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Curing disease through human gene therapy.

A Vision of Gene Therapy

Ever since they first learned the chemical nature of heredity, a few medical scientists have shared a dazzling vision: someday, faulty genes would be replaced to treat and cure disease. Today that dream is becoming reality. It is called gene therapy.

The first gene therapy patients are being treated at the National Institutes of Health (NIH) in Bethesda, Maryland. Their treatment is the result of a notable collaboration between the National Heart, Lung, and Blood Institute's (NHLBI) Molecular Hematology Branch, led by Dr. W. French Anderson(1) and scientists from the National Cancer Institute (NCI), led by Dr. R. Michael Blaese, chief of NCI's Cellular Immunology Section, and Dr. Steven A. Rosenberg, chief of surgery at NCI.

This scientific team created the new era of gene therapy on September 14, 1990, when they, along with Dr. Kenneth W. Culver of the NCI, began treating a 4-year-old girl with a rare defect in a single gene. Untreated, most children with her disorder die in early childhood. Today, she lives an essentially normal life.

"She has shown remarkable progress," Dr. Anderson said some months after the historic first treatment. "We could not be more pleased."

The child has a rare deadly disease called severe combined immunodeficiency (or SCID). For her, even the most minor infections were dire threats. It is essentially the same disease that doomed the "Bubble Boy" of Houston, Texas, to live all of his short life inside a plastic bubble to keep him isolated from the world's germs.

In the young girl's case, the disorder stems from a flawed gene that is the genetic blueprint for making a substance, adenosine deaminase (ADA), that plays a crucial role in immune defenses. It is one of the many enzymes that serve as biological catalysts to do the body's chemical work. The disorder is known as ADA deficiency.

The gene therapy treatment is deceptively simple. Each session takes about one hour in which the child receives a steady drip of fluid into a vein. In that pint or less of fluid are more than 10 billion of her own white blood cells. These white cells of the variety known as T lymphocytes, or simply T-cells, were removed earlier and treated so that they contain good copies of the ADA gene for making the crucial enzyme that the girl lacks. In the scientists' term, the cells are "gene-corrected." The infusions are repeated every couple of months. The child is also receiving injections of a drug, PEG-ADA, that supplies some ADA in a durable form so that it is not destroyed immediately by the body's chemistry. Before she entered the study, PEG-ADOX had helped the child somewhat, but she still experienced serious abnormalities in her immune defenses.

Human ADA Gene Therapy

1) ADA gene copy is inserted into a specially engineered virus.

2) A type of white blood cells called T-cells are withdrawn from the patient's blood.

3) These T-cells are grown in large numbers in tissue culture plates. The cultured T-cells are then mixed with the virus.

4) T-cells with ADA gene copy are multiplied further in gas-permeable bags.

5) The cells are then injected back into the patient to help form a working immune system.

From Genetic Code to Gene Therapy

Gene therapy became possible in concept in 1953 when Drs. James D. Watson and Francis F. C. Crick, then at Cambridge University, discovered the double helix structure of DNA and showed that its chemistry served as the alphabet and the language of heredity. Before that discovery, genes had been little more than intellectual abstractions. After Watson and Crick, genes were facts of life with physical and chemical identities. Only a few years later, Drs. Marshall Nirenberg and Heinrich Matthaei of the National Heart Institute, now the NHLBI, broke the genetic code and showed how the messages of heredity were spelled out and could be translated. Dr. Nirenberg received a Nobel prize in 1968 for his research on the genetic code.

A torrent of research followed. Now that the code was understood, the messages of the genes could be translated and even written artificially in the laboratory. A drumfire of rapid advances raised hopes that someday genes might be manipulated, perhaps even repaired. That was by no means a main-stream idea in the late 1950's. Few discussed it in public. Many thought it was ridiculous, but there were a few who believed in it. Among them was W. French Anderson, then a pre-medical student at Harvard University. In the early 1940's, scientists at the Rockefeller Institute had demonstrated that DNA was the substance of the genes, using bacteria to show that traits changed when one bacterium took up DNA from another. Dr. Anderson wrote his senior thesis on a new aspect of this work. He also saw implications -- if genes could be transferred in bacteria, it might be done someday in humans too.

After graduating, he studied at Cambridge University with Dr. Crick and later returned to Harvard. Since graduating from Harvard Medical School in 1963, most of Dr. Anderson's medical career has been dedicated to the goal of gene therapy. His first post at the NIH was in Dr. Nirenberg's laboratory at what was then the National Heart Institute. Under Dr. Nirenberg. Dr. Anderson took part in the historic research through which the genetic code was translated completely. He also made important contributions to understanding the vital process by which proteins are manufactured under the guidance and regulation of RNA (ribonucleic acid, chief partner of DNA in the chemistry of heredity).

