IP sample were first normalized using GAPDH, and then expressed as m 6 A IP / input calculation m 6 A enrichment.

[0214] Example 1: Construction of ALKBH5 knockout human pluripotent stem cell line

[0215] To date, there have been no reports on ALKBH5 gene knockout human pluripotent stem cell lines and the role of ALKBH5 in the differentiation of human pluripotent stem cells. First, Western blotting was used to detect changes in ALKBH5 expression during the differentiation of human pancreatic β cells. Figure 1 a) It was found that the expression level of ALKBH5 gradually increased during the differentiation of stem cells into pancreatic islet progenitor cells, and then decreased, with the highest expression level in the pancreatic islet progenitor stage (PP).

[0216] Using the CRISPR-Cpf1 system recently established by the inventors, precise gene editing was performed in human pluripotent stem cells to construct an ALKBH5 gene knockout (KO) human pluripotent stem cell line. Figure 1 b and Figure 2 a).

[0217] The ALKBH5 knockout human pluripotent stem cell line was labeled A5-KO, and the wild-type human pluripotent stem cell line was labeled WT. PCR genotyping and Sanger sequencing results showed that ALKBH5 knockout cell lines (A5-KO and A5-KO2) were successfully constructed. Figure 2 b, Figure 2 c and Figure 3a).

[0218] Next, Western blot results confirmed the absence of ALKBH5 protein in the homozygous A5-KO cell line. Figure 1 c and Figure 3 b).

[0219] During long-term in vitro culture, the A5-KO human pluripotent stem cell line exhibited typical human pluripotent stem cell morphology and could proliferate normally. Immunostaining results showed that both WT and A5-KO human pluripotent stem cells expressed the pluripotent stem cell marker genes OCT4 and NANOG. Figure 2 d and Figure 3 c). RT-qPCR analysis showed no significant difference in the expression levels of marker genes OCT4, SOX2, and NANOG in WT and A5-KO human pluripotent stem cells. Figure 2 e).

[0220] These results indicate that ALKBH5 is not essential for the survival and self-renewal process of human pluripotent stem cells.

[0221] Example 2: ALKBH5 is essential for the differentiation of human pancreatic lineage cells.

[0222] First, human pluripotent stem cells were cultured using a human pluripotent stem cell culture medium. This medium contained 88% DMEM, 10% KSR, 1% NEAA, a mixture of 1% penicillin and streptomycin, 0.055 mM β-mercaptoethanol, and 10 ng / mL bFGF. The human pluripotent stem cell lines used included the MEL1 human embryonic stem cell line, the H9 human embryonic stem cell line, and the human induced pluripotent stem cell line (hiPSC).

[0223] Subsequently, human pluripotent stem cells were differentiated into pancreatic islet precursor cells using 2D or 3D differentiation methods.

[0224] The ability of WT and A5-KO human pluripotent stem cells to differentiate into endoderm lineages was then tested. Figure 2 d). Human pluripotent stem cells were differentiated into definitive endoderm cells (DE) by treatment with the Wnt signaling pathway activator CHIR99021 and high concentrations of Activin A. Both WT and A5-KO human pluripotent stem cells were effectively differentiated into definitive endoderm cells co-expressing SOX17 and FOXA2. Immunostaining and fluorescence activated cell sorting (FACS) showed that the differentiation efficiencies of the two groups were comparable (both groups had greater than 90%). Figure 2 d, Figure 2 f, Figure 2 g, Figure 3 c, Figure 3 d and Figure 3 e).

[0225] No differences were observed between WT and A5-KO at the final endoderm stage, indicating that the absence of ALKBH5 does not affect differentiation into final endoderm cells.

[0226] Next, the ability of WT and A5-KO shaped endoderm cells to differentiate into hindbrain endoderm cells (PF) was examined. Figure 2 d) Immunostaining and FACS results showed that both WT and A5-KO cell lines produced more than 80% PDX1-positive hindgut endoderm cells. Figure 2 d and Figure 2 h).

[0227] Therefore, the differentiation of hindgut endoderm cells does not depend on ALKBH5.

[0228] Subsequently, immunostaining was performed at the islet progenitor cell stage, and the results showed that the number of PDX1 and NKX6.1 double-positive cells in A5-KO islet progenitor cells was significantly lower than that in WT islet progenitor cells. Figure 1 e and Figure 3 c).

