CN116574674A

CN116574674A - Activation of exosomal driven somatic cell reconstitution pluripotency using CD3254

Info

Publication number: CN116574674A
Application number: CN202310501307.9A
Authority: CN
Inventors: 祝赛勇; 金燕
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2023-04-27
Filing date: 2023-04-27
Publication date: 2023-08-11

Abstract

The present invention relates to the use of CD3254 to activate exoRNA to drive somatic cell reconstitution pluripotency. In particular, the invention provides a method of inducing pluripotent stem cells from mammalian non-pluripotent cell production comprising contacting a starter cell with a combination of agents comprising an RXRa activator, promoting cytochemical reprogramming, activating exosomal driven somatic cell reconstitution pluripotency.

Description

Activation of exosomal driven somatic cell reconstitution pluripotency using CD3254

Technical Field

The invention relates to the field of biological medicine, in particular to a CD 3254-activated exoRNA (ribonucleic acid) -driven somatic cell reconstruction method.

Background

Induced pluripotent stem cells have infinite self-renewal capacity and polyblastic differentiation potential similar to embryonic stem cells, and are widely used in disease modeling, drug development and regenerative medicine. Currently, there are mainly 2 ways to obtain induced pluripotent stem cells: transcription factor reprogramming and chemical reprogramming.

The reprogramming of transcription factors is mainly carried out by a transgenic method, and the integration of exogenous transcription factors into the genome of an initiating cell can cause potential safety hazards. Chemical reprogramming has unique advantages over transcription factor reprogramming.

Chemical reprogramming is mainly realized by virtue of small molecule combination, and small molecules cannot be integrated into somatic cell genome, so that the safety is higher. And secondly, the small molecules are easy to penetrate cell membranes, the treatment is reversible, the treatment time and the dosage are easy to control, the small molecules can be combined at will, and the cost is lower.

Finally, small molecules have been used in the treatment of human diseases. Thus, small molecule chemical reprogramming may be more readily accepted for clinical treatment. At present, cytochemical reprogramming has the problems of low efficiency, unclear mechanism and the like, and seriously hinders the application of the cytochemical reprogramming. Chemical reprogramming has evolved to date, still a slow and inefficient process, in 3 stages, requiring approximately 40 days.

It is therefore desirable to find new small molecules to achieve rapid and efficient chemical reprogramming.

Disclosure of Invention

The object of the present invention is to provide a method for achieving a fast and efficient chemical reprogramming by small molecule modulation.

In a first aspect of the invention there is provided the use of an active ingredient comprising an agent which is an RXRa activator for the preparation of a composition or formulation for:

activating the exorna complex;

promoting Sall4 gene expression;

Promoting cell reprogramming;

promoting expression of a pluripotent marker of induced pluripotent stem cells; and/or

Treating or reducing inflammatory response.

In another preferred embodiment, the promotion of cell reprogramming is to significantly increase the efficiency of cytochemical reprogramming.

In another preferred embodiment, the exosome complex comprises 11 subunits: exosc1, exosc2, exosc3, exosc4, exosc5, exosc6, exosc7, exosc8, exosc9, exosc10 and Dis3.

In another preferred embodiment, the activating exorna complex comprises promoting expression of genes or proteins of Exosc1, exosc2, exosc3, exosc4, exosc5, exosc6, exosc7, exosc8, exosc9, exosc10 and Dis 3; preferably includes promoting expression of genes or proteins of Exosc3, exosc7, exosc8 and Dis3.

In another preferred embodiment, the activating exosome complex comprises:

promoting the expression or activity of Exosc3 gene or protein to be improved by more than or equal to 2 times, preferably more than or equal to 4 times;

promoting the expression or activity of Exosc7 gene or protein to be improved by more than or equal to 1 time, preferably more than or equal to 3 times;

promoting the expression or activity of Exosc8 gene or protein to be improved by more than or equal to 1 time, preferably more than or equal to 3 times; and/or

Promote the expression or activity of Exosc8 gene or protein to be improved by more than or equal to 1 time, preferably more than or equal to 3 times.

In another preferred embodiment, the activating exosome complex comprises: promoting degradation of transposon RNAs such as MMVL 30.

In another preferred embodiment, the RXRa activator is CD3254 or a CD3254 like compound.

In another preferred embodiment, the active ingredient further comprises:

(i) HDAC inhibitors;

(ii) GSK-3 alpha/beta inhibitors;

(iii) TGF-beta-RI/ALK 5 inhibitors;

(iv) An adenylate cyclase activator;

(v) Rarα agonists.

In another preferred embodiment, the activator is a substance capable of increasing the activity and/or content of the RXRa gene or protein thereof in vivo or in vitro; the substance may be an artificial or natural compound, protein, nucleotide, etc.

In another preferred embodiment, the RXRa activator comprises a substance that activates RXRa expression.

In another preferred embodiment, the RXRa activator comprises a RXRa protein activator and/or a RXRa gene activator.

In another preferred embodiment, the promotion of Sall4 gene expression refers to an increase in Sall4 gene or protein expression or activity of 1-fold or more, preferably 2-fold or more, more preferably 5-fold or more.

In another preferred embodiment, the pluripotent marker of induced pluripotent stem cells comprises Oct4, sox2 or Nanog genes.

In another preferred embodiment, the promoting the induction of expression of a pluripotent marker of a pluripotent stem cell comprises:

Promoting the expression or activity of Oct4 gene or protein to be improved by more than or equal to 2 times, preferably more than or equal to 4 times;

promoting the expression or activity of Sox2 gene or protein to be improved by more than or equal to 1 time, preferably more than or equal to 2 times; and/or

Promoting the expression or activity of Nanog gene or protein to be improved by more than or equal to 1 time, preferably more than or equal to 3 times.

In another preferred embodiment, the promotion of Oct4, sox2 or Nanog gene expression refers to an increase in Sall4 gene or protein expression or activity of 1-fold or more, preferably 2-fold or more, more preferably 5-fold or more.

In another preferred embodiment, the HDAC inhibitor is VPA (Valproic Acid).

In another preferred embodiment, the GSK-3 alpha/beta inhibitor is CHIR99021.

In another preferred embodiment, the TGF-beta-RI/ALK 5 inhibitor is RepSox (616452).

In another preferred embodiment, the adenylate cyclase activator is Forskolin.

In another preferred embodiment, the rarα agonist is AM580.

In another preferred embodiment, the combination of reagents comprising an RXRa activator is used to:

upregulation of early multipotent genes (e.g., sall4, lin28a, esrrb, klf4, cMyc) and epithelial genes (e.g., cdh1, cldn4, tjp 3); and/or

Down-regulate mesenchymal genes (e.g., zeb, twist1, snail 1).

In another preferred embodiment, the combination of agents comprising RXR activators and/or RXR antagonists is used to:

Up-regulating stem cell population maintenance, chromatin organization, RNA metabolic processes and DNA replication;

down-regulated extracellular matrix, epithelial to mesenchymal transition, TGF- β and MAPK signaling pathways.

In another preferred embodiment, the cell is a mammalian non-pluripotent cell.

In another preferred embodiment, the mammal comprises a human or a non-human mammal.

In another preferred embodiment, the non-human mammal comprises a rodent (e.g., mouse, rat, rabbit), primate (e.g., monkey).

In another preferred embodiment, the inflammation is IFN-gamma-or TNF-alpha mediated inflammation; including secretion of IFN-gamma by double-stranded DNA, activation of IFNGR receptor and downstream JAK-STAT signaling pathway, activation of inflammatory response; and promote secretion of TNF- α, activate TNFR1 receptor and downstream IKK-NFkB signaling pathway, activate inflammatory response.

In a second aspect of the invention, there is provided a method of screening for potential compounds that promote the formation of induced pluripotent stem cells (ipscs) in a mammalian non-pluripotent cell, the method comprising:

(a) Providing a test group and a blank control group, taking a culture system added with a test object as the test group, and culturing non-pluripotent cells in the presence of the test object;

Taking a culture system without adding a test compound as a blank control group, and culturing non-pluripotent cells under the condition without adding the test compound, wherein the blank control group has the same other conditions as the test group;

(b) Detecting degradation conditions of transposon RNA in a test group and a blank control group, wherein the expression level of the transposon RNA in the test group is marked as C1, and the expression level of the transposon RNA in the blank control group is marked as C0; and

(c) Comparing the level of transposon RNA degradation of the test group and the blank control group, if the expression level of the transposon RNA of the test group is significantly lower than the expression level of the transposon RNA of the blank control group; the compound is suggested to be a potential compound capable of promoting the formation of non-pluripotent mammalian cells to induce pluripotent stem cells (ipscs).

In another preferred embodiment, the starting cells are embryonic fibroblasts (MEFs).

