Engineering of a mouse model of choline acetycholinesterase deficients using CRISPR/Cas9 gene editing

胆碱乙酰转移酶缺乏症小鼠模型的建立 CRISPR/CAS9基因编辑 科学摘要： 胆碱乙酰转移酶(ChAT)是一种从 乙酰辅酶A和胆碱在外周和中枢神经系统多种类型神经元胞体的分布 (CNS)。ChAT基因突变(ChAT)导致一种可能致命的人类先天性肌无力 综合征(CMS)是指与发作性呼吸暂停相关的CMS。聊天敲除(KO)鼠标是不能移动的 并在出生时死亡，条件Chat KO(Chatflx/FLOX/CRE-ERT2)小鼠在诱导ChAT几周后死亡 通过腹腔注射他莫昔芬进行消融。然而，致命的Chatflx/Flox/Cre-ERT2表型可以是 大多数情况下，在出生后第一天一次脑内注射2x1013Vg/kg腺相关药物即可获救 携带人类ChAT基因的9型病毒(AAV9-ChAT)。此外，注射的小鼠可以到达成年。 生活不会发展出明显的弱点。虽然这些结果令人鼓舞，但石油资源的普遍枯竭 在条件性基因敲除模型中诱导的Chat不是人类疾病的适当模型，也不是 在生命的第一天注射治疗剂反映了最有可能的治疗干预时间 在人类身上。因此，为了完善由于ChAT缺乏而导致的CMS模型，我们计划创建敲门(KI)小鼠 两个先前发现的人类ChAT突变，p.Val136 Met和p.Arg207His的纯合，使用 CRISPR/Cas9基因编辑。然后我们将尝试通过注射纠正ChatKI/Ki小鼠的表型 出生后早期脑池内AAV9-Chat。该项目的总体目标是产生一只动物 将尽可能真实地复制人类疾病的模型，并就 AAV9-ChAT介导的基因治疗对CMS动物模型的疗效 聊天变异。

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.