But Dr. Anderson's medical training had been in pediatrics and his great commitment was to the treatment of patients. He began studying patients with beta-thalassemia, a serious disorder of hemoglobin production. Scientists had discovered that beta-thalassemia resulted from gene flaws that disrupted the production of normal hemoglobin, the oxygen carrying pigment in blood. Patients with the most serious form of beta-thalassemia -- called Cooley's anemia -- seldom lived to see their 15th birthdays.

In 1968, Dr. Anderson admitted a young brother and sister, Nick and Judy Lambis(2), who suffered from that disease. They were the first two patients in what would soon become an active hematology service at the Institute. Dr. Anderson treated them for the rest of their relatively short lives. Many other patients were admitted into the hematology research program.

"We had all these thalassemia kids and gene therapy was what I wanted to do in the long run to help them," recalls Dr. Anderson, "but I had to do something short term because they were dying in front of us."

One of the patients' crucial problems was the accumulation of iron from the rapid breakdown of their red blood cells -- an effect of many blood transfusions. Dr. Anderson developed a treatment to rid thalassemia patients of their iron overload by using the drug desferioxamine, a chemical that grabs iron and takes it safely out of circulation.

Meanwhile, an attempt at something close to what is now called gene therapy had been made in 1970 by Dr. Stanfield Rogers of Oak Ridge National Laboratory in Tennessee collaborating with doctors in West Germany. Two young German girls suffered epileptic seizures and were risking serious brain damage because genetic flaw robbed them of one particular enzyme. Dr. Rogers saw a possible remedy: he deliberately infected the sisters with a virus -- the Shope papilloma virus. It was best known for causing warts in rabbits, but it was also thought to have a gene for making an enzyme equivalent to the one the girls lacked. The virus infections were attempts to treat the girls by the transfer of foreign genes so that their cells would manufacture the product of those genes. Unfortunately, Dr. Rogers' attempt failed. Some scientists criticized his medical experiment, while others lauded him for having the imagination and the courage to try.

The experiments brought the concept of gene therapy to the attention of Congress where there had already been much discussion of possible dangers and benefits of gene splicing research. The incident led to a clamor for national regulations to govern any future attempts at genes therapy.

Meanwhile, Dr. Anderson was already giving much thought, not only to how gene therapy might be done, but also to the ethical concerns that must be faced. A case that illustrated both issues made headlines in 1980.

There had been no known attempts for a decade. Then, in 1980, Dr. Martin Cline, of the University of California at Los Angeles (UCLA) decided to try gene transfers to treat severe cases of thalassemia. He applied for permission at UCLA, but was refused. He took the idea abroad. The countries around the Mediterranean have many cases of beta thalassemia. Dr. Cline got permission from local doctors to try his experimental treatment on a patient in Israel and another in Italy,

News of the attempts soon leaked out. The technique Dr. Cline used violated NIH guidelines that had been drafted several years earlier. These rules were binding on essentially all American universities and their scientists. The episode received a great deal of adverse publicity. Dr. Cline was severely reprimanded both by the NIH and UCLA. The treatments apparently produced neither benefits nor harm, but the incident evoked a powerful movement to strengthen the role of the NIH in overseeing any future attempts to apply recombinant DNA technology to human beings.

It was clear that there were issues of ethics as well as science. Dr. Anderson spelled out his own conclusions in 1980 and repeatedly thereafter. There were no ethical bars to the use of gene therapy to treat disease, he argued, so long as the genes were transplanted only into specific somatic cells, the body cells, but not into the person's germ cells. Given that basic constraint, he said gene therapy offered the possibility of enormous good. The real question was when to start. In Dr. Anderson's view, this should come only after animal research had demonstrated some key points: that the new gene could be put into the intended target cells and would remain there capable of functioning that the body would regulate the transplanted gene appropriately and that it would not harm the cell. Only after those crucial points have been demonstrated in animals, he argued,should human gene therapy be attempted.

While human gene therapy was on hold, so to speak, the transfer of genes into animals became a major enterprise by itself. This research has made enormous strides helping to advance knowledge about human health and recently even contributing to clinical medicine. Much of this has been accomplished through the development, study and use of what are now called transgenic animals. They are animals of many species that carry foreign genes. The importance of this new field burst on the public consciousness in the early 1980's when Drs. Ralph L. Brinster of the University of Pennsylvania and Richard D. Palmiter of the University of Washington made mice grow twice the size of their littermates by injecting foreign growth hormone genes into their embryos. Others among the many who did important early work in this field included Drs. Frank H. Ruddle and J. W. Gordon of Yale, Frank Constantini of Columbia and a collaboration between Elaine G. Diacumakos and Dr. Anderson.

