Zinc-Finger Nuclease- Mediated Repair of Disease-Causing Triplet CAG Repeats

描述（由申请人提供）：亨廷顿病（HD）是一种由HTT基因第一外显子中CAG重复序列扩增引起的可变发作神经退行性疾病。重复是多态性的，特别是在纹状体和皮质;并编码多聚谷氨酰胺道，这是神经元毒性和死亡的病理基础。目前还没有治愈这种毁灭性疾病的方法。在这种显性疾病中，CAG重复序列长度主要决定HD的发作和严重程度，从超过35-39个CAG重复序列的重复开始。通过直接靶向CAG重复序列减少神经元毒性和死亡预期将降低HD和其他三联体重复序列疾病的严重程度或至少延迟其发作。所提出的研究直接集中在作为治疗靶点的扩增的CAG重复序列上，并寻求在基因组水平上永久破坏或缩小扩增的CAG重复序列。为了实现这一目标，将使用锌指核酸酶（ZFN）将DSB特异性地引导至扩增的HTT重复等位基因，目的是通过诱导移码插入缺失将重复缩短至良性长度或消除其表达。在 第一个目标是，这种基于ZFN的疗法将在来自亨廷顿病患者的原代细胞中进行测试。下一代测序将用于确定ZFN诱导的HTT重复序列变化的全谱。总之，拟议的研究将测试ZFN介导的治疗策略的关键方面，以缩小或破坏诱导神经变性并导致亨廷顿病和其他神经系统疾病的扩展CAG重复等位基因。 公共卫生相关性：扩增的CAG/CTG重复序列是十几种遗传性神经系统疾病的遗传基础，包括亨廷顿病、强直性肌营养不良和几种脊髓小脑共济失调。尽管这些疾病的病理基础众多，但它们都有共同的病因学：CAG/CTG重复序列从约30个重复序列的短良性片段扩展到可延伸数百个重复序列的较长致病长度。我们将在人类患者细胞和亨廷顿病动物模型中验证一种新的治疗策略，该策略针对这一共同特征-扩增的重复序列。

DESCRIPTION (provided by applicant): Huntington's disease (HD) is a variable onset neurodegenerative disorder caused by the expansion of a CAG repeat sequence in the first exon of the HTT gene. The repeat is polymorphic, especially in the striatum and cortex; and encodes a polyglutamine tract that is the pathological basis for neuronal toxicity and death. There is currently no cure for this devastating disorder. CAG repeat length in this dominant disorder principally determines the onset and severity of HD starting with repetitions that exceed 35-39 CAG repeats. Reducing neuronal toxicity and death by directly targeting the CAG repeat tract would be expected to reduce the severity or at least delay the onset of HD and other triplet repeat disorders. The proposed studies focus directly on the expanded CAG repeat tract as the target of therapy and seek to permanently disrupt or shrink the expanded CAG repeat at the genomic level. To achieve this goal, zinc finger nucleases (ZFNs) will be used to direct DSBs specifically to the expanded HTT repeat allele, with the intent of either shrinking the repeat to benign lengths or eliminating its expression through the induction of frame-shifting indels. In the first aim, this ZFN- based therapy will be tested in primary cells derived from a Huntington's patient. Next-generation sequencing will be used to determine the full spectrum of ZFN-induced changes to the HTT repeat. In conclusion, the proposed research will test key aspects of a ZFN-mediated treatment strategy for shrinking or disrupting expanded CAG repeat alleles that induce neurodegeneration and lead to Huntington's disease and other neurological disorders. PUBLIC HEALTH RELEVANCE: Expanded CAG/CTG repeat tracts are the genetic basis for more than a dozen inherited neurological disorders including Huntington's disease, myotonic dystrophy, and several spinocerebellar ataxias. Despite the multitude of pathologies underlying these disorders, they all share common etiology: the expansion of CAG/CTG repeats from short, benign tracts of about 30 repeats to longer, pathogenic lengths that can extend for several hundred repeats. We will validate a novel therapeutic strategy that targets this common feature-expanded repeats-in human patient cells and in animal models for Huntington's disease.