CRISPR/Cas9 Targeted Disruption of Herpes Simplex Virus type 1 in a Rabbit Latency Model Reduces Viral Reactivation and Associated Corneal Pathology

Abstract

Purpose : Recurrent herpes simplex virus (HSV) ocular keratitis, a result of latent virus reactivation within the trigeminal ganglia (TG), is considered the leading cause of infectious corneal blindness worldwide. Current standard of care fails to block latent virus reactivation and, for many patients, is contraindicated. To address this unmet medical need, we have developed a gene editing approach using CRISPR-associated protein-9 (Cas9) nuclease and guide RNAs (gRNAs) to target latent HSV-1 genomes in a rabbit model of recurrent HSV-1 keratitis.

Methods : gRNAs (N=411) assembled into ribonucleoproteins were screened against HSV-1-specific sequences in high throughput in vitro assays. For the rabbit model of recurrent HSV-1 keratitis, HSV-1 strain 17Syn+ was applied topically to scarified corneas of New Zealand white rabbits and allowed to establish latency in the TG over four weeks. Rabbits were then treated with adeno-associated virus (AAV) serotype 8 vectors expressing Cas9 and gRNAs targeting GFP (negative control) or HSV-1 genes, applied individually or in combinations, to re-scarified corneas at a total of 1.0E+11 viral genomes/eye. Latent HSV-1 was reactivated in each rabbit using epinephrine iontophoresis. Post-reactivation, tear swabs were collected daily and slit lamp examinations were conducted every 2-3 days. HSV-1 virions were detected in tear films using a plaque assay. Viral genomes in TG were quantified using qPCR. Statistical analyses utilized one-way ANOVA.

Results : Of the 411 gRNAs screened, 56 hits targeting 13 essential viral genes were selected for follow up. In rabbits, gRNAs targeting UL48 or RL2 inhibited HSV-1 production in tear films up to 64% (p>0.05) and corneal lesions were suppressed by up to 56% (p>0.05); a combination of gRNAs targeting UL48 and RL2 inhibited HSV-1 virions in tear films by 75% (p=0.03) and reduced corneal lesions by 91% (p<0.01), compared to the control group.

Conclusions : This study is the first to establish an in vivo proof of concept for a topically applied AAV expressing CRISPR/Cas9 editing machinery for controlling recurrent HSV ocular keratitis. Efficacy was greatest using combinations of gRNAs to reduce secreted virions and associated corneal pathology, favoring a multiplexed gene editing approach.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.