Project 4: Therapeutic Gene Editing for Rett Syndrome

项目4（Rett） Rett综合征（RTT）是一种由MECP 2基因突变引起的X连锁神经发育障碍。 大多数RTT是由第三和第四突变热点中相同的8个重复发生的突变引起的。 第四外显子。这四个错义突变和四个无义突变（R106 W，R133 C，T158 M，R168 X， R255 X、R270 X、R294 X和R306 C）占所有Rett病例的70%。RTT主要影响女性， 发生率为1- 10，000例活产女婴，并呈现为 6-18个月的年龄。Rett的标志性特征包括有目的的手部技能的逐渐丧失， 以及语言退化、步态异常和刻板的手部运动。疾病的其他特征 包括头部生长减速、睡眠障碍、呼吸异常和癫痫发作率高。 Rett患者的预期寿命通常在40-50岁左右。里程碑式的研究， Mecp 2的条件性小鼠模型，以打开内源性Mecp 2小鼠基因后， 令人信服地证明，与Mecp 2丢失相关的神经功能缺损是可逆的， ℃下到目前为止，临床试验集中在调节下游机制的小分子上， MECP 2。随着更多的25+这样的试验，有限的成功已被证明只有适度的行为 没有FDA批准的RTT药物。基因和蛋白质替代策略受到阻碍 关于需要精确滴定MECP 2的受控剂量影响，如Mecp 2在 细胞是有毒的，并且进一步被疾病的X连锁性质与MECP 2的细胞间变异所混淆 由染色体X连锁失活引起的表达。基于这些原因， 直接纠正内源性基因突变并将MECP 2恢复到内源性细胞水平是一种有效的方法。 极具吸引力的RTT治疗策略。 在这个追随者项目中，我们的目标是纠正错义和无义突变，以拯救疾病。 Rett患者的进展。具体而言，我们的目标是：（1）设计，测试和优化基础编辑，总理编辑 （2）在小鼠模型中测试这些策略，所述小鼠模型含有： 具有工程化错义和无义突变的人源化外显子。(3)执行临床前IND启用 评估安全性和有效性的研究。我们将与基因编辑核心紧密合作，开发最新的基地 编辑和/或主要编辑技术。我们将与基因编辑核心合作， 确保我们的基因组编辑工具最大限度地提高靶向编辑效率， 本发明提供了一种新的生物技术，其可以减少副产物和脱靶编辑事件，并最大限度地提高与潜在的体内递送方法的相容性。 治疗相关性

PROJECT SUMMARY PROJECT 4 (Rett) Rett syndrome (RTT) is an X-linked neurodevelopmental disorder caused by mutations in the gene MECP2. The majority of RTT is caused by the same d eight reoccurring mutations in mutational hotspots of the 3rd and 4th exons. These four missense mutations and four nonsense mutations (R106W, R133C, T158M, R168X, R255X, R270X, R294X, and R306C) make up 70% of all Rett cases. RTT predominantly affects females, occurring at an incidence of 1-10,000 live female births and presents as a regression in milestones between the ages of 6-18 months. Hallmark characteristics of Rett include progressive loss of purposeful hand skills, speech and language regression, gait abnormalities, and stereotypic hand movements. Other features of the disorder include decelerated head growth, sleep disturbances, breathing abnormalities, and a high incidence of seizures. The life expectancy for patients with Rett is typically around 40-50 years of age. Landmark studies using conditional mouse models of Mecp2 to turn on the endogenous Mecp2 mouse gene post-symptomatically have convincingly demonstrated that neurological deficits associated with loss of Mecp2 is reversible to a significant degree. To date, clinical trials have focused on small molecules that modulate mechanisms downstream of MECP2. With more the 25+ such trials, limited success has been demonstrated with only modest behavior modifications and no approved FDA drugs for RTT. Gene and protein replacement strategies have been stymied with regards to controlled dosage affects that require precise titration of MECP2, as overexpression of Mecp2 in cells is toxic and are further confounded by the X-linked nature of the disease with cell to cell variation of MECP2 expression resulting from chromosome X-linked inactivation. For these reasons, base editing strategies that directly correct the endogenous genetic mutation and restore MECP2 to endogenous cellular levels are an extremely attractive therapeutic strategy for RTT. In this follower project, we aim to correct missense and nonsense mutations to enable rescue of disease progression in Rett patients. Specifically, we aim to: (1) Design, test and optimize Base Editing, Prime editing and Twin Editing strategies for 5 different mutations; (2) Test these strategies in mouse models containing humanized exons with engineered missense and nonsense mutations. (3) Perform pre-clinical IND enabling studies to assess safety and efficacy. We will work closely with the Gene Editing Core to develop the latest base editing and/or prime editing technologies in RTT model systems. We will iterate with the Gene Editing Core to ensure that our genome editing tools maximize on-target editing efficiencies, minimize undesirable gene editing byproducts and off-target editing events, and maximize compatibility with in vivo delivery methods of potential therapeutic relevance.