Gene therapy posed to reinvent medicine

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On May 22, 1989, doctors at the National Cancer Institute in Bethesda, Md., hovered over the bedside of their patient, a 52-year-old man dying of cancer. A nurse hooked up a plastic intravenous bag containing a milky fluid.

The man and his wife held hands. The IV dripped. The minutes ticked by.

It was a critical moment in science's long march toward gene therapy, which seeks to cure disease by altering the molecular instructions for life.

For the first time, genes were being transplanted into a human being. If the genes worked, medical science would never be the same.

Dripping into the IV were the man's own white blood cells, which had been removed from his tumors and supercharged in the lab to increase their cancer-fighting power a million-fold. Spliced into these cells were bacterial genes that would serve as tracer bullets to see if the cells were finding their targets.

Even though the genes would play only a passive role in the cancer treatment, nobody knew with certainty what would happen when alien genes were introduced into a person. They might even kill him.

However, the blood-cell therapy represented the man's last hope. All other efforts had failed to arrest the black-mole cancer-malignant melanoma-that had spread uncontrollably throughout his body.

The man's doctor, NCI chief of surgery Steven A. Rosenberg, projected the upbeat confidence he had displayed as Ronald Reagan`s surgeon during the former president's bouts with colon cancer.

"Today is the first ever!" he declared jubilantly for posterity, in honor of the gene transfer.

But Rosenberg`s partner at the National Institutes of Health, gene splicer Dr. W. French Anderson, was less self-assured. He had been dreaming about this morning for years and he felt as though he was carrying the revolution in molecular medicine on his shoulders. Anderson had been up all night finishing preparations on the five plastic bags of IV fluids the patient would receive, and obsessively writing down everything that happened.

As he silently observed the bedside drama, Anderson was afraid to breathe.

"The patient seemed serene," he recalls. "Hopeful, unafraid. But I was scared to death, of course. My worst fear was that the impossible would happen-that after everything all of us had been through, he would suffer a heart attack and die, right then and there."

One minute passed . . . "He`s OK."

Three minutes passed..."Absolutely uneventful," noted Anderson.

Five minutes . . . "Perfectly normal."

The participants relaxed a little.

After 45 minutes, the doctors moved on to other patients in the intensive care unit, their faces professionally masking feelings of overwhelming relief. "So far, so good," Anderson remembers thinking, emphasizing that the first experiment represents merely "a toe in the water; a foot in the door."

While important to the development of gene therapy, the experiment did not actually alter the patient`s genetic makeup and is a far cry from the classic goal of gene therapists: Permanently curing victims of inherited diseases by introducing healthy genes into their cells, or modifying genes already in place, or removing faulty genes and replacing them with good ones.

But the apparently safe gene transfer in the cancer experiment quickly led gene therapists to the next step: Two weeks ago, Anderson and his colleagues became the first scientists in history to seek permission to try genetic engineering in humans.

The NIH researchers told the government subcommittee that oversees recombinant DNA research that they want to insert working genes in blood cells of children born with adenosine deaminase deficiency (ADA), a rare genetic defect that wipes out their immune systems. If authorized, the experiments could begin as early as this year.

In ADA-deficient children, toxins build up in the blood and destroy the key white blood cells of immunity. In the past, youngsters never survived infancy, unless they underwent bone marrow transplants, or like David the famous Houston "Bubble Boy," were confined in sterile plastic enclosures and protected from all viruses and bacteria.

The experiments would be designed to see if new genes would gradually build a permanent new immune system for the children.

The National Cancer Institute program is being expanded as well. Since the initial treatment last May, seven NCI metatastic melanoma patients have received Rosenberg`s therapy as part of a 10-person clinical trial. All seven patients, aged 26 to 52, absorbed the marker genes with no side effects, Rosenberg reports. Even though these patients had failed to respond to any other therapy, a third of them were helped by the experimental treatment.

