Using Genome Engineering to Understand Huntington's Disease

Huntington's disease (HD) is a fatal, dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the Huntingtin (HTT) 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 beta (TGF-beta) 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.