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Stanford Report, October 22, 2003

Glowing protein may cast light on potential muscular dystrophy treatment


A mouse with a glowing leg muscle may signal early success for researchers hoping to use gene therapy to treat muscular dystrophy. The work, presented Tuesday at the American Neurological Association’s annual meeting in San Francisco, is a proof-of-principle model for a promising new gene therapy technique.

"The long-term goal is to deliver a therapeutic gene to muscles to treat muscular dystrophy," said study leader Thomas Rando, MD, PhD, associate professor of neurology and neurological sciences at the School of Medicine.

The most common forms of muscular dystrophy are caused by a mutation in a gene called dystrophin. In muscle cells, this gene produces a protein that is needed for normal muscle function.

Although the idea behind gene therapy is simple — let a functional replacement gene take over for a mutated gene — its implementation has been riddled with problems. One significant hurdle has been inserting the gene into a cell’s DNA. Some researchers have taken advantage of viruses whose normal role is to insert their own genes into host DNA. The problem is that the virus itself may cause disease or may damage neighboring genes as it wedges in the therapeutic DNA.

Rando and a postdoctoral scholar in his lab, Thurman M. Wheeler, MD, hope to sidestep the viral problem using a technique developed by fellow Stanford researcher Michele Calos, PhD, associate professor of genetics.

Using her approach, the researchers slip a circular piece of DNA containing the dystrophin gene and an "insert me" signal into a cell, along with DNA producing a protein called integrase.

The integrase then inserts the therapeutic DNA into one of a few highly specific genetic hot spots in the cell’s DNA where the gene begins churning out protein and, the researchers hope, treating the disease.

The first step to using Calos’ technique to treat muscular dystrophy was to show that a gene could be inserted into the muscle DNA. Rather than using the dystrophin gene, Rando and Wheeler inserted a gene called luciferase. The protein made by this gene emits light that can be detected by a sensitive camera, giving a visual signal that the gene is producing protein.

Wheeler inserted the luciferase gene in the two hind legs of mice. In one leg he inserted the gene alone and in the other leg he inserted both the luciferase and integrase genes.

Although both leg muscles showed visible signs of luciferase soon after the injection, the signal in the leg lacking integrase dimmed over time. The gene had not integrated and degraded slowly. The other leg muscle continued to show luciferase protein throughout the experiment, evidence that the gene had successfully integrated into the cell’s DNA, Rando said.

He said these results are hopeful but warns that many steps lie between this experiment and a gene therapy cure for muscular dystrophy. The team must still show that the dystrophin gene can integrate into muscle DNA, and they must devise a way of delivering the gene to all muscles in the body.

Other researchers who participated in this study are Christopher Contag, PhD, assistant professor of pediatrics and of microbiology and immunology; Eric Olivares, PhD, graduate student in genetics; postdoctoral scholar Carmen Bertoni, PhD; and research assistant Sohail Jarrahain.

New gene therapy technique sharply reduces risks (10/16/02)

Rando to head geriatric center at Palo Alto VA (5/24/00)