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，主要表现在没有 有效的治疗。RTT是由甲基CpG结合蛋白2基因功能缺失突变引起的 (MeCP2)位于X染色体上。大多数RTT患者是MECP2突变杂合子的女性， 在发育过程中随机X染色体失活(XCI)使大约50%的神经元没有 发挥功能的MeCP2蛋白，从而造成细胞自主神经元功能障碍。基因中的相应突变 半合子男性会导致严重的新生儿脑病和过早死亡。携带MeCP2零等位基因的小鼠 与患者的症状非常相似，包括呼吸不规律，肢体运动和 缩短了寿命，因此是RTT的忠实典范。MeCP2基因缺失的雄性小鼠开始出现症状 早在30-60天龄，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，神经功能障碍延迟(~6个月)，严重程度高度不同。一个 RTT研究的重大突破是证明了成年小鼠的RTT样症状可以是 通过基因或病毒恢复MeCP2蛋白而逆转。因此，沉默的野生型(WT)等位基因的重新激活 来自不活跃的X染色体的MECP2(Xi)提供了一个令人兴奋的攻击根部的治疗机会 通过恢复MeCP2的功能来引起这种疾病。我们的初步数据表明， MECP2启动子去甲基化足以重新激活人RTT ESCs和 在体外和体内RTT小鼠大脑中的神经元。这是使用dCas9-Tet1蛋白完成的 通过sgRNA定位到MECP2基因座，sgRNA是由Shawn Liu博士首创的DNA甲基化编辑工具。我们 假设从XI重新激活MECP2将挽救小鼠的RTT相关表型。我们有 开发了两种新的转基因小鼠模型(1，2)和两种新的传递表观遗传编辑的方法(3，4) 对于MECP2在体内的重新激活，包括：1)XI连接的MeCP2-纳米荧光素酶-td番茄双报告小鼠， 这使得能够高灵敏度地检测和量化MeCP2重新激活；2)MeCP2-空杂合子 携带WT MeCP2的重度RTT女性模型 测量重新激活诱导的救援，绕过评估MeCP2的延迟和可变表型 杂合子随机XCI；3)Cre重组酶依赖的dCas9-Tet1转基因系使高效 和体内组织特异性DNA甲基化编辑；以及4)dCasMini-Tet1，其中笨重的dCas9(4.1kb)是 替换为紧凑的dCasMini(1.6kb)，用于通过单个AAV9载体传递甲基化编辑器。这 转基因模型的组合，以测量重新激活效果(1)和救援(2)与那些使 通过遗传手段(3)和单载体AAV9介导的递送(4)进行的有效体内编辑包括 评估MECP2再激活策略治疗RTT和其他疾病的体内可行性的ART工具包 女性中的X连锁障碍。

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.