Characterization of the Prion Protein Binding Properties of Antisense Oligonucleotides

Abstract

Antisense oligonucleotides (ASOs) designed to lower prion protein (PrP) expression in the brain through RNase H1-mediated degradation of PrP RNA are in development as prion disease therapeutics. ASOs were previously reported to sequence-independently interact with PrP and inhibit prion accumulation in cell culture, yet in vivo studies using a new generation of ASOs found that only PrP-lowering sequences were effective at extending survival. Cerebrospinal fluid (CSF) PrP has been proposed as a pharmacodynamic biomarker for trials of such ASOs, but is only interpretable if PrP lowering is indeed the relevant mechanism of action in vivo and if measurement of PrP is unconfounded by any PrP–ASO interaction. Here, we examine the PrP-binding and antiprion properties of ASOs in vitro and in cell culture. Binding parameters determined by isothermal titration calorimetry were similar across all ASOs tested, indicating that ASOs of various chemistries bind full-length recombinant PrP with low- to mid-nanomolar affinity in a sequence-independent manner. Nuclear magnetic resonance, dynamic light scattering, and visual inspection of ASO–PrP mixtures suggested, however, that this interaction is characterized by the formation of large aggregates, a conclusion further supported by the salt dependence of the affinity measured by isothermal titration calorimetry. Sequence-independent inhibition of prion accumulation in cell culture was observed. The inefficacy of non-PrP-lowering ASOs against prion disease in vivo may be because their apparent activity in vitro is an artifact of aggregation, or because the concentration of ASOs in relevant compartments within the central nervous system (CNS) quickly drops below the effective concentration for sequence-independent antiprion activity after bolus dosing into CSF. Measurements of PrP concentration in human CSF were not impacted by the addition of ASO. These findings support the further development of PrP-lowering ASOs and of CSF PrP as a pharmacodynamic biomarker.

Prion protein lowering is a disease-modifying therapy across prion disease stages, strains and endpoints

“…Active ASO 5 was not tolerated at this timepoint (Figure 2D , Supplementary Table S2 ), replicating the ASO-specific, disease stage-dependent toxicity reported previously ( 22 , 28 ). Across both prophylactic and delayed treatment paradigms, non-targeting control ASO 4 conferred no survival benefit (Figure 2A , D ), replicating control ASO 3 results ( 28 ) and confirming PrP lowering as the mechanism of action by which ASOs antagonize prion disease ( 28 , 58 ). In both of these experiments, with blinded assessments (see Materials and Methods), we recapitulated our previous findings, demonstrating that ASO-mediated PrP lowering extended survival and delayed disease course, in both prophylactic and delayed treatment paradigms.…”

“… ASOs 1, 2, 3, 4 and 6 have been previously described ( 58 , 86 ). Color code for ASO chemical modifications: black = unmodified deoxyribose (2′H).…”

Multimodal small-molecule screening for human prion protein binders

“…Similarly, a range of anionic polymers 33,[39][40][41][42] also bind PrP and show in vivo antiprion activity in certain contexts 41,43,44 . However, these binding events may not be monomeric 45 nor specific to PrP 35,46 . Still other compounds with demonstrated antiprion activity exhibit interaction with PrP only at concentrations orders of magnitude above their effective concentration in cell culture 47,48 .…”

Cellular Prion Protein (PrPc): Putative Interacting Partners and Consequences of the Interaction

“…It should also be considered that some proteins in biological samples may interact with the surface of their containers. It is also noteworthy that both physical adsorption (physisorption) and chemical adsorption (chemisorption) phenomena may exist with regards to analytes and, in particular, proteins including PrPc [ 300 ]. The nature of PrPc interactions can be weak or short-lived or temporary, which makes their interactions very difficult to study with the limitations in analytical techniques.…”