University Times

Pitt reports best gene delivery of protein missing in Duchenne Muscular Dystrophy

Pitt scientists have engineered perhaps the best gene therapy to date for Duchenne Muscular Dystrophy (DMD), the catastrophic muscle-wasting disease that strikes thousands of boys in the United States each year.

The Pitt team whittled down the largest gene ever found (14 kilobases) and packaged its most important components (less than 4.2 kilobases) into the smallest — and arguably the safest — viral vector ever used for gene therapy.

With this work the investigators demonstrated, for the first time, the minimal amount of the dystrophin gene needed to achieve functional muscle. The gene vector used was a genetically engineered form of adeno-associated virus, or AAV.

When injected into the calf muscles of mice unable to naturally produce the dystrophin protein, the "mini-gene" construct resulted in the expression of functional dystrophin protein in almost 90 percent of the muscle tissue treated. The dystrophin expression lasted at least one year — the duration of the experiments.

"This gives us great hope that we can use this gene therapy strategy in a larger animal model of DMD and eventually treat patients within several years," said Xiao Xiao, assistant professor in Pitt's molecular genetics and biochemistry department.

"With this work, Dr. Xiao and his laboratory have really taken the AAV vector to a new level in its application to the treatment of DMD," said Joseph Glorioso, chairperson of the Department of Molecular Biochemistry and Genetics at Pitt and vice president of the American Society of Gene Therapy. "At the same time, he has shown exactly what components of the dystrophin gene product are necessary for its localization and functional activity in muscle cell membranes."

The mini-gene AAV and a new technology recently developed in Xiao's laboratory may also be applicable to other genetic disorders involving unusually large genes, according to Pitt researchers.

Considered the most common genetic ailment, Duchenne Muscular Dystrophy strikes one of every 3,500 boys worldwide. It causes progressive muscle weakening and death, usually before age 20. No effective therapy exists for the disease.

Central to this research accomplishment was the work of research associate Bing Wang and senior research associate Juan Li. Wang and Li treated DMD mice with one of three mini-gene constructs. Each mini-gene construct contained a truncated version of the full dystrophin gene. Based on investigations performed previously in other university laboratories, Xiao's group selected certain sections of the dystrophin gene known to produce regions of the protein essential to its function. DMD mice treated with the mini-gene constructs showed evidence of stable, functional dystrophin, and their muscles did not degenerate, as typically is the case. Although all three mini-gene constructs were effective in terms of reducing signs of muscle regeneration, one mini-gene in particular was best at entering the greatest number of muscle cells.

In a next step, Xiao's laboratory will collaborate with other investigators in testing the AAV mini-gene construct in a dog model of DMD. Other research initiatives include developing the best ways of delivering the DMD mini-gene systemically, as opposed to performing localized injections.

Support for the research came from the National Institute of Arthritis and Musculoskeletal and Skin Diseases and from the Parent Project, a national organization founded by parents whose children have Duchenne or Becker Muscular Dystrophy.

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UPMC to test gene therapy for blood vessel growth

The University of Pittsburgh Medical Center is one of six centers participating in a Genzyme Surgical Products-sponsored clinical trial of a gene therapy designed to promote the growth of new blood vessels from existing blood vessels around blocked coronary arteries.

The Phase 1 study will evaluate the safety and efficacy of a gene therapy based on an engineered form of the gene known as hypoxia inducible factor one alpha. HIF-1a has been shown in laboratory studies to turn on the expression of many proteins associated with the natural process of blood vessel formation called angiogenesis.

While angiogenesis takes place when restricted vessels cause tissues to be deprived of oxygen, the process often is not sufficiently robust for adequate blood flow. The HIF-1a product will be administered to patients with coronary artery disease undergoing bypass surgery who have an area of the heart that is not suitable for surgical revascularization. It's hoped that the gene will stimulate a more stable and robust process of blood vessel formation.

Kenneth R. McCurry, assistant professor of surgery in the division of cardiothoracic surgery at Pitt's School of Medicine, is principal investigator at the Pittsburgh study site.

The HIF-1a trial is distinct among cardiovascular gene therapies in development because HIF-1a turns on a cascade of proteins associated with blood vessel formation rather than single proteins. In addition, Genzyme researchers have engineered HIF-1a to increase its potency.

The six centers involved in the study, including Pitt, will enroll 20-25 patients in the double-blind clinical trial in which patients, during their bypass surgery, will be randomized to receive either a placebo medication or one of four doses of the study medication consisting of an adenovirus vector with the gene that codes for HIF-1a.

Co-investigators working with McCurry are Bartley P. Griffith, the Henry T. Bahnson Professor of Surgery and chief of cardiothoracic surgery; Joon Sup Lee, assistant professor of medicine, division of cardiology; William P. Follansbee, professor of medicine, division of cardiology, and Diana Zaldonis, the study's coordinator.

In addition to Pitt, other study sites are Evanston Northwestern Medical Center in Evanston, Ill.; Washington Hospital Center in Washington, D.C.; Cedars Sinai Medical Center in Los Angeles; Case Western Reserve University, and Duke University Medical Center.

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Tamoxifen for breast cancer prevention has no effect on heart

Tamoxifen, a drug used to prevent breast cancer, does not increase cardiovascular risk in healthy women or in women with coronary heart disease, according to a study published in the Jan. 3 Journal of the National Cancer Institute.

The study, the largest clinical trial to assess the cardiovascular risk of tamoxifen, is part of the National Surgical Adjuvant Breast and Bowel Project Breast Cancer Prevention Trial.

"We found that cardiovascular event rates were not statistically significantly different between women assigned to tamoxifen or placebo, independent of pre-existing coronary heart disease," said Steven Reis, principal investigator in the study, associate professor of medicine and director of the Women's Heart Center at UPMC Health System's Cardiovascular Institute.

Cardiovascular events included fatal and non-fatal myocardial infarction, unstable angina and severe angina.

Between 1992 and 1997, 13,388 women at increased risk for breast cancer were randomly assigned to receive tamoxifen or a placebo. After four years, cardiovascular follow-up was available for 13,194 women. Of them, 1,048 had a prior history of clinical coronary heart disease such as heart attack or angina and 12,146 women reported no such history.

The study found that women who had coronary heart disease when they entered the study had a higher rate of cardiovascular events than the women without heart problems. However, within each group, there was no difference in the rate of cardiovascular events between those receiving tamoxifen and those receiving a placebo.

"Although the event rates in the group of women with a prior history of heart disease are greater than those observed in the group of women without a history of heart disease, these data demonstrate that assignment to tamoxifen did not affect cardiovascular events in this high risk population," Reis reported.