Single nucleus mouse visual cortex data for N=16 animals, N=4 per group treated vehicle or zinc finger repressors of Prnp packaged in AAV PHP.B with the CMV, GfaABC1D, or hSyn promoters. Study Abstract: Prion disease is a rapidly progressing and invariably fatal neurodegenerative disorder with no approved treatment. The disease is caused by the self-templated misfolding of the prion protein (PrP) into toxic species, ultimately leading to neurodegeneration and death. We evaluated a novel epigenetic regulation approach using Zinc Finger Repressors (ZFRs) to ablate PrP expression at the transcriptional level. When delivered using adeno-associated virus (AAV), ZFRs potently and specifically reduced prion mRNA expression by >95% in vitro and to near undetectable levels within single neurons in vivo. In wildtype mice, ZFRs stably lowered neuronal PrP expression throughout the central nervous system for at least 17 months. In mice inoculated with misfolded PrP, AAV-ZFRs given at either early or late disease stages profoundly extended lifespan, significantly reduced PrP in the brain, and improved an array of molecular, histological, biomarker, and behavioral readouts. Finally, we delivered a ZFR targeting the human prion gene (PRNP) to cynomolgus monkeys using a novel blood-brain-barrier penetrant AAV capsid. Extensive bulk and single-cell assessments revealed widespread ZFR expression and PRNP repression in all 35 brain regions assessed, providing the first demonstration of epigenetic regulation across the nonhuman primate neuraxis following a single intravenous (IV) dose. These results highlight the potential of a one-time IV administered ZFR treatment for prion disease and other neurological disorders.