CN112680407B - Compound for promoting muscle stem cell differentiation and application thereof - Google Patents

Abstract

The invention provides a compound for promoting muscle stem cell differentiation and application thereof; the invention also provides a multi-gene knockout experimental model for preparing the type 1 myotonic dystrophy (DM 1), a preparation method and application thereof. The experimental model of the invention can be used for researching type 1 myotonic dystrophy and can be used for screening and testing tests of specific medicines, gene therapy or other treatment schemes. The small molecular compound for promoting the muscle stem cell differentiation function provides a new way for developing a medicine for treating DM1 diseases.

Description

Compounds for promoting differentiation of muscle stem cells and their application

Technical Field

The present invention belongs to the field of cell biology and pharmacy, and more specifically, the present invention relates to a compound for promoting differentiation of muscle stem cells and application thereof.

Background Art

Myotonic dystrophy (DM) is a dominant hereditary disease characterized by muscle stiffness, weakness and atrophy, with multiple organ involvement. It is mainly divided into two types: myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2).

Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy, which can cause multi-system lesions. The disease is caused by abnormal amplification of CTG trinucleotides at the 3' end of the myotonic dystrophy protein kinase (DMPK) gene, but the molecular mechanism of abnormal amplification of CTG causing lesions is still unclear. DM1 is generally more severe in distal muscle phenotypes. Compared with DM2 patients, the phenotype of DM1 patients is more serious, and some patients will experience sudden death as the disease progresses. The present invention only relates to the DM1 disease model.

The organs and tissues that are affected by DM1 disease include muscles (skeletal muscles), heart (myocardium), eyes, brain, digestive system (smooth muscle) and endocrine system. In order to further deepen the understanding of the disease and find an effective cure. Scientists have constructed some DM1 disease mouse models. First of all, since the onset of DM1 disease is caused by the abnormal extension of the CTG sequence at the 3' end of the DMPK gene, the mutant RNA of DMPK cannot be normally transported to the cytoplasm, resulting in a decrease in its protein expression level. The mRNA of DMPK aggregates in the nucleus and cannot complete the RNA-to-protein coding. Therefore, it was first speculated that the DMPK gene is responsible for the occurrence of the disease. Subsequently, a laboratory constructed a DMPK knockout mouse model and found that the DMPK knockout mouse could not simulate the occurrence of the disease well. It would show phenotypes such as muscle fiber degeneration, increased cross-sectional size differences and weakened muscle strength, and some more typical DM1 phenotypes did not appear. This shows that the occurrence of the disease is not entirely caused by abnormal expression of the DMPK gene.

By testing the patient's muscle tissue, it was found that the abnormal lengthening of the CTG sequence at the 3' end of the DMPK gene not only affected the expression of DMPK, but also the expression of the upstream gene SIX5 and the downstream gene DMWD of the DMPK gene, which were reduced to a certain extent. At the same time, it was also found that the CUG repeat sequence transcribed by the mutated DMPK would form a neck ring structure, which could recruit some RNA splicing proteins, such as the Muscleblind-like family (MBNL family, including three genes MBNL1, MBNL2, and MBNL3) and CUGBP1, and then form aggregation points in the cell nucleus. After a large number of splicing proteins were "ineffectively recruited", the amount of RNA splicing proteins that could function normally in the cell was reduced, so a large number of abnormally spliced proteins would accumulate in the cell, thereby accelerating the occurrence and development of the disease. Among them, the mRNA of genes such as Clcn1 in muscle tissue showed abnormal splicing, abnormal muscle contraction and excitement, leading to the generation of stiffness, and the mRNA splicing of genes such as Ldb3 in heart tissue was abnormal, resulting in phenotypes such as arrhythmia. In response to these phenomena, two laboratories first constructed SIX5 knockout mice in 2000. Cataract phenotypes appeared in the knockout mice. The phenotype of homozygous knockout was more severe than that of heterozygous knockout. Different genetic backgrounds of mice also affected the occurrence of the disease. In 2004, Sita Reddy et al. analyzed the phenotype of SIX5 knockout and found that SIX5 knockout male mice were infertile, which was similar to male infertility in DM1 disease. Professor Charles A. Thornton of the University of Rochester and others added about 250 CTG repeats to the 3' non-coding region of the HSA gene expression fragment and constructed a transgenic mouse model. They found that transgenic mice had DM1 disease-related phenotypes, including myotonia and muscular atrophy. Pathological sections also found muscle nuclear intranslocation, sarcoplasmic masses and ring-like muscle fibers. However, many phenotypes such as heart block and cataracts did not appear.

Since there are a large number of abnormally spliced RNAs in mature muscles. In 2003, Professor Maurice S. Swanson's laboratory constructed MBNL1 knockout mice. MBNL1 homozygous knockout mice showed phenotypes such as muscle stiffness and cataracts, and many genes including Clcn1, Tnnt2, and Tnnt3 were detected to have abnormal RNA splicing, which is similar to clinical data. Many nuclear intrusion phenomena were also observed in muscle pathological sections. In 2008, Fabian Chen et al. constructed MBNL2 knockout mice again, and observed the occurrence of rickets during mouse development. At the same time, muscle pathological sections also showed nuclear intrusion, smaller muscle fiber cross-sections, and abnormal splicing of some RNAs. In 2012, Professor Maurice S. Swanson et al. found that the brain development of MBNL2 knockout mice was abnormal, and there were a large number of RNA splicing abnormalities, which are similar to some phenotypes of DM1 disease. In 2013, Professor Maurice S. Swanson's laboratory knocked out both MBNL1 and MBNL2, and the phenotype of the mutant mice was worse than that of a single gene mutation. In 2016, Seta Reddy et al. analyzed MBNL3 knockout mice and found that the mice had abnormal glucose metabolism and heart function.

Although many disease-related mouse models have been constructed, these models can only partially simulate the occurrence of the disease. No model can fully display the patient's manifestations. At the same time, clinical data show that the genes involved are not completely devoid of functional proteins. Therefore, the phenotype of homozygous knockout cannot well explain the actual situation of the disease.

In summary, this field still needs to establish more ideal animal models that are closer to clinical characteristics, and to screen and obtain useful drugs based on such models.

Summary of the invention

The purpose of the present invention is to provide a compound for promoting the differentiation of muscle stem cells and its application.

The present invention also aims to provide a multi-gene knockout experimental model for myotonic dystrophy type 1 (DM1), a preparation method thereof and its application. The experimental model includes an animal model and a cell model.

In the first aspect of the present invention, there is provided a use of a compound having a parent core structure as represented by formula (I) or an isomer, derivative, solvate or precursor thereof, or a pharmaceutically acceptable salt thereof, for preparing a composition for promoting muscle stem cell differentiation, or for preparing a composition for alleviating or treating a disease associated with abnormal muscle stem cell differentiation;

In a preferred embodiment, the solvate is a hydrate.

In another preferred embodiment, the composition is a pharmaceutical composition.

In another preferred embodiment, the composition is a culture medium composition.

In another preferred embodiment, the abnormal differentiation of muscle stem cells includes: decreased differentiation ability of muscle stem cells or no differentiation.

In another preferred embodiment, diseases associated with abnormal muscle stem cell differentiation include: sarcopenia, muscle aging or muscular dystrophy; preferably, the muscular dystrophy includes myotonic dystrophy type 1.

