Antiviral Targeting of Varicella Zoster Virus Replication and Neuronal Reactivation Using CRISPR/Cas9 Cleavage of the Duplicated Open Reading Frames 62/71

Abstract

Varicella Zoster Virus (VZV) causes Herpes Zoster (HZ), a common debilitating and complicated disease affecting up to a third of unvaccinated populations. Novel antiviral treatments for VZV reactivation and HZ are still in need. Here, we evaluated the potential of targeting the replicating and reactivating VZV genome using Clustered Regularly Interspaced Short Palindromic Repeat-Cas9 nucleases (CRISPR/Cas9) delivered by adeno-associated virus (AAV) vectors. After AAV serotype and guide RNA (gRNA) optimization, we report that a single treatment with AAV2-expressing Staphylococcus aureus CRISPR/Cas9 (saCas9) with gRNA to the duplicated and essential VZV genes ORF62/71 (AAV2-62gRsaCas9) greatly reduced VZV progeny yield and cell-to-cell spread in representative epithelial cells and in lytically infected human embryonic stem cell (hESC)-derived neurons. In contrast, AAV2-62gRsaCas9 did not reduce the replication of a recombinant virus mutated in the ORF62 targeted sequence, establishing that antiviral effects were a consequence of VZV-genome targeting. Delivery to latently infected and reactivation-induced neuron cultures also greatly reduced infectious-virus production. These results demonstrate the potential of AAV-delivered genome editors to limit VZV productive replication in epithelial cells, infected human neurons, and upon reactivation. The approach could be developed into a strategy for the treatment of VZV disease and virus spread in HZ.