CN-122012612-A - Friable X syndrome animal model, construction and application thereof

Abstract

The invention provides a fragile X syndrome animal model, and construction and application thereof. The invention discloses a target which is matched with a non-human primate and is suitable for genetic modification, namely a third exon and/or a fourth exon of an FMR1 gene, and the target is subjected to down regulation to obtain a fragile X syndrome animal model. The animal model has stable and controllable state, is easy to realize observation, can show typical disease symptoms more than animal models of other species of genes, and has good application prospect.

Inventors

• XIONG ZHIQI
• SUN QIANG
• ZHANG RUNZE
• ZHUANG LING
• ZHANG XIAOLEI
• KONG DELUN

Assignees

• 中国科学院脑科学与智能技术卓越创新中心

Dates

Publication Date

20260512

Application Date

20241112

Claims (12)

1. 1. A method of preparing a fragile X syndrome animal comprising down-regulating an FMR1 gene of the animal, wherein the down-regulation targets a third exon and/or a fourth exon of the FMR1 gene, wherein the animal is a non-human primate.
2. 2. A method of making an animal cell comprising down-regulating an FMR1 gene in an animal cell, the down-regulation targeting a third exon and/or a fourth exon of the FMR1 gene, the animal being a non-human primate, preferably the cell comprises a fertilized egg.
3. 3. The method of claim 1 or 2, wherein the downregulating comprises: targeting the fourth exon of the FMR1 gene to the target sequence SEQ ID NO 3, and/or Targeting the target sequence SEQ ID NO. 2 and/or SEQ ID NO.1 in the third exon of the FMR1 gene.
4. 4. The method of claim 3, wherein said down-regulating comprises targeting the target sequence of SEQ ID NO.3 in the fourth exon of said FMR1 gene and targeting the target sequence of SEQ ID NO.2 in the third exon of said FMR1 gene.
5. 5. The method of claim 3, wherein down-regulating comprises knocking out using a CRISPR gene editing method, preferably with sgRNA as a guide, wherein knocking out is performed: the nucleotide sequence of the sgRNA targeting the target sequence SEQ ID NO. 3 is shown as SEQ ID NO. 6; The sgRNA targeting the target sequence SEQ ID NO. 2 has a nucleotide sequence shown as SEQ ID NO. 5; the sgRNA targeting the target sequence SEQ ID NO. 1 has the nucleotide sequence shown as SEQ ID NO. 4.
6. 6. The method of claim 5, wherein the sgRNA and Cas mRNA or a construct capable of forming the sgRNA and Cas9 mRNA are introduced into fertilized eggs of an animal, preferably further comprising developing fertilized eggs to obtain the fragile X syndrome animal.
7. 7. The method of claim 1, wherein the fragile X syndrome animal has a phenotype selected from the group consisting of increased total activity and repetitive notch activity, increased anxiety, increased mood, reduced social desire, memory impairment, reduced cognitive flexibility, and attention deficit.
8. 8. Use of a fragile X syndrome animal prepared by the method of any one of claims 1-7 for: An animal model for screening candidate drugs or therapeutic agents for alleviating or treating fragile X syndrome; As an animal model for studying fragile X syndrome, or And (5) performing drug metabolism and toxicology detection.
9. 9. A method of screening for a candidate drug or therapeutic agent for alleviating or treating fragile-X syndrome, the method comprising: (1) Preparing a fragile X syndrome animal model by the method of any one of claims 1-7; (2) And (2) administering the candidate substance to the animal model in the step (1), observing whether the candidate substance has an alleviating or treating effect on fragile X syndrome, and if the symptom of fragile X syndrome of the animal model is observed to be alleviated, the candidate substance is a substance for alleviating or treating fragile X syndrome.
10. 10. The method of claim 9, wherein observing whether the candidate substance has an alleviating or treating effect on fragile X syndrome comprises analyzing a phenotype of activity, repetitive notch activity, vocalization, social intent, working memory, cognitive flexibility, attention; Preferably, the candidate substance is a substance that alleviates or treats fragile X syndrome if the amount of activity is varied less, the repeated activities of the clapping are varied less, the gurgling sounds are varied less or the total sounds are increased less, the social willingness is increased less, the working memory is enhanced, the cognitive flexibility is enhanced, and the attention tends to be concentrated.
11. 11. The method of claim 9, further comprising providing a control animal, wherein the control animal is not administered the candidate substance, when performing the observation.
12. 12. The method of claim 9, further comprising performing further cellular and/or animal experiments on the obtained potential substances to further select and determine substances from the candidate substances that are useful for alleviating or treating fragile X syndrome.