In another preferred embodiment, there is at least one substituent R on the phenyl group of the compound of the core structure represented by formula (I), and R is independently selected from the group consisting of hydrogen, C1-C4 alkyl, hydroxyl, C2-C4 alkenyl, C2-C4 alkynyl, and halogen.

In another preferred embodiment, R is independently selected from: hydrogen, hydroxyl, C1-C2 alkyl.

In another preferred embodiment, the compound is:

In another aspect of the present invention, a pharmaceutical composition or culture medium composition for promoting muscle stem cell differentiation is provided, which contains a compound with a core structure shown in formula (I) or its isomers, derivatives, solvates or precursors, or pharmaceutically acceptable salts thereof as an active ingredient, and a pharmaceutically or food acceptable carrier.

In a preferred embodiment, there is at least one substituent R on the phenyl group of the compound of the core structure represented by formula (I), and R is independently selected from: hydrogen, C1-C4 alkyl, hydroxyl, C2-C4 alkenyl, C2-C4 alkynyl, halogen; preferably, R is independently selected from: hydrogen, hydroxyl, C1-C2 alkyl; more preferably, the compound is:

In another aspect of the present invention, a method for preparing a composition for promoting muscle stem cell differentiation or alleviating or treating diseases related to abnormal muscle stem cell differentiation is provided, comprising: mixing a compound having a parent core structure shown in formula (I) or its isomers, derivatives, solvates or precursors, or pharmaceutically acceptable salts thereof with a pharmaceutically or food acceptable carrier.

In another aspect of the present invention, a method for promoting muscle stem cell differentiation is provided, comprising: treating muscle stem cells with a compound having a core structure as shown in formula (I) or its isomers, derivatives, solvates or precursors, or pharmaceutically acceptable salts thereof.

In another preferred embodiment, the method is an in vitro method.

In another preferred embodiment, the method is not a method for therapeutic purposes.

In another preferred embodiment, the method is a method for promoting the differentiation of muscle stem cells in muscle stem cell culture.

In another preferred embodiment, there is at least one substituent R on the phenyl group of the compound of the core structure represented by formula (I), and R is independently selected from: hydrogen, C1-C4 alkyl, hydroxyl, C2-C4 alkenyl, C2-C4 alkynyl, halogen; preferably, R is independently selected from: hydrogen, hydroxyl, C1-C2 alkyl; more preferably, the compound is

In another aspect of the present invention, a method for preparing an animal model of myotonic dystrophy type 1 is provided, comprising: modifying the Dmpk, Six5, Mbnl1 and Dmwd genes of the animal so that these genes exhibit the phenotypes of Dmpk+/-, Six5+/-, Mbnl1+/- and Dmwd+/-.

In a preferred embodiment, the animals include: rodents and non-human primates; preferably, the rodents include mice (such as mice or rats).

In another preferred embodiment, the non-human primate includes a macaque or a marmoset.

In another preferred embodiment, the modification includes: gene knockout; preferably, includes knockout using CRISPR gene editing method.

In another preferred embodiment, the animal is derived from haploid embryonic stem cells; preferably, by combining haploid technology with CRISPR technology, an animal model exhibiting Dmpk+/-, Six5+/-, Mbnl1+/- and Dmwd+/- phenotypes can be obtained in a one-step method.

In another aspect of the present invention, a method for preparing an animal cell model is provided, comprising: modifying the Dmpk, Six5, Mbnl1 and Dmwd genes in the cells so that these genes present the phenotypes of Dmpk+/-, Six5+/-, Mbnl1+/- and Dmwd+/-.

In a preferred embodiment, the cells are muscle cells.

In another preferred embodiment, the cells are separated and isolated cells.

In another preferred embodiment, the cell comprises a cell culture.

In another aspect of the present invention, a method for screening substances (including potential substances) that promote muscle stem cell differentiation is provided, the method comprising: (1) treating cells or cell cultures having Dmpk+/-, Six5+/-, Mbnl1+/- and Dmwd+/- phenotypes in their genomes with candidate substances; and (2) detecting the differentiation of cells having Dmpk+/-, Six5+/-, Mbnl1+/- and Dmwd+/- phenotypes; if the candidate substance statistically promotes the differentiation of the cells, it indicates that the candidate substance is a potential substance that promotes muscle stem cell differentiation.

In a preferred embodiment, step (1) includes: in the test group, adding the candidate substance to cells or cell cultures with Dmpk+/-, Six5+/-, Mbnl1+/- and Dmwd+/- phenotypes; and/or step (2) includes: detecting the differentiation of cells in the test group system and comparing it with that of the control group, wherein the control group is cells or cell cultures with Dmpk+/-, Six5+/-, Mbnl1+/- and Dmwd+/- phenotypes to which the candidate substance is not added; if the differentiation efficiency of the cells in the test group is significantly higher than that in the control group, it indicates that the candidate substance is a potential substance for promoting muscle stem cell differentiation.

In another preferred embodiment, the candidate substance is a small molecule compound, an interfering molecule, a gene editing reagent, a small RNA, etc.

In another aspect of the present invention, a method for screening substances (including potential substances) for alleviating or treating myotonic dystrophy type 1 is provided, comprising: (1) treating an animal model having Dmpk+/-, Six5+/-, Mbnl1+/- and Dmwd+/- phenotypes in its genome with a candidate substance; and (2) detecting morphological or symptom changes presented by the animal model of (1); if the candidate substance statistically improves the animal's myotonic dystrophy type 1, it indicates that the candidate substance is a substance for alleviating or treating myotonic dystrophy type 1.

Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. DSM-TKO semi-cloned mice and skeletal muscle-related phenotype analysis. a, Schematic diagram of DSM-TKO semi-cloned mice; b, DSM-AGH cell line; c, postnatal growth curve of mice. d, DSM-TKO semi-cloned mice showed ricket phenotype; e, EMG detection; f, treadmill test statistics of mice; g, front paw grip analysis of mice; h, Rotarod analysis of mice; i, dystrophin staining, intact muscle structure; j, H&E tissue staining of tibialis anterior muscle; k, cross-sectional statistics of muscle fibers of semi-cloned mice.

Figure 2. CDM1 respiratory system-related phenotypes of DSMD-QKO semi-cloned mice. a, DSMD-QKO-DAH cell line. b, Some DSMD-QKO semi-cloned mice died at birth; c, Some newborn DSMD-QKO semi-cloned mice sank to the bottom of the water. d, H&E staining of mouse diaphragm (P1); e, statistics of diaphragm muscle fiber cross-sections; f, staining and statistics of diaphragm neuromuscular junctions.

Figure 3. Other phenotypes associated with CDM1. a-b, DSMD-QKO semi-cloned mouse fetuses grow slowly during subsequent feeding. c, DSMD-QKO semi-cloned mice have abnormal metatarsal development (P1). d, H&E staining of muscles (P2). e, DSMD-QKO semi-cloned mice have obvious developmental delay (P5). f, muscle tension analysis of DSMD-QKO semi-cloned mice. g, H&E staining of the heart.