[0229] Furthermore, FACS results also confirmed that the proportion of PDX1 and NKX6.1 double-positive cells was significantly reduced in A5-KO. Figure 1 f and Figure 3 f). RT-qPCR results showed that the expression levels of pancreatic islet precursor cell marker genes PDX1, NKX6.1, HNF6, and SOX9 in A5-KO were significantly lower than those in WT. Figure 1 g and Figure 3 g). Western blotting results also yielded the same conclusion: NKX6.1 expression was significantly decreased in A5-KO cells, while PDX1 expression was slightly decreased. Figure 1 h and Figure 2 i). Next, Ki67 staining was used to detect cell proliferation. The results showed no significant difference in proliferation capacity between the WT and A5-KO cell lines. Figure 2 d).

[0230] RNA-seq results showed that after ALKBH5 knockout, 965 genes were significantly upregulated and 811 genes were significantly downregulated. Figure 1 i). These downregulated genes include PDX1, SOX9, HNF6, MNX1, and NKX6.1, all of which are crucial for pancreatic cell differentiation. Meanwhile, other endoderm lineage marker genes, such as hepatocyte genes ALB and AFP, were significantly upregulated. Figure 1 This phenomenon has also been demonstrated in immunofluorescence staining experiments. Figure 5 a).

[0231] GO analysis revealed that these downregulated genes are associated with biological processes such as RNA modification, regulation of cell morphogenesis, and pancreatic development. Figure 1 On the other hand, the upregulated genes are mainly associated with digestive system function and lipid homeostasis, which may be due to cell differentiation into other endoderm lineages. Figure 1 k).

[0232] In summary, these data indicate that ALKBH5 is essential for the formation of PDX1 and NKX6.1 double-positive pancreatic islet precursor cells.

[0233] Example 3: ALKBH5 regulates the production of human pancreatic organoids.

[0234] To investigate the role of ALKBH5 in human islet development, WT and A5-KO islet progenitor cells were further differentiated into human islet organoids. Figure 4 a).

[0235] like Figure 4 As shown in b, the production of pancreatic β cells (INS-GFP positive cells) was significantly suppressed after ALKBH5 knockout. FACS results also confirmed this conclusion. Figure 4 d and Figure 5 b). Immunostaining results showed that PDX1 was present in A5-KO human pancreatic islet organoids. + NKX6.1 + INS + The number of cells was significantly reduced. Figure 4 c). Next, RNA-seq analysis was performed on WT and A5-KO human islet organoids to further investigate the effect of ALKBH5 on human islet organoid development.

[0236] RNA-seq results showed that endocrine genes, such as INS, GCG, SST, and PPY, were downregulated in A5-KO human pancreatic islet organoids. Figure 4 e). Furthermore, many marker genes for pancreatic β cells, such as NKX6.1, MNX1, NKX2.2, GCK, PCSK1, and PCSK2, were also significantly downregulated. Figure 4 e). Furthermore, some liver-related genes are significantly upregulated in A5-KO cells ( Figure 4 e and Figure 5 c). Kyoto Encyclopedia of Genetics and Genomes (KEGG) analysis showed that downregulated genes were associated with adolescent diabetes, type 2 diabetes, calcium signaling pathways, and glucagon signaling pathways. Figure 4 f). Upregulated genes are mainly associated with drug metabolism and fatty acid degradation, etc. Figure 4 f).

[0237] Finally, the physiological function of pancreatic β-cells was assessed using a glucose-stimulated insulin secretion assay (GSIS). Human pancreatic organoids from WT and A5-KO cells were stimulated with high concentrations of glucose, and their insulin secretion capacity was detected using an enzyme-linked immunosorbent assay (ELISA). Figure 4 As shown in g, the function of pancreatic β cells in human pancreatic organoids derived from A5-KO to sense changes in glucose concentration and secrete insulin is severely impaired.

[0238] In summary, we can conclude that ALKBH5 can regulate the expression levels of many genes that are crucial for human pancreatic development, which are essential for the differentiation of pancreatic lineage cells, the development of pancreatic organelles, and the function of pancreatic β cells.

[0239] Example 4: The demethylase activity of ALKBH5 is crucial for the differentiation process of the human pancreas.

[0240] To rule out off-target effects and determine whether the enzyme activity of ALKBH5 is a key factor affecting this phenotype, we examined whether the phenotype could be restored by re-expressing ALKBH5 in A5-KO human pluripotent stem cells.

