‘Second-Generation’ Gene Transfer

Dongsheng Duan is tweaking dystrophin genes to improve their therapeutic efficacy, designing better viral vectors in which to package the genes, and aims to treat the heart with gene transfer

"The field is moving faster, and we're getting there, says Dongsheng Duan. "We're in a very good spot right now."
Article Highlights:
  • Duan is developing highly miniaturized dystrophin genes that include a section that sticks to nitric oxide synthase; including that part of the gene is expected to help regulate blood flow to exercising muscles.
  • Duan wants to develop specialized gene delivery vehicles using “test-tube evolution”; he says the best vehicles would hold a functional dystrophin gene, target skeletal and heart muscle cells (and not other cells), and not disrupt existing DNA.
  • Duan believes unwanted immune responses to gene transfer therapy can probably be circumvented with medications to suppress the immune system.
  • This article is part of the Quest series titled Gene Therapy: The Next Generation (see below for the other stories included in the special package).
by Margaret Wahl on January 9, 2014 - 9:17am

Quest Winter 2014

By the time Dongsheng Duan finished medical school at West China University of Medical Sciences in Sichuan Province in the late 1980s, he knew he wanted to study biological sciences in the United States. Political turmoil in China temporarily derailed his plans, but in 1993, he left his home country. By the following year, he was at the University of Pennsylvania, working with Katherine High, an investigator who was developing gene-transfer therapy for hemophilia (a disease involving insufficient clotting of blood).

"I did a rotation in her lab, and that's when I got exposed to gene therapy," Duan recalls. "That was very hot and very new, cutting-edge." Duan was immediately attracted to the new field of transferring therapeutic genes via delivery vehicles derived from viruses (viral "vectors") to treat disease and began to focus on it for his graduate studies.

In 1997, having earned a doctoral degree in biology, Duan moved to the University of Iowa, at first working on gene-transfer therapy for the lung disease cystic fibrosis and then switching to gene therapy for Duchenne muscular dystrophy (DMD). In 2002, he joined the faculty at the University of Missouri, Columbia, where he's now a full professor in the Department of Molecular Microbiology & Immunology.

"I love muscle research," Duan says, "and I feel attached to kids with Duchenne. It's part of my life." The attachment has become even more compelling since he became acquainted with a young boy with DMD, the brother of a research colleague.

Recently, he's been concentrating on three main areas: tweaking the gene for dystrophin, the protein missing in DMD-affected muscles, so that miniaturized versions of it used for gene transfer have optimal function; designing better viral vectors in which to package and deliver miniaturized dystrophin genes; and improving heart function with gene transfer therapy.

Second-generation genes

"At the time when we made the first microdystrophins [highly miniaturized dystrophin genes], our knowledge of dystrophin was still relatively limited," Duan says. "But the field has evolved, and now we are trying to include all the recent developments in new, second-generation microdystrophins."

For example, he says, it's been known since the mid-1990s that a protein called NOS (nitric oxide synthase) that helps regulate blood flow to exercising muscles normally sticks to the dystrophin protein inside muscle fibers. When dystrophin is missing or abnormally structured, however, NOS can't find its usual anchor, and that adversely affects blood flow regulation in muscles.

It was Duan's team that first described, in 2012, exactly which parts of the dystrophin protein are needed to supply an anchor for NOS and showed how the DNA codes for these parts can be included in a microdystrophin gene used for gene therapy.

Designer vectors

For viral vectors to deliver the dystrophin gene to muscles, Duan, like most others in the field, prefers to use adeno-associated viruses (AAVs).

Here too, he says, "the field has changed a lot" since the early days. Today, many different types of AAVs are available to researchers. Some are natural, and others are engineered in laboratories. "AAV2 [a natural AAV] is the oldest vector," Duan says, noting that some investigators have also used natural subtypes AAV1 and AAV8 in gene transfer experiments, as well as the laboratory-tweaked AAV2.5. But now, he says, "We have ways to do 'test-tube evolution' of AAV vectors, so that you can get different kinds that include specific properties."

The ideal AAV vector would be one that can hold a functional, though miniaturized, dystrophin gene; efficiently target skeletal and heart muscle cells but not other cells in the body; and, once inside a cell, not disrupt existing DNA.

Today, Duan says, there are more than 100 subtypes of AAVs. "We’ve been trying to capitalize on the newest developments and make the best vectors," he says.

Targeting the heart

Targeting the heart muscle is a crucial matter for Duan, as heart function is crucial for health. Heart muscle deterioration — cardiomyopathy — is common in DMD, but it usually develops relatively late in the disease course. The same is true in dystrophin-deficient mice that have a DMD-like disease, prompting Duan and his team to conduct studies in aged mice with cardiomyopathy.

Duan's group has found that treating older mice with cardiomyopathy with gene-transfer therapy is effective, but that once they reach a very advanced age — 21 months, which is ancient for a mouse — the gene therapy can't rescue heart function, even though it can enter the heart.

"We got very good gene transfer into the heart," he says of these experiments. "Unfortunately, though, the heart function was not improved. This tells us that we need to treat the heart as early as possible. If it's too late, it may not work."

Getting around the immune system

Duan says muscle is a hard target for gene-transfer therapy, in part because there's so much of it that high doses of therapy are needed, potentially alarming the body's immune system. But he doesn't let that discourage him.

In a recent set of experiments in dystrophin-deficient dogs, Duan found that giving two commercially available drugs that suppress the immune system allowed the dogs to tolerate the gene therapy very well and to benefit from it. "In organ transplantation, they have been doing immunosuppression for many years," he says, noting that the level of suppression of the immune system that will likely be needed for dystrophin gene therapy is "almost nothing" compared to what has been used for people with organ transplants.

"The field is moving faster, and we're getting there," Duan says of muscle-directed gene therapy. "We're in a very good spot right now."

This article is part of a special series titled Gene Therapy: The Next Generation, which includes these additional articles profiling researchers who are pursuing next-generation gene therapy for DMD:

For more information about Duan's gene transfer research, be sure to read:

No votes yet
MDA cannot respond to questions asked in the comments field. For help with questions, contact your local MDA office or clinic or email publications@mdausa.org. See comment policy