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疾病，其特征是 出现一阵阵瘫痪，常伴有发育异常。ATP1A3基因内的突变 编码Na/K ATPase神经元亚型的基因是AHC最常见的原因。AHC是 很罕见，目前还没有治愈方法。有两个因素是造成在以下方面缺乏进展的重要原因 治疗方法的发展。首先，与大多数罕见的遗传疾病一样，资源不足， 私营部门的财务动力有限。第二，基因突变导致 疾病是不被理解的。AHC突变是显性隐性的，目前尚不清楚Na/K突变是如何 ATPase干扰野生型版本，造成比预期更高的生理缺陷。对于任何 对于遗传性疾病，最直接的治疗方法将是纠正潜在的突变。从理论上讲，这可能是 通过编辑基因或其编码的信使RNA来完成。对于神经紊乱，基因编辑是 不切实际，因为使用CRISPR技术的最先进的系统在神经元中不能很好地工作。在……里面 此外，它们很难在体内传递，因为它们是基于细菌成分，而细菌成分很可能 会产生免疫并发症。最近，用于编辑mRNAs的新系统，称为定点RNA 编辑(SDRE)，为遗传病的治疗提供了明显的优势。首先，他们可以在 神经元，它们是基于人类自然产生的酶。另一个优势是它们是 相对简单，由连接到人类RNA编辑的小寡核苷酸引导RNA组成 酵素。由于遗传信息在不同细胞中的不同RNA之间以相同的方式编码，因此它 无论它在哪里表达，都可以以大致相同的方式进行编辑。这使得SDRE成为一种半通用的方法 不同的遗传病。在这项工作中，SDRE组件将被优化以高效和选择性地 纠正最常见的AHC(ATP1A3 D801N)突变。将确定顶级指南RNA 通过迭代选择程序从数十亿名随机候选者中选出。然后，这些将是 结合不同版本的工程RNA编辑酶在细胞中进行测试。这些试剂会 然后被包装成病毒颗粒，这样它们就可以有效地传递到细胞。同时， ATP1A3 D801N突变改变Na/K ATPase功能的机制将在以下两个方面进行研究 含有突变的酶和野生型的酶。这些实验将提供更好的 了解AHC的生理基础，并有助于估计 必须加以纠正，以抵消功能性赤字。综上所述，SDRE试剂的发展耦合 随着对AHC突变引起的异常生理的清楚了解，我们将能够开始 开发出治疗这种疾病的第一种疗法。

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.