Development of prion protein-lowering divalent siRNA therapy for prion disease Project Summary / Abstract Prion disease is a fatal, untreatable, rapidly progressive neurodegenerative disease caused by a single protein, the prion protein or PrP. In disease, PrP converts into a pathogenic ?prion? conformation that spreads across the brain by conformationally corrupting other PrP molecules. This process is strictly reliant on PrP expression, and reduction of PrP, either genetically or pharmacologically, dose-dependently prion delays disease in animals. Critically, even with sustained 50% knockdown of PrP, all animals eventually succumb to fatal disease. PrP knockout models indicate that total ablation of PrP is fully protective against prion disease, and that even transient clamp suppression of >90% could allow the brain to clear prions, resulting in a one-time ?cure.? To date, no drug modality has been available that is a) capable of reducing levels of a single pathogenic protein, b) active in, and realistically deliverable to, the whole brain, and c) potent enough to test the above hypothesis pharmacologically. However, recently described divalent si-RNAs (di-siRNAs) now offer the first such modality. Di-siRNAs are chemically modified linked siRNA duplexes capable of reaching the whole rodent and non-human primate brain following delivery into CSF, and reducing levels of specific disease-causing proteins such as Huntingtin by >99%. Di-siRNAs preliminarily pre-screened against both mouse and human PrP RNA will be synthesized and provided by our collaborator Dr. Anastasia Khvorova at UMass. In the R61 award phase, we will: 1) Validate potency of PrP-targeting di-siRNA molecules in cells, by characterizing PrP RNA and protein knockdown, and dose-responsiveness of effect, in mouse and human cells. 2) Characterize leading di-siRNA in vivo PK/PD properties in uninfected mice, by assessing in vivo potency, tolerability, correlation between RNA and protein reduction, dose-responsiveness, brain distribution, and time to washout in wild-type and ?humanized? (human-PrP expressing) transgenic mice. 3) Characterize time to pathology and terminal disease in humanized mice, by infecting a novel transgenic humanized mouse model with human prions and tracking time to biomarker changes, symptoms and endpoint. In the R33 award phase, we will perform survival experiments in mice infected with both mouse and human prions to assess multiple clinically relevant treatment paradigms. 1) Survival following prophylactically initiated chronic dosing. We will model preventive treatment of individuals at risk for genetic prion disease by initiating chronic di-siRNA dosing before prion infection, and assess impact on survival; 2) Survival following a single dose at a pre-pathological timepoint. We will assess the impact of a single bolus of di-siRNA early in prion infection on both brain prion titer and survival. 3) Survival following a single dose at a symptomatic timepoint. We will model symptomatic-stage treatment by delivering a single bolus dose of di-siRNA at an advanced disease timepoint, and will assess impact on prion titer and survival. Together, these aims will determine whether PrP- lowering di-siRNAs should be advanced to the clinic for prevention and/or treatment of human prion disease.
Development of prion protein-lowering divalent siRNA therapy for prion disease Project Narrative Prion disease is a rapidly deadly, currently untreatable disease that depends on expression of a single protein, the prion protein or PrP. Chronic reduction of PrP levels delays disease in animals, and genetic proofs of concept indicate that aggressive brain-wide suppression of PrP expression, even transiently, has the potential to halt disease and allow complete clearance of prions from the brain. Divalent siRNAs (di-siRNAs), a powerful new RNA-targeting technology capable of reducing levels of specific disease-causing proteins by >99% across the brain, offers the first opportunity to suppress PrP to levels that may offer full protection from disease and to test the potential for a one time ?cure,? offering a new path forward against prion disease and perhaps many other brain diseases.