By David N. Leff

From the volume button on your TV remote to the dimmer switch on a light fixture, modern life is largely controlled by voltage regulators called rheostats.

Pioneer gene therapist James Wilson, at the University of Pennsylvania, in Philadelphia, looked for molecular rheostats to govern the actions of genes and their protein products in human patients. Wilson directs the university's Institute for Human Gene Therapy.

"Using a unique combination of innovative technologies," he told BioWorld Today, "we and our collaborators have demonstrated the ability to introduce therapeutic genes into the body, and then precisely control their activity with a drug that could be given as a simple pill. It's a very precise gene switch, like a rheostat."

Wilson is senior author of a paper reporting this feat in the current issue of Science, dated Jan 1, 1999. Its title is "Regulated delivery of therapeutic proteins after in vivo somatic cell gene transfer."

Gene therapy begins with the vector that delivers a DNA sequence of interest to its cellular site of expression. Wilson and his collaborators struggled for years with adenoviral vectors, which consistently failed to confer stable expression.

"What has happened to change that," he recounted, "is that now there's one vector - possibly two - that seem to work very well at conferring stable expression. The one we've focused on, adeno-associated virus [AAV], has moved along. Our problem, in evaluating its potential, was in producing the AAV - manufacturing it. So, we spent two years developing a method for large-scale production, which we now have.

"With that vector in hand," Wilson said, "we moved forward in many different applications of in vivo gene therapy. And what really struck me in finally achieving that goal was the problem of unregulated expression. Theoretically, we anticipated this, but until you actually have practiced it in animal models, it doesn't quite sink in as to how critical regulating the gene would be for safe and effective therapy in human patients.

"One of the first experiments that we then did," he recalled, "collaboratively with Kathy High at the Institute, was Factor IX for hemophilia. I'm very excited about the potential for that of her work." (See BioWorld Today, Jan. 5, 1999, p. 1.)

Transcription Factor Broken, Dimerized

"But, when we look critically at other applications," Wilson said, "in particular the use of this approach for secreting proteins that go into the blood, virtually every other use is going to require gene regulation. For example, if insulin is too high, you've got to turn it down. Interferons too high are very toxic. If erythropoietin (EPO) is too high, the red-blood cell counts go sky high, and patients run the risk of having a stroke.

"Furthermore, ultimately you would need to turn down or stop the treatment in some situations," he added. "The way that gene therapy has been practiced up to now is that the vector goes into the muscle, the liver, or whatever the organ, and if it's stable it's going to be there as long as those cells are there. To turn it off, you'd literally have to remove that organ.

"So, it was with our experience with AAV vectors," Wilson said, "using genes that were not regulated, that we hooked up with Mike Gilman and his colleagues at Ariad [Pharmaceuticals Inc., of Cambridge, Mass.] to think about regulation."

Biochemist Michael Gilman, chief scientific officer at Ariad, is a co-senior author of the Science paper.

The company calls its patented strategy, to which Wilson turned, ARGENT, short for Ariad Regulated Gene Expression Technology.

"It uses small molecules," Gilman told BioWorld Today, "to induce protein-protein interactions, and turn whole complex pathways on inside cells. The application described in the Science paper uses the general strategy of controlling the activity of a transcription factor - a protein that turns genes on and off. Here, the idea was to break the transcription factor into two pieces, then use a small-molecule, orally available drug called rapamycin to bring those pieces back together again, that is, dimerize them. When we did that, we fully reconstituted the transcription factor's activity. The concentration of rapamycin then determined the transcription rate of a suitably engineered target gene - in this case, the EPO gene."

Gilman recounted the genesis of ARGENT: "This whole idea of small-molecule protein dimerization was invented in 1993 by Stuart Schreiber, at Harvard University, and Gerald Crabtree, at Stanford. Their technology was licensed exclusively worldwide by Ariad over four years ago. We put that quite brilliant but academic invention to practical use, and the current Science paper is its culmination to date."

The first U.S. patent for the technology issued on November 3, 1998, titled "Regulated transcription of targeted genes and other biological events," and numbered 5,830,462.

Rapamycin is a natural bacterial product, identified as an antifungal compound in the mid-1970s and subsequently found to be a potent immunosuppressive drug in humans

After injecting their vector/EPO construct into the muscles of mice, then rhesus monkeys, the co-authors gave their animal models repeated doses of rapamycin over time.

"We've done a total of six monkeys," Gilman said. "The EPO secretion has persisted from a low of three months to one animal that's still going strong at seven months." In fact, the red-blood-cell-boosting effect is so vigorous, he observed, that the animals "have a hematocrit so high that they must be bled periodically."

On To Clinical Trials - Stepwise

Wilson said the focus now is on developing a greater experience in large non-human animals. "We are still focussing on EPO, and our initial plan is to test the vector alone in humans without the regulated system - and do that soon," he said. "We expect to do that sometime this winter for muscular dystrophy patients. Once we have that experience behind us, we would move to the next clinical trial, utilizing the same vector, but incorporating the regulatory system. My goal is to start those trials in a little less than two years, realizing that we have to do this in steps. Our plan is to stay with EPO for the treatment of thalassemia, which is an inherited and severe form of anemia."

Wilson concluded: "We first have to make sure that we understand the safety and the biology of the vector alone, and only then incorporate the bells and whistles." *