Infectious diseases were once the scourge of the human race, felling the majority of their victims before they even reached reproductive age. That changed with improved public health programs, effective immunization procedures, and the discovery of antibiotics. Before the AIDS epidemic, it was rare (although certainly not unknown) for someone to die of an infectious disease in industrialized countries. But when all of the diseases caused by microbial pathogens are accounted for, human beings still find themselves assaulted by a wide range of crippling, even lethal maladies. These diseases are idiopathic, arising because of some defect in the myriad molecules involved in the construction and operation of the enormously complex human body.

A significant portion of human idiopathic diseases are genetic in origin. Humans have on the order of 100,000 different genes, instructions embedded in DNA for the production of the proteins used to organize and direct every aspect of human physiology. Defects in these genes can have disastrous consequences for the individual inheriting them. The resulting 4,000 or so human genetic diseases can be every bit as devastating as infectious disease, and in one way they are much worse: we pass them on to our children,

generation after generation after generation. Science and medicine have provided us with clues to the treatment of some of them, but by the very nature of these diseases they have never been considered curable.

That is about to change through one of the most profound revolutions in modern medicine: gene therapy, a branch of the new field of molecular medicine. Progress in molecular biology has made it possible to isolate normal, healthy copies of human genes, and to restore them to those unfortunate individuals who have inherited damaged or functionless genes. This same technology, turned around on itself, can also be used to introduce deliberately "bad" genes to attack and destroy unwanted cells, such as cancer cells or cells infected with the AIDS virus.

The first human genetic disease to be treated by gene therapy was a form of SCID - severe combined immunodeficiency disease. In this disease, children are born without a functioning subset of white blood cells called T cells. Without functional T cells, vast portions of the immune repertoire are unable to work properly. T cells are the cells destroyed by HIV, the AIDS virus; in fact, children born with SCID are clinically equivalent to patients entering the final stages of AIDS, and they have a similar life expectancy.

Available therapies, such as bone marrow transplantation and drugs, are able to save less than half of the children born with SCID. One of the most famous SCID patients was David the Bubble Boy, who was kept in sterile isolation for over twelve years in the hope his T cells would recover on their own. They didn’t. He was finally removed from his bubble and given a bone marrow transplant from his sister. The transplant failed to take, and he died of transplant-related complications a few months later.

One form of SCID is caused by a defect in the gene encoding adenosine deaminase (ADA), an enzyme critical for T cell function. The human gene for ADA had been cloned, and was used in late 1990 by Dr. W. French Anderson to treat ADA-SCID in a delightful young lady named Ashanti DeSilva. Ashanti had been treated with a drug called "PEG-ADA", which occasionally helps children with this form of SCID. However, the drug was not working well for her, and Ashanti’s only remaining hope seemed to be a risky bone marrow transplant. But Dr. Anderson had just received federal approval to try gene therapy for this disease. So it was that this shy, smiling four-year-old girl made medical history by becoming the first human being ever treated with gene therapy to correct an inherited genetic defect.

Before they could begin, Dr. Anderson and his team had to develop a suitable delivery vector for their gene. In most cases this is a virus. Viruses have many advantages for delivering genes to cells, not the least of which is that they have been selected over millions of years of evolution to do just that - to work their way into a cell and transfer their own genes into the host cell nucleus. It is relatively easy to insert an additional "passenger" gene into the viral DNA; the resulting hybrid is called a recombinant delivery vector. For Ashanti, a healthy human ADA gene was incorporated into a retrovirus, a virus that readily infects blood cells. Retroviruses are the preferred delivery vector for use in gene therapy because they insert their genes directly into a host-cell chromosome. The virus is crippled so that it cannot itself replicate and cause disease, but any passenger genes will be read and used just as if they were part of the host cell’s own DNA.

The second task is to get the delivery vector with its passenger gene into cells. In the case of blood cells, this is relatively easy. Preclinical experiments with mice had demonstrated that retroviruses readily carry human genes into blood cells, and that the genes function well in their new environment. So some of Ashanti’s T cells were removed from her blood, exposed for several days to the recombinant vector containing the ADA gene, and returned to her body through a simple i.v. drip. This procedure went very smoothly, and today, after a half dozen or so treatments, Ashanti’s T cells and immune system are almost completely normal. Modifications of this basic procedure have now been used to treat over a dozen more youngsters with this disease as part of an extended clinical trial.

A total of nearly twenty human genetic diseases are in various stages of development for treatment by gene therapy (Table); clinical trials for eight of these are already in progress. One of the first diseases tackled after SCID was cystic fibrosis (CF), another devastating genetic disease of childhood. The gene defective in CF encodes a protein causing the production of mucus too thick to carry out its function of sweeping inert particles and bacteria from the airways. Eventually the thickened mucus clogs the airways, making breathing very difficult, and the patient may suffocate. CF patients only rarely survive beyond age 30.

