The possibility that the virus had introduced its gene for arginase into the scientists was a curiosity, nothing more–until 1969, when the Lancet published a paper by Heinz-Georg Terheggen, a pediatrician in Cologne, Germany, and colleagues. Two little girls had been brought to Terheggen, deeply mentally retarded and suffering from a form of cerebral palsy, the British journal reported. Tests showed they had high levels of arginine, while very little of the enzyme arginase was detectable. This was a new genetic disease.
Rogers went to Terheggen to urge that he and his colleagues be permitted to inject the girls with Shope virus, hoping to give them a functioning gene for arginase. As an essential precaution, they did try inoculating the virus in a tissue culture of cells from one of the girls. They reported in the Journal of Experimental Medicine that they found arginase activity, apparently from the virus-introduced gene. But in the trial, there was no response, no reduction of arginine, no evidence of arginase activity. After an interval, they gave one child a larger dose. Still no response. The general consensus was that Rogers had made a premature attempt, with inadequate scientific understanding. That judgment was not wrong.
In the spring of 1972, Theodore Friedmann and Richard Roblin published the first extended study of the possibility of treating genetic diseases through gene transfer. “Gene Therapy for Human Genetic Disease?” appeared in Science. Disease by disease and therapy by therapy, the researchers warned of formidable technical problems; much that they laid out was prescient. They were the first to analyze the potential risks that gene therapy posed to patients and the grave ethical concerns it raised.
Nonetheless, the paper was a work of advocacy. With a medical degree from the University of Pennsylvania, Friedmann had spent three years in the 1960s at NIH, where, in the laboratory of Jay Seegmiller, he had begun to work on Lesch-Nyhan disease. Seegmiller had discovered that the disease is caused by the absence of the enzyme hypoxanthine phosphoribosyltransferase, or HPRT, owing to a defect in its gene. Friedmann hoped to find a way to put the correct gene into Lesch-Nyhan cells in culture, perhaps using a virus. His imagination had been caught by the prospect of gene transfer. Indeed, as an assistant professor of pediatrics at the University of California, San Diego, in the early 1970s, he introduced the term “gene therapy.”
In January 1983, Friedmann and colleagues announced that they had isolated the normal gene for HPRT. Inder Verma, with whom Friedmann had struck up a collaboration in the early 1980s, had a potential viral vector: in this case, a type of retrovirus–one for a mouse leukemia. In August 1983, the two researchers reported that they had built the vector and used it successfully to introduce a functioning gene for human HPRT into rodent cells in vitro.
After that initial glimpse of success, Verma says, “very quickly we asked, ‘Can we do it in vivo?’” They began experiments on hemophilia in live mice. The gene defects causing hemophilia were known: the lack of a single protein could prevent blood from clotting. Working in vitro, adding the correct gene to cells in culture, “we could produce the protein forever,” Verma says. “And this is where the first surprise came.” The moment the cells were put back into the mice, “they instantly stopped making the protein. And this is the first limitation we recognized: retroviruses can only introduce genes when the cells are dividing.” Verma adds, “We could take [the cells] out, grow them in vitro, transfuse them with the virus, put them back–but when we put them back, they shut off.” Why? “We still really have no idea,” he says.