Fixing the Genes

By LEON JAROFF

Eight years after the heart-bypass operation that saved his life, Floyd Stokes was in deep trouble again. His angina had returned with a vengeance. He was gulping nitroglycerine tablets and was virtually incapacitated, unable to do simple chores on his Seminole, Texas, ranch. Too far gone for another bypass, he had a choice, as he puts it, of "just waiting for death or trying to do something about it."

Stokes chose to survive. He volunteered to take part in a novel clinical trial about to be conducted on heart patients by Dr. Jeffrey Isner at the St. Elizabeth Medical Center in Boston. To his surprise, he was accepted. Last May he flew to Boston, where a solution containing billions of copies of a gene that triggers blood-vessel growth was injected directly into his heart.

Within three weeks, Stokes was feeling better and now, at 58, he is back at work on a normal, nitroglycerine-free routine. "I ride horses and I run tractors," he says. "You have to be in pretty good shape to do what I do." As it turned out, all 16 heart patients in Isner's trial showed improvement, and six are entirely free of pain.

The St. Elizabeth clinical trial is one of some 300 similar types of procedures being performed today on more than 3,000 patients around the world. These numbers reflect a growing optimism that gene therapy, a medical discipline that emerged with great fanfare in the early 1990s but fell out of favor during its adolescence, is finally coming of age. "Twenty years from now gene therapy will have revolutionized the practice of medicine," predicts Dr. W. French Anderson, director of gene therapy at the University of Southern California medical school, who is perhaps the most outspoken champion of this slowly maturing medical art. "Virtually every disease will have gene therapy as one of its treatments."

Gene therapy, simply defined, is the placement of beneficial genes into the cells of patients. By introducing the gene and consequently the protein it produces, says Inder Verma, a professor at the Salk Institute in La Jolla, Calif., "you either eliminate the defect, ameliorate the defect, slow down the progression of the disease or in some way interfere with the disease."

The initial goals of gene therapists were to cure relatively straightforward genetic disorders, such as Huntington's disease and sickle-cell anemia, that are caused by a single defective gene. The strategy was simple: substitute a normal gene for a faulty one. But scientists quickly realized that adding genes to cells could also impart new functions to those cells. That may lead to the genetic treatment of a host of other disorders, including heart disease and many forms of cancer.

But how do you get a new gene into the nucleus of a cell? The trick, researchers discovered early on, is to take advantage of the infectious power of viruses; burrowing into cells is second nature to them. A virus is nothing more than a tiny strip of DNA or RNA crammed into a protein envelope. Using the tools of molecular biology, scientists render the virus harmless by deleting some or all of its genes, splicing the therapeutic gene into the remaining genetic material and, in a laboratory Petri dish, mixing it with human cells. The altered virus, now called a carrier or vector, can deliver the therapeutic gene into the nucleus with great dispatch.

"You can do spectacular things with cells in a laboratory dish," explains Anderson. "You can easily get the genes in, change the cell's properties and do other things that ought to enable you to treat disease successfully." That is precisely what Anderson and his colleagues did eight years ago in the first approved use of gene therapy, when they removed blood cells from a young patient, genetically altered them with a viral vector and infused them back into her bloodstream. (See box.)

But could the same be done directly to cells within the human body? "That's where we hit the wall in the early 1990s," recalls Dr. James Wilson, director of the Institute for Human Gene Therapy at the University of Pennsylvania. One problem was that the body's immune system regarded the viral carriers as foreign invaders, and its response caused inflammation and swelling at the injection site. The antibodies that developed in response to the virus caused further difficulties. "In a very unfortunate turn of events," Wilson explains, "the patients would become immune against the therapy."

In an early gene-therapy trial for cystic fibrosis, inflammation caused by the viral carrier, an altered adenovirus, was so severe that the FDA ordered a halt to the effort, casting a pall over all the other trials--and the field in general. More problems plagued the researchers. In many cases the implanted genes failed to "turn on," or express themselves, and were unable to command the cells to produce the protein they were supposed to provide. Some operated for a while and then inexplicably shut down.

As a result, many gene-therapy trials failed during what the FDA calls Phase I, in which the safety of the procedure is evaluated on a handful of patients. Others proved ineffective and faltered during Phase II trials, which test a larger group to determine the efficacy of the therapy. And apparently only one trial has so far weathered Phase III, which calls for a larger number of patients and a statistical analysis of the results before the FDA gives its approval for general use.

That trial, being conducted by GTI-Novartis in Gaithersburg, Md., uses an ingenious technique to attack brain tumors. After re-engineering a retrovirus--an RNA virus that invades only cells that are in the process of dividing--the doctors outfitted it with a gene from the herpes virus and injected it into the brain. Because virtually the only cells that divide in the brain are tumor cells, the retroviruses infected them alone, inserting the herpes gene into their nuclei. As this gene expressed itself, it made the tumor cells sensitive to the herpes drug ganciclovir. When the drug was then administered to the patient, says Anderson, it "made the tumor cells commit suicide." But here there were troublesome side effects.

Clearly, gene therapy is not yet a panacea. Anderson concedes that except for reports of individual patients being helped, "there is still no conclusive evidence that a gene-therapy protocol has been successful in the treatment of a human disease."