In another preferred embodiment, the expression level C1 of transposon RNA in the test group is substantially lower than that of the control group RNA by a ratio of (C1/C0) 1/2, preferably 1/3, more preferably 1/4.

In another preferred embodiment, the transposon RNA is VL30 ERV1 family; preferably MMVL30.

In another preferred embodiment, the compound that promotes the transformation of mammalian non-pluripotent cells to induced pluripotent stem cells is a rxrα -specific agonist.

In another preferred embodiment, the rxrα specific agonist is CD3452 or a CD 3452-like compound.

In another preferred embodiment, the CD 3452-like compound is capable of promoting transposon MMVL30 degradation.

In another preferred embodiment, in (a), the kit further comprises a positive control group, wherein the positive control group and the test group have the same experimental conditions, and the expression level of the detected transposon RNA is marked as C2 in the presence of a positive control CD 3452;

if the ratio of the expression level C1 and the expression level C2 (C1/C2) of transposon RNA in the test group is smaller, the degradation effect of the substance to be screened on the transposon RNA is suggested to be larger.

In another preferred embodiment, the method further comprises (d) performing a test experiment on the selected compound in combination with a compound selected from the group consisting of:

(i) HDAC inhibitors;

(ii) GSK-3 alpha/beta inhibitors;

(iii) TGF-beta-RI/ALK 5 inhibitors;

(iv) An adenylate cyclase activator; and

(v) Rarα agonists.

In a third aspect of the invention, there is provided a method of screening for a compound that promotes up-regulation of the Sall4 gene in a non-pluripotent mammalian cell, the method comprising:

(c) Comparing the level of transposon RNA degradation of the test group and the blank control group, if the expression level of the transposon RNA of the test group is significantly lower than the expression level of the transposon RNA of the blank control group; the compound is suggested to be a potential compound capable of promoting up-regulation of the Sall4 gene in non-pluripotent mammalian cells.

In another preferred embodiment, the compound that promotes up-regulation of the Sall4 gene in a mammalian non-pluripotent cell is a RXR alpha-specific agonist.

In a fourth aspect of the invention there is provided a compound having CD3254 activity obtainable by the screening method of the second aspect of the invention.

In a fifth aspect of the invention, there is provided a method of inducing pluripotent stem cells from mammalian non-pluripotent cell cultures by culturing mammalian non-pluripotent cells under conditions suitable for growth of the cells and in the presence of an RXRa activator to obtain induced pluripotent stem cells.

In another preferred embodiment, the culture system further comprises one or more agents selected from the group consisting of:

(i) HDAC inhibitors;

(ii) GSK-3 alpha/beta inhibitors;

(iii) TGF-beta-RI/ALK 5 inhibitors;

(iv) An adenylate cyclase activator; and

(v) Rarα agonists.

In another preferred embodiment, the method comprises:

(S1) providing a starting cell, contacting the cell with a first medium, and culturing for a time D1; obtaining a first induced pluripotent stem cell;

the first medium comprises a first combination of reagents comprising: an RXRa activator, and one or more agents selected from the group consisting of:

(i) HDAC inhibitors;

(ii) GSK-3 alpha/beta inhibitors;

(iii) TGF-beta-RI/ALK 5 inhibitors;

(iv) An adenylate cyclase activator; and

(v) Rarα agonists.

In another preferred embodiment, the culturing time D1 is 10 to 20 days; preferably 12-15 days.

In another preferred embodiment, the HDAC inhibitor is VPA (Valproic Acid).

In another preferred embodiment, the GSK-3 alpha/beta inhibitor is CHIR99021.

In another preferred embodiment, the adenylate cyclase activator is Forskolin.

In another preferred embodiment, the rarα agonist is AM580.

In another preferred embodiment, the RXRa activator is CD3254, having the structure shown below:

in another preferred embodiment, the number of the starting cells is 3X 10 ⁵ -4×10 ⁵ /plate。

In another preferred embodiment, the concentration of CD3254 is between 0.1 and 1mM; preferably 0.25-0.75mM.

In another preferred embodiment, the VPA (Valproic Acid) concentration is from 0.1 to 1mM; preferably 0.25-0.75mM.

In another preferred embodiment, the concentration of CHIR99021 is 5-30 μm; preferably 10-15. Mu.M.

In another preferred embodiment, the concentration of RepSox is 5 to 30. Mu.M; preferably 10-15. Mu.M.

In another preferred embodiment, the Forskolin is at a concentration of 1-50 μm; preferably 5-20. Mu.M.

In another preferred embodiment, the concentration of AM580 is between 0.1 and 1mM; preferably 0.25-0.75mM.

In another preferred embodiment, the first induced pluripotent stem cell expresses an early pluripotent gene marker selected from the group consisting of Sall4, cdh1, epcam and Lin28a; preferably Sall4.

In another preferred embodiment, the first induced pluripotent stem cell:

20% -60% of the cells express the early multipotent gene marker Sall4.

In another preferred embodiment, the method further comprises:

(S2) culturing the first induced pluripotent stem cells obtained in (S1) in a second medium for a time D2 to obtain second induced pluripotent stem cells.

In another preferred embodiment, the second induced pluripotent stem cell expresses an early pluripotent gene marker selected from the group consisting of Sall4, cdh1, epcam and Lin28a.

In another preferred embodiment, the second induced pluripotent stem cells:

40% -80% of cells express the early multipotent gene marker Sall4;

40% -80% of the cell expression phase multipotent gene marker Cdh1;

40% -80% of the cell expression phase multipotent gene markers Epcam; and

40% -80% of the cells express the phase multipotent gene marker Lin28a.

In another preferred embodiment, the incubation time D2 is 12-20 days; preferably 12-18 days.

In another preferred embodiment, the second medium comprises or does not comprise an RXRa activator.

In another preferred embodiment, the second medium comprises a combination of reagents selected from the group consisting of:

GSK-3 alpha/beta inhibitor, preferably CHIR99021;

TGF-beta-RI/ALK 5 inhibitors, preferably RepSox (616452);

an adenylate cyclase activator, preferably Forskolin;

monoamine oxidase inhibitors, preferably Parnatte/Tranylcypromine;

the rarα activator, preferably AM580;

inhibitors of histone methyltransferase EZH2, preferably DZNep.

DOT1L methyltransferase inhibitors, preferably SGC0946.

In another preferred embodiment, the method further comprises:

(S3) culturing the second induced pluripotent stem cells obtained in (S1) in a third medium for a time D3 to obtain embryonic stem cell-like cells.

In another preferred embodiment, the embryonic stem cell-like cells express a multipotent marker selected from the group consisting of Oct4, sox2, and Nanog.

In another preferred embodiment, the embryonic stem cell-like cells are:

20% -60% of cells express the multipotential marker Oct4;

20% -60% of cells express a multipotential marker Sox2; and

20% -60% of the cells express the multipotential marker Nanog.

In another preferred embodiment, the incubation time D3 is 12-20 days; preferably 12-18 days.

In another preferred embodiment, the third medium comprises a combination of reagents selected from the group consisting of:

GSK-3 alpha/beta inhibitor, preferably CHIR99021;

MEK inhibitors, preferably PD0325901.

In a sixth aspect of the invention, there is provided a pharmaceutical composition comprising an active ingredient, and a pharmaceutically acceptable carrier, wherein the active ingredient comprises an RXRa activator and one or more agents selected from the group consisting of:

(i) HDAC inhibitors;

(ii) GSK-3 alpha/beta inhibitors;

(iii) TGF-beta-RI/ALK 5 inhibitors;

(iv) An adenylate cyclase activator;

(v) Rarα agonists.

In another preferred embodiment, the pharmaceutical composition is for

Promoting Sall4 gene expression;

promoting cell reprogramming;

activating the exorna complex;

Treating or reducing inflammatory response.

In another preferred embodiment, the inflammation is IFN-gamma-or TRAIL-mediated inflammation.

In a seventh aspect of the invention, there is provided a kit comprising

(1) RXRA activator reagent, and

(2) One or more agents selected from the group consisting of:

(i) HDAC inhibitors;

(ii) GSK-3 alpha/beta inhibitors;

(iii) TGF-beta-RI/ALK 5 inhibitors;

(iv) An adenylate cyclase activator; and

(v) Rarα agonists.

In another preferred embodiment, the kit is for:

promoting Sall4 gene expression;

promoting cell reprogramming;

activating the exorna complex;

Treating or reducing inflammatory response.