[0241] Previous studies have found that the H204A mutation leads to the loss of demethylase activity of ALKBH5. The inventors constructed A5-KO+A5 by transfecting lentiviruses carrying wild-type ALKBH5 and mutant ALKBH5. WT and A5-KO+A5 Mut Human pluripotent stem cell lines. Furthermore, using the N-terminal sequence of GFP (GFP... N Lentiviral-infected WT and A5-KO human pluripotent stem cells were used as controls. Then, the four human pluripotent stem cell lines were applied to a pancreatic differentiation system. Figure 6 a).

[0242] First, the expression level of ALKBH5 in these cell lines was examined, and A5-KO+A5 was observed. WT and A5-KO+A5 Mut Human pluripotent stem cell lines restored ALKBH5 expression. Figure 6 b). Western blot results also showed the recovery of ALKBH5 at the protein level. Figure 6 c).

[0243] Detection of intracellular m using mass spectrometry 6 The content of A was shown in the results as A5-KO+A5. WT m in human pluripotent stem cells 6 Compared to the A5-KO control group and A5-KO+A5, the A content was... Mut The group showed a significant decrease ( Figure 6d), Explanation of A5 WT The role of m 6 The function of A demethylase and A5 Mut Because it lacks enzyme activity, it cannot reduce m 6 Level A.

[0244] Next, these pluripotent stem cell lines were differentiated into pancreatic lineage cells using the methods described above. On day 14 of differentiation, immunostaining was used to detect the production of pancreatic islet precursor cells, and A5 was found. WT The ability of A5-KO human pluripotent stem cells to differentiate into pancreatic islet precursor cells can be restored, proving that the inability of A5-KO human pluripotent stem cells to effectively differentiate into pancreatic islet precursor cells is due to the absence of ALKBH5, rather than off-target effects. Figure 6 e). On the other hand, A5 Mut The inability to salvage pancreatic differentiation disorders caused by ALKBH5 deficiency indicates that ALKBH5's function depends on its enzyme activity. Figure 6 e).

[0245] FACS experiments were conducted to detect the proportion of PDX1 and NKX6.1 double-positive cells in each group, and the same conclusion was reached. Figure 6 f). RT-qPCR results of pancreatic islet progenitor cell marker genes, including PDX1, NKX6.1, SOX9, and HNF6, further validated this conclusion. Figure 6 g).

[0246] In summary, these results indicate that ALKBH5 with intact demethylase activity is essential for the differentiation of human pluripotent stem cells into pancreatic islet precursor cells.

[0247] Example 5: Identification of potential ALKBH5 targets during human pancreatic differentiation

[0248] From the pancreatic islet progenitor cell stage, significant phenotypic differences were observed between WT and A5-KO. To elucidate the role of ALKBH5 in human pancreatic differentiation, m... 6 A-seq analysis. m 6 A-seq data show that the vast majority of m 6 Peak A is distributed in the CDS and 3'UTR regions of the transcript. Figure 7 a, Figure 7 b and Figure 7 c). ALKBH5 as m 6 The demethylase of A, after its gene knockout, is present in the 3'UTR region and near the stop codon. 6 Peak A increased significantly ( Figure 7 a).

[0249] A5-KO pancreatic islet progenitor cells were found to be highly methylated compared to WT, with 7903 m molecules corresponding to 4343 genes. 6 Peak A was significantly upregulated ( Figure 8 (a and 8b). Among them, 281 genes were significantly downregulated in the RNA-seq results, which may mean m 6 A modification can affect mRNA degradation. Figure 8 b). Gene ontology (GO) analysis showed that these genes are mainly associated with the regulation of cell morphogenesis, stem cell development, and pancreatic development. Figure 8 c).

[0250] Interestingly, several transcription factors important for pancreatic lineage differentiation, such as MNX1, SOX9, HNF6, and NKX6.1, were identified as potential targets for ALKBH5. Figure 8 a). Analysis of other published pancreatic differentiation data showed that ALKBH5 expression was significantly positively correlated with the expression of MNX1, SOX9, PDX1, and NKX6.1. Figure 7 d).

[0251] Next, through m 6 A-RIP-qPCR confirmed the mRNA levels of PDX1, NKX6.1, MNX1, and SOX9 after ALKBH5 deletion. 6 A modifier level increased ( Figure 8 d).

