By Jonathan D. Grinstein, PhD

When Brian Cosgrove, PhD, Principal Scientist at Tune Therapeutics, was thinking of what to go after with epigenetic editing, generating first-in-class viral therapeutics—in particular, the hepatitis B virus (HBV)—wasn’t necessarily the first thing that came to mind.

“We started linking up some of the biology and said, ‘HBV—there’s a vaccine and a treated population,’” Cosgrove told GEN Edge. “Then, on a couple levels, you realize what the patient population is that actually has chronic hepatitis B worldwide, and it’s gigantic—it’s in the hundreds of millions.”

HBV causes chronic disease in two interesting ways: viral DNA integrates into the host genome, and a covalently closed circular DNA (cccDNA) forms and becomes a minichromosome in the nucleus of hepatocytes.

“It’s cool! It’s a Frankenstein-like chromatin structure that forms—half human, half viral,” said Cosgrove. “What really captured all of us is that here is a disease that has very unique epigenetic regulation and one that we really felt like there was a value add for this platform to come in and make a huge difference in the lives of patients and hopefully reach a functional cure for this disease.”

Emerging from stealth in 2021, Tune Therapeutics is one of the leaders in the rapidly growing arena of epigenome editing. The scientific co-founders are Charles Gersbach, PhD (Duke University) and Fyodor Urnov, PhD (Innovative Genomics Institute). GEN Edge recently conducted an exclusive interview with Urnov, during which he spoke at length about Tune’s founding and philosophy.

For the first time, Tune Therapeutics made data available to support their chronic HBV program, showing that it can safely silence key viral persistence and replication mechanisms in various model systems.

On day 4 of the 2023 HepDART hepatology conference in Cabo San Lucas, Mexico, the epigenetic editing company showed how their TEMPO platform silences HBV via the epigenome and does not cut, damage, or alter the genomic DNA.

One guide to rule them all

Based on these data, Cosgrove says that TUNE-401 safely turns off both the viral DNA integrated into host chromosomes and the extra-chromosomal cccDNA “viral factories” needed for HBV to stay in the body.

The effect also lasts a long time. It has stopped almost all viral DNA replication in primary human hepatocytes and cell lines grown in the lab. It has worked for over 550 days and more than 275 cell divisions.

“From one single, transient delivery, we have been out to 550 days and counting therapeutically viable levels of repression,” said Cosgrove. “This example of an in vitro cell dividing every other day and still seeing repression is exciting. It’s just not something that we’ve seen! The joke is, ‘Which birthday are we going to get these cells out to?’”

The Tune team has simplified their targeting method only to need one guide RNA (sgRNA) to go after both DNA depots: the integrated viral DNA and the cccDNA. This single sgRNA targets both DNA depots with almost complete repression (90–95% peak repression). Cosgrove said that this sgRNA targets a very conserved sequence that they hope will be penetrant for human patients. Cosgrove also said they found minimal background epigenetic changes compared to controls, suggesting very specific targeting of the sgRNA.

Human chimeric liver mice

The goal then became to show that this targeting strategy could translate into in vivo models. As HBV infects only human cells, the team at Tune had to come up with a model using human chimeric liver mice where the rodent hepatocytes get ablated and replaced by engrafted human hepatocytes. Cosgrove said that human cells could make up the vast majority of the liver, around 90%, and, upon injecting HBV into the animals, 97% of them get infected and show clinically relevant levels of the virus and cccDNA.

Cosgrove said that when they compared TUNE-401 to the standard-of-care nucleoside analogues (NAs), which work by inhibiting the activity of the HBV DNA polymerase from reducing viral replication, the NAs don’t target the cccDNA, which is integral to the virus’s persistence and resistance to antivirals. So, in essence, NAs can stabilize patients but not rid them of the viral depots.

Cosgrove says the main problem with the human chimeric liver mice model is that any living mouse hepatocytes soak up any lipid nanoparticles (LNPs) put into these systems. Tune researchers showed that TUNE-401 was delivered to an average of 63% of the human hepatocytes. Cosgrove said their data suggests near-complete repression of HBV that extends throughout the lifespan of the animal.

“We’re excited that this is a very good model in terms of the cccDNA and the epigenetic regulation with high levels akin to humans, and we’re getting near complete repression in all the cells that we could get it to,” said Cosgrove.

Finding the right large animal model

Although it’s crucial to demonstrate safety and efficacy in a large animal model, the only other species that can contract HBV besides humans are chimpanzees, which are no longer used for modeling.

Cosgrove and his Tune colleagues used non-human primates (NHPs) as a substitute model and changed the sgRNA to one that targets PCSK9 for methylation-driven promoter silencing, but they left the delivery vehicle and payload the same. Cosgrove said that they have data showing PCDK9 repression for nine-plus months now and counting with a single LNP infusion.

“I hope that the decisions that we’ve made in our modeling really help us push it to the clinic faster,” said Cosgrove. “We’ve set that high bar for ourselves.”

Tune’s goal is for TUNE-401 to be in the clinic by the end of 2024.

“We have a new modality with durable repression without the need for immune reactivation and just relying on that for the genetic silencing of the viral DNA,” said Cosgrove. “I’m hopeful for how fast that development will go.”

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