In another preferred embodiment, the kit further comprises mammalian cells.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

Fig. 1. Retinoid X receptor subtype α (rxrα) agonist CD3254 significantly promotes chemical reprogramming in mice. (A) Schematic of compound screening during chemical reprogramming in mice. (B) Small molecule screening results Sall4 immunostaining was performed on day 12 and the number of Sall4 positive clones was counted. (C) Fluorescence images of Sall4 positive clones after DMSO and CD3254 treatment. Scale bar, 100 μm. (D) Sall4 positive clone number treated with CD 3254. (E) schematic of the cloning procedure for obtaining pOct 4-GFP. (F) Fluorescence images of pOct4-GFP clones after DMSO and CD3254 treatment. Scale bar, 100 μm. (G-H) concentration (G) and stage (H) testing of CD 3254. (I) RT-qPCR analysis of Sall4, cdh1, epcam and Lin28a gene expression in MEFs, intermediate cells on d12 treated with DMSO and CD3254, and R1 mESCs. Data are presented as mean ± SD of three independent experiments. * P <0.01, P <0.001, P <0.0001.

FIG. 2 identification of chemically induced pluripotent stem cells (cipCs). (A) schematic drawing of the CiPSC system is established. (B) Clear field and pOct4-GFP pictures of ciPSC treated with DMSO and CD3254 at stage 1. (C) Immunofluorescent staining results of pluripotency markers in DMSO-cisCs and CD 3254-cisCs. Scale bar, 50 μm. (D) Real-time fluorescent quantitative PCR (RT-qPCR) analysis was performed on Oct4, sox2 and Nanog gene expression in Mouse Embryo Fibroblasts (MEFs), DMSO-cisCPC, CD 3254-cisCPC and R1 mouse embryonic stem cells (mESCs). (E) Comparing the overall transcript levels between MEFs and DMSO-ciPSC, MEFs and CD3254-ciPSC, DMSO-ciPSCs and CD3254-ciPSC, R1 mESCs and DMSO-iPSCs, and R1 mESCs and CD 3254-ciPSCs. (F) Bisulfite sequencing analysis DNA CpG methylation at Oct4 and Nanog promoter sites. (G) karyotyping of ciPSCs. (H) Immunofluorescent staining results of three germ layer differentiation markers. Scale bar, 100 μm. (I) Hematoxylin and eosin staining (H & E staining) showed ectodermal, mesodermal and endodermal components from ciscs teratomas. Scale bar, 100 μm. Data are presented as mean ± SD of three independent experiments. * P <0.01, P <0.001, P <0.0001. (J) Schematic representation of healthy mice produced by ciscs

FIG. 3 transcriptional analysis of the effect of CD3254 on chemical reprogramming. (A) A scatter plot of the overall gene expression pattern between CD3254 and control DMSO cells was compared. Red (n=1330) and blue (n=1330) points represent genes differentially expressed up (FC > 1.5) and down (FC < 0.67), respectively, with statistical significance (adjusted P value < 0.05). (B-C) bubble diagrams of KEGG pathways enriched for up-regulated (B) and down-regulated (C) genes in CD3254 compared to DMSO cells. The period size represents the base factor in each channel and the orange or blue gradient represents the adjusted P value for each channel. (D-G) expression heatmap of early pluripotency (D), mesenchymal (E), epithelial (F) and TGF-beta signaling pathways. (G) genes related to CD3254 and DMSO cells. (H) Functional enrichment analysis of CD3254 with up (left) and down (right) regulated genes in DMSO cells.

Fig. 4, retinoid X receptor alpha subtype (rxrα) enhances chemical reprogramming in mice. (A) a graph showing the Rxra knockdown process. (B) RT-qPCR analysis of Rxra expression in MEFs infected with shCTR and shRxra viruses. (C) Immunofluorescence of Sall4 in reprogramming intermediates infected with shCTR and shRxra. Scale bar, 100 μm. (D) RT-qPCR analysis of pluripotent gene expression in cells treated with shCTR and shRxra. (E) A graph showing the reprogramming process of an infected Rxra-OE virus. (F) Immunofluorescence of Sall4 in control cells and of Rxra overexpressing cells on d 12. Scale bar, 100 μm. (G) RT-qPCR analysis of pluripotent gene expression in cells treated with CTR and Rxra-OE. Data are presented as mean ± SD of three independent experiments. * P <0.01, P <0.001, P <0.0001.

FIG. 5 CD3254-RXR alpha axis transcription activated exosomes. (A) Comparing the scatter plot of the global gene expression pattern between Rxra-OE and control cells. Red (n=1953) and blue (n=2417) represent genes differentially expressed up (FC > 1.5) and down (FC < 0.67), respectively, with statistical significance (adjusted P value < 0.05). (B) Bubble diagram of KEGG pathway for enrichment of up-regulated genes in Rxra-OE versus control cells. RNA degradation pathways are highlighted in red font. (C) model of an exo-RNA complex. This nine subunit core exosome consists of a top three subunit cap (Exosc 1/2/3) and a middle six subunit ring (Exosc 4-9). The two active ribonuclease subunits Dis3 and Exosc10 will contact the bottom and top of the structure. (E-F) real-time fluorescent quantitative PCR (RT-qPCR) analysis of Exosc3, exosc7, exosc8 and Dis3 expression in CD3254 treated (E) and Rxra-OE treated cells (F). (G) The rxrα binding motif (H) is shown to show rxrα peaks at Exosc3 and Dis3 sites. (I) Predicted rxrα binding sites at the Exosc3 and Dis3 promoters. (J) qPCR analysis of the binding capacity of RXR alpha at Exosc3 and Dis3 gene loci. Data are presented as mean ± SD of three independent experiments. * P <0.05, < P <0.01, < P <0.001, < P <0.0001.

FIG. 6. Exosomes are essential in the process that reprogramming is pluripotent. (A) A graph showing Exosc3 knockdown process. (B) RT-qPCR analysis of Exosc3 expression in MEFs infected with shCTR and shExosc3 virus. (C) Immunofluorescence of Sall4 in intermediate body weight programming infected with shCTR and shaxosc 3. Scale bar, 100 μm. (D) RT-qPCR results of pluripotent gene expression in cells treated with shCTR and shExosc 3. (E) Comparing the scatter plot of the global gene expression pattern between the shoxosc 3 and the shrt cells. Red (n=314) and blue (n=1093) points represent genes differentially expressed up (FC > 1.5) and down (FC < 0.67), respectively, with statistical significance (adjusted P value < 0.05). (F) Expression heatmaps of differentially expressed mRNA in shaxosc 3 and shactr cells. (G-H) enrichment up-regulated (G) and down-regulated KEGG pathway bubble patterns. (I) Functional enrichment analysis of the genes upregulated (left) and downregulated (right) in shoxc 3 and shrt cells. Data are presented as mean ± SD of three independent experiments. * P <0.01, P <0.001, P <0.0001.

FIG. 7 exosomes target transposon-related RNA for degradation. (A) The volcanic plot representing log 2-fold changes in different types of differentially expressed RNAs (B) shows retrotransposons differentially expressed in shaxosc 3 and shCTR cells. Red dot: 3403 up-regulated repeat RNAs (FC >1.5, adjusted P value < 0.05), blue dot: 35 down-regulated repeat RNAs (FC <0.67, adjusted P value < 0.05). The MMVL30-int repeat item is highlighted in red font. (C) Comparing the differential expression reverse transcription element (FC >1.5 or <0.67, adjusted P value < 0.05) of the shaxosc 3 with the shactr cells. The MMVL30-int repeat item is highlighted in red font. (E) The Venn diagram shows overlapping differentially expressed MMVL30-int transposons in shExosc3 and shCTR (up-regulated), CD3254 and DMSO (down-regulated) and Rxra-OE and CTR (down-regulated) cells. (F) Density and heat maps of RNA abundance of 13 overlapping MMVL30-int transposons. RNA abundance on MMVL30-int transposons is classified by intensity. (G) It was shown that some representative MMVL30-int transposons (MMVL 30-int_dup945 to dup 947) were expressed in CD3254 treated and Rxra-OE cells decreased and in shExosc3 cells increased. (H) RT-qPCR analysis of MMVL30-int expression in CD3254 treated Rxra-OE and shExosc3 cells. (I) RIP-qPCR analysis of MMVL30 conjugated to Exosc 3. (J) decay curve of MMVL 30-int. (K) Sall4 positive clones in intermediate cells infected with shCTR and shMMVL30 were reprogrammed. Data are presented as mean ± SD of three independent experiments. * P <0.05, < P <0.01, < P <0.001.