One woman, in fact, who had dozens of melanoma lesions throughout her body, has enjoyed a nearly-complete remission, Rosenberg says.

With this response, and the fact that the marker gene demonstrated that the supercharged white blood cells were actually functioning, Rosenberg and Anderson plan to treat another 50 volunteers with a variety of end-stagecancers.

Since 1969, Rosenberg has been inventing treatments that can sometimes activate a patient`s own immune system to destroy cancer cells while sparing normal tissue. His therapies use specialized white blood cells called tumor-infiltrating lymphocytes (TILs), which have their tumor-killing powers greatly enhanced in the lab. Then they`re unleashed against an individual's cancer.

Early experiments with victims of melanoma showed remarkable tumor shrinkage in 12 patients out of 20. But no one knew why the other patients weren't helped and until now Rosenberg has been flying blind. He has been unable to tag the TILs to determine how many of the cells survive, for how long, and if they even reached the cancers.

But gene splicers like Anderson have long employed techniques to detect whether gene transfer has occurred in cells, giving Rosenberg a way to track his supercharged TIL cells after he reinjects them.

The key feature is a marker, a gene that in chemical tests will glow inside a cell like a firefly. A favored marker gene comes from the common intestinal bacterium, E. Coli, and that's the one used in the NIH experiment.

To deliver the bacterial marker genes into the tumor-fighting cells, Anderson deployed a mouse leukemia retrovirus. Unlike other viruses, whose goal in life is to blindly invade cells and reproduce, a retrovirus exists only to get in somebody else's genes and subvert them to its own use. AIDS, for example, is caused by a retrovirus. When retroviruses are genetically altered to render them harmless, they become superb delivery trucks to haul new genes into cells.

The mouse leukemia retrovirus had been altered by the top virus rehabbers in the world and was presumed to be a safe delivery vehicle. Still, no medical experiment in history had received more official scrutiny before having been allowed to proceed-stormy debate before seven committees of the NIH and Food and Drug administration, and a court challenge. All told, the proposed experiment was reviewed, re-reviewed and chewed over by experts 15 times.

Despite the fears, the genetically altered retroviruses did their jobs precisely as programmed. They ferried in sufficient numbers of the new bacterial genes for later testing and then stopped cold and didn't reproduce, convincing the scientists that foreign genes circulating in blood cells can be safely implanted into humans.

"The next step," says Rosenberg, "is to put genes into the TILs that will make them more effective in fighting cancer. I plan on seeking permission to do that soon."

Similarly, Anderson says that in addition to the ADA gene therapy experiment, he soon may propose human experiments using three different genes- against cancer, AIDS and heart disease.

A restless, driven man, the 53-year-old Anderson is a pediatrician, molecular biologist, specialist in blood diseases and director of the molecular hematology division of the National Heart, Lung, and Blood Institute. He works off excess energy by sparring with fellow masters of the martial art Taekwon do.

But in science, Anderson seems to function like some sort of master enzyme. He keeps catalyzing gene therapy, pushing it along, seizing opportunities, collaborating with everybody (currently 20 scientists across the nation), and enduring criticism from more conservative colleagues that he is too intent on jamming new genes into people before science is ready.

"Yet there`s a certain genius behind the cancer experiment," contends University of Michigan physician-virologist James Wilson, a young scientist dedicated to gene therapy.

"Those patients are dying, but not from anything French Anderson did to them," Wilson says. "He wasn't trying to do gene therapy but merely to insert a presumably harmless marker gene and see if it took hold. The next step is to add new genes to see if they will help fight disease. And before you know it, French has snuck up on you and gene therapy suddenly is a reality."

Anderson long has been grappling with problems that confound would-be gene therapists: how to get healthy new genes into patients` bodies and make them work once they get there. One can't just rub genes into the skin or swallow them. Genes must enter the body at the cellular level, where they will be absorbed by the chromosomes. Nor is it enough to get them into a few cells. They must infiltrate whole legions of cells.

