Developing treatments for prion diseases

When normal prion proteins misfold in the brain, they cause a chain reaction of prion protein misfolding. These form toxic clumps that kill brain cells and eventually lead to death. Prion diseases include Creutzfeldt-Jacob disease and familial fatal insomnia in people, mad cow disease in cattle, scrapie in sheep, and chronic wasting disease in deer, elk, and moose.

Prion misfolding can be spontaneous or the result of genetic changes. It can also result from infection with misfolded prion protein, such as from contact with infected animals.

No treatments for prion diseases yet exist. But prion protein isn’t essential for survival. Previous research has shown that mice completely lacking the gene for prion protein resist infectious prions. And reducing prion protein in neurons after infection can halt and even reverse disease in mice with minimal side effects. This suggests that reducing prion protein levels in neurons is a potential strategy for treating prion diseases.

One way to reduce prion protein levels is to turn off the gene coding for the prion protein. This can be done via a DNA modification called methylation. This is a type of epigenetic modification—one that affects how DNA is used without changing the genetic sequence itself. In a new NIH-funded study, a research team led by Drs. Sonia Vallabh at the Broad Institute and Jonathan Weissman at MIT and the Whitehead Institute developed an improved method to target the prion gene. Their results appeared in Science on June 28, 2024.

Earlier, Weissman’s group had developed a genetic tool for turning genes off in cultured cells. The tool, called CRISPRoff, combined the Cas9 protein from CRISPR gene editing technology, which guides the tool to a target gene, with an enzyme that methylates DNA. But CRISPRoff is not well-suited to therapeutic use. CRISPRoff is too big to fit into the harmless virus, called adeno-associated virus (AAV), that’s typically used to deliver gene therapies into the brain.

To design a system for therapies, the team replaced Cas9 with a zinc finger protein. Like Cas9, zinc finger proteins can be designed to target specific DNA sequences. But zinc finger proteins are much smaller than Cas9. They also occur naturally in human cells and so are less likely to trigger an immune response.

The next challenge the team faced is that too much of the DNA-methylating enzyme can be toxic to cells. So they designed the tool to recruit and activate a DNA-methylating enzyme already found in cells, instead of bringing in an outside enzyme. They called the new tool Coupled Histone tail for Autoinhibition Release of Methyltransferase (CHARM).

When the team injected mice with AAV carrying CHARM, the prion protein gene was turned off throughout the mouse brains. Prion protein levels decreased by up to 80% after six weeks. This is much more than earlier studies found was needed for a therapeutic effect. To further reduce the chance of side effects, the team modified CHARM so that it would turn itself off after turning off the prion protein gene.

The results suggest that CHARM can safely suppress prion protein levels in brain cells. It thus could form the basis for potential prion disease treatments. Other neurodegenerative diseases, such as Huntington’s, are also caused by a buildup of toxic misfolded proteins. CHARM might help with treating these diseases as well.

“CHARMs are an elegant solution to the problem of silencing disease genes,” Weissman says, “and they have the potential to have an important position in the future of genetic medicines.”

—by Brian Doctrow, Ph.D.