MDA SCIENTISTS DESTROY FLAWS IN CELLS;
MUSCULAR DYSTROPHY ADVANCE
MAY APPLY TO OTHER DISORDERS

TUCSON, Ariz., April 22, 2003 — Scientists funded by the Muscular Dystrophy Association have successfully destroyed genetic material that carries disease-causing flaws (mutations), with minimal damage to normal cellular structures.

A three-country research team deleted excess genetic material in human cells harboring the mutation that causes type 1 myotonic muscular dystrophy (MMD1). Most “gene therapy” strategies have focused on adding, rather than subtracting, genetic material.

The ability to target and destroy specific genetic material may have implications for other diseases, including a second form of myotonic dystrophy, some other muscular dystrophies and some genetic forms of amyotrophic lateral sclerosis (Lou Gehrig’s disease). It may also have relevance to cancer.

The technique applies to genetically dominant diseases (those that can be inherited from one parent). In dominant disorders, the flaw generally results from the presence of something harmful in the genetic code, rather than something missing.

MMD1 (see "Facts about MMD") results from the presence of extra DNA in a gene called DMPK on chromosome 19.

“Dominant disorders have always posed a problem for researchers attempting gene therapy, as a very precise kind of molecular surgery is required to remove only harmful portions of a gene,” said Sharon Hesterlee, MDA’s director of Research Development.
MDA research grantee Jack Puymirat of the University of Laval in Ste-Foy, Quebec, Canada, was part of a team from the United States, Canada and France, which published results of the MMD1 experiments in today’s issue of the journal Gene Therapy.

The team used a strategy called “antisense.” They created a molecule that sticks to the sections of genetic material known to make up the myotonic dystrophy gene flaw. Once the antisense sticks to and tags the genetic material — in this case, RNA — cellular machinery comes in and destroys the tagged molecules.

Some normal DMPK RNA was also destroyed in the process, Puymirat says, but on balance, the results were encouraging.

At least two cellular functions known to be disrupted by the MMD1 mutation improved. One function has to do with how the cells take up sugar and the other with how the cells change from immature muscle cells to mature muscle fibers.

For a second set of experiments, published in the March 25 online edition of Molecular Therapy, Puymirat teamed up with researchers from the Beckman Research Institute of the City of Hope National Medical Center in Duarte, Calif.

This group tried a different strategy to destroy the same mutation. They used a laboratory-modified “ribozyme,” a natural cellular chemical that can cut RNA into pieces.

Normally, DMPK RNA moves quickly out of the cell’s nucleus into the cell’s main compartment. But the extra-long DMPK RNA that leads to MMD1 gets stuck in the nucleus, where it forms clumps. Previous research suggests that this traffic jam is a major cause of the problems that occur in myotonic dystrophy.

The investigators reasoned that they could take advantage of this bottleneck by adding a molecular “on switch” to the ribozyme that caused it to work only in the cell’s nucleus.
The cells treated with this special ribozyme showed better sugar uptake mechanisms and fewer abnormal clumps of stuck RNA.

“To my knowledge, our work is the first example showing the efficiency of antisense and ribozymes to restore normal functions in a dominant neuromuscular disease,” Puymirat said.
Puymirat is doing experiments to determine whether antisense or ribozymes could be used to correct defects in mice with MMD1. He also notes that these strategies would probably be useful in type 2 MMD, which is also a dominant muscular dystrophy.

“If these experiments are successful, we may expect to conduct the first human trials in three to five years,” Puymirat said.

MDA is a voluntary health agency working to defeat more than 40 neuromuscular diseases.

Facts About Myotonic Muscular Dystrophy

Most people with myotonic dystrophy who have been tested have extra DNA on chromosome 19 as the underlying cause of their disorder.

The chromosome 19 form of MMD, called type 1:

• is a common inherited neuromuscular disease throughout the world
  affects both sexes
• usually begins to show itself in adolescence or later, but there’s a severe form that appears at birth (congenital MMD)
• shows worsening of disease severity as extra DNA is passed from one generation to the next and enlarges
• affects several areas of the body

Type 1 MMD involves the following signs and symptoms:

• Voluntary muscles weaken and have difficulty relaxing at will.
• Breathing muscles and brain control of breathing can be affected.
• Heartbeat can be irregular, which can be life-threatening and may require a pacemaker.
• Digestive organs and uterus can have abnormal muscle activity.
• Cataracts are very common.
• There’s a high risk for diabetes.
• There’s a high risk for adverse reactions to anesthesia.
• Mental retardation is common in infant-onset patients; less severe cognitive difficulties sometimes occur in later-onset patients.

A minority of people tested have a chromosome 3 form of myotonic dystrophy (type 2) that’s probably less common than type 1. The chromosome 3 form is also caused by extra DNA and closely resembles the chromosome 19 form in most aspects, except that:

• It doesn’t seem to worsen as it’s passed to next generation.
• No severe infant form has been seen so far.