As gene scientists learned more about their trade, it became obvious that tinkering with a few genes in organisms was a lot more frustrating than anyone had envisioned. The organisms usually responded by dying.

There is a reassuring side to this: The complexity of controlling genes in even the simplest forms of life means that the chances of a few altered genes seizing control of an entire organism and doing something dangerous are exceedingly remote.

Until now, the experimental tissue of choice for gene doctors has been not blood, but its source-bone marrow, the spongy substance that occupies the cavities of the larger bones. It is the body's blood-making factory and, with skin, the only self-renewing tissue that doctors can remove, treat in some manner, and put back in the body to cure a disease.

Hundreds of classic inherited blood diseases-among them hemophilia and several lethal forms of anemia-occur because of minuscule genetic errors in bone marrow cells, and these diseases have long been considered good candidates for gene therapy. Although remarkable progress is being made on several fronts, the delicate biochemical regulation required by these genes has stymied scientists for a long time.

If science could cure just one of these diseases by gene therapy, it would open a very large door. More than 1,600 diseases are caused by defects in single genes.

Success hinges on finding a way to replenish the blood system by targeting genes to the relatively few "stem" cells in bone marrow that give rise to all the different blood cell types. In the mouse, for example, it takes only one good stem cell to make good blood, scientists have found.

Human stem cells have basically eluded science, but the field heated up in 1988 when a Stanford University team led by pathologist Irving Weissman developed a highly successful technique to purify the stem cells of mice. And by last spring, Weissman had succeeded in nearly transferring his stem cell identification method from mice to men. "We're within sight of it," he says, referring to what is commonly called the "holy grail" of blood research.

Although the TIL and proposed ADA experiments are the only human trials that involve new genes, several strategies should become possible within the next few years. Anderson's ADA proposal shows how researchers are working on short-term therapies, which would provide patients with temporary new genes encased in special cells. These would be administered periodically to provide patients with the missing proteins or hormones their own genes should be making.Some examples:

