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）氨基末端的聚谷氨酰胺扩增引起。它的特点是进行性运动障碍，认知能力下降和精神障碍。死亡发生在发病后大约20年。目前还没有有效的治疗HD的方法。HTT是一种约3144个氨基酸的可溶性蛋白，与其他蛋白没有序列同源性。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普遍表达，在中枢神经系统神经元和睾丸中含量最高。由于涉及单一致病基因和简单的基因检测，HD和其他由CAG-repeat扩增引起的疾病提供了独特的治疗机会。HD突变被认为是一种“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，目前被认为是治疗HD的有希望的策略。这些药物可能会导致突变型和野生型HTT等位基因失活或损害正常功能。因此，单等位基因治疗仅针对病理等位基因是特别感兴趣的。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方法，可以特异性修复扩增的病理等位基因，而不会对正常等位基因产生功能影响。这项技术不仅可以恢复显性基因中被纠正的mRNA的表达，还可以恢复相应蛋白质的产生。该技术的优点包括可以利用靶向突变基因内外显子序列的小校正RNA序列，具有高特异性以及基因表达的自然调控。在具体目标1中，我们将生成并在功能上优化反式剪接结构。这些构建体的效率将使用二元细胞转染系统进行测试，该系统由反式剪接模块和HTT迷你基因组成。在具体目标2中，将进一步测试所选择的优化反式剪接构建体修复内源性HTT pre-mRNA的能力。此外，我们将评估成功修复HTT后细胞功能的恢复。未来的工作将致力于将最有效的结构转移到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HD基因疗法。

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.