Figure 4. Phenotypes of DSMD-QKO semi-cloned mice in adulthood. a. DSMD-QKO semi-cloned mice show ankylosing phenotype. b. Statistics of treadmill test of mice; c. Statistics of forepaw grip test of mice; d. Statistics of Rotarod test of mice; e. No difference in dystrophin staining; f. H&E staining of tibialis anterior muscle; g. Statistics of muscle fiber cross-section; h. ATPase staining of tibialis anterior muscle and analysis of type I muscle fibers; i. Cataract phenotype.

Figure 5. Phenotypes of MuSCs in DSM-TKO and DSMD-QKO. a, Immunofluorescence of Pax7 protein in tibialis anterior muscle. b, Stem cell statistics after Pax7 staining in tibialis anterior muscle. c, MuSCs proliferation assay in vitro. d, MuSCs differentiated in vitro for 2 days. e, MuSCs differentiated in vitro for 2 days.

Figure 6. Expression of muscle-related genes during differentiation. a, Heat map of transcriptome sequencing of muscle-related genes. b, qRT-PCR detection of muscle differentiation-related genes.

Figure 7. MuSCs in vitro differentiation system combined with small molecule library. a, screening process; b, compound structure; c, small molecule compounds promoted the differentiation of DSM-TKO muscle stem cells.

DETAILED DESCRIPTION

After in-depth research, the inventors have combined semi-cloning technology mediated by haploid embryonic stem cells and gene editing technology to identify some genes closely related to the occurrence and development of myotonic dystrophy type 1 (DM1), which are Dmpk, Six5, Mbnl1 and Dmwd genes. The inventors have modified them to obtain animals or cells showing Dmpk+/-, Six5+/-, Mbnl1+/- and Dmwd+/- phenotypes, and the animals show a typical phenotype of myotonic dystrophy type 1. The animal model can be used for the study of myotonic dystrophy type 1, and can be used for the screening and testing of specific drugs, gene therapy or other treatment options.

Using the constructed cell model, the inventors also screened a small molecule compound that has the function of promoting muscle stem cell differentiation, providing a new approach for the development of drugs for the treatment of DM1 disease.

Model and its construction

The inventors have conducted genetic research at the gene level and after repeated research and demonstration, have identified a series of pathogenic genes closely related to DM1 disease, and prepared a DM1 experimental model by modifying the target genes. The experimental model includes an animal model or a cell model.

It has been found in previous work that mice with single knockout of genes such as Dmpk, Six5, Mbnl1 and Dmwd cannot simulate the typical symptoms of DM1. Therefore, the inventors speculate that DM1 is caused by abnormal expression of multiple genes caused by CTG repeats. In order to verify this hypothesis, the inventors used semi-cloning technology mediated by haploid embryonic stem cells to knock out Dmpk, Six5, Mbnl1 and Dmwd in haploid cells, and then obtained heterozygous mice carrying different combinations of mutations in one step by injecting them into oocytes, and then analyzed the phenotype to obtain an ideal mouse model that can simulate DM1. Through phenotypic analysis, the inventors found that the model mouse can well simulate the relevant symptoms of DM1 disease. In order to further explore its pathogenesis, the inventors also isolated the corresponding muscle stem cells to provide tools for in-depth exploration of related mechanisms.

According to the new discovery of the present invention, the present invention provides a method for preparing a DM1 disease experimental model, which comprises: genetically modifying the experimental subject so that the subject has the phenotype of Dmpk+/-, Six5+/-, Mbnl1+/- and Dmwd+/-.

The idea of preparing the experimental model can be applied to the preparation of cell models and animal models. The cell model includes a mammalian cell model or other eukaryotic cell models, such as human cells or mouse cells. The animals include but are not limited to: rodents (including rats, mice, hamsters, etc.), non-human primates (including crab monkeys, macaques, marmosets, etc.) and other model animals. Preferably, the animal is a rodent, including but not limited to: rats and mice. It should be understood that the core of the protection of the present invention lies in the model design and implementation strategy of the correlation between Dmpk, Six5, Mbnl1 and Dmwd gene loss-of-function mutations leading to DM1 disease, including but not limited to the schemes recorded in the subsequent embodiments of the present invention.

A variety of methods can be used to form Dmpk+/-, Six5+/-, Mbnl1+/- and Dmwd+/- phenotypes in animals or cells, including: gene knockout by stem cell targeting (homologous recombination, transposon site-directed inactivation, etc.); or by gene editing technology (including CRISPR/cas9 (or spf1), TALEN, zinc finger nuclease, Base editing technology, etc.), etc. A variety of methods known in the art for downregulating expression or inactivating genes or polypeptides are also available.

As a preferred embodiment of the present invention, semi-cloning technology mediated by haploid embryonic stem cells and gene editing technology are combined to form the phenotypes of Dmpk+/-, Six5+/-, Mbnl1+/- and Dmwd+/-. Preferably, by combining animals from haploid embryonic stem cells with CRISPR technology, an animal model showing the phenotypes of Dmpk+/-, Six5+/-, Mbnl1+/- and Dmwd+/- can be obtained in a one-step method. In a more specific embodiment, gene editing is performed using the CRISPR/Cas9 system, and high gene editing efficiency can be achieved by selecting appropriate sgRNA target sites.

In a preferred embodiment of the present invention, a preferred target site is designed and provided. SgRNA or a nucleic acid capable of forming the sgRNA, Cas9 mRNA or a nucleic acid capable of forming the Cas9 mRNA are co-introduced into a fertilized egg of an animal (such as a mouse) to obtain a gene-edited animal (such as a mouse). Cas9 mRNA and sgRNA can be obtained by in vitro transcription, or the nucleic acid capable of forming the sgRNA can be a nucleic acid construct or an expression vector, or the nucleic acid capable of forming the Cas9 mRNA is a nucleic acid construct or an expression vector, and these expression vectors are introduced into cells, thereby forming active sgRNA and Cas9 mRNA in the cells. The introduction method is preferably microinjection for fertilized eggs, and preferably electroporation or liposome transfection for cells, but other existing effective introduction methods are not excluded.

As another alternative, the Cre and loxp methods can also be used to selectively knock out, reduce or inactivate the expression of related genes in the genome of animals or cells.

The experimental model constructed by the present invention can be used for screening and testing of specific drugs, and can be used as a powerful tool for scientific research and new drug evaluation, and has good stability.

The present invention also provides a method for screening candidate drugs or therapeutic agents for treating DM1 disease using the experimental model of the present invention (including animal models or cell models), comprising administering a candidate substance to the experimental model and observing whether the experimental model has disease alleviation or improvement. If so, the candidate substance is a potential drug for DM1 disease.

In the present invention, candidate drugs or therapeutic agents refer to substances that are known to have certain pharmacological activities or are being tested and may have certain pharmacological activities, including but not limited to nucleic acids, proteins, carbohydrates, chemically synthesized small molecules or macromolecular compounds, cells, natural product extracts, and composite components of the above substances. The administration of candidate drugs or therapeutic agents can be oral, intravenous, intraperitoneal, subcutaneous, gavage, etc.

Based on the method of the present invention, the present invention also provides a kit for preparing a DM1 disease experimental model, the kit comprising: reagents for forming the phenotypes of Dmpk+/-, Six5+/-, Mbnl1+/- and Dmwd+/- in the genome. The kit may also include instructions for implementing the method of preparing a DM1 disease animal model/cell model of the present invention, so as to facilitate application by those skilled in the art.

