Durable HTT silencing using non-evolved dCas9 epigenome editors in patient-derived cells

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a trinucleotide repeat expansion in exon 1 of the huntingtin (<i>HTT</i>) gene. Nuclease-deficient Cas9 protein (dCas9) epigenetic editing for targeted gene regulation is a promising therapeutic approach for HD through downregulation of the causative gene, <i>HTT</i>. A screen of several dCas9 variants with expanded PAM recognition was fused to KRAB and DNMT3A/L to assess the ability to downregulate total <i>HTT</i>. Surprisingly, only <i>S</i> <i>p</i>dCas9 could significantly downregulate <i>HTT</i>, while expanded PAM recognition variants dxCas9 and dCas9-VQR were less efficient or unable to reduce <i>HTT</i> expression. Using our lead construct with <i>S</i> <i>p</i>dCas9, DNA methylation changes were assessed through reduced representation bisulfite sequencing, showing high on-target increases in DNA methylation and few off-targets. In addition, <i>HTT</i> silencing was mitotically stable for up to 6 weeks in a rapidly dividing cell line. Finally, significant downregulation of <i>HTT</i> was achieved in patient-derived neuronal stem cells, showing the efficacy of this system in a disease-relevant cell type. This approach represents a novel therapeutic pathway for the treatment of HD.