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CN-121992041-A - Method for establishing angel syndrome animal model by targeting UBE3A allele and application

CN121992041ACN 121992041 ACN121992041 ACN 121992041ACN-121992041-A

Abstract

The invention provides a method for establishing an angel syndrome animal model by targeting UBE3A maternal alleles and application thereof. On the basis of extensive and intensive research and screening for the sequence of primate genomic UBE3A genes, targets suitable for genetic engineering are obtained, namely upstream introns and/or downstream introns of maternal UBE3A gene exon5, and angel syndrome animal models can be obtained by downregulating the targets. The animal model has stable and controllable state, typical disease phenotype and easy realization of observation.

Inventors

  • XIONG ZHIQI
  • SUN QIANG
  • ZHUANG LING
  • ZHANG XIAOLEI
  • LI CHUNYANG
  • NIE YANHONG
  • ZHANG RUNZE
  • 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
20260508
Application Date
20241106

Claims (13)

  1. 1. A method of preparing an animal with angel's syndrome comprising down-regulating a parent UBE3A gene of the animal, said down-regulation targeting an upstream intron and/or a downstream intron of the parent UBE3A gene exon5, wherein said animal is a non-human primate.
  2. 2. A method of making an animal cell comprising down-regulating a parent UBE3A gene in an animal cell, said down-regulation targeting an upstream intron and/or a downstream intron of the parent UBE3A gene exon5, said animal being a non-human primate, preferably said cell comprising a fertilized egg.
  3. 3. The method of claim 1 or 2, wherein down-regulating comprises targeting a target sequence of SEQ ID NO 3, SEQ ID NO 1 or SEQ ID NO 2 in an upstream intron of the parent UBE3A gene exon5 and/or targeting a target sequence of SEQ ID NO 4 or SEQ ID NO 5 in a downstream intron of the parent UBE3A gene exon 5.
  4. 4. The method of claim 1 or 2, wherein the down-regulation targets the target sequence SEQ ID NO. 4 in the downstream intron of the parent UBE3A gene exon5 and the target sequence SEQ ID NO. 3, SEQ ID NO.1 or SEQ ID NO.2 in the upstream intron.
  5. 5. The method of claim 4, 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. 8; the sgRNA targeting the target sequence SEQ ID NO.1 has a nucleotide sequence 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. 7; The sgRNA targeting the target sequence SEQ ID NO. 4 has a nucleotide sequence as shown in SEQ ID NO. 9, or The sgRNA targeting the target sequence SEQ ID NO. 5 has the nucleotide sequence shown as SEQ ID NO. 10.
  6. 6. The method of claim 5, wherein the sgRNA and the Cas mRNA or a construct capable of forming the sgRNA and the Cas mRNA are introduced into fertilized eggs of an animal, preferably further comprising developing the fertilized eggs to obtain the angel syndrome animal.
  7. 7. For the preparation of sgRNA of an Angel syndrome animal, which targets the target sequence SEQ ID NO 3, SEQ ID NO 1 or SEQ ID NO 2 in the upstream intron of the parent UBE3A gene exon5 of the animal and/or targets the target sequence SEQ ID NO 4 or SEQ ID NO 5 in the downstream intron of the parent UBE3A gene exon 5.
  8. 8. The sgRNA of claim 7, wherein the sgRNA is a combination of sgRNAs comprising a target sequence SEQ ID NO. 4 targeted to a downstream intron of the parent UBE3A gene exon5 and a target sequence SEQ ID NO.3, SEQ ID NO. 1 or SEQ ID NO. 2 in an upstream intron.
  9. 9. The sgrnas for use in the preparation of angel syndrome animals of claim 8, wherein the nucleotide sequence of the sgrnas is shown as SEQ ID NO. 8, the nucleotide sequence of the sgrnas is shown as SEQ ID NO.6, the nucleotide sequence of the sgrnas is shown as SEQ ID NO. 2, the nucleotide sequence of the sgrnas is shown as SEQ ID NO. 7, the nucleotide sequence of the sgrnas is shown as SEQ ID NO. 4, the nucleotide sequence of the sgrnas is shown as SEQ ID NO. 9, or the nucleotide sequence of the sgrnas is shown as SEQ ID NO. 10.
  10. 10. The use of the angel syndrome animal prepared by the method of any one of claims 1-6 for: An animal model for screening candidate drugs or therapeutic agents for alleviating or treating angel syndrome; as an animal model for studying Angel syndrome, or And (5) performing drug metabolism and toxicology detection.
  11. 11. A kit for preparing an angel syndrome animal, comprising the sgRNA for preparing an angel syndrome animal according to any one of claims 6 to 9.
  12. 12. The kit of claim 11, wherein the kit further comprises a Cas mRNA or a construct capable of forming a Cas mRNA.
  13. 13. A method of screening for a candidate drug or therapeutic agent for alleviating or treating angel's syndrome, the method comprising: (1) Preparing an angel syndrome animal model using any of the methods described above; (2) The candidate substance is administered to the animal model of (1), and whether the candidate substance has an alleviation or treatment effect on angel's syndrome is observed, and if the symptom of angel's syndrome of the animal model is observed to be alleviated, the candidate substance is a substance for alleviating or treating angel's syndrome, and preferably, the observation includes analysis of phenotypes such as motor function and cognitive function.