Figure 8. Exosomes degrade MMVL30 and reduce inflammatory responses to promote chemical reprogramming. (A) Differential expression of cis-or trans-transcripts of ERVs in shCTR and shExosc3 samples (FC>1.5 or<0.37, adjusted P value<0.05 A) an expression heat map. (B) The log 2-fold change in the transcript of many representative bidirectional ERVs in the shaxosc 3 and shactr cells is demonstrated. (C) CD3254 treatment down-regulates dsRNAs in reprogrammed intermediate cells. (D) CD3254 reduces dsRNAs on the MMVL30 region. (E) Expression of Ifngr1, tnfrsf1a and Tnfrsf10b in Rxra-OE and shoxosc 3 cells treated with CD 3254. (F) Receiving IFN-gamma (20 ng ml) ^-1 ) And TNF-alpha (20 ng ml) ^-1 ) Treated Sall4 positive clones. (G) RT-qPCR analysis was performed on Sall4, cdh1 and Zeb2 expression with IFN-gamma and TNF-alpha treatments. (H) Sall4 positive clones after treatment with the specific JAK inhibitor Upadacitinib (5. Mu.M) and the IKK inhibitor BMS-345541 (1. Mu.M). (I) RT-qP for Sall4, cdh1 and Zeb2 expression with JAKi and IKKi treatmentsCR analysis. (J) the mechanism of the present invention. Data are presented as mean ± SD of three independent experiments. * P (P)<0.05，**P<0.01，****P<0.001，****P<0.0001。

Detailed Description

The inventors have conducted extensive and intensive studies and, for the first time, have unexpectedly found that RXRa activator small molecule CD3254 is capable of significantly promoting cytochemical reprogramming, transcriptional activation of exosomes, modulating degradation of transposon TEs (e.g., MMVL 30) and reducing MMVL30 mediated inflammatory responses. The present invention has been completed on the basis of this finding.

Specifically, in the present invention:

1. the non-preferential small molecule drug screening is carried out in the early stage of chemical reprogramming, and the CD3254 which is the specific activator of the transcription factor RXRalpha is found and definitely capable of remarkably and stably promoting the chemical reprogramming. Meanwhile, the direct overexpression of Rxra can obviously improve the reprogramming efficiency to 10 times, and further proves that CD32540-RXR alpha plays a key role in chemical reprogramming.

2. CD3254 treatment was found by RNA-seq analysis to be able to activate the 11 genes associated with the exosomes as a whole by over-expression of Rxra (Exosc 1-10 and Dis 3). CUT & Tag sequencing and CUT & Tag-qPCR confirm that the transcription factor RXR alpha can be combined with a promoter region of an exonuclease related gene, and the CD3254-RXR alpha axis can be used for overall transcriptional activation of an exonuclease complex for the first time.

3. After further analysis of lncRNA-seq data, we found that after knocking down exosome key gene Exosc3 during reprogramming, transposon-related RNAs expression was significantly increased, which was mainly the class MMVL30-int, and it was revealed for the first time that CD3254-rxrα -exosome axis mainly regulated MMVL 30-int-like transposon RNAs, as well as demonstrating for the first time that MMVL30 is an obstacle in cytochemical reprogramming.

4. Further analysis found that CD 3254-RXRalpha-exosome signaling axis in reprogramming reduced MMVL 30-mediated inflammatory responses, the first reported IFN-gamma and TNF-alpha pathways associated with inflammatory responses were the barriers to cytochemical reprogramming.

Terminology

Unless defined otherwise, 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 belongs.

The term "about" may refer to a value or composition that is within an acceptable error of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or measured.

As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

Retinoid X Receptor (RXR)

Nuclear receptors are key transcription factors in organisms, and can directly convert chemical signals into different physiological effects, playing an important role in development and physiology. Retinoid X Receptors (RXRs) can heterodimerize with other nuclear receptors, regulating multiple, inter-interlaced signal networks. Current studies indicate that retinoid X receptor signaling plays a key role in the development processes of blood, brown fat, and pancreatic differentiation. In addition, retinoid X receptors have been shown to be involved in tissue repair and regeneration processes. However, the precise molecular mechanism of retinoid X receptors is not clear and requires further exploration.

There are three subtypes of RXR proteins, rxrα, rxrβ and rxrγ in mammals. Human rxrα, rxrβ and rxrγ proteins share 97.4%,94.75% and 98.27% homology with the mouse counterpart proteins, respectively. Rxrα is mainly localized to the nucleus, and is also present in the cytoplasm and mitochondria. Rxrα is widely expressed mainly in liver, kidney and brain tissues. Rxrα expression is quite common during development, with higher expression in the epidermis of the skin during late gestational development.

RNA exosomes

An exosome is a complex of 11 subunits in total and is involved in RNA degradation. As a ubiquitous RNA monitoring system in organisms, the RNA monitoring system can accurately regulate and control RNA steady state and ensure normal development. Among them, the exosomes are key components of RNA monitoring systems, a polyprotein complex with 3 'to 5' ribonuclease activity, comprising 11 subunits (Exosc 1-Esosc10 and Dis 3). The rapid degradation and synthesis of large amounts of RNA is accompanied by a cell fate shift, requiring comprehensive RNA monitoring.

At present, the contribution of RNA monitoring to cell reprogramming is not known. In addition, how to better manipulate cell fate using RNA monitoring is worth studying and elucidating. In addition, the whole expression condition of the exosomes of the RNA is regulated and controlled by using chemical small molecules, so that the method has wide application value.

CD 3254-RXRalpha-RNA exosome shaft

In the invention, the inventor discovers a brand new passage for the first time: CD 3254-RXRalpha-exosomal axis. Specifically, in mammalian cells, the CD3254-RXR alpha axis can transcriptionally activate all 11 components of the exosome (Exosc 1-10 and Dis 3). It was further found that the exosomes predominantly modulate the degradation of transposon TEs (predominantly MMVL 30-int) and reduce MMVL30 mediated inflammatory responses (IFN- γ and TNF- α pathways).

Activator and pharmaceutical composition

As used herein, a "CD3452 or CD 3452-like compound" refers to a series of small molecule compounds capable of activating rxrα expression, transcriptionally activating an exorna, modulating the degradation of transposon TEs, significantly promoting cytochemical reprogramming.

As used herein, the term "RXRa activator", "RXRa activators of the present invention" refers to substances, especially small molecule compounds, capable of activating RXRa expression.

One representative activator is CD3254, which has the structure shown below:

it can significantly promote cytochemical reprogramming efficiency and can activate the exo-RNA complex by transcription as a whole.

The invention also provides a pharmaceutical composition comprising a safe and effective amount of an activator (e.g., a small chemical molecule) of the invention and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. 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 sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, about 1 microgram-10 milligrams per kilogram of body weight per day.

The main advantages of the invention include

(1) The invention discovers that the novel small molecule CD3254 can obviously promote the cytochemistry reprogramming efficiency.

(2) The invention discovers that CD3254 can activate the exosomal driving somatic cell reconstruction pluripotency and break down the potential molecular mechanism of chemical reprogramming.

(3) The small molecule CD3254 of the invention is capable of overall transcriptional activation of an exorna complex, and is expected to be useful in models of lesions and cancers.

(4) The small molecule CD3254 can be used as an exosome activator of cancer cells to promote RNA degradation of related cancer genes, and can be further used as a medicament for treating related diseases.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, in which the detailed conditions are not noted in the following examples, is generally followed by routine conditions such as Sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Examples

The experimental method comprises the following steps:

animals

Mating of OG2 mice and 129 mice produced offspring carrying the Oct4 promoter-driven GFP reporter (OG 2X129 mice). Mouse Embryonic Fibroblasts (MEFs) were obtained from E13.5 embryos of OG2X129 mice. Mice were housed in pathogen free facilities at the university of Zhejiang laboratory animal center and fed a normal food diet. Animal-related procedures used in the present invention have been approved by the laboratory animal ethics committee of the university of Zhejiang.

Cell culture

MEF medium: the DMEM basic culture medium is added with 10% FBS and 1 Xpenicillin/streptomycin double antibody, and the mixture is preserved at 4 ℃ after being uniformly mixed.

Mouse pluripotent stem cell medium (2 i): DMEM basic medium was added with 10% FBS,10% KSR,1 XNEAA solution, 1 XP/S diabody, 0.055mM beta-mercaptoethanol, 1000U/mL LIF, (3. Mu.M) CHIR99021 and PD0325901 (0.2. Mu.M), and after mixing, stored at 4 ℃.

Mouse transcription factor reprogramming media: DMEM basic medium was supplemented with 10% FBS,10% KSR,1 XNEAA solution, 1 XP/S diabody, 0.055mM beta-mercaptoethanol, 1000U/mL mLIF and 2. Mu.g/mL doxycycline (Dox).

Embryoid Body (EB) differentiation medium: DMEM basic medium was added with 10% FBS,10% KSR,1 XNEAA solution, 1 XP/S diabody and 0.055mM beta-mercaptoethanol, and stored at 4℃after mixing.

Mouse Embryonic Fibroblasts (MEFs) and Human Embryonic Kidney (HEK) 293T cells were cultured in MEF medium. Mouse ESCs and cisCs were cultured in mouse pluripotent stem cell medium containing CF1 feeder cells. All cells were maintained at 37℃and 5% CO ₂ Is placed in a humidification incubator.