Most researchers in the field agree that the adenovirus and retrovirus vectors are imperfect, to say the least. In addition to having immunological side effects, both lack the carrying capacity to accommodate the larger, more complex genes that would be useful in therapy. "There are only three problems in gene therapy," says Salk's Verma, "delivery, delivery and delivery. It isn't going to be a problem to make gene therapy work--if we have an appropriate set of tools to deliver the genes."

For his heart-patient trial, St. Elizabeth's Isner found a novel way around the delivery problem. Eschewing virus carriers, he fashioned a construct called "naked DNA." It consists of part of a human gene called VEG-F, which stimulates the growth of blood vessels, and includes its signal segments. These segments, Isner explains, "order the cell, once it has manufactured the gene product, to export it from the cell."

In his Phase I trial, Isner injected a saline solution containing his naked DNA through a small "keyhole" incision in the chest of his heart patients and directly into their heart muscle. A few weeks later, tests on everyone in the trial group showed greatly improved blood flow to the heart muscle though tiny new blood vessels that bypassed clogged arteries.

How does the naked DNA, without viral assistance, penetrate the walls of the heart-muscle cells? "To be perfectly honest," Isner confesses, "no one really understands how it gets there." But unlike most other therapeutic genes, which must find their way into millions of cells to have a therapeutic effect, VEG-F needs to invade only relatively few. Its protein product, issuing from the cell, can act on untold numbers of surrounding, untreated cells. Quips Isner in a parody of the Marine Corps slogan, "All we're looking for are a few good cells."

The fact that the VEG-F gene seems to turn off after three or four weeks makes little difference in this trial because the new blood vessels have already sprouted and remain in place. Still, for this and other reasons, the naked-DNA approach is applicable to only a handful of disorders.

For the vast majority of other trials, scientists are hard at work developing a new generation of viral vectors. One promising candidate, says Pennsylvania's Wilson, is the AAV (adeno-associated virus), a small, benign human virus that does not seem to cause any disease. "It doesn't elicit the same kind of inflammatory response that the other vectors do," Wilson explains. "It's somehow evolved the way to get around that." The AAV also efficiently insinuates itself into nondividing cells and, in tests with monkeys and mice, has enabled the therapeutic gene engineered into it to express itself for more than two years.

Wilson expects Phase I trials using AAV to begin later this year, first for the treatment of hemophilia and later for a form of muscular dystrophy, a liver metabolic disease and retinitis pigmentosa, an eye disorder. "It's kind of a new wave," he says.

The other new vector is being fashioned by Salk's Verma. "What we want," he says, "is a virus that is easy to make, that delivers genes at very high efficiency, that can infect a nondividing cell and that enables its therapeutic gene to become part and parcel of the chromosome."

Seeking the best candidate, Verma zeroed in on the most notorious of the retroviruses--HIV, the virus that causes AIDS. He eliminated the protein envelope that allows the virus entry into T cells, substituted one enabling it to infect a greater variety of cells, and removed the six genes that make the virus dangerous.

Can the lentivirus, as Verma dubbed his creation, ever recombine to generate a virus that has the ability to cause disease? "We have done 115 such preparations," he says reassuringly, "and to date we have never seen a virus that is capable of infecting new cells." Later this year he plans to ask the FDA for permission to begin a Phase I trial for hemophilia.

Gene therapists are looking even further ahead. Pennsylvania's Wilson predicts that the next advance will be a mechanism built into the vector to regulate the expression of a therapeutic gene, turning it on or off. "Most diseases and most drugs require modifying the dose," he explains, "but the genes carried into cells by currently used vectors are either on or off."

This means gene therapy cannot now be used to treat, for example, diabetics. If they were provided with a normal insulin gene that was always turned on, their insulin level would soon be dangerously high. "But the mechanism we have in mind," Wilson says, "will be like a genetic rheostat. The gene will not work until you take a pill, and the more pills you take, the more the gene will be expressed--and if you want to cut off the supply, you simply stop taking the pill."

Some researchers look forward to the day when gene therapy is used to repair damaged genes. With the new vectors, they would infect cells with small molecules that combine DNA and RNA. These hybrid molecules would seek out and bind to the defective gene, enabling it to function normally. "It would be like a repair mechanism," Wilson explains, "rather than a replacement."

French Anderson, ever pushing the envelope, last September asked the National Institutes of Health to begin considering gene therapy in the womb for fetuses found to be afflicted with a hemoglobin deficiency that would kill them before birth and for fetuses with ADA deficiency, the "bubble boy" disorder he treated in his pioneering 1990 trial.

To critics of gene therapy dismayed by what seems to be the slow pace of progress, Anderson urges patience. "People don't understand that the development of an ordinary drug from time of concept to product is 10 years," he says. "We're talking about a revolutionary approach to therapy, and we're only eight years into it."

Floyd Stokes, recovered, vigorous and hard at work on his Texas ranch last week, needs no convincing. "Dr. Isner and these fellows had to do some really far-out thinking to come up with this treatment," he says. "I owe my life to them."

--With reporting by Alice Park/New York

With reporting by Alice Park/New York