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

描述（由申请人提供）：亨廷顿氏病（HD）是一种可变发病的神经退行性疾病，由HTT基因第一外显子CAG重复序列扩增引起。重复是多态的，特别是在纹状体和皮层；并编码一个聚谷氨酰胺束，这是神经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重复长度主要决定了HD的发病和严重程度，从重复超过35-39 CAG重复开始。通过直接靶向CAG重复序列来减少神经元毒性和死亡，有望降低HD和其他三联体重复序列疾病的严重程度或至少延迟发病。拟议的研究直接关注扩大的CAG重复序列作为治疗目标，并寻求在基因组水平上永久破坏或缩小扩大的CAG重复序列。为了实现这一目标，锌指核酸酶（ZFNs）将用于引导dsb特异性地指向扩增的HTT重复等位基因，目的是将重复基因缩小到良性长度或通过诱导移帧索引消除其表达。在

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.