The invaders politely avoided staring any monkey in the face, a perilous faux pas in simian society, but there was no disguising their intent: to make one of the residents surrender a sample of her blood.
The monkeys are press-ganged pioneers in a bold medical frontier, the treatment of disease by reprogramming the body's genetic instructions.
Gene therapy, as it is hopefully known, has long been a field of high promise and low fulfillment. The promise is evident: all diseases, even infections, have a genetic component, and the best possible treatment in many cases would be to repair the genetic defect that permits the disease.
But inserting genes into human cells in full working order has proved to be exasperatingly difficult.
More than 230 clinical trials of genetic engineering techniques are now in progress, but most are at a preliminary stage and none has yet led to an approved therapy. Along with a batch of recent experiments at other universities, the Penn monkeys represent one of the few rays of hope in a long series of setbacks. Not only do six of them now carry a newly inserted gene that makes a therapeutic protein, but the gene can be switched on at will simply by giving the monkey a pill.
The monkeys belong to the university's Institute for Human Gene Therapy, directed by a youthful-looking veteran of the gene therapy wars, James M. Wilson. Dr. Wilson has corralled the resources to pursue a broad-based strategy.
Not for him the gamble of going after a single disease, with heavy odds of failure. He is pursuing a wide range of maladies with a variety of methods. "I will allow nothing technical to get in our way," he says firmly in a late-evening conversation in his office.
Mouse rooms and monkey colonies, where gene insertion techniques must be tested before being tried in patients, do not come cheap. Dr. Wilson's virus manufacturing center cost $5 million to build. Merely walking into it costs $20 -- the price of the disposable clothes worn to prevent people from contaminating the growing viruses.
Viruses are both the hope and despair of genetic engineers. Viruses can focus in on specific kinds of cells, penetrate their membranes and insert their genes in working order into the cell's DNA.
Most approaches to gene therapy use viruses as the vehicle to convey genes to target cells. The virus's harmful genes are stripped out and a therapeutic gene inserted in their place.
In the mid-1980's, when Dr. Wilson joined the field, a disabled mouse virus was everyone's favorite candidate. But the virus infected only cells that were dividing. The next candidate was adenovirus, a cause of the common cold. Adenovirus is a fine vehicle; it inserts new genes into many kinds of human cell, and the genes produce protein.
But only for eight weeks or so. That is how long it takes for the body's immune system to identify and root out every Trojan horse cell where an adenovirus, even a disabled one, is lurking.
In 1993, Dr. Wilson left the University of Michigan and a Howard Hughes fellowship -- a munificent but nontransferable kind of research grant -- to move to Penn. The dean of its medical school, Dr. William N. Kelley, made it a crusade to get gene therapy off the ground and persuaded Dr. Wilson, his former Ph.D. student, to head the new institute.
Two years later the whole community of genetic engineers was plunged into a crisis of confidence. The National Institutes of Health, finding that $200 million, some 2 percent of its budget, was going into gene therapy research, commissioned a critical review of the field. The review's authors chided gene therapists for promising too much and delivering too little.
The many exaggerated claims for gene therapy "threaten confidence in the integrity of the field," the institutes' report said.
A shakeout followed, but Dr. Wilson escaped damage.
"Personally it helped us, because it eliminated a lot of the hacks," he said. "So we contrived to grow after that." His institute, the largest of its kind, has a staff of 187 people and an annual budget of $25 million, half of which comes from the National Institutes of Health and the rest from industry and foundations.
"It's a very impressive operation and he has been combining a lot of basic science with applications," said Dr. Theodore Friedman, a gene therapy expert at the University of California, San Diego.
Dr. Wilson is now working with a new gene delivery system, one based on a small virus called adeno-associated virus. It has only two genes, both of which can be removed, leaving just its head and tail as a shell to carry therapeutic genes into target cells.
The little virus does not greatly provoke the body's immune system.
Dr. Wilson has also incorporated an important device that genetic engineers had neglected, a switch for regulating the inserted genes. Cells usually activate genes by having an agent that serves as a switch bind to a stretch of DNA called a promoter region, which lies upstream of a target gene. Once the switch is in place, the gene's program is executed.
Working with Ariad Pharmaceuticals of Cambridge, Mass., Dr. Wilson has inserted genes for a two-part on-switch into adeno-associated virus. The two parts assemble into a working switch only in the presence of the drug rapamycin. This means a gene can be inserted but will work only when a rapamycin pill is taken.
Dr. Wilson reported last month that the system had proved successful in mice and in rhesus monkeys. He used the gene for a powerful hormone known as erythropoietin, which spurs the bone marrow to churn out more red blood cells. Adeno-associated viruses carrying the rhesus version of the erythropoietin gene and the new switch system were injected into the animals' muscle cells. The gene was silent until the monkeys were given a rapamycin pill.
