Researchers identify a a master epigenetic switch microbiologystudy

Biomedical engineers at Duke University have demonstrated a promising new approach that could be used to treat a rare and complex class of genetic diseases caused by defects in a relatively large region of the genome.

By identifying and activating a master epigenetic switch using CRISPR, the researchers showed they can turn on many naturally suppressed genes from one parent to compensate for defects in the same genes provided by the second parent.

The specific disease targeted in the research is Prader-Willi syndrome, which causes a wide range of physical, mental and behavioral problems, most notably a constant sense of hunger. While the proof-of-concept work in this study was limited to treating stem cells and neurons grown from those cells in a lab, the researchers hope it can eventually have a clinical impact.

The results appear February 12 online in the journal Cell Genomics.

Prader-Willi syndrome’s impacts on patients are multifaceted because it stems from a person missing an entire region of a chromosome containing many different genes with many different functions. It causes people to want to eat all the time because their appetite is never satiated, which leads to a host of other issues associated with weight gain.

The disease also causes defects in growth and physical development, cognitive impairment, speech problems, distinct facial features and many more effects.

These genes, however, are only missing from the father’s side of the genetic equation. People with Prader-Willi syndrome still carry the requisite genes from their mother, but those genes are naturally silenced in healthy people through a mechanism called imprinting.

“There are many examples of these imprinted regions of the genome, where one copy of a set of genes from either parent is normally silenced,” said Charles Gersbach, the John W. Strohbehn Distinguished Professor of Biomedical Engineering at Duke.

“But problems occur when a mutation causes people to lose the complementary active genes from the other parent. There aren’t really any therapies for this right now, but these people already have copies of all the genes they need, we just need to figure out a way to turn them on.”

Gersbach’s laboratory and collaborators across Duke have spent over a decade developing ways of using CRISPR to modulate epigenetic activity. Originally discovered as a bacterial defense system against viruses, CRISPR targets very specific gene sequences.

While the original system carries a protein called Cas9 that slices and dices the targeted viral genomes, the DNA-targeting part of the system can operate independently.

By removing this cutting function to “defang” CRISPR, it can instead be used to target and perform other manipulations to genetic material while leaving the underlying DNA sequence unchanged. For example, it can bind to genes and stop them from carrying out their normal functions. Or it can chemically manipulate the proteins that package DNA, activating or silencing expression of genes through the web of biomolecules called the epigenome.

“Though CRISPR is most widely known for changing DNA sequence, our focus on regulating the epigenome is much more akin to how biology regulates genetic activity naturally,” Gersbach explained.

“Think about how our genes get regulated across all different types of cells and tissues, and how their activity changes when we age or regenerate or respond to drugs. None of these effects change our underlying genetic sequence, but they have profound effects on how our genes are regulated.”

Gersbach and his laboratory have already demonstrated specific methods for silencing individual genes or sending them into hyperdrive. But activating an entire region of genes silenced through imprinting is a different challenge. The researchers weren’t even sure it was possible.

To find out, Gersbach mentored two Ph.D. students, Josh Black, who is now a staff scientist at Duke, and Dahlia Rohm, who recently graduated and is beginning post-doctoral studies at the University of Toronto.

The team of researchers screened thousands of genomic targets for responses to epigenetic changes that might affect the whole chromosomal region. This effort was made easier by the fact that CRISPR targeting sequences are fast and easy to produce, and thousands can be tested for effects in millions of cells in a single experiment.

The researchers’ efforts eventually paid off, revealing specific sites that act like a main power switch for the entire genomic region being silenced.

The researchers tried two approaches to turning on these switches—one that recruits the cellular activation machinery directly to its doorstep and another that essentially frees it from its repressive biomolecular chains.

They discovered that the second approach, called DNA demethylation, permanently activated the necessary maternal genes in stem cells that eventually grew into neurons that maintained expression of the necessary genes.

“That was probably the most exciting result,” Rohm said.

“That we could use CRISPR as a transient exposure but get a permanent, stable effect. That leads us to hope that we can eventually translate this into a durable therapy, not just for Prader-Willi syndrome patients, but for other rare genetic diseases that occur by a similar mechanism.”

There is still much research to be done before that hope becomes a reality, however. To work in human patients, the CRISPR demethylation system would need to be delivered to their neurons across large brain regions, which is a challenge that many in the CRISPR field are currently working toward overcoming.

It also needs to be shown that these epigenetic changes and their effects can take root and remain stable in already mature neuronal cells.

These are the next steps that Gersbach and his colleagues are already working on by pursuing animal studies with existing CRISPR delivery mechanisms. And with a large amount of activity and progress in this field, there will likely be many more delivery techniques available in the coming years.

“If you wanted to treat this disease through conventional gene therapy, you’d have to deliver many different genes and RNAs,” Black said.

“Our approach to manipulate a master controller of all of these genes that are already present in the imprinted region is a much more straightforward option. This is really an ideal use case for the epigenome editing technology we have been focused on.”