CRISPR antiviral inhibits neurotrophic JC polyomavirus in 2D and 3D culture models through dual-gRNA excision by SaCas9.

Abstract

Without an effective antiviral, JC virus (JCV) has persisted throughout multiple epochs of immunosuppression, causing the opportunistic demyelinating disease, progressive multifocal leukoencephalopathy (PML). This study proposes a novel therapy using a dual-gRNA, SaCas9, CRISPR antiviral targeting JCV transcription factor, large tumor antigen (LT-Ag), and capsid protein, viral protein 1 (VP1). This treatment was validated using traditional two-dimensional cell culture. A recombinant cell line was produced from SVG astrocytes (SVGA) via lentiviral inoculation and puromycin selection. Following infection, sanger sequencing identified uniform excision of the circular dsDNA genome of JCV, significantly reducing viral load per genomic copy number on qPCR, viral proteins on western blot, and infectivity of viral progeny on adoptive transfer. Following this proof-of-concept using cell lines, translatability of results was advanced using three-dimensional, heterogeneous cerebral organoids (COs). COs were infected and treated with the lentivirus-packaged CRISPR antiviral. As observed in monolayer culture, a truncated genome was confirmed with sequencing, reducing viral load per genomic copy number on qPCR, protein levels on immunofluorescent imaging, and infectivity on adoptive transfer. The high efficacy of this JCV-targeting CRISPR antiviral in the context of cerebral organoids expounds on its value for the currently untreatable JCV and PML.