Correction of Mutations Underlying Alternating Hemiplegia of Childhood by Site-Directed RNA Editing

项目摘要 儿童交替性偏瘫（AHC）是一种毁灭性的神经系统疾病，其特征是 麻痹发作，并经常伴有发育异常。ATP 1A 3内的突变 编码Na+/K+ ATP酶的神经元亚型的基因是AHC的最常见原因。AHC是 罕见，目前还没有治愈方法。有两个因素在很大程度上造成了这方面缺乏进展。 治疗的发展。首先，与大多数罕见的遗传疾病一样，资源不足， 私营部门的财政动机有限。第二，突变引起突变的机制。 疾病不了解。AHC突变是显性隐性的，目前还不清楚突变的Na+/K+ ATP酶干扰野生型产生高于预期的生理缺陷。任何 对于遗传性疾病，最直接的治疗方法是纠正潜在的突变。理论上，这可能是 通过编辑基因或其编码的信使RNA来完成。对于神经疾病，基因编辑是 这是不切实际的，因为使用CRISPR技术的最先进系统在神经元中效果不佳。在 此外，它们难以在体内递送，因为它们是基于细菌组分， 产生免疫并发症。最近，新的编辑mRNA的系统，称为定点RNA， 基因编辑（SDRE）为治疗遗传疾病提供了明显的优势。首先，他们可以在 神经元，它们是基于人体内天然存在的酶。另一个优点是， 相对简单，由一个小的寡核苷酸指导RNA偶联到一个人RNA编辑 酵素由于不同细胞中不同RNA之间的遗传信息编码方式相同， 无论在哪里表达，都可以用大致相同的方式进行编辑。这使得SDRE成为一种半通用方法， 不同的遗传疾病在这项工作中，SDRE组件将被优化，以有效地和有选择地 纠正AHC最常见的突变（ATP 1A 3 D801 N）。将鉴定最佳指导RNA 从数十亿随机候选人的池中通过迭代选择程序。这些将是 在细胞中与不同版本的工程RNA编辑酶组合进行测试。这些试剂将 然后被包装成病毒颗粒，以便它们可以有效地递送到细胞。同时， 将研究ATP 1A 3 D801 N突变改变Na+/K+ ATP酶功能的机制， 含有突变的酶和野生型酶。这些实验将提供更好的 了解AHC的生理基础，并帮助提供突变体的比例估计， 必须纠正以弥补功能缺陷。综上所述，SDRE试剂的发展与 对AHC突变引起的异常生理学有了清楚的了解，将使我们开始 开发出第一种治疗这种疾病的药物。

Project Summary Alternating Hemiplegia of Childhood (AHC) is a devastating neurological disorder that is characterized by bouts of paralysis and is often accompanied by developmental abnormalities. Mutations within the ATP1A3 gene, which encodes a neuronal isoform of the Na+/K+ ATPase, are the most common cause of AHC. AHC is rare, and at present there is no cure. Two factors significantly contribute to the lack of progress in the development of treatments. First, as with most rare genetic disorders, there are insufficient resources and limited financial motivation in the private sector. Second, the mechanisms by which the mutations cause the disease are not understood. AHC mutations are dominant recessive, and it is unclear how mutant Na+/K+ ATPases interfere with wild type versions to create physiological deficits that are higher than expected. For any genetic disorder, the most direct treatment would be to correct the underlying mutation. In theory, this could be accomplished by editing the gene or the messenger RNA that it encodes. For neural disorders, gene editing is not practical because the most advanced systems using CRISPR technology don’t work well in neurons. In addition, they are difficult to deliver in vivo because they are based on bacterial components which will likely generate immunological complications. Recently, new systems for editing mRNAs, called site-directed RNA editing (SDRE), offer distinct advantages for the treatment of genetic diseases. First, they can operate in neurons, and they are based on enzymes that occur naturally in humans. Another advantage is that they are relatively simple, being composed of a small oligonucleotide guide RNA coupled to a human RNA editing enzyme. Because genetic information is encoded the same way between different RNAs in different cells, it can be edited in much the same way wherever it is expressed. This make SDRE a semi-generic approach for different genetic disorders. In this work, SDRE components will be optimized to efficiently and selectively correct the most frequent mutation that underlies AHC (ATP1A3 D801N). Top guide RNAs will be identified from pools of billions of randomized candidates through an iterative selection procedure. These will then be tested in cells in combination with different versions of engineered RNA editing enzymes. These reagents will then be packaged into virus particles so that they can be efficiently delivered to cells. Simultaneously, the mechanisms by which the ATP1A3 D801N mutation alters Na+/K+ ATPase function will be studied, both in enzymes that contain the mutation and in wild type enzymes. These experiments will provide a better understanding of the physiological basis of AHC and help provide estimates of the proportion of mutants that must be corrected to offset functional deficits. Taken together, the development of SDRE reagents coupled with a clear understanding of the aberrant physiology caused by AHC mutations will allow us to begin to develop the first therapeutics for this condition.