[0252] Furthermore, ALKBH5-RIP-qPCR experiments confirmed that ALKBH5 can bind to transcripts of PDX1, NKX6.1, MNX1, and SOX9. Figure 8 e).

[0253] In summary, the inventors have discovered that some key pancreatic differentiation marker genes can serve as targets for ALKBH5, including PDX1, NKX6.1, MNX1, and SOX9.

[0254] Example 6: ALKBH5 regulates key genes in human pancreatic differentiation and development. 6 A modification to regulate human pancreatic cell differentiation

[0255] Because m 6 A modification affects mRNA stability, and the half-life of mRNA in pancreatic islet precursor cells of WT and A5-KO was further investigated.

[0256] First, the RNA-seq results were analyzed, and it was found that there was no m 6 A-modified mRNA transcripts are generally more common than those with m... 6 A-modified mRNA is more stable. Figure 8 f).

[0257] Next, WT and A5-KO pancreatic islet precursor cells were treated with the transcription inhibitor actinomycin D (Act D), and samples were collected at 0, 1, 2, and 3 hours, respectively. The half-life of mRNA was then detected by RNA-seq.

[0258] Interestingly and unexpectedly, a decrease in overall mRNA stability was observed in A5-KO cells (median mRNA half-life in A5-KO cells was 3.11 hours; median mRNA half-life in WT cells was 7.81 hours, p < 2.2e-16, Mann-Whitney U test). Figure 8 g).

[0259] Further analysis showed that in the A5-KO sample, there was no m 6 A-modified mRNA is usually more resistant to mRNA than mRNA with m-modification. 6 A-modified mRNAs are more stable (p<2.2e-16, Mann-Whitney U test) Figure 8 h). Furthermore, with the absence of m 6 Compared to A-modified mRNA, ALKBH5 deletion results in mRNAs with m... 6 A-modified mRNAs have a shorter lifespan (approximately 38% on average) (p = 1.377e-11, Mann-Whitney U-test). Figure 8 i).

[0260] Notably, ALKBH5 deletion significantly shortened the half-life of 1555 transcripts, while only a few (22) transcripts showed an increased half-life. Figure 8 j). Gene ontology (GO) analysis showed that genes with shortened half-lives were mainly associated with stem cell maintenance, Notch signaling, and pancreatic development. Figure 8 k).

[0261] Finally, the half-life of the target mRNA was detected by RT-qPCR, and it was observed that ALKBH5 deletion significantly shortened the half-life of PDX1, NKX6.1, MNX1, and SOX9 mRNA. Figure 8 l).

[0262] In summary, these results confirm that ALKBH5 regulates the m-molecules of key genes involved in human pancreatic differentiation and development. 6 A modification affects mRNA stability, thereby regulating the differentiation of human pancreatic cells.

[0263] Example 7: Effect of YTHDF2 gene knockdown on human pancreatic cell differentiation

[0264] m 6 The function of A modification requires the cooperation of various cell-specific reading proteins. It is known that m... 6 The A-reading protein YTHDF2 can specifically recognize m 6 A and resulting in m 6 A's mRNA is rapidly degraded.

[0265] RNA-seq analysis revealed that YTHDF2 expression was significantly higher than other reading proteins during the pancreatic islet progenitor cell stage of pancreatic differentiation. Figure 9 a and Figure 9 b). Based on this background knowledge and analysis results, it was believed that YTHDF2 might play an important role in this process, and relevant tests were subsequently conducted.

[0266] First, using RIP-qPCR of YTHDF2, it was verified that the transcripts of PDX1, NKX6.1, MNX1, and SOX9 can interact with YTHDF2. Figure 10 a).

[0267] Subsequently, YTHDF2 was knocked down using shRNAs (shDF2-1 and shDF2-2), and its effect on pancreatic differentiation was examined. Figure 10 b and 10c). For example... Figure 10 As shown in d, shDF2 can partially salvage pancreatic differentiation disorder caused by A5-KO.

[0268] qPCR results showed that shDF2 partially restored the expression levels of PDX1, NKX6.1, MNX1, and SOX9. Figure 10 e and Figure 11 b). Furthermore, the results of immunofluorescence staining also yielded the same conclusion ( Figure 11 a).

[0269] Example 8 α-ketoglutaric acid-ALKBH5-m 6 A regulation promotes human pancreatic differentiation.

[0270] To date, there are very few known activators specific to and effective against ALKBH5. α-Ketoglutarate (αKG) is a cofactor of ALKBH5; therefore, in this embodiment, the effect of αKG on human pancreatic differentiation was tested.