Drug screening method

Existing work has shown that there are abnormalities in the differentiation of muscle stem cells (MuSCs) isolated from DM1 patients. In order to verify whether the constructed model mice have abnormal MuSCs differentiation, the inventors used the previously established muscle stem cell culture method to first isolate and culture MuSCs from the DM1 mouse model, and then evaluated the function and gene expression of the muscle stem cells to analyze whether there are differences with normal muscle stem cells. Then, the inventors screened a series of small molecule libraries provided by LifeArc in the UK, trying to find small molecule compounds that promote muscle stem cell differentiation.

After obtaining the above experimental model (including animal model or cell model), potential substances that promote muscle stem cell differentiation can be screened based on the characteristics. According to the principle, the substances that promote muscle stem cell differentiation can also potentially be used for sarcopenia, muscle aging or muscular dystrophy; preferably, the muscular dystrophy includes myotonic dystrophy type 1. Drugs that are truly useful for improving or treating diseases can be found from the substances.

Therefore, the present invention provides a method for screening potential substances that promote muscle stem cell differentiation, the method comprising: (1) treating cells or cell cultures having Dmpk+/-, Six5+/-, Mbnl1+/- and Dmwd+/- phenotypes in their genome with candidate substances; and (2) detecting the differentiation of cells having Dmpk+/-, Six5+/-, Mbnl1+/- and Dmwd+/- phenotypes; if the candidate substance statistically promotes the differentiation of the cells, it indicates that the candidate substance is a potential substance that promotes muscle stem cell differentiation.

In a preferred embodiment of the present invention, during screening, a control group may be set up to more easily observe changes in cell differentiation. The control group may be a system with Dmpk+/-, Six5+/-, Mbnl1+/- and Dmwd+/- phenotypes without the addition of the candidate substance.

As a preferred embodiment of the present invention, the method further comprises: conducting further cell experiments and/or animal experiments on the obtained potential substances to further select and determine substances that are truly useful for improving or promoting muscle stem cell differentiation.

In an embodiment of the present invention, a MuSCs in vitro differentiation system is used to screen small molecules that are effective for MuSCs-related diseases and sarcopenia in the elderly. In the DM1 disease mouse model constructed by the inventors, the inventors used a heterozygous knockout of DM1-related genes to carry out research, which is closer to the clinic. At the same time, the inventors observed abnormal differentiation of MuSCs in the mouse model for the first time, and proposed that the MuSCs in vitro differentiation system can simulate the process of differentiation in vivo, and then use the in vitro differentiation system to screen out small molecule compounds that promote MuSCs differentiation. Such a differentiation system can also be used for the study of other diseases. For example, sarcopenia exists in the elderly population, skeletal muscle is the power of the human motor system, and muscle aging and atrophy are important signs of human aging, which are very likely to cause fractures and joint injuries, etc. At the same time, there are articles that prove that the differentiation ability of MuSCs in the elderly population is significantly reduced. Therefore, combined with the MuSCs in vitro differentiation system, small molecule compounds that promote the differentiation of MuSCs can be screened, thereby delaying muscle aging.

Compounds that promote muscle stem cell differentiation

The present invention first provides a compound having a core structure shown in structural formula (I):

Wherein, R is independently selected from: hydrogen, hydroxyl, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, halogen. Preferably, R is independently selected from: hydrogen, hydroxyl, C1-C2 alkyl.

The present invention also includes isomers, solvates, precursors, or pharmaceutically acceptable salts of the above-mentioned compounds having the parent core structure shown in formula (I), as long as they also have the same or substantially the same function as the compound having the parent core structure shown in formula (I). The "pharmaceutically acceptable salt" refers to a salt formed by the reaction of a compound with an inorganic acid, an organic acid, an alkali metal or an alkaline earth metal. These salts include (but are not limited to): (1) salts formed with the following inorganic acids: such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid; (2) salts formed with the following organic acids, such as acetic acid, oxalic acid, succinic acid, tartaric acid, methanesulfonic acid, maleic acid, or arginine. Other salts include salts formed with alkali metals or alkaline earth metals (such as sodium, potassium, calcium or magnesium) in the form of esters, carbamates, or other conventional "prodrugs". The compounds have one or more asymmetric centers. Therefore, these compounds can exist as racemic mixtures, individual enantiomers, individual diastereomers, diastereoisomer mixtures, cis or trans isomers.

The "precursor of a compound" refers to a compound having a parent core structure shown in structural formula (I) or a salt or solution composed of a compound having a parent core structure shown in chemical formula (I) which undergoes metabolism or chemical reaction in the patient's body after being taken by an appropriate method.

As a preferred embodiment of the present invention, the compound is:

As an isomer of the above compound, it has the following structure:

Those skilled in the art should understand that, after knowing the structure of the compound of the present invention, the compound of the present invention can be obtained by a variety of methods well known in the art, using well-known raw materials, such as chemical synthesis or extraction from organisms (such as animals or plants) or modification based on extraction, and these methods are all included in the present invention.

The compounds of the present invention can be synthesized by known methods; the synthesized compounds can be further purified by column chromatography, high performance liquid chromatography, etc. In addition, the compounds of the present invention can also be obtained by commercial means.

The inventors have found in their research that the compound with the parent nucleus structure shown in formula (I) of the present invention has a significant effect on promoting the differentiation of muscle stem cells. Based on the new discovery of the inventors, the present invention provides the use of the compound shown in formula (I) or its isomers, solvates, precursors, or pharmaceutically acceptable salts thereof for preparing a composition for promoting the differentiation of muscle stem cells, or for preparing a composition for alleviating or treating diseases related to abnormal differentiation of muscle stem cells. The abnormal differentiation of muscle stem cells includes: decreased differentiation ability of muscle stem cells or no differentiation. Diseases related to abnormal differentiation of muscle stem cells include: sarcopenia, muscle aging or muscular dystrophy; preferably, the muscular dystrophy includes myotonic dystrophy type 1.

The present invention also provides a composition containing an effective amount of the compound of the parent core structure shown in formula (I), or its isomer, solvate, or precursor.

In the present invention, the term “contains”, “has” or “includes” includes “comprises”, “mainly consists of”, “substantially consists of”, and “consisting of”; “mainly consists of”, “substantially consists of” and “consisting of” are subordinate concepts of “contains”, “has” or “includes”.

As a preferred embodiment, the composition is a pharmaceutical composition, which further contains a pharmaceutically acceptable salt and/or a pharmaceutically acceptable carrier or excipient.

As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit/risk ratio. A "pharmaceutically acceptable carrier" is a pharmaceutically or food-acceptable solvent, suspending agent, or excipient for delivering the compounds of the present invention to animals or humans. The carrier can be a liquid or a solid.

In the present invention, the pharmaceutical composition contains 0.001-50% by weight of the compound represented by the parent core structure represented by formula (I) or a pharmaceutically acceptable salt thereof. Preferably, the pharmaceutical composition contains 0.05-30% by weight of the compound represented by the parent core structure represented by formula (I) or a pharmaceutically acceptable salt thereof; more preferably, the pharmaceutical composition contains 0.01-20% by weight of the compound represented by the parent core structure represented by formula (I) or a pharmaceutically acceptable salt thereof. Those skilled in the art should understand that other weight ratios are also feasible according to actual clinical needs or drug design methods in pharmacy.

