The quest for a viral victory
By Lindsay Chung

OTTAWA — All around the world, young boys are slowly wasting away from Duchenne Muscular Dystrophy. There is still no cure for this debilitating neuromuscular disease, and scientists have been working feverishly for years to come up with a way to combat the effects that leave most patients in a wheelchair before the age of 10.

Adenoviruses can be used to inject genes into cells to produceprotein.

Dr. Robin Parks has muscled his way into the arena with his innovative research into viral vectors.

Parks, a molecular scientist at the Ottawa Health Research Institute, studies the use of adenoviruses as a way to deliver therapeutic genes into animal models of genetic or acquired diseases.

An adenovirus is a non-enveloped, spherical virus containing double-stranded DNA. Adenoviruses, which cause respiratory diseases such as the common cold, are attractive vectors, or vehicles, for delivering foreign genes into animal cells because they have an increased cloning capacity.

Since he started his own lab at the Research Institute in 1999, he has worked on many aspects of gene therapy, including a way to reverse the effects of Duchenne Muscular Dystrophy (DMD).

Parks is also an assistant professor in the University of Ottawa’s department of medicine and biochemistry. In 1996, while working on his post-doctoral fellowship with Dr. Frank L. Graham in the department of biology and pathology at McMaster University, Parks started using adenovirus vectors in gene therapy and continued it once he joined the Research Institute.

The disorder

DMD, a degenerative muscle disorder that affects about one in 3,500 men, is caused by genetic malfunctions. People born with DMD have a mutation in their dystrophin gene, which results in a lack of dystrophin protein. This protein is important for the normal function of cells that are required during muscle contraction and stretching. Since dystrophin is missing, the muscle cells are weaker and tear faster than the body can repair them. As their muscles waste away, patients end up in wheelchairs and eventually succumb to respiratory or cardiac failure because the muscles of the diaphragm and heart stop working.

According to George Henderson, national manager of firefighter relations and communications for Muscular Dystrophy Canada, the diagnosis generally occurs at the ages of two or three, he says. Life expectancy for those suffering from the disorder has changed dramatically over the years.

“In 1954, when we were first formed, expectations were until the early teens,” he says. “The great progress in the last 50 years has improved their mobility and quality of life and has extended their life to allow these individuals to live much longer lives.”

“We now have a number of clients in their 40s,” he adds. “They are completely paralyzed and require breathing assistance.” Life expectancy for DMD patients is now well into the 20s. However, there is still no cure for the disease.

The research

This is where Dr. Parks comes in. Parks had previously been working with viruses before he became interested in muscle disease. He says there are a lot of people at the Research Institute and the University of Ottawa who are very knowledgeable about muscle diseases, so he and his research partners latched onto this group.

Parks and his team are working on adenoviruses that have been stripped of all their viral genes, known as helper-dependent adenoviruses.

Parks' assistant Robert Lanthier says to get rid of viral RNA, the virus is run on a gradient, which is a grainy solution that is put into a tube with the virus. The tube spins really fast in a centrifuge, and the virus is separated into various components based on size.

When the virus is injected into the animal, it binds onto the outside of the cell by attaching to the cell’s receptor proteins. It then moves inside to take over the cell and replicate itself.

The researchers give the virus a week or so to run its course, and then they kill the mouse and harvest some of its cells. They compare the cells from a mouse infected with the disease and a healthy one, in order to see if the injected viral genes have had any benefit and whether they have succeeded in producing the missing protein.

A virus is full of genes, which code for proteins, so if a person has a bad copy of the gene they are not able to make a certain protein, Lanthier explains. When the virus takes over the cell, it exploits the cell’s machinery for making the protein, and the virus forces the cell to make protein for it. Once the virus has made its way into the cell, it breaks apart and exposes its DNA, and there are proteins in the cell that make copies using the virus’ RNA.

As Lanthier explains, DNA is like the whole recipe book, made up of smaller fragments. The genes are the recipes, and they are each a code for something specific. The cell reads the recipe, and the final product is a protein. Researchers hope that this protein will be as successful as a well-baked chocolate cake.

“If a virus makes more copies of itself, the immune system can tell something is going on, so we remove all the viral genes, and that way the system doesn’t detect it,” Parks explains.

“In a variety of disease models, it seems to work fairly well,” he says. “It’s not perfect, but much better than other virus vectors that people are using.”

The helper-dependent adenoviruses have potential in gene transportation because they have a large capacity for cloning and they may allow for the simultaneous delivery of multiple genes.

Parks has also removed the essential genes, known as early region 1 (E1), from the adenovirus. These E1 genes are required for normal virus replication; therefore, the virus cannot cause disease in humans because it can only grow in the lab.

“In animal models of DMD, these viruses have shown to work quite well and can reverse the DMD state,” Parks says.

The catch

However, there is a catch. A person’s immune system is very good at detecting when a cell has been infected with a virus, Parks explains. While the adenovirus lacks the E1 genes, there are still numerous viral genes present, and they produce a bit of virus protein that is detected by the body’s immune system.

The immune system will attack and eliminate the cell that has been infected by the virus, regardless of whether the virus is producing a therapeutic gene or not. To counteract this, Parks has removed all the viral genes from the virus so that there is nothing for the immune system to detect. This is what makes his research unique among the various scientists studying gene therapy for DMD.

“Any cell infected with these new and improved cells will no longer be detected as being infected, and, hopefully, the dystrophin gene will provide enough protein to correct the DMD state,” he says, adding that these new viruses have proven to be much more effective in animals than previous viruses.

Along the way, Parks has come up against many obstacles.

“We’ve discovered a few things about how the cell responds (to the virus),” Parks says. “Not only the immune system responds, but all the cells respond by signals.”

“We’re now trying to figure out how we blunt some of these signals so the virus hides easier.”

Parks says his research has uncovered some interesting facts. He has noticed how viruses have evolved to try to take over a cell and the cell, in turn, has advanced to withstand the virus.

“It’s interesting how the two have co-evolved to try and combat each other,” he says.

The community

Parks stresses that he is only one researcher in a whole community of scientists looking at DMD. They share a common goal of finding new therapies for the disorder. He says his lab keeps in contact with Muscular Dystrophy Canada, as well as the Muscular Dystrophy Association of the United States in order to keep up to date and help one another.

For example, Dr. Parks and Dr. Jonathan Bramson, who works out of the Centre for Gene Therapeutics at McMaster University, recently received a grant from Muscular Dystrophy Canada for their project “Building a Better Vector.”

“We keep in touch with people affected with the disease,” he says. “It more or less keeps it real for us, so we know what we’re trying to cure.”

Teren Clarke, national director of programs and services at Muscular Dystrophy Canada, is in charge of providing research grants to scientists. She says projects such as Parks’s have been receiving a lot of money lately because the hope for hereditary neuromuscular disorders like DMD lies in gene therapy or stem cell therapy.

In order to receive a grant, the research proposal must be peer-reviewed and must be judged as high quality science with relevance to people with neuromuscular disorders, Clarke explains.

Parks has to submit a report each year. Clarke says they have been very satisfied with his work, but they know a cure is still a long way off.

“He’s continuing to add to the body of knowledge,” she says. “But we’re certainly not close to a cure or therapy. It could be considered as a small step in the journey to a cure.”

"The more (the scientists) learn, the more they uncover that we don’t know.”