  • Genes that dissolve blood clots have been transferred into cells that line blood vessels and the airways of the lungs of animals.About 350,000 coronary artery bypass operations are done each year in the U.S., and nearly a third of them are plagued by blood-clotting problems. Tiny fibrous sponges or Gore-Tex fibers containing anti-clotting genes or factors crucial to blood vessel growth are undergoing experiments in hopes they eventually can provide protection in heart surgery patients until healing occurs.
  • Genes have been introduced in certain skin cells-called fibroblasts and keratinocytes-that secrete clotting factors and other proteins needed by victims of blood and autoimmune diseases such as hemophilia and diabetes. It is hoped that such skin patches can be grafted, or placed inside the body, if some way can be found to make them survive. Skin cells are simple to harvest and grow in the lab, but they last only a few weeks.
  • Genes have been transplanted into blood cells to produce CD-4, the protein "target" on immune cells that AIDS viruses use to invade the cell, eventually destroying the immune system. Various versions of the CD-4 molecule are in clinical trials to determine if, when injected into the bloodstream, they can decoy the AIDS virus away from the immune cells and stop its spread in infected people. These are the first drugs designed specifically against AIDS, but they must be injected repeatedly. Anderson believes the genes that produce CD-4 could be inserted in different kinds of cells and implanted in the body, whereupon the genes would secrete the proteins continuously for months.
  • Genes can be aerosolized. Nasal sprays can deliver missing genes that make such proteins as alpha-1 antitrypsin to treat emphysema patients and save their lungs, according to Dr. Ronald Crystal, chief of pulmonary medicine at the National Heart, Lung, and Blood Institute. The newly discovered protein that cystic fibrosis victims lack perhaps could be delivered in this manner as well, says the University of Michigan`s Dr. Francis Collins, the co-discoverer of the cystic fibrosis gene last September with the University of Toronto`s Lap-Chee Tsui.
  • Liver cells could be given genes to make receptors on the cell surface that clear cholesterol from the bloodstream. First aimed at patients who lack the receptor necessary for cholesterol metabolism and hence are prone to early heart attacks, the technique has broad implications for a wide range of liver diseases.
  • Muscle cells may have the unique ability to accept and use new genes after simple injections.A University of Wisconsin team led by Dr. Jon Wolff reported recently that he and his colleagues had injected raw genes into the muscles of mice and were amazed to discover that the genes obligingly entered the muscle cells and began performing their protein-making duties. If the work is borne out, Wolff says, it may be a lot simpler to deliver healing genes to Duchenne muscular dystrophy victims, as well as other bodies wracked by disease, than previously thought.
  • Cancers sometimes arise when genes inadvertently get lost during cell division, or stop working. Pioneering gene splicer Theodore Freedmann's group at the University of California in San Diego is pondering ways to engineer viruses to resupply cells with the lost genes that stop cancerous growths. Friedmann and his colleague, Fred H. Gage, recently demonstrated that it may be possible to engineer genes to stop the deterioration of such brain disorders as Parkinson's and Alzheimer's disease. Even as French Anderson and his colleagues are struggling to bring gene therapy to the bedside, other researchers are leapfrogging toward easier techniques to transfer genes that they hope may be available in the near future.They are trying to find ways of delivering genes into cells without the use of viruses. Michigan's James Wilson, for example, has temporarily cured a form of heart disease in rabbits by delivering new genes directly into liver cells.Undeniably effective at invading cells, viruses insert the new genes willy-nilly into chromosomes. This raises the possibility that the new genes might disturb the genes already there. If disrupted in this manner, the cell might turn malignant and start growing a cancer."Up to now the best we can do is put a new gene into cells and stand back and pray," says Stanford Nobelist Paul Berg.A possible solution to gene targeting was revealed in 1985 when researchers Oliver Smithies, then at the University of Wisconsin, and the University of Illinois` Raju Kucherlepati made an amazing discovery: The genetic machinery, or DNA, of a cell is sometimes willing to absorb a transplanted gene and then use the new gene as a spare part to repair orreplace a defective resident gene.The phenomenon, called homologous recombination, represents the keys to the kingdom of gene therapy, and occupies many of the top minds in the field.Homologous recombination occurs frequently in simpler organisms like bacteria and yeast, says Stanford's Berg. "If you just introduce a piece of DNA, it finds its matching partner (its homologue) wherever it sits on the chromosome. It doesn't ever get integrated into a wrong spot."However, in the cells of more complex organisms like mammals, "foreign DNA goes wild," Berg says. "It gets recombined into chromosomes literally everywhere. Our problem is how to control where it goes."It has taken years of hard slogging in the lab, but in 1988 two Berg graduate students at Stanford made major advances.Maria Jesin showed that she can target a single transplanted gene to recombine with its native counterpart on the proper chromosome, even though the transplanted gene may be absorbed in myriad wrong places as well. And David Strehlow demonstrated that a cell can repair one of its genes with help from a transplanted gene, even if the transplant itself is defective. The native gene merely scavenges what it needs.A few months later, another veteran gene-targeting pioneer, Mario Capecchi at the Howard Hughes Medical Institute of the University of Utah, successfully transplanted new genes into mouse cells at the precise sites of defective genes-whereupon the healthy new genes busily went about fixing the old, or swapping places.The immediate use, Capecchi says, is that scientists now can create mutant mouse models for virtually any disease they choose. But, he hastens to add, such developments suggest that gene therapy might someday consist of dispatching healthy new genes into the body like molecular paramedics. The ailing gene would simply reach out and grab what it needs to get better."What we're after is an effective way to correct any gene that's causing disease, so that it behaves normally," Capecchi observes. "I wouldn't be surprised if we were starting to bring these techniques to patients within the next five years."
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