Characterization of the Prion Protein Binding Properties of Antisense Oligonucleotides.

Andrew G Reidenbach,Alison J Leed,Michael F Mesleh,Hien T Zhao,Eric Vallabh Minikel,Holly B Kordasiewicz,Stuart L Schreiber,Sonia M Vallabh,Stacy G Guzman

doi:10.3390/biom10010001

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.

Highlights

Prion disease is a fatal, incurable neurodegenerative disease caused by misfolding of the prion protein (PrP) [1]
[21] Antisense oligonucleotides (ASOs) with MOE and/or cEt modifications employed more recently [13]. 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 sequence-independent manner with nanomolar affinity and to antagonize misfolded PrP accumulation in cell culture with nanomolar efficacy [6,11,12], yet in vivo, we have reported that only antisense sequences targeting the PrP RNA are effective at extending survival in prion-infected mice [13]

Summary

Introduction

Prion disease is a fatal, incurable neurodegenerative disease caused by misfolding of the prion protein (PrP) [1]. Antisense oligonucleotides (ASOs) in preclinical development for prion disease aim to lower PrP expression in the brain, a therapeutic strategy supported by strong genetic proof of concept [2]. These ASOs are being designed to trigger RNase H1 cleavage of. PS ASOs have been reported to bind PrP in vitro with nanomolar affinity [11] in a sequence-independent manner, lower PrP expression in cultured cells with micromolar potency [6,12], and even strongly inhibit the accumulation of misfolded PrP in prion-infected cultured cells, with low nanomolar potency [6,11,12].

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Conclusion