Cytochemical reprogramming

Mice were chemically reprogrammed to first Stage medium (Stage 1, see table 1): DMEM basic medium was supplemented with 10% FBS,10% KSR,1 XNEAA solution, 1 XP/S diabody, 0.055mM beta-mercaptoethanol, 20ng/mL bFGF and chemical small molecule VPA (0.5 mM), CHIR99021 (20. Mu.M), 616452 (10. Mu.M), parnate (5. Mu.M), forskolin (50. Mu.M), AM580 (0.5. Mu.M), EPZ004777 (5. Mu.M) and Vc (250. Mu.M) and stored at 4℃after mixing.

Mice were chemically reprogrammed to phase two media (Stage 2): DMEM basic medium was supplemented with 10% FBS,10% KSR,1 XNEAA solution, 1 XP/S diabody, 0.055mM beta-mercaptoethanol, 20ng/mL bFGF and chemical small molecule VPA (0.5 mM), CHIR99021 (10. Mu.M), 616452 (10. Mu.M), parnatte (5. Mu.M), forskolin (10. Mu.M), AM580 (0.5. Mu.M), DZNep (0.05. Mu.M), 5-aza-dC (0.5. Mu.M), SGC0946 (5. Mu.M) and Vc (250. Mu.M) and stored at 4℃after homogenization.

Mice were chemically reprogrammed to phase three medium (Stage 3): 47% DMEM/F12, 47% Neurobasal medium, 100 XN 2, 50 XB 27 supplement, 1% NEAA, 1% P/S, 0.055mM beta-mercaptoethanol, 1000U/mL LIF, small molecule CHIR99021 (3. Mu.M) and PD0325901 (0.2. Mu.M), and stored at 4℃after mixing.

Will be about 4 x 10 ⁵ Mouse Embryonic Fibroblasts (MEFs) were resuscitated and inoculated into a plate and cultured overnight.

The original MEF medium was replaced with the mouse chemistry reprogramming first Stage medium (Stage 1), fresh medium was replaced every 4 days, and culture was continued for 12 days.

On day 12, the medium was replaced with mouse chemistry reprogramming phase two medium (Stage 2), fresh medium was replaced every 4 days, and culture was continued for 12 days.

On day 24, the medium was replaced with mouse chemistry reprogramming phase three medium (Stage 3), fresh medium was replaced every 4 days, and culture continued for 12 to 16 days.

Finally, ipscs were purified and amplified by means of either subcloning or passaging.

TABLE 1 important small molecules of Stage 1

Small molecules	Target(s)
		VPA	Also known as Valproic Acid, HDAC inhibitors
CHIR99021	Selection ofSex GSK-3 alpha/beta inhibitors
		616452	Also known as RepSox, TGF-beta-RI/ALK 5 inhibitors
Forskolin	Adenylate cyclase activators
		AM580	Selective RARα agonists

Small molecule drug screening

Approximately 200 small molecule screens were performed in the first stage of chemical reprogramming. Mouse Embryonic Fibroblasts (MEFs) were inoculated into 24-well plates, one small molecule was added to each well, DMSO wells were set up as controls for each plate, cultured until day 12, sall4 immunostained, and the number of Sall4 positive clones per well was observed and counted under a fluorescent microscope.

Mouse transcription factor reprogramming

Mice Embryo Fibroblasts (MEFs) were transfected twice with viruses expressing Oct4, sox2, klf4 and Nanog and passaged into 24 well plates. After 24 hours, MEF medium was replaced with mouse transcription factor reprogramming medium, the fluid was changed every 4 days, and the number of GFP positive clones was counted on day 8 of reprogramming.

Immunostaining experiments

Removing culture medium by adhering cells, adding 4% Paraformaldehyde (PFA) to cover cells, and shaking at room temperature for 10min; PFA is removed, and the mixture is washed 3 times by PBST buffer solution for 5min each time; removing PBST, adding immunofluorescence staining sealing liquid, and treating at room temperature for 30-60min; removing the sealing liquid, adding a primary antibody (anti-Sall 4,1:1000; anti-Sox2,1:2000; anti-Oct4,1:500; anti-Nanog,1:500; anti-Tuj1,1:5000; anti-alpha-SMA, 1:2000; anti-Foxa2, 1:1000) diluted with the sealing liquid, and placing in a refrigerator at 4 ℃ for overnight treatment; recovering primary antibody, and washing cells by PBST for 3 times for 15min each time; adding a second antibody (also diluted with a blocking solution, 1:2000) diluent with corresponding resistance, placing the mixture in a shaking table, and performing light-shading treatment at room temperature for 1h; removing the secondary antibody, and cleaning the cells for 2 times by PBST for 10min each time, and performing light-shielding treatment; adding 1:5000 diluted Hoechst solution, placing in a shaking table, and treating in dark place for 10min; removing Hoechst diluent, cleaning cells for 2 times by PBST, and performing light-shielding treatment for 10min each time; cells were observed using a fluorescent inverted microscope.

Real-time fluorescent quantitative PCR (RT-qPCR)

Total RNA was isolated and purified using a total RNA extraction kit, and then reverse transcribed into cDNA using a reverse transcription kit. And performing real-time fluorescence quantitative PCR according to the real-time fluorescence quantitative PCR kit instruction. The primer sequences are listed in Table 2.

Plasmid construction and viral packaging

Rxra-, exosc3-, dis 3-and MMVL 30-targeting or control shRNA were ligated to the pLKO.1 vector. The Rxra CDS was amplified from the cDNA library and ligated with pSin vector. The plasmid was transfected into HEK293T cells containing PEI and the MEF medium was removed from the double antibody P/S. Viruses were collected and filtered 48 and 72 hours post-transfection.

Gene knockout and over-expression experiments in chemical reprogramming

Mouse Embryonic Fibroblasts (MEFs) were seeded onto 24-well plates and cultured in phase 1 media. On day 2, 5. Mu.g ml was used with shRNA virus or pSin-overexpressing virus ^-1 polybrene treated cells for 4 hours. On day 12, sall4 positive clones were counted as reads.

Embryoid body differentiation test

Embryoid Body (EB) differentiation medium: DMEM medium was added with 10% FBS,10% KSR,1 XNEAA solution, 1 XP/S diabody and 0.055mM beta-mercaptoethanol, and stored at 4℃after mixing.

Chemically induced pluripotent stem cells (ciscs) of mice were inoculated into a low-adsorption cell culture plate and cultured in embryoid-like body (EB) differentiation medium for 7 days to form suspension embryoid-like bodies, with liquid changes every 3 days. Then, the embryoid bodies are inoculated into a common culture plate for adherent culture in a ratio of 1:6, liquid is changed every 3 days, and immunofluorescence staining is carried out after about 2 weeks.

Sodium bisulfite sequencing method for detecting DNA methylation

Extracting genome DNA by using a genome extraction kit, modifying and purifying the obtained genome DNA by using a DNA methylation kit, amplifying the modified DNA by PCR, connecting a PCR product to a T vector, and sequencing and analyzing.

Chemically induced pluripotent stem cell (ciscs) karyotype analysis

In the invention, 7 th-generation iPSC cells are normally cultured to 80% density and sent to a prenatal diagnosis center of Hangzhou women and child health care department for nuclear detection.

Western blot (Western blot) experiment

Cells were treated with lysis buffer containing 1% phenylmethylsulfonyl fluoride (PMSF). The cell extract was then centrifuged and the supernatant collected. Cell lysates were separated on a 10% acrylamide gradient SDS-PAGE gel and transferred to NC membrane. The membranes were blocked with blocking solution for 2 hours, incubated overnight at 4℃with primary antibody (anti-Exosc 3,1:1000; anti-Dis3, 1:1000), then incubated with secondary antibody for 1 hour at room temperature, and finally immunoblotted with high sensitivity ECL chemiluminescent detection kit or high sensitivity ECL detection kit.

RNA sequencing analysis

The total RNA of the sample is extracted by using an RNA kit and is used for RNA sequencing. cDNA library construction and high throughput sequencing were performed by Beijing norelvan source technologies Co. Double-ended sequencing was performed using an Illumina HiSeq 2500 read length of 150 bp. Adapter sequences and reads of low sequencing quality in the raw data were first removed by software fastp (v0.20.1) [151], and aligned with the mouse genome (mm 10) using software HISAT2 (v2.1.0). The above procedure was the same for strand specific RNA-seqs (lncRNA-seq) data knockdown Exosc 3. Transcripts (including mRNAs and lncRNAs) were quantified by software stringtie (v 2.0) and their FPKM calculated. For analysis of transposon TEs, FPKM for each TE transcript was calculated and they were subjected to integration calculation. Finally, differential expression analysis was performed using the R-package DESeq2 (http:// www.bioconductor.org /) from the Bioconductor subject group.

GO and KEGG enrichment analysis was done by R package clusterifier from the Bioconductor topic group [155]. The subject chooses an adjusted P value of less than 0.05 as statistically significant.

