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By Denise Trunk

GAINESVILLE, Fla. - University of Florida researchers have used a common gel to successfully deliver gene therapy to the diaphragm muscle of mice with inherited respiratory weaknesses, enabling them to breathe easier.

The technique, described in the current issue of Molecular Therapy, could eventually lead to a method to correct genetic conditions in humans that cause diaphragm weakness and respiratory failure &Mac178; a leading cause of death in tens of thousands of patients with forms of muscular dystrophy, including Pompe's disease. Thousands of Americans with muscle-weakening diseases are placed on ventilators each year, according to the Muscular Dystrophy Association.

"The heart and diaphragm are two critical muscles for sustaining life," said study researcher Barry Byrne, M.D., Ph.D., director of the UF Powell Gene Therapy Center and associate chairman of UF's department of Pediatrics. "This approach essentially makes the genetic background of the muscle normal again and could significantly improve the quality of life for people on ventilators and those who care for them."

People with muscular dystrophy inherit a mutated gene unable to produce a critical enzyme, which causes their muscles to become increasingly weak as the disease progresses. In Pompe's disease, the weakness leads to respiratory failure and is fatal.

Cathryn Mah, Ph.D., the study's principal investigator and a UF research assistant professor of pediatrics, collaborated on the research with several UF scientists, including Byrne, a professor of pediatrics and of molecular genetics and microbiology, and Tom Fraites, Ph.D., a former doctoral student. The study was funded by grants from the National Institutes of Health, and the Florida and Puerto Rico affiliate of the American Heart Association.

In the current study, UF researchers applied a glycerin-based polymer gel they modified to contain corrective copies of the gene to the diaphragms of mice sick with a disorder that mimics Pompe's disease, thereby strengthening the muscle. This approach was the first time transferring a corrective gene to mouse diaphragm muscle cells was efficient and had a sustained effect, said Byrne, a member of the UF Genetics Institute.

Until now, scientists have been stymied by the mouse diaphragm's extreme thinness, which prevents direct injection of genes into the muscle. Infusing or injecting genes into veins or arteries also was problematic. In the two-part study, UF researchers first compared the gel-delivery method with a saline rinse used to deliver copies of a gene that stained the cells blue when the cell accepted them.

Next, they tested the gel's ability to deliver the gene therapy to the weakened muscle cells using the apparently harmless recombinant adeno-associated virus, or rAAV.

Byrne said the polymer gel was crucial to the study's success because it clung to muscle cells longer than the saline. The gel acted as a time-release mechanism, increasing the muscle's exposure to the rAAV, which was modified to deliver copies of the gene that produces the missing enzyme, acid alpha-glucosidase. The enzyme also is known as GAA, or acid maltase.

Scientists elsewhere have studied the effectiveness of a water-based gel in certain tissues but found it interfered with the stability of the virus commonly used to carry copies of therapeutic genes into cells. Other gels dissipate too quickly, before enough corrective genes can be transferred to yield beneficial results, researchers said.

"We tested this gel for ease of handling and for its ability to retain its consistency at certain temperatures," Mah said. "It had the right properties to make it useful for this study."

Cells normally must use GAA to break down the carbohydrate glycogen to create energy. The 24 mice in the study were missing the GAA enzyme, causing massive amounts of glycogen to accumulate in their muscle cells. This accumulation disrupts the cells' architecture, disabling their ability to power efficient breathing, Mah said. Researchers painted the gene therapy gel onto the underside of the thin diaphragm muscle in the live study mice, then took tissue samples six weeks later.

"Stain a section of muscle tissue and one can see the accumulated glycogen with a reagent that turns glycogen bright pink," Mah said. "After the treatment, we no longer saw the accumulated glycogen &Mac178; no bright pink granules in the cells &Mac178; meaning that the cells were able to break down the stored glycogen. In addition, we were able to detect that the GAA enzyme itself was present in those cells."

Because the treated cells eliminated the excess stored glycogen, the diaphragm could work normally and breathing was repaired, the researchers said.

Other studies to address the adult and infantile form of Pompe's disease is in various stages of development, Mah said. Byrne, who has a long-standing interest in the ailment, is currently participating in second-stage clinical trials to develop an enzyme replacement therapy to administer by infusion. Other researchers are in the early stages of working to modify the outer protein shell of the delivery virus so that it could be injected into a vein and target a specific area or organ.

Jeffrey Chamberlain, Ph.D., a professor of neurology and director of the Muscular Dystrophy Cooperative Research Center at the University of Washington School of Medicine, said the findings could have important implications for a variety of neuromuscular disorders as well as for Pompe's disease.

"The methods described by the Byrne lab are a new and exciting approach that have led to a significant level of gene delivery to this important muscle," Chamberlain said. "Unfortunately, it is not a very accessible muscle, and it has proved quite difficult to deliver new genes to this muscle for the purpose of gene therapy interventions. In the short term, this work will help advance research into studying these diseases, and in the long term should help accelerate the ability to treat a variety of very serious diseases."