Description

Friable X syndrome animal model, construction and application thereof Technical Field The invention belongs to the fields of medicine and biology, and more particularly relates to a fragile X syndrome animal model, and construction and application thereof. Background Fragile X syndrome (Fragile X Syndrome, FXS) is a hereditary intellectual disorder disease with a incidence next to down syndrome, and is also the most common type of autism spectrum disorder, with a male incidence of 1/2500-1/5000 and a female incidence of 1/4000-1/6000.FXS patients have various clinical manifestations, mainly representing moderate to severe mental disorder, and often have IQ values of 20-60, and FXS patients also have abnormal appearance characteristics, such as forehead protrusion, large and protruding mandibular, large ears, high palate arch, thick lips, protruding lower lips, etc., and male males have large testes in puberty. Some patients exhibit behavioral disorders such as hyperactivity, autism (e.g., aggressive, social and pragmatic behaviors, etc.), and 20% have sporadic seizures. FXS is caused by mutation of FMR1 gene, the gene is located on an X chromosome q27.3, CGG repeated sequences exist in a 5 '-untranslated region (5' -UTR) of FMR1 gene, the number of CGG trinucleotide repeated sequences of normal FMR1 gene is generally between 6 and 54, the number of CGG trinucleotide repeated sequences of individuals with pre-mutation is 55-200, the copy number of CGG of women carrying the pre-mutation is unstable, and the CGG is easy to be converted into full mutation in the process of transferring to offspring. When the number of CGG trinucleotide repeats exceeds 200, referred to as total mutation, an epigenetic change occurs in the FMR1 gene, and the CGG repeats and the promoter of FMR1 are methylated, resulting in silencing of FMR1 gene expression. Over 99% of patients are caused by additional expansion (> 200 copies) of CGG trinucleotide repeats in the 5' -UTR of FMR1 gene, the remaining 1% being due to FMR1 gene point mutations and gene duplication/deletion. The FMRP protein coded by the FMR1 gene is expressed in various tissues of mammals, is particularly rich in brain and testis, and is expressed in brain neurons and various glial cells. FMRP is an RNA-binding protein whose expression can be regulated by binding to mRNA, and whose selectivity for RNA targets is largely dependent on two central KH domains and a C-terminal arginine-rich glycine domain. It is currently believed that FMRP is primarily responsible for the negative regulation of neuronal local protein expression, most of which are associated with dendritic growth and receptor signaling pathways, and that these regulated proteins are increased in expression following the absence of FMRP. At present, the specific mechanism of FXS caused by FMRP deficiency is not clear, and no specific medicine for treating FXS is clinically available, mainly for symptomatic treatment and supportive treatment. Animal models are an important basis for human disease mechanism research and new drug development. The current FXS animal models are drosophila model, zebra fish model, mouse model and rat model. But these animals also show great limitations in mimicking FXS disease. The drosophila homology gene of the FMR1 gene is Dfmr.sup.1. DFMRP-deleted Drosophila showed several FXS patients Associated phenotypes, including sleep problems, memory deficits, social interaction deficits, and neurodevelopment deficits. But they did not exhibit increased activity and anxiety phenotypes nor typical repetitive plating. The zebra fish model also exhibited some phenotypes in some FXS patients, including behavioral characteristics such as anxiety and hyperactivity, which were exhibited when Fmr knockout zebra fish were transferred to new fish tanks. Craniofacial dysplasia was also observed in Fmr knocked-out zebra fish larvae. But it does not exhibit cognitive impairment and social behavioral abnormalities nor typical repetitive plating behavior. In addition, the absorption and metabolism modes of the drug of the drosophila and the zebra fish are very different from those of human beings, so that the application of the drosophila and the zebra fish in FXS drug development has very large limitation. The Fmr knockout mouse model is the most used FXS animal model, the Fmr knockout mouse presents phenotypes of some FXS patients, has abnormal development of neuron dendritic spines and large testes, the Fmr knockout mouse also has abnormal behaviors such as overactivity, auditory epileptic seizure (not only sporadic seizure) and the like, but does not reach the typical repeated notch behavior. Furthermore, the autism-like phenotype common in FXS patients was not observed in mice of a few strains, the mouse model also lacked the most typical cognitive impairment in FXS patients, and the manifestation of social behavior (e.g. social impairment) was also lacked. Another non-negligible problem is that