[0271] The results showed that during cell differentiation (DE→PP), the addition of cell-permeable dimethyl-αKG (dm-αKG) to the culture medium significantly increased the proportion of PDX1 and NKX6.1 double-positive pancreatic islet precursor cells. Figure 10 f and Figure 11 c).

[0272] RT-qPCR showed that dm-αKG upregulated the expression of genes such as PDX1, NKX6.1, SOX9, and MNX1. Figure 10 g).

[0273] In addition, m 6 A-RIP experiments demonstrated that the use of dm-αKG can reduce the m-αKG level on PDX1, NKX6.1, SOX9, and MNX1 mRNA. 6 A content ( Figure 10 h).

[0274] In summary, αKG-ALKBH5-m 6 The A-YTHDF2 pathway can be used to promote human pancreatic cell differentiation. Figure 10 i) This is beneficial for its application in translational medicine.

[0275] discuss

[0276] Over the past few decades, studies of model organisms such as zebrafish, frogs, and mice have depicted the normal developmental process of the pancreas. The differentiation of human pluripotent stem cells into the pancreatic lineage depends on a complex regulatory network involving transcription factors, epigenetic regulators, and signaling pathways. Research in this area can help us understand how human pancreatic lineage cells gradually form, and also help us obtain functional human islet cells for diabetes-related research and treatment. Early embryonic development research is subject to many ethical restrictions; therefore, researchers have focused on differentiating human pluripotent stem cells in vitro into insulin-secreting pancreatic β cells. This research field has made significant progress in recent years. Current differentiation methods can progressively differentiate human pluripotent stem cells into definitive endoderm cells (DE), posterior foregut cells (PF), pancreatic islet precursor cells (PP), and finally, human islet organoids (hILO). The pancreatic differentiation system based on human pluripotent stem cells is an effective platform for studying human pancreatic biology. Furthermore, precise gene editing and high-throughput sequencing methods provide powerful tools for research on pancreatic differentiation and development. Recently, these methods have been successfully applied to the study of many key genes for pancreatic development and genes that are susceptible to diabetes, including PDX1, NGN3, RFX6, GATA6, GLIS3, HNF1A, and MAFB.

[0277] N6-adenosine methylation modification (m 6 A) is the most common modification within mammalian messenger RNA (mRNA). This modification is dynamic; a methyltransferase complex composed of METTL3, METTL14, WTAP, and other accessory proteins is responsible for adding the modification, while demethylases FTO and ALKBH5 help remove methylation modifications.6 A has multiple recognition proteins, including YTHDF1-3, YTHDC1-2, and IGF2BP1-3, which can specifically bind to m 6 A-modified mRNAs regulate the processing, export, stability, and translation of these mRNAs. Recent studies have shown that mRNA m... 6 A modification can regulate various life processes, including circadian rhythms, sperm production, neural development, cellular pluripotency, and immunity. Furthermore, mounting evidence suggests that the occurrence of various diseases, such as tumors and inflammation, is related to m... 6 Metabolic disorders in A are also somewhat related. However, m 6 The role of A modification in pancreatic differentiation and development remains unknown.

[0278] ALKBH5 is a member of the α-ketoglutarate-dependent dioxygenase AlkB family and is a major m 6 One of the A demethylases. Besides tumorigenesis, the functions of ALKBH5 in other biological processes are unclear. To date, the role of ALKBH5 in pancreatic differentiation has not been investigated. Furthermore, there is room for further improvement in the efficiency of stem cell differentiation into pancreatic systems.

[0279] This invention utilizes a pancreatic differentiation system based on human pluripotent stem cells, collecting samples at the stages of human pluripotent stem cells (hPSCs), definitive endoderm cells (DE), posterior foregut cells (PF), pancreatic islet precursor cells (PP), and finally, human islet organoids (hILO), for m... 6 A mRNA immunoprecipitation assay combined with next-generation sequencing (mRNA immunoprecipitation assay) 6 A-seq and RNA-seq analyses were used to study m in detail. 6 A modification regulates gene expression during pancreatic cell differentiation, and further revealed that the demethylase ALKBH5 plays an important regulatory role in pancreatic lineage differentiation and islet organ development.

