Ovine recombinant PrP as an inhibitor of ruminant prion propagation in vitro

Abstract

Prion diseases are fatal and incurable neurodegenerative diseases of humans and animals. Despite years of research, no therapeutic agents have been developed that can effectively manage or reverse disease progression. Recently it has been identified that recombinant prion proteins (rPrP) expressed in bacteria can act as inhibitors of prion replication within the in vitro prion replication system protein misfolding cyclic amplification (PMCA). Here, within PMCA reactions amplifying a range of ruminant prions including distinct Prnp genotypes/host species and distinct prion strains, recombinant ovine VRQ PrP displayed consistent inhibition of prion replication and produced IC50 values of 122 and 171 nM for ovine scrapie and bovine BSE replication, respectively. These findings illustrate the therapeutic potential of rPrPs with distinct TSE diseases.

Recombinant ovine prion protein can be mutated at position 136 to improve its efficacy as an inhibitor of prion propagation

“…To further quantify the level of inhibition for each rPrP, IC50 values were determined as previously described 24 . The rVRQ IC50 was estimated to be 114±25 nM (from 3 repeat experiments, Figure 2 and data not shown)) which corresponded well with the previously reported value of 122 nM 24 . For rRRQ, rCRQ, rLRQ, rYRQ, rHRQ, rKRQ, rPRQ and rERQ the values were estimated to be 16, 112, 17, 45, 90, 2, 2 and 95 nM, respectively from single experiments (Figures 2 and S2).…”

“…Yuan and co-workers demonstrated that homologous human rPrP could inhibit prion conversion in PMCA and this was more effective than heterologous mouse rPrP 23 . Furthermore, rVRQ inhibited propagation of PrP Sc in PMCA for the conversion of substrate with a range of ovine PrP C genotypes and bovine PrP C , for distinct ruminant prion seeds and where substrate/seed combinations had non-matching PRNP genotypes 24 . It has been suggested that rPrP may act as an effective inhibitor of PrP Sc propagation as it lacks the GPI anchor and any glycosylation and so may be an ineffective conversion substrate 23 .…”

“…Potential therapeutics have included small molecules [11][12][13][14] , anti-PrP C /PrP Sc antibodies [15][16][17] , and antisense oligonucleotides to lower PrP C levels 18,19 . The development of therapeutics has used in vitro assays of prion replication such as protein misfolding assays [20][21][22][23][24] , cell models of infection 11,17,21,23,25−27 , animal models of infection 28,29 and several drugs have made it into human clinical trials. The latter therapeutic candidates have included quinacrine and pentosan polysulphate (PPS).…”

“…It is well established that in in vitro and in animal models the presence of a heterologous PrP C can block or delay disease or prion replication 20-24, 29,40-42 . A further approach is to use heterologous recombinant PrP (rPrP) as the therapeutic and studies have shown that such an approach can prevent prion replication in in vitro assays [20][21][22][23][24] , in cell culture 21,23,40,41 and also in rodent models 29,42 . Peptides of PrP have also been reported to be effective at inhibiting prion replication, including in cell culture models 20,21,23 .…”

“…Here, we determined whether mutations at an amino acid residue in ovine PrP that is crucial in dictating susceptibility to scrapie can provide a mutant rPrP that is more effective at blocking prion replication than the natural variants previously reported 24 . The study used protein misfolding cyclic ampli cation (PMCA) as the main model of prion convertion.…”