UC Davis

Huntington’s Disease (HD) is an autosomal dominant neurodegenerative disorder caused by a trinucleotide repeat in exon 1 of the Huntingtin (HTT) gene. This expansion leads to protein misfolding that causes widespread cellular dysfunction and ultimately leads to neuronal cell death. The advent of nuclease-deficient CRISPR-Cas9 (CRISPR-dCas9) gene regulation technologies allows targeting of the causative gene and subsequent downregulation via fused effector domains that induce heterochromatin at the epigenetic level through DNA methylation (DNMT3A/L) and H3K9me3 deposition (KRAB), blocking transcription. Therefore, we propose using dCas9 epigenetic editing to downregulate HTT as a therapeutic approach for HD. HTT has large haplotype blocks that allow for allele-specific targeting based upon the presence of heterozygous single nucleotide polymorphisms (SNPs). These SNPs are in genomic regions devoid of NGG PAM sites, a requirement for spdCas9 binding. To address this bottleneck, a screen was conducted of multiple dCas9 variants fused to KRAB and DNMT3A/L with increasingly expanded PAM targeting to initially assess the ability to downregulate total HTT. Surprisingly, only spdCas9 was able to significantly knockdown HTT, while expanded PAM site variants dxCas9 and dCas9-VQR were less efficient in reducing HTT expression. ChIP-qPCR of the HTT promoter showed a decrease in the binding efficiency of dCas9 variants, likely leading to the decreased efficiency of HTT downregulation. We further investigated DNA methylation changes through reduced representation bisulfite sequencing, showing high on-target increases in DNA methylation and few off-targets. In addition, we demonstrate mitotically stable HTT silencing of up to 6 weeks in vitro in a rapidly dividing cell line. We then assessed total HTT knockdown in HD patient-derived fibroblasts and neuronal stem cells. We identified significant downregulation of HTT in our treatment group compared to an unguided control. RNAseq was used to identify differential gene expression between treatment and controls, and gene ontology analysis was performed to identify the rescue of biological processes involved in HTT molecular pathogenesis. Off-targets were assessed through overlaying genome-wide changes in H3K9me3 enrichment, dCas9 binding, and differential gene expression. An additional bottleneck is the delivery of large dCas9 epigenome editors to the CNS. Current studies are addressing the ability to do large scale-ups of AAV for transgene delivery, showing we can produce clinical levels of AAV for decreased costs compared to current methodologies. This approach holds great promise for those suffering from HD.