Gene enrichment analysis (GSEA) and visualization were done with R-packets msigdbr and enrichplot from the Bioconductor topic group, respectively.

Targeted shear and transposase experiments (CUT & Tag)

The OG2 mouse embryonic fibroblasts were transfected with pSin-Flag-Rxra lentivirus 2 times and cultured continuously with the first stage medium reprogrammed in mouse chemistry for 12 days. The procedure was according to CUT & Tag kit instructions.

For CUT & Tag-qPCR experiments, related qPCR primers were designed first. And predicting possible binding sites of transcription factors in relevant gene promoter regions on a JASPAR website, and designing qPCR forward and reverse primers at sequences of 100bp before and after the binding sites. The amplified DNA was diluted 10-fold and subjected to RT-qPCR experiments.

The final DNA product of CUT & Tag was sent to Beijing Nodejingyuan technologies Inc., and double-ended sequencing was performed using an Illumina HiSeq 6000 read length of 150 bp. Raw data were processed using software fastp (v0.20.1) and data were matched to the mouse genome using software Bowtie2 (v2.2.5) [158]. The CUT & Tag peaks were then visualized using software IGV (v2.6.3) and deepTools (v3.4.3) [159]. The sequencing peaks were annotated with the R package ChIPseeker from the Bioconductor problem group.

Motif enrichment analysis was performed using the software HOMER (v 4.11).

The Cistrome database (http:// Cistrome. Org /) was used to predict the likelihood that transcription factors regulate a particular gene.

RNA immunoprecipitation experiments (RIP)

The procedure was performed according to the RIP kit instructions. The resulting RNA samples were reverse transcribed and the cDNA products were diluted 10-fold for real-time fluorescent quantitative PCR (RT-qPCR).

mRNA stability test

Samples from day 12 of reprogramming were treated with 10 μg/mL actinomycin D and cells were collected at different time points at 0h,4h and 8 h. Total RNA from the samples was extracted using a total RNA extraction kit and sent to the company for high throughput sequencing.

Flow cytometry analysis (FACS) experiments

The reprogrammed intermediate cells were dissociated into single cells using 0.25% trypsin. Cells were fixed with 4% Paraformaldehyde (PFA) at 4 ℃ for 30min and washed 3-5 times with PBST, then centrifuged at 1500rpm for 5 min. Next, cells were blocked with blocking buffer and incubated with dsRNA antibodies (1:200) overnight at 4 ℃. After 3 washes with PBST, cells were incubated in secondary antibody for 1 hour at room temperature.

Double-stranded RNA (dsRNA) detection assay

5. Mu.g total RNAs were extracted from the corresponding cells, diluted to 46. Mu.L with deionized water, added with 3.5. Mu.L of 5M sodium chloride solution, and thoroughly mixed to prepare the same 2-tube solution. To the 2 tubes, 0.5. Mu.L of 10mg/mL RNaseA enzyme solution and 0.5. Mu.L of deionized water were added as a blank group, respectively, and the total volume was 50. Mu.L, and the reaction was performed at 37℃for 30 minutes. The reaction products were purified using a PCR product purification kit according to the instructions. After purification, the obtained dsRNAs were subjected to denaturation at 95℃for 5min, and reverse transcribed into cDNA by a reverse transcription kit. The RNA transcript region of selected MMVL30 was measured by RT-qPCR with action as an internal reference. The enrichment fold of dsRNAs in MMVL30 region was calculated using the (retrotransposon/action) RNase-A/(retrotransposon/action) blank formula.

Example 1 screening small molecule CD3254 and retinoid X receptor alpha subtype (RXRalpha) were found to promote cytochemical reprogramming

Cells were treated with reprogramming phase 1 medium from day 0 to day 12 using OG2 Mouse Embryonic Fibroblasts (MEFs) as starting cells, and small molecules were screened using Sall4 positive clone numbers at phase 1 (fig. 1A). Interestingly, CD3254 reprogramming was most efficient in small molecules, which can be further demonstrated (FIGS. 1B-D). Next, the reprogramming process was extended to stage 2, and an increase in the number of Oct4-GFP positive clones was observed (FIGS. 1E and F). The optimal concentration of CD3254 was 0.5 μm, and furthermore, CD3254 acted primarily at stage1 (fig. 1G and H).

Using real-time quantitative PCR (RT-qPCR), CD3254 was observed to induce expression of early multipotent genes, including Sall4, cdh1, epcam and Lin28a (fig. 1I). From the screening results, other Retinoid X Receptor (RXR) agonists Bexarotene and SR11237 were also found to significantly enhance reprogramming (fig. 1A and B).

The first 5 compounds of the small molecule screen are: CD3254, UNC0646, bexarotene, SR11237, a-366. The molecular formula is shown in Table 2.

RXR antagonist HX531, on the other hand, can block the action of CD3254 (fig. 1C and D). Shortening Stage1 time, the greatest effect of CD3254 was found to be to increase reprogramming efficiency, rather than to accelerate the chemical reprogramming process (fig. 1E). The effect of CD3254 on mouse Transcription Factor (TFs) reprogramming was further tested and found that CD3254 failed to promote TF-induced reprogramming (fig. 1F).

In general, through chemical screening, a novel small molecule CD3254 is determined, which is a specific and effective retinoid X receptor alpha subtype (RXRalpha) agonist, can remarkably improve the efficiency of chemical reprogramming and is helpful for solving the problem of inefficiency of chemical reprogramming. Based on these results, the following study was focused on CD3254, which was used as an entry point to better understand chemical reprogramming.

After 12-16 days of further culture of the 24-day reprogramming intermediates, several Oct4 positive chemically induced pluripotent stem cells (ciPSC) lines were established and identified in detail (fig. 2A). These Oct4-GFP positive cells had typical mouse embryonic stem cell morphology (fig. 2B). Immunostaining showed that these cisscs expressed typical multipotential markers including Oct4, sox2 and Nanog (fig. 2C).

RT-qPCR showed stable expression of multipotent genes such as Oct4, sox2 and Nanog (FIG. 2D). Transcriptional analysis showed that DMSO-cisCs and CD 3254-cisCs were close to R1 mESCs, away from MEFs (FIG. 2E). Bisulphite sequencing analysis showed that Oct4 and Nanog promoters of these cisccs were largely demethylated, providing further evidence for successful epigenetic reconfiguration (fig. 2F). The results of the karyotyping analysis showed that these cisccs remained normal karyotypes (fig. 2G). All three germ layer cells, such as Tuj1 positive ectodermal cells, α -SMA positive mesodermal cells, and Foxa2 positive endodermal cells, were observed by embryoid body differentiation experiments (fig. 2H). Teratoma experiments showed that these ciPSCs can produce typical teratomas, which contain derivatives of all three germ layers, including ectoderm, mesoderm and endoderm (fig. 2I). Chimeric experiments showed that these cisscs could produce healthy mice (fig. 2J).

The above identification shows that these cisccs are similar in molecule and function to mescs, demonstrating the successful multipotent reprogramming by chemical means.

Table 2 first 5 Compounds and molecular formula in Small molecule screening

TABLE 3RXR antagonist HX531

Example 2 influence of CD3254 on the level of transcription of chemical reprogramming

To explore the molecular mechanism of CD3254 in chemical reprogramming, reprogrammed intermediate cells that were treated for 12 days with CD3254 were RNA sequenced. Sequencing data were better reproducible (fig. 2A). Next, differential gene expression analysis was performed on the two processed data. After CD3254 treatment, there were 1330 genes up-regulated, 1330 genes down-regulated (fold change >1.5 or <0.67, P-value after adjustment < 0.05) (fig. 3A and 2B).

Next, a Kyoto gene and genome encyclopedia (KEGG) pathway enrichment analysis was performed. The KEGG pathway in the up-regulated gene includes DNA replication, homologous recombination, spliceosome and RNA degradation (fig. 3B). Down-regulated KEGG pathways include steroid biosynthesis, MAPK signaling, adhesion and TGF- β signaling (fig. 3C). In particular, early multipotent genes (e.g., sall4, lin28a, esrrb, klf4, cMyc) and epithelial genes (e.g., cdh1, cldn4, tjp 3) were up-regulated, while mesenchymal genes (e.g., zeb1, twist1, snail 1) were down-regulated (fig. 3D-F). Down-regulation of key genes involved in TGF-beta signaling pathways (e.g., tgfb1, grem1, smad 3) was also observed (FIG. 3G). In addition, gene Ontology (GO) analysis shows up-regulation terms including stem cell population maintenance, chromatin organization, RNA metabolic processes and DNA replication. The down-regulated GO pathways include extracellular matrix, epithelial to mesenchymal transition, TGF- β and MAPK signaling pathways (fig. 3H).

The results of the Gene Set Enrichment Analysis (GSEA) further suggested that ribosomes, spliceosomes, DNA repair, upregulation of pyrimidine metabolism, lysosomes, cytokine-cytokine receptor interactions, downregulation of hedgehog signaling pathways (fig. 2C).

