Engineering Protective PrPC Variants to Suppress and Reverse Prion Infection in Cell Models

Introduction: Natural evolution of the prion protein (PrP^C) in humans has revealed the protective effects of specific sequence variants—such as those at codons 129 and 127—against prion disease.

Objectives: We sought to identify additional PrP^C mutations that suppress prion propagation in cell culture as potential gene therapy targets.

Methods: Using the scrapie cell assay, we examined the propagation capacity of select PrP^C mutants.

Results/Discussion: In mouse cell lines lacking endogenous PrP^C, de novo expression of PrP^C variants carrying alanine substitutions within the flexible helix-2 (H2) region markedly reduced prion infection. However, when both wild-type and H2-alanine mutant PrP^C were co-expressed, prion propagation persisted. In contrast, alanine mutations within or near the helix-1(H1) region, situated in the N-terminal lobe of the amyloid, blocked the continuation of infection both in PrP^C knockout cells and in cells co-expressing wild-type and mutant PrP^C. Similar inhibition was observed with alanine substitutions in the initial N-terminal loop region. Notably, in chronically infected lines, H1 mutants demonstrated a “curing” effect. Collectively, these findings suggest that specific mutant PrP^C substrates may bind to existing fibrils, acting as caps to halt further propagation. Moreover, mutations within the H1 region appear to actively dissolve existing prion fibrils—whether by inhibiting their seeding capacity, accelerating degradation, or engaging other mechanisms that facilitate clearance.

Conclusion: Further investigation into how these mutations affect toxicity and perform in human cell lines will provide valuable insight; nonetheless, these findings highlight the prospects of engineering targeted PrP^C mutations as an innovative gene therapy approach.