Gene Therapy: The Next Generation

A look at how today's gene therapy research for DMD is overcoming obstacles

Article Highlights:

In this series focusing on gene therapy:

  • Among the challenges to development of gene transfer therapy for the dystrophin-deficient disorders Duchenne or Becker muscular dystrophies are unwanted immune responses to the therapy; the enormous size of the dystrophin gene; and the potential impermanence of gene transfer.
  • MDA-supported researchers developing gene transfer therapy are developing ways to outwit the immune system; miniaturize the dystrophin gene so  that maximal functionality is maintained; use utrophin genes as a substitute for dystrophin genes; and combine gene therapy with stem cell therapy.
  • MDA-supported researchers are also developing gene therapy approaches that do not involve inserting new genes but instead aim to modify and improve the function of existing dystrophin genes.
by Margaret Wahl on January 9, 2014 - 9:29am

Quest Winter 2014

The year was 1986, and the Duchenne muscular dystrophy (DMD) community was more excited than it had been for decades. A group of MDA-supported researchers had identified the genetic cause of the disorder — any of a number of different flaws (“mutations”) in the gene for a newly identified muscle protein, one that would come to be called dystrophin. 

It would soon be known that dystrophin plays an important role in skeletal muscles and in the heart. Located near the membrane that surrounds each muscle cell, it provides structural support to contracting skeletal-muscle fibers and constantly beating heart-muscle cells.

Mutations in the dystrophin gene that lead to a complete absence of the protein cause DMD, while mutations that result in a protein that’s either diminished in quantity or less than fully functional result in the less severe Becker muscular dystrophy (BMD)

Dystrophin, it was learned, is also one of the largest proteins in the body, with a correspondingly large gene. What remained unclear was whether dystrophin genes could be used to treat disease. 

A new approach

By the early 1990s, scientists and patients alike were asking a new question: Now that we know that DMD and BMD are caused by a lack of dystrophin, why not just add dystrophin genes to DMD-affected muscles to treat the disease?

Simple in concept, but extremely complex in execution, the idea of inserting therapeutic genes into tissues was dubbed “gene therapy,” also known as gene transfer or gene replacement, although there was no plan to get rid of a patient’s existing genes, making “replacement” somewhat of a misnomer.

Experiments in cells, mice, dogs and monkeys showed that dystrophin genes and other muscle protein genes might be used to treat genetic disorders caused by muscle protein deficiencies. Among the many disorders in this category, besides DMD and BMD, are limb-girdle muscular dystrophy (LGMD), congenital muscular dystrophy (CMD) and myotubular myopathy (MTM). (Gene transfer strategies for DMD are likely to have implications for at least some BMD patients.)

Challenges and dead ends

But challenges — expected and unexpected — soon followed. The response of the immune system to the therapy, the enormous size of the dystrophin gene, the need to target the vast territory of skeletal muscle tissue in the human body as well as the crucial heart muscle, and the potential impermanence of gene transfer all became apparent.

By the mid-1990s, altered viruses became the method of choice for transporting genes into muscles. Known as viral vectors, these tiny transport vehicles are the shells of viruses — without the ability to cause viral disease or replicate in the body, but with the advantage of attaching to docking sites on muscle cells and thereby gaining entry into the cell’s interior.

Once inside cells, a new dystrophin gene could, researchers found, be “read” by the cell and used to make dystrophin protein.

An early challenge surfaced when it was found that a popular viral vector — the AV — caused an immune response in patients in a gene therapy trial for a metabolic disorder. 

Unfortunately, the other front-running viral vector — known as the AAV and less likely to cause an immune response — wasn’t large enough to hold the huge dystrophin gene.

To fit inside an AAV vector, the dystrophin gene had to be miniaturized — parts of it removed, with the hope that the remaining parts would lead to a protein that was still functional. Today, MDA scientists continue in their quest to create miniaturized dystrophin genes that provide maximal function to muscle cells.

Another potential obstacle was that the new genes didn’t always last very long in muscle cells. For instance, if a damaged muscle fiber is repaired by muscle stem cells (a natural process), the new genes can be lost along the way. One solution may be to target muscle stem cells rather than mature muscle cells, but there may be other approaches as well.

In 2006, MDA-supported researchers at Nationwide Children’s Hospital in Columbus, Ohio, began testing the safety and feasibility of dystrophin gene transfer therapy in a small group of boys with DMD. They used a miniaturized dystrophin gene inside an AAV vector — both developed by an MDA-supported company called Asklepios BioPharmaceutical — and injected it into a biceps muscle in each boy. (The biceps muscles on the other arm received a placebo injection, for comparison.)

Although the treatment was safe, the result was less than ideal: Very little dystrophin was produced. Further analysis revealed that the culprit was probably an immune response, apparently against the newly made dystrophin protein, and possibly against the AAV vector as well. Today, several MDA-supported researchers are investigating ways to get around unwanted immune responses to dystrophin gene therapy. Some propose using a different protein — utrophin — which resembles dystrophin but appears less likely to cause an unwanted immune response.

MDA grantee Jeffrey Chamberlain at the University of Washington, Seattle, is interested in outwitting the immune system by using medications that suppress it or by using utrophin instead of dystrophin genes. He’s also interested in combining gene transfer therapy with treatments like anti-inflammatory drugs and stem cell transplants, to get the best solution possible for DMD.

Dongsheng Duan, an MDA grantee at the University of Missouri, Columbia, is interested in tweaking the miniaturized dystrophin genes used for gene transfer as well as the viral vectors used to deliver them. He’s particularly interested in ensuring that a section of the dystrophin gene needed to help regulate blood flow to muscle tissue is included. Duan says his team is trying to capitalize on the newest developments in the field.

And Hansell Stedman of the University of Pennsylvania, who has MDA support, is probing the nuances of the “inflammatory environment” that exist in dystrophin-deficient muscle fibers, and wants to use this knowledge to accomplish utrophin-based gene transfer that will “fly below the immune system’s radar.” He says his group is gearing up to “move forward into rational and appropriate clinical trials. 

Beyond gene transfer

Gene transfer therapy for DMD is still an important strategy in development for the disease, and there also are several MDA grantees actively working to advance research in this area.

But the term “gene therapy” has expanded recently to mean not only inserting new genes but also blocking existing ones or changing their structure. So some MDA grantees are pursuing strategies that change the structure of existing dystrophin DNA rather than inserting new dystrophin genes to treat DMD or BMD.

At the University of California, Los Angeles, Carmen Bertoni is repairing flawed dystrophin genes using laboratory-engineered compounds that trigger a natural cellular editing mechanism. The approach is working in mice, and Bertoni hopes to take it into clinical trials in the not-too-distant future.

And at Duke University, Charles Gersbach is correcting error-containing dystrophin DNA sequences using enzymes called nucleases. He and his colleagues have recently succeeded in correcting the dystrophin gene in human muscle cells in a lab dish. He now plans to take the strategy into laboratory animals and ultimately into the clinic.

This article is part of a special series that includes these additional articles profiling five researchers who are pursuing next-generation gene therapy for DMD:

And, for more on gene therapy, be sure to read these past Quest articles:

Search for these stories and more in the Quest research news archive

Margaret Wahl, R.N., B.S.N., is MDA’s medical and science editor and a frequent contributor to Quest.

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