After 1980 and Dr. Cline's attempt, no one would be ready to try human gene therapy for years, but many still thought beta-thalassemia was the best target. Unfortunately, the chemistry of that disease is a maze of complications. The derangement of hemoglobin is an imbalance that develops in the crucial blood pigment as it is formed through an intricate ballet of molecules. Every part of the complex arrangement must mesh perfectly with all the others. A single genetic error could bring on disease, but correcting such an error would be immensely complicated.

Dr. Anderson was among the first to see the difficulties. He argued that a much simpler system should be attacked first.

"Early attempts at gene therapy will almost certainly be done with genes for enzymes that have a simple `always-on' type of regulation," he wrote in 1984. That ruled out beta-thalassemia, but ADA deficiency fit the description perfectly. In Bethesda and across the country, scientific teams pursing gene therapy research agreed that ADA deficiency would be a good first target.

Dr. Blaese, a recognized authority on the disorder, had his office within easy walking distance of Dr. Anderson's. The two decided to combine forces.

"We started working on it together and pushed it as hard as we could," Dr. Blaese said noting that the collaboration is still vigorous. "His lab and my lab meet each week around the table, present our data and fight about experiments. It has been a terrific collaboration." Through that collaboration, a new era has been born.

Signs of Success

In the first several months of her treatment the main goal was to show that the young patient could take the treatment safely. She was watched anxiously for signs of fever, pain, or other bad reactions to the infusions -- even though many animal studies had found this to be an unlikely prospect. She showed no signs of problems, and, in fact, began to show signs of improvement after several infusions.

By the time she had received eight infusions, the child had a substantial increase in the level of circulating T-cells in her blood. Tests also showed increased ADA activity in her peripheral blood T-cells. As evidence of her improved immune system function, the child has also had positive skin tests to diphtheria, tetanus, and candida. In addition, since she began gene therapy, the child has had fewer infectious illnesses.

While the 4-year-old girl is far too young to understand talk of T-cells and immune defenses, she is one of the most important pioneers of human gene therapy.

In early 1991, another child became a pioneer: a 9-year-old girl with ADA deficiency was also enrolled in the gene therapy program. As the study progresses, the scientists will continue to add patients.

Soon after the ADA study began, a second gene therapy experiment got under way at the NIH. Dr. Steven A. Rosenberg, chief of surgery of the NCI, in collaboration with Drs. Anderson and Blaese, began gene therapy on two cancer patients suffering from advanced malignant melanoma, a deadly form of skin cancer.

The immediate goal of this cancer study and the ADA experiment is to save lives and reduce suffering. The long-range goal is to prove that gene therapy has a role in the future of medicine.

The Next Targets

ADA deficiency is but one of at least 4,000 known disorders that result from different single genetic flaws. Some of these genetic disorders degrade the immune defenses. Others affect essential tissues or organs, including the blood, liver, brain, heart, kidneys, or bones. These genetic disorders add up to a major public health problem and today there is no cure for any of them. Some of these disorders will be among the next targets of gene therapy, building on what scientists have learned about genes and their functions in the complex orchestration of life.

But the grand strategy of gene therapy also envisages a much broader use of the new techniques, to include assaults on heart disease, diabetes and other major health problems that are influenced by the functioning of genes.

Genes are the individual determinants of all hereditary traits; not only obvious things like the color of the hair and the shape of the nose, but all vital life processes. In chemical terms, genes are segments of DNA (deoxyribonucleic acid) that serve as the blueprints used by cells to guide the manufacture of every substance and structural part that the body needs and can make. Humans have an estimated 100,000 genes. A flaw in almost any one of them could result in a disease.

The objective of gene therapy is to correct such faults by giving the patient healthy copies of a flawed gene. That is what scientists in the Molecular Hematology Branch, and their collaborators, are doing with these first gene therapy patients.

Treating these patients is possible today because of the immense advances scientists have made in recent years in understanding the chemistry of the genes. They have learned the chemical language of heredity and often have been able to identify not only the specific genes that are at fault in a disease, but also the precise spelling errors in the genetic message that ruins those genes. In laboratory cell cultures they can even correct some of the flaws -- supplying good genes to remedy the error. This has been done in fruit flies, mice, and human cells.

An Exchange of Ideas

It would not have been possible to leap so quickly from correcting genetic flaws in tissue culture to gene therapy in people without the interlaboratory cooperation and cross-fertilization of ideas that have been the hallmark of gene therapy research at the NIH. Besides Drs. Anderson, Blaese, and Rosenberg, and members of their staffs (most notably Dr. Kenneth W. Culver, a senior clinical investigator with Dr. Blaese), other NIH scientists made significant contributions. These scientists include Dr. Arthur W. Nienhuis, former chief of NHLBI's Clinical Hematology Branch, Dr. Ronald G. Crystal, former chief of NHLBI's Pulmonary Branch, and Dr. Harvey Klein, director of the NIH Blood Bank. With these men of diverse experience and talents working together, gene therapy became a reality much faster than it otherwise would have.

