使用 CRISPR/Cas9 基因编辑构建胆碱乙酰胆碱酯酶缺陷小鼠模型-猫眼课题宝

使用 CRISPR/Cas9 基因编辑构建胆碱乙酰胆碱酯酶缺陷小鼠模型

批准号：

10511979

负责人：

RICARDO Anibal MASELLI

金额：

$15.98万

依托单位：

UNIVERSITY OF CALIFORNIA AT DAVIS

依托单位国家：

美国

项目类别：

财政年份：

2022

资助国家：

美国

项目状态：

已结题

起止时间：

2022-07-01 至 2024-06-30

来源：

https://reporter.nih.gov/project-details/10511979

关键词：

Ablation Acetyl Coenzyme A Acetylcholine Address Adult Affect Albuterol Animal Model Animals Apnea Biodistribution Biological Birth CRISPR/Cas technology Canis familiaris Categories Cessation of life Characteristics Child Choline Choline O-Acetyltransferase Cholinesterase Inhibitors Collection Communities Confocal Microscopy Congenital Myasthenic Syndromes Data Dependovirus Development Diagnosis Disease Disease model Dose Engineering Enzymes Ephedrine Evaluation Experimental Models Family Feasibility Studies Foundations Future Gene Mutation Genes Goals Hand Strength Human Immunohistochemistry Impairment Injection of therapeutic agent Injections Intraperitoneal Injections Investigational Therapies Knock-in Knock-in Mouse Knock-out Knockout Mice Life Location Measures Mediating Medical Mind Modeling Motor Neurons Mouse Strains Mus Muscle Mutant Strains Mice Mutate Mutation Neuraxis Neurons Neurotransmitters Online Mendelian Inheritance In Man Pathogenesis Pathogenicity Patients Peripheral Persons Pharmaceutical Preparations Pharmacological Treatment Phenotype Prevalence Proteins Rare Diseases Reporting Research Single-Gene Defect Spinal Sympathomimetics Synapses Tamoxifen Testing Therapeutic Intervention Time Toxic effect Transgenic Mice Transgenic Organisms Translating Treatment Efficacy Tropism Variant Viral Vector Western Blotting amifampridine base conditional knockout efficacy evaluation gene therapy genetic pedigree genome editing human disease in vivo in vivo evaluation motor deficit mouse model neuronal cell body postnatal postsynaptic presynaptic pyridostigmine repository response stillbirth therapeutically effective

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ENGINEERING OF A MOUSE MODEL OF CHOLINE ACETYLTRANSFERASE DEFICIENCY USING CRISPR/CAS9 GENE EDITING Scientific Abstract: Choline acetyltransferase (ChAT) is the enzyme that resynthesizes the neurotransmitter acetylcholine, from acetyl-CoA and choline at the soma of multiple types of neurons of the peripheral and central nervous system (CNS). Mutations in the ChAT gene (CHAT) result in a potentially fatal form of human congenital myasthenic syndrome (CMS) referred to as CMS associated with episodic apneas. Chat knockout (KO) mice are immobile and die at birth, and conditional Chat KO (Chatflox/flox/Cre-ERT2) mice die a few weeks after the induction of Chat ablation by an intraperitoneal injection of tamoxifen. However, the lethal Chatflox/flox/Cre-ERT2 phenotype can be rescued in most cases by a single intracranial injection at the first day of life of 2x1013 vg/kg adeno-associated virus type 9 (AAV9) carrying the human CHAT gene (AAV9-CHAT). Furthermore, injected mice can reach adult life without developing obvious weakness. While these results are encouraging, the widespread depletion of Chat induced in the conditional knock out model is not an adequate model of the human disease, nor the injection of the therapeutic agent at the first day of life reflects the most likely time of therapeutic interventions in humans. Thus, to refine the model of CMS due to ChAT deficiency, we plan to create knock in (KI) mice homozygous for two previously identified human CHAT mutations, p.Val136 Met and p.Arg207His, using CRISPR/Cas9 gene editing. We will then attempt to correct the phenotype of ChatKI/KI mice by injecting intracisternally AAV9-CHAT during early postnatal life. The overall goal of the project is to generate an animal model that will reproduce as faithfully as possible the human disease and to conduct a pilot feasibility study on the efficacy of AAV9-CHAT mediated gene therapy in this newly developed animal model of CMS caused by CHAT mutations.

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