Rapid generation of prion disease models using AAV ‐delivered PrP variants in knockout mice

The study of prion biology has traditionally relied on transgenic mouse models, which, while valuable, require significant time and resources to develop. Here, we present a rapid and flexible alternative using adeno‐associated virus (AAV) vectors to express modified prion proteins in PrP‐knockout (PrP‐KO) mice. Through systematic evaluation of multiple AAV constructs, we optimized vector design by comparing different CNS‐specific promoters and regulatory elements to generate prion disease models capable of faithfully propagating the inoculated prion strain. We identified an optimized AAV construct incorporating the human synapsin promoter, MVM enhancer, and WPRE posttranscriptional regulatory element encapsidated in the AAV9P31 serotype to drive neuron‐specific expression of modified mouse PrP (W144Y epitope) and bank vole I109 PrP (W145Y epitope). Following intravenous administration, we achieved brain‐wide expression at levels comparable to or even exceeding endogenous PrP in some regions. When challenged with mouse‐adapted RML prions or human Gerstmann‐Sträussler‐Scheinker (GSS‐A117V) disease‐causing prions, AAV‐PrP mice developed characteristic signs of prion disease with accelerated kinetics (58–106 days post‐inoculation for RML; 105–112 dpi for GSS‐A117V), displaying features typical of each strain. Serial transmission of AAV‐generated RML prions to wild‐type mice confirmed preservation of strain‐specific properties (165 ± 4 dpi), validating the authenticity of prion propagation in this system. This approach provides a versatile platform for rapidly generating and studying prion variants in an authentic brain environment. By reducing model generation time from months to weeks, this system enables accelerated investigation of prion structure–function relationships, strain properties, and therapeutic strategies, with potential applications extending to other protein misfolding diseases.