[0280] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of an N6-adenosine methylation inhibitor of mRNA, characterized in that, For use in the preparation of compositions or formulations for (1) promoting the differentiation of human pluripotent stem cells into pancreatic islet precursor cells (PP), said pancreatic islet precursor cells being PDX1 and NKX6.1 double-positive islet precursor cells; and / or (2) preventing and / or treating insulin-dependent diabetes mellitus; The N6-adenosine methylation inhibitor of the mRNA is selected from the group consisting of: (a) ALKBH5 Accelerators; and / or (b) YTHDF2 Inhibitors; Among them, the ALKBH5 The promoter is ALKBH5 protein promoter and / or ALKBH5 Gene promoter, wherein the amino acid sequence of the ALKBH5 protein is shown in SEQ ID NO: 1, the... ALKBH5 The gene encodes the protein shown in SEQ ID NO: 1; The aforementioned YTHDF2 The inhibitor is a YTHDF2 protein inhibitor and / or YTHDF2 Gene inhibitor, wherein the amino acid sequence of the YTHDF2 protein is shown in SEQ ID NO:

2. YTHDF2 The gene encodes the protein shown in SEQ ID NO:

2.

2. The use as described in claim 1, characterized in that, The ALKBH5 The accelerator is dimethyl-α-ketoglutaric acid or α-ketoglutaric acid.

3. The use as described in claim 1, characterized in that, The composition or formulation is also used for the following purposes: (1) Reduce the RNA m of pancreatic islet precursor cell marker genes 6 A content; and / or (2) Increase the content of PDX1 and NKX6.1 double-positive pancreatic islet precursor cells.

4. A method for preparing pancreatic islet precursor cells in vitro, characterized in that, Including the following steps: In (a) ALKBH5 Accelerators; and / or (b) YTHDF2 In the presence of inhibitors, human pluripotent stem cells are cultured in a culture system to differentiate into pancreatic islet precursor cells, thereby obtaining the pancreatic islet precursor cells. The pancreatic islet precursor cells are PDX1 and NKX6.1 double-positive pancreatic islet precursor cells; The aforementioned ALKBH5 The promoter is ALKBH5 protein promoter and / or ALKBH5 Gene promoter, wherein the amino acid sequence of the ALKBH5 protein is shown in SEQ ID NO: 1, the... ALKBH5 The gene encodes the protein shown in SEQ ID NO: 1; The aforementioned YTHDF2 The inhibitor is a YTHDF2 protein inhibitor and / or YTHDF2 Gene inhibitor, wherein the amino acid sequence of the YTHDF2 protein is shown in SEQ ID NO:

2. YTHDF2 The gene encodes the protein shown in SEQ ID NO:

2.

5. A method for screening candidate compounds, said candidate compounds being (a) candidate compounds for treating insulin-dependent diabetes mellitus; and / or (b) candidate compounds for promoting the differentiation of human pluripotent stem cells into islet precursor cells, said islet precursor cells being PDX1 and NKX6.1 double-positive islet precursor cells, characterized in that, include: (i) The test compound was added to the cell culture system as the test group, and the cell culture system without the test compound was used as the control group; (ii) Comparison test group ALKBH5 The expression level and / or activity of the gene or its protein, E1, compared with the expression level and / or activity, E0, in the control group; Among them, when E1 is significantly higher than E0 in the test group, it indicates that the tested compound is (a) a candidate compound for treating insulin-dependent diabetes mellitus; and / or (b) a candidate compound for promoting the differentiation of human pluripotent stem cells into pancreatic islet progenitor cells; Among them, the ALKBH5 The gene encodes the protein shown in SEQ ID NO:

1.

6. The method as described in claim 5, characterized in that, The method further includes the steps of: (iii) The test compound was added to the human pluripotent stem cell culture system as the test group, and the human pluripotent stem cell culture system without the test compound was used as the control group; (iv) Compare the proportion of human pluripotent stem cells that transform into pancreatic islet precursor cells in the test group to determine whether the compound is (a) a candidate compound for treating insulin-dependent diabetes mellitus; and / or (b) a candidate compound for promoting the differentiation of human pluripotent stem cells into pancreatic islet precursor cells; Specifically, if the proportion (T1) of human pluripotent stem cells transforming into pancreatic islet precursor cells in the test group is significantly higher than that in the control group (T0), then the test compound is (a) a candidate compound for treating insulin-dependent diabetes mellitus; and / or (b) a candidate compound for promoting the differentiation of human pluripotent stem cells into pancreatic islet precursor cells.

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