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

通过定点 RNA 编辑纠正儿童交替性偏瘫的突变

• 批准号: 10354983
• 负责人: JOSHUA J.C. ROSENTHAL
• 金额: $ 45.65万
• 依托单位: MARINE BIOLOGICAL LABORATORY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10354983
• 关键词: ATP1A3 gene; Address; Adenosine; Affinity; Animal Model; Bacterial Proteins; Binding; Biological Assay; Brain; CRISPR/Cas technology; Capsid; Catalytic Domain; Cells; Childhood; Codon Nucleotides; Complement; Coupled; DNA Sequence Alteration; DRADA2b protein; Dangerousness; Deamination; Development; Disease; Dominant-Negative Mutation; Electrophysiology (science); Engineering; Enzymes; Event; Evolution; Family; Foundations; Genes; Genetic; Genetic Diseases; Guanosine; Guide RNA; Homeostasis; Human; Immune response; Immunologics; Impairment; In Vitro; Inflammatory; Inosine; Instruction; Ion Transport; Ions; Ligands; Mediating; Messenger RNA; Mitotic; Motivation; Mutation; Na(+)-K(+)-Exchanging ATPase; Neurodevelopmental Disorder; Neurons; Oligonucleotides; Outcome; Paralysed; Phenotype; Physiological; Physiology; Plasmids; Positioning Attribute; Private Sector; Procedures; Process; Protein Isoforms; Pump; RNA; RNA Editing; Randomized; Reagent; Resources; Sensory; Site; Structure; System; Testing; Therapeutic; Translations; Virion; Virus; Work; Workplace; advanced system; alternating hemiplegia; autosomal dominant mutation; base; cognitive function; deep sequencing; design; disease-causing mutation; dsRNA adenosine deaminase; effective therapy; experimental study; gene therapy; genetic information; in vivo; inhibitory neuron; motor impairment; mutant; mutation correction; nervous system disorder; novel therapeutics; operation; rare genetic disorder; relating to nervous system; targeted treatment; theories; therapeutically effective; therapy development; transcriptome

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.

项目摘要 童年的交替偏瘫（AHC）是一种毁灭性的神经系统疾病，其特征是 瘫痪的回合，通常伴有发育异常。 ATP1A3中的突变 编码Na+/K+ ATPase的神经元同工型的基因是AHC的最常见原因。 AHC是 罕见，目前无法治愈。两个因素显着导致缺乏进展 治疗的发展。首先，与大多数罕见的遗传疾病一样，资源不足和 私营部门的财务动机有限。第二，突变引起的机制 疾病尚不清楚。 AHC突变是主要隐性的，目前尚不清楚突变的Na+/K+如何 ATPases干扰野生型版本以创建物理定义的定义高于预期。任何 遗传疾病，最直接的治疗方法是纠正潜在的突变。从理论上讲，这可能是 通过编辑其编码的基因或信使RNA来完成。对于神经疾病，基因编辑是 不实用，因为使用CRISPR技术的最先进的系统在神经元中无法很好地工作。 此外，它们很难在体内输送，因为它们基于细菌成分 产生免疫学并发症。最近，用于编辑mRNA的新系统，称为位置定向RNA 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，它们基于人类自然发生的酶。另一个优点是它们是 相关简单，由与人RNA编辑耦合的小寡核苷酸指南RNA组成 酶。因为在不同细胞中不同RNA之间将遗传信息编码为相同的方式，所以 无论表达何处，都可以以几乎相同的方式进行编辑。这使SDRE成为半世代的方法 不同的遗传疾病。在这项工作中，SDRE组件将被优化，以有效，有选择地 纠正基于AHC的最常见突变（ATP1A3 D801N）。将确定顶级指南RNA 通过迭代选择程序的数十亿个随机候选者的池。这些将是 在细胞中与不同版本的RNA编辑酶结合进行测试。这些试剂会 然后将其包装到病毒颗粒中，以便将它们有效地传递到细胞中。同时， ATP1A3 D801N突变会改变Na+/K+ ATPase函数的机制，都将在 包含突变和野生型酶的酶。这些实验将提供更好的 理解AHC的物理基础并帮助估计突变体的比例 必须校正以抵消功能缺陷。综上所述，SDRE试剂的开发耦合 有了清楚地了解由AHC突变引起的异常生理学，将使我们开始 开发这种情况的第一个理论。