Potential treatment for deadly prion disease

That's why when Vallabh began collaborating with Jonathan Weissman at the Whitehead Institute, he was excited to find that Weissman's group likes to work at full speed. In less than two years, Weissman, Vallabh, and their collaborators have developed a set of molecular tools, called CHARMs, that can turn off disease-causing genes such as the prion protein gene, as well as genes that code for many other proteins implicated in neurodegenerative and other diseases, and are refining these tools to make them suitable candidates for use in human patients. Although the researchers still have many hurdles to overcome before they know if it works as a therapeutic, the team is encouraged by the speed with which they've developed the technology so far.

“From the beginning, the spirit of this collaboration was not to wait for red tape,” Vallabh says. “Once we realized we were both excited about doing this project, everything started moving.”

Co-corresponding authors Weissman and Vallabh, along with co-first authors Edwin Neumann, a graduate student in Weissman's lab, and Tessa Bertozzi, a postdoc in the Weissman lab, describe their CHARM (Connected Histone Tail for Autoinhibited Release of Methyltransferases) in their paper published in the journal Nature. Science June 27th.

“With the Whitehead and Broad Institutes nearby, I can't think of a better place for motivated people to work quickly and flexibly in the pursuit of academic science and medical technology,” says Weissman, a professor of biology at MIT and an HHMI investigator. “CHARM is an elegant solution to the problem of silencing disease genes and could have an important place in the future of genetic medicine.”

Targeting genes to treat genetic diseases

Prion diseases lead to rapid neurodegeneration and death and are caused by the deformation of the prion protein. These trigger a chain reaction in the brain. The defective prion protein deforms other proteins, which together not only fail to function properly but also form toxic aggregates that kill nerve cells. The best-known prion disease, colloquially known as mad cow disease, is infectious, while others can develop spontaneously or be caused by a defective prion protein gene.

Most conventional drugs work by targeting proteins. But CHARM works further upstream, turning off the gene that codes for the defective protein so that the protein is not produced in the first place. CHARM does this through epigenetic editing, which adds chemical tags to DNA to turn off or silence the targeted gene. Unlike gene editing, epigenetic editing does not change the underlying DNA, and the gene itself remains intact. But like gene editing, epigenetic edits are stable, and genes turned off by CHARM should stay off. This means that patients only need to take CHARM once, as opposed to protein-targeted drugs, which must be taken periodically as cellular protein levels are restored.

Animal studies suggest that the prion protein is unnecessary in healthy adults, and that in those with the disease, removing the protein improves symptoms or eliminates them entirely. For people who don't yet have symptoms, removing the protein should prevent the disease entirely. In other words, epigenetic editing could be an effective approach to treating genetic disorders such as inherited prion diseases. The challenge is to generate new types of treatments.

Fortunately, the team had a good template for CHARM: a research tool called CRISPRoff that Weissman's group had previously developed for gene silencing. CRISPRoff uses components of the CRISPR gene editing technology, including the guide protein Cas9, which directs the tool to the target gene. CRISPRoff silences the target gene by adding a methyl group, a chemical tag that prevents a gene from being transcribed, or read, into RNA and expressed as a protein. When the researchers tested CRISPRoff's ability to silence the prion protein gene, they found it to be effective and stable.

But some of its properties prevented CRISPRoff from being a good candidate for therapy. The researchers' goal was to create a tool based on CRISPRoff that was equally effective but safe for use in humans, small enough to be delivered to the brain, and designed to minimize the risk of silencing the wrong genes or causing side effects.

From research tool to drug candidate

Researchers led by Newman and Bertozzi set out to design and apply a new epigenetic editor. The first problem they had to tackle was size: the editor must be small enough to be packaged and delivered to specific cells in the body. Delivering genes to the human brain is difficult. Many clinical trials use adeno-associated viruses (AAVs) as gene delivery vehicles, but these are small and can only contain a small amount of genetic code. CRISPRoff is too big: the Cas9 code alone takes up most of the available space.

Researchers in the Weissman lab decided to replace Cas9 with much smaller zinc finger proteins (ZFPs). Like Cas9, ZFPs act as guide proteins that direct the tool to a target site in DNA. ZFPs are also commonly found in human cells, making them less likely to trigger an immune response against the host than bacterial Cas9.

Next, the researchers had to design the part of the tool that would silence the prion protein gene. At first, they used a part of methyltransferase, DNMT3A, a molecule that adds methyl groups to DNA. But in the particular configuration needed for the tool, this molecule was toxic to cells. The researchers focused on a different solution. Instead of delivering external DNMT3A as part of the treatment, the tool could recruit the cell's own DNMT3A to the prion protein gene. This freed up valuable space inside the AAV vector and prevented toxicity.

The researchers also needed to activate DNMT3A. Inside the cell, DNMT3A is normally inactive until it interacts with a specific partner molecule. This default inactivity prevents it from accidentally methylating genes that need to stay on. Newman came up with an ingenious way to combine sections of DNMT3A's partner molecules and connect these to a ZFP that directs them to the prion protein gene. When the cell's DNMT3A encounters this combination of parts, it activates and silences the gene.

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

Tests in mice showed that the ZFP-induced CHARM could remove more than 80% of the prion protein in the brain, but previous studies have shown that removing just 21% can improve symptoms.

After the researchers knew they had a powerful gene silencer, they addressed the problem of off-target effects. The genetic code for CHARM delivered to cells would continue to produce copies of CHARM indefinitely. But this offered no benefit after the prion protein gene was turned off, other than prolonging the time for side effects to occur. So the researchers tweaked their tool so that it would turn itself off after turning off the prion protein gene.

Meanwhile, a complementary project from the lab of Broad Institute scientist and collaborator Benjamin Deverman is focusing on gene delivery throughout the brain. Science The announcement, made May 17, brings the CHARM technology one step closer to being ready for clinical trials. Naturally occurring types of AAV have been used in human gene therapy before, but they cannot efficiently enter the adult brain, making them unable to treat brain-wide diseases like prion diseases. To address the delivery problem, Deberman's group engineered an AAV vector that can enter the brain more efficiently, by harnessing the pathway that naturally transports iron to the brain. With such an engineered vector, therapies like CHARM are one step closer to reality.

Thanks to these ingenious solutions, the researchers now have a highly effective epigenetic editor that is small enough to be delivered to the brain and has been shown to have low toxicity and limited off-target effects in cell culture and animal studies.

“It has been an honor to be part of this project; it is very rare to go from basic research to therapeutic application in such a short time,” Bertozzi said. “I think the key was to develop a collaboration that leveraged the tool-building experience of the Weissman lab, the deep disease knowledge of the Vallabh and Minikel labs, and the gene delivery expertise of the Deverman lab.”

Looking to the future

With the key elements of the CHARM technology worked out, the team is now fine-tuning the tool to make it more effective, safer, and easier to produce at the scale needed for clinical trials. The tool is already modular, with different parts interchangeable, so future CHARMs won't have to be programmed from scratch. CHARM is also currently being tested as a therapeutic agent in mice.

The road from basic research to clinical trials is long and winding, and researchers know there is still a long way to go before CHARM can become a viable medical option for patients with prion diseases like Valabu disease, as well as other diseases with a similar genetic component. But with a strong treatment design and promising lab results at hand, the researchers have every reason to be hopeful. They are committed to developing the technology so that it can help save patients' lives as soon as possible, rather than someday.