Expert Uses Webinar to Explain the Science Behind Gene Therapies for Duchenne MD

Scientists are increasingly looking at gene therapies as potential treatments for Duchenne muscular dystrophy.

But there are a lot of ways to do them, and an expert in the field shed light on the subject in a webinar arranged by Parent Project Muscular Dystrophy (PPMD).

Dr. Timothy Cripe discussed the science of gene therapies, including the types of viruses used to deliver healthy versions of genes to cells. Cripe is a pediatric specialist at Nationwide Children’s Hospital in Columbus, Ohio.

Gene therapies are being tested in three ongoing clinical trials as a possible way of treating Duchenne MD,  including two at Nationwide Children’s. A fourth trial was placed on hold this week.

Cripe, a former chair of the U.S. Food and Drug Administration (FDA) Cellular, Tissue and Gene Therapy Advisory Committee, explained several concepts of gene therapies. The approach’s overall aim is to correct a genetic defect in a cell, like a mutation or DNA deletion that leads to the disease. The defect can cause a gene to produce a protein that doesn’t work as it should or not produce it at all.

“With gene therapy, we can correct that defect in the DNA and restore the function of a protein in a cell, turning a diseased cell into a healthy cell,” Cripe said.

A cure for DMD could involve researchers being able to correct the gene defect in a patient’s cells, promoting each cell’s transition from a diseased state to a healthy one.

“That’s the goal here, really taking a direct aim at the heart of the problem — and that is the mutations in dystrophin [ the gene],” Cripe said.

The therapy’s basic principle is to use any nucleic acid — a piece of DNA or RNA — to modify a cell by replacing a defective gene. It can also be used to introduce a new gene into the cells, forcing the cell to generate a protein that it wouldn’t normally make, thereby changing the cell’s behavior.

Gene therapy is already being used to treat people with other diseases. The U.S. Food and Drug Administration has approved Luxturna to treat congenital retinal dystrophy — a chronic and progressive disorder of visual function — and Kymriah to treat leukemia patients whose cancer has returned.

Duchenne and gene therapy

In Duchenne patients, researchers need to correct the genetic defect in muscle cells. For that they need to deliver the dystrophin (or DMD) gene into muscle cells. Scientists refer to a replacement gene as a transgene — “trans” for transferred gene.

The dystrophin transgene has no introns — parts of DNA sequences that are unimportant to dystrophin protein production. This means that it’s shorter than the dystrophin gene found normally in cells. The shorter versions are called mini- and micro-dystrophin.

One way of delivering the transgene is by virus, which know how to deliver genetic material — DNA or RNA genomes — into cells.

Current Duchenne gene therapies use the adeno-associated virus or AAV, a non-disease-carrying virus.

Muscle cells that are the target of Duchenne MD gene therapies are around 70 micrometers in size, Cripe said, while an AAV that carryies the transgene is around 20 nanometers, or more than 1,000 times smaller than a muscle cell.

This means that researchers need a lot of transgene-carrying viruses to get the therapy in the nuclei of muscle fibers in sufficient numbers to correct the genetic defect underlying Duchenne’s.

But complications, such as muscle fibrosis, can impede the viruses’ access to muscle cells. “This is probably one of the reasons why gene therapy trials are starting with young patients that still don’t have a lot of fibrosis,” or tissue scarring, Cripe said.

A question looming over  the application of these therapies is whether “there’s an age or a disease stage” that they are “going to work best at,” he said.

Researchers tweaked the DNA of the AAV virus to carry the dystrophin transgene along with a promoter. The promoter is a DNA sequence associated with the genes in our cells that acts as a switch to turn a gene on and off.

A promoter determines how much protein a gene will produce. Promoters can also be used to determine which of the cells will have their gene turned on, so that the treatment is tissue-specific.

Since viruses have a fixed size, researchers can fit only a certain amount of genetic material into them — and the dystrophin gene is the largest known human gene.

