RNAi therapeutics for Friedreich ataxia

描述（由申请人提供）： 弗里德赖希共济失调（FRDA）是一种常染色体隐性遗传神经和心脏退行性疾病，目前尚无有效的治疗方法。 1996 年该疾病基因的鉴定以及随后对编码蛋白 frataxin 功能的阐明，为可能的治疗方法（包括传统的高通量药物筛选）打开了大门。该提案描述了一种新颖的、补充性的方法来开发 FRDA 疗法。我们设计了一种方法来构建编码随机短发夹环 RNA (shRNA) 的文库。该文库可用于鉴定具有治疗、治疗靶向和/或生物学意义的 shRNA 序列。鉴定基于功能选择，通过 PCR 从在特定条件下存活或表现出预定表型的细胞中检索有效序列。该设计允许有效序列的命中优化，并重新选择具有改进效果的序列。我们使用原代 FRDA 成纤维细胞，基于线粒体功能障碍在 FRDA 体征和症状中的关键作用以及 FRDA 细胞对氧化应激的敏感性，开发了筛选和选择测定法。通过我们的活/死选择测定，我们可以使用我们的随机 shRNA 编码库来识别对 FRDA 细胞有益的 shRNA 序列。通过我们对线粒体功能的筛选分析，我们可以确认并优先考虑这些序列。该提案的主要目标是确定潜在的 shRNA 疗法。第二个目标是开始了解我们确定的 shRNA 的作用机制。具体目标是： 1. 鉴定可使原代 FRDA 成纤维细胞在致死但对正常对照细胞不致死的条件下存活的 shRNA 序列。 2. 优化目标 1 中确定的 shRNA 序列。 3. 确认优化的 shRNA 并确定其优先顺序，并开始了解作用机制。该提案描述了一种使用随机 shRNA 库开发 Friedreich 共济失调新疗法的方法。这种方法对其他遗传疾病以及传染病的治疗方法的开发具有潜在的影响，因此与公共卫生高度相关。

DESCRIPTION (provided by applicant): Friedreich ataxia (FRDA) is an autosomal recessive neuro- and cardio-degenerative disorder for which there are currently no established effective treatments. The identification of the disease gene in 1996 and the subsequent elucidation of the function of the encoded protein, frataxin, have opened the door to possible therapeutic approaches, including conventional high-throughput drug screening. This proposal describes a novel, complementary approach to the development of therapeutics for FRDA. We devised a method to construct a library that encodes random, short-hairpin-loop RNAs (shRNAs). This library can be used to identify shRNA sequences of therapeutic, therapeutic-targeting, and/or biological interest. The identification is based on functional selection, with effective sequences retrieved by PCR from cells that survive a particular condition or exhibit a predetermined phenotype. The design allows for hit-optimization of effective sequences, with re-selection for sequences with improved effects. Using primary FRDA fibroblasts, we developed screening and selection assays based on the critical role of mitochondrial dysfunction in the signs and symptoms of FRDA and on the sensitivity of FRDA cells to oxidative stress. With our live/dead selection assays, we can use our random shRNA-encoding library to identify shRNA sequences of benefit to FRDA cells. With our screening assays of mitochondrial function, we can confirm and prioritize these sequences. The primary objective of this proposal is to identify potential shRNA therapeutics. A secondary objective is to begin to understand the mechanisms of action of the shRNAs we identify. The Specific Aims are: 1. To identify shRNA sequences that allow survival under conditions lethal to primary FRDA fibroblasts but non-lethal to normal control cells. 2. To optimize the shRNA sequences identified in Aim 1. 3. To confirm and prioritize optimized shRNAs and begin to understand mechanisms of action. This proposal describes an approach to develop novel therapeutics for Friedreich ataxia using a random shRNA library. This approach has potential implications for the development of therapeutics for other genetic diseases, as well as for infectious diseases, and is therefore highly relevant to public health.