International breast cancer experts convene to discuss state of diagnosis and treatment

By Melanie Fridl Ross GAINESVILLE, Fla. - Researchers are heralding improved methods of assessing a woman's breast cancer risk and of detecting and treating the disease early if it does occur. And while a cure remains a long way off, women have more reasons than ever to hold onto hope, say world leaders in clinical practice and breast cancer research who gathered recently at the 9th Annual Multidisciplinary Symposium on Breast Disease. Newer screening techniques such as breast magnetic resonance imaging mammography are particularly intriguing practitioners who treat women considered at high risk because they carry gene mutations linked with the disease's to development, researchers reported. Studies are evaluating whether MRI might better identify the earliest-stage malignancies in these patients. Other advances also are paving the way for optimizing prevention as well as improving diagnosis and better tailoring treatment. "At this time it's known that breast cancer is still the second-leading cause of mortality in American women, and it continues to frighten them," said Thomas Julian, M.D., associate director of the Allegheny General Breast Care Center in Pittsburgh and an associate professor of human oncology at Drexel University School of Medicine. "But certainly there's been a decrease in mortality thanks to screening mammography, adjuvant chemotherapy and hormonal agents, and we have a very strong ability to assess risk and intervene with therapy and treatments previously not available. I think this is an exciting time. "Obviously we would like to intervene at the earliest possible time," Julian added. "Imaging often gives us a very late start in beginning treatment. Usually by then (the cancer) has been progressing for at least five years." The symposium, sponsored by the University of Florida Health Science Center Jacksonville in association with the Susan G. Komen Breast Cancer Foundation and the UF Shands Cancer Center, is the brainchild of Shahla Masood, M.D., a professor and associate chair of the department of pathology at UF, and is designed to foster a multidisciplinary approach to breast cancer education and research. Mutations in the two breast cancer susceptibility genes, identified in 1994, are associated with up to an 80 percent chance of developing breast cancer before the age of 70 and up to a 60 percent chance of developing ovarian cancer. In contrast, women without an inherited predisposition for these cancers have a one in eight chance over their lifetime. Those who discover they are at high risk of developing a malignancy may opt to have their breasts or ovaries surgically removed to lower their risk. Others elect more frequent screenings or are vigilant about conducting self-exams. Researchers at the symposium discussed many promising technologies, including refined diagnostic tests and new medications that target cancer at the molecular level. Pathologists at UF and elsewhere also are working to identify biological markers that can predict breast cancer's behavior before the disease can be detected through standard breast self-examination or traditional mammography. "I think at the end of the day we'll be more successful at preventing breast cancer than treating it once it has developed," said D. Craig Allred, M.D., a professor of pathology at the Baylor College of Medicine in Houston and a featured speaker at the symposium. MRI mammography is still evolving, but experts said it often finds growths earlier, when the cancer is most curable. And they added that the false-positive rate for breast MRI will likely drop as more physicians are taught how to properly interpret the images, and as equipment is standardized. At the moment, breast MRI mammography is primarily used to gauge the extent of existing disease, to localize malignant tissue for biopsy and to screen people that have dense breast tissue or who are at especially high risk of developing cancer, Masood said. "I know the amount of energy and science that is incorporated into making breast MRI a complementary methodology for early breast cancer detection and also assessment of residual disease or extent of the disease," said Masood, a member of an international MRI Working Group. "I think that's incredibly important for people to understand, the promise that this technology will give not as a screening procedure but as a procedure that answers very specific questions. We need to use it appropriately. With respect to identifying early lesions, that again is in an investigational mode, but this is being used more and more, and there are trials under way that ultimately will bring light on these." For women at average risk, researchers said, traditional mammography should remain the first choice. Breast MRI can be so efficient at pinpointing small abnormalities that it uncovers many benign growths as well. As a result, women may end up facing needless worries and undergoing unnecessary tests or even surgery. And because these women often begin undergoing mammography while they are still in their 30s, they ultimately receive more radiation over time than women who begin screening later, potentially heightening their risk. Accurately assessing risk, meanwhile, is no small matter, Masood said, and many women have the false perception that they are at greater risk than they actually are. "Given the fact that none of us has a crystal ball, it's really the art of probability and statistics more than anything," Masood said. "Understanding all the factors involved is not as simple as it seems. One of the most important considerations in any kind of preventive mode is to try to be able to identify people that are at extreme risk for breast cancer, and that leads to importance of individualized risk assessment programs." Masood has established such a program at the Breast Center at Shands Jacksonville, which offers a comprehensive analysis of a woman's family history and genetic testing. Included in this service is the use of ductal lavage, a technology that enables physicians to sample breast epithelial cells to screen for early changes that signal cancer. "Fundamentally you just try to plan something that is absolutely designed for that specific patient," she said. "It's done in a confidential, comfortable manner, with people who really have the understanding of the science and psychology of breast cancer. All these things revolve around my single message, that the more educated we all become about the latest scientific information and the more we understand the psychology of the disease as a whole and become a patient advocate as a physician, the better we are going to serve our patients."