The dosage form of the pharmaceutical composition of the present invention can be varied, as long as it is a dosage form that can effectively allow the active ingredient to reach the mammalian body. For example, it can be selected from: gel, aerosol, tablet, capsule, powder, granule, syrup, solution, or suspension. According to the type of disease treated by the compound of the present invention, those skilled in the art can choose a dosage form that is convenient for use. From the standpoint of ease of preparation and administration, the preferred pharmaceutical composition is a solid composition, especially tablets and solid-filled or liquid-filled capsules. The compound of the present invention or its pharmaceutical composition can also be stored in a sterile device suitable for injection or instillation. The effective dosage of the parent core structure compound shown in formula (I) as the active ingredient can vary with the mode of administration and the severity of the disease to be treated.

As another preferred embodiment, the composition is a culture medium composition, which can be used to culture muscle stem cells in vitro/ex vivo to promote their differentiation into desired muscle stem cells. Other components in the culture medium composition can be substances that provide conventional nutrients for cells, which are easily available to those skilled in the art.

The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

Materials and methods

1. Backbone plasmid and strain

pMD19-T vector (Takara);

px330-mcherry (Addgene);

DH5α: Escherichia coli.

2. Cell lines

Mouse embryonic fibroblast (MEF);

Androgenetic haploid embryonic stem cells (AG-haESCs) (Yang H, Shi L, Wang BA, Liang D, Zhong C, Liu W et al (2012) Generation of genetically modified mice by oocyte injection of androgenetic haploid embryonic stem cells. Cell 149: 605–617).

3. Antibodies

Anti-Muscleblind-like 1 antibody (Abcam, cat.no.ab108519);

Anti-SIX5 antibody (Abcam, cat. no. ab113064);

Anti-DMWD antibody (Santa cruz, cat. no. sc-167638);

Anti-DMPK antibody (Santa cruz, cat. no. sc-13612).

4. Experimental animals

C57/BL6 mice;

B6D2F1 mice: obtained by mating C57/BL6 female mice with DBA/2 male mice;

ICR mice;

IG-DMR knockout mice (4.15 kb) were obtained from Cambridge University;

H19 DMR knockout mice (3.8 kb) were obtained from the University of Pennsylvania;

All mouse experiments were in accordance with the rules and regulations of the Shanghai Institute of Biochemistry and Cell Biology Laboratory Animal Experiment Management Committee, and all experimental mice were kept in the SPF room of the Shanghai Institute of Biochemistry and Cell Biology Laboratory Animal Platform. All mice were kept in a 12-hour day and 12-hour night cycle.

5. Preparation of Mouse ESCs Feeder Cells

The feeder layer cells used were inactivated mouse embryonic fibroblasts (12.5dpc). Take the 12.5dpc ICR mother mouse, dislocate the neck and kill it, and remove the fetus. Wash the fetus several times with PBS, remove the fetal head, limbs and internal organs, cut the fetal trunk into pieces, add an appropriate amount of 0.05% Trysin-EDTA and mix well, place it in a 37℃ water bath for digestion for 10min, shake it from time to time during the digestion process to prevent the tissue from sinking to the bottom and hindering the digestion, then terminate the trypsin digestion, filter out the undigested large tissue blocks with a 70μm filter, spread it into a 10cm culture dish, digest it with 0.05% Trysin-EDTA after the fibroblasts are full, and pass it at a ratio of 1:5. After 2-3 passagings, it can be inactivated by radiation and cryopreserved in separate tubes. The inactivated mouse fibroblasts can be frozen at -80℃ for one month. If you want to store them for a long time, you need to transfer them to liquid nitrogen.

6. Collection of MⅡ stage oocytes

8-10 week old B6D2F1 female mice were injected with 5-6 IU PMSG intraperitoneally, and 48 hours later, 5-6 IU hCG was injected. Egg retrieval can be performed 14 hours after the hCG hormone injection. Before the egg retrieval operation, prepare CZB droplets and KSOM droplet culture dishes, each with 10-20 μl. After the mouse was killed by cervical dislocation, the oviduct was removed and the egg mass in the oviduct bulge was cut out. Subsequently, the cumulus cells were removed with Hy working solution, and after washing several times with HCZB droplets and CZB droplets, they could be placed in CZB droplet culture dishes and briefly cultured in a constant temperature 37°C _CO2 incubator until the end of the experiment.

7. Establishment, culture and genetic manipulation of AG-haESCs

AG-haESCs are derived from the haploid blastocysts obtained by injecting the tail-broken sperm into the enucleated oocytes. After obtaining the haploid embryos in the blastocyst stage, the zona pellucida is first digested with TSA, and then transferred to a 96-well plate with feeder cells, and one blastocyst is placed in each well. 2i culture medium is placed in the wells in advance. After growing for 4-5 days, 0.05% trypsin-EDTA digestion is performed and the cells are passaged to another well of the 96-well plate. After 3-5 days, the wells with visible clones are further passaged to 48-well plates and then to 24-well plates. During the passage in the 24-well plate, some cells can be tested for haploidy and sorted. Before FACS sorting, ESCs need to be stained with Hoechst33342, which can bind to double-stranded DNA. During FACS sorting, Hoechst 33342 can be excited by ultraviolet light to release fluorescence at a wavelength of 461nm. The fluorescence intensity is proportional to the Hoechst 33342 content in the cell. Therefore, cells with different chromosome ploidy can be distinguished based on the fluorescence intensity.

After the haploid line is established, genetic manipulation can be performed.

Design of sgRNA plasmid:

px330-SIX5: Six5-sgRNA-f: CACCGTCTGATGGGAACCCCACTA (SEQ ID NO: 1); Six5-sgRNA-r: TAGTGGGGTTCCCATCAGAC (SEQ ID NO: 2), synthesized sgRNA sequence, and inserted into the DBSI restriction site of px330-mcherry.

px330-DMPK: Dmpk-sgRNA-f: CACCGCCCATTGCAGCAAGGCTTA (SEQ ID NO: 3); Dmpk-sgRNA-r: AAACTAAGCCTTGCTGCAATGGGC (SEQ ID NO: 4), synthesized sgRNA sequence, and inserted into the DBSI restriction site of px330-mcherry.

px330-MBNL1: Mbnl1-up-sgRNA-f: CACCGCTGCAACACTATCTGTGAT (SEQ ID NO: 5); Mbnl1-up-sgRNA-r: AAACATCACAGATAGTGTTGCAGC (SEQ ID NO: 6); Mbnl1-down-sgRNA-f: CACCGGTACATGGTACTCTAAATT (SEQ ID NO: 7); Mbnl1-down-sgRNA-r: AAACAATTTAGAGTACCATGTACC (SEQ ID NO: 8). Two sgRNA sequences were synthesized and inserted into the DBSI restriction site of px330-mcherry.

px330-DMWD: Dmwd-sgRNA-f: CACCGGCTTCTACAAGCTGCTCCC (SEQ ID NO: 9); Dmwd-sgRNA-r: AAACGGGAGCAGCTTGTAGAAGCC (SEQ ID NO: 10), synthesized sgRNA sequence, and inserted into the DBSI restriction site of px330-mcherry.