In the treated monkeys, the gene remains active for several days, requiring the red blood cell count to be checked regularly. If the red cell count gets too high, the monkeys are bled to reduce the risk of stroke.
People with low blood counts, like dialysis patients, have regular injections of erythropoietin itself. A better therapy, if feasible, might be to insert the erythropoeitin gene in their muscle cells, and flick it on every month or so with a rapamycin pill.
The erythropoietin project is just one of many that Dr. Wilson and his colleagues are pursuing.
"We are pursuing a blitz of activity across the board on a range of diseases," he says. He has projects for delivering corrective genes to the liver, the eye and the lung as well as to muscle cells.
A second group of projects is focused on cancers, including those of the skin, lung and brain. The cancer projects still rely on adenovirus as the delivery vehicle. Because the goal is to insert genes that will kill the cancerous cells, the vehicle's limited period of effectiveness is not an issue.
Dr. Wilson is switching all his genetic repair projects from adenovirus to the more promising adeno-associated virus. One of these projects, designed by his colleague Dr. Jean Bennett, is an attack on a form of retinitis pigmentosa.
The disease, a degeneration of retinal cells, is caused by an inherited defect in one of the enzymes that helps convert light into a nervous signal.
The defect, at present incurable, affects mice and certain breeds of dogs, like Irish setters, as well as people. Dr. Bennett has prepared viral vehicles that carry a correct version of the gene for the dysfunctional enzyme. The virus can be injected into the fluid-filled compartment behind the retina, where it will infect retinal cells.
Having proved the concept in mice, she then tried it in dogs and found that the treatment restored a dog's sight.
"It was so exciting to see the dog follow my fingers with one eye, while the other eye was still searching," she said. One eye had been left untreated, to serve as a comparison for the treated eye.
The improvement lasted for only seven weeks or so because the virus vehicle was the short-lived adenovirus.
Dr. Bennett is now repeating the experiment with adeno-associated virus. If that works, she said, she will seek approval to try the method in patients.
Dr. Wilson and his team face numerous pressures. "We get 50 calls a week from parents of children with awful diseases," said Dr. Nelson A. Wivel, deputy director of the institute. The money must be kept flowing with constant grant applications. Even when a genetic engineering technique should prove successful, there are many scientific and regulatory problems that remain unresolved.
One is that a virus and its cargo of improving genes might inadvertently get into the patient's eggs or sperm. The virus could insert itself in the middle of a gene, disrupting the gene's function and causing an inherited defect. Or it could insert correctly, remedying a genetic disease in the patient's descendants. Though that might not seem like a problem, making inheritable changes in the human genome is a bridge that no one is yet ready to cross.
Another awkward problem is the extreme xenophobia of the body's immune system, which instinctively assails any substance that it did not grow up with. Gene therapy aims to provide the correct genes to people who inherit some genetic defect, but the normal protein produced by the correct gene may nevertheless be treated as foreign by the patient's immune system, which has never seen the normal protein before.
The Food and Drug Administration has a staff of some 20 people who review applications to try genetic engineering techniques on patients. The agency has liberally allowed first-phase clinical trials but in giving final approval for a therapy may require more assurance on a range of issues. One is whether the virus vehicles, especially a disabled form of the AIDS virus, which some gene therapists favor, might not find occasion to reacquire their missing genes. "Here is where we are beginning to get into difficult areas," said Dr. Philip D. Noguchi, head of the agency's gene therapy unit.
A different danger on the gene therapists' horizons is that of being overtaken by the new technology of stem cells, the multi-purpose cells from which each organ is thought to repair itself. Inserting corrective genes into these cells, once they can be identified, could prove more effective than messing with viruses and other provocations to the immune system. But stem cell technology is in its infancy, and Dr. Wilson says he is not concerned that it will put him out of business. "It doesn't worry me at all," he said, noting that part of an organ must be destroyed to make room for any new tissue derived from stem cells.
Back in the monkey colony, a female rhesus, known only by a number, is deftly removed from her cage and placed in an open box. The edges of a permanent collar she wears slip neatly into grooves in the box, keeping her immobile.
However human the monkeys may seem, they are wild animals and require sophisticated handling.
Dr. Michael A. Schnell, the supervisor of the monkey center, feeds her from a child's pack of Kellogg's Fruit Loops while his colleague Ernest Glover scratches her back in the grooming motions that are simian peace gestures. After several minutes, when she seems sufficiently calm, Mr. Glover deftly inserts a needle into a tiny leg vein to remove blood. The monkey soon starts to moan, fretful to escape from the constraining box. She is returned to her cage, just in time for the afternoon round of orange segments.
The whole elaborate procedure will result in a single point on a graph recording the erythropoietin gene's state of activity. Evolution's programming took 3.5 billion years to develop; learning to fix the bugs in it is not going to happen overnight.