Vollum Institute, Oregon Health and Science University, Portland, OR 97239.
Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR 97239.
Proc Natl Acad Sci U S A. 2017 Oct 31;114(44):E9395-E9402. doi: 10.1073/pnas.1715320114. Epub 2017 Oct 16.
Rett syndrome (RTT) is a debilitating neurological disorder caused by mutations in the gene encoding the transcription factor Methyl CpG Binding Protein 2 (MECP2). A distinct disorder results from gene duplication, suggesting that therapeutic approaches must restore close to normal levels of MECP2. Here, we apply the approach of site-directed RNA editing to repair, at the mRNA level, a disease-causing guanosine to adenosine (G > A) mutation in the mouse MeCP2 DNA binding domain. To mediate repair, we exploit the catalytic domain of Adenosine Deaminase Acting on RNA (ADAR2) that deaminates A to inosine (I) residues that are subsequently translated as G. We fuse the ADAR2 domain, tagged with a nuclear localization signal, to an RNA binding peptide from bacteriophage lambda. In cultured neurons from mice that harbor an RTT patient G > A mutation and express engineered ADAR2, along with an appropriate RNA guide to target the enzyme, 72% of mRNA is repaired. Levels of MeCP2 protein are also increased significantly. Importantly, as in wild-type neurons, the repaired MeCP2 protein is enriched in heterochromatic foci, reflecting restoration of normal MeCP2 binding to methylated DNA. This successful use of site-directed RNA editing to repair an endogenous mRNA and restore protein function opens the door to future in vivo applications to treat RTT and other diseases.
雷特综合征（RTT）是一种由编码转录因子甲基化 CpG 结合蛋白 2（MECP2）的基因突变引起的致残性神经发育障碍。由于基因重复导致了一种截然不同的疾病，这表明治疗方法必须使 MECP2 接近正常水平。在这里，我们应用靶向 RNA 编辑的方法，在信使 RNA 水平上修复小鼠 MECP2 DNA 结合域中引起疾病的鸟嘌呤到腺嘌呤（G > A）突变。为了介导修复，我们利用腺苷脱氨酶作用于 RNA（ADAR2）的催化结构域，该结构域将 A 脱氨为肌苷（I）残基，随后将其翻译为 G。我们将 ADAR2 结构域与来自噬菌体 lambda 的 RNA 结合肽融合，并标记核定位信号。在携带 RTT 患者 G > A 突变并表达工程化 ADAR2 的培养神经元中，有 72%的 mRNA 被修复。MeCP2 蛋白水平也显著增加。重要的是，与野生型神经元一样，修复后的 MeCP2 蛋白在异染色质焦点中富集，反映了正常 MeCP2 与甲基化 DNA 的结合得到了恢复。这种成功地使用靶向 RNA 编辑修复内源性 mRNA 并恢复蛋白功能为未来治疗 RTT 和其他疾病的体内应用开辟了道路。