The Cas9 plasmid px330-mcherry carrying the sgRNA targeting the target gene can be transfected into AG-haESCs using Lipofectamine 2000. The next day after cell transfection, FACS sorting is performed based on the fluorescence of the transfected plasmid and Hoechst33342 staining, and positive haploid cells are collected. Then, they are spread at a low density in a 6-well plate (no more than 10,000 cells per well), grown for 6-8 days, and a single ESCs clone is picked for digestion. Half of them are transferred to a new 96-well plate, and half are lysed for target gene mutation identification. After determining the genotype of the cell line, haploid sorting is performed. For cell lines with multi-gene manipulation, after determining that one gene manipulation is completed, other genes are manipulated. If multiple genes are located on different chromosomes, or if the same chromosome is far away, multi-gene mutation manipulation can also be performed simultaneously.

8. ROSI and ICAHCI experiments

ROSI (Round spermatid injection): Round spermatid injection. The testicles of adult male mice were cut into pieces and digested. After Hoechst 33342 staining, haploid spherical sperm were sorted out by FACS. Activate MⅡ stage oocytes with ^{Sr 2+} 30 minutes in advance, and then inject the spherical sperm nuclei into the activated MⅡ stage oocytes. The operation time for each group should not exceed 30 minutes. After all the operations were completed, continue Sr ²⁺ activation for 3 hours, and then continue to culture in KSOM droplets. After the reconstructed embryos developed to the two-cell stage, they were transplanted into the oviducts of ICR female mice with pseudopregnancy of 0.5 days (20-25 reconstructed embryos/female mouse), and the mice matured at 19.5 days.

ICAHCI: Colchicine was added to AG-haESCs with a relatively suitable clone size 1 day in advance to block the cells in the metaphase of the cell cycle. The cells were digested for 8-12 hours (more than 80% of the cells were in the metaphase), added to the operation drop, and the haploid cells in the metaphase (without nucleus and small cells) were selected and injected into the oocytes. The operation time for each group did not exceed 30 minutes. After the end of one group, it was washed several times and placed in CZB droplets. After the operation was completely completed, the reconstructed embryos were activated by Sr ²⁺ for 5 hours, and then placed in KSOM droplets for continued culture. After the reconstructed embryos developed to the two-cell stage, they were transplanted into the oviduct of ICR female mice with a pseudopregnancy of 0.5 days (30-40 reconstructed embryos/female mouse). The mice matured at 19.5 days. If the birth did not take place at the maturity date, a cesarean section could be performed.

9. Frozen sections and immunofluorescence

After the mouse neck was dislocated, the skin covering the lower limbs was removed, the tibialis anterior muscle was taken out and embedded in OCT, then quickly frozen in liquid nitrogen for 15s and stored at -80℃ until sectioning. For longitudinal sectioning of the heart, the newly taken heart was first washed in PBS, and the blood in the heart was removed by pressing, then fixed in 4% PFA for 30min, dehydrated with 30% sucrose, embedded in OCT, quickly frozen in liquid nitrogen and stored at -80℃ until sectioning. The thickness of the section is generally 8-10μm. The sections for immunofluorescence staining were first fixed with 4% PFA, washed with PBS, incubated with primary antibody at 37℃ for 1h, washed with PBS, incubated with secondary antibody at 37℃ for 1h, and then labeled with DAPI for nuclei and sealed for observation. All fluorescent photos were taken with a Leica SP8 fluorescence confocal microscope. The neuromuscular junctions (NMJs) of the diaphragm of newborn mice were stained. The diaphragm frozen sections were labeled with Alexa Fluor 594-conjugated bungarus toxin to label acetylcholine receptors (muscle fibers), and neurofilament H was used to label neurons.

10. Histological analysis

For the pathological analysis of muscle, diaphragm, small intestine and heart, the eosin-methylene blue staining method was used. Muscle, diaphragm and heart were stained with frozen sections, and small intestine was stained with paraffin sections. For the analysis of fast and slow muscles, the ATPase staining method was used. That is, the frozen sections were incubated in an acidic environment of pH 4.6 for 5-15 minutes, and then stained with ATP solution. After being treated with 1% CaCl ₂ , 2% CoCl ₂ , 5mM barbituric acid and 1% (NH ₄ ) ₂ S, the sections were dehydrated with ethanol gradient, and slow muscle fibers appeared dark gray, while white was fast muscle. For small intestinal tissue sections, freshly isolated tissues were flushed with PBS to remove intestinal contents, and then fixed with 4% PFA at 4°C overnight. After ethanol gradient dehydration, they were embedded in paraffin and then sectioned. Both samples and sections can be stored at room temperature, and the section thickness is 5μm.

11. Mouse front paw strength test and Rotarod experiment

First, the experimental mice were placed on a uniformly moving roller to adapt for 5 minutes, and then the speed of the roller was adjusted, gradually increasing from 4rpm to 40rpm within 5 minutes. The time it took for the roller to fall was recorded. If the mouse did not fall for more than 5 minutes, it was recorded as 300s. Before the formal experiment, the mice were trained for 3 days in advance.

12. Mouse electromyography experiment

Electromyography (EMG) experiments can be used to detect whether the mouse muscles have a tonic phenotype. The experimental mice are anesthetized with isoflurane, and the soft hair on the abdomen and legs of the mice is removed with depilatory cream. Electrodes are attached to the abdomen of the mice, and electrode needles are inserted into the legs to detect tonic potentials for electromyography (EMG).

13. RNA extraction and fluorescence quantitative PCR

After cell digestion and DPBS washing, add Trizol to lyse at room temperature for several minutes to extract RNA. The newly obtained tissues (leg muscles and heart) are placed in liquid nitrogen to inactivate RNase, and then add Trizol to shake and lyse to fully extract RNA. Add chloroform to the Trizol lysis solution, shake vigorously and centrifuge, take the supernatant into a new EP tube, add an equal amount of isoamyl alcohol, invert and mix, centrifuge to remove the supernatant, add 75% ethanol to wash the precipitate, discard the supernatant after centrifugation, dry and add ddH ₂ O to dissolve.

Take 0.5 μg RNA and first add the reverse transcription kit buffer 1 to remove genomic DNA, then add buffer 2 to reverse transcribe into cDNA. After transcription, use Toyobo SYBR Green for gene expression detection. Each sample was repeated three times, 20 μl system: SYBR Green (10 μl), primer (X μl, 5 μM), sample (1 μl), ddH ₂ O [(9-2X) μl]. PCR program: 94℃ denaturation for 4 min, cycle 94℃ denaturation for 10 s, 60℃ annealing for 20 s, 72℃ extension for 20 s program 40 times, 72℃ extension for 4 min. GADPH expression was used as the internal reference for gene expression.

14. Cell culture related reagents

ES DMEM (Millipore, cat. no. SLM-220-M).

DMEM (Thermo Fisher Scientific, cat. no. 11965118).

Opti-MEM (Thermo Fisher Scientific, cat. no. 51985042).

ES FBS (Thermo Fisher Scientific, cat. no. 16000044).

FBS (Excell, cat. no. FSP500).

Nucleotides (100×) (Millipore, cat. no. ES-008-D).

Non-Essential Amino Acids (100×) (NEAA; Millipore, cat. no. TMS-001-C).

L-Glutamine Solution (100×) (Millipore, cat. no. TMS-002-C).

2-Mercaptoethanol (100×) (Millipore, cat. no. ES-007-E).