A Strategy for Gene Therapy

When Dr. Anderson set out to develop gene therapy he devised a three-part strategy:

- Find cells that would accept the transplanted genes and put them to use;

- Develop a delivery system to put the foreign genes into the patient's cells; and

- Show that these genetically engineered cells actually function in the intended way so that they might do the patient some good and not cause harm.

Only after these points had been proven in laboratory experiments and in animals would a trial of gene therapy in humans be justifiable.

Some people saw gene therapy as a totally new and frightening concept. Genes are the most personal, individual and private embodiments of a human life. To address the public's concerns about gene therapy and to provide guidance to gene therapy researchers, the human gene therapy subcommittee of NIH's Recombinant DNA Advisory Committee (RAC) devised a set of guidelines. These "points to consider" established the rules linder which gene therapy could be conducted and they bore a strong imprint from Dr. Anderson's views on ethics.

Once these rules were in place, the scientists could proceed with their research strategy. It was a formidable task.

A Delivery System

The NIH team's challenge was not only to identify "target" cells that would accept the transplanted genes but also to find a way to deliver the genes to those cells. Bone marrow cells, the ultimate source of all blood cells, appeared to be the best choice for the "target cell." If genes could be transferred into the patient's own bone marrow, doctors might hope to cure their patients in one procedure.

With this goal in mind, the scientists concentrated on a "delivery system." There were several known ways of putting foreign genes into mammalian cells, but most of them were not applicable to present-day gene therapy. The one possibility that seemed practical was the use of viruses. By the mid-1980's, many researchers believed that this was a method to pursue. Viruses would be employed to introduce new genes into certain of the patient's cells. In the scientists' jargon, viruses would be the "vectors" for delivery of foreign genes.

Viruses are ready-made guided missiles for gene delivery. In nature, they are little more than infectious packages of genes honed by evolution to be adept at getting these genes reproduced in the cells the viruses invade.

But viruses could never be used in their natural state. Many cause disease and some contribute to cancer. For use in gene therapy, the viruses would 4ave to be rebuilt.

The problem with which scientists were grappling was this: How to rebuild a virus so it would carry useful genes into human cells, but be unable to cause harm? Many of the experts chose a class of viruses called retroviruses. Their genetic material is in the form of RNA (ribonucleic acid), a companion substance to DNA in the chemistry of heredity. Retroviruses force infected cells to translate the virus RNA into the DNA form. But retroviruses can be dangerous. They would have to be totally disarmed, re-equipped and turned into self-destroying packages that vanish as soon as they deliver their cargo of therapeutic genes. This process took years to achieve. Here is the strategy:

The virus genetic material is stripped of the genes that carry the instructions for making complete new virus particles, leaving only the genes to be transplanted for purposes of therapy and the instructions that prompt an infected cell to reproduce those genes. This package of genetic instructions is then incubated in a special breed of cells that have already been infected with a replication-incompetent (i.e., defective) "helper" virus. The helper virus carries the instructions for fabricating new virus outer coats, but cannot make copies of the virus' own genes.

Inside the "helper" cells, the two sets of genetic instructions come together to produce new virus particles. They can deliver their intended cargo of foreign genes into new cells, but then they vanish because they lack the complete set of instructions for making new virus particles.

The next and final step is to expose the patient's cells to these engineered viruses so that the useful genes are transplanted. The technique requires care in order to keep any helper viruses from the patient's cells. Once this is achieved, the system allows the transfer of the desired genes and nothing else.

Dr. Anderson recruited many imaginative and hard working young scientists to join his team in order to get gene therapy clinical protocols started. In addition, he set up collaborations with other institutions where a few specialists were also groping their way forward toward gene therapy. One member of the team, Dr. Phillip W. Kantoff, took on the job of making retroviral vectors that could be used clinically. He began working feverishly on the retroviral problem and collaborated on this with Dr. Gilboa and his staff then at Princeton.

The Molecular Hematology Branch: A Training Ground

Many scientists throughout the world have contributed to the science of gene therapy, but if any single place could be judged the birthplace of the new era, it would bet the Molecular Hematology Branch of the National Heart, Lung, and Blood Institute. The emphasis there is on team effort. Most of one wall of Dr. Anderson's crowded office is a portrait gallery of researchers who have contributed and gone on to important scientific posts at other institutions or other laboratories of the NIH. Appropriately, one of the portraits is of a monkey, a species that has contributed greatly to the advent of human gene therapy.

Dr. Anderson also set up a collaboration with Dr. John Hutton of the Cincinnati Pediatric Research Hospital who had been among the most successful in producing clones of the ADA gene -- the intended genetic payload to be carried by the remodeled retroviruses.