Overall, CD3254 treatment can facilitate the reconfiguration of gene regulatory networks, accelerating MET and pluripotency induction.

Example 3 retinoid X receptor alpha subtype (rxrα) overexpression significantly facilitates chemical reprogramming

To further analyze whether CD3254 facilitates chemical reprogramming of mice via its cognate target rxrα, the function of Rxra knockdown and overexpression was tested (fig. 4A and B). shRNA knockdown Rxra inhibited Sall4 positive clone numbers, as well as expression of early pluripotency marker genes, such as Sall4, cdh1, epcam, and Lin28a (fig. 4C and D).

Unexpectedly, the effect of Rxra overexpression was very pronounced, increasing the number of Sall4 positive clones by about 10-fold, further indicating that the CD3254-RXR alpha axis plays a key role in chemical reprogramming (FIG. 4E-G). Furthermore, rxra overexpression was found not to promote TF-induced reprogramming (fig. 1F).

Likewise, using RNA-seq, the change in gene expression of Rxra overexpression was assessed. After Rxra overexpression, there were a total of 1953 genes up-regulated, 2147 genes down-regulated (fold change >1.5 or <0.67, P-value after adjustment < 0.05) (fig. 5A, fig. 3A and B). Upregulation classes included stem cell population maintenance, chromatin remodeling, DNA replication, RNA degradation, and mRNA monitoring pathways (fig. 5B and 3D).

On the other hand, down-regulated genes are among lysosomes, adhesion, MAPK signaling pathway, TGF- β signaling pathway, autophagy and hypoxia response (fig. 3C and D). Similar to CD3254 treatment, early multipotent and epithelial genes were found to be up-regulated, while mesenchymal genes and key genes involved in TGF- β signaling were down-regulated (fig. 3F). GSEA further found up-regulation of homologous recombination, cytokine-cytokine receptor interactions, lysosomes, down-regulation of chemokine signaling pathways (fig. 3E).

Overall, rxra overexpression also enhances (even more efficient than CD 3254) the rearrangement of the gene regulatory network from the somatic state to the pluripotent state.

EXAMPLE 4CD3254-RXR alpha axis direct transcription up-regulates RNA exons

Integrating the RNA-seq data of CD3254 treatment and Rxra overexpression, it was found that the exosome component genes were significantly induced. An exosome is a complex of 11 total subunits and is involved in RNA degradation (fig. 5B and C). The role of exosomes in cell reprogramming has not been described, and remains largely unknown. As shown in FIG. 5D, both CD3254 treatment and Rxra overexpression increased the expression of all 11 subunits of the exosomes, including Exosc1-10 and Dis3. Further confirmation of CD3254 treatment and Rxra overexpression was performed using real-time fluorescent quantitative PCR (RT-qPCR) with upregulation of Exosc3, exosc7, exosc8 and Dis3 (fig. 5E and F). In addition, both CD3254 treatment and Rxra overexpression enhanced Exosc3 and Dis3 protein levels (fig. 3G).

The Transcription Factor (TF) regulatory potential index analysis was performed on the exosome component genes using the cistome database, and retinoid X receptor alpha subtype (rxrα) was identified as predictive upstream regulatory TF of the exosome component genes (fig. 4A and B). To determine if the exo-RNA subunit gene is a direct downstream target of rxrα, CUT & Tag experiments were performed. In fact, it was observed that rxrα could bind directly to the genomic regions of all 11 exosome constituent genes (fig. 5G and H and fig. 4C). Next, direct binding of RXR alpha to the genomic regions of the exosome component genes Exosc3 and Dis3 was further confirmed by CUT & Tag-qPCR assay (FIGS. 5I and J).

Taken together, the present invention suggests that rxrα is a novel direct upstream regulator of all constituent genes of the exosomes.

Example 5 exosomes are necessary for chemical reprogramming

Next, the function of the exosomes in chemical reprogramming was tested. Exosomes were knocked down using shRNA as core component Exosc3 (fig. 6A and B). Exosc3 knockdown was found to reduce the number of Sall4 positive clones and the expression of the early multipotent genes Sall4, cdh1, epcam and Lin28a (FIGS. 6C and D). However, exosc3 knockdown had no effect on transcription factor-induced reprogramming, indicating that the CD3254-rxrα -exosome signaling axis was not involved in transcription factor-induced reprogramming (fig. 1F). In addition, another key component of the knockdown exorna, dis3, exhibited a similar phenotype (fig. 5A-D). Using RNA-seq, the change in gene expression after extoc 3 knockdown was evaluated. After extoc 3 knockdown, there were a total of 314 genes up-regulated, 1093 genes down-regulated (fold change >1.5 or <0.67, P-value after adjustment < 0.05) (fig. 6E and F). Up-regulated genes belong to ECM-receptor interactions, responses to injury, and cytokine-cytokine receptor interactions (fig. 6G and I). On the other hand, down-regulated pathways include DNA replication, chromatin tissue, glycine, serine and threonine metabolism, and signaling pathways that regulate stem cell pluripotency (fig. 6H and I).

In conclusion, it was determined that exornas are novel upregulators of chemical reprogramming.

Example 6 exosomes primarily target transposon RNAs for degradation

Exons are an RNA degradation complex, but the overall number of down-regulated mRNAs is much higher after Exosc3 knockdown (314 up-regulated vs 1093 down). This phenomenon is also different from previous observations in other biological systems. In addition to mRNAs, non-coding RNAs (ncRNAs), including long non-coding RNAs (lncRNAs), enhancer RNAs (eRNAs), promoter-associated RNAs (paRNAs) and transposon RNAs (repeat RNAs), have also been demonstrated to be substrates for exosomes. To investigate the effect of exosomes on ncRNAs, the expression changes of lncRNA, enona and paRNA at Exosc3 knockdown were quantified. Overall, the expression levels of these ncRNAs were on a decreasing trend in shoxosc 3, similar to mRNA (fig. 6A, E and F). Furthermore, it was observed that the expression pattern of some lncRNAs was correlated with the expression pattern of neighboring genes, indicating the modulation of expression of lncRNAs on their neighboring genes. For example, after Exosc3 knockdown, expression of Gm14261 upstream of Sall4 was decreased, while expression of Gm10638 downstream of Siah1a was increased (fig. 6B-D).

Unexpectedly, expression of the repeat RNA increased significantly following Exosc3 knockdown, indicating degradation of the exosomes primarily targeting the repeat RNAs during reprogramming (FIG. 7A). Most differentially expressed repeat RNAs were found to be up-regulated (n=3403) while only a few were down-regulated (n=35) (fig. 7B). Increased repeat RNAs include most transposon classes, such as Endogenous Retroviruses (ERV) containing Long Terminal Repeats (LTRs) and non-LTR elements, including long and short interspersed nuclear elements (LINE) (figure 7C). Interestingly, by precisely studying the expression changes of the repeat RNAs, 8 of the first 10 upregulated repeat RNAs (arranged in log2 fold changes and P values) were found to be MMVL30-int belonging to the VL30 ERV1 family (FIGS. 7B and C). Furthermore, the proportion of upregulated MMVL30-int transposons is significantly higher than that of all expressed TEs, which means that MMVL30-int is the main target of the exorna (fig. 7D). To further confirm this hypothesis, changes in expression of repetitive RNAs following CD3254 treatment and Rxra overexpression were studied. Likewise, MMVL30-int is significantly enriched in down-regulated TEs (fig. 6G and 6H). Comprehensive analysis of these RNA-seq datasets showed that 13 MMVL30-int transposons were knocked down by Exosc3, CD3254 treatment and modulation of Rxra overexpression (fig. 7E). The expression pattern of these transposons showed different patterns in CD3254 treatment, rxra overexpression and Exosc3 knockdown, which means that the exosomes were indeed responsible for MMVL30-int transcript degradation (fig. 7F and G).

RT-qPCR confirmed the down-regulation of MMVL30 expression by CD3254 treatment and Rxra overexpression, and the up-regulation of MMVL30 expression by Exosc3 knockdown (FIG. 7H). In addition, exosc3 RNA immunoprecipitation was performed using RIP-qPCR, an RNA immunoprecipitation assay, and it was verified that Exosc3 protein could bind directly to MMVL30 transcript (FIG. 7I). Next, RNA-seq data of the reprogrammed intermediate treated with the transcription inhibitor actinomycin D (Act D) were analyzed and collected at 0h,4h and 8h, respectively. The stability of MMVL30-int was found to be significantly reduced in CD3254 treated cells (fig. 7J). Functionally, MMVL30 knockdown was found to increase the number of Sall4 positive clones (FIGS. 7K and 7A-C) and promote induction of early multipotent genes, including Sall4, cdh1, epcam and Lin28a (FIG. 7D). Thus, MMVL30 has proven to be a new obstacle to chemical reprogramming.

