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Can Gene-Editing Tools Cure Chronic Hepatitis B?

Researchers from Duke and UNC team up to find an answer, with funding from the two institutions' NIH Clinical and Translational Science Awards (CTSA).

For hundreds of millions of people around the world with chronic hepatitis B infection, anti-viral treatments do a good job of keeping the virus under wraps. Anti-viral treatments are essential in slowing damage to the liver, reducing the chance of liver cancer, and helping people live longer. But in the vast majority of cases, there is no end to the infection. If a patient stops medication for any reason, the hepatitis B virus (HBV) re-emerges. The cause of its resilience is circular bits of DNA, called covalently closed circular DNA, or cccDNA, that lurk in the liver cells of infected patients.

Bryan Cullen, PhD (Duke) and Lishan Su, PhD (UNC)
Bryan Cullen, PhD (Duke) and Lishan Su, PhD (UNC)

"Treatment blocks new viral replication," said Lishan Su, a professor of microbiology and immunology at the University of North Carolina, Chapel Hill. "It doesn't touch this cDNA." When treatment stops, new virus is produced from these cccDNA templates, he continued. As a result, "you need almost lifetime (or very long-term) treatment."

That reality makes viral cccDNA a prime target for the development of new and more effective treatments with the potential to knock HBV infections out for good. Now, with support from the Clinical and Translational Science Awards (CTSAs) at Duke and UNC, Su and Bryan Cullen, in Duke's Department of Molecular Genetics & Microbiology, think they are on the right track to producing just such a treatment.

Cut It Out

Their effort builds on major advances in recent years in the development of a gene-editing tool called CRISPR/Cas. The CRISPR/Cas system uses tiny snippets of "guide RNA" to direct the scissor-like enzyme Cas9 to just the right spot in the genome. The technology has gained considerable attention for its potential to cure genetic conditions such as sickle cell disease, which trace to a single, well-known typo in the DNA sequence. Once the DNA is cut, cells can be directed to replace mutated sequence with a healthy sequence or, in some instances, to edit in new functions.

But getting CRISPR/Cas9 to work against HBV and specifically those circular ccDNA is actually much easier than all that. The researchers think they can destroy the viral DNA by cutting it alone.

As Cullen says, "we're just trying to whack the cccDNA and destroy it." There's no need to do anything more.

In fact, Cullen and Su hope to use CRISPR/Cas to do exactly what it was designed to do over the course of millions of years of evolution. CRISPR originates from the adaptive immune systems of bacteria, which rely on it to snip and destroy the DNA genomes of bacterial viruses, called bacteriophages, that infect them.

"This is what it was made for," Cullen said. "It's a bacterial defense mechanism that's specific for DNA bacteriophage and it is designed to cleave the DNA of the bacteriophage and destroy them. So we're repurposing a bacterial antiviral defense mechanism to try to treat a virus in a human setting."

Fit it In

In fact, Su and Cullen have already shown that the approach works well in tissue culture. They are working now on ways to deliver CRISPR/Cas to the liver, first in mice with humanized livers and ultimately in people. The researchers aim to use a viral vector used in many older approaches to gene therapy to get Cas9 where it's needed in the liver. They know from years of experience by other researchers in the field that it's feasible to get a viral vector carrying a therapeutic load of DNA into nearly every liver cell.

The challenge, as Su and Cullen explain it, is to find a way to package CRISPR into viral vectors for delivery. The best-studied form of CRISPR/Cas comes from Streptococcus pyogenes and it's too big to fit. The researchers are exploring the possibility of using a Cas9 derived instead from Staph aureus, which offers the same basic function in a more compact form.

Partners on the path from mice to mankind

"We've done a lot of experiments in vitro and the question then is whether we can get a good readout in an in vivo model system where we're actually trying to treat HBV in human hepatocytes in an animal model," Cullen said. "Hopefully if that works well then we can interest an industrial partner in taking this forward in some way."

"We have been interested in working toward an HBV cure for many years and the CRISPR technology offers a very exciting possibility to either completely get rid of the cccDNA or at least impair it's ability to produce more viral proteins," Su said.

Either way, they're getting close to what looks like the first HBV cure.

Su and Cullen met some 20 years ago, long before Su made the move to UNC. They make a great pair. Su is an expert in working with HBV and humanized liver mice and Cullen brings expertise in molecular biology including gene-editing tools to the table.

"It was a no-brainer that we should work together for this project," Su said. "But it wouldn't have happened without the Duke/UNC CTSA Consortium pilot funding."

Tags: Duke/UNC Collaboration

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