Brain-wide AAV-mediated silencing of prion protein by a compact epigenetic editor

Prion disease is caused by misfolding of the prion protein (PrP) into pathogenic self-propagating conformations, leading to rapid onset dementia and death. However, elimination of endogenous PrP can halt prion disease progression. Here, we describe CHARM, a compact, enzyme-free epigenetic editor capable of silencing transcription through programmable targeted DNA methylation. Using a histone H3 tail-Dnmt3l fusion, CHARM recruits and activates the endogenous DNA methyltransferases, thereby reducing transgene size and bystander effects. When delivered to the mouse brain by an adeno-associated viral (AAV) vector, PRNP-targeted CHARM ablates PrP expression across the brain. We temporally limit editor expression by implementing a kinetically-tuned self-silencing approach. CHARM represents a broadly applicable strategy to programmably prevent expression of pathogenic proteins, including those implicated in other neurodegenerative diseases. Overall design: Neuro-2a cells were transduced with ZFcharm Kv1 and CRISPRcharm Kv1 lentiviral constructs. CISPRcharm Kv1 was introduced into cells already expressing either a non-targeting sgRNA or a sgRNA targeting Prnp. An empty lentiviral vector was used as a no-editor control. Each transduction was done in triplicate. Cells were dislodged from 6-well plates using Trizol and total RNA was extracted using the Direct-zol RNA Miniprep Kit (Zymo, R2051). Libraries were prepared using the KAPA RNA HyperPrep Kit with RiboErase (HMR) (Roche, KK8560) and sequenced as 50 bp single-end reads on a NovaSeq 6000 (Illumina). Raw sequencing reads were aligned to the mouse genome (mm39) using STAR 2.7.1a and quantified using featureCounts. Differential expression analysis was carried out using DESeq2.