Dr. A. Dusty Miller in Seattle was also working on gene therapy. His research was crucial to the final stages of producing remodeled viruses -- suitable for use in humans.

At the opposite pole from viruses and their genetic payload was the experimental animal that would first receive the foreign genes. The animal model system became the province of Dr. Martin A. Eglitis, a mouse embryologist. He set to work doing bone marrow studies and transplants in mice to provide cells that would be good recipient of the virus payload. Once the virus system was developed, its effectiveness eclipsed previous methods of putting new genes into living animals. The success was almost a shock when the first set of DNA samples were analyzed. The team was stunned to see how many animals had taken up the foreign genes. "Where I was accustomed to seeing one or two positives on a film of 20 or 30 samples," Dr. Eglitis recalls, "I developed the first film with 24 samples on it and 20 of them were positive."

The result was so good that neither he nor his colleagues could believe it. But they tried it again and again and got the same impressive results. The genetically engineered retroviruses -- retroviral vectors -- were the way to go. That hurdle was crossed.

Then in the summer of 1985, Drs. Anderson, Blaese and their colleagues did the first gene transfers in cell cultures using blood cells from an ADA patient as their target. It worked.

"It was one of those beautiful experiments," Dr. Blaese recalls. "It just absolutely cured the population of cells by the transfer of the ADA gene.

The next step was to try the experiments in primates -- induce some of the animals' bone marrow cells to take up the ADA gene and then put the cells back into the animals. These crucial experiments in monkeys, done with Drs. Richard O'Reilly at Memorial Sloan-Kettering and Arthur Nienhuls in the NHLBI, were only partially successful.

"Those experiments were critically important," Dr. Anderson recalls, "because they demonstrated two vital components of gene therapy: first, that a functional human gene could be successfully inserted into the blood cells of a monkey, and second, that gene transfer using retroviruses was not harmful. The level of activity was too low to be clinically useful, however.

For the NIH team, gene transfer into bone marrow hovered stubbornly just short of potential clinical application.

Then, in the summer of 1987, the team tried putting genes into white blood cells -- lymphocytes.

"I put Ken Culver on the project to try to put genes into lymphocytes and his experiments worked beautifully, Dr. Blaese remembers.

"He showed that lymphocytes would hold the genes and would express for long periods of time. Put them back into the animal and they would continue to express. It worked.

The research team's enthusiasm soared. But there were other problems. By general consensus, gene therapy would be acceptable, at the start, only for patients near death and bereft of any other hope.

A Life and Death Need

By now, the PEG-ADA treatment had been developed, removing the specter of immediate death from the very patients who would be the best test of gene therapy. Was it ethical to subject these young children to an experimental treatment that had never been tested in any humans? If not, what patients could be considered for testing something so drastically new? There had to be a life and death need.

That need existed in another part of the NIH Clinical Center where Dr. Steven A. Rosenberg of the NCI was using a new method of treating patients desperately ill with malignant melanoma. His treatment was based on a new concept for bolstering the patient's own immune defense system. He gave these cancer patients a population of their own immune cells that had been grown in the laboratory to increase their numbers and their potency in fighting cancer. The cells used in this innovative cancer treatment are called TIL cells, for tumor-infiltrating lymphocytes. The TIL cells are obtained from the patient's own cancer. Buried in that cancer tissue are lymphocytes that had invaded the tumor to fight it. The doctors collect these natural cancer-fighting cells and grow large quantities of them in the laboratory. After these cells are primed for combat against cancer by exposure to the lymphokine called interleukin 2, a stimulant of T-cell growth, they are put back into the patient.

Dr. Rosenberg calls his treatment with the special lymphocytes "adoptive immunotherapy." This therapy has produced some remarkable effects including the complete disappearance of cancer in patients who are near death. But many patients had no detectable benefit from the treatments.

It was a reasonable guess that some of the lymphocytes fought cancer better than others, but how could they be identified? Perhaps the treated cells could be labeled with a foreign gene. That thought led to the collaboration between the research teams of Rosenberg, Blaese and Anderson. If a harmless, but easily detectable, gene could be transferred into the cancer patients' TIL cells, Dr. Rosenberg would have the identifying label he needed and Dr. Anderson would have ethically acceptable patients to be his first human recipients of an intentionally transplanted foreign gene. Once the idea was broached, its logic was compelling. Dr. Blaese arranged a meeting on January 17, 1988, between Drs. Rosenberg, Anderson, and himself. "And at that meeting, within an hour, we had mapped out the entire protocol and it basically never changed," Dr. Anderson remembers.

Patients would be given their own TIL cells containing a foreign gene. When samples of lymphocytes were removed from the patients for testing, the "labeled" ones could easily be identified.

