In preclinical work recently published in Nature Communications, Fred Hutch Cancer Center scientists used a genetic “chain reaction” to transform herpes simplex virus DNA during an HSV infection. The proof-of-concept study, which used a CRISPR gene editing tool to change the color of fluorescent viruses, potentially opens the door to a treatment that uses HSV-based gene therapy to cure HSV.
“This paper is really about establishing that [this genetic strategy] could be something that could work in the future,” said Fred Hutch virologist Marius Walter, PhD, the staff scientist in the Jerome Lab who led the project. “It’s proof of concept for a new technology, but we don't know yet if it will work and how much it will work.”
Walter capitalized on a phenomenon called “gene drive,” which can push a gene variant through a population, to retool HSV DNA lurking in infected neurons. His strategy also used engineered HSV virions to carry gene editing technology to the neurons where latent HSV DNA hides out.
While Walter didn’t alter HSV infectivity (that’s the subject of future work), he did show that engineered HSV can co-infect neurons with non-engineered HSV, which makes gene drive possible.
An HSV-based gene therapy built off this concept could act as a sheep in wolf’s clothing: a defanged HSV able to infiltrate infected cells and inactivate lurking viral DNA, rendering it toothless.
If successful, it would be a second gene therapy strategy to target HSV developed by Keith Jerome, MD, PhD, and his team.
“It’s always good to have multiple shots on goal — because this is a big goal,” Jerome said.
Fred Hutch file photos
HSV has been infecting humans for six million years. The World Health Organization estimates that nearly 65% of people under age 50 carry HSV, which can cause sores around the lips (usually HSV-1) or genitals (HSV-1 or HSV-2). The virus targets neurons; after an initial infection, it sets up a lifelong, latent infection and HSV DNA remains dormant in the form of DNA circles called episomes.
During the latent phase of infection, the virus can reactivate and produce new virions that travel through the nerves toward the skin, causing herpes outbreaks. Though many people experience no or tolerable symptoms, others find the infection to have debilitating effects on their health and quality of life. In rare cases, HSV can cause meningitis, encephalitis, blindness and, even more rarely, death. Herpes encephalitis is particularly dangerous in newborns who contract HSV from HSV-positive mothers during childbirth.
Herpes symptoms can be managed with antiviral therapy, but there is currently no vaccine or cure. And while the HSV episomes lurking in neurons are a source of new virus, they also pose an attractive target for potentially curative gene therapies.
Jerome’s team has spent years investigating strategies to cure or minimize this latent, lingering HSV DNA in neurons, as a way to minimize or perhaps even cure HSV infections.
“But it’s always good to have other options, more tools,” Jerome said.
Walter had previously developed a gene drive strategy that works in cytomegaloviruses, a member of the larger Herpesviridae virus family to which HSV belongs. Both Walter and Jerome were excited at the prospect of exploring the approach in HSV.
“I always loved the concept of gene drives in Marius’ work when he was at his previous institution, so the opportunity to have him come join us … and move it into model organisms was incredibly exciting,” Jerome said.
Gene drive enables a genetic trait to spread more rapidly through a population than average. If the “drive” is strong enough, the phenomenon can ensure that nearly all members of that population carry the trait.
“The concept of the gene drive is really just like a chain reaction that gets amplified,” Walter said.
It’s been studied as a way to help control populations of disease-carrying animals. Scientists have tested it in mosquitos as a way to reduce malaria transmission by heavily biasing mosquito sex ratios away from biting females and toward non-biting males.
Though viruses don’t mate like mosquitos do, some species can mingle their genetic information when individual viruses meet inside the same cell. In this case, gene drive could be used to inactivate HSV DNA.
Walter needed to show that gene drive pushes genetic changes through HSV DNA in infected cells. He also wanted to discover whether DNA from different HSV virions would mix in the same neurons during infection.
To do this, he used HSV as the delivery and targeting vehicle for his gene drive strategy.
The bonus to using HSV as the gene drive carrier is that it naturally goes where natural infection occurs, Walter said. (An HSV-based therapeutic already has precedent: earlier this year an oncolytic HSV therapy was fast-tracked by the U.S. Food and Drug Administration to treat head and neck cancer.)
“You’re basically using herpes to target herpes,” he said. “The idea is that it would be super specific and maybe will reach the relevant cells with a high efficiency.”
He set out to answer two questions: would his gene drive strategy work for HSV as it did for CMV, and were conditions during infection conducive to using an HSV-based gene therapy?
Walter had previously developed a CRISPR-based gene drive method that takes advantage of natural viral DNA mingling (also known as recombination) to insert a gene for a red fluorescent protein into viral DNA. This allows him to see gene drive in action: because the HSV he’s targeting naturally glows yellow, a shift to orange indicates gene drive has occurred.
For the current study, Walter engineered HSV-1 to carry the DNA editing strategy to neurons, where HSV infects and hangs out during latency.
He quickly showed that in lab dishes, engineered and non-engineered HSV virions could easily co-infect the same cells and undergo genetic modification.
“In a Petri dish, this is easy because you end up having a limited amount of cells and tons of virus, so a lot of the viruses are going to infect the same cells,” Walter said. “The big question that we spend a lot of time in this paper addressing is, ‘How much do you have co-infection happening in vivo?’”
He addressed the question using two scenarios: an acute infection and a reactivated latent infection. In a mouse model of acute HSV-1 encephalitis, he showed that in some areas of the nervous system, as many as 80% of the viruses showed evidence of gene drive.
Walter then used a mouse model of HSV-1 latency and reactivation (HSV doesn’t naturally reactivate in mice, so scientists use a drug to turn it back on). He found several examples of recombination with the non-engineered viruses, showing that both co-infection and gene drive can occur.
“We showed that you often have two viruses that infect the same cell,” Walter said. “This is where the paper brings in a lot of interesting basic biology, because it shows that [co-infection is] actually happening at a high frequency, which nobody had really shown in that way before.”
Now that he and Jerome have evidence that gene drive can occur during latent HSV infections, they’re working on moving that insight further, Walter said.
“We’re trying to see if we can use the technique as a way to prevent or cure disease,” he said.
It’s a tricky goal.
“You need an engineered virus that by itself does not cause disease, but needs to be able to meet and recombine with the wild-type virus to inactivate it,” Walter said. “So we need to find the balance between having an engineered virus that in itself doesn’t cause disease, but gets where it needs to go.”
To hopefully discover where that balance lies, Walter is moving his method to a preclinical model that better mimics the course of human HSV infection. He’s also working to address key questions about the factors that influence how efficiently gene drive occurs during latent HSV infection. What Walter learns in models of HSV-1 he will also translate to HSV-2, he said.
Whether gene drive could underpin a therapy that works during HSV dormancy, or would need to be administered during a reactivation phase, will become more clear with more work.
“The goal is to reduce the level of viral shedding and disease, but if we only reduce the level of symptomatic disease, it’s already a big win,” Walter said.
Jerome also noted that insights from Walter’s work complemented the other gene therapy approaches being studied in his lab, and vice versa. The team immediately use lessons learned from one approach to improve the other, he said.
“It’s really sped up the overall work and that's really the goal here: To provide something new to help people who are living with the virus,” Jerome said.
This work was funded by Fred Hutch Cancer Center, the National Institutes of Health and the Buck Institute for Aging Research.
Sabrina Richards, a staff writer at Fred Hutchinson Cancer Center, has written about scientific research and the environment for The Scientist and OnEarth Magazine. She has a PhD in immunology from the University of Washington, an MA in journalism and an advanced certificate from the Science, Health and Environmental Reporting Program at New York University. Reach her at srichar2@fredhutch.org.
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