Herpes simplex virus (HSV) infects a large proportion of the population, many of whom are asymptomatic. HSV infection is acquired at the mucosal surface where the virus accesses sensory nerve endings and travels down the axons to the neuronal bodies. Once in the neuronal body the virus establishes a productive latent infection. Periodically, the virus will reactivate traveling back down the axon creating an actively replicating viral infection with viral shedding (roughly 28% of time after infection) and sometimes symptoms (on average 2-7 times per year). This viral shedding resulting from the reactivation event increases transmission even in asymptomatic hosts. Current therapies shorten the duration of infections and reduce symptomatic recurrence. However, the drugs have no effect on persistent, or latent HSV, only treating the symptoms of infection not the viral pathogen itself. In an article published in Journal of Clinical Investigtions insight, Dr. Aubert and her colleges in the Jerome lab (Vaccine and Infections Disease Division) have been working towards developing targeted endonucleases that cleave viral DNA and trigger DNA mutagenesis at specific sites, possibly disrupting persistent HSV in neurons.
Image provided by Dr. Jerome
In order to harness the power of targeted homing endonucleases, the Jerome lab worked to optimize a useful delivery system for both in vivo and in vitro studies. They used an adeno-associated virus (AAV) delivery system to target mouse trigeminal ganglia with either of two HSV specific endonucleases: HSV1m5, which is designed to cleave the virion protein VP5 gene, and HS1m8 ,which targets the gene encoding DNA polymerase catalytic subunit. Creating mutations in either of these genes causes disruption of the viral genome and prevents virion production. In order to further increase the effects of the treatment Trex-2, a 3’-5’ endonuclease, which has been shown to increase directed mutagenesis, was co-transduced. After establishing the system, the lab started in vitro work focusing on neurons, the biologically relevant cell type. Cultures of neurons were co-transduced with HSV1m5 or 8 and Trex-2 and then infected with HSV. Treated cultures showed a 50% reduction in viral titer as well as the presence of mutations in HSV by the T7 Endonuclease 1 assay and clonal sequencing. Once disruption of lytic infection was established the researchers started looking at effects on the different stages of HSV infection. Using infected cultures of neurons with HSV and subsequent treated with HSV1m5 or 8 with Trex-2 at different time points the researchers could simulate the various stages of infection. They treated cultures at 7 days (acute phase), 14 days (late acute/ early latent phase), and 32 days (latent phase) and found no change in effect regardless of viral phase. This suggests that HSV is similarly susceptible to endonuclease mutagenesis regardless of viral phase. The levels of target-site mutations ranged from 4.5-7.6% and 1.1-6.6% for HSV1m5 and HSV1m8 respectively. Currently, this is the first study to look at endonuclease activity in latently infected neurons.
Encouraged by the in vitro results, the researchers performed in vivo studies using mice infected with HSV followed 32 days later by AAV (expressing HSV1m5 or 8 and Trex-2) injection. Neurons from the mice were sampled 30 days after AAV injection for viral titer, viral load, and HSV genome mutations. The group showed 1-2% mutations in 2/4 mice for HSV1m5 and in 3/4 mice for HSV1m8. However the viral load of these animals was similar between control and treated animals. Viral shedding was delayed by one day in treated animals but reached similar levels over time compared to untreated. Upon reflecting on the results Dr. Jerome said, “This is the first time an actual latent viral infection has been established in an animal, and then disabled, even partially, using endonuclease therapy. So it represents a critical step toward bringing such therapies to human application. The main challenge now is to increase the potency of the therapy, so that all or nearly all of the latent virus is genetically disabled. We’re investigating newer, more efficient nucleases like CRISPR/Cas, and evaluating new methods of delivering the nucleases to infected neurons in vivo.” In the future, this therapy may lead to a new way of thinking about treating latent diseases such as HSV and HIV that we currently view as incurable.
Funding for this research was provided by the National Institutes of Health and the Canadian Foundation.
Aubert M,Madden EA,Loprieno M,DeSilva Feelixge HS,Stensland L,Huang ML,Greninger AL,Roychoudhury P,Niyonzima N,Nguyen T,Magaret A,Galleto R,Stone D,Jerome KR. 2016. In vivo disruption of latent HSV by designer endonuclease therapy. JCI Insight, 1(14).