Monoallelic repair of expanded huntingtin by trans-splicing

描述（由申请人提供）：亨廷顿病（HD）是一种常染色体显性神经退行性疾病，由亨廷顿蛋白（HTT）氨基末端的多聚谷氨酰胺扩增引起。其特征是进行性运动障碍、认知能力下降和精神障碍。发病后约20年死亡。目前尚无有效的HD治疗方法。 HTT是一种约3，144个氨基酸的可溶性蛋白质，与其他蛋白质没有序列同源性。越来越多的证据表明，HTT作为一种分子支架，能够组织各种信号复合物。它在人类和啮齿类动物中广泛表达，在CNS神经元和睾丸中发现的水平最高。由于涉及单个致病基因和简单的基因检测，由CAG重复扩增引起的HD和其他疾病提供了独特的治疗机会。HD突变被认为是“功能获得”突变。因此，基于减少突变HTT表达的治疗方法，如RNA干扰，目前被认为是有前途的HD治疗策略。这些试剂可能会导致突变型和野生型HTT等位基因失活或损害其正常功能。因此，仅靶向病理等位基因的单等位基因疗法是特别感兴趣的。 我们建议进行概念验证研究，以证明剪接体介导的反式剪接是一种有效的手段，专门修复扩大，病理等位基因，而不影响正常等位基因的功能。该技术不仅将恢复显性基因的校正mRNA的表达，而且还将恢复相应蛋白质的产生。该技术的优点包括利用小的校正RNA序列的可能性，所述小的校正RNA序列以高特异性靶向突变基因内的外显子序列以及基因表达的天然调节。在具体目标1中，我们将产生并功能优化反式剪接构建体。将使用由反式剪接模块和HTT小基因组成的二元细胞转染系统测试这些构建体的效率。在具体目标2中，将进一步测试所选优化的反式剪接构建体修复内源性HTT前mRNA的能力。此外，我们将评估HTT修复成功后细胞功能的恢复情况。未来的工作将旨在将最有效的构建体转移到HD动物模型中。在这个项目中获得的知识可能最终导致一种新的基因治疗HD。 公共卫生相关性：亨廷顿氏病是一种进行性和致命的神经退行性疾病，目前尚无治愈方法。遗传性突变导致大脑神经元死亡。该项目探索修复突变并恢复正常细胞功能的可能性。

DESCRIPTION (provided by applicant): Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder which is caused by polyglutamine expansion in the amino-terminus of huntingtin (HTT). It is characterized by progressive movement disorder, cognitive decline and psychiatric disturbances. Death ensues approximately 20 years after onset of the disease. No effective therapy currently exists for HD. HTT is a soluble protein of ~3,144 amino acids that has no sequence homology with other proteins. Increasing evidence indicates that HTT functions as a molecular scaffold that is able to organize a variety of signaling complexes. It is expressed ubiquitously in humans and rodents, with the highest levels found in CNS neurons and the testes. Because of the involvement of a single causal gene and simple genetic testing, HD and other diseases caused by CAG-repeat expansions offer unique therapeutic opportunities. The HD mutation is considered to be a "gain of function" mutation. Therefore, therapeutic approaches that are based on reducing mutant HTT expression such as RNA interference are currently considered to be promising strategies for HD treatment. It is likely that these agents will cause inactivation or impair normal function of both mutant and wild type HTT alleles. Therefore, monoallelic therapies targeting only the pathological allele are of particular interest. We propose to conduct proof-of-concept studies to demonstrate that spliceosome-mediated trans-splicing is an efficient means to repair specifically the expanded, pathological allele, without functionally affecting the normal allele. This technology will restore not only the expression of the corrected mRNA from the dominant gene but also the production of the corresponding protein. Advantages of this technology include the possibility of utilizing small corrective RNA sequences that target exonic sequences within the mutated gene with high specificity as well as the natural regulation of gene expression. In specific aim 1 we will generate and functionally optimize trans-splicing constructs. The efficiency of these constructs will be tested using a binary cell transfection system, consisting of the trans-splicing module and an HTT minigene. In specific aim 2, selected optimized trans-splicing constructs will be further tested for the ability to repair endogenous HTT pre-mRNA. In addition, we will assess restoration of cellular functions as a result of successful HTT repair. Future work will be aimed at transferring the most efficient constructs into HD animal models. The knowledge gained in this project might eventually lead to a novel gene therapy for HD. PUBLIC HEALTH RELEVANCE: Huntington's Disease is a progressive and deadly neurodegenerative disease for which there is no cure. An inherited mutation causes the death of neurons in the brain. This project explores the possibility of repairing the mutation and to restore normal cellular functions.