Gene Therapy: Clinical Gains Yield A Wealth Of Research Opportunities

investigation that will carry advances forward The transfer of genetic materials into humans to correct diseases--gene therapy--is a new medical enterprise, barely three years old in the clinic. But in the short time since a research team at the National Institutes of Health in Bethesda, Md., first treated a young girl's genetically compromised immune system with a transfusion of her own DNA-corrected white blood cells on Sept. 14, 1990, gene therapy has grown to command considerable public and scientific attention. While the public focuses on the small but growing number of clinical successes, scientists also predict an exponential growth in multi-disciplinary research opportunities linked to the young field. Disciplines that will be called on to contribute, these researchers say, include virology, immunology, cell biology, pathology, genetics, biochemistry, and molecular biology. "The fun thing about gene therapy is that it requires a wide variety of skills,"says Richard C. Boucher, director of the division of pulmonary diseases at the University of North Carolina, Chapel Hill. Planned trials there to treat cystic fibrosis received approval from NIH's Recombinant DNA Advisory Committee (RAC) in March. "It spans a lot of disciplines, and the groups that move fast are the ones able to make those disciplines interact." Two research institutes devoted exclusively to gene therapy studies were launched just this year--the first under James M. Wilson at the University of Pennsylvania in Philadelphia, and the other under Kenneth W. Culver at the Iowa Methodist Medical Center, Des Moines. Increasingly, academic institutions are organizing research efforts around the concepts of gene therapy. "Most universities now are setting up their genetic medicine or gene therapy divisions," says Culver, executive director of the Iowa center's Human Gene Therapy Research Institute and a member of the NIH team that designed the first gene therapy protocol. "And many of them are in the process of trying to recruit." The number of biotechnology companies specifically targeting related technologies, though still small, also is growing. One of the first entrepreneurial entries in this field was Genetic Therapy Inc., Gaithersburg, Md. This 1986 startup has been involved in developing the viral vectors used to deliver selected genes to brain tumor cells in several RAC-approved protocols proposed by NIH researchers. "The biotechnology infrastructure certainly is growing to support gene therapy, and this whole area won't move forward without that," says Gary Nabel, an associate professor of internal medicine and biological chemistry and a Howard Hughes Medical Institute investigator at the University of Michigan Medical Center, Ann Arbor. Nabel's research team has developed protocols for treating melanoma tumors and AIDS that have been approved by RAC and NIH. But Nabel also emphasizes that basic researchers in academic settings will have critical roles to play in the development of gene therapy. For example, he says, most early gene therapy protocols developed in nonprofit settings have tended to treat orphan diseases with little market potential. These rare-disease efforts, however, will be crucial in developing the fundamental tools that will lead to broader success. "These are diseases that may not be commercially attractive to pursue," Nabel says, "but that provide very important paradigms for what's to come." The early studies, in many cases, have been designed to demonstrate the basic principle of gene therapy, that it is possible to successfully infect specific cells or tissues with genes that will then express therapeutic proteins. They are what Boucher calls "proof-of-concept studies." This aspect of these proj-ects--that the early studies have implications beyond immediate efficacy--is one reason, he adds, that they've attracted such widespread attention. Still, there are important basic science hurdles to jump, researchers agree, before gene therapy can realize its full therapeutic potential. These include the development of improved vectors for delivery of genetic materials to target cells and tissues, better understanding and control of gene regulation, and more detailed knowledge about the immune system and its responses to the introduced genes and their products. Optimizing Viral Vectors For now, most gene therapy protocols have taken advantage of the ability of viruses to introduce and integrate their DNA into cells. "What we're really trying to do in gene therapy is to learn how to deliver genes, regulate their expression, and maintain them at specific sites in patients," says Nabel. "And that's something viruses learned to do long ago." The two types of viruses used most often in experiments are adenoviruses, which are often accompanied by cold symptoms in humans, and retroviruses, especially murine retroviruses. They each have advantages and disadvantages, and it is in this area-- development of vectors--that scientists say the most research needs to be done for the field to move forward. "There's a tremendous amount of vector development going on," says Edward Oldfield, chief of the surgical neurology branch of NIH's National Institute of Neurological Disorders and Stroke. Oldfield, along with Zvi Ram, Michael Blaese, and Culver, helped develop an experimental brain tumor protocol in the NIH clinics. "The biggest problem with gene therapy right now is one of targeted delivery, successful delivery to specific tissues," Oldfield says. Oldfield and colleagues take advantage of the fact that retroviruses target only dividing cells, such as tumor cells, while healthy brain cells are nondividing. The virus is engineered to carry the gene for producing thymidine kinase, a herpes simplex product, and is injected un- der general anesthesia directly into the tumor region. Tumor cells that accept the virus and its genetic payload become susceptible to the antiviral drug ganciclovir. So far, the procedure has produced encouraging results. For now, the characteristic of retroviruses that Oldfield's group is putting to use, the fact that they enter only dividing cells, is also a limitation in other areas. Researchers would like to be able to introduce genes into stem cells, the relatively quiescent marrow cells that give rise to blood cells. This is an area in which basic research in cell biology and other disciplines will be very important. "If we can genetically alter the hematopoietic stem cell sufficiently well to get good expression in peripheral cells in the blood," says Culver, "then that will accelerate the pace of gene therapy unbelievably. Theoretically, we could treat a variety of infectious diseases--HIV being one--and it would allow us a good vehicle