To assess the feasibility of allele-specific RNA interference (RNAi) as a treatment strategy for genetic prion diseases.

Genetic Prion diseases (PrD) are autosomal dominant fatal neurodegenerative disorders that result from the accumulation of pathogenic misfolded prion protein (PrP). Selective suppression of the mutated allele, while preserving the normal allele, is predicted to limit potential deleterious cognitive, behavioral, and motoric effects of long-term reduction of wild-type PrP observed in aged PrP knockout mice.

Using the A117V human PrP gene mutation linked to GSS as the target, we generated and screened six siRNAs spanning the mouse homolog in Cos-7 cells stably expressing mouse PrP^116V or wild-type PrP^116A. The most selective 19-mer (guide strand mismatch at position 18) was cloned as a short-hairpin RNA into a lentiviral vector (pLKO.1-puro-UbC-tGFP) used for intra-cerebellar injections of 2-month-old transgenic mice expressing mouse PrP^116V compared with mice injected with a control vector. The disease course, PrP expression, and amyloid plaque deposition were assessed.

In vitro, the 19-mer siRNA reduced mutant PrP mRNA by 92 ± 2% while sparing wild-type PrP. In vivo, the lentiviral vector suppressed the sequence-specific expression of PrP mRNA in the cerebellum at 11 days (38.7±3.3%, p<0.01) and at 4 months (37.7 ± 6.8%, p<0.01) post-injection. Additionally, fewer PrP plaques were present at 4 months (27.8 ± 2% p<0.01). Mice receiving lentivirus carrying the active shRNA survived slightly, but not significantly, longer than control mice (164.4 ± 16.6 d vs. 159.5 ± 21.3 d, p > 0.05. Work in progress utilizing AAV delivery of allele-specific shRNA targeting PrP^116V results in improved cell transduction and brain distribution.

These results support allele-selective knockdown as a potential strategy for GSS and other autosomal-dominant prion disorders.