As soon as the CF gene was cloned in 1989, plans for its use in gene therapy got underway. Scientists decided to take advantage of a virus called adenovirus to carry the CF gene into crippled cells. Adenovirus has a natural affinity for human lung and airway tissue, causing mild flu-like symptoms. The virus was again crippled before inserting the CF gene, to prevent even these relatively harmless side effects, but its airway-seeking portions were left intact. The first patient to be treated with the recombinant CF gene-adenovirus vector was a 23-year-old man with fairly advanced disease; he received his first treatment in April, 1993; since then, several dozen other CF patients have been entered into clinical trials for CF gene therapy.

The initial results from the CF trials are encouraging, but also demonstrate some of the problems that remain to be solved in this challenging field. The CF gene found its way into the airway cells, and produced the missing protein. But unlike retroviruses, adenoviruses do not insert their genes directly into the host DNA; the passenger gene remains as a floating episomal or "satellite" DNA in the host nucleus. Eventually these satellite genes disappear, necessitating repeat administrations. Multiple injections are not in themselves harmful, but they eventually provoke an immune response to the virus; the resulting antibodies neutralize future incoming recombinant vectors before they can deliver their genes.

Scientists are currently working to solve this problem, and are coming up with some highly imaginative solutions. One reason the less immunogenic (immunity-provoking) retroviruses have not been used in the CF trials is that retroviruses only infect cells that can be made to divide, like blood cells removed from the body. The one exception among retroviruses is HIV and its relatives, which can invade non-dividing cells such as those found in human airway tissue. It is now possible to render HIV completely harmless by removing key genes from its DNA. What is left is a highly efficient, relatively non-immunogenic vector like the ones that have worked so well in SCID. Delivery vectors of this type may well find their way to the clinic within the year. (For psychological more than health reasons, relatives of HIV unable to cause disease in humans may actually be used.)

One of the most exciting new possibilities is something called a human artificial chromosome (HAC.) Each chromosomes normally contains hundreds of millions of genes, plus the machinery necessary for replicating and optimally utilizing the chromosome and its genes. Scientists have recently succeeded in assembling only the working elements of human chromosomes, leaving out the normal complement of genes. They can then insert a single gene of their choosing into this tiny synthetic chromosome, which will greatly enhance the gene’s ability to function in human cells. The HACs will be embedded in tiny lipid spheres called liposomes, which do not provoke immune reactions by the recipient. The liposomes can be coated with just those viral proteins involved in recognizing and binding to human cells. Researchers will, in effect, have created artificial viruses, carrying a tiny human chromosome!

In gene therapy for human genetic disorders, the object is to deliver a good gene to cells lacking that specific gene, in order to save the cell. But the same technology can be used to deliver a gene to a cell that will result in that cell’s destruction. One of the most intriguing examples of this kind of gene therapy involves one of the most deadly forms of brain cancer: glioblastoma multiforme (GBM). GBM is resistant to virtually every weapon in the oncologist’s standard repertoire, including surgery, radiation and chemotherapy; mortality for this cancer is currently 100 percent.

Two members of Dr. Anderson’s team, Drs. Ken Culver and Michael Blaese, stumbled on a treatment strategy for GBM while trying to avoid a potential problem with retroviruses for delivering genes to human patients. One of the worst nightmares of physician-scientists like Culver and Blaese, who use retroviral vectors in gene therapy, is that somehow the retrovirus might insert into the host DNA near an oncogene - one of the genes involved in converting a normal cell to a cancer cell. Such insertions had been shown in mouse cells to result in occasional activation of an oncogene, leading to development of a tumor. This has not been observed in human cells, but the possibility that it might haunts everyone who uses them.

The scheme Blaese and Culver developed for this emergency, should it arise, was to embed a suicide gene into the delivery vector. They added a gene from the common virus Herpes simplex to the retroviral vector used to deliver the passenger gene. This Herpes gene, called Tk (for thymidine kinase), produces a protein capable of converting the harmless drug acyclovir into a deadly poison. They reasoned that if signs of cancer appeared in the cells treated with the retroviral vector, they could simply administer acyclovir to the patient; only those cells that had taken up the retroviral vector containing the Tk gene, plus possibly a few surrounding cells, would be killed.

It was a short jump to realize that their scheme could be converted into a general plan for attacking cancer cells. The final assembly of retroviruses takes place in something called a packaging cell, which makes endless copies of the retroviral genome and its passenger genes, and packages them into their normal protein coat (See Figure). Some of these packaging cells were introduced into GBM tumors in rats, using a highly sophisticated imaging system and a stereotaxic device for guiding a thin delivery needle through the brain to the center of the tumor. The packaging cells immediately began producing recombinant retroviruses and releasing them into the surrounding tumor. When the researchers treated the rats with acyclovir, all of them experienced rapid shrinkage of their brain tumors; eighty percent of them were completely cured. Clinical trials using this same technique in human GBM patients are now underway at several institutions around the world.

