Specificity assessment of allele selective Zinc Finger Protein Repressors in hESC-derived HD neurons

Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range, while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and at least 9 months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological, and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD. Four treatments (ZFP-A, ZFP-B, ZFP-C, GFP) were tested in neurons derived from HD hESCs (GENEA020). Each ZFP was tested in a different batch with the GFP control. For each batch of treatment and control, 4 replicate transfections were conducted yielding 4 biological replicates. Cells were harvested 24 hours later and processed for total RNA isolation and Affymetrix sample Prep. The same control GFP treatment was used for each batch.

亨廷顿舞蹈症（Huntington’s disease, HD）是一种显性遗传性神经退行性疾病，由亨廷顿基因（huntingtin gene, HTT）中的CAG三核苷酸重复扩增引发，该基因编码致病性突变型HTT（mutant HTT, mHTT）蛋白。鉴于正常HTT对脑功能至关重要，我们设计了锌指蛋白转录因子（zinc finger protein transcription factors, ZFP-TFs），以靶向致病性CAG重复序列，选择性降低mHTT水平作为治疗策略。我们利用患者来源的成纤维细胞和神经元，证明ZFP-TFs在宽剂量范围内可选择性抑制超过99%的HD致病等位基因，同时保留超过86%的正常等位基因的表达。其他含CAG的基因受影响极小，且病毒递送的ZFP-TFs在HD神经元中培养超过100天、在小鼠脑中至少9个月时仍保持活性且耐受性良好。我们使用三种HD小鼠模型，证明其在一系列分子、组织病理学、电生理及功能终点上均得到改善。本研究结果支持等位基因选择性ZFP-TF用于HD治疗的进一步开发。我们在源自HD人类胚胎干细胞（human embryonic stem cells, hESCs，株系GENEA020）的神经元中测试了四种处理方式（ZFP-A、ZFP-B、ZFP-C与绿色荧光蛋白（green fluorescent protein, GFP））。每种ZFP均设置GFP对照并在不同批次中进行测试。每个处理与对照批次均设置4次重复转染，得到4个生物学重复样本。转染24小时后收集细胞，进行总RNA提取与Affymetrix样本制备流程。所有批次均使用相同的GFP对照处理。