Gene-edited murine cell lines for propagation of chronic wasting disease prions

Abstract

Prions cause fatal infectious neurodegenerative diseases in humans and animals. Cell culture models are essential for studying the molecular biology of prion propagation. Defining such culture models is mostly a random process, includes extensive subcloning, and for many prion diseases few or no models exist. One example is chronic wasting disease (CWD), a highly contagious prion disease of cervids. To extend the range of cell models propagating CWD prions, we gene-edited mouse cell lines known to efficiently propagate murine prions. Endogenous prion protein (PrP) was ablated in CAD5 and MEF cells, using CRISPR-Cas9 editing. PrP knock-out cells were reconstituted with mouse, bank vole and cervid PrP genes by lentiviral transduction. Reconstituted cells expressing mouse PrP provided proof-of-concept for re-established prion infection. Bank voles are considered universal receptors for prions from a variety of species. Bank vole PrP reconstituted cells propagated mouse prions and cervid prions, even without subcloning for highly susceptible cells. Cells reconstituted with cervid PrP and infected with CWD prions tested positive in prion conversion assay, whereas non-reconstituted cells were negative. This novel cell culture platform which is easily adjustable and allows testing of polymorphic alleles will provide important new insights into the biology of CWD prions.

An astrocyte cell line that differentially propagates murine prions

“…Interestingly, these were mostly established cell lines that allowed persistent propagation of mouse-adapted scrapie prion strains like 22L, RML, or ME7 ( 24 , 25 ). By using genetically engineered cells, it seems possible to expand the range of prions which can be propagated in such cell lines ( 26 – 28 ). There still is no cell line that can persistently propagate human or bovine prions ( 25 ).…”

From Cell Culture to Organoids-Model Systems for Investigating Prion Strain Characteristics

“…CAD5 cells expressing bank vole PrP (bvCAD5 cells) were also able to propagate these strains of CWD, but with lower efficiency. These bvCAD5 cells could also be infected with 22L prions [ 33 ]. Additionally, Bourkas et al, demonstrated that CAD5 cells expressing hamster PrP could propagate the 263K, hyper (HY) and 139H strains of hamster-adapted prions, but not the drowsy (DY) strain [ 32 ].…”

Cellular models for discovering prion disease therapeutics: Progress and challenges

“…Successful infection of cells is typically assessed by detecting PK-resistant PrP in cell lysates, although this can also be done using intact cells adhered to a membrane (Bosque & Prusiner, 2000). More sensitive techniques such as RT-QuIC can also be used to detect low levels of cellular prion infection (Walia, Ho, Lee, Gilch, & Schatzl, 2019). It is important to note that, generally, infection of immortalized cell lines with prions does not appear to produce any major cytotoxic effects.…”

“…Recently, CWD prion infection has been achieved in murine fibroblast lines that were genetically engineered to lack endogenous mouse PrP C expression using CRISPR/Cas9 gene editing technology (Walia et al, 2019). Upon expression of cervid PrP C , these cells could be infected by CWD prions, but infection could only be detected using the ultrasensitive RT-QuIC technique.…”

“…Interestingly, CJD passaged in bank voles appears to retain the characteristics of the original CJD isolates, suggesting that BVPrP-adapted CJD prions may be an excellent proxy for authentic CJD prions (Nonno et al, 2006;. The recent development of a PrP-knockout version of prion-permissive CAD5 cells may be valuable for examining the ability of human prions to propagate in cultured cells expressing BVPrP or other PrP molecules (Bourkas et al, 2019;Walia et al, 2019). Chimeric mouse/human PrP molecules have been identified that permit the rapid propagation of human prions in transgenic mice (Giles et al, 2010), and such PrP variants may be particularly useful for propagating human prions in immortalized cells.…”