In 1990, 28-year-old Emanuela Gussoni arrived at Stanford University in California, having completed a doctoral degree in neuroscience from the University of Milan in Italy and postgraduate studies in neuromuscular disease at Milan’s Carlo Besta Neurological Institute.
The gene for dystrophin, the protein missing in Duchenne MD, had been identified only four years earlier, by MDA grantee Louis Kunkel and colleagues at Harvard-associated Children’s Hospital in Boston, and doctors were gearing up to make use of the new discovery.
In California, as elsewhere, the plan was to undertake myoblast transfer, transplanting cells from close relatives into boys with DMD, with the goal of replacing missing dystrophin and rescuing muscle tissue from destruction.
Gussoni joined a team that included MDA grantees Larry Steinman and Helen Blau at Stanford, and Robert Miller at California Pacific Medical Center in San Francisco.
“My role was to detect any immune response between donor and patient,” she says, “and to design an assay to detect donor transcripts [dystrophin from donors].”
She detected both. In three out of eight boys in the San Francisco trial, she found evidence of dystrophin production from donor cells, although the amount was miniscule compared to the number of injected myoblasts. About 1 percent of the muscle fibers in the injected area showed some normal dystrophin one month after the transfer.
Then, in 1992, just before Gussoni was scheduled to return to Italy, she received an MDA grant that allowed her to stay at Stanford for two more years. After that she moved to Harvard to work with Kunkel, who also had conducted myoblast transfer experiments.
Gussoni and Kunkel asked themselves questions about the myoblast transfer trials, none of which had harmed anyone, but none of which had shown much dystrophin production or even much cell survival.
In 1997, with Kunkel and Blau, she published an updated analysis of the fate of the transplanted myoblasts in the San Francisco trial of the early ‘90s.
By then molecular detection techniques had improved. “We re-examined the muscle biopsies using a new method,” Gussoni says. “We had originally injected 80 million to 100 million myoblasts and gotten few dystrophin-positive fibers, so the question was, Where did the cells go?”
Using the new techniques, they found a lot of the cells still there, in fact, more than they had originally thought. “Many were not expressing dystrophin, but they were there,” she says.
Gussoni and others theorized that only the slowly dividing cells survived. “That provided a hint that maybe we should use a different kind of progenitor cells, not the classic ‘myoblasts’ we used.”
For Gussoni and many others, muscle “side population” cells are a major focus. These cells reside in the muscles themselves and probably give rise to satellite cells, as well as integrating directly into damaged fibers at times. (The term side population cells comes from methods used to isolate them.)
In addition to the status of the donor cells, the status of the recipient’s muscle also has to be taken into account, Gussoni notes.
“We have to pay attention to what cells we put in, but also how to condition the muscle to accept the transplant.” Radiation has been shown to help the process in mice, but that has obvious drawbacks and probably can’t be used in people.
“Immune parameters are important,” Gussoni says. “The children in our clinical trial were immunosuppressed, and now we know that some immunosuppressant medications interfere with differentiation [maturation] of muscle cells. Cyclosporine [the drug they used] does that.”
But just matching fathers or siblings to patients isn’t enough to prevent an immune response.
“These days we would not have gone to human myoblast trials from the mouse data that we had,” she says.
For Gussoni, a crucial question is, What forces determine cell integration? “The cell has to have something on it that can be recognized by a fiber,” she says. “I don’t think the process is random. When you look at muscle fibers after cell delivery, there are groups of cells that have engrafted. It’s not a random uptake.”
The role of intercellular signals that say “repairs needed” and “repairs offered,” once understood, Gussoni believes, will clarify where we go from here.