Using Genome Engineering to Understand Huntington’s Disease

Abstract

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 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.

Molecular Targets and Therapeutic Strategies in Spinocerebellar Ataxia Type 7

“…In any case, for siRNA, ASO or viral vector delivery, safety and efficacy issues related to biodistribution and durability still need to be evaluated [186,187]. The recent years have seen the development of a powerful technologies to target and edit specific disease genes, such as TALEN, Zinc finger endonucleases, and CRISPR/Cas9 systems [188][189][190]. Currently under development for the most frequent polyQ diseases, CRISPR/Cas9 strategies have the potential to have great success in clinical trials for all polyQ neurodegenerative disorders.…”

Advances in Modeling Polyglutamine Diseases Using Genome Editing Tools

“…The limitations of this method are the reduced efficiency at the targeted site resulting from the chromatin organization or epigenetic changes, as well as potential immunogenicity and a high off-target probability [47,49]. The advancement in this technology was the introduction of different effector domains, allowing for gene activation (VP64), silencing (KRAB) or methylation (DNMT1) [52]. No polyQ models have been created using ZFNs, but this technology was used to reduce huntingtin expression in the R6/2 mouse brain [53] and to study CAG repeats' instability in cell lines [54,55].…”

“…TALENs contain a programmable DNA-binding domain with 34 amino acid repeated units fused with a DNA-cleavage domain of the FokI enzyme [56,57]. The highly variable amino acids on the 12th and 13th positions, called repeat-variable diresidues (RVDs), are responsible for nucleotide recognition [46,52,58]. The lengths of target sequences for TALENs extend from 50 to 60 bp (including a 14-18 bp spacer, where FokI acts as a dimer and cleaves opposite binding sites for paired TALENs) [59].…”

New Perspectives of Gene Therapy on Polyglutamine Spinocerebellar Ataxias: From Molecular Targets to Novel Nanovectors

“…The emergence of genome editing technology, including clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs) and zinc-finger nuclease (ZFN) platforms, has allowed targeting and editing polyQ-disease genes [ 92 , 128 , 129 ] ( Figure 3 D–F). An allele-specific CRISPR/Cas9-mediated strategy was successfully applied to HD [ 130 ].…”