Bailus Barbara, Zhang Ningzhe, Ellerby Lisa M.
Buck Institute for Research on Aging, 8001 Redwood Blvd, Novato, CA, 94945, USA
Huntington’s disease (HD) is a fatal, dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the gene, leading to an expanded polyglutamine (polyQ) region in the encoded protein HTT. We have used homologous recombination (HR) to genetically correct HD patient-derived induced pluripotent stem cells (iPSCs) and found that this reversed HD disease phenotypes. We have utilized exploited genome editing tools including TALENs (Transcription like activator effectors) and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 technology to carry out genetic correction or expansion, and we were able to detect HR without selection in human cells. The overall goal is to use this technology to model HD-relevant cell types and better understand disease progression by leveraging system biology approaches. To understand the disease progression, isogenic iPSC lines were created. We found that the disease phenotypes only manifested in the differentiated neural stem cell (NSC) stage, not in iPSCs. Transcriptomic analysis of HD iPSCs and HD NSCs compared to isogenic controls was utilized to understand the molecular basis for the CAG repeat expansion-dependent disease phenotypes in NSCs. Differential gene expression and pathway analysis identified transforming growth factor β (TGF-β) signaling, netrin-1 signaling and medium spiny neuron (MSNs) maturation and maintenance as the top dysregulated pathways in HD NSCs. The ability to create additional isogenic cell lines through CRISPR-mediated HR will further enhance our understanding of HD progression. These lines can be manipulated with CRISPR to understand the effects of common SNPs (single nucleotide polymorphism) that modulate disease onset in HD, allowing the identification of new pathways and helping to elucidate potential therapeutic targets for HD. Beyond drug discovery, the CRISPR system could eventually be optimized to use in vivo, correcting a patient’s disease-causing mutation, in the asymptomatic stages of HD.
亨廷顿舞蹈症（HD）是一种致命的、显性遗传的神经退行性疾病，由该基因中的CAG三核苷酸重复扩增引起，导致编码蛋白HTT中出现扩展的多聚谷氨酰胺（polyQ）区域。我们利用同源重组（HR）对HD患者来源的诱导多能干细胞（iPSC）进行基因校正，发现这逆转了HD疾病表型。我们利用了包括转录激活样效应因子核酸酶（TALENs）和规律成簇间隔短回文重复序列（CRISPR）/Cas9技术在内的基因组编辑工具来进行基因校正或扩增，并且我们能够在不进行筛选的情况下在人类细胞中检测到HR。总体目标是利用该技术对与HD相关的细胞类型进行建模，并通过利用系统生物学方法更好地理解疾病进展。为了理解疾病进展，我们创建了同基因iPSC系。我们发现疾病表型仅在分化的神经干细胞（NSC）阶段出现，而在iPSC中不出现。通过对HD iPSC和HD NSC与同基因对照进行转录组分析，以了解NSC中CAG重复扩增依赖性疾病表型的分子基础。差异基因表达和通路分析确定转化生长因子β（TGF-β）信号通路、netrin-1信号通路以及中等棘状神经元（MSN）的成熟和维持是HD NSC中失调最严重的通路。通过CRISPR介导的HR创建更多同基因细胞系的能力将进一步增强我们对HD进展的理解。这些细胞系可以用CRISPR进行操作，以了解调节HD疾病发作的常见单核苷酸多态性（SNP）的作用，从而识别新的通路并有助于阐明HD的潜在治疗靶点。除了药物研发之外，CRISPR系统最终可能会被优化用于体内，在HD的无症状阶段校正患者的致病突变。