After a long and detailed review process at the NIH, the project was approved early in 1989. The first successful gene transplants in humans were done at the NIH in May, June, and July. This was a watershed advance. The gene transplants helped refine the strategy of gene transfer and demonstrated for the first time that foreign genes could be transplanted into patients' cells without ill effects.

It still required months to get approval for the ADA gene therapy clinical protocol, but with the evidence from the cancer patients at hand, the proposal to use ADA gene-corrected T-cells as therapy against ADA deficiency in children could no longer be denied.

Because T-cells do not live forever, this therapy would not provide a lasting cure. However, Drs. Anderson, Blaese, and Culver in collaboration with Dr. Craig Mullen, a senior clinical investigator at NCI; Dr. Cynthia Dunbar, a senior clinical investigator at NHLBI, and

Dr. Lauren Chang, a senior staff fellow at NHLBI, have recently embarked on a new phase of the ADA study which may prove to be a cure. In this part of the study, the research team inserted the gene into the patients' stem cells. Stem cells are the parent cells that produce all cellular blood elements.

The procedure involves a new technique that detects and isolates stem cells in the bloodstream (rather than stem cells in the bone marrow, which are more difficult to obtain). These cells were "gene-corrected" and then infused into the patients. In addition, two teams of physicians in California have also begun stem cell studies. The scientists -- at Children's Hospital in Los Angeles and the University of California, San Francisco -- are treating two babies who were diagnosed prenatally with ADA deficiency. If the stem cell studies are successful, patients may have continuous production of ADA-normal T-cells.

A Partnership: Pooling Resources to Advance Research Goals

An agreement between NHLBI, Dr. Anderson(*) and a private company is providing a mechanism for research and development that is highly useful to gene therapy. The agreement between the Institute and Genetic Therapy, Inc. (GTI) is called a Cooperative Research and Development Agreement or CRADA. CRADAs were established under the Federal Technology Transfer Act of 1986.

A CRADA enables government scientists to collaborate with private industry in order to develop scientific and technical knowledge into useful, marketable products. This partnership results in a pooling of intellectual and material resources and serves to strengthen research efforts. In a CRADA, private companies can own licensing rights to patients held by government agencies. In return, both the Federal scientists and their agencies are allowed to benefit monetarily from their research.

The CRADA between NHLBI and GTI allows the private company to serve as an adjunct laboratory to the Molecular Hematology Branch. For example, GTI is working on the development of safe and effective vectors for gene transfer. The company collaborated with Dr. Anderson on the engineering of the vector used in the ADA study (derived from the Moloney murine leukemia virus). One of the GTI/NHLBI projects is the development of "injectable vectors," a concept which would make the tools of gene therapy more widely available and easy to use.

Other Applications

While the ADA study continues, the NHLBI team is exploring an ever broadening range of projects related to gene therapy and its underlying science.

Could a form of gene transfer help the success of heart and vascular surgery? Could it help protect blood vessels from being closed off by clots after surgery? Dr. David A. Dichek, a physician specialist in cardiology, had come to Dr. Anderson's laboratory from the Massachusetts General Hospital to explore just such questions.

Obstructive blood clots are a major problem in transplanting blood vessels to improve circulation to the heart muscle or to the legs, and in the use of artificial devices called stents. Stents are tiny flexible metal coils that help keep blood vessels open following angioplasty.

Dr. Dichek and his coworkers devised an ingenious two-part study that explored the use of gene therapy as. a means of producing powerful localized effects in the body where they were most needed. First, they demonstrated that a gene could be introduced into endothelial cells that line blood vessels. Then they showed that these cells could be grown on stents in the laboratory and -- most importantly -- that they would stay in place when the stents were expanded with the aid of balloon catheters.

The researchers are now exploring the use of such gene transfers to prevent dangerous clotting in blood vessel grafts in the period after surgery. The key element in such genetic modifications is the gene for the anti-clotting substance t-PA (tissue plasminogen activator) specially configured so that it would be secreted at levels potentially high enough to prevent clot formation inside a blood vessel graft. Although temporary, this kind of gene therapy might be useful in bringing patients safely through postoperative periods. If successful, this therapy could save thousands of lives.

NHLBI scientists are also focusing on possible solutions to the worldwide AIDS epidemic.

Scientists have already learned a great deal about the virus, HIV, that causes this grim disease. They know that a protein called CD4 on the surface of some white blood cells -- T-cells -- is the natural target of the AIDS virus. The attack against those important T-cells is what cripples the AIDS patient's immune defense system.

This knowledge has led to a new strategy to fight AIDS. Researchers speculate that if a patient's blood could be flooded with excess CD4 molecules, they might sop up most of the virus, leaving the natural target cells unharmed. Such treatment might not cure the deadly disease, but could keep its ravages in check.

