Muscular Dystrophy
Credit: © Studio Pookini/Fotolia

Researchers at UPenn have found a way to modify gene expression without modifying genome coding, opening the door for future possibilities of treatment for patients with Duchenne muscular dystrophy (DMD).

The team of researchers have identified a group of small molecules that may aid in the  development of new therapies in DMD, an as-of-yet incurable disease that results in devastating muscle weakening and loss of bodily functions in patients over time.

The molecules tested by the team from the Perelman School of Medicine work by easing the repression of a specific gene, utrophin. This gene codes for a protein expressed in muscle cells. When the suppressor mechanism is removed from this gene, cells are free to produce excess utrophin protein—a protein that can substitute dystrophin, the protein whose absence causes DMD. These novel findings were published this month in Scientific Reports.

“We’re trying to find therapies that will restore a patient’s muscle function without resorting to gene therapy,” explained the study’s senior author Tejvir S. Khurana, M.D., Ph.D., a professor of Physiology and member of the Pennsylvania Muscle Institute. “Increasing utrophin is a major focus of muscular dystrophy research. While, ideally, we would replace the missing dystrophin in patients, there are a number of technical and immunological problems associated with this approach.”

Introducing dystrophin through gene therapy has been a challenge for scientists for two main reasons: on one hand, the dystrophin gene is extremely large. It requires extensive down-sizing and conversion into a micro-dystrophin to fit the Adeno-associated viral vectors currently being used in clinical gene therapy. One the other hand, the immune system presents a challenge. Because the patient’s body has never produced dystrophin, it would interpret any of the newly synthesized micro-dystrophin proteins as a foreign, hostile invader and launch an autoimmune attack, which may lead to adverse events and nullify any benefits.

“We’re using an approach that attempts to increase utrophin levels in the body because it has functional characteristics and a genetic structure similar to dystrophin. Since the body already produces it, the immune system recognizes the protein as the body’s own and does not attack it or the cells producing it, even when overexpressed,” Khurana explained.

There have been a few other attempts to use utrophin as a substitute for dystrophin-using drugs, but those methods have focused on boosting utrophin through activating the “promoter,” a genetic sequence, usually upstream of the genetic sequence of DNA that codes for a protein, that tells RNA polymerase how much of a gene to transcribe. It can be used to increase the expression levels of the protein that gene codes. Using the metaphor of trying to move a car, Khurana said that this approach is like pressing the gas pedal.

However, there are additional mechanisms within the cell that limit the expression levels of proteins, which make stimulating more utrophin production similar to pressing a vehicle’s gas pedal while the brake is on: there may be some movement, but not a lot, because it is being counteracted by an opposing signal.

Khurana and his team, including first author Emanuele Loro, Ph.D., a Physiology research associate at Penn Medicine, decided to try an approach that would be akin to releasing the parking brake. They believed that by overpowering the repression with drugs, the body would naturally produce higher levels of the utrophin it was already making. The process is referred to as “upregulation,” and they hoped it would result in enough utrophin being produced that it could also compensate for the missing dystrophin.

The researchers tested a library of small molecules in a utrophin cellular assay they developed to test for effects.  The assay identified 27 promising “hits,” or drugs that would result in an increase in utrophin protein expression. After ranking their effectiveness with an algorithm developed by the lab called ‘Hit to Lead Prioritization Score’ (H2LPS), 10 molecules were extensively tested in mouse muscle cell lines, and the top-scoring molecule, trichostatin A (TSA), was tested in a mouse model of muscular dystrophy.  The results of this study show that when this molecule was given to the test mice, it would lead to significant improvements in muscle structure and function.

With the potential molecules identified, Khurana and his team believe they are on the right path to discovering and developing therapies to treat DMD patients. Testing is still in early stages, but Khurana is very excited about the doors this discovery will open.

“Our next steps here will be to do more screenings to identify new hits [of small molecules that can serve a similar purpose] using chemically diverse libraries,” Khurana said. “This is a completely new approach to increase utrophin for this condition, and we’re very keen to test it further and eventually bring it to clinical trials.”

When these small molecules make it to human clinical trials, the results will undoubtedly be of great interest to the scientific and patient community.

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