MeCP2 reactivation from the inactive X chromosome as treatment for Rett syndrome

项目摘要 Rett综合征（RTT）是一种严重的X连锁神经发育障碍，主要表现在女孩身上， 有效治疗。RTT是由甲基CpG结合蛋白2基因的功能缺失突变引起的 （MECP 2）在X染色体上。大多数RTT患者是MECP 2突变杂合子的女性， 其中，在发育过程中随机X染色体失活（XCI）使~50%的神经元没有 功能性MeCP 2蛋白，从而产生细胞自主神经元功能障碍。相应的突变 半合子雄性导致严重的新生儿脑病和早期死亡。携带Mecp 2无效等位基因的小鼠 与患者的症状非常相似，包括呼吸不规则，刻板的肢体运动， 寿命缩短，因此是RTT的忠实模型。雄性Mecp 2缺失小鼠开始表现出症状， 早在30-60日龄，只有一半的动物存活超过75天。杂合子女性有 接近正常的寿命，神经功能缺损延迟（约6个月），严重程度差异很大。一 RTT研究的一个重大突破是证明了成年小鼠的RTT样症状可以 通过MeCP 2蛋白的遗传或病毒恢复来逆转。因此，沉默的野生型（WT）等位基因的再激活 来自失活X染色体（Xi）的MECP 2的表达提供了一个令人兴奋的治疗机会， 通过恢复MeCP 2功能来治疗这种疾病。我们的初步数据显示， MECP 2启动子的去甲基化足以在人RTT ESC中从Xi重新激活MECP 2， 在体外以及在RTT小鼠脑中的体内的神经元。这是使用dCas 9-Tet 1蛋白完成的 通过sgRNA靶向MECP 2基因座，这是Shawn Liu博士开创的DNA甲基化编辑工具。我们 假设来自Xi MECP 2的再活化将拯救小鼠中的RTT相关表型。我们有 开发了两种新的转基因小鼠模型（1，2）和两种新的外遗传编辑器递送方法（3，4） 用于体内MECP 2再活化的小鼠包括：1）X1-连接的Mecp 2-纳米荧光素酶-tdTomato双报告基因小鼠， 其使得能够高灵敏度检测和定量Mecp 2再激活; 2）MeCP 2缺失杂合 携带WT Mecp 2的X染色体完全失活的重度RTT女性模型， 测量再活化诱导的拯救，其避免评估Mecp 2中的延迟和可变表型 与随机XCI杂合; 3）Cre重组酶依赖性dCas 9-Tetl转基因系使得能够高效地 和体内组织特异性DNA甲基化编辑;以及4）dCasMini-Tetl，其中大体积dCas 9（4.1 kb）被 用紧凑的dCasMini（1.6kb）替换，用于经由单个AAV 9载体递送甲基化编辑器。这 将用于测量再激活功效（1）和拯救（2）的转基因模型与能够 通过遗传手段的有效体内编辑（3）和单一载体AAV 9介导的递送（4）包括一种 评估用于治疗RTT和其他疾病的MECP 2再激活策略的体内可行性的现有技术工具包 女性的X连锁疾病。

Project Summary Rett syndrome (RTT) is a severe X-linked neurodevelopmental disorder that mainly manifests in girls without effective treatment. RTT is caused by loss-of-function mutations of the Methyl CpG-binding Protein 2 gene (MECP2) on the X chromosome. The majority of RTT patients are females heterozygous for MECP2 mutation, in which random X chromosome inactivation (XCI) during development leaves ~50% of neurons without functional MeCP2 protein, thereby creating cell-autonomous neuronal dysfunction. Corresponding mutations in hemizygous males lead to severe neonatal encephalopathy and early death. Mice carrying null alleles of Mecp2 closely mimic symptoms seen in patients, including irregular breathing, stereotypical limb movements and shortened lifespan, and are thus faithful models of RTT. Male Mecp2-null mice start exhibiting symptoms as early as 30-60 days of age, with only half of the animals surviving beyond 75 days. Heterozygous females have a near-normal lifespan with neurologic deficits that are delayed (~6 months) and highly variable in severity. A major breakthrough in RTT research was the demonstration that RTT-like symptoms in adult mice can be reversed by genetic or viral restoration of MeCP2 protein. Thus, reactivation of the silenced wild type (WT) allele of MECP2 from the inactive X chromosome (Xi) presents an exciting therapeutic opportunity that attacks the root cause of this disease by restoring MeCP2 function. Our preliminary data demonstrate that targeted demethylation of the MECP2 promoter is sufficient to reactivate MECP2 from the Xi in human RTT ESCs and neurons in vitro as well as in vivo in the RTT mouse brain. This was accomplished using a dCas9-Tet1 protein targeted to the MECP2 locus via sgRNA, a DNA methylation editing tool pioneered by Dr. Shawn Liu. We hypothesize that reactivation of MECP2 from the Xi will rescue RTT-associated phenotypes in mice. We have developed two new transgenic mouse models (1,2) and two novel methods of delivery of epigenetic editors (3,4) for MECP2 reactivation in vivo including: 1) Xi-linked Mecp2-NanoLuciferase-tdTomato dual reporter mice, which enables high sensitivity detection and quantification of Mecp2 reactivation; 2) MeCP2-null heterozygous female model of severe RTT with exclusive inactivation of the X-chromosome harboring the WT Mecp2 for measuring reactivation-induced rescue which circumvents evaluating delayed and variable phenotypes in Mecp2 heterozygous with random XCI; 3) Cre recombinase-dependent dCas9-Tet1 transgenic line enables efficient and tissue-specific DNA methylation editing in vivo; and 4) dCasMini-Tet1, in which the bulky dCas9 (4.1 kb) is replaced with a compact dCasMini (1.6 kb) for delivery of a methylation editor via a single AAV9 vector. This combination of transgenic models to measure reactivation efficacy (1) and rescue (2) with those that enable efficient in vivo editing via genetic means (3) and single vector AAV9-mediated delivery (4) comprise a state-of- the-art tool kit to evaluate the in vivo feasibility of a MECP2 reactivation strategy for treatment of RTT and other X-linked disorders in females.