A therapy candidate for fatal prion diseases turns off disease-causing gene

Prion diseases lead to rapid neurodegeneration and death and are caused by misshapen versions of the prion protein in the brain. There are currently no treatments, but researchers from the Whitehead Institute for Biomedical Research and Broad Institute of MIT and Harvard have developed an approach that could one day be used to turn off the gene encoding this protein throughout the brain to treat or even prevent prion disease.

In a paper published in today’s Science, the team describes their technology: a set of molecular tools that are delivered to the brain and adds a chemical tag to the gene for the prion protein to prevent the protein from being produced by cells. Unlike gene editing, this “epigenetic” editing does not modify the underlying DNA sequence, but it should switch the gene off permanently, which means that this could be a one-time treatment. Research in animals suggests that the prion protein isn’t necessary in a healthy adult, so epigenetic editing that silences the gene for this protein could be an effective approach for treating prion diseases.

The researchers, led by Jonathan Weissman of the Whitehead and Sonia Vallabh of the Broad, showed that their system, called CHARM, in a single intravenous injection, could be delivered across the brain in mice and eliminate more than 80 percent of the prion protein. Previous research has shown that as little as 21 percent elimination of the protein can improve symptoms.

To deliver CHARM throughout the brain, the team collaborated with scientists in the lab of Ben Deverman, senior director of vector engineering at the Broad and a co-author of the study. They used an engineered adeno-associated virus (AAV) that crosses the blood-brain barrier after intravenous administration.

“With the Whitehead and Broad Institutes right next door to each other, I don’t think there’s any better place than this for a group of motivated people to move quickly and flexibly in the pursuit of academic science and medical technology,” said Weissman, co-senior author of the study who is also a professor of biology at the Massachusetts Institute of Technology, a Howard Hughes Medical Institute investigator, and an affiliate of the Broad. “CHARMs are an elegant solution to the problem of silencing disease genes, and they have the potential to have an important position in the future of genetic medicines.”

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

The study co-first authors are Edwin Neumann, a graduate student, and Tessa Bertozzi, a postdoc, both in Weissman’s lab.

Therapy search

Vallabh, along with her husband Eric Minikel, leads a lab at Broad focused on developing drugs to prevent and treat prion diseases. They switched careers and became researchers 12 years ago after Vallabh’s mother passed away from a rare form of prion disease called fatal familial insomnia. Vallabh soon found out that she inherited the same disease-causing mutation in the prion protein gene. 

Vallabh and Minikel’s lab has been working urgently to develop prion disease treatments, so Vallabh was excited to discover that Weissman’s group also likes to work at full throttle. In less than two years, Weissman, Vallabh, and their collaborators developed the CHARM system that can turn off disease-causing genes such as the prion protein gene, as well as, potentially, genes coding for many other proteins implicated in neurodegenerative and other diseases. 

The team says epigenetic editing could be an effective approach for treating genetic diseases such as inherited prion diseases. They are refining those editing tools to be good candidates for use in patients. Prion disease research in animals suggests that removing the prion protein could improve or even eliminate disease symptoms, and removing it before symptoms develop should prevent disease altogether. 

Although the CHARM tools still have many hurdles to pass before the researchers will know if they work as therapeutics, the team is encouraged by the speed with which they have developed the technology thus far.

To develop CHARM as a potential therapy, the team had a good template: a research tool called CRISPRoff that Weissman’s group previously developed for silencing genes. CRISPRoff uses building blocks from CRISPR gene editing technology, including the guide protein Cas9 that directs the tool to the target gene. CRISPRoff silences the targeted gene by adding methyl groups, chemical tags that prevent the gene from being transcribed or read into RNA and so from being expressed as protein. When the researchers tested CRISPRoff’s ability to silence the prion protein gene, they found that it was effective and stable.
 
Several of its properties, though, prevented CRISPRoff from being a good candidate for a therapy. The researchers’ goal was to create a tool based on CRISPRoff that was just as potent but also safe for use in humans, small enough to deliver to the brain, and designed to minimize the risk of silencing the wrong genes or causing side effects.

