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

Abstract

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a trinucleotide repeat expansion in exon 1 of the huntingtin (HTT) 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, HTT. A screen of several dCas9 variants with expanded PAM recognition was fused to KRAB and DNMT3A/L to assess the ability to downregulate total HTT. Surprisingly, only S pdCas9 could significantly downregulate HTT, while expanded PAM recognition variants dxCas9 and dCas9-VQR were less efficient or unable to reduce HTT expression. Using our lead construct with S pdCas9, DNA methylation changes were assessed through reduced representation bisulfite sequencing, showing high on-target increases in DNA methylation and few off-targets. In addition, HTT silencing was mitotically stable for up to 6 weeks in a rapidly dividing cell line. Finally, significant downregulation of HTT 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.