for attacking autoimmune disease and for treatment of malignancy." A. Dusty Miller, associate member in the program in molecular medicine at the Fred Hutchinson Cancer Research Institute in Seattle, and a member of RAC, sees this area as crucial, also. Researchers there are working on genetic therapies for Gaucher's disease, HIV, and other diseases with genetic components. "If you could isolate a liver stem cell, let's say," Miller says, "you could then repopulate large portions of the liver [with genetically corrected cells]. So, understanding the basic biology of organs and developmental biology will be important." A number of research groups have chosen to work with cystic fibrosis, including Boucher at UNC and the University of Pennsylvania's Wilson. One reason this disease has attracted research attention is that direct delivery to the affected cells lining the lung or nasal passages is relatively straightforward. In the protocols for cystic fibrosis, an adenovirus is engineered so that it cannot cause illness and so that it carries the normal, functioning cystic fibrosis gene missing in the disease victims. Under Wilson's protocol, performed on a 32-year-old New Jersey woman September 3, the virus is introduced to the lungs using a bronchoscope. The virus infects the lung epithelial cells with the normal gene, after which the infected cells should begin producing the lacking protein. `Pluses And Minuses' Although the virus has been altered so that the patient will not contract a cold, per se, there are safety concerns associated with the procedure that Wilson intends to monitor. Such worries, specifically fears of an inflammation response in the lung, are among the reasons Boucher plans to treat only the nasal passages of patients in his procedure treating cystic fibrosis. He says adenoviruses, while efficient delivery vehicles for genes, have, "pluses and minuses"--they are not necessarily ideal vectors. "We're not comfortable that the safety aspects are well enough worked out in the lung yet," says Boucher. "So, the strategy is [to treat] the cells that line the nasal mucosa, which are virtually precisely like the ones in the lower lung, and they express the same defect. If there's inflammation, we can see it, and it's easier to treat." Worry over the safety of some current viral vectors is one reason investigators say that continued research in this area is critical to moving gene therapy forward. Penn's Wilson has also developed an ex vivo protocol to treat familial hypercholesterolemia, a genetic deficiency in which the liver lacks the receptor for low-density lipoprotein, leading to extremely high cholesterol levels and severe heart and circulatory problems. While at the University of Michigan, Wilson treated a 29-year-old Canadian woman for the deficiency by removing approximately a tenth of her liver cells, infecting those cells in the laboratory with a retrovirus carrying the missing gene, and then returning the cells to the woman's body through the portal vein leading back to the liver. The cells, it was hoped, would lodge in the liver and produce the necessary receptor. Since the operation, the woman's cholesterol levels have indeed dropped significantly. Wilson traces his interest in gene therapy to the case of Stormie Jones, a Texas girl who died at the age of 13 after surgeon Thomas Starzl at the University of Pittsburgh attempted a heart- and-liver transplant to correct her familial hypercholesterolemia. "I thought that there had to be a more elegant approach to treating this disease," Wilson says. "Her own liver cells functioned normally. They were only dysfunctional with respect to one gene. So, I kept thinking about how to utilize a gene- transfer technology." Getting genes to the sites in the body where they are needed will continue to be the biggest challenge for gene therapy, say researchers. Epithelial cells, those that line cavities and vessels in the body, will be relatively accessible, they say, but reaching other tissues, including many tumors, will be more difficult. "The difficulty is going to be delivery to solid tissues," says Oldfield. "If that can be overcome, the field will successfully develop new therapies, and it may occur relatively rapidly." Another limitation that will have to be confronted is the relatively small carrying capacity of current viral vectors, says Jeffrey Chamberlain, an assistant professor of human genetics at the University of Michigan. Chamberlain's team announced in August that they had cured Duchenne muscular dystrophy in transgenic mice through insertion of the correct dystrophin gene into embryos. But the embryos were sometimes damaged in the procedure. To treat people, a way of introducing the healthy gene to muscle cells will have to be found--and the gene involved is the largest gene yet known, Chamberlain says. Adenoviruses and retroviruses can accept genetic inserts of about 7,000 to 8,000 base pairs, after some viral removal. The dystrophin gene, by contrast, is about 2.5 million base pairs. Even the cDNA, although shorter, is about 14,000 base pairs, still double the capacity of current vectors. The difficulties of working with the dystrophin gene illustrate why basic research in gene regulation and other areas of basic biology must be part of progress in gene therapy, says Chamberlain. His lab is working to trim the gene down for insertion, while trying to identify and retain all sections of code necessary for functionality. The medical and scientific payoff, he says, could be dramatic and not only in terms of muscular dystrophy. "People have already shown that there are probably a lot of genes that, if you get them into the muscle, will secrete their protein products directly into the bloodstream," says Chamberlain. "So, finding ways to efficiently deliver genes to muscle could go well beyond the muscular dystrophies and be applicable to a lot of systemic diseases as well." As research continues in gene therapy and in understanding the immune system, scientists anticipate that recombinant vaccines for cancer, infectious diseases, and autoimmune disorders may eventually be added to the tool kit of genetic medicine. Already, the pace of progress in gene therapy has been encouraging--even astounding--to its advocates, and it shows few signs of slowing. "In 1987, I went to NIH to work there on gene therapy until it could be done outside the NIH, and then I was going to leave," says Culver. "Then, between 1987 and 1993, in that six-year period, that all happened. Not only did we start [therapies] in 1990, but it was just a couple of years later when it was reasonable to do it just about anywhere. That's quite remarkable."

Gene Therapy: Clinical Gains Yield A Wealth Of Research Opportunities

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