This means an entire dystrophin gene cannot fit into an AAV virus. Researchers have to either pick another virus — which may not be as safe or have other  limitations — or use a smaller gene with the potential to correct the defect.

In DMD, their choice is to use a micro-dystrophin gene, a shorter version of the dystrophin gene.

Viruses carrying the transgene bind to the cell’s surface through a protein or molecule on that surface.“Think about that as a lock and key,” Cripe said. The binding unlocks the cells targeted — in the case of Duchenne, they are muscles cells — allowing the virus to enter.

Once inside, the virus moves to the cell’s nucleus, where it unloads its DNA and the transgene it carries.

Duchenne trials and therapy questions

One of the clinical trials of potential gene therapies for DMD is a Phase 1/2 study (NCT03333590) at Nationwide Children’s Hospital that is recruiting by invitation. Another is a Phase 1/2 trial (NCT03375164), also at Nationwide, that’s recruiting infants and children. The third is a Phase 1 trial (NCT03362502) that Pfizer is sponsoring at Duke University, which is enrolling children.

The goal of the invitation-only trial is not to correct the gene defect in DMD. Instead it is to modify cells’ behavior so they are more resistant to the damage associated with lack of dystrophin protein, Cripe said.

Patients will receive the therapy by injection in both legs to “help increase the number of viruses that can reach” muscle cells there, he said. The trial, in DMD patients ages 4 and older, will test two different therapy doses. It will conclude in November 2020.

The Pfizer-sponsored trial is delivering by single intravenous injection a mini-dystrophin gene that can enter both limb and heart muscle cells.

The third trial will consist of two groups. One will be children ages 3 months to 3 years, and the other children ages 4 to 7. It will involve intravenous delivery of a micro-dystrophin gene.

“I think is a very exciting time, but this is very early,” Cripe said, noting that all of the trials are initial muscular dystrophy gene therapy studies.

Gene therapies have problems and limitations, mostly linked to immune responses, he added.

Chief among them is that some patients can have an immune reaction to an AAV because of antibodies they produce against proteins present in the virus shell.

For this reason, patients in DMD clinical trials are screened for AAV antibodies. If they have  antibodies against the virus being used to deliver the gene therapy, they can’t take part in the study.

“Now, that’s not to say that will always have to be the case,” Cripe said.

A patient’s immune system may also regard the dystrophin protein introduced through the therapy as an invader and initiate an immune attack against it, diminishing the therapy’s effectiveness, he said.

“We currently don’t really know why some patients may develop a strong immune response while others don’t,” he added.

The U.S. Food and Drug Administration placed a Phase 1/2 trial using a micro-dystrophin gene on hold March 14 after the first patient dosed was hospitalized with a potentially serious reaction.

The trial, sponsored by Solid Biosciences (NCT03368742), was taking place at the University of Florida. For now, enrollment and further dosing in the trial (NCT03368742) has been suspended until the cause of the boy’s reaction is understood.

Ways to circumvent a possible immune reaction against gene therapy is to suppress patients’ immune system, which decreases its response to perceived invaders, or to filter patients’ blood to remove antibodies that could hamper the treatment.

“So there might be ways around for some of these problems, but the best ways around are yet to be explored,” Cripe said.

In concluding his presentation, Cripe highlighted that gene therapy holds considerable promise, but “we are still at the beginning. There’s still a lot to learn, a lot of research to be done.”

Unknowns fall into the “how can we make gene therapy better” category, he said.

For DMD, the questions include:

Are the shorter dystrophin gene versions that are being used sufficient to correct the defect, or will scientists have to try longer versions? Is the viral load high enough for the transgene to enter enough muscles cells, improving walking and other functions?

Are the doses being used adequate, and if they are, how long will the benefits last? Is there an optimal age or a disease stage at which gene therapies are most effective? Can they benefit patients regardless of the underlying mutation?

All this, and more, await discovery, Cripe said.

“We don’t really know the answer to these questions, and so that’s why we need to do these research studies,” he said.