Hsv1-specific Meganuclease May Reduce Ocular Infection In A Mouse Model Of Herpes Keratitis

Abstract

Purpose: : Current anti-viral treatments of Herpes simplex virus type 1 (HSV1) inhibit the viral replication but do not impair the latent form of the virus. This could be addressed by rare cutting endonucleases, such as meganucleases. The aim of this study was to demonstrate the antiviral activity of a HSV1-specific meganuclease in a mouse model of herpes keratitis.

Methods: : Three weeks after bilateral subconjunctival inoculation of 10¹⁰ particles of recombinant associated-adenovirus (rAAV) encoding either a HSV1-specific meganuclease or a reporter gene (GFP), the cornea of mice was scarified and inoculated with the wild-type HSV1 strain SC16 (100 pfu). At either 6 or 28 days post-inoculation (dpi), mice were sacrificed and cornea, trigeminal ganglion (TG), superior cervical ganglion, and spinal cord were analyzed for the presence of HSV1 genome and viral transcripts (namely LAT, TK and UL18 transcripts).

Results: : Among the mice treated with either GFP or meganucleases and sacrificed at 6 dpi (time of acute infection), there was no clinical difference in the rate acute herpetic keratitis (11/20 eyes and 10/20, respectively), nor in the amounts of viral genomes or viral transcripts in the tested tissues. In contrast, in mice sacrificed at 28 dpi (expected stage of HSV1 latency), the rate of clear cornea (no ongoing keratitis and no scar) was more important in the mice treated with meganuclease-encoding rAAV (18/22 eyes versus 10/20 in the GFP group, p = 0.03). Accordingly, the amounts of viral genome and UL18 viral transcripts (which are found in case of ongoing viral replication) were significantly less important in the meganuclease treated mice (p<0.02) in all the tested tissues, showing a significant reduction in the viral activity.

Conclusions: : This study shows that the subconjunctival inoculation of r-AAV coding a HSV1-specific meganucleases in the ocular tissues is associated with an improvement of corneal recovery, and a reduction in the viral load and replication in the cornea and the neurological tissues, especially in the TG. These results suggest that specific meganucleases could be qualified as a new class of antiviral agent, with the potential to address replicative as well as latent viral DNA.

Keywords: herpes simplex virus • antiviral drugs • keratitis