A new epigenetic editing tool shows promise as a therapy for prion disease. The approach for silencing prion protein (PrP), is both specific and can lead to wide-spread knockdown expression of PrP using the epigenetic silencer, Coupled Histone tail for Autoinhibition Release of Methyltransferase, or CHARM.

The collaboration between researchers at the Whitehead and Broad Institutes has been published in Science in the paper titled, “Brain-wide silencing of prion protein by AAV-mediated delivery of an engineered compact epigenetic editor.”

“The spirit of the collaboration since the beginning has been that there was no waiting on formality,” said Sonia Vallabh, PhD, senior group leader at the Broad Institute. “As soon as we realized our mutual excitement to do this, everything was off to the races.”

The joint team, led by Vallabh and Jonathan Weissman, PhD, a core member at the Whitehead Institute, combined their labs’ expertise to develop a novel way to target prion diseases. While mad cow disease is a widely known infectious prion disease, there are also inherited prion diseases. In both cases, the misfolded prion proteins trigger a cascade of misfolding events in the brain resulting in neurodegeneration and neuron death. In genetic prion disease, that Vallabh herself is currently asymptomatic for, misfolding of the PrPs leads to profound dementia and inability to sleep, culminating in the death of the affected individual (human or otherwise).

Current treatments target the proteins themselves, but Vallabh, Weissman, and their team endeavored to take a step back, both in their strategy and in the cellular production process of proteins to target protein precursors. CHARM works on an epigenetic level to silence the PrP gene so that the protein that may eventually become a prion will not be produced in the first place.

The project was led by a Whitehead Institute graduate student, Edwin Neumann and Tessa Bertozzi, PhD, a postdoc in the Weissman lab. The lab had previously developed the technology to silence specific genes using the tool CRISPRoff. CRISPRoff functions by adding methyl groups to specific target genes to prevent transcription. The gene itself remains unedited, but its ability to function is inhibited.

The next hurdle to clear was delivery of the CRISPRoff into the brain. CRISPRoff was initially designed to use Cas9, however the use of adeno-associated viruses (AAV) is limited by the size of Cas9. The team replaced Cas9 with a smaller zinc finger protein (ZFP) to target the correct genes. An added bonus is that in humans, ZFPs are less likely to cause an immune response compared to bacterially derived Cas9 and alleviate off target effects seen with the previous Cas9 system.

Another innovation to the system was the use of the cells’ own methylation mechanism. Originally the technique involved the inclusion of part of a methyltransferase, however the team found that they could reduce cellular toxicity and further reduce the content load of AAVs by using using the cells’ own enzyme, DNMT3A.

“From the perspectives of both toxicity and size, it made sense to recruit the machinery that the cell already has; it was a much simpler, more elegant solution,” Neumann said. “Cells are already using methyltransferases all of the time, and we’re essentially just tricking them into turning off a gene that they would normally leave turned on.”

Using mouse models, they tested ZFP-guided CHARMs, finding that over 80% of the prions in the brain were eradicated. Previous research has indicated that a much smaller reduction in prions, approximately 21%, will improve symptoms in patients. The reduction in PrPs prior to misfolding is also not an overt concern. The current research indicates that these proteins are not essential for healthy adults and their removal therefore should not cause adverse effects in those without symptoms. On the contrary, removing the prions may halt symptom progression or prevent development of symptoms in asymptomatic genetic carriers.

With CHARM, there is a potential for a safe, efficient, and effective treatment and preventative therapy for treating prion diseases. Within a short time, this collaboration resulted in an adaptable tool that, with appropriate tests and scalability trials, may move toward the clinic as a viable treatment option. Bertozzi commented, “It’s been a privilege to be part of this; it’s pretty rare to go from basic research to therapeutic application in such a short amount of time.”

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