Scientists at several institutions have attempted to use the CD4 molecule directly by injecting large quantities into the bloodstream of AIDS patients. So far, this tactic has not been successful. However, Dr. Richard Morgan, a Ph.D. from Johns Hopkins on Dr. Anderson's staff, together with Dr. Robert Gallo in the NCI, have begun work on another approach: using gene transfer to produce lymphocytes that would circulate in the patient and continue to secrete large quantities of CD4 into the blood.

"We began work on this project to do the molecular cloning by manipulating the CD4 gene and putting it into retroviral vectors and then making the virus," Dr. Morgan recalls. They had to make viruses -- the familiar retroviral vectors -- to carry the gene. Then they had to prove that the transplanted gene actually made the CD4 and that the lymphocytes would shed it into the blood. They also had to show that the HIV virus would indeed bind to this decoy molecule.

"All those experiments were positive," Dr. Morgan said.

"That took a good year or so. "

The system is now reaching the stage where it could soon be considered for trial in patients. Meanwhile, the scientists have also begun to develop new ways of attacking the HIV itself. One of these is to genetically engineer lymphocytes to produce large amounts of the antiviral substance alpha interferon. Another strategy is to alter some of HIV's own proteins to make them into genetic mutants that would disrupt the virus' life cycle.

While that work is progressing, Dr. Morgan is tackling another important project -- research aimed at treating the blood disorder hemophilia by gene therapy. This would involve transfer into patients of the genes for clotting factors VIII or IX -- the clot-promoting substances that are lacking in the various kinds of hemophilia.

"I don't think it is unreasonable to think about protocols to treat hemophilia within 2 to 3 years," Morgan said.

Another major area of current research at the NHLBI is gene therapy of cystic fibrosis (CF). The first gene therapy study of CF began in April 1993. A modified adenovirus -- a cold virus -- was used to transfer a normal cystic fibrosis gene into the cells lining the nose and airways of a 23-year-old man with CF. The patient came through the procedure with no ill effects. Ten patients will participate in this study, which will evaluate the safety and effectiveness the adenovirus vector.

Gene therapy's promise for the future includes the development of an injectable vector: a gene therapy virus that can be taken off the shelf, so to speak, and injected directly into the patient. These special delivery vectors would have to home in exclusively on just the cells where new genes were needed. The viruses would have to deliver their cargo of therapeutic genes and vanish as the present day vectors do. Once these injectable vectors are developed, gene therapy will not require the present procedure of removing cells, treating them in the laboratory and reintroducing them into the patients.

"Injectable vectors would enormously broaden the usefulness of gene therapy," Dr. Anderson says, "and that is why I am focusing my primary efforts on this problem."

According to Dr. Anderson, "Twenty years from now gene therapy will have revolutionized the way many diseases are treated.

The vision and dedication of a team of scientists at the National Heart, Lung, and Blood Institute will help bring the treatment of these diseases into the 21st century.


Adenosine Deaminase (ADA) - An enzyme important to the immune defense system.

Antibody - Protein produced by the body to protect against invading germs and other foreign substances.

Amino Acids - The structural sub-units or chemical building blocks of all proteins.

B Cell - One of the major classes of white blood cells. It includes the cells that produce antibodies.

Bone Marrow - The material within the bone cavities that constitutes the body's main organ for blood production. Bone marrow cells include the stem cells which are the progenitors of all classes of blood cells.

Carcinogen - Cancer-causing substance.

Chromosome - Threadlike strand DNA and protein that contains genes. Humans have 23 pairs of chromosomes; 22 pairs called autosomes and one pair for the sex chromosomes -- two X chromosomes in females; one X and one Y chromosome in males.

Cloning - The production of many copies of a gene, usually by use of microorganisms as cellular "factories."

Cystic Fibrosis - One of the most common of the serious genetic diseases that stem from an error in a single gene. The patient's lungs, pancreas, and sweat glands are particularly affected. The specific gene responsible for the disease was identified in 1989 after a long international search.

Cytokine - A chemical that serves as a messenger through which one cell influences the performance of another.

DNA - Deoxyribonucleic acid, the substance that is the archive of genetic information in all forms of life. DNA is the information-carrying substance of the genes.

Duchenne Muscular Dystrophy - A grave, wasting disease of muscles that results from flaws in a single gene. The specific gene was discovered in 1986. Through knowledge of the gene, the protein for which it is the genetic blueprint was discovered a year later.

Dystrophy - Deterioration.

Enzyme - A protein that acts as a biological catalyst by speeding up a specific chemical reaction in the body. Virtually every chemical event in a living organism depends on one or more enzymes. Most of the known genes are genetic blueprints for producing one or another enzyme.

Expression - In genetics terminology it means activation. A gene that is said to "express" or to be expressed is "switched on" to do the job for which nature designed it.

