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

描述(申请人提供)：亨廷顿病(HD)是一种由HTT基因第一外显子中CAG重复序列扩展引起的变发性神经退行性疾病。该重复序列是多态的，特别是在纹状体和皮质中；它编码了一个多谷氨酰胺束，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重复长度主要决定HD的发病和严重程度，从重复超过35-39个CAG重复开始。通过直接靶向CAG重复束来减少神经元毒性和死亡有望减轻或至少推迟HD和其他三联体重复疾病的发病。建议的研究直接集中在扩大的CAG重复序列作为治疗的目标，并寻求在基因组水平永久干扰或缩小扩大的CAG重复序列。为了实现这一目标，锌指核酸酶(ZFN)将被用来将DSB特异性地定向到扩展的HTT重复等位基因，目的是要么将重复序列缩小到良性长度，要么通过诱导移码插入来消除其表达。在 首先，这种基于ZFN的疗法将在一名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重复序列的全谱变化。总之，这项拟议的研究将测试ZFN介导的治疗策略的关键方面，该策略用于缩小或破坏扩展的CAG重复等位基因，这些等位基因会导致神经退化并导致亨廷顿氏病和其他神经疾病。

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.