In Hutch’s herpes experiments, AAVs are found in clusters of nerves harboring inactive or latent herpesviruses, and then the scissors go to work, targeting and cutting certain segments of viral genes. For their latest experiments, the Hutch team selected a different combination of AAVs and improved their results.
Previously, Jerome and Aubert reported that the drug can eliminate more than 90% of latent herpesvirus in nerve clusters near the faces of mice injected with the enzyme-carrying AAVs. In this new study, they describe how they have tested the therapy for the first time to treat infections in a group of nerves called the dorsal root ganglia, near the genital tract of mice. They found that the experimental therapy reduced the latent virus there by 97%.
Jerome said that for technical reasons, measuring latent infections in the dorsal root ganglia is extraordinarily difficult, but Aubert was able to achieve it.
“We didn’t know how well our therapy worked in these nodes, and the response was that it worked best of all, which is very good news,” Jerome said.
Until this latest report, Jerome and Aubert did not have a good method to assess whether the reduction of latent virus also reduced the level of viral spread. This is because the mice used in these studies do not naturally reactivate latent herpes infections, the source of the alarming and periodic outbreaks of painful sores that recur in humans.
The team solved this problem by using a drug that stimulates recurrences of herpes simplex type 1, or HSV-1, in mice. The results showed what might have been expected, but had never been demonstrated: Those mice whose latent virus had been reduced by gene therapy shed far less virus than controls whose latent virus had not. done
An analysis combining the five mouse experiments found a statistically significant reduction in both the frequency and amount of viral shedding among all treated animals compared to controls. Importantly, the levels of reduction depended on both the dose and the duration of the treatments.
“Spill is the biggest thing, and we see a big difference here,” Jerome said.
Concerns about potential toxicities
The Hutch scientists note, however, that the good news is tempered by recent concerns within the gene therapy field about the potential for therapies using AAV to cause liver and nerve damage.
The issue of toxicity has been raised by other gene therapy researchers based on their own experiments. It does not appear to involve the gene-cutting enzymes used in Hutch’s herpes studies, where these molecular scissors work as expected. Instead, it focuses on the AAVs used to package and transport them.
“AAV vectors have generally been considered quite safe. … However, emerging work has suggested that AAV vectors may not be as innocuous as previously assumed,” the Hutch authors wrote in the bioRxiv paper.
They observed liver toxicity in some of their mice, but said it appears to be the result of very high concentrations of AAV, far above what is now known to be necessary.
“We know what the toxic dose is, and we get good results below that,” Jerome said.
Neurotoxicity is potentially more concerning. The Hutch team reported “subtle evidence of neuronal injury” in mice when tissue samples were examined under a powerful microscope. However, they also saw no evidence in these animals of changes in behavior or mobility, such as impaired gait or balance, after gene therapy.
“We saw some toxicity in the slides in the first experiments, and maybe that’s something we can handle very easily,” Jerome said. “But we just don’t know. We want to know what exactly is causing this.”
A priority in the next round of research will be to solve this mystery and, if necessary, find a way to avoid it. Researchers are confident that the problem can be solved.
“Like any scientific project, we always stumble on some things. It’s actually how we make discoveries,” Aubert said.
“It rarely goes well. There’s always something you didn’t expect to come up,” he said. “It’s like working with a recipe. ‘Oh, I put too much salt! Let’s try to step back and put in less and see how it goes.’ Or maybe there’s a little ingredient missing and I just need to figure out what’s missing to make it tastier.”
Ensuring safety before starting human trials
The researchers are conducting additional preclinical studies of the therapy in guinea pigs, which, unlike mice, have naturally occurring recurrent outbreaks of latent herpes infections. As with mice, the initial focus of this research is on HSV-1, which is primarily associated with cold sores. However, Dr. Anna Wald, a researcher at Fred Hutch, notes that recent studies, including one she published with colleagues at the University of Washington, hint at a change.
HSV-1 is becoming, especially in first infections in adults under 30, the leading cause of genital herpes, the most feared disease traditionally associated with HSV-2. Regardless, if the experimental therapy works for HSV-1, the researchers are confident it can be adapted relatively easily to target HSV-2.
Wald, who also directs UW’s Division of Allergy and Infectious Diseases, has been researching vaccines to prevent or treat HSV infection since 1991. He said Jerome and Aubert’s work is a change from paradigm in the field, because the hypothesis has been that once a person is infected with a herpesvirus, and there are eight members of that family, it’s impossible to get rid of that particular strain.
“It’s a completely new approach, where you can take a member of the herpes group of viruses and eradicate it from the host. As a proof of principle, the fact that it can be done even in a them it’s kind of mind-blowing,” he said.
Jerome and Aubert still hope to get FDA approval to test the therapy in humans in an early-stage clinical trial designed primarily to ensure it’s safe before the end of 2023. If toxicity issues are more discouraging, the expected human studies will have to wait until these questions are resolved.
The work was funded by the National Institutes of Health, the Caladan Foundation and more than 1,600 individual donors. Cellectis developed the meganucleases used in this research.
Note: Fred Hutch scientists played a role in the development of these discoveries, and Fred Hutch and some of its scientists may benefit financially from this work in the future.