Penicillin-Streptomycin (Thermo Fisher Scientific, cat. no. 15140122).

Leukemia Inhibitory Factor (LIF; Millipore, cat. no. ESG1107).

DPBS (Thermo Fisher Scientific, cat. no. 14190-144).

0.05% Trypsin-EDTA (Thermo Fisher Scientific, cat. no. 25300-112).

G418 (Sigma, cat. no. A1720).

Demecolcine solution (Sigma, cat. no. D1925).

Dimethyl sulfoxide (DMSO; Sigma, cat. no. D8418).

Recovery ^TM Cell Culture Freezing Medium (Thermo Fisher Scientific, cat. no. 12648-010).

Lipofectamine 2000 (Thermo Fisher Scientific, cat. no. 12648-010).

15. Isolation and culture of mouse muscle stem cells

Take the muscle of the mouse (such as the tibialis anterior muscle) and cut it into pieces, add an appropriate amount of 0.05% Trysin-EDTA and mix it, place it in a 37°C water bath for digestion for 10 minutes, shake it from time to time during the digestion process to prevent the tissue from sinking to the bottom and hindering the digestion, then terminate the trypsin digestion, spread it into a 10cm culture dish, digest it with 0.05% Trysin-EDTA after the muscle stem cells are full, and pass it in an appropriate proportion. In addition to the basic culture medium (DMEM+10%FBS+5%Penicillin-Streptomycin), the cell culture medium is added with cytokines including at least IL1, IL4, IL13, TNF alpha, IL2 and IFN gamma, each at a concentration of 1ng/ml. The sum of the concentrations is not less than 6ng/ml, preferably 50-1250ng/ml.

Example 1. Acquisition and phenotypic analysis of DSM-TKO heterozygous mutant mice

After obtaining the AG-haESCs cell line that can effectively support the birth of semi-cloned mice, the inventors tried to see if this technology could be used to obtain mice with multiple gene modifications in one step. First, a single gene knockout AG-haESCs cell line, namely the ΔDMPK-AGH cell line (frameshift mutation), was constructed using CRISPR technology. Gene knockout operations of SIX5 and MBNL1 were performed on the ΔDMPK-AGH cell line (Figure 1a). The cell morphology and growth of the triple gene knockout cell line (DSM-AGH cell line) were normal (Figure 1b). After the ICAHCI experiment, the growth of semi-cloned mice was also relatively normal (Figure 1c). However, a ricket phenotype appeared in adult mice (Figure 1d). EMG experiments were performed on triple-gene knockout DSM-TKO semi-cloned mice, and the occurrence of spasticity was detected (Figure 1e); their forepaw grip strength was also significantly weakened compared with the control; when conducting treadmill experiments, their overall motor ability was significantly reduced; in the rotarod test, DSM-TKO semi-cloned mice were more likely to fall (Figure 1f-h), all of which indicate that the muscle strength of DSM-TKO semi-cloned mice was weakened and their muscle function was affected to a certain extent.

The inventors then performed histological analysis on mouse skeletal muscle, diaphragm, heart and small intestine. In terms of skeletal muscle, dystrophin staining was performed on tibialis anterior muscle sections, and the results showed that the entire muscle structure was relatively intact. H&E staining of the sections did not reveal obvious nuclear intranslocation, but phenotypes such as sarcoplasmic masses, nuclear aggregation and vacuolar fibers could be observed. At the same time, statistics were performed on the cross-sections of the muscle fibers, and it was found that the cross-sections of the muscle fibers were smaller as a whole (Figure 1i-k), indicating the occurrence of muscle atrophy.

So far, the inventors have obtained a new DM1 disease mouse model by combining several genes involved in DM1. In this model, it can be seen that there are certain abnormalities in skeletal muscle, myocardium and small intestine, which are consistent with the clinical results. However, some phenotypes are not obtained. First, the ankylosing phenotype of DSM-TKO semi-cloned mice is not as obvious as that of several other DM1 mouse models. Second, in the analysis of muscle histological sections, no obvious muscle fiber nuclear involution was observed, but this phenomenon is very common in clinical patients (some patients do not have obvious nuclear involution). Third, no obvious phenotypes in cardiac structure and function were observed, and cardiac problems are the main cause of sudden death in classic DM1 patients. Fourth, there is no large amount of abnormally spliced RNA in mouse muscle and heart tissue. These points show that there are other genes that have not been paid attention to in this disease model that still play an important role.

Example 2: Construction of CDM1 mouse model

From the above experiments, it can be seen that although the triple-gene knockout mice showed some phenotypes of DM1 disease, some important phenotypes were not simulated, including a series of phenotypes of congenital DM1. No abnormal heart function, cataracts and other phenotypes appeared in adult mice, which indicates that other genes may be involved in the occurrence of the disease.

The inventors further knocked out DMWD on the DSM-TKO-DAH (DAH refers to haploid stem cells) cell line, and obtained the DSMD-QKO-DAH cell line, which has relatively normal cell morphology and growth (Figure 2a), and can effectively support the birth of semi-cloned mice when performing ICAHCI experiments. However, it was found that about 20% of the mice died quickly after birth (Figure 2b). Phenotypic analysis of the obtained DSMD-QKO semi-cloned mice found that the lungs of surviving mice floated on the water surface when placed in clear water, while the lungs of dead mice sank to the bottom of the water (Figure 2c), which shows that the dead mice did not breathe after birth. Since patients with congenital DM1 are likely to survive the earliest stage, but still have breathing problems in the follow-up, the inventors took the diaphragm of mice that were just born one day ago and stained them. It was found that compared with control mice, some areas of the diaphragm structure of DSMD-QKO semi-cloned mice were not complete (Figure 2d), and the cross-section of their muscle fibers was significantly reduced (Figure 2e). Neuromuscular junction (NMJ) staining revealed that the mature junction connections in DSMD-QKO semi-cloned mice were significantly reduced compared with the controls (Figure 2f).

During the subsequent feeding process, the fetuses of DSMD-QKO semi-cloned mice were significantly lighter and had developmental delays compared to control mice (Figure 3a-b). Alcian blue and Alizarin Red S staining of mice born 1 day ago showed that the metatarsal development of DSMD-QKO semi-cloned mice was relatively slow (Figure 3c), which to some extent explains why newborns with CDM1 disease suffer from clubfoot. It may be because the metatarsal calcification is slow during the development of the fetus, so it may be more susceptible to external influences and deformity in the late pregnancy of the mother. HE staining was performed on the hind limbs of mice 2 days after birth. The inventors found that DSMD-QKO semi-cloned mice had many muscle fibers with central nuclei (Figure 3d), which once again proved that DSMD-QKO semi-cloned mice were developmentally delayed. On the fifth day of the growth of semi-cloned mice, the inventors could already see that some DSMD-QKO semi-cloned mice were obviously different from other mice and grew more slowly (Figure 3e). The muscle tension of the newborn mice at this time was tested. Compared with the control, the DMSM-QKO semi-cloned mice took longer to turn over under supine conditions (Figure 3f). In the subsequent nursing process, there was still a certain mortality rate in two-week-old mice. This sudden death was similar to the phenotype of CDM1 patients and was suspected to be caused by abnormal heart rhythm conduction. Echocardiography experiments on mice also showed that some mice did have abnormal heart rhythm conduction, and H&E staining of heart sections also showed structural abnormalities (Figure 3g).