Overall, by further data exploration, CD3254-rxrα -exo-RNA axes were found to promote degradation of transposon RNAs (predominantly MMVL 30) during cell fate switching.

Example 7 exornas mediate transposable element degradation and attenuate inflammatory response to facilitate chemical reprogramming

Recent studies have shown that accumulation of MMVL30 transcripts can stimulate cytoplasmic nucleotide sensing pathways, followed by stimulation of interferon responses that play a role in tumorigenesis, development and tissue repair. However, the role of transposons in cell reprogramming remains difficult to determine. Therefore, it was decided to further investigate the consequences of imbalanced MMVL30 during chemical reprogramming. CD3254 treatment was the same as in example 1.

Overall, it was found that upon Exosc3 knockdown, many ERVs were induced in the form of cis and trans transcripts (fig. 8A). These up-regulated ERVs, including MMVL30-int, are expressed in both forward and reverse directions and can form double stranded RNAs (dsRNAs) (FIG. 8B). Indeed, fluorescence Activated Cell Sorting (FACS) analysis found that CD3254 treatment can reduce overall dsRNAs levels (fig. 8C). A significant reduction in dsRNAs formed by the MMVL30 region was also observed (fig. 8D). Accumulation of dsRNAs can trigger inflammatory and immune responses. Indeed, several inflammation-associated genes were observed, including Ifngr1, tnfrsf1a and Tnfrsf10b, were down-regulated in CD3254 treated, rxra over-expressed and MMVL30 knockdown samples, but up-regulated in Exosc3 knockdown samples (fig. 8E). To further test the effect of IFN-gamma and TNF-alpha pathways, IFN-gamma and TNF-alpha were added during reprogramming, which were found to indeed reduce the number of Sall4 positive clones (FIG. 8F). Meanwhile, the expression of the early multipotent gene Sall4 and the epithelial gene Cdh1 is reduced, and the expression of the mesenchymal gene Zeb is increased. On the other hand, the JAK inhibitors upadacrinib and IKK inhibitor BMS-345541 may promote reprogramming (fig. 8H and I). The relationship between inflammation and cell fate depends on the environment. Preliminary studies have shown that activation of TLR3 or IL6 mediated innate immunity is necessary for efficient multipotent reprogramming. Later, some studies showed that IFN-gamma-or TRAIL-mediated inflammation is a barrier to transcription factor induced pluripotency. Here, studies have shown that IFN-gamma and TNF-alpha pathways are obstacles to chemical reprogramming, which has not been reported before.

Taken together, by no preference for chemical screening, rxrα specific agonist CD3254 was found to significantly promote chemical reprogramming. Further mechanism studies suggest that the CD3254-RXR alpha axis can transcriptionally activate all 11 components of the exosome (Exosc 1-10 and Dis 3). Upon further analysis of the relevant data, it was observed that the exosomes predominantly regulate the degradation of transposon TEs (predominantly MMVL 30-int) and reduce inflammation (e.g. IFN- γ and TNF- α pathways).

Notably, these studies provided new insights about cell fate transitions and better strategies to control cell fate (fig. 8J).

Discussion of the invention

Since the 2006 Yamanaka factor was reported, the field of cell reprogramming has been vigorously developed, with significant achievements, and is expected to be used for exploring disease mechanisms and enhancing disease treatments. Currently, the contribution of somatic reprogramming technology to disease modeling, cell transplantation, drug screening, and the like has been widely accepted. Combining with new technologies such as direct cell reprogramming and gene editing, the iPSC technology will contribute to future personalized, predictive and accurate therapies. Compared with exogenous transcription factors which suddenly force cells to undergo fate transition, the chemical micromolecules can reduce manual operation by triggering endogenous innate mechanisms of the cells, and the clinical prospect is wider. However, chemical reprogramming still has 2 fundamental problems of inefficiency and unknown exact molecular mechanisms, which limit its true clinical progress.

In order to try to solve the current problems in the chemical reprogramming field, the invention firstly establishes a mouse somatic cell chemical reprogramming system and performs no-preference compound screening. Through screening of more than 200 compounds, the RXR alpha activator CD3254 and the RXR activators Bexarotene and SR11237 of the same type can be found to effectively improve the reprogramming efficiency, wherein the CD3254 promoting effect is most remarkable, and the promoting effect of the CD3254 on reprogramming is not reported before.

The invention takes CD3254 as an access point to study the reprogramming mechanism. In combination with CD3254 treatment and 2 sets of RNA-seq data over-expressing Rxra, it was found that both 2 treatments could facilitate the reconfiguration of the gene regulatory network, accelerating MET and pluripotency induction. Furthermore, it was found that both CD3254 treatment and up-regulated differential gene over-expression of Rxra were enriched for the pathway of RNA degradation by KEGG pathway enrichment analysis. Through further RT-qPCR and CUT & Tag experiments, it was determined that the CD3254-RXR alpha axis was able to transcriptionally activate all 11 genes of the exosome complex (Exosc 1-10 and Dis 3). The exoRNA body plays an important role in an RNA monitoring system, ensures that the steady-state balance of RNA in cells is accurately controlled in a space-time regulation mode, and ensures normal development. It is highly advantageous to induce exons by non-integrated small molecules rather than genetic means. Testing whether CD 3254-activating exosomes can be used in other biological systems, such as injury and cancer models, is advantageous for further development of drugs for CD3254 as a variety of diseases.

In the prior art, exosomes were studied using mRNAs as their primary substrates. However, in the present invention, it was found that the RNA, including mRNAs, lncRNAs, eRNAs and paRNAs, knockdown the exosome key gene Exosc3, showed a decreasing overall transcript level, which was inconsistent with expectations. Through deeper bioinformatics analysis, it was found that exons of the invention mainly regulate the degradation of transposon TEs, most of which belong to the MMVL30 family. Further functional experiments demonstrated that MMVL30 is a new obstacle to cell reprogramming.

In addition, the decrease in MMVL 30-mediated inflammatory responses (IFN-gamma and TNF-alpha pathways) helps to promote chemical reprogramming. Several studies have reported a relationship between inflammatory response and cell fate before this time. For example, preliminary studies have shown that TLR 3-mediated activation of innate immunity is essential for efficient multipotent reprogramming. Thereafter, two other efforts have shown that injury-induced inflammation and aging can also regulate reprogramming in vivo. Unlike these studies, inflammatory responses were found to be an obstacle to chemical reprogramming of mice. Thus, the role of the inflammatory response in the cell fate transition depends on the environment in which it is located.

In summary, the present invention not only finds new small molecules for improving chemical reprogramming techniques, but also finds new mechanisms that facilitate further exploration in the iPSC field.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. Use of an active ingredient, characterized in that the active ingredient comprises an agent of an RXRa activator, for the preparation of a composition or formulation for:

activating the exorna complex;

promoting Sall4 gene expression;

promoting cell reprogramming;

Treating or reducing inflammatory response.

2. A method of screening for potential compounds that promote the formation of induced pluripotent stem cells (ipscs) in a mammalian non-pluripotent cell, comprising:

3. The method of claim 2, wherein the transposon RNA is of the VL30 ERV1 family; preferably MMVL30.

4. The method of claim 3, wherein the compound that promotes the transformation of mammalian non-pluripotent cells to induced pluripotent stem cells is a rxrα -specific agonist; preferably CD3452 or CD 3452-like compounds.

5. A method of screening for a compound that promotes up-regulation of the Sall4 gene in a non-pluripotent mammalian cell, comprising:

6. A compound having CD3254 activity obtainable by the screening method of claim 1.

7. A method of inducing pluripotent stem cells from mammalian non-pluripotent cell culture, wherein the mammalian non-pluripotent cells are cultured under conditions suitable for growth of the cells and in the presence of an RXRa activator to obtain induced pluripotent stem cells.

8. A pharmaceutical composition comprising an active ingredient, and a pharmaceutically acceptable carrier, wherein the active ingredient comprises a RXRa activator and one or more agents selected from the group consisting of:

(i) HDAC inhibitors;

(ii) GSK-3 alpha/beta inhibitors;

(iii) TGF-beta-RI/ALK 5 inhibitors;

(iv) An adenylate cyclase activator;

(v) Rarα agonists.

9. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition is for

Activating the exorna complex;

promoting Sall4 gene expression;

promoting cell reprogramming;

Treating or reducing inflammatory response.

10. A kit for promoting reprogramming of cells, comprising

(1) RXRA activator reagent, and

(2) One or more agents selected from the group consisting of:

(i) HDAC inhibitors;

(ii) GSK-3 alpha/beta inhibitors;

(iii) TGF-beta-RI/ALK 5 inhibitors;

(iv) An adenylate cyclase activator; and

(v) Rarα agonists.