Characterizing Determinants of Species Barrier in Prion Infection Using Gene-edited Cell Culture Models

Abstract

Prion diseases, fatal neurodegeneraƟve disorders, are caused by the misfolded isoform of the prion protein (PrPSc), which can propagate and induce prion disease across different species. The primary aim of this study was to establish a cell model for invesƟgaƟng the prion propagaƟon in various species, including camel and cervids. A major obstacle in studying prion diseases is the "species barrier," where prion transmission between species is inhibited due to structural mismatches between the normal host prion protein (PrPC) and the infecƟous isoform, PrPSc. Overcoming this barrier is crucial for studying emerging prion diseases, such as Chronic WasƟng Disease (CWD) and Camel prion disease. Another key obstacle is “dominant-negaƟve inhibiƟon of prion replicaƟon”, where endogenous PrP, such as mouse PrP, can negaƟvely interfere with exogenous prion infecƟon. Therefore, it is essenƟal to first confirm the complete knockout (KO) of endogenous PrP in our cell culture system, then transduce species-specific PrP into the KO cells. To achieve this, we first confirmed the complete knockout of endogenous mouse PrP in a CAD5 subclone by performing bacterial gene cloning, sequencing, and INDEL idenƟficaƟon. Then, we used lenƟviral transducƟon to introduce PrP genes from mouse, bank vole, cervid, and camel into the neuronal CAD5-KO cells. Immunoblot analysis confirmed successful PrP expression in these gene-edited cells, thereby overcoming the species barrier. Fluorescent imaging and Fluorescence- AcƟvated Cell SorƟng (FACS) were used to further characterize and enrich the cells for high PrP expression. Subsequently, we challenged these engineered cell models with brain homogenates from prion-infected animals to study prion infecƟon. Notably, for the first Ɵme, we report the detecƟon of PrPSc in camel-PrP-expressing cells infected with camel prions. AddiƟonally, cervid-PrP-expressing cells infected with CWD prions showed the presence of PrPSc bands in immunoblot analysis, validaƟng prion propagaƟon in our cervid model. This is the first evidence of successful camel prion propagaƟon in a cell model, demonstraƟng the ability of these engineered cells to overcome the species barrier. Overall, this model provides a novel plaƞorm for studying prion biology and cross-species transmission.