Epigenetic Editor CHARMs Prion Disease

New epigenetic editing tools are promising therapeutic approaches for prion diseases. Approaches to silence the prion protein (PrP) are specific and can lead to widespread knockdown expression of PrP using the epigenetic silencer Binding Histone Tail for Autoinhibitory Release of Methyltransferase (CHARM).

The joint research project by researchers from the Whitehead Institute and the Broad Institute is Science “Brain-wide prion protein silencing by AAV-mediated delivery of an artificial compact epigenetic editor.”

“From the beginning, the spirit of this collaboration has been not to wait for red tape,” said Dr. Sonia Vallabh, senior group leader at the Broad Institute. “The moment we sensed our mutual excitement about this endeavor, everything started moving.”

A collaborative team led by Vallabh and Whitehead Institute core member Dr. Jonathan Weissman combined the expertise of their respective laboratories to develop a new method to target prion diseases. Mad Cow Disease is a well-known infectious prion disease, but there are also hereditary prion diseases. In both cases, a misfolded prion protein triggers a series of misfolding events in the brain, leading to neurodegeneration and neuronal death. In hereditary prion diseases, of which Vallabh himself is currently asymptomatic, the misfolding of PrP leads to severe dementia and insomnia, and ultimately to the death of the affected individual (human or otherwise).

While current treatments target the protein itself, Vallabh, Weissman and their team have taken a step back in both strategy and the process of protein production within cells, seeking to target the protein precursor: CHARM works at the epigenetic level to silence the PrP gene, preventing the protein that could eventually become a prion from being produced in the first place.

The project was led by Whitehead graduate student Edwin Newman and Tessa Bertozzi, PhD, a postdoctoral researcher in the Weissman lab, who had previously developed techniques to silence specific genes using a tool called CRISPRoff, which adds methyl groups to specific target genes to prevent their transcription, so the gene itself remains unedited but its function is silenced.

The next hurdle to overcome was delivering CRISPRoff to the brain. CRISPRoff was initially designed to use Cas9, but the use of adeno-associated viruses (AAVs) is limited by the size of Cas9. The team replaced Cas9 with smaller zinc finger proteins (ZFPs) to target the correct genes. An added benefit is that ZFPs are less likely to trigger an immune response in humans compared to bacterial Cas9, reducing the off-target effects seen with previous Cas9 systems.

Another innovation of the system is the use of the cell's own methylation machinery: initially, the technique required the inclusion of a portion of a methyltransferase, but the team found that by using the cell's own enzyme, DNMT3A, they could reduce cytotoxicity and further reduce the content load of the AAV.

“From both a toxicity and size standpoint, it made sense to use the machinery that cells already have. It was a much simpler, more elegant solution,” Newman says. “Cells already use methyltransferases all the time, and we're basically just tricking them into turning off genes that would normally be left on.”

The researchers tested ZFP-induced CHARM using mouse models and found that it eliminated over 80% of prions in the brain. Previous studies have shown that a much smaller reduction in prions, around 21%, can still improve patients' symptoms. Also, the reduction in pre-misfolded PrP is not an obvious concern. Current research shows that these proteins are not essential in healthy adults, and therefore their removal should not have any adverse effects on asymptomatic individuals. Conversely, removing prions may halt the progression of symptoms or prevent the onset of symptoms in asymptomatic gene carriers.

CHARM has the potential to deliver safe, efficient and effective treatment and prevention therapies for prion diseases. In a short space of time, the collaboration has produced a highly adaptable tool that, with proper testing and scalability studies, could potentially be introduced into the clinic as a viable treatment option. “It's an honor to be part of this project. It's very rare to go from basic research to therapeutic application in such a short time,” said Bertozzi.