PROJECT SUMMARY Herpes simplex virus (HSV) type 2 is the leading cause of recurrent genital herpes which can have severe physical and psychosocial consequences. The mainline therapy to treat HSV infection, acyclovir, is still inadequate as it only shortens the duration of lesions during primary or recurrent disease by a few days at best. Furthermore, while long-term therapy can limit the frequency of lesions, it only reduces the likelihood of transmission to a sexual partner by about 50%. Most importantly, acyclovir only suppresses viral replication, but it does not eliminate latent HSV harbored by ganglionic neurons, which is the source for recurrent disease. Thus, once treatment is stopped viral replication can begin again. We have developed a potentially curative therapy for latent HSV infection, based on gene editing using HSV-specific meganucleases. Our original work focused on the treatment of infections and pathogenesis caused by HSV type 1 (HSV-1) and demonstrated that adeno-associated virus (AAV)-mediated delivery of anti-HSV-1 meganucleases eliminated up to 97% of latent HSV DNA from ganglia in animal models of latent HSV-1 infection. Furthermore, we showed that this reduction in ganglionic viral load led to a reduction or even elimination of peripheral virus shedding. In this Phase I STTR proposal, we plan to extend our anti-HSV gene editing strategy to target HSV-2, the leading cause of genital herpes. After in vitro selection of a highly active and specific anti-HSV-2 meganuclease, we will demonstrate its activity in an in vivo guinea pig model of HSV-2 infection, and carefully evaluate the safety and tolerability of this approach. There is tremendous patient interest in potentially curative therapies, and with approximately 20 million people infected with HSV-2 in the US alone, the potential impact of a successful therapy is enormous. Specific Aim 1. Select a lead anti-HSV-2 meganuclease variant. We used our established informed randomized mutagenesis and selection approach to generate 6 lead candidate meganucleases recognizing HSV-2 as determined by yeast surface display and in vitro cleavage assays. These candidates will be further assessed and validated using a mammalian cell reporter system for on- and off-target activity, to select a lead meganuclease for further in vivo development. Specific Aim 2. Demonstrate meganuclease antiviral activity in the guinea pig model of HSV-2 infection. The meganuclease showing the best activity and specificity will then be evaluated in the guinea model of HSV-2 infection for its ability to reduce ganglionic load and HSV shedding and symptomatic disease. We will also carefully examine treated animals for general tolerability and evidence of therapy-associated genotoxicity, specifically off-target activity, chromosomal translocations, and integration of sequences in host genomes.