Allele-Specific Reduction of the Mutant Huntingtin Allele Using Transcription Activator-Like Effectors in Human Huntington's Disease Fibroblasts.
Allele-Specific Reduction of the Mutant Huntingtin Allele Using Transcription Activator-Like Effectors in Human Huntington's Disease Fibroblasts.
复制标题
在人类亨廷顿氏病成纤维细胞中使用转录激活效应子对突变体亨廷顿等位基因的特定于等位基因特异性减少。
DOI：
10.3727/096368916x690863
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发表时间：
2016
影响因子：
3.3
通讯作者：
Nolta JA
中科院分区：
文献类型：
--
作者：
Fink KD;Deng P;Gutierrez J;Anderson JS;Torrest A;Komarla A;Kalomoiris S;Cary W;Anderson JD;Gruenloh W;Duffy A;Tempkin T;Annett G;Wheelock V;Segal DJ;Nolta JA
亨廷顿病(HD)是一种常染色体显性遗传性神经退行性疾病，由CAG重复序列异常扩张引起。虽然其致病机制被认为是由于多聚谷氨酰胺的扩张，但亨廷顿蛋白的潜在功能机制还没有被阐明。有研究表明，通过蛋白质干扰或条件性基因敲除来减少突变的亨廷顿蛋白在出生后可能被证明是治疗HD患者的一种有效方法。对于等位基因特异性靶向，转录激活子样效应器(TALE)被设计成靶向突变等位基因中的单核苷酸多态(SNP)，并包装到含有KRAb的载体骨架中，以促进疾病相关等位基因的转录抑制。更多的TALS被包装成含有异源二聚体FokI的载体骨架，并被设计用于核酸酶(TALEN)，以导致突变等位基因的CAG崩溃。用每种TALE-SNP或TALEN处理人HD成纤维细胞。用SNP基因分型试验检测等位基因表达，用免疫印迹法检测抗泛素突变蛋白聚集性。与对照组相比，TALE-SNP和TALEN显著降低了突变等位基因的表达(p<；0.05)，而不影响非疾病等位基因的表达。这项研究证明了使用TALE蛋白进行等位基因特异性基因修饰的潜力，并为亨廷顿氏病或其他与基因相关的疾病患者的靶向治疗提供了基础。
Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by an abnormal expansion of CAG repeats. Although pathogenesis has been attributed to this polyglutamine expansion, the underlying mechanisms through which the huntingtin protein functions have yet to be elucidated. It has been suggested that postnatal reduction of mutant huntingtin through protein interference or conditional gene knockout could prove to be an effective therapy for patients suffering from HD. For allele-specific targeting, transcription activator-like effectors (TALE) were designed to target single-nucleotide polymorphisms (SNP) in the mutant allele and packaged into a vector backbone containing KRAB to promote transcriptional repression of the disease-associated allele. Additional TALEs were packaged into a vector backbone containing heterodimeric FokI and were designed to be used as nucleases (TALEN) to cause a CAG-collapse in the mutant allele. Human HD fibroblasts were treated with each TALE-SNP or TALEN. Allele-expression was measured using a SNP-genotyping assay and mutant protein aggregation was quantified with Western blots for anti-ubiquitin. The TALE-SNP and TALEN significantly reduced mutant allele expression (p < 0.05) when compared to control transfections while not affecting expression of the nondisease allele. This study demonstrates the potential of allele-specific gene modification using TALE proteins, and provides a foundation for targeted treatment for individuals suffering from Huntington’s or other genetically linked diseases.