CRISPR-Cas9 Mediated Gene-Silencing of the Mutant Huntingtin Gene in an In Vitro Model of Huntington’s Disease

Abstract

Huntington’s disease (HD) is a fatal neurodegenerative genetic disease characterized by a loss of neurons in the striatum. It is caused by a mutation in the Huntingtin gene (HTT) that codes for the protein huntingtin (HTT). The mutant Huntingtin gene (mHTT) contains extra poly-glutamine (CAG) repeats from which the translated mutant huntingtin proteins (mHTT) undergo inappropriate post-translational modifications, conferring a toxic gain of function, in addition to its non-functional property. In order to curb the production of the mHTT, we have constructed two CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR associate protein) plasmids, among which one nicks the DNA at untranslated region upstream to the open reading frame (uORF), and the other nicks the DNA at exon1-intron boundary. The primary goal of this study was to apply this plasmid into mesenchymal stem cells (MSCs) extracted from the bone-marrow of YAC128 mice, which carries the transgene for HD. Our results suggest that the disruption of uORF through CRISPR-Cas9 influences the translation of mHTT negatively and, to a lesser extent, disrupts the exon1-intron boundary, which affects the translation of the mHTT. These findings also revealed the pattern of the nucleotide addition or deletion at the site of the DNA-nick in this model.

Recent Overview of the Use of iPSCs Huntington’s Disease Modeling and Therapy

“…Subsequent analyses revealed a large 44-kb deletion leading to the complete inactivation of mHTT without affecting the normal allele. According to the aforementioned studies and several others [67][68][69], CRISPR/Cas9 has the ability to solely inhibit mHTT expression in a specific brain region and thus has rapidly become the most promising gene-editing tool for neurodegenerative diseases such as HD. However, a question remains regarding the safety of the CRISPR/Cas9 system, which is now being tested in animal models to establish an effective and completely safe procedure before its application in humans [70].…”

Prion-like mechanisms in neurodegenerative disease: Implications for Huntington’s disease therapy

“…The deletion mutations created following CRISPR‐Cas9‐based editing showed reduced CAG repeat expansions in the HD cells. Further analysis showed that mHTT was reduced compared with controls …”

Strategie wyciszania ekspresji zmutowanego genu w terapii choroby Huntingtona

“…Badania przedkliniczne sugerują zwiększoną efektywność wprowadzenia zmian w DNA, w skutek jego przecięcia przez system CRISPR-Cas9, poprzez mechanizm niehomologicznego łączenia końców (NHEJ, ang. non-homologous end joining) [53]. Przy pomocy tej technologii można wpłynąć na transkrypcję HTT na wiele sposobów począwszy od wycięcia powtórzeń CAG czy poprzez przesunięcie ramki odczytu i wystąpienie w ten sposób przedwczesnych kodonów stop [33,54,55].…”