From tool to drug 

Led by Neumann and Bertozzi, the researchers began engineering a new epigenome editor. The first problem they had to tackle was the editor’s size, because the editor needs to be small enough to be packaged and delivered to specific cells in the body. Delivering genes into the human brain is challenging; many clinical trials have used adeno-associated viruses (AAVs) as gene-delivery vehicles, which are small and can only contain a small amount of genetic code. CRISPRoff is too big; the code for Cas9 alone takes up most of the available space.
 
The Weissman lab researchers decided to replace Cas9 with a much smaller zinc finger protein (ZFP), which fits in an AAV. Like Cas9, ZFPs can serve as guide proteins to direct the tool to a target site in DNA. ZFPs are also common in human cells, meaning they are less likely to trigger an immune response against themselves than the bacterial Cas9.
 
Next, the researchers had to design the part of the tool that would silence the prion protein gene. At first, they used part of a methyltransferase, a molecule that adds methyl groups to DNA, called DNMT3A. However, in the particular configuration needed for the tool, the molecule was toxic to the cell. The researchers focused on a different solution: instead of delivering outside DNMT3A as part of the therapy, the tool is able to recruit the cell’s own DNMT3A to the prion protein gene. This freed up precious space inside of the AAV vector and prevented toxicity.

The researchers also needed to activate DNMT3A. In the cell, DNMT3A is usually inactive until it interacts with certain partner molecules. This default inactivity prevents accidental methylation of genes that need to remain turned on. Neumann came up with an ingenious way around this by combining sections of DNMT3A’s partner molecules and connecting these to ZFPs that bring them to the prion protein gene. When the cell’s DNMT3A comes across this combination of parts, it activates, silencing the gene.
 
“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.”
 
Once the researchers knew that they had a potent gene silencer, they turned to the problem of off-target effects. The genetic code for a CHARM that gets delivered to a cell will keep producing copies of the CHARM indefinitely. However, after the prion protein gene is switched off, there is no benefit to this, only more time for side effects to develop, so they tweaked the tool so that after it turns off the prion protein gene, it then turns itself off.

Meanwhile, a complementary project from Deverman’s lab, focused on brain-wide gene delivery and published in Science in May, has brought the CHARM technology one step closer to being ready for clinical trials. Although naturally occurring types of AAV have been used for gene therapy in humans before, they do not enter the adult brain efficiently, making it impossible to treat a whole-brain disease like prion disease. Tackling the delivery problem, Deverman’s group has engineered an AAV vector that can get into the brain more efficiently by grabbing on to a protein that naturally shuttles iron into the brain. Engineered vectors like this one make a therapy like CHARM one step closer to reality.
 
Thanks to these creative solutions, the researchers now have a highly effective epigenetic editor that is small enough to deliver to the brain, and that appears in cell culture and animal testing to have low toxicity and limited off-target effects.
 
“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,” Bertozzi said. “I think the key was forming a collaboration that took advantage of the Weissman lab’s tool-building experience, the Vallabh and Minikel lab’s deep knowledge of the disease, and the Deverman lab’s expertise in gene delivery.”

 
Looking ahead

With certain major elements of the CHARM technology solved, the team is now fine-tuning their tool to make it more effective, safer, and easier to produce at scale, which will be necessary for clinical trials. They have already made the tool modular, so that its various pieces can be swapped out and future CHARMs won’t have to be programmed from scratch. CHARMs are also currently being tested as therapeutics in mice.  
 
The path from basic research to clinical trials is a long and winding one, and the researchers know that CHARMs still have a way to go before they might become a viable medical option for people with prion diseases, including Vallabh, or other diseases with similar genetic components. However, with a strong therapy design and promising laboratory results in hand, the researchers have good reason to be hopeful. They continue to work at full throttle, intent on developing their technology so that it can save patients’ lives not someday, but as soon as possible.

Adapted from a Whitehead Institute news story