Another fascinating molecular medicine approach to treating cancer is something called adoptive immunotherapy. There is good reason to believe that many potential cancers are detected by the immune system and eliminated before they become a problem. The tumors arising in our bodies are the ones that somehow managed to avoid being spotted by our immune defenses. Immune responses are strongly stimulated by tiny chemical messages called cytokines, many of which are produced by T cells. In adoptive immunotherapy, a few of the patient’s tumor cells are removed, and genes encoding one or more cytokines are introduced into them. The cells are irradiated and returned to various sites throughout the body. The radiation prevents them from developing into new tumors, but the cytokines they produce greatly stimulate elements of the immune system, and the response they provoke will act against tumor cells anywhere in the body.

Similar strategies have been devised for treating AIDS. Scientists are exploiting the peculiarities of HIV replication inside cells to either stop it in its tracks, or to kill the cell it has infected. For example, HIV carries a copy of a gene that is used to initiate expression of its own genes inside a human T cell (Figure). Without expression of this gene, called TAT, the rest of the HIV genes cannot be "read" to produce HIV proteins. In one strategy about to reach the clinic, all of the body’s T cells will be provided with a suicide Tk gene whose expression is also initiated by TAT. If an individual becomes HIV-infected, acyclovir will be administered, and any HIV-infected T cells will be killed; Tk is not expressed in the other T cells, and so they are unharmed.

Another anti-HIV strategy under development involves introducing a faulty copy of a critical HIV gene into all T cells of the body. The gene called Rev produces a protein used by HIV to process messages read from its DNA. The corresponding Rev protein is multimeric, meaning it is composed of a number of individual Rev subunits grouped together in a single structure. In the dominant-negative strategy, a defective copy of Rev is introduced into T cell DNA. The protein produced from this gene will be incorporated into multimeric Rev, but renders it unable to carry out its function. Even one defective Rev monomer exerts its negative effect on the entire Rev multimer, bringing HIV production to a halt. A clinical trial using this strategy was recently initiated in Los Angeles involving five HIV-infected children.

What we have learned from gene therapy clinical trials so far is that genes prepared in the laboratory can be introduced into human cells, either in vivo or in vitro, and that the transferred genes can function in their new surroundings for periods of weeks to years, depending on the gene, the target cell and the delivery vector involved. Importantly, all of the data collected so far suggest that gene therapy as currently practiced is safe. Larger numbers of patients, treated over many more years, will be required to confirm this absolutely, but none of the so patients treated so far have suffered more than mild and transient side effects. The vast majority have suffered no ill effects at all.

The available evidence also suggests that the transferred genes function in a manner indistinguishable from their counterparts in normal human cells. There is thus good reason to believe that if enough genes can be transferred into enough cells, and if their expression can be maintained over a long enough period of time, for a great many inherited genetic diseases gene therapy will be able to correct the underlying defect. The new delivery vectors about to appear in the clinic will go a very long way toward making that happen. In the case of cancer, it is unlikely that gene therapy will ever entirely replace conventional methods of treatment, but almost certainly it will became a major new addition to the cancer specialist’s treatment armamentarium. For AIDS, where each knew treatment seems eventually to succumb to the ability of HIV to mutate to a resistant form, the gene therapy strategies currently being developed may be the best - and possibly the only - long-term hope.

Molecular medicine will be a major part of our lives in the new millennium. Genes are no longer a mystery. We know how genes work, and why they sometimes fail. The technology to isolate healthy genes from human DNA, and to deliver them to human cells, is now essentially routine. These surrogate genes function perfectly normally in their new cellular environment, particularly if they insert into the host cell DNA. Almost certainly, by the end of this century the remaining technical problems associated with gene therapy will have been overcome, and we will be witnesses to a truly profound revolution in human health care.

Like any medical revolution, gene therapy may seem frightening at first. We can all identify with the fear that must have been felt by the first mother whose child was deliberately injected with an altered form of a deadly disease-causing bacterium to ward off future disease. The early years of immunization must have seemed like black magic incarnate. Yet today’s toddlers are routinely immunized with a wide spectrum of such pathogens. The notion of introducing foreign genes into our DNA may seem equally frightening. But that in itself is nothing new: viruses have been inserting their DNA into ours for millions of years. We will simply be asking them to carry along a small piece of hope for our medical future.

Some common single-gene hereditary diseases.

Molecular medicine and cancer. The clinical trials indicated below represent a partial list of those currently in progress.

Malignant Melanoma Neuroblastoma

Prostate Glioma

Leukemia Breast

Ovarian Bladder

Colon Head and Neck Carcinoma

Myeloma Hodgkin’s Lymphoma

Renal carcinoma

WILLIAM R. CLARK is Professor and Chair, Emeritus, in the Department of Molecular, Cell and Developmental Biology of the University of California at Los Angeles. His research specialization is cell-mediated immunity, and he taught in the field of immunology for thirty years. He is the author of numerous scientific publications and academic textbooks, as well as At War Within: The Double-edged Sword of Immunity (1995); Sex and the Origins of Death (1996); and The New Healers: Molecular Medicine in the Twenty-first Century (1997), all published by Oxford University Press. He is currently writing a book on the molecular basis of aging.

http://www.wrclarkbooks.com