Antisense oligonucleotides extend survival of prion-infected mice

Abstract

Prion disease is a fatal, incurable neurodegenerative disease of humans and other mammals caused by conversion of cellular prion protein (PrPC) into a self-propagating neurotoxic conformer (prions; PrPSc). Strong genetic proofs of concept support lowering PrP expression as a therapeutic strategy. Antisense oligonucleotides (ASOs) can provide a practical route to lowering 1 target mRNA in the brain, but their development for prion disease has been hindered by 3 unresolved issues from prior work: uncertainty about mechanism of action, unclear potential for efficacy against established prion infection, and poor tolerability of drug delivery by osmotic pumps. Here, we test ASOs delivered by bolus intracerebroventricular injection to intracerebrally prion-infected WT mice. Prophylactic treatments given every 2–3 months extended survival times 61%–98%, and a single injection at 120 days after infection, near the onset of clinical signs, extended survival 55% (87 days). In contrast, a nontargeting control ASO was ineffective. Thus, PrP lowering is the mechanism of action of ASOs effective against prion disease in vivo, and infrequent — or even single — bolus injections of ASOs can slow prion neuropathogenesis and markedly extend survival, even when initiated near clinical signs. These findings should empower development of PrP-lowering therapy for prion disease.

Characterization of the prion protein binding properties of antisense oligonucleotides

“…All eight ASOs and heparin showed isotherms consistent with a saturable binding event driven by enthalpy, despite a decrease in entropy ( Figure 1C-E). Calculated affinity (Kd) values were similar, in the low-to mid-nanomolar range ( Figure 1C) for all tested ASOs including a fully phosphorothioated 20mer used as a previous generation of control ASO 6 , and the mixed phosphorothioate/phosphodiester (PS/PO) backbone 15 ASOs with MOE and/or cEt modifications employed more recently 12 . This suggested that the lack of in vivo efficacy of non-PrP-targeting ASOs in recent studies was not due to chemical differences in PrP affinity between ASO chemistries.…”

“…Phosphorothioate oligonucleotides have been reported to bind PrP in vitro in a sequenceindependent manner with nanomolar affinity and to antagonize misfolded PrP accumulation in cell culture with nanomolar efficacy 6,10,11 , yet in vivo, we have reported that only antisense sequences targeting the PrP RNA are effective at extending survival in prion-infected mice 12 . To reconcile these observations, here we revisited the binding of ASOs and PrP.…”

“…We used isothermal titration calorimetry (ITC) to estimate the thermodynamic parameters of interactions between full-length recombinant human prion protein (HuPrP23-230) and eight previously described 2,6,12 ASOs (Table 1) as well as heparin, a polyanionic positive control 8 (Figure 1). All eight ASOs and heparin showed isotherms consistent with a saturable binding event driven by enthalpy, despite a decrease in entropy ( Figure 1C-E).…”

“…Given that the biophysical parameters of ASO-PrP interaction appeared similar across ASOs, we sought to replicate, with ASOs recently tested in vivo 12 , the previous reports of sequenceindependent lowering of misfolded PrP accumulation in cell culture. Chronically RML prioninfected mouse neuroblastoma (ScN2a) cells 18 were exposed to ASOs in a gymnotic system (without transfection or electroporation) and harvested for PrP mRNA quantification by qPCR after 24h, or for PrP quantification with or without proteinase K digestion after 72h.…”

“…PS ASOs have been reported to bind PrP in vitro with nanomolar affinity 10 in a sequence-independent manner, lower PrP expression in cultured cells with micromolar potency 6,11 , and even strongly inhibit the accumulation of misfolded PrP in prion-infected cultured cells, with low nanomolar potency 6,10,11 . In recent studies, PrP-lowering ASOs delivered by intracerebroventricular bolus injection potently extended survival of prioninfected mice, whereas a non-PrP-lowering control ASO conferred no survival benefit 12 . These findings establish RNA-mediated PrP lowering as the mechanism of action of ASOs against prion disease in vivo.…”