Erythrocyte - Red blood cell, the oxygen-carrying cell of the blood.

Factor VIII - This blood substance and blood factor IX are proteins vital to the proper clotting of blood. The two most common kinds of hemophilia result from lack of one or the other of these blood factors.

Gamma Globulin - Blood proteins important in the immune defense system; the class of proteins that includes antibodies.

Gene - The fundamental unit of heredity; a segment of DNA that carries the genetic information to make a protein.

Genetic Engineering - The manipulation of genetic material, usually the DNA, to produce specific changes in the characteristics or performance of a cell or an organism.

Germline Gene Therapy (or Gene Transfer - The introduction of foreign genes into the reproductive cells of animals or humans to produce hereditary genetic changes (as contrasted with somatic cell gene transfers which are not passed on from one generation to the next). Germline gene transfers have been successful in animal research, but are neither practical nor considered ethically acceptable for use in humans.

Interleukin 2 - Known originally as T-cell growth factor, it is a control substance important for the functioning of the immune defense system.

Lymphocyte - A cell of the immune defense system, a white blood cell.

Lymphokine - A substance produced by an immune defense cell to perform a defensive function or to help activate or mobilize other elements of the defense system; a subclass of cytokines.

NIH - National Institutes of Health, Headquartered in Bethesda, MD, it is the Federal Government's main agency for sponsoring and conducting biomedical research.

PEG-ADA - A drug that has proven very helpful to some patients who suffer from a lack of the enzyme adenosine deaminase. Full name of the drug is polyethylene glycol-adenosine deaminase.

Protein - A long chain (or a group of linked chains) of amino acids. Proteins are the fundamental buildings blocks of all biological structures. Some proteins, acting as enzymes, function as the universal agents that build up, break down and carry out the metabolic tasks of all tissues.

RAC - Recombinant DNA Advisory Committee. The committee advises the director of the NIH on issues related to gene splicing research and gene therapy. It is the main Federal advisory committee overseeing this field.

Retrovirus - A virus that carries its genes in the form of RNA. Such a virus forces the infected cell to translate the viral RNA into the DNA form for use by that cell's genetic machinery.

RNA - Ribonucleic acid, companion substance to DNA in the chemistry of heredity. RNA is the messenger that translates the genetic information of the DNA into the production of working proteins by cells.

Somatic Cell Gene Therapy - Gene therapy that alters a specific trait or function by the introduction of an effective new gene into a specific class of somatic cells (body cells). All current uses of gene therapy involve somatic cell gene transfers.

T-cell - Immune defense cells, also known as T lymphocytes. There are several varieties of T-cells, each with specific functions in the immune system.

Thalassemia - Any of a group of hereditary blood diseases that result from various flaws in the production of hemoglobin. Some of the varieties are among the most serious known blood disorders.

TIL - Tumor-infiltrating lymphocyte, an immune defense cell that invades tumor tissue to bring about its destruction.

Transduction - As gene therapists use the term, it means using a virus to add a foreign gene or genes to a cell to alter that cell's characteristics or performance.

Vector - A carrier or agent used to transmit something such as a gene. In gene therapy usage, a viral vector is the carrier that transmits a gene into a cell.


Under provisions of applicable public laws enacted by Congress since 1964, no person in the United States shall, on the grounds of race, color, national origin, handicap or age, be excluded from participation in, be denied the benefits of, or be subjected to discrimination under any program or activity, (or, on the bases of sex, with respect to any educational program or activity) receiving Federal financial assistance.

In addition, Executive Order 11141 prohibits discrimination on the basis of age by contractors and subcontractors in the performance of Federal contracts, and Executive Order 11246 states that no federally funded contractor may discriminate against any employee or applicant for employment because of race, color, religion, sex, or national origin. Therefore, the National Heart, Lung, and Blood Institute must be operated in compliance with these laws and Executive Orders.

(1) Dr. W. French Anderson resigned from the NHLBI, effective September 10, 1992. He is now professor of biochemistry and pediatrics at the University of Southern California School of Medicine, Los Angeles. He is also directing a gene therapy program at USC Dr. Anderson will continue his NHLBI research as a special volunteer. He will remain a principal investigator on the adenosine deaminase gene therapy study.

(2) Their familes have approved use of their names.

(*) The CRADA is currently between NHLBI, Dr. Safer, and GTI.



Public Health Service

National Institute of Health

NIH Publication No. 93-2888

July 1993
COPYRIGHT 1993 National Heart, Lung, and Blood Institute
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 1993, Gale Group. All rights reserved. Gale Group is a Thomson Corporation Company.

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Publication:Pamphlet by: National Heart, Lung, and Blood Institute
Article Type:Pamphlet
Date:Jul 1, 1993
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