Description

Method for establishing angel syndrome animal model by targeting UBE3A allele and application Technical Field The invention belongs to the field of medicine, and more particularly relates to a method and application of targeting UBE3A alleles to establish angel syndrome animal models. Background Angel syndrome (Angelman syndrome, AS) is a neurogenetic disorder, and patients mainly show symptoms such AS intellectual impairment, language deficiency, sleep disorder, dyskinesia, seizures, and happy expression. It is estimated that angel's syndrome is about 1/20,000-1/12,000 and that men and women are similar in prevalence. Their life expectancy is near normal and full-time home care is required. There is no approved therapy dedicated to the treatment of AS, mainly intervention training therapy and symptomatic therapy in clinic, mainly aimed at reducing seizures and improving sleep. In 2018, AS was recorded in the "first few rare diseases catalog" of our country. AS is due to a deletion or defective expression of the UBE3A parent allele in the q11-13 region of chromosome 15. In neurons of the central nervous system, the UBE3A gene is affected by genomic imprinting, in which maternal alleles are expressed and paternal alleles are not. There are various molecular genetic mechanisms leading to AS, one due to the deletion of a large fragment (6 Mb in size) of the q11-13 region of chromosome 15 (containing the UBE3A gene) of the parent source, one due to the point mutation of the UBE3A allele of the parent source, one due to the point mutation of the parent source, one due to the parent-diploid, one due to the inheritance of two epigenetic silenced parent UBE3A alleles, and one due to the microdeletion of the center of the imprinting of the q11-q13 region of chromosome 15 of the parent source, which is approximately 2-4% of cases. The deletion of the UBE3A gene is sufficient to cause the core phenotype of AS disease, therefore UBE3A is considered a key gene leading to AS. At present, AS animal models mainly comprise a drosophila model, a mouse model and a rat model. Drosophila Ube3a (Dube 3 a) is highly homologous to humans in amino acid sequence, suggesting that their function may be conserved, and researchers have found that knockout of Dube3a does not affect survival of Drosophila. However, loss of Dube3a activity reduces the dendritic branches of sensory neurons in the peripheral nervous system and slows down the progression of terminal dendrite elaboration. Another study showed that Drosophila with the Dube3a protein deleted exhibited abnormal motor behavior and circadian rhythm, AS well AS defective long-term memory, and that Dube3a protein exhibited loss of function after introducing missense mutations found in AS patients in Drosophila Dube3a gene. The AS mouse model which is established at present comprises a mouse model for knocking out the parent Ube3A gene exon2, a mouse model for knocking out the parent Ube3A gene exon15-16, a UBE3A-Gabrb large fragment knocked-out mouse model and the like. Wherein a relatively large number of AS mouse models were created by knocking out Ube3a gene exon2, the mouse Ube3a gene exon2 being conserved in the major isochrom, the expression cassette being frameshifted after the knocking out such that Ube3a protein ceases expression prematurely, ube3a m-/p+ heterozygous mice of the model exhibit reduced brain weight, ataxia, dyskinesia and abnormal EEG patterns. Another AS mouse model was created by knocking out the corresponding exon15-16 of the human UBE3A gene, and researchers introduced LacZ reporter gene after deletion site to facilitate detection of truncated protein expression. The truncated protein ubiquitin ligase activity is lost and the mouse model shows motor deficits, learning and memory disorders, abnormal EEG patterns and sleep-wake cycle disorders, but no seizures. Although the deletion of the UBE3A gene is sufficient to elicit the core phenotype of AS, the deletion of the large fragment of maternal chromosomes 15q11-q13 is most common in patients, and such patients have a more severe clinical phenotype, probably due to the single dose deficiency of neighboring genes such AS GABRB and ATP 10A. To create a mouse model simulating 15q11-q13 deficient patients, researchers knocked out the sequence of the 1.6Mb region of the 15q11-q13 female source of the mice, containing three genes Ube3a, atp10a and Gabrb, using Cre/loxP and Hprt (hypoxanthine-guanine phosphoribosyl transferase) techniques, AS well AS mice deleted by Ube3a alone, the large fragment deleted mice also exhibited AS-related defects, including abnormal EEG patterns, seizures, and motor and cognitive dysfunction, unfortunately the large fragment deleted mice did not exhibit a more severe phenotype than mice deleted by Ube3a alone. Although some animal models exist in the art that mimic to some extent the classical characteristics associated with many AS patients, and provide a tool to study molecular pat