The Clearance of Human Cytomegalovirus Using CRISPR/Cas9 RNA Lipid Nanoparticles

Abstract Human cytomegalovirus (HCMV), a DNA virus, poses significant health risks to immunocompromised and immunosuppressed individuals and newborns. Clinical antiviral drugs like ganciclovir inhibit viral replication but have toxicities, are ineffective against drug-resistant strains, and cannot destroy HCMV DNA. CRISPR/Cas9 can cleave DNA, but has not been used therapeutically to target and degrade HCMV DNA in cells post-infection. We developed an all-in-one CRISPR/Cas9 RNA lipid nanoparticle (LNP) that clears established HCMV infections, permits rapid updating to combat resistance and is effective against multiple strains. Bioinformatic analyses identified essential, conserved viral genes as CRISPR/Cas9 targets. A delivery material screen revealed that antiviral activity was dependent on the ionizable lipid and LNP composition. Our lead formulation, βN2-40, inhibited up to 93.5% of HCMV infection with a single treatment. Furthermore, multitargeting βN2-40 LNPs demonstrated antiviral kinetics and a safety profile similar to ganciclovir, making it a compelling alternative to existing small-molecule antiviral drugs. Competing Interest Statement OFK, YMAL, MQJ and the University of Toronto have filed an invention disclosure related to this work.