Based on the above phenotypic description, the inventors obtained a congenital DM1 disease mouse model. The skeletal muscle, heart and respiratory system all have phenotypes similar to those in the clinic. At the same time, using this disease model, it was also concluded that CDM1 respiratory disorders may be related to the diaphragm structure. The inventors observed that the diaphragm of the DSMD-QKO semi-cloned mice had structural incompleteness, insufficient diaphragm muscle fiber strength and fewer mature neuromuscular junctions, which made up for the defects of in-depth research in the clinic due to the difficulty in obtaining samples.

Rickets phenotype also appeared in DSMD-QKO adult mice. EMG experiments detected that DSMD-QKO semi-cloned mice had the appearance of stiffness (Figure 4a). The grip of the mouse forepaws was significantly weakened. At the same time, DSMD-QKO semi-cloned mice were more likely to fall during the rotarod experiment. The treadmill experiment also confirmed that their overall motor ability was significantly reduced (Figure 4b-d). Some mice showed cataract phenotypes (Figure 4i). Subsequently, histological analysis of mouse skeletal muscle, diaphragm, heart and small intestine was performed. Similar to DSM-TK semi-cloned mice. Tibialis anterior muscle sections did not show obvious nuclear inward migration, but phenotypes such as sarcoplasmic masses, nuclear aggregation and vacuolar fibers were observed (Figure 4f), the cross-section of muscle fibers became smaller (Figure 4g), and anti-dystrophin staining showed that the muscle structure was relatively normal (Figure 4e). ATPase staining was performed on its muscle sections, and the inventors found that the number of type I muscle fibers increased significantly, while their cross-sectional area decreased (Figure 4h).

In the disease model constructed by the inventors, mice exhibited the DM1 phenotype, indicating that DM1 is not just an RNA disease, but that abnormalities in multiple genes including SIX5, DMPK, and DMWD lead to the occurrence of the disease.

Example 3. Abnormal in vitro differentiation of muscle stem cells (MuSCs) in mouse disease models

Furthermore, the present inventors detected whether there was abnormality of MuSCs in the aforementioned constructed DM1 disease models (DSM-TKO, DSMD-QKO).

First, muscle tissue sections of the tibialis anterior muscle of mice were obtained and Pax7 immunofluorescence detection was performed, and it was found that the number of stem cells in the mice did not decrease (Figure 5a-b). The FACS test results also showed that there was no significant difference in the number of MuSCs in the muscle tissue of DSM-TKO semi-cloned mice, DSMD-QKO semi-cloned mice and control semi-cloned mice. Through FACS sorting or cell adhesion, the inventors separated MuSCs from the body for in vitro culture. Compared with the MuSCs of the control semi-cloned mice, the MuSCs of DSM-TKO or DSMD-QKO isolated from the body had normal in vitro proliferation and no significant difference. Subsequently, the inventors conducted an in vitro differentiation experiment on MuSCs and found that the differentiation ability of MuSCs of DSM-TKO or DSMD-QKO was significantly weakened. The statistical results showed that the ratio of muscle fibers with more than 5 nuclei was significantly reduced, and its fusion ability was reduced during the differentiation process of MuSCs (Figure 5d-e).

In order to further study why there is such a difference? The inventors took cells before differentiation, cells differentiated for one day and two days for deep transcriptome sequencing. Analysis of genes related to muscle development structure found that compared with one day and two days of differentiation, the difference between MuSCs with three or four genes heterozygous knockout before differentiation and the control was greater (Figure 6a), and qPCR detection of the corresponding genes obtained similar results (Figure 6b). This shows that the stemness of heterozygous knockout MuSCs cultured in vitro is reduced, and the reduction in stemness directly leads to the inability to produce enough cells in the differentiation system for subsequent muscle fiber fusion. Therefore, the inventors observed that the fused muscle fibers during the differentiation of MuSCs with three or four genes heterozygous knockout were thinner and had fewer nuclei.

2. Screening of small molecules using MuSCs from DM1 model mice

The abnormality of MuSCs differentiation ability leads to the occurrence of diseases to a certain extent. Therefore, the inventors used the MuSCs differentiation system to conduct small molecule compound screening experiments to screen drugs that can alleviate or even cure diseases. First, the inventors used a 384-well plate differentiation system to screen the small molecule library, and tested DSM-TKO-MuSCs and DSMD-QKO MuSCs heterozygous knockout cell lines (muscle stem cells were isolated from the tibialis anterior muscle of mice) and control group cell lines, cultured the cell lines and treated with compounds. The screening process is shown in Figure 7a.

The inventors first separated WT, DSM-TKO and DSMD-QKO muscle stem cells from the tibialis anterior muscle of mice, and plated 3500 cells per well into a 384-well cell culture dish. After two days of normal culture, the compound to be tested was added at a concentration of 10uM (the compound was dissolved in DMSO). After two days of culture, the muscle stem cells were observed under a microscope and statistically analyzed for differentiation, including the number of nuclei in the cells. After the muscle stem cells differentiated, the number of nuclei in each cell changed. The number of nuclei in each group of cells was observed and counted. A large number indicated that cell differentiation was promoted, which also indicated that the cell differentiation was better.

The inventors screened nearly 10,000 small molecules and obtained 17 small molecules that promoted the differentiation of the heterozygous knockout cell lines DSM-TKO-MuSCs and DSMD-QKO MuSCs. Among them, the most effective small molecule was T5381948 (Enamine; https://enaminestore.com/catalog) (Figure 7b), and its structural formula is as follows:

The results after treatment with the above-mentioned small molecule T5381948 are shown in Figure 7c. After treatment with T5381948, the differentiation of DSM-TKO muscle stem cells was significantly better than that of the control group, as shown by: the number of muscle cells with an average of 10 or more nuclei was significantly greater than that of the control group; ***, P<0.001. This result shows that the small molecule T5381948 can significantly promote the differentiation of muscle stem cells and is useful for treating diseases related to abnormal differentiation of muscle stem cells.

All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Sequence Listing

<110> Shanghai Institutes for Life Sciences, Chinese Academy of Sciences

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Claims (6)

1. Use of the compound represented by formula (I) or a pharmaceutically acceptable salt thereof for preparing a composition for promoting muscle stem cell differentiation, or for preparing a composition for alleviating or treating a disease of abnormal muscle stem cell differentiation; the disease of abnormal muscle stem cell differentiation includes: decreased differentiation ability of muscle stem cells or no differentiation;

(I). 2. The use according to claim 1, characterized in that the diseases caused by abnormal differentiation of muscle stem cells include: sarcopenia, muscle aging or muscular dystrophy. 3. The use according to claim 2, characterized in that the muscular dystrophy comprises myotonic dystrophy type 1. 4. The use according to claim 1, characterized in that the composition for promoting muscle stem cell differentiation is a culture medium composition. 5. The use according to claim 1, characterized in that the composition for alleviating or treating diseases of abnormal muscle stem cell differentiation further comprises a pharmaceutically acceptable carrier. 6. A method for promoting differentiation of muscle stem cells, wherein the method is not for therapeutic purposes, characterized in that the method comprises: treating muscle stem cells with a compound represented by formula (I) or